Nutritional Oncology

NUTRITIONAL ONCOLOGY Second Edition EDITOR-IN-CHIEF David Heber Center for Human Nutrition University of California, ...

Author: George L. Blackburn | Vay Liang W. Go | John Milner | David Heber

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NUTRITIONAL ONCOLOGY Second Edition

EDITOR-IN-CHIEF David Heber Center for Human Nutrition University of California, Los Angeles Los Angeles, California

SENIOR EDITORS George L. Blackburn Beth Israel Deaconess Medical Center Harvard Medical Center Boston, Massachusetts Vay Liang W. Go David Geffen School of Medicine University of California, Los Angeles Los Angeles, California John Milner Nutritional Science Research Group National Cancer Institute Rockville, Maryland

NUTRITIONAL ONCOLOGY Second Edition Editor-in-Chief

David Heber Center for Human Nutrition University of California, Los Angeles Los Angeles, California

AMSTERDAM • BOSTON • HEIDELBERG • LONDON • NEW YORK • OXFORD PARIS • SAN DIEGO • SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier

Academic Press is an imprint of Elsevier 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, California 92101-4495, USA 84 Theobald’s Road, London WC1X 8RR, UK This book is printed on acid-free paper. Copyright © 2006, Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, E-mail: [email protected]. You may also complete your request on-line via the Elsevier homepage (http://elsevier.com), by selecting “Support & Contact” then “Copyright and Permission” and then “Obtaining Permissions.” Library of Congress Cataloging-in-Publication Data Nutritional oncology / David L. Heber . . . [et al.].—2nd ed. p. cm. Includes bibliographical references and index. ISBN 0-12-088393-7 (alk. paper) 1. Cancer–Nutritional aspects. 2. Cancer–diet therapy. I. Heber, David. RC258.45.N886 2006 616.99′40654—dc22 2005059095 British Library Cataloging-in-Publication Data A catalogue record for this book is available from the British Library. ISBN 13: 978-0-12-088393-6 ISBN 10: 0-12-088393-7 For information on all Academic Press publications visit our Web site at www.books.elsevier.com Printed in the United States of America 06 07 08 09 10 11 9 8 7 6

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Contents

3. Genetics and Epigenetics in Cancer Biology 25

Contributing Authors xiii Preface xvii Introduction: The Principles of Nutritional Oncology xix

ANNA VELCICH, LIDIJA KLAMPFER, JOHN MARIADASON, HELENA SMARTT, SANDRA GUILMEAU, SANDRA MAIER, WANCAI YANG, JUDITH CHRISTMAN, BARBARA HEERDT, AND LEONARD AUGENLICHT

DAVID HEBER, GEORGE BLACKBURN, VAY LIANG W. GO, AND JOHN MILNER

Introduction 25 Fundamental Cell Biology of Malignant Transformation in the Colon 26 Gene Expression Profiling 29 Signaling Pathways and Transcriptional Factors in Intestinal Cell Maturation, Tissue Homeostasis, and Tumorigenesis 30 Epigenetic Mechanisms of Altered Gene Expression 41 Mitochondria: A Mechanism of Regulation and Integration 43 References 45

I. Biology of Nutrition and Cancer 1. Fundamentals of Human Nutrition 1 DAVID HEBER AND SUSAN BOWERMAN

Introduction 1 Classification of Food and Dietary Quality 1 Energetics and Obesity 2 Protein 2 Quality of Lipids, Fats, and Fatty Acids 4 Fatty Acid Structure and Classification 4 Quality of Carbohydrates 6 Functional Foods 8 Building Diets for Interventional Studies 10 Conclusion 10 References 10

4. Metabolic Networks in Cancer Cells 57 LASZLO G. BOROS AND WAI-NANG PAUL LEE

2.

Nutrigenomics and Nutrigenetics 15

Introduction 57 Metabolic Hypothesis of Tumor Growth 60 Applying Metabolic Control Analysis 64 Metabolic Profiling of Cell Proliferation and Differentiation 64 Relative Roles of Genomics, Proteomics, and Metabolomics in Cancer Research 65 Future Directions 66 References 66

JOHN MILNER

Introduction 15 Nutrigenetic Response 17 Nutritional Epigenetics 19 Transcriptomics and Microarray Technologies Beyond Nutrigenomics 21 Conclusions 22 References 22

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Contents

5. Energy Balance and Cancer

69

STEPHEN D. HURSTING, CONNIE J. ROGERS, SOMDAT MAHABIR, NOMELI P. NUNEZ, J. CARL BARRETT, SUSAN N. PERKINS, AND MICHELE R. FORMAN

Introduction 69 Physical Activity and Energy Balance 70 Exercise and Energy Balance in Carcinogenesis 70 Future Research Directions and Conclusions 76 References 79

II. Epidemiology of Nutrition and Cancer 6. Nutritional Epidemiology

85

MARJORIE MCCULLOUGH AND EDWARD GIOVANNUCCI

Introduction 85 Study Designs 85 Special Issues in Nutritional Epidemiology 92 Gene–Diet Interactions 93 Interpretation of Epidemiological Studies 94 Summary 94 References 94

9. Obesity and Cancer Risk

185

CATHERINE L. CARPENTER AND LESLIE BERNSTEIN

Introduction 185 Etiology of Obesity 185 Obesity and Cancer Risk of Specific Sites 187 Cancer Sites Suspected to Be Associated with Obesity 190 Summary 193 References 193

10. Nutrition and Tobacco-Related Cancers 199 KARAM EL-BAYOUMY, JOSHUA E. MUSCAT, AND DIETRICH HOFFMANN

Introduction 199 Tobacco Carcinogenesis 200 Nutrition and Tobacco-Related Cancers Summary 209 References 210

11. Alcohol and Cancer

202

219

ELISA V. BANDERA AND LAWRENCE H. KUSHI

7. Fruit and Vegetable Consumption and Cancer 97 STEPHANIE A. SMITH-WARNER, JEANINE GENKINGER, AND EDWARD GIOVANNUCCI

Published Reviews 100 Individual Cancer Sites 101 All Cancer 153 Methodological Issues 153 Summary 161 References 162

8. Are Whole Grains Protective Against a Variety of Cancers? 175 DAVID M. KLURFELD

Introduction 175 Whole Grains and Cancer 176 Whole Grains and Gastrointestinal Tract Cancers 177 Whole Grains and Nonintestinal Cancers 180 Conclusions 181 References 182

Introduction 219 Alcohol Consumption: Trends and Prevalence 219 Alcoholic Beverages Composition 220 Alcohol Metabolism 220 Challenges in Studying Alcohol and Cancer 223 Summary of the Epidemiological Evidence 224 Alcohol Drinking and Cancer Survival 260 Conclusions and Public Health Implications 264 References 265

12. Environmental Toxins, Nutrition, and Cancer 273 RAMUNE RELIENE AND ROBERT H. SCHIESTL

Introduction 273 Tobacco Smoke 273 Particulate Matter 276 Diesel Exhaust Particles 276 Arsenic 277 Chromium 278 Conclusions 279 References 279

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Contents

III. Biological Approaches to Investigating Nutrition and Cancer 13. Endocrine and Paracrine Factors in Carcinogenesis 283 DAVID HEBER AND PINCHAS COHEN

Introduction 283 The IGF Axis and Energy Balance 283 Estrogens and Cancer 285 Androgens and Cancer 287 Nuclear Receptor Superfamilies and Orphan Receptors 288 Lipid Sensor Receptors and Lipid Metabolism 289 Steroid and Xenobiotic Receptors 291 Feed-Forward Loops and Upregulation of Metabolic Enzymes 291 A Theoretical Model for Xenobiosis and Oxidant Stress in Cancer 291 References 292

14. Oxidation and Antioxidation in Cancer 297

Thiols in Redox Signaling and Control 311 Systems Act Independently to Increase the Specificity of Redox Control 314 Oxidative Stress and Carcinogenesis 314 Glutathione Plays Opposing Roles in Carcinogenesis 314 Thioredoxin is Overexpressed in Cancer 316 Summary 317 References 317

16. Principles of Tumor Immunology

321

BENJAMIN BONAVIDA

Introduction 321 Immune Surveillance of Tumors 323 Do Tumor Cells Express Tumor-Associated Antigens? 323 Immune Response to Cancer 324 Immunotherapy 325 Future Considerations for Successful Anticancer Immunotherapy 328 References 329

PAUL DAVIS, DAVID HEBER, AND LESTER PACKER

Introduction 297 Antioxidant Mechanisms Conclusion 304 References 304

17. Animal Models in Nutritional Oncology Research 333

298

15. Thiols in Cancer

JIN-RONG ZHOU

307

JASON M. HANSEN AND DEAN P. JONES

Introduction 307 Sulfur Has Multiple Biological Roles Affecting Cancer 307 Thiols in Cancer Chemoprevention 308 Most Dietary and Chemical Carcinogens Are Bioactivated to Reactive Mutagens 309 The GSH System Protects Against Cancer by Detoxification of Reactive Electrophiles 309 The Liver Functions to Maintain Systemic Cysteine Supply Through an Enterorgan GSH/Cysteine Cycle 310 Optimal Sulfur Amino Acid Intake in Humans Remains Uncertain 310 Dietary GSH Provides a Means to Decrease Risk from Direct-Acting Dietary Carcinogens 310 Thiols in the Diet Function to Protect Against Direct-Acting Carcinogens in the Diet 311

Introduction 333 An Overview of Animal Models 334 Organ-Specific Animal Tumor Models 335 Future Research Directions 343 Conclusions 344 References 344

IV. Gene–Nutrient Interaction and Cancer Prevention 18. The Challenge of Nutrition in Cancer Prevention 349 PETER GREENWALD AND JOHN MILNER

Overview of the Nutrition–Cancer Relationship Discovery 352 Development 357 Delivery 360 References 361

349

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19. Dietary Assessment

367

CATHERINE L. CARPENTER

Introduction 367 Study Design and Assessment 367 Methods of Dietary Assessment 368 Nutritional Biomarkers 370 Summary 373 References 373

20. Prostate Cancer

23. Colon Cancer

377

Introduction 377 International Epidemiological Observations 378 Prostate Anatomy, Physiology, and Pathogenesis of Prostate Cancer 378 Gene–Nutrient Interaction in Prostate Cancer 379 Hormones, Obesity, and Prostate Cancer 379 Conclusion and Future Directions of Research 388 References 388

393

DAVID HEBER AND GEORGE BLACKBURN

Introduction 393 Breast Anatomy and Physiology 393 Epidemiological Studies 394 Endocrinology of Breast Cancer 395 Exogenous Hormones and Breast Cancer Risk 396 Macronutrient Intakes, Diet, Energy Balance, and Breast Cancer Risk 397 Insulin Resistance and Breast Cancer: Proposed Mechanisms 398 Abdominal Obesity and Breast Cancer Recurrence and Survival 398 Alcohol and Folate Intake in Breast Cancer 399 Genetics, Nongenetic Factors, and Gene–Nutrient Interaction in Breast Cancer 400 References 402

22. Skin Cancer

412

423

LEO TREYZON, GORDON OHNING, AND DAVID HEBER

HOWARD PARNES, ASHRAFUL HOQUE, DEMETRIUS ALBANES, PHILIP TAYLOR, AND SCOTT LIPPMAN

21. Breast Cancer

Clinical Studies of Nutritional Effects Conclusions 417 References 418

405

HOMER S. BLACK

Introduction 405 The Nature of Ultraviolet Radiation 406 The Etiological Role of UV Radiation in Skin Cancer 407 Experimental Data on Nutritional Effects 408 UV-Induced Skin Cancer and Dietary Modification 409 Possible Mode of Action of Dietary Fatty Acids in Modulation of UV Carcinogenesis 411

Introduction 423 The Multistep Process of Colorectal Carcinogenesis 424 Inherited and Spontaneous Colon Cancer Nutritional Factors in Colon Cancer 426 Nutrient–Nutrient Interactions 430 Antioxidants and Phytochemicals 431 Conclusion 431 References 432

24. Gastric Cancer

425

437

NAI-CHIEH YUKO YOU AND ZUO-FENG ZHANG

Introduction 437 Background 437 Dietary Factors and Gastric Cancer 438 Micronutrients 441 Prevention Trials for Gastric Cancer 443 Conclusion and Future Perspectives 444 References 444

25. Pancreatic Cancer

449

DIANE M. HARRIS, MANISH C. CHAMPANERIA, AND VAY LIANG W. GO

Introduction 449 Background 449 Genes Altered in Sporadic Ductal Adenocarcinomas 450 The Importance of Diet 456 Metabolic Hypothesis for Pancreatic Cancer Future Directions 465 References 467

26. Bladder Cancer

465

475

ALLAN J. PANTUCK, RON LIEBERMAN, KELLY KAWAOKA, OLEG SHVARTS, AND DONALD LAMM

Background 475 Smoking and Bladder Cancer 475 Intrinsic Genetic Susceptibility Factors in Bladder Cancer 476 Chemoprevention of Bladder Cancer 477 The Future of Bladder Cancer Chemoprevention: A Rational Strategy for the Identification and Testing of New Agents 483 Conclusions 486 References 486

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27. Differentiation Induction in Leukemia and Lymphoma 491 SVEN DE VOS AND H. PHILLIP KOEFFLER

Introduction 491 Retinoids 492 Retinoid Therapy of Myelodysplastic Syndrome 497 Adult T-Cell Leukemia and Retinoids 498 Cutaneous T-Cell Lymphoma and Retinoids 498 1,25-Dihydroxyvitamin D3 498 Vitamin K 501 Ascorbic Acid 501 Future Perspective 501 References 502

V. Bioactive Food Components and Botanical Approaches to Cancer

The Translation Initiation Connection Conclusion 541 References 541

536

31. Calcium and Vitamin D 545 JOELLEN WELSH

Overview: Calcium, Vitamin D, and Cancer 545 Vitamin D and Calcium Metabolism: Interplay of Endocrinology and Nutrition 545 Cellular Mechanisms of Vitamin D Action 547 Modulation of Cell Turnover and Carcinogenesis by Vitamin D In Vivo: Animal Studies 550 Calcium and Cancer Risk 551 Epidemiological and Clinical Studies on Vitamin D, Calcium, and Cancer 551 Summary 553 References 553

28. Dietary Supplements in Cancer Prevention and Therapy 507 MARY FRANCES PICCIANO, BARBARA E. COHEN, AND PAUL R. THOMAS

Introduction 507 Prevalence of Dietary Supplement Usage Among People with Cancer and the General Population 507 Role of Dietary Supplements in Cancer Prevention and During Therapy 513 Conclusions 516 References 517

29. Dietary Fiber and Carbohydrates

521

MARÍA ELENA MARTÍNEZ AND ELIZABETH T. JACOBS

Introduction 521 Carbohydrate Classification and Metabolism Dietary Fiber 521 Carbohydrates and Cancer 523 Simple Sugars and Cancer 523 Glycemic Index and Cancer 524 Fiber and Cancer 524 Summary and Conclusions 527 References 528

30. Dietary Lipids

32. Soy Isoflavones

559

STEPHEN BARNES, JEEVAN PRASAIN, TRACY D’ALESSANDRO, CHAO-CHENG WANG, HUANG-GE ZHANG, AND HELEN KIM

Introduction 559 History of Soy 559 Food Materials Derived from Soybeans 559 Chemistry and Biosynthesis of Isoflavones 560 Soy and Human Health 565 Preclinical Experiments Exploring the Soy–Cancer Prevention Hypothesis 565 Genistein and Breast Cancer Models 566 Timing of Exposure to Soy in Humans 567 Soy, Isoflavones, and Preexisting Breast Cancer 567 Conclusion 568 References 569

521

531

HUSEYIN AKTAS, MICHAEL CHOREV, AND J.A. HALPERIN

Introduction 531 Fatty Acid Biosynthesis 531 Epidemiological, Prospective, and Experimental Studies 533

33. Selenium and Cancer Prevention

573

CLEMENT IP, JAMES MARSHALL, YOUNG-MEE PARK, HAITAO ZHANG, YAN DONG, YUE WU, AND ALLEN C. GAO

Introduction 573 Breast Cancer 573 Colon Cancer 574 Selenium and Prostate Cancer 574 Microarray Methods Searching for Potential Se Targets 577 Redox Modification of Proteins by MSA 577 Future Directions 578 Conclusion 579 References 579

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Contents

34. Glucosinolates

583

RADHA M. BHEEMREDDY AND ELIZABETH H. JEFFERY

Introduction 583 Occurrence and Distribution of Glucosinolates in Edible Plants 583 Epidemiological Investigations Relating Glucosinolates and Cancer Prevention 586 Chemoprevention Studies in Animal Models 587 Mechanisms of Cancer Prevention by Glucosinolate Derivatives 589 Clinical Studies with Glucosinolate Derivatives 591 Conclusion 593 References 594

35. Green Tea

597

JANELLE M. LANDAU, JOSHUA D. LAMBERT, AND CHUNG S. YANG

Introduction 597 Tea Chemistry and Oxidation-Reduction Properties 597 Inhibition of Carcinogenesis in Animal Models Mechanisms of Cancer Prevention 599 Bioavailability and Metabolism 601 Epidemiological Studies 602 Intervention Studies 603 Conclusions 604 References 604

598

HUANBIAO MO AND CHARLES E. ELSON

Introduction 629 Isoprenoids: Pure and Mixed 629 Part A: Postulated Isoprenoid-Mediated Action: Suppression of Farnesyl Protein Transferase Activity 632 Part B: Postulated Isoprenoid-Mediated Action: Suppression of HMG CoA Reductase Activity 634 Cancer Prevention 636 Summary 637 References 637

VI. Nutritional Assessment and Support of the Cancer Patient 645

DAVID HEBER AND N. SIMON TCHEKMEDYIAN

Introduction 645 Malnutrition and Cachexia in Cancer 645 Relative Roles of Anorexia and Metabolic Abnormalities 646 Cancer Anorexia 651 Approaches to the Treatment of Anorexia and Cachexia 653 Clinical Recommendations for Anorexia and Cachexia Prevention and Treatment 654 References 656

JOHN MILNER

37. Berries

38. Isoprenoids and Novel Inhibitors of Mevalonate Pathway Activities 629

39. Cancer Anorexia and Cachexia

36. Garlic 607 Introduction 607 Bioactive Food Components and Their Metabolites 608 Free Radicals and Garlic 609 Epigenomics and Garlic 609 Garlic and Transcriptomics 610 Garlic and Proteomics 610 Multiple Targets 611 Interaction with Other Food Components References 613

Bioavailability and Metabolism of Berry Bioactives 624 Conclusions and Future Directions 625 References 625

40. Weight Management in the Breast Cancer Survivor 661 ANNE MCTIERNAN

613

615

NAVINDRA P. SEERAM

Introduction 615 Structural Types of Berry Bioactives 615 Distribution of Berry Bioactives 618 Chemoprevention Studies with Berry Bioactives Dietary Intake of Berry Bioactives 623

622

Introduction 661 Obesity and Breast Cancer Mortality: Nonpatient Populations 661 Overweight, Obesity, and Breast Cancer Prognosis 662 Weight Gain After Diagnosis and Breast Cancer Outcomes 666 Obesity and Development of Comorbidities in Breast Cancer Patients 667 Obesity and Quality of Life in Breast Cancer Patients 667

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Contents

Potential Mechanisms for an Adverse Prognostic Effect of Obesity 667 Methods for Weight Loss and Maintenance for Breast Cancer Patients 672 Summary 674 References 674

VII: Nutritional Implementation Guidelines and Practice 45. Modern Statistical Methods in Clinical Nutrition 721 ROBERT M. ELASHOFF

41. Nutrition Support of the Adult Cancer Patient 679 HEIDI J. SILVER, EVE CALLAHAN, AND GORDON L. JENSEN

Introduction 679 Enteral Nutrition Support 679 Parenteral Nutrition Support 683 Meeting Nutrient Goals 687 Home Nutrition Support 688 References 691

42. Assessing Endocrine Effects of Cancer and Ectopic Hormone Syndromes 695 DAVID HEBER

Endocrine Abnormalities in the Cancer Patient 695 Paraneoplastic Syndromes 696 Ectopic Hormone Syndromes 697 Ectopic Pituitary, Hypothalamic, and Placental Hormones 698 Conclusion 701 References 702

43. Counseling the Cancer Survivor

705

LINDA A. JACOBS AND ELLEN GIARELLI

Introduction 705 Survivorship 706 Discussion and Future Directions References 710

710

44. Nutritional Support and Quality of Life 713 N. SIMON TCHEKMEDYIAN, DAVID CELLA, AND DAVID HEBER

Introduction 713 Definition of Quality of Life 714 Purpose of Quality of Life Measurement Quality of Life and Nutrition 718 Exercise and Quality of Life 718 Conclusion 718 References 719

716

Introduction 721 Description of the WINS Study 721 Design Issues 722 Important Statistical Issues 723 Conduct of the Trial 724 Statistical Analysis of the Trial Data 725 Results of the Statistical Analysis of the Trial Data Summary 736 Conclusions 737 References 737

727

46. Evidence-based Practice Management in Cancer Prevention and Treatment 739 GEORGE L. BLACKBURN

Introduction 739 Cancer Rates and Causation 739 Grading of Scientific Evidence 742 History of Evidence-based Medicine 743 Hierarchies of Evidence 746 The Roles of RCTs and Observational Studies 748 Methodology Advances in Observational Studies 748 Factors Promoting Evolution in Hierarchies of Evidence 748 Evolutions in Evidence-based Assessments of Research Quality 749 Adapting to Uncertainty 750 An Emerging State of the Art 750 References 751 Appendix 755

47. Dietary Guidelines in Cancer Prevention 757 JOHANNA DWYER

Introduction: History of Nutrition Guidelines in Public Health 757 Current Nutrition Guidelines 760 Individualizing the Radiant Pyramid 765 Cancer-Specific Guidelines 767 Nutrition Guidelines and the Future 772 Conclusions 777 References 777

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Contents

48. Dietary Interventions

779

LALITA KHAODHIAR AND GEORGE L. BLACKBURN

Upper Aerodigestive Tract Cancers (Head and Neck Cancers) 780 Esophageal Cancer 782 Gastric Cancer 783 Colorectal Cancer 784 Breast Cancer 788 Lung Cancer 790 Prostate Cancer 792 Conclusions 796 References 796

49. Future Directions in Cancer and Nutrition Research: Gene–Nutrient Interactions, Networks, and the Xenobiotic Hypothesis 807 DAVID HEBER, JOHN MILNER, GEORGE BLACKBURN, AND VAY LIANG W. GO

Introduction 807 Gene–Nutrient Interaction and the Xenobiotic Hypothesis of Cancer 808 Conclusion 809 Index

811

Contributing Authors

Susan Bowerman, Center for Human Nutrition, University of California, Los Angeles, Los Angeles, California 90095

Huseyin Aktas, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115 Demetrius Albanes, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, Maryland 20892

Eve Callahan, Vanderbilt University Medical Center, Nashville, Tennessee 37232

Leonard Augenlicht, Department of Oncology, Montefiore Medical Center, Albert Einstein Cancer Center, Bronx, New York 10467

Catherine L. Carpenter, Center for Human Nutrition, University of California, Los Angeles, Los Angeles, California 90095

Elisa V. Bandera, The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, New Brunswick, New Jersey 08901

David Cella, Institute for Health Services Research and Policy Studies, Northwestern University, Chicago, Illinois 60611

Stephen Barnes, Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama 35294

Manish C. Champaneria, Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California 90095

J. Carl Barrett, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892

Michael Chorev, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115

Leslie Bernstein, Keck School of Medicine, University of Southern California, Los Angeles, California 90089

Judith Christman, Department of Oncology, Montefiore Medical Center, Albert Einstein Cancer Center, Bronx, New York 10467

Radha M. Bheemreddy, Department of Food Science and Human Nutrition, University of Illinois, Urbana, Illinois 61801

Barbara E. Cohen, Division of Cancer Control and Population Sciences, National Cancer Institute, Bethesda, Maryland 20892

Homer S. Black, Department of Dermatology, Baylor College of Medicine, Houston, Texas 77042

Pinchas Cohen, Mattel Children’s Hospital, University of California, Los Angeles, Los Angeles, California 90095

George L. Blackburn, Beth Israel Deaconess Medical Center, Harvard Medical Center, Boston, Massachusetts 02115

Tracy D’Alessandro, Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama 35294

Benjamin Bonavida, Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, California 90095

Paul Davis, Department of Nutrition, University of California, Davis, Davis, California 95616

Laszlo G. Boros, Harbor UCLA Medical Center, University of California, Los Angeles, Los Angeles, California 90095

Sven de Vos, Department of Hematology and Oncology, University of California, Los Angeles, Los Angeles, California 90095

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Yan Dong, Department of Cancer Prevention and Population Sciences, Roswell Park Cancer Institute, Buffalo, New York 14263

Dietrich Hoffmann, Division of Cancer Etiology and Prevention, American Health Foundation, Valhalla, New York 10595

Johanna Dwyer, Tufts University School of Medicine, New England Medical Center, Boston, Massachusetts 02111

Ashraful Hoque, MD Anderson Cancer Center, University of Texas, Houston, Texas 77030

Robert M. Elashoff, Department of Biomathematics, University of California, Los Angeles School of Medicine, Los Angeles, California 90095 Karam El-Bayoumy, Penn State Cancer Institute, Pennsylvania State University, Hershey, Pennsylvania 17033 Charles E. Elson, Department of Nutritional Sciences, University of Wisconsin, Madison, Wisconsin 53706 Michele R. Forman, MD Anderson Cancer Center, University of Texas, Houston, Texas 77030 Allen C. Gao, Departments of Medicine and Pharmacology & Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263 Jeanine Genkinger, Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts 02115 Ellen Giarelli, University of Pennsylvania School of Nursing, Philadelphia, Pennsylvania 19104 Edward Giovannucci, Department of Nutrition and Epidemiology, Harvard School of Public Health, Boston, Massachusetts 02115 Vay Liang W. Go, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California 90095 Peter Greenwald, Division of Cancer Prevention, National Cancer Institute, Bethesda, Maryland 20892 Sandra Guilmeau, Department of Oncology, Montefiore Medical Center, Albert Einstein Cancer Center, Bronx, New York 10467 J.A. Halperin, Laboratory of Membrane Transport, Harvard Medical School, Boston, Massachusetts 02115

Stephen D. Hursting, Department of Human Ecology, College of Natural Sciences, University of Texas, Austin, Texas 78712 Clement Ip, Department of Cancer Chemoprevention, Roswell Park Cancer Institute, Buffalo, New York 14263 Elizabeth T. Jacobs, Arizona Cancer Center, University of Arizona, Tucson, Arizona 85724 Linda A. Jacobs, University of Pennsylvania Cancer Center, Philadelphia, Pennsylvania 19104 Elizabeth H. Jeffery, Department of Food Science and Human Nutrition, University of Illinois, Urbana, Illinois 61801 Gordon L. Jensen, Vanderbilt Center for Human Nutrition, Vanderbilt University, Nashville, Tennessee 37212 Dean P. Jones, Division of Pulmonary, Allergy, and Critical Care, Department of Medicine, Emory University, Atlanta, Georgia 30322 Kelly Kawaoka, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii 96813 Lalita Khaodhiar, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, 02115 Helen Kim, Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama 35294 Lidija Klampfer, Montefiore Medical Center, Albert Einstein Cancer Center, Bronx, New York 10467 David M. Klurfeld, Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland 20705

Jason M. Hansen, Department of Medicine, Emory University, Atlanta, Georgia 30322

H. Phillip Koeffler, Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, California 90095

Diane M. Harris, Center for Human Nutrition, University of California, Los Angeles, Los Angeles, California 90095

Lawrence H. Kushi, Division of Research, Kaiser Permanente, Oakland, California 94611

David Heber, Center for Human Nutrition, University of California, Los Angeles, Los Angeles, California 90095 Barbara Heerdt, Department of Medicine, Montefiore Medical Center, Albert Einstein Cancer Center, Bronx, New York 10467

Joshua D. Lambert, Department of Chemical Biology, Rutgers University, Piscataway, New Jersey 08854 Donald Lamm, BCG Oncology, Phoenix, Arizona 85032 Janelle M. Landau, Department of Chemical Biology, Rutgers University, Piscataway, New Jersey 08854


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Wai-Nang Paul Lee, University of California, Los Angeles School of Medicine, Los Angeles, California 90095

Susan N. Perkins, Division of Cancer Prevention, National Cancer Institute, Bethesda, Maryland 20892

Ron Lieberman, Division of Cancer Prevention, National Cancer Institute, Bethesda, Maryland 20892

Mary Frances Picciano, Office of Dietary Supplements, National Institutes of Health, Bethesda, Maryland 20892

Scott Lippman, MD Anderson Cancer Center, University of Texas, Houston, Texas 77030

Jeevan Prasain, Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama 35294

Somdat Mahabir, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892 Sandra Maier, Montefiore Medical Center, Albert Einstein Cancer Center, Bronx, New York 10467 John Mariadason, Department of Oncology, Montefiore Medical Center, Albert Einstein Cancer Center, Bronx, New York 10467 James Marshall, Department of Cancer Prevention and Population Sciences, Roswell Park Cancer Institute, Buffalo, New York 14263

Ramune Reliene, Department of Pathology, University of California, Los Angeles, Los Angeles, California 90089 Connie J. Rogers, Laboratory of Tumor Immunology and Biology, National Cancer Institute, Bethesda, Maryland 20892 Robert H. Schiestl, Department of Pathology, University of California, Los Angeles, Los Angeles, California 90089

María Elena Martínez, Arizona Cancer Center, University of Arizona, Tucson, Arizona 85724

Navindra P. Seeram, Center for Human Nutrition, University of California, Los Angeles, Los Angeles, California 90095

Marjorie McCullough, Department of Epidemiology and Surveillance Research, American Cancer Society, Atlanta, Georgia 30329

Oleg Shvarts, UCLA Medical Center, University of California, Los Angeles, Los Angeles, California 90095

Anne McTiernan, Fred Hutchinson Cancer Research Center, University of Washington, Seattle, Washington 98109

Heidi J. Silver, Vanderbilt Center for Human Nutrition, Vanderbilt University, Nashville, Tennessee 37232

John Milner, Nutritional Science Research Group, National Cancer Institute, Rockville, Maryland 20852 Huanbiao Mo, Department of Nutrition and Food Sciences, Texas Women’s University, Denton, Texas 76204 Joshua E. Muscat, Penn State Cancer Institute, Pennsylvania State University, Hershey, Pennsylvania 17033 Nomeli P. Nunez, Department of Human Ecology, College of Natural Sciences, University of Texas, Austin, Texas 78712 Gordon Ohning, Department of Digestive Diseases and Gastroenterology, UCLA Medical Center, Los Angeles, California 90095 Lester Packer, School of Pharmacy, Molecular Pharmacology, and Toxicology, University of Southern California, Los Angeles, California, 90089 Allan J. Pantuck, Department of Urology, University of California, Los Angeles School of Medicine, Los Angeles, California 90095

Helena Smartt, Montefiore Medical Center, Albert Einstein Cancer Center, Bronx, New York 10467 Stephanie A. Smith-Warner, Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts 02115 Philip Taylor, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892 N. Simon Tchekmedyian, UCLA School of Medicine, University of California, Los Angeles, Los Angeles, California 90095 Paul R. Thomas, Office of Dietary Supplements, National Institutes of Health, Bethesda, Maryland 20892 Leo Treyzon, Center for Human Nutrition, University of California, Los Angeles, Los Angeles, California 90095 Anna Velcich, Department of Medicine, Montefiore Medical Center, Albert Einstein Cancer Center, Bronx, New York 10467

Young-Mee Park, Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, New York 14263

Chao-Cheng Wang, Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama 35294

Howard Parnes, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

JoEllen Welsh, Department of Biological Sciences, University of Notre Dame, South Bend, Indiana 46556

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Yue Wu, Department of Cancer Chemoprevention, Roswell Park Cancer Institute, Buffalo, New York 14263 Chung S. Yang, Department of Chemical Biology, Rutgers University, Piscataway, New Jersey 08854 Wancai Yang, Montefiore Medical Center, Albert Einstein Cancer Center, Bronx, New York 10467 Nai-chieh Yuko You, Department of Epidemiology, School of Public Health, University of California, Los Angeles, Los Angeles, California 90095

Haitao Zhang, Department of Cancer Prevention and Population Sciences, Roswell Park Cancer Institute, Buffalo, New York 14263 Huang-Ge Zhang, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294 Zuo-Feng Zhang, University of California, Los Angeles School of Public Health, Los Angeles, California 90095 Jin-Rong Zhou, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115

Preface

stage of carcinogenesis, from the initiation of the cancer cell to its metastatic spread to other areas of the body. This text is divided into the following sections: First, the biology of nutrition and cancer including fundamentals of human nutrition, nutrigenomics and nutrigenetics, genetics and epigenetics, metabolic networks, and energy balance in cancer are described. Second, the evidence from the nutritional epidemiology of cancer is explored, including fruit and vegetable consumption, whole grains, obesity, tobaccorelated cancers, alcohol, and environmental toxins. Third, biological approaches to investigating cancer are described, including endocrine and paracrine factors, oxidants and antioxidants, thiols, tumor immunology, and animal models of cancer. Fourth, gene–nutrient interaction in cancer is explored, including an overview of the challenges facing the field, methodological issues in dietary assessment, and the evidence for specific cancers, including prostate cancer, breast cancer, skin cancer, colon cancer, gastric cancer, pancreatic cancer, bladder cancer, leukemia, and lymphoma. Fifth, bioactive food components are examined in detail, including dietary supplements, fiber and carbohydrates, dietary lipids, calcium and vitamin D, soy isoflavones, selenium, glucosinolates, green tea, garlic, berries, and isoprenoids. The sixth section deals with the nutritional assessment and support of the patient with cancer, including cancer anorexia and cachexia, weight management of the breast cancer survivor, nutritional support of the adult patient with cancer, endocrine effects of cancer and ectopic hormone syndromes, counseling the cancer survivor, and nutritional support in quality of life. Finally, the seventh section tackles the problem of implementing nutrition in guidelines and clinical practice, including an analysis of modern statistical methods, evidence-based practice management in prevention and treatment, dietary guidelines in

Nutritional oncology is an interdisciplinary field that bridges two areas of metabolic homeostasis—nutrition and cancer. Increasingly there are critical links being found between normal metabolic control, normal inflammation, normal genetic and epigenetic homeostasis, DNA repair, and the abnormal processes leading to the multistep process of carcinogenesis. The sequencing of the human genome and the rapidly expanding areas of molecular epidemiology, molecular endocrinology, and cellular signaling have provided new pathways for the study of the abnormal regulatory steps involved in initiating and maintaining carcinogenesis and the steps involved in the progression to metastatic spread. From these insights it is clear that there are many genetic predispositions that can interact with a Western dietary pattern in experimental models and in human populations to lead to common forms of cancer. The Western dietary pattern does not exist in isolation but is found nested within a sedentary lifestyle and an excess of dietary fats, starches, and refined carbohydrates often combined with excess use of tobacco and alcohol. In the past few decades, the ability to modify environmental factors through nutritional counseling and exercise instruction has been refined, but the ability to modify the behavior of populations through public health measures and dietary guidelines remains a major challenge. Medical oncologists, nurses, and dietitians will have an increasing opportunity to enhance their care of patients with cancer and those at risk by using the principles and information in this text. Basic scientists moving from a focus on molecular biology, genetics, and metabolism will have an opportunity to integrate basic science with questions important in clinical medicine. In these ways, the field of nutritional oncology aims to modify the environmental factors influencing the genetic changes and their expression at every

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Preface

cancer prevention, dietary interventions for cancer prevention, and the future of nutritional oncology with a detailed analysis of the prospects for personalized medicine based on gene–nutrient interaction. We and our contributors have attempted to provide a comprehensive summary of the field of nutritional oncology from basic principles to the growing edge of research. We would like to thank Susan Bowerman, M.S., R.D., for her excellent editorial assistance and contributions to the organ-

ization of the writing efforts of so many authors, and Ms. Tari Paschall and her colleagues at Academic Press for their patience and support of this important publication. David Heber, M.D., Ph.D. (Los Angeles, California) George L. Blackburn, M.D., Ph.D. (Boston, Massachusetts) Vay Liang W. Go, M.D. (Los Angeles, California) John Milner, Ph.D. (Rockville, Maryland)

Introduction: The Principles of Nutritional Oncology DAVID HEBER, GEORGE L. BLACKBURN, VAY LIANG W. GO, AND JOHN MILNER

WHAT IS NUTRITIONAL ONCOLOGY?

of all breast cancer patients (Finn and Slamon, 2003). This observation led to the development of targeted antibodybased therapies that bind to the receptor for this ligand and concentrate chemotherapies on receptor-bearing cells. Although this approach has resulted in some modest improvements in survival in patients with advanced breast cancer, it has minimally affected overall breast cancer morbidity or mortality statistically. On the other hand, the discovery of prostate-specific antigen (PSA) has led to a reduction in prostate cancer mortality largely through the employment of surgery and radiation at an earlier stage in the disease (Anscher, 2005). Just as both targeted therapy and early biomarkers have an important place in the war on cancers, so does nutritional oncology, in which nutritional factors become an important focal point for research and practice. The very idea that nutrition could affect a serious condition such as cancer was viewed to be both ridiculous and irrelevant by many oncologists 2 decades ago, and this attitude unfortunately persists today among many cancer clinicians and biologists. Increased exposure of medical students and other health professionals to the preventive and therapeutic potential of modern nutrition may ultimately result in an improved appreciation of the proper role of nutrition by those caring for cancer patients. The important role of nutrition in cancer is supported by many signal achievements in nutritional sciences over the last few decades as the result of the advances in human genetics applied to population studies; basic science studies in animals; cellular and molecular discoveries, which demonstrate the effects of nutrients on gene expression (Nowell et al., 2004); and even some limited intervention studies in humans demonstrating the effects of nutritional interventions on the incidence of

Nutritional oncology is an interdisciplinary field in which cancers are investigated as both systemic and local diseases originating by changes in the genome and progressing through a multistep process that may be modified by nutritional factors. These points of nutritional preemption are fundamental for the prevention of cancer, the quality of life of cancer patients, and the reduction in cancer recurrence. Traditional medical oncology in the middle part of the last century sought to kill cancer cells with radiation, surgery, and chemotherapy drugs through a disruption in normal cellular processes, with the premise that the cancer cell is more susceptible to these approaches than the normal tissues, and thus provided a window of therapeutic efficacy. Although this approach succeeded in producing significant cures in childhood leukemia, testicular carcinoma, and Hodgkin’s lymphomas, it has brought with it morbidity in patients being treated for more common forms of cancer (Albers et al., 2004; Bonadonna et al., 2005; Redaelli et al., 2005). Empowering the patient and being aware of the multitude of factors affecting quality of life often necessitates that physicians and other health care providers become involved with nutritional issues in cancer patients undergoing treatment. Finally, nutrition has a role to play, not simply in the primary prevention of cancer, but also in the prevention of cancer recurrence, which is of utmost importance in determining survival. Medical oncology in the postgenomic era has sought to identify targeted treatments that act at a single and/or process-specific site. There has been some success with this approach, as exemplified by the discovery of the overexpression of the Her-2/neu oncogene, which occurs in ~30%

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precancerous precursors such as colon polyps (Wallace et al., 2004). These advances have established the role of nutrition at many levels in the multistep process of carcinogenesis and set the stage for advances in this rapidly expanding interdisciplinary field. Integrating information from various sources, including population studies, basic science studies, and human intervention trials, is critical for scientists in nutritional oncology to develop testable hypotheses that can be used for personalized nutrition approaches for cancer prevention and therapy. These studies are able to examine changes in dietary patterns, physical activity, prevention and treatment of obesity, and the use of plant-derived bioactive substances (not as drugs but as supplements) to modify the natural history of cancer in such a way as to prevent its onset or reduce the risk of cancer recurrence following primary therapy with surgery, radiation, or chemotherapy. The National Institutes of Health has developed interdisciplinary study sections for the first time, which are devoted to the review of scientific applications dealing with diet and the chemoprevention of cancer. Nutritional oncology is an integrative medical specialty that embraces the best of orthodox and alternative approaches in combination while recognizing the value of proven therapies for cancer. This second edition will revisit and focus on the achievements in nutrition and cancer in relation to the basic mechanism and clinical applicability of the results from human nutrition, intervention, and clinical studies, as well as the evidence provided by the epidemiological case–control and cohort studies as reviewed by experts of their respective field. Through the influence of these scientific achievements on clinical practice, it is hoped that nutrition will soon be integrated into the routine care of the cancer patient in two situations. First, in the diagnosed cancer patient, nutritional support may help to minimize side effects of treatment and improve responses to therapy. Second, in the growing population of cancer survivors, nutrition may help to prevent cancer recurrence through the modulation of hormonal and inflammatory factors that promote angiogenesis and change the invasive and metastatic potential of tumors. However, despite the potential of nutrition, it is not a substitute for definitive therapies of proven curative or preventive benefit. Nutrition can be used as an adjunct but should not be used to delay or avoid appropriate diagnosis and treatment of common forms of cancer.

HISTORY AND FUTURE OF NUTRITIONAL ONCOLOGY In the early 1920s, prior to the development of chemotherapy, Shields Warren (1932) surmised that cancer killed its victims by depleting the tumor cell’s nutrients. This insight led several generations of researchers to investigate the effects of tumors on host metabolism. Nobel laureates,

including Warburg and the Coris, discovered much about carbohydrate metabolism while in search of the metabolic aberrations induced by cancer. Decades later, Waterhouse and others confirmed that there were clear abnormalities of carbohydrate metabolism in the cancer patient (Terepka and Waterhouse, 1956; Holroyde et al., 1975). Protein was also lost from the host and the “nitrogen trap” hypothesis was developed by Fenninger and Midler (1954) in the 1950s to explain the net transfer of protein from tumor to host. In the 1980s, investigations by a number of laboratories, using radioactive and stable isotope tracers of glucose, fatty acids, and amino acids, quantitatively identified increased glucose production, proteolysis, and lipolysis in cancer patients in the 1970s and 1980s (Heber et al., 1986). The discovery that tumor necrosis factor and other cytokines may account for these metabolic changes in the 1980s and 1990s led ultimately to the concept that the nutritional changes were part of an immune response of the host to the tumor (Shaw and Wolfe, 1987; Lang et al., 1992). Cytokines were shown to exert central nervous system (CNS) effects, which explained in part the anorexia associated with cancer in many patients. Furthermore, cytokines were demonstrated to stimulate tumor growth under certain circumstances. More recently, investigators have found that the Warburg hypothesis is supported by observations of early changes in the metabolic pathways operative in cancer cells, including an increased rate of aerobic glycolysis and a truncated Krebs cycle mediated by the C-myc oncogene and the hypoxiainducible factor, Hif-1, which induce the expression of specific glycolytic enzymes (Shaw and Wolfe, 1987). Studies of colonies of cancer cells growing as small spheres have indicated that cancer cell metabolism is also affected by oxygenation (Rempel et al., 1996; Younes et al., 1996). Tumor angiogenesis is relatively inefficient so that the interior of tumor masses carry out anaerobic metabolism, while a 150micron outer shell carries out enhanced aerobic metabolism. There is a decreasing gradient of oxygen toward the anaerobic core and a decreasing gradient of glucose while lactate increases. All of these metabolic changes based on the induction of specific genes are related to the need of the cancer cell to proliferate and avoid apoptosis. These stunning discoveries, together with an enhanced understanding of the biology of carcinogenesis, have led to the concept that cancer is a chronic disease of the genome. It begins with genetic changes during tumor cell initiation and progressively moves from tumor growth to tissue invasion to metastasis with many opportunities for nutritional modulation. Many food substances traditionally characterized as nutrients or micronutrients affect DNA replication and cellular differentiation. Studies of populations based on global prospects (World Cancer Research Fund, 1997) indicate that differences in


diet and lifestyle have pronounced effects on tumor incidence, prevalence, and natural history. The promise of nutritional oncology is that it will augment traditional therapies to prolong and improve the life of cancer patients. Clearly, the principles of evidence-based medicine must be developed, demonstrating that these interventions are worthwhile. Healthful changes in diet and lifestyle have benefits for other chronic diseases, including heart disease. Cancer patients often welcome the opportunity to become involved in a meaningful way in their own care. The existing evidence of the impact of nutritional medicine on the quality of life of cancer patients cannot be overestimated. Since the first edition of this text in 1999, the idea that there is a single gene pathway or single drug cure for cancer has given way to the general view that dietary/environmental factors have an impact on the progression of genetic and cellular changes in common forms of cancer. This broad concept can now be investigated within a basic and clinical research context for specific types of cancer. This book attempts to cover the available knowledge in this new field of nutritional oncology and has been written by invited experts.

GENE–NUTRIENT INTERACTION IN CANCER Unlike genetic diseases, such as sickle cell disease, cancer is not usually the result of a single mutation and most forms of cancer are unlikely to respond to a single agent targeting a single gene or gene product (Kaiser, 2005). Several genes must be defective before a normal cell will become an invasive cancer cell. Advances in genomics and molecular biology have revealed that alterations in three types of genes are responsible for carcinogenesis, including oncogenes, tumor suppressor genes, and stability genes. Mutations in the oncogenes and tumor suppressor genes increase tumor cell number by stimulating cell proliferation, inhibiting cell death or apoptosis, or providing increased nutrients by stimulating angiogenesis. Stability genes are DNA repair genes that induce the expression of enzymes that carry out multistep processes that repair both spontaneous and mutagen-induced alterations in DNA (Scherer et al., 2005). Mutations in these genes can occur in the germline, resulting in a familial predisposition to cancer or in somatic cells leading to sporadic cancers. Although inherited cancers are much less common than acquired cancers, germline mutations in DNA repair genes have been shown to confer an increased lifetime risk of cancer. Since only one allele is typically abnormal at birth in the BRCA1 in breast cancer patients, the second mutation must trigger the cancer (Kotsopoulos et al., 2005). Undeniably, lifestyle and diet can influence the onset of even these less common inherited cancers, as demonstrated for the BRCA1 mutation and breast cancer (Kotsopoulos et al.,

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2005). Although the BRCA1 mutations confer a 90% lifetime risk of breast cancer, the age at onset of breast cancer in a group of such women was shown to be delayed in those who were more physically active during adolescence (King et al., 2003). In addition, studies show that the effects of obesity on breast cancer in postmenopausal women are more easily demonstrated in those with a family history of breast cancer (Carpenter et al., 2003). Therefore, genes and nutrients are not in competition with each other, so a strong genetic predisposition would not necessarily mask or marginalize the response to diet and lifestyle. Rather, there is a gene–nutrient interaction so that genetic predisposition uncovers the influence of nutritional factors. This model of gene–nutrient interaction can be extended to the most prevalent human cancers, including breast cancer, prostate cancer, ovarian cancer, head and neck cancer, pancreatic cancer, and uterine cancer, among others. In these various cancers, the biochemical, pathological, and genetic markers are under active investigation for the discovery, development, and delivery of appropriate nutrition strategies for prevention or therapy. Each cancer differs in its natural history, in the factors promoting progression, and in the specific surrogate endpoint biomarkers that may be useful for its study. The emergence of nanotechnology and systems biology—and their application to cancer research—promises to provide a more detailed picture that will ultimately benefit the field of nutritional oncology to a greater extent than classical pharmacological fields looking for a single target, as opposed to multiple effects occurring simultaneously.

AGING AND CANCER With aging, the risks of many common forms of cancer increase as the amount of oxidant stress in cells increases (Wallace, 2005). The generation of oxygen radicals from aging mitochondria and the DNA damage that follows may mediate age-related changes in cancer incidence. Targeted antioxidation continues to be studied, but many phytochemicals have effects beyond this characteristic. Overnutrition and obesity increase oxidant stress and inflammation, while caloric restriction increases lifespan in sedentary experimental animals and delays the onset of age-associated diseases, including atherosclerosis, diabetes, and cancer. Caloric restriction is recognized to affect the levels of insulin, insulin-like growth factor 1 (IGF-1), and SIRT1, the mammalian analogue of Sir2 protein discovered in the budding yeast Saccharomyces cerevisiae. Aging is associated with increased rates of stress-induced apoptosis, and SIRT1 promotes cell survival by inhibiting stress-induced cellular apoptosis. In addition to caloric restriction, small molecules called STACs can stimulate SIRT1; the most potent of these recently discovered is resveratrol, a stilbene

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phytochemical found in grapes and wine (Wood et al., 2004). Although known as an antioxidant, these effects of resveratrol are independent of its actions as an antioxidant. The case of resveratrol is not an isolated one, as other phytochemicals, including lycopene from tomatoes and other flavonoids such as quercetin (found in fruits and vegetables), can stimulate DNA repair and demonstrate evidence of inhibiting carcinogenesis in various experimental model systems (Heber, 2004). In the first edition of this text in 1999, we originated the concept of the “Xenobiotic Hypothesis of Cancer,” in which the interactions beyond oxidation of phytochemicals trigger protective responses in cells, inhibiting the development of cancer. Research conducted since 1999 has demonstrated that two nuclear receptors function in this metabolic cascade to regulate detoxification and elimination. The constitutive androstane receptor (CAR) mediates the response to a narrow range of phenobarbital-like inducers. In contrast, the human steroid xenobiotic receptor (SXR) responds to many prescription drugs, environmental contaminants, steroids, and toxic bile acids (Xie et al., 2000). Consistent with their role as xenobiotic sensors, both receptors are expressed primarily in the liver and in the small intestine. The CYP 3A enzyme is responsible for metabolizing and clearing >60% of clinically prescribed drugs, and its induction plays a pivotal role in the clearance of hepatotoxic bile salts. CYP 3A gene expression is induced by a large variety of xenobiotic compounds through SXR activation. Two xenobiotic transporters, ABCB1 (or MDR1) and ABCC2 (or MRP2), are also upregulated in hepatocytes and intestinal cells by SXR activators, including a number of phytochemicals. Taken together, the xenobiotic activation of CAR and SXR induces a positive feed-forward loop that aids in clearance of foreign chemicals, including those from plant-based foods, and this process thereby resets the xenosensors for another round of signaling. Mammalian cells can mirror some events in the plant world, and plant cells contain many homologous genes and gene products. Humans evolved in a plant-based environment where restricted water and light or increased heat is known to lead to increased production of protective colorful phytochemicals that inhibit oxidation. Modern diets in developed countries are typically deficient in these compounds, as fruit, vegetable, and whole grain intakes have declined from those typical in traditional diets. The loss of these substances may lead to a reduction in the activity of cancer-preventive xenobiotic defense mechanisms as outlined earlier. In summary, massive evidence has accumulated since 1999 that indicates that cancer is the result of a genetic– environmental interaction where nutrition likely has a critical role. A strong association of common forms of cancer with diet and lifestyle suggests that primary prevention of

cancer may be possible, and large clinical trials are examining this possibility. It is now appreciated that the process of cancer progression and metastasis may also be modifiable through nutritional intervention. Enhanced understanding of the biology of angiogenesis, tumor invasion of surrounding stroma, stromal–epithelial cell interactions, and factors modifying metastatic spread create new opportunities to modify the natural history of human cancers.

OBESITY AND CANCER Obesity is the most common nutritional disorder in the United States today, resulting from an imbalance in energy expenditure and intake superimposed on a genetic predisposition to accumulate excess body fat. Obesity has been clearly associated with many forms of cancer, as documented in a number of epidemiological studies (McTiernan, 2005). In common with other chronic diseases, including cancer, it is rare for obesity to be the expression of a pure genetic or medical disorder. In most cases, obesity results from a combination of physiological, psychological, and environmental factors. The endocrine system, which is critically involved in many common forms of cancer, has also been shown to translate environmental influences into excess adiposity. Fat cells have been found to synthesize and secrete numerous peptides called adipocytokines, including leptin and vascular endothelial growth factor (VEGF), which promote angiogenesis, and interleukins, which in turn promote inflammation and oxidation. Obesity is an independent risk factor for heart disease and this increased risk is associated with increased levels of systemic markers of inflammation, including C-reactive protein (CRP), that promote inflammation in the heart (Rose et al., 2004). The possibility that such inflammation and oxidation could promote the development of common forms of cancer is being investigated for a number of common forms of cancer. Many classic hormones involved in obesity also play a role in the initiation and promotion of cancer at cellular, paracrine, and systemic levels. For example, reproductive hormones are critically involved in both obesity and cancer. In patients with primary anorexia nervosa and malnutrition, reproductive hormone secretion is reduced, resulting in both infertility and a reduced risk of breast cancer. At the same time, women who are very athletic during adolescence have a markedly reduced lifetime risk of breast cancer, as do women who have a later onset of puberty. On the other hand, excess secretion of reproductive hormones (e.g., estrogens) has been observed in overweight and obese women and implicated in the etiology of breast cancer and other reproductive tract cancers (Stephenson and Rose, 2003; Rose et al., 2004).

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It is well established that age at menarche, age at menopause, parity, and age at first full-term pregnancy are important determinants of breast cancer risk. Asian women living a traditional lifestyle have lower estrogen levels both before and after menopause, and these lower levels of estrogen have been considered important markers of the differences in breast cancer incidence observed in international epidemiological studies. Increased daily alcohol intake has been associated with increased risk of breast cancer and increased levels of endogenous estrogens in premenopausal women. A number of aspects of dietary intake, physical activity, and body composition affect endogenous estrogen levels. Increased estrogen production in women with excess fat compared to lean women is probably due to increased peripheral conversion of androstenedione to estrone by fat tissue. Obesity has been associated repeatedly with more advanced breast cancer at the time of diagnosis, higher rates of recurrence, and shorter survival times, even after controlling for tumor size and stage of disease at diagnosis. There is emerging evidence that insulin and insulinrelated hormones, such as IGF-1, may stimulate breast tumor growth. Upper body obesity and a family history of diabetes mellitus increase the risk of developing postmenopausal breast cancer. Therefore, overnutrition, through its stimulation of peptide and steroid hormones, may stimulate tumor growth and progression. Although there is less known about the influence of nutrition on prostatic cancer, ovarian cancer, uterine cancer, head and neck cancer, and pancreatic cancer, many hypotheses developed using the models of colon cancer and breast cancer are under study.

MICRONUTRIENTS, FIBER, AND CANCER One of the natural results of eating a modern diet is that along with excess calories and fat comes a deficiency of dietary fiber and the many micronutrients and phytochemicals found in fruits and vegetables (Heber and Bowerman, 2001). It has been estimated that only 10% of the U.S. population eats the five servings per day of fruits and vegetables recommended by the National Cancer Institute. Antioxidants such as vitamin E, vitamin C, and carotenoids are found in fruits and vegetables. In fact, the bright colors of most fruits and vegetables are indicators of their content of antioxidants, where alternating double bonds result in light absorption in the visible spectrum and bright colors. It has been postulated that these antioxidants were developed to protect plants from oxidation by the oxygen produced through photosynthesis. In fact, the concentration of antioxidants can be increased by raising plants under stressful conditions, such as reduced light or water.

Other, less visible phytonutrients, such as organosulfurs in garlic, isoflavones in soy beans, and limonene and geraniol derived from citrus fruit skins, have been reported to have anticancer effects. In some cases, it has been proposed that an accident of nature has resulted in the anticancer activity of substances originally evolved to protect plants from pathogens. Ancient humans ate a variety of fruits, vegetables, cereals, and grains. Although there are 50,000–100,000 edible plant species on earth, modern humans obtain >60% of all plant-derived proteins from a handful of plant species, such as corn, wheat, rice, and soy. Our genetic makeup was designed to complement a high intake of fruits, vegetables, cereals, and grains. The role played by micronutrients in controlling oxidant damage to DNA, mutagenesis, and carcinogenesis may be a significant factor in accounting for the protective effects of these foods observed in cancer epidemiology studies.

NUTRITIONAL ONCOLOGY: THE NEED FOR CONSENSUS Skeptics demand clinical trial evidence that increasing fruit and vegetable intake reduces cancer risk, and some early evidence from intervention trials is emerging to support this view. However, it is our view that such beneficial maneuvers as improving dietary patterns with more fruits and vegetables and combating obesity with increased physical activity and dietary modification are reasonable at our current state of knowledge and should not wait for the ultimate evidence from definitive clinical trials. While the research is being accumulated to support the use of nutritional interventions for the reduction of morbidity and mortality, cancer patients continue to demand consensus-based recommendations from their physicians to improve their quality of life and their sense of control over their lives. This book attempts not only to provide the theoretical and research basis for nutritional oncology, but also to offer the medical oncologist and other members of multidisciplinary groups treating cancer patients practical information on nutrition assessment and nutritional regimens, including micronutrient and phytochemical supplementation. The editors hope that this volume will stimulate increased research, education, and patient application of the principles of nutritional oncology.

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1 Fundamentals of Human Nutrition DAVID HEBER and SUSAN BOWERMAN

INTRODUCTION

beyond the classic understanding of food elements in terms of macronutrients. Diets are made up of numerous foods in varied proportions that are prepared in many ways but ultimately contribute energy to the body to support basic cellular energy needs. How that energy is provided as foods, which are made up of the basic macronutrients—protein, carbohydrate, and fat—plays a major role in determining the impact of dietary patterns on the risk of cancer. Within each category of macronutrient, there are marked differences in how different food sources are digested, absorbed, and metabolized. It is critical both for epidemiological investigations of the role of nutrition in the etiology of cancer and for efforts at modifying dietary behaviors in intervention trials to understand the impact of the specific food sources of these macronutrients.

There is accumulating evidence that foods, or their component bioactive substances (Kris-Etherton et al., 2004), offer benefits beyond basic nutrition. Macronutrients alone do not provide complete information about food intake. The types and sources of fats, proteins, and carbohydrates influence dietary patterns, which are composites of the component foods. Dietary patterns characterized as Western or prudent have been linked to the risk of common forms of cancer (Milner, 2002). Obesity, associated with Western diet patterns, has also been associated with an increased risk of common forms of cancer (Calle and Thun, 2004). A significant body of evidence suggests that increased consumption of fruits and vegetables along with a reduced intake of fats, especially animal fats, may be related to a reduced risk of cancer (Steinmetz and Potter, 1996; Fung et al., 2003). These effects have been attributed to changes in the pattern of dietary fats, phytochemicals from fruits and vegetables, fiber, and other elements in the diet. To date, there is no evidence that increasing the amount of fruits and vegetables without making other changes in the diet will have an impact on body fat or body weight (Tohill et al., 2004). However, ongoing research is investigating the impact of specific dietary patterns and physical activity on energetics and obesity. The contribution of specific macronutrients to satiety, especially the impact of increased dietary protein intake together with high-volume/lowcaloric density fruits and vegetables, may provide an additional key to controlling energy intake. To understand the rationale for the aforementioned proposed impacts of dietary patterns on cancer prevention and risk, it is necessary to go

Nutritional Oncology

CLASSIFICATION OF FOOD AND DIETARY QUALITY Foods can be grouped according to their content of macronutrients combined with their traditional use in an ethnic or societal geographic cuisine. Food groupings such as the basic four food groups [(1) Fruits and Vegetables, (2) Grains and Cereals, (3) Dairy, and (4) Meat, Beans, Nuts, and Cheese] classify foods of very different composition together such as red meat and ocean-caught fish or muffins and whole-grain bread. However, considerations of chemical structure, digestibility, metabolism, and functionality contribute to what is called the quality of the diet overall, as well as of individual macronutrients.

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Chapter 1/Fundamentals of Human Nutrition

The quality of dietary macronutrients, such as the ratio of n-3 fatty acids to n-6 fatty acids or of whole grains to refined grains complicates the basic considerations of the effects of diet on cancer incidence and efforts to organize dietary interventions designed to reduce cancer risk. An additional and important consideration is the presence of thousands of phytochemicals in fruits, vegetables, and whole grains, leading to their designation in some cases as “functional foods.” The term functional food indicates the presence of bioactive substances that affect physiology or cellular and molecular biology. The term quality implies that a value judgment is being leveled against a particular food. Although there is a hierarchical ranking of fats, carbohydrates, and proteins common to the disease prevention literature, the mechanisms underlying the differences among foods that provide protein, fat, and carbohydrate to the diet are simply analyzed in light of fundamental principles of nutrition. Taken together, these aspects of foods contribute to the assessment of the quality of the diet. The lowest quality foods are called junk foods because they are high in energy density but low in nutrient density (e.g., French fries). It has been said that there are no junk foods but simply “junk diets.” Obviously, if one combines enough junk foods, it leads to a junk diet.

ENERGETICS AND OBESITY Because humans are well-adapted to starvation and poorly adapted to excess energy intake, the balance of energy taken in as food and energy expenditure through metabolism and exercise has been emphasized as a major factor in cancer etiology (McCullough and Giovannucci, 2004). Evidence that obesity contributes to many common forms of cancer has also accumulated. Among species, animals with a smaller surface area such as mice burn more energy at rest per unit body mass than large mammals such as elephants. Children have higher metabolic rates than adults per unit body mass. Within the same species, there can be significant variations in metabolic rates. For example, the sedentary and overfed laboratory rat has a higher metabolic rate than the desert rat that is better adapted to starvation (Kalman et al., 1993). Energy efficiency may vary as well among humans. There is evidence that the post-obese adult may have a lower metabolism than a never-obese individual of the same size. However, the impact of excess energy is modulated by the location of excess body fat and its effects on hormones and inflammatory cytokines. Therefore, although energy balance is critical, it is not sufficient for an understanding of the effects of nutrition on cancer. Because obesity results from an imbalance of energy intake and expenditure, certain dietary factors have been identified as contributing to obesity. These include hidden

processed fats in foods, added refined sugars in foods, and a high glycemic load (GL) diet rich in refined carbohydrates. Therefore, the quality of the diet in terms of nutrient density can contribute to the tendency of a dietary pattern to promote the development of obesity in genetically susceptible individuals. Low-energy-density foods include all fruits and vegetables, generally because of their high water content. High-energy-density foods include red meats, fats, cheeses, pastries, cookies, cakes, ice cream, snack chips, some fruit juices, and refined grains.

PROTEIN Roles of Protein Proteins are involved in the growth, repair, and replacement of tissue and serve numerous functions in the body both as enzymes, antibodies, hormones, and regulators of fluid and acid–base balance and as integral parts of most body structures including skin, muscle, and bone. Within each cell, there is a continuous process of synthesis and breakdown of proteins in the body, referred to as protein turnover. The rate of protein turnover affects organ protein mass, body size, and ultimately the body’s protein and amino acid requirements (Matthews, 1999; Fuller, 2000). The amino acids are the basic units in protein metabolism, and all have the same basic structure with a central carbon atom with a hydrogen, an amino group, and an acid group attached to it. Attached to the fourth site on the carbon atom is a distinct side chain, which defines the amino acid. Cells link these amino acids in an infinite variety to create proteins that become metabolically essential compounds. There are 21 amino acids in human proteins, and 12 of these are synthesized by the body and are, therefore, known as nonessential amino acids. The nine remaining amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine) are either not made by the body or not made in sufficient quantities to meet needs and are, thus, termed essential amino acids. The proper balance and sufficient intake of essential amino acids, along with an adequate amount of nitrogen for the production of nonessential amino acids, is required for proper protein nutriture (Berdanier, 2000).

Protein Quality To manufacture proteins, cells require all the needed amino acids simultaneously with adequate nitrogencontaining amino groups for the manufacturing of the nonessential amino acids. The amino acid composition of a food can vary widely and determines the nutritional quality of the dietary protein. Foods that contain essential amino acids at levels that facilitate tissue growth and repair are known as

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Protein

complete proteins and are supplied in the diet from animal sources and soy protein. There are several ways of measuring protein quality. Most commonly, the term biological value is used, which is a measure of the efficiency of a given protein in supporting the body’s needs. Complete proteins have a high biological value, which is an expression of the amount of nitrogen absorbed relative to the amount of nitrogen retained by the body. All protein sources are compared with egg white, which provides the most complete protein and has the highest biological value of 100, indicating that 100% of the nitrogen absorbed is retained. A low concentration of one or more essential amino acids in a food lowers its biological value. With the exception of soy, most plant proteins are deficient in one or more essential amino acids and are, therefore, regarded as incomplete. However, the biological value of incomplete proteins can be improved by combining two proteins that are complementary so that those essential amino acids lacking or deficient in one protein are provided by the other when they are combined. In this way, the two complementary proteins together provide all the essential amino acids in ratios ideal for human protein utilization (Lappe, 1971; Kreutler and Czajka-Narins, 1987; Matthews, 1999). For example, the combination of corn (limited in lysine) with beans (limited in methionine) results in a high-quality protein food combination. Thus, the requirement for adequate essential amino acids can be met in a vegetarian diet by mixing foods of complementary amino acid composition (Lappe, 1971; Committee on Diet and Health, 1989; Berdanier, 2000).

Protein Requirements The U.S. food supply can provide an average of 102 g of protein per person per day (Nationwide Food Consumption Survey, 1984). Actual daily protein consumption ranges from 88 to 92 g for men and from 63 to 66 g for women (McDowell et al., 1994). Animal products provide ~75% of the essential amino acids in the food supply, followed by dairy products, cereal products, eggs, legumes, fruits, and vegetables (McDowell et al., 1994). The recommended daily allowance (RDA) for protein of high biological value for adults, based on body weight, is ~0.8 g/kg (National Research Council, 1989) or 0.36 g/lb. However, the RDA is set to meet the needs of a defined population group as a whole rather than indicating individual requirements. In a report concerning dietary reference intakes (DRIs), the acceptable macronutrient distribution range (AMDR) was set at 10–35% of total calories from protein. The AMDR is defined as the acceptable range of intakes for protein associated with reduced risk of chronic disease while providing intakes of essential nutrients (Barr et al., 2003). This range was largely set so that the intake of other macronutrients in the diet would be in an acceptable range.

There are many conditions in which extra protein is needed, including periods of growth, pregnancy, lactation, intense strength and endurance training and other forms of physical activity, and possibly in the elderly (Campbell et al., 1994). Additionally, there is research into the role of protein in the regulation of long-term energy balance, maintenance of body weight, and satiety (see the section “Role in Satiety,” later in this chapter).

Optimum Protein Intake Given the variation in the needs for protein throughout the life cycle, there is an individual optimal intake that exists based on lean body mass and activity levels. However, optimal intakes are difficult to determine based on the existing science base in nutrition. In 1977, Garza et al. studied a small number of healthy volunteers and found that 0.8 g/kg/day resulted in positive nitrogen balance. Subsequent studies in endurance athletes found that >1 g/kg/day was required for positive nitrogen balance (Tarnopolsky, 2004), and studies in weightlifters indicated that >2 g/kg/day was needed to achieve positive nitrogen balance (Tarnopolsky et al., 1992). Therefore, although the DRI, which is the same as the RDA, is set at 56 g/day for men consistent with the 1977 study, the allowable range of macronutrient intake is broad (10–35% of total calories), enabling some individual adjustment for optimal intakes both to control hunger and to provide support to lean tissues.

Role in Satiety In comparison with carbohydrate or fat, protein provides a stronger signal to the brain to satisfy hunger. Although the mechanism of action is unknown, it has been suggested that either single amino acids or small peptides enter the brain to elicit their effects, and several amino acids, including tryptophan, phenylalanine, and tyrosine, have been theorized to affect the hunger control mechanisms once they cross the blood–brain barrier. Small differences in the rates at which proteins release their amino acids into the bloodstream may also affect satiety. In subjects consuming highprotein meals compared with high-carbohydrate meals fed ad libitum, a voluntary reduction in energy consumption has been observed. Researchers in the Netherlands (Westerterp-Plantenga et al., 1999) have studied the effects of protein on hunger perceptions by studying two groups of subjects in a wholebody energy chamber under controlled conditions for >24 hours. Subjects were fed isocaloric diets, which were either high protein/high carbohydrate (protein/carbohydrate/fat, percentage of calories 30/60/10) or high fat (protein/ carbohydrate/fat, percentage of calories 10/30/60). Significantly more satiety was reported by subjects on the highprotein/high-carbohydrate diet. At the same time, hunger,

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appetite, desire to eat, and estimated quantity of food eaten were significantly lower in this group, with less hunger both during and after the high-protein meals. The level of protein in the diet may also have an impact on maintenance of body weight after weight loss. After following a very low energy diet for 4 weeks, subjects who consumed a 20% higher intake of protein than controls (15% vs 18% of energy) showed a 50% lower body weight regain, only consisting of fat-free mass, with increased satiety and decreased energy efficiency during a 3-month maintenance period (Westerterp-Plantenga et al., 2004). Similar studies have reported improved weight loss and fat loss in subjects consuming a high-protein diet versus a control diet (25% vs 12% energy from protein) ad libitum, because of a reduction in daily calorie intake of ~16% (Skov et al., 1999) and improved utilization of body fat with maintenance of lean body mass in subjects consuming 32% of energy from protein compared with controls who consumed 15% of calories as protein (Layman et al., 2003). A similar study comparing diets with 15% versus 30% of calories from protein found that although weight loss in the two groups was similar over the 6-week trial, diet satisfaction was significantly greater in those consuming the higher protein diet (Johnston et al., 2004). A meta-analysis of studies (Eisenstein et al., 2002) concluded that, on average, high-protein diets were associated with a 9% decrease in total calorie intake. Although the role of protein in affecting overall calorie intake and regulating body weight in comparison to fat and carbohydrate needs further investigation, the evidence is strong that protein affects hunger signaling mechanisms in the brain, induces thermogenesis, and contributes to the building and maintenance of lean body mass.

QUALITY OF LIPIDS, FATS, AND FATTY ACIDS Fats are a subset of the lipid family, which includes triglycerides (fats and oils), phospholipids, and sterols. Fats play an extremely important role in energy balance by enabling efficient storage of calories in adipose tissue. It is possible for the mythical 70-kg man to carry 130,000 calories in 13.5 kg of fat tissue compared with 54,000 calories stored as protein in an equivalent weight of lean tissue. This efficient storage is accomplished both by largely excluding water from adipose tissues and by storing energy in the chemical bonds of very long chain fatty acids. The typical fatty acids found in digested and stored fat range between 16 and 22 carbons in length. Triglycerides are the chief form of fat in the diet and the major storage form of fat in the body and are composed of a molecule of glycerol with three fatty acids attached. The principal dietary sources of fat are meats, dairy products,

poultry, fish, nuts, and vegetable oils, as well as fats used in processed foods. Vegetables and fruits contain only small amounts of fat, so vegetable oils are only sources of fat due to processing of vegetables. The most commonly used oils and fats for salad oil, cooking oils, shortenings, and margarines in the United States include soybean, corn, cottonseed, palm, peanut, olive, canola (low erucic acid rapeseed oil), safflower, sunflower, coconut, palm kernel, tallow, and lard. These oils contain varying compositions of fatty acids, which have particular physiological properties. The fats stored in tissues reflect to a certain extent the fats in the diet. Humans synthesize saturated fats (e.g., palmitic acid) from carbohydrates, but the polyunsaturated essential fats (linoleic and linolenic acids) must be taken in from the diet and the balance of these fats and the metabolic products of these fats reflect short-term and long-term dietary intake. There is a statistically significant but poor correlation between adipose tissue fatty acid profiles and dietary fatty acid intake as measured on a food frequency questionnaire (London et al., 1991). Red blood cell membranes change their composition in about 3 weeks. However, it is clearly possible to change the amount of fatty acids in tissues (Bagga et al., 1997), and total quantitative fatty acids can be altered by dietary intervention. The quality of fats in the diet is defined as that ratio of fatty acids that can be measured in plasma and tissues.

FATTY ACID STRUCTURE AND CLASSIFICATION Fatty acids are organic compounds composed of a carbon chain with hydrogens attached at one end and an acid group at the other. Most naturally occurring fatty acids have an even number of carbons in their chain, up to 24 carbons in length, with 18-carbon chains the most abundant fatty acids in the food supply. Saturated fatty acids are completely saturated with hydrogens. Those fatty acids lacking two hydrogen atoms and containing one double bond are monounsaturated fatty acids, and polyunsaturated fatty acids contain two or more double bonds in the carbon chain. The degree of saturation influences the texture of fats so that, in general, polyunsaturated vegetable oils are liquid at room temperature and the more saturated fats, most of which are animal fats, are solid. Some vegetable oils such as palm and coconut oils are highly saturated, and liquid oils can be hydrogenated in the presence of a nickel catalyst to produce a firmer fat. The nomenclature of fatty acids is based on location of the double bonds: an omega-3 fatty acid has its first double bond three carbons from the methyl end of the carbon chain. Similarly, an omega-6 fatty acid has its double bond six carbons from the methyl end. Fatty acids are also denoted by the length of the carbon chain and the number of double

Fatty Acid Structure and Classification

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Fatty Acids in Dietary Fats Lauric (C12:0) Saturated Myristic (C14:0) Palmitic (C16:0) Stearic (C18:0) Monounsaturated Oleic (C18:1 n-9) Unsaturated

Linoleic (C18:2 n-6)

Linolenic (C18:3 n-3) Polyunsaturated Arachidonic (C20:4 n-6) Eicosapentaenoic (C20:5 n-3) Docosahexaenoic (C22:6 n-3)

FIGURE 1 Fatty acids in dietary fats.

bonds they contain, such that linoleic acid is an 18 : 2 fatty acid, which contains 18 carbons and two double bonds. The human body requires fatty acids and can manufacture all but two essential fatty acids: linoleic acid and linolenic acid (18 : 3) (Figure 1). Omega-3 fatty acids possess anti-inflammatory, antiarrhythmic, and antithrombotic properties and have been shown to reduce the risk for sudden death caused by cardiac arrhythmias and decrease mortality from all causes in patients with coronary heart disease. Conversely, the omega6 fatty acids, obtained in the diet primarily from vegetable oils such as corn, safflower, sunflower, and cottonseed, are proinflammatory and prothrombotic. Fish and fish oils are the richest sources of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and are also present in algae. Green leafy vegetables, nuts, seeds, and soybeans contain the omega-3 fatty acid alpha-linolenic acid (AHA). The increased consumption in the United States of omega-6 fats from vegetable oils and grain-fed animals has led to a drastic increase in the ratio of omega-6 to omega-3 fatty acids in the diet from an estimated 1 : 1 in early human diets to a ratio exceeding 10 : 1 today (Simopoulos, 2001).

Fatty Acids as Cellular Signals Increasing evidence from animal and in vitro studies indicates that omega-3 fatty acids, especially the long-chain polyunsaturated fatty acids EPA and DHA, present in fatty fish and fish oils inhibit carcinogenesis (Karmali et al., 1984; Lindner, 1991; Rose et al., 1991; Tsai et al., 1998; Boudreau et al., 2001; Narayanan et al., 2001). Several molecular mechanisms have been proposed for the influences on the process, including suppression of arachidonic acid–derived eicosanoid biosynthesis (Okuyama et al., 1996; Rose and

Connolly, 1999), and influences on transcription factor activity, gene expression, and signal transduction pathways (Bartsch et al., 1999). The peroxisome proliferator–activated nuclear receptors (PPARs; α, δ, γ) are activated by polyunsaturated fatty acids, eicosanoids, and various synthetic ligands (Willson et al., 2000). Consistent with their distinct expression patterns, gene-knockout experiments have revealed that each PPAR subtype performs a specific function in fatty acid homeostasis. More than a decade ago, PPARα was found to respond to hypolipidemic drugs, such as fibrates. Subsequently, it was discovered that fatty acids serve as their natural ligands. Together with the analyses of PPARα-null mice, these studies established PPARα as a global regulator of fatty acid catabolism. PPARα target genes function together to coordinate the complex metabolic changes necessary to conserve energy during fasting and feeding. In the fatty acid metabolic cascade, PPARα activation upregulates the transcription of liver fatty acid–binding protein, which buffers intracellular fatty acids and delivers PPARα ligands to the nucleus (Wolfrum et al., 2001). In addition, expression of two members of the adrenoleukodystrophy subfamily of ABC transporters, ABCD2 and ABCD3, is similarly upregulated to promote transport of fatty acids into peroxisomes (Fourcade et al., 2001) where catabolic enzymes promote beta-oxidation. The hepatocyte CYP4A enzymes complete the metabolic cascade by catalyzing gamma-oxidation, the final catabolic step in the clearance of PPARα ligands (Lee et al., 1995). PPARγ was identified initially as a key regulator of adipogenesis, but it also plays an important role in cellular differentiation, insulin sensitization, atherosclerosis, and cancer (Rosen and Spiegelman, 2001). Ligands for PPARγ

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include fatty acids and other arachidonic acid metabolites, antidiabetic drugs (e.g., thiazolidinediones), and triterpenoids. In contrast to PPARα, PPARγ promotes fat storage by increasing adipocyte differentiation and transcription of a number of important lipogenic proteins. Ligand homeostasis is regulated by governing expression of the adipocyte fatty acid–binding protein (A-FABP/aP2) and CYP4B1(Way et al., 2001). In macrophages, PPARγ induces the lipid transporter ABCA1 through an indirect mechanism involving the LXR pathway, which in turn promotes cellular efflux of phospholipids and cholesterol into high-density lipoproteins (Chawla et al., 2001; Chinetti et al., 2001).

QUALITY OF CARBOHYDRATES As with proteins and fats, one can consider the quality of carbohydrates based on the source of the carbohydrates (fruits, vegetables, or whole grains vs refined grains and simple sugars) and their digestibility (soluble vs insoluble fiber). A quantitative approach to the analysis of dietary carbohydrate has been developed based on glycemic index (GI) and GL, as discussed in the following section.

Sugars and Starches Simple carbohydrates are present in foods as monosaccharides or disaccharides and are naturally present in such foods as fruit and milk. Glucose, fructose, and galactose are the most common monosaccharides in the human diet and combine to form the disaccharides sucrose (glucose + fructose), lactose (glucose + galactose), and maltose (glucose + glucose). Oligosaccharides are short chains of 3–10 sugar molecules, and the most common ones, raffinose and stachyose, are found in beans, peas, and lentils. Polysaccharides are starches that contain >10 sugar molecules, found in wheat, rice, corn, oats, legumes, and tubers. Starches form long chains that are either straight (amylose) or branched (amylopectin). Amylose and amylopectin occur in a ratio of about 1 : 4 in plant foods. Although there are several dietary factors that contribute to obesity, a dietary pattern that is rich in sugars and starches is considered a risk factor for obesity, whereas a high intake of nonstarch polysaccharides in the form of dietary fiber is considered protective (Swinburn et al., 2004). The typical Western diet is high in refined starches and sugars, which are digested and absorbed rapidly, resulting in a high GL and increased demand for insulin secretion. This in turn promotes postprandial carbohydrate oxidation at the expense of fat oxidation. Both acute (Ludwig et al., 1999; Febbraio et al., 2000) and short-term studies (Howe et al., 1996; Agus et al., 2000) indicate that a dietary pattern that produces a high glycemic response affects appetite and promotes body fat storage.

However, diets based on high-fiber foods that produce a low glycemic response can enhance weight control because they promote satiety, minimize postprandial insulin secretion, and maintain insulin sensitivity (Brand-Miller et al., 2002). This is supported by several intervention studies in humans in which energy-restricted diets based on low-GI foods produced greater weight loss than equivalent diets based on high-GI foods. Long-term studies in animal models have also shown that diets based on high-GI starches promote weight gain, visceral adiposity, and higher concentrations of lipogenic enzymes than isoenergetic diets with a low-GI, which are macronutrient controlled.

Soluble and Insoluble Fiber Insoluble dietary fibers such as cellulose and lignins are not digested in the intestine and pass in the stool intact. These fibers trap water, increase fecal weight, and accelerate transit time in the gastrointestinal tract, thus promoting regularity. Soluble carbohydrates such as pectin, gums, and β-glucans are digested by bacteria in the colon. These fibers delay glucose absorption and are able to bind bile acids in the gastrointestinal tract, thus reducing serum cholesterol levels. Ancient humans ate a great deal of fiber, estimated at >50 g/day, whereas modern humans consume on average 10–15 g/day. Epidemiological evidence suggests that the risk of colorectal and breast cancers may be decreased by increasing the intake of foods high in fiber such as vegetables, fruits, cereals, and whole grains (Hill, 1998; Jacobs et al., 1998; Martínez et al., 1999), but the findings have not been entirely consistent. Based on 25 years of follow-up data for men in the Seven Countries Study, a 10-g/day increase in fiber intake was associated with a 33% lower 25-year colorectal cancer mortality risk (Jansen et al., 1999). However, 16 years of follow-up data from the Nurses’ Health Study showed no association between dietary fiber intake and colorectal cancer risk in women (Fuchs et al., 1999). In the prospective Cancer Study II Nutrition Cohort, higher intakes of plant foods or fiber were not related to lower risk of colon cancer, but it was concluded that very low intakes of plant foods may increase risk (McCullough et al., 2003). Diets high in fiber have been hypothesized to protect against breast cancer, perhaps because of inhibition of the intestinal reabsorption of estrogens normally excreted in the bile (Goldin et al., 1982). In a meta-analysis of 10 case–control studies, a statistically significant reduction in breast cancer risk with a 20-g/day increase in dietary fiber was observed (Howe et al., 1990), yet prospective studies have been unevenly supportive. In a Canadian prospective study including 519 cases (Rohan et al., 1993), a marginally significant inverse association between dietary fiber and breast cancer risk was seen. In a subsample of >11,000 postmenopausal women in the Malmo Diet and Cancer cohort,

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Quality of Carbohydrates

high fiber intakes were associated with a lower risk of postmenopausal breast cancer for the highest quintile of fiber intake compared with the lowest quintile, and the combination of high fiber and low fat intakes had the lowest risk (Mattisson et al., 2004). In other prospective cohorts, however, with 344 and 650 cases, no suggestion of a protective effect was found (Graham et al., 1992; Verhoeven et al., 1997). In the Nurses’ Health Study, the association between total dietary fiber intake and subsequent breast cancer incidence (1439 cases) was very close to null (Willett et al., 1992). The evaluation of the role of dietary fiber in cancer prevention is complicated by the varying composition of fiber from different food sources, variations in fiber measurement techniques and dietary assessment, and the possible effects of other substances in high-fiber foods, such as micronutrients and phytochemicals. There are potentially numerous antimutagenic compounds in fruits, vegetables, and whole grains, and fibers may provide one of the simplest mechanisms in their ability to reduce mutagen uptake (Ferguson et al., 2004).

Glycemic Index and Glycemic Load Conventional approaches to weight loss have focused on decreasing dietary fat because of its high-calorie density. However, the relationship between dietary fat and obesity has been brought into question for several reasons. Low-fat diets have been shown to produce only modest weight loss, and prospective epidemiological studies have not been able to consistently correlate dietary fat intake with weight. Despite a decrease in fat consumption as a percentage of total calories and widespread availability of low-fat and fat-free foods, obesity prevalence in the United States has risen dramatically since the 1970s (Putnam and Allshouse, 1999). At the same time, carbohydrate consumption has increased, and most of this increase has been in the form of refined starches and concentrated sweets with a high GI and/or GL. In 1981, Jenkins et al. introduced the GI as a system for classifying carbohydrate-containing foods based on their effect on postprandial glycemia. The glycemic response to the ingestion of 50 g of available carbohydrate from the test food is compared with the response from the ingestion of 50 g of the reference food (glucose or white bread), and the GI is expressed as the area under the glucose response curve for the test food divided by the area under the curve for the standard, multiplied by 100. However, the amount of carbohydrate in 50 g of a given food will vary depending on the food, and this observation led to the introduction of the concept of GL. This is an expression of the GI of the food, multiplied by the carbohydrate content of the food, and takes into account the differences in carbohydrate content among foods (Liu, 1998). Foods with a high index but relatively

low total carbohydrate content, such as carrots, have a low GL. In general, fruits, nonstarchy vegetables, nuts, and legumes have a low GI (Table 1). The intake of high-GI/GL meals induces a sequence of hormonal changes, including an increased ratio of insulin to glucagon, which limit the availability of metabolic fuels in the postprandial period and promote nutrient storage (Ludwig, 2002) and would be expected to stimulate hunger and promote food intake. Short-term feeding studies have demonstrated less satiety and greater voluntary food intake after consumption of high-GI meals as compared with

TABLE 1

Glycemic Index and Glycemic Load (GL) Values for Foods

Low GI ( 20 mM, with total SCFA content of > 200 mM (Roediger, 1980; Cummings, 1983). It has been demonstrated that these SCFAs play a critical role in colonic epithelial cell maturation pathways in vivo (Glotzer et al., 1981; Roediger, 1988; Harig et al., 1989; Augenlicht et al., 1999a). This is likely through their role as the principal energy source of colonic epithelial cells (Roediger, 1980, 1982), where they enter the mitochondria, perhaps through porin (Schulz, 1985), and undergo β-oxidation. Despite the mitochondrial abnormalities that characterize colonic carcinoma cells, we have shown that sodium butyrate significantly increases mitochondrial gene expression and mitochondrial enzymatic activity without altering expression of nuclear genes encoding proteins destined for mitochondrial import. Furthermore, inefficiently oxidized derivatives of butyrate are ineffective in modulating mitochondrial function, again suggesting that the effects of butyrate are linked to its β-oxidation (Heerdt and Augenlicht, 1990, 1991). In mice, the homozygous deletion of the gene for short-chain acyl dehydrogenase, an initial enzyme of mitochondrial SCFA oxidation, abrogates apoptosis in the colonic mucosa, which is further evidence for the necessity of β-oxidation of butyrate in triggering an apoptotic pathway in the mucosa (Augenlicht et al., 1999a).

A critical component in many apoptotic pathways is disruption of the ∆ψm and the consequent release of factors from the mitochondria (Hennet et al., 1993, 1998, 2000; Decaudin et al., 1997; Ferri and Kroemer, 2001). We have shown that an intact ∆ψm is required not only for butyrateinduced apoptosis but also for its initiation of growth arrest of colonic epithelial cells (Heerdt et al., 1994, 1996, 1997, 1998, 2000). Moreover, there is a link between the intrinsic level of the ∆ψm of a cell and the extent to which butyrate induces cell cycle arrest and apoptosis (Heerdt et al., 2003), making it likely that the ∆ψm plays a critical role in integrating proliferation and apoptotic pathways (Heerdt et al., 1997, 1998) and as a consequence impacting the probability of colonic tumor formation and progression (Augenlicht and Heerdt, 2001). As will be reviewed later, there is ample evidence suggesting that another major determinant of the ∆ψm is dietary intake of PUFAs, thereby linking diet directly to this biochemical function at the heart of probability of tumor development.

Dietary Polyunsaturated Fatty Acids, Mitochondrial Function, and the Modulation of Butyrate Induction of Cell Maturation Extensive epidemiological evidence has demonstrated an inverse relationship between the risk of colon tumorigenesis and fish consumption, particularly fatty cold-water fish, which are rich in the n-3 PUFAs eicosapentaenoic acid (EPA; C20: 5n-3), and docosahexaenoic acid (DHA; D22: 6n-3) (Fernandez et al., 1999; Larsson et al., 2004). Clinical studies have shown that dietary n-3 PUFAs decrease intestinal proliferation in healthy subjects (Bartram et al., 1993) and in those at risk for colon cancer (Anti et al., 1992, 1994) and inhibit the promotion and progression of colonic carcinogenesis in model systems (Paulsen et al., 1997; Chang et al., 1998). n-3 fatty acid–mediated protection against colonic tumorigenesis has been linked to inhibition of proliferation (Ramos and Colquhoun, 2003), promotion of differentiation (Chang et al., 1997), decreased expression of Bcl-2 (Chang et al., 1997; Hong et al., 2003), increased production of ROS, and induction of apoptosis (Hong et al., 2002). In contrast, high consumption of dietary n-6 PUFAs, such as linoleic acid (LA; C18: 2n-6), found in corn, safflower, and sunflower seed oils, is linked to inhibition of apoptosis and the promotion and progression of colonic tumors (Nelson et al., 1988; Iigo et al., 1997; Meterissian et al., 2000; Rao et al., 2001). n-3, but not n-6, PUFAs enhance the effects of butyrate on colonic epithelial cells. The combination of n-3 fatty acids and dietary fiber significantly decreased the formation of aberrant crypt foci induced in rats by AOM (Coleman et al., 2002). In vitro, butyrate-mediated production of ROS,

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Mitochondria: A Mechanism of Regulation and Integration

lipid peroxidation, ∆ψm dissipation, cytochrome c release, caspase-3 and caspase-9 activation, PARP cleavage, and apoptosis were enhanced by n-3 PUFAs, while expression of antiapoptotic Mcl-1 protein was decreased (Hong et al., 2002; Hofmanova et al., 2004; Sanders et al., 2004). This link of the effects of butyrate to n-3 fatty acids may be a key mechanism by which dietary PUFAs modulate tumor formation. Approximately 80% of the lipids in mammalian mitochondrial membranes are phospholipids, with phosphatidylcholine (PC), phosphatidylethanolamine (PE), and diphosphatidylglycerol (cardiolipin [CL]) accounting for ~45%, 35%, and 20%, respectively, of total mitochondrial phospholipids. However, CL is unique in that it is exclusively found in the mitochondria, where its synthesis is completed in the inner mitochondrial membrane (Gennis, 1989). As a specific lipid component of the mitochondrial inner membrane, CL is integrated into the quaternary structure and is essential for the activity of many of the mitochondrial enzyme complexes involved in electron transport (Fry and Green, 1980, 1981; Hoch, 1992; Jiang et al., 2000; Haines and Dencher, 2002). It is also involved in the retention of cytochrome c in the intermembranous space and its release during apoptosis (Ott et al., 2002; Poot et al., 2002; Hardy et al., 2003), the function of the adenine nucleotide translocase (Schlame et al., 1991; Ostrander et al., 2001), and the mitochondrial import and activation of nuclear-encoded peptides (Hoch, 1992). Although the fatty acyl groups of CL in mammalian tissues are almost exclusively 18-carbon fatty acids, the majority of which are LA (18:2n-6) (Schlame et al., 1993), CL has the most diet-responsive and changeable fatty acid composition among phospholipids (Watkins et al., 1998). There is extensive evidence that the fatty acyl chain composition and saturation index of CL can be modified both in vitro and in vivo by n-3 and n-6 fatty acids (Robblee and Clandinin, 1984; Malis et al., 1990; Groden et al., 1991; Yamaoka-Koseki et al., 1991; Berger et al., 1992; Watkins et al., 1998; Gaposchkin et al., 2000; Chapkin et al., 2002; Hong et al., 2002; Ramos and Colquhoun, 2003; Valianpour et al., 2003). Compared with n-6 fatty acids, n-3 fatty acids decrease the amount of LA and increase the quantity of EPA and DHA that are incorporated into colonic epithelial cell mitochondrial phospholipids (Malis et al., 1990; Chapkin et al., 2002; Hong et al., 2002). n-3 fatty acids also increase mitochondrial phospholipid unsaturation (Watkins et al., 1998; Hong et al., 2002), which is associated with decreased ∆ψm, elevated production of ROS, and increased cytochrome c release (Watkins et al., 1998; Chapkin et al., 2002; Piccotti et al., 2004). Moreover, n-3 fatty acids inhibit migration and decrease metastasis of colonic carcinoma cells (Iigo et al., 1997; Iwamoto et al., 1998; Gaposchkin et al., 2000; Kontogiannea et al., 2000; Ramos and Colquhoun,

2003) while n-6 PUFAs are linked to increased metastatic potential (Nelson et al., 1988; Iigo et al., 1997). Thus, dietary PUFAs, one of the clearest dietary influences on risk for colon cancer, may alter risk through their modulation of composition and function of the mitochondrial membrane, which in turn, through complex interactions with SCFA metabolism and interaction with the binding and function of multiple effectors of signaling pathways that trigger growth or apoptosis, influence the probability of tumor formation.

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4 Metabolic Networks in Cancer Cells LASZLO G. BOROS AND WAI-NANG PAUL LEE

Like other living cells, tumor cells possess the potential for proliferation, differentiation, growth cycle arrest, and apoptosis. There is a specific metabolic phenotype associated with each of these functions. Tumor cells lock themselves into the proliferative state by employing a metabolic network that is dominated by nonoxidative anabolic reactions and macromolecule synthesis. Therefore, their transformation and growth are associated with the activation of intermediary metabolic enzymes that facilitate glucose carbon utilization for nucleic acid synthesis. The metabolic network possesses many control points composed of metabolic enzymes with high control properties on metabolic reactions that sustain malignant cell proliferation and tumor growth.

include frequent mutations of the K-ras, p53, p16, and Smad4 genes and are reported to be associated with accelerated disease progression and poor prognosis (Sakai et al., 2000; Yatsuoka et al., 2000; Comin et al., 2001; Boros et al., 2002a; Cascante et al., 2002). Genetic abnormalities influencing cellular responses to hormonal growth regulators and their signaling pathways have been reported in connection with all major tumor types (Issa, 2000; Jung and Messing, 2000; Largaespada, 2000; Martin and Weber, 2000; Ozen and Pathak, 2000; Szabo et al., 2000). However, it is well established through molecular genetic studies that in response to environmental changes, nutrition, lifestyle, and age, there is great variation in the expression of human genes, resulting in variability of the phenotypes. Thus, possible mechanisms for the development of common human malignancies include tumor-inducing genes, growthmodulating signal transduction pathways, and nutritional and environmental factors. The diverse mechanisms of tumor induction have in common their resultant influence on metabolism (altering the respective metabolic constraints), thus altering normal potential for differentiation, cell cycle arrest, and apoptosis. Figure 2 illustrates the transition of cells from one phenotype to another as changes in metabolic solution spaces under proliferation or differentiation signals (constraints). The metabolic network consisting of all functional metabolic pathways gives rise to the “general solution space,” which represents all possible metabolic phenotypes (solutions) for the given metabolic network. Environmental and signaling constraints further narrow the space for possible phenotypes of proliferation, differentiation, and apoptosis. Many potential growth-modulating factors have been identified and characterized through their signal transduc-

INTRODUCTION Decades of intensive research have identified many possible mechanisms for the transformation of normal regulated cells to those with unregulated proliferation characteristic of cancer cells. The cause for development of common human malignancies is multifactorial, which includes tumorinducing genes, environmental factors, and signal transduction pathways. In the constraint-based model of cell proliferation, these factors constitute genomic, metabolic, and environmental constraints (Figure 1). The current understanding of malignant cell behavior places heavy emphasis on genetic regulation of human cell functions. Human cancer is thought to arise from a dysregulation of gene expression resulting from the presence of growth-promoting oncogenes or the absence of tumor suppressor genes. Genetic alterations in cancer reported to date


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FIGURE 1 Constraint-based model of cell proliferation. FIGURE 3 Metabolism of tumors is affected by oxygen tension caused by poor blood flow, so lactate increases, whereas glucose, oxygen, and pH level decrease inside a 150-µ thick well-oxygenated shell.

Apoptosis

FIGURE 2 Transition of cells from one phenotype to another with changes in metabolic solution spaces under proliferation or differentiation signals (constraints).

tion pathways. These belong to two major signal types: one that acts on intracellular receptors and influences gene expression and the other that acts on cell surface receptors and influences multiple enzyme activities by protein phosphorylation.

Metabolic Alterations in Tumor Tissues Normal tissues depend on diffusion of oxygen from blood vessels and demonstrate an oxygen gradient across a distance of 400 µ from a blood supply. On the other hand, human tumors and tumor xenografts are hypoxic, and cells adjacent to capillaries have a mean oxygen concentration of 2%. Cells located 200 µ from the nearest capillary have a mean oxygen concentration of only 0.2% (Helmlinger et al., 1997) (Figure 3). This restrictive environment selects for cells that are adapted to chronic hypoxia. In normal cells, a critical response to hypoxia is the induction of the hypoxiainducible transcription factor HIF-1 (Wang et al., 1995).

Interestingly, HIF-1β is also known as the aryl hydrocarbon receptor nuclear translocator (ARNT) (Wang et al., 1995). HIF-1 binds to the DNA sequence 5′-RCGTG-3′ and increases the expression of genes that encode glycolytic enzymes, including aldolase A, enolase 1, lactate dehydrogenase A (LDH-A), phosphofructokinase L, phosphoglycerate kinase 1, and pyruvate kinase M, as well as the vascular endothelial growth factor (VEGF) gene, which mediates tumor angiogenesis (Firth et al., 1995; Semenza et al., 1996; Carmeliet et al., 1998; Iyer et al., 1998; Ryan et al., 1998). Changes in glucose concentration can also activate many glycolytic enzyme genes through the carbohydrate-response element (ChoRE; 5′-CACGTG-3′), which matches the consensus binding-site sequences for MYC and HIF-1 (Towle, 1995; Grandori and Eisenman, 1997). Studies of transgenic mice have demonstrated that HIF-1 regulates glycolytic enzyme gene expression via changes in the concentration of cellular oxygen and glucose, respectively (Iyer et al., 1998; Ryan et al., 1998; Vallet et al., 1998). Activation of the HIF1 pathway may mediate the adaptive responses to hypoxia and hypoglycemia in cancer cells. The activation of oncogenes or loss of tumor suppressors by somatic mutation in tumor cells can also lead directly to nonphysiological alterations of cellular metabolism and provide a selective advantage in hostile metabolic environments. The epigenetic changes and those secondary to metabolic signaling may both mediate the ultimate changes observed in tumor cells when they provide a selective growth advantage. Elegant studies by Warburg (1930) >70 years ago demonstrate that the vast majority of human and animal tumors display a high rate of glycolysis under aerobic conditions. Prior to World War II and the discovery of DNA, this phenomenon, called Warburg effect, was thought to be causally related to cancer. Although Warburg’s basic proposal that defective oxidative metabolism was the cause of increased

Introduction

glycolysis via futile cycling was not supported, his original observation has been confirmed repeatedly (Gatenby, 1995). Magnetic resonance spectroscopy (MRS) and positron emission tomography (PET) studies with 2-[18F]fluoro-2deoxy-glucose have consistently demonstrated that different clinical tumors show about an order of magnitude more glucose uptake in vivo than normal tissue (Gatenby, 1995). Furthermore, glucose uptake correlates with tumor aggressiveness and prognosis, and the expression of the glucose transporter GLUT1 is also increased in cancer cells (Younes et al., 1996). Increases in glucose transport and type II hexokinase activity in cancer cells cause the increased flux of glucose through the cancer cells (Rempel et al., 1996). Type II hexokinase plays a role in initiation and maintenance of high rates of glucose catabolism in rapidly growing tumors. The enzyme converts glucose to glucose-6-phosphate, the initial phosphorylated intermediate of the glycolytic pathway. The gene that encodes type II hexokinase has been shown to be amplified fivefold in a hepatoma cell line (Rempel et al., 1996). Its promoter contains a potential glucoseresponse element and putative p53-responsive elements, and type II hexokinase expression is markedly decreased in HIF1α–deficient embryonic stem (ES) cells (Iyer et al., 1998). A mutated p53 allele stimulates transcription of the type II hexokinase promoter, which suggests that mutant p53 plays a role in tumor metabolism (Mathupala et al., 1997). The ability of HIF-1α to interact with and stabilize p53 protein suggests that p53 plays a direct role as a transcription factor in response to hypoxia, although p53 protein is induced by near anoxic conditions ( 20 pounds in both men and women between the early 1960s and 2002, while during the same time period mean height increased by ~1 in. (Ogden et al., 2004). Over the same years, mean BMI increased ~3 BMI units in both men and women as well (Ogden et al., 2004). Although this trend has appeared across all ethnic groups and genders, it has occurred disproportionately in members of specific ethnic groups, particularly African Americans, Hispanic whites, and American Indians. Of particular concern is that these same trends have also appeared in children, with ethnic group disparities similar to those in adults (Hedley et al., 2004).


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course and their influence on energy balance and mortality. An International Agency for Research on Cancer (IARC) Working Group on the Evaluation of Weight Control and Physical Activity (2003) concluded that the avoidance of weight gain reduces the risk of developing cancers of the colon, breast (in postmenopausal women), endometrium, kidney, and esophagus based on epidemiological studies of overweight and/or obese compared with leaner individuals. The IARC Working Group Report also concluded that there is consistent epidemiological evidence for a protective effect of physical activity for some cancers. However, the report stated that the relationship between energy balance and cancer is poorly understood. Furthermore, obesity prevention and treatment regimens are difficult, and research on cancer risk in people who have lost weight is extremely limited (Ogden et al., 2003; Calle et al., 2004). In addition, surprisingly little is known about the mechanisms through which caloric restriction or physical activity (the major lifestyle-based strategies for reducing/maintaining weight) exert their anticancer effects (Rundle et al., 2005).

PHYSICAL ACTIVITY AND ENERGY BALANCE The positive health benefits from physical activity and high fitness are clear (U.S. DHHS, 1996). The Healthy People 2000 goals described in that U.S. DHHS report recognized the importance of physical activity for the nation’s health. There is a growing body of evidence that physical inactivity and low physical fitness are associated with increased risk of several cancers, and the underlying physiological changes associated with the healthful effects of exercise are beginning to be characterized. The evidence for an inverse relationship between physical inactivity is conclusive for colon cancer and quite strong for cancers of the breast (although menopausal status does influence this effect, as reported by Friedenreich, 2004), endometrial, ovarian, prostate, and testicular cancers (IARC, 2003). Work since the IARC report was published is further clarifying the exercise–breast cancer link. A meta-analysis of physical activity in adolescence and young adult years had an overall summary risk estimator of 0.81 for those in the highest compared with those in the lowest category of physical activity in observational epidemiological studies, thereby suggesting a protective effect of physical activity on breast cancer risk (Lagerros et al., 2004). This protection due to physical exercise in youth did not vary by study design (cohort vs case–control) or by menopausal status of the women. A report by Holmes et al. (2005) also suggests that increased physical activity after breast cancer diagnosis may decrease risk of death from the disease in breast cancer patients.

One of the issues in the analysis of physical activity and cancer risk is illustrated in an earlier review of the association with ovarian cancer (Bertone et al., 2001). Older birth cohorts of women might not have experienced a protective effect of physical activity in adult years on ovarian cancer because their birth cohort did not have the variation in and intensity of sports or regular exercise to the extent of more recent birth cohorts of women (Bertone et al., 2001). Few studies on other cancers are available, but serious work in this area has been undertaken.

EXERCISE AND ENERGY BALANCE IN CARCINOGENESIS Studies of the effects of exercise on carcinogenesis have used a variety of animal models including chemically induced tumors, transplantable tumors, spontaneous tumor models, and transgenic and induced-mutant mice, in addition to experimental metastasis models (Hoffman-Goetz, 2003). Many of the studies have identified some form of protective effect of either voluntary or involuntary exercise on carcinogenesis, although other studies have been less consistent (reviewed in Shephard and Futcher, 1997; Woods, 1998). Colon cancer has been the best-studied type of cancer using exercise regimens in animal models, and a consistent inverse association has been observed. To the detriment of the field, the number of animal studies has not increased dramatically, and perhaps more importantly, few studies have used the newer genetically altered models of cancer or have focused on potential mechanisms that might explain the exercise effects. Mechanisms most commonly cited as potential mediators include enhanced antioxidant defense, reductions in body fat, decreases in reproductive hormones, enhanced immunity, or a change in insulin resistance and the insulin-like growth factor-1 (IGF-1) pathway, including its related binding proteins (IARC, 2002). There are also sitespecific mechanisms for various cancers such as a decreased colonic transit time in relation to colon cancer. Many of the mechanisms that have received more attention in humans (estrogen, IGF-1) have not received much focus in exercised animal models and vice versa (e.g., immunity and inflammation in animal models). Some of the more intriguing proposed mechanisms are examined in the following sections.

Calorie Restriction and Cancer The best-studied perturbation of energy balance in experimental cancer systems is on the energy-intake side of the equation, specifically involving obesity prevention through calorie restriction (CR). CR is the only established intervention that extends mean and maximal lifespan in mammals (Weindruch and Walford, 1988; Roth et al., 2001). The antiaging effects of CR have been observed in diverse

Exercise and Energy Balance in Carcinogenesis

organisms, including protozoa, yeast (Saccharomyces cerevisiae), nematode (Caenorhabditis elegans), several insect species including fruit fly (Drosophila melanogaster), mouse (including transgenic and knockout models of cancer susceptibility), rat, hamster, guinea pig, dog, cow, and preliminarily in several nonhuman primate species (Pinney et al., 1972; Weindruch and Walford, 1988; Defossez et al., 2001; Roth et al., 2001; Kealy et al., 2002). Thus, the mechanisms underlying the survival extension in response to CR, imposed using various dietary compositions, feeding strategies, and levels of restriction, appear to be evolutionarily conserved. This suggests that a better understanding of these mechanisms will reveal important clues about the biology of aging, as well as preventive strategies for diseases of aging, including cancer. CR inhibits a variety of spontaneous neoplasias in experimental cancer model systems (Hursting and Kari, 1998), including tumors arising in several genetically altered mouse models, such as p53-deficient mice, APCmin mice, and Wnt-1 transgenic mice (Hursting et al., 2005). CR also suppresses the carcinogenic action of several classes of chemicals in rodents, including polycyclic aromatic hydrocarbons, for example, benzo(a)pyrene (Tannenbaum, 1940, 1942) and DMBA (Andreous and Morgan, 1981; Boissonneault et al., 1986); alkylating and methylating agents, for example, diethylnitrosamine (Lagopoulous and Stadler, 1987); and aromatic amines, such as p-cresidine (Dunn et al., 1997). In addition, CR inhibits several forms of radiation-induced cancers (Gross and Dreyfuss, 1984, 1990). Thus, the inhibitory action of CR on carcinogenesis is effective in several species for a variety of tumor types and for both spontaneous tumors and chemically induced neoplasias. In our view, the central goals of contemporary CR research are to apply its findings to human health concerns and to understand the biological mechanisms of CR’s effects. Observational studies suggest that CR has beneficial effects on longevity in humans. These studies include natural experiments, such as a study of Spanish nursing home residents, suggesting that reduced caloric intake reduces morbidity and mortality (Roth et al., 1999). Moreover, physiological changes analogous to those observed in CR rodents and monkeys, including high-density lipoprotein (HDL) cholesterol increases, are reported in Muslims who fast during the daylight hours of the holy month of Ramadan (Temizhan et al., 2000). In addition, inhabitants of Okinawa, Japan, who are known to consume fewer calories than residents of the main Japanese islands, display lower death rates from cancer and vascular diseases (Kagawa, 1978). The relationship between reduced calories and reduced mortality in Okinawa, relative to the rest of Japan, could be confounded by other factors such as genetic differences or other dietary differences, but these observations are intriguing. Data from certain historical events, such as the Dutch famine during World War II, are also suggestive of decreased mor-

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tality from cancer and other age-related diseases following extended reductions in calorie intake, although such observations are difficult to interpret because of confounding factors such as malnutrition (van Noord and Kaaks, 1991). Another category of studies in humans includes more controlled demonstrations of the antiaging effects of CR, such as occurred with Biosphere 2 and the ongoing Netherlands Toxicology and Nutrition Institute study. Biosphere 2, which took place in a closed ecosystem in Arizona from 1991 to 1993, involved four men and four women who experienced, on average, a 30% restriction in calorie intake relative to their usual intake. Although this sample was too small and uncontrolled to allow clear conclusions, many of the physiological parameters associated with the anticancer effects of CR in rodents and nonhuman primates were observed in these subjects (Walford et al., 1992). The TNO study was more controlled, with 8 ad libitum (AL) control subjects and 16 subjects on a 20% CR regimen. As in Biosphere 2, the TNO study subjects on the CR regimen, relative to the controls, displayed positive effects, including decreased fat mass and lowered blood pressure (Velthuis et al., 1994). In the past decade, four longevity studies involving longterm CR versus AL feeding have been initiated in several nonhuman primate species (Roth et al., 1999; Lane et al., 2001). It remains to be seen whether the extension of lifespan and/or reduction in tumor development demonstrated consistently in rodents will be replicated in nonhuman primates. However, preliminary reports on chronic diseaseassociated markers and initial tumor incidence and mortality data suggest that monkeys on CR regimens are less likely to develop diabetes, cardiovascular disease, obesity, autoimmune diseases, and cancer than their AL-fed counterparts (Roth et al., 1999; Lane et al., 2001).

Selected Potential Mechanisms Because perturbations in energy balance act broadly with respect to species, mode of induction, and type of tumor effected, and because CR, obesity, and physical activity each influences the levels of a number of hormones and growth factors (Weindruch and Walford, 1988), several investigators (including us) have suggested that globally active circulating factors may be the key mediators of the energy balance and cancer link (reviewed in Weindruch and Walford, 1988; Hursting et al., 2003). A number of inherited endocrinological disorders may contribute to obesity, although the prevalence of even the most common syndromes is very low. Several central nervous system factors, including neuropeptide Y, proopiomelanocortin, 5-hydroxytryptamine, noradrenaline, dopamine, and corticotrophinreleasing factor, have been shown to play a role in the regulation of normal body weight in rodents (Schwartz et al., 2000; Altman, 2002; Bray, 2004). These neurochemicals interact with the receptors on specific neurons in the arcuate

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nucleus (ARC) in the hypothalamus (Altman, 2002). The ARC has neurotransmitters and modulators that play a critical role in receiving and transmitting signals that regulate energy intake (Schwartz et al., 2000; Zigman and Elmquist, 2003; Seely et al., 2004). However, the roles of these factors in humans (particularly with respect to cancer risk) are unclear, primarily because of the tremendous technical challenges involved in measuring these neurochemicals. Numerous other hormones are involved in body weight regulation as satiety signals or as regulators of energy metabolism, such as gherelin, cholecystokinin, glucagon, glucagon-like peptide-1, adiponectin, resistin, and bombesin (deGraaf et al., 2004), although the relationships between these factors and cancer have not been well characterized. Finally, several other hormones serve as intermediate and long-term communicators of nutritional state throughout the biosystem and have been implicated in both energy balance and carcinogenesis. These hormones include IGF-1, as well as insulin, leptin, and several factors associated with inflammation and oxidative stress, and are the focus of the following mechanistic discussion.

Potential Role of IGF-1 in Cancer The possible involvement of IGF-1 in cancer was first suspected when in vitro studies consistently showed that IGF-1 enhances the growth of various cancer cell lines. These include prostate, bladder, breast, lung, colon, stomach, esophagus, liver, pancreas, kidney, thyroid, brain, ovarian, cervical, and endometrial cancer cell lines (Macauley, 1992; LeRoith et al., 1995; Fenton et al., 2005; Singh et al., 1996). There is now abundant epidemiological evidence, reviewed elsewhere in this book, supporting the hypothesis that IGF-1 is involved in several types of human cancer. IGF-1 acts directly on cells via the IGF-1 receptor (IGF-1R), which is overexpressed in many tumors, or indirectly through its action with other cancer-related molecules. For example, IGF-1 and the p53 tumor suppressor appear to function together in a regulatory network. The p53 gene regulates the expression of IGF-binding protein-3 (IGFBP-3) (Buckbinder et al., 1996), and IGF-1–induced mitogenesis is associated with phosphorylation and translocation of the p53 protein from the nucleus to the cytoplasm (Takahashi and Suzuki, 1993). Findings in diverse species, including yeast (S. cerevisiae), nematode (C. elegans), fruit fly (D. melanogaster), and mouse (Mus spp.), suggest that IGF-1 is part of an evolutionarily conserved neurosecretory pathway that regulates animal aging in response to food availability (Gems and Partridge, 2001). For example, increased longevity is observed in Drosophila Chico mutants and InR mutants that display altered insulin/IGF-1 signaling (Clancy et al., 2001; Tatar et al., 2001). In C. elegans, mutants in the daf (dauer formation) gene, which is part of an IGF-like signal transduction

pathway, demonstrate increased change to stresses such as crowding, starvation, and thermal, as well as increased lifespan (Murakami and Johnson, 1996, 1998; Barsyte et al., 2001). A markedly increased average and maximal lifespan is also observed in several strains of mutant or genetically modified mice that suffer defects in the production of growth hormone or IGF-1 or in responsiveness to growth hormone (and hence express significantly lower levels of circulating IGF-1). The “little” mouse, with its defective response to hypothalamic growth hormone–releasing hormone, lives 20–25% longer than wild-type mice (Flurkey et al., 2001). Laron mice, with a disruption in the growth hormone receptor/binding protein gene, have higher circulating levels of growth hormone than wild-type mice but much lower serum IGF-1 levels, and they live 38–55% longer than wild-type mice (Coschigano et al., 2000). Mice with primary deficiencies in growth hormone, prolactin, and thyrotropin, caused by failure of the pituitary to differentiate during fetal development, live still longer, 40–64% longer than wildtype mice. These latter examples include the Snell and Jackson dwarf mice, which have a point mutation in the homeotic transcription factor, Pit1 (Flurkey et al., 2002), and the Ames dwarf mouse, which fails to express Pit1 because of an inactivating point mutation in the Prop1 transcription factor (Brown-Borg et al., 1996). As seen with CR, these mutations appear to reduce the onset and/or rate of aging and age-associated cancers, resulting in an extended lifespan (Anisimov, 2001). In addition, mice displaying an ~75% reduction of circulating IGF-1 levels, due to a liverspecific knockout of IGF-1, show reduced tumor development and growth (Yakar et al., 2005). In contrast, tissue-specific overexpression of IGF-1 via the keratin 5 promoter increases spontaneous tumor development and susceptibility to carcinogens (DiGiovanni et al., 2000).

IGF-1, Growth, and Cell Cycle Regulation The complex process of growth involves the coordination of environmental signals (including nutritional cues) with programmed neuroendocrine responses that determine body size and composition. IGF-1 is a central component of a complex network of molecules that includes growth hormone, IGF-2, insulin, six (at least) IGFBPs that regulate IGF-1 ligand activity, leptin, adrenal steroid hormones, and several cell surface receptors. IGF-1 (a 70–amino acid polypeptide growth factor that shares ~50% homology with insulin) and pituitary-derived growth hormone are key regulators of an endocrine, paracrine, and autocrine signaling network that controls long bone growth and energy metabolism (LeRoith et al., 2001). The circulating level of IGF-1 is mainly determined by hepatic synthesis, which is regulated by growth hormone and influenced by nutrient intake, particularly intake of energy and protein (Hursting et al.,


1993). Regulation of IGF-1 in extrahepatic tissues is more complex, involving growth hormone, other hormones and growth factors, and IGFBPs, which determine the systemic half-life and local availability of IGF-1 (LeRoith et al., 2001). IGF-1 also regulates mitogenic and antiapoptotic signaling in many types of normal and cancer cells. IGF-1 has been identified as a cell cycle progression factor based on its ability in many normal and cancer cell types to stimulate progression through the cell cycle from G1 to S phase, purportedly by activating the phosphatidylinositol-3 kinase/Akt signal transduction pathway and modulating cyclin-dependent kinases. In vivo, IGF-1 may also be required for efficiently traversing the later stages of the cell cycle (LeRoith et al., 2001).

IGF-1 and Apoptosis IGF-1 can suppress apoptosis in various cell types, and cells overexpressing the IGF-1 receptor (IGF-1R) show decreased apoptosis (Resnicoff et al., 1995; Dunn et al., 1998). In addition, apoptosis is induced by reducing the cellular levels of IGF-1R using antisense oligonucleotides or by expression of a dominant-negative IGF-1R (D’Ambrosio et al., 1996). IGF-1 in many cell types stimulates phosphorylation of phosphatidylinositol-3 kinase, resulting in active phospholipid phosphatases that activate Akt (LeRoith et al., 2001). Activated Akt can suppress apoptosis by inhibiting the activation of interleukin-1β–converting enzyme (ICE)–like proteases. Other pathways may also play a role in IGF-1–dependent suppression of apoptosis. The apparently ubiquitous suppression of apoptosis by IGF-1 suggests that it is part of an important regulatory mechanism for maintaining tissue homeostasis that is usurped during the neoplastic process. It is primarily these effects on proliferation and apoptosis that make IGF-1 a plausible target for therapeutic and preventive strategies against cancer.

IGF-1 as a Mediator of the Anticancer Effects of CR There is mounting evidence that IGF-1 mediates at least some of the anticancer effects of CR through its role in an evolutionarily conserved regulatory pathway that is responsive to energy availability (Gems and Partridge, 2001). In various mouse and rat models, we and others have shown that serum IGF-1 levels are consistently decreased in proportion to the severity of the CR regimen imposed (reviewed in Hursting et al., 2005). For example, we reported that a CR regimen (25% reduction in calorie intake) reduces serum IGF-1, decreases in situ leukemia cell proliferation, and inhibits spontaneous and transplanted mononuclear cell leukemia in F344 rats (Hursting et al., 1993) and p53deficient mice (Hursting et al., 1994). Furthermore, restora-

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tion of serum IGF-1 concentrations to control levels in CR-treated rats by infusion via miniature osmotic pumps restores in situ leukemia cell proliferation rates to near control levels (Hursting et al., 1993). We also demonstrated that a moderate CR regimen (20% reduction in calorie intake) decreases serum IGF-1, increases the ratio of apoptotic versus proliferating preneoplastic urothelial cells, and suppresses p-cresidine–induced bladder carcinogenesis in p53-deficient mice (Dunn et al., 1997). Restoration of serum IGF-1 concentrations (again with miniature osmotic pumps) to control levels in p-cresidine–treated CR mice reversed the effects of CR on urothelial cell proliferation, apoptosis, and bladder tumor development (Dunn et al., 1997). Taken together, these studies suggest that IGF-1 mediates at least some of the antiproliferative, proapoptotic, and anticancer effects of CR. This conclusion does not exclude other mediators, which may be regulated by IGF-1 or function independently of IGF-1. In fact, our studies of CR in K5-IGF-1 transgenic mice, which constitutively overexpress IGF-1, and in liver-specific IGF-1–deficient mice, which have only 25% of normal circulating IGF-1 levels, suggest that IGF-1 accounts for some but certainly not all of the effects of CR (Hursting et al., unpublished observations).

Physical Activity and IGF-1 Levels in Men and Women Because physical activity induces changes in body fat, especially intraabdominal fat, with changes in insulin and insulin sensitivity, as well as the IGF axis (McTiernan, 1998), several physical activity intervention projects have been conducted in women aged 30–50 years (Schmitz et al., 2002) and in postmenopausal women (McTiernan et al., 2004). Interestingly, none of these intervention studies demonstrated a difference in preintervention and postintervention IGF-1 levels even though the intensity of physical activity and duration of the intervention varied by study. Because IGF-1 concentrations in humans decline from late adolescence on and serum levels of IGF-1 are related to age, perhaps the inability to detect differences in IGF-1 levels from exercise may relate to the limited range in age and large interindividual variation in IGF-1 levels in adults (O’Connor et al., 1998). However, using age-matched, inbred mice, which have only modest interindividual variation in IGF-1 levels, we have not observed differences in circulating IGF-1 levels in response to various levels of treadmill (Colbert et al., 2003) or voluntary wheel running (Colbert et al., unpublished observations). These findings are consistent with little or no long-term effects of exercise on circulating IGF-1 levels, as suggested by the human studies. However, we are seeing exercise effects on IGFBPs, so levels of bioavailable IGF-1 may be altered by physical activity. Another issue described by Rundle et al. (2005) is that there are no available biomarkers of exposure to or biolog-

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ically effective doses of physical activity on cancer risk or biomarkers of intermediate effects from physical activity training by level and duration.

Other Possible Mechanisms Underlying the Energy Balance and Cancer Association Insulin: Chronic hyperinsulinemia and insulin resistance increase risk of cancer at several sites (Calle and Kaaks, 2004), although it is unclear whether the tumor-enhancing effects of insulin are due to direct effects via the insulin receptor on preneoplastic cells or alternatively due to indirect effects via stimulation of IGF-1, estrogens, or other hormones. There is certainly important crosstalk between these hormonal pathways that is only now beginning to be understood, and it is clear that high circulating levels of insulin promote the hepatic synthesis of IGF-1 and decrease the production of IGFBP-1, thus increasing the biologic activity of IGF-1 (Calle and Kaaks, 2004). Furthermore, both insulin and IGF-1 act in vitro as growth factors to promote cancer cell proliferation and decrease apoptosis (Yakar et al., 2005). Insulin resistance, a state of reduced response of tissues to the physiological actions of insulin, results in a compensatory rise in plasma insulin levels and is affected by both adiposity and physical activity. Intraabdominal obesity is associated with insulin resistance (Abate, 1996), although physical activity improves insulin sensitivity (Grimm et al., 1999). A growing body of epidemiological evidence suggests that type II diabetes, which is usually characterized by hyperinsulinemia and insulin resistance for long periods, is associated with increased risks of endometrial, colon, pancreas, kidney, and postmenopausal breast cancers (Calle and Kaaks, 2004). Leptin is a 16-kDa peptide hormone secreted from adipocytes that is involved with appetite control and energy metabolism through hypothalamic influence. In the nonobese state, rising leptin levels result in decreased appetite and increased energy metabolism through a series of neuroendocrine changes. The obese state is associated with high circulating leptin levels (Lohnquist et al., 1997; Montague et al., 1997; Woods et al., 1998; Zhang and Leibel, 1998), suggesting that the obese may be leptin resistant. This resistance appears to explain much of the inability of exogenous leptin administration to prevent weight gain. The mechanisms underlying this apparent leptin insensitivity in the obese are not fully understood, although they may be due to defects in leptin signal transduction and/or transport across the blood–brain barrier, resulting in a higher “setpoint” of body weight (Wilding et al., 1997). The limited number of studies are suggestive of an association between circulating leptin levels and cancer risk, with the most consistent findings thus far for colon (Stattin et al., 2004) and prostate cancer (particularly progression of prostate cancer, as suggested by Chang et al. [2001] and Saglam et al.

[2003]). In vitro, leptin stimulates proliferation of multiple types of preneoplastic and neoplastic cells (but not “normal” cells, as reported by Fenton et al., 2005) and in animal models appears to promote angiogenesis and tumor invasion (Bouloumie et al., 1998). The primary physiological role of leptin may be regulating energy homeostasis by providing a signal to the central nervous system regarding the size of fat stores, and circulating leptin levels correlate strongly with adipose tissue levels in animals and humans (Ostlund, 1996). Findings reviewed by Rajala and Scherer (2003) also suggest that leptin exerts its metabolic effects, at least in part, by activating 5′-AMP–activated protein kinase (AMPK) in muscle and liver, thus decreasing several anabolic pathways (including glucose-regulated transcription and fatty acid and triglyceride synthesis) and increasing several ATP-producing catabolic pathways (including glucose transport, glycolysis, and mitochondrial biogenesis). In addition, there is significant crosstalk among the leptin pathway, which involves the Jak/Stat family of transcription factors, the insulin/IGF-1/Akt pathway, and AMPK. Furthermore, leptin plays a role in regulating the hypothalamus– pituitary–adrenal axis and, thus, influences IGF-1 synthesis (Rajala and Scherer, 2003). Finally, leptin functions as an inflammatory cytokine and appears to influence immune function (Loffreda et al., 1998). In fact, the immunosuppression associated with acute starvation is reversed by leptin administration (Rajala and Scherer, 2003). Thus, though not well studied, leptin is certainly positioned as a central player in the energy balance and cancer association. Sex steroid hormones, including estrogens, androgens, and progesterone, also reportedly play a role in the relationship between energy balance and certain types of cancer. Adipose tissue is the main site of estrogen synthesis in men and postmenopausal (or otherwise ovarian hormone–deficient) women, through the ability of aromatase (an enzyme present in adipose tissue) to convert androgenic precursors produced in the adrenals and gonads to estrogens (Calle and Kaaks, 2004). In addition, adipose tissue is the second major source of IGF-1 (liver is the primary source). The increased insulin and bioactive IGF-1 levels that typically accompany increased adiposity can provide feedback to reduce sex hormone binding globulin, resulting in an increased fraction of bioavailable estradiol in men and women (Calle and Kaaks, 2004). This can also result in higher bioavailable testosterone in women, and extreme obesity can lead to polycystic ovary disease, which manifests as hyperandrogenism, chronic anovulation, and progesterone deficiency. The epidemiological literature clearly suggests that the increased bioavailability of sex steroids that accompanies increased adiposity is strongly associated with risk of endometrial and postmenopausal breast cancers (Endogenous Hormones and Breast Cancer Collaborative Group, 2002; Kaaks et al., 2002) and may impact colon and other cancers as well.


The estrogen and androgen receptors, through which the sex steroids exert most of their proliferative and pro-cancer effects, are part of a nuclear receptor superfamily. A review by Ronald Evans (2005) suggests that this family of nuclear receptors has exquisitely evolved to acquire, absorb, distribute, store, and use energy. Examples include (1) dietary and endogenous fats that involve the lipid-sensing peroxisome proliferator-activated receptors (PPARs), which are emerging as key regulators of lipid and glucose homeostasis (Evans et al., 2004); (2) sugar mobilization involving the glucocorticoid receptor; (3) cholesterol through the liver X receptor; and (4) maintenance of metabolic rate through the thyroid hormone receptor. Evans et al. (2004) suggest that these “fuel management–related” receptors interact with gonadal steroid receptors (estrogen, androgen, progesterone receptors) to link nutritional status and fertility. Inflammation, which is the third key component regulated by the nuclear receptor superfamily, is also linked to energy balance through these same pathways that coordinately manage energy and inflammation. For example, the glucocorticoid receptor pathway is capable of mobilizing massive amounts of energy and suppressing inflammation, and the retinoic acid receptors, liver X receptors, PPARα and PPARγ, and the vitamin D receptor can also protect against inflammation, which can have an impact on cancer risk. Inflammation: The association between chronic inflammation and cancer is widely accepted (reviewed by Coussens and Werb, 2002). The effect of exercise on inflammatory processes has been better characterized than the effects of obesity, CR, or other energy balance perturbations. However, obesity is clearly associated with increased inflammation, and we and others have shown that CR decreases certain inflammatory markers. The cytokine response to exercise (reviewed by Petersen and Pedersen, 2005) does not involve an increase in the proinflammatory cytokines, tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β) but is initiated by IL-6 and then is followed by an increase in the anti-inflammatory mediators, IL-1ra, sTNF-R, and IL-10 (Ostrowski et al., 1999, 2000). Circulating levels of IL-6 have been shown to significantly increase (up to 100-fold) with exercise and decline in the postexercise period, with the relative increase in IL-6 in relation to exercise intensity, duration, and endurance capacity (Pedersen and Hoffman-Goetz, 2000; Pedersen et al., 2001; Suzuki et al., 2002; Febbraio et al., 2004). Interestingly, the main source of the exercise-induced increase in IL-6 is skeletal muscle (Penkowa et al., 2003; Steensberg et al., 2003; Hiscock et al., 2004) rather than monocytes (Ullum et al., 1994; Moldoveannu et al., 2000; Starkie et al., 2001). This exercise-induced increase in IL-6 is intriguing for several reasons. First, several studies have shown that IL-6 can inhibit the production of the proinflammatory cytokines, TNF-α, and IL-1β. IL-6 has been shown to inhibit

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lipopolysaccharide (LPS)-induced TNF-α production in vitro (Schindler et al., 1990). In both IL-6 knockout mice (Mizuhara et al., 1994) and mice treated with anti-IL-6 antibodies (Matthys et al., 1995), the circulating levels of TNF-α are significantly increased following either bacterial or an injury stimulus. Furthermore, the administration of recombinant human IL-6 to healthy volunteers inhibited the endotoxin-induced increase in TNF-α (Starkie et al., 2003). Second, IL-6 has also been shown to stimulate the production of anti-inflammatory mediators, such as IL-1ra, sTNFR, and IL-10 (Steensberg et al., 2003). Combined, these data suggest that exercise increases systemic IL-6 and that this exercise-induced increase in IL-6 may result in a reduction of chronic inflammation by reducing proinflammatory mediators and elevating anti-inflammatory mediators. However, additional studies are needed to determine whether regular moderate exercise can significantly reduce systemic inflammation and subsequently affect tumor development. Oxidative stress: Reactive oxygen species (ROS), involved in the inflammatory response, are important in a variety of normal processes within the body, including energy metabolism, direction of the immune response against pathogens, apoptotic regulation, intracellular signaling, and control of vascular permeability. However, the accumulation of ROS as byproducts of normal energy metabolism and in response to inflammatory conditions or ROS-generating environmental exposures (e.g., to particulates in tobacco smoke) has been associated with the pathogenesis of cancer and nonneoplastic age-related pathologies in rodents, nonhuman primates, and humans (Ames et al., 1993; Marnet et al., 2000). ROS can act as both initiators and promoters of tumors by damaging critical cellular macromolecules, such as DNA, proteins, and lipids, and by acting as cell-signaling molecules, in the manner of nitric oxide (Marnet et al., 2000; Hursting et al., 2005). There are few studies that have examined ROS-related damage or relevant antioxidant enzymes in the context of exercise in a cancer model, although exercise has been shown to effect oxidative stress (Ji, 1999; Fehrenbach et al., 2001; Radak et al., 2001). A 10-week treadmill training program in rats led to increased levels of superoxide dismutase (SOD) and catalase (CAT) in the lung, whereas UDP-glucuronosyl transferase was upregulated in both liver and lung (Duncan et al., 1997). Radak et al. (2004) also reported a decrease in ROS production in the liver following 8 weeks of treadmill training in Fischer 344 rats. Thus, evidence is accumulating that chronic exercise training promotes beneficial effects in many animal antioxidant systems (Radak et al., 2001). One study has examined wheel running in a tumorbearing rat model, and its effects on coenzymes Q9 and Q10 in skeletal muscle, as exercise in this same model had been found to delay cancer-induced cachexia, which interestingly is mediated by the inflammatory cytokine TNF-α (Daneryd

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et al., 1995). Both enzymes appeared to be upregulated in anterior tibialis muscle and Q9 also in the soleus. CR is known to decrease oxidative stress and inflammation (Sohal and Weindruch, 1996), and the CR-induced reduction in IGF-1 levels may play a role in these effects. Evidence from diverse species suggests a link among defects in the IGF-1 pathway, increased tolerance to oxidative and other stresses, and extended lifespan. For example, Daf gene mutants in C. elegans, described earlier because of their defect in IGF-like signaling and extended lifespan, also demonstrate increased tolerance to several types of stress (Murakami and Johnson, 1998). In Drosophila, InR mutants mimic C. elegans Daf-2 mutants (e.g., altered IGF-like signaling, increased lifespan, and tolerance to stress), whereas overexpression of antioxidant proteins, including superoxide dismutase, catalase, and glutathione reductase, also extends lifespan and increases resistance to oxidative stress (Tatar et al., 2001). Furthermore, CR in S. cerevisiae increases Sir2 activity, which extends lifespan and increases genomic stability and stress resistance (Defossez et al., 2001). Mice in which the p66 splice variant of the shc protooncogene has been ablated by gene targeting appear not only to be resistant to oxidative stress but also to live 30% longer than wildtype mice (Migliaccio, 1999). Finally, Ames dwarf mice, which are growth-hormone/IGF-1 deficient because of a mutation in Prop1, exhibit an upregulation of antioxidative defenses and a significant extension of lifespan relative to wild-type siblings (Brown-Borg et al., 2002). CR may exert some of its antitumor effects by decreasing ROS production and enhancing antioxidant defenses. CR decreases the rate of accumulation of oxidized DNA and protein that accompanies aging in rodents (Youngman et al., 1992). In addition, a number of intracellular antioxidant defense systems, including SOD, CAT, and glutathione peroxidase, which decline with age, are reportedly maintained or even enhanced by CR (Weindruch and Walford, 1988). CR also reduces nitric oxide production in peritoneal macrophages in p53-deficient and wild-type mice (Mei et al., 1998). Thus, evidence is mounting that CR may decrease oxidative stress by decreasing oxidant production and enhancing antioxidant capacity, although the exact mechanisms involved have yet to be fully established. The reduction in IGF-1 in response to CR may again play a role because cultured hepatocytes treated with IGF-1 demonstrate decreased activity and expression of CAT, glutathione peroxidase, and manganese SOD and, thus, have a decreased ability to counter oxidative stress (Brown-Borg et al., 2002).

FUTURE RESEARCH DIRECTIONS AND CONCLUSIONS Although the associations between overweight/obesity and several cancers are becoming well defined, the causal

relationships between energy balance and cancer risk are not well established. For example, key unanswered questions include the following: (1) Does reversal of obesity through diet, exercise, or pharmacological regimens decrease cancer risk or impact existing cancers? (2) Are there important differences between antiobesity regimens (calorie restriction, exercise, drugs) in terms of anticancer effects, or is weight reduction/maintenance the key irrespective of the means? (3) Which, if any, of the hormonal changes (i.e., IGF-1, insulin, leptin, and sex steroids) or biosystem changes (i.e., weight, adiposity, insulin resistance, inflammation, hypoxia, and alterations in energy metabolism) accompanying energy imbalance are causally linked to carcinogenesis? (4) What is the impact of different energy balance states and their associated effects on physiology (i.e., lean vs obese; insulin resistant vs insulin sensitive; high vs low postmenopausal estrogen levels) on the response to cancer prevention or cancer therapy regimens? As illustrated in Figure 1, progress in this area will require a multidisciplinary approach, and these and other key questions will only be answered through well-designed studies in both animals and humans that incorporate molecular, genetic, and metabolic/nutritional tools and expertise. As discussed in this chapter, the components of several interacting pathways associated with aging and carcinogenesis are altered by perturbations in energy balance, such as physical activity and CR. One approach to deciphering the complex network of mediators underlying the energy

FIGURE 1 The transdisciplinary nature of mechanism-based energy balance and cancer prevention research. Progress in the translation of mechanism-based findings in energy balance and carcinogenesis to cancer prevention strategies in humans requires the integration of observational epidemiological and clinical findings, the development and use of relevant animal models that can model the key events of both energy balance and carcinogenesis, the characterization of basic mechanisms at the molecular, cellular, and metabolic level, and the ultimate application of these findings to the clinic and community.

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Future Research Directions and Conclusions

TABLE 1

Selected Microarray Studies of Gene Expression in Mice in Response to Aging and Calorie Restriction (CR) Age of “aged” animals

Age, proportional to maximal longevitya

No. of genes significantly altered by aging

Percentage of genes changed by aging

Gastrocnemius muscle: male C57BL/6 mice Brain: male C57BL/6 mice Liver: female C3B10RF1 mice Brain: male C57BL/6 mice

30 months

~0.95

6,347

113

1.8

29% completely 34% partially

30 months

~0.95

6,347

110

1.7

27 months

~0.75

~5,300

46

0.7

30% completely 75% partially 59% completely

30 months

~0.95

6,347

114 (neocortex) 108 (cerebellum)

Liver: female Ames homozygous (Dwarf) vs heterozygous (normal) littermates

6 months

~0.19

12,252

212 (Dwarf vs normal); 77 (CR only)

~1 (neocortex and cerebellum similar) —

Tsuchiya et al., 2004

Dhabi et al., 2004

Liver: male B6C3F1 mice, with CR begun at 7 months of age

34 months

~0.98

12,422

—

—

Higami et al., 2004

Adipose: male C57BL/6 mice

11 months

~0.33

~11,200

—

—

Reference Lee et al., 1999

Lee et al., 2000 Cao et al., 2001 Prolla, 2002

Tissue: model

No. of genes measured

Percent ameliorated by CRb,c

30% completely or partially; similar patterns in neocortex and cerebellum 77 genes independently affected by CR; 212 independently altered by dwarfism; 95 additively affected by CR and Dwarfism (including several in IGF-1 pathway) 6% (123) genes changed in response to CR begun at seven months vs ad lib throughout life 5.5% (345) of total libgenes expressed in adipose from controls altered by long-term (9-month) CR

a

Maximal longevity data obtained from Weindruch and Walford, 1988. Ameliorated = either a change occurred in aged AL-fed animals but not in aged CR-fed animals, or changes occurred in both groups but in opposite directions. c CR also induced multiple gene alterations different from those occurring with age. d Investigated gene expression changes in response to aging over time: 7, 18, 23, 28, 42, and 47 days. b

balance and cancer link is to incorporate genomic and proteomic (and very soon metabolomic) analyses into these studies. Weindruch; Prolla and colleagues; and Spindler, Dhabi, and colleagues were among the first to use microarray technology to investigate the antiaging effects of CR, and a selection of these studies is summarized in Table 1. For example, Weindruch’s and Prolla’s initial experiments involved the comparison of global gene expression patterns in muscle (Lee et al., 1999) and brain (Lee et al., 2000) of young (3-month-old) AL-fed mice, aged (30-month-old) AL-fed mice, and aged CR-fed mice. Their findings linked aging with a gene expression pattern indicative of decreased metabolism and biosynthesis in muscle, increased inflammatory response in brain, and an increased stress response

in both tissues (Weindruch, et al., 2002). CR either completely or partially attenuated many of these changes (Table 1). A similar reversal of age-related gene expression changes was evident in a comparative whole-genome transcript profiling of AL-fed and CR-fed aged D. melanogaster (Pletcher et al., 2002). Microarray analysis of gene expression in livers of both short- and long-term CR mice indicates that CR also reverses many of the gene expression changes associated with aging in the livers of C3B10RF1 female mice (Cao et al., 2001), and the reversal of these age-related gene expression patterns by CR is rapid (Dhabi et al., 2004). AL-fed aged, relative to AL-fed young, rhesus monkeys demonstrated significant upregulation in skeletal muscle of transcripts involved in inflammation and oxidative stress, as

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well as downregulation of genes involved in mitochondrial electron transport, consistent with the age-related gene expression changes observed in mice. In addition, adultonset CR in rhesus monkeys decreased the expression of skeletal muscle genes involved in mitochondrial bioenergetics and upregulated several cytoskeletal-related genes, but it did not reverse the age-related changes in gene expression (Kayo et al., 2001). A review by Han and Hickey (2005) of the nearly 25 reports of microarray analyses in various organisms (mostly mice) that included some aspect of CR in their study suggests that no specific genes were altered across all the studies, possibly because of differences in microarray platform, species, strain and tissue differences, and other study differences. However, several functional categories of genes universally emerge as responsive to CR, including energy metabolism, stress responses (such as heat shock and oxidative), immune/inflammation pathways, and transcriptional regulation. To our knowledge, no studies using microarray or proteomic approaches to directly investigate the anticancer effects of CR have been published. Because carcinogenesis is a multistage process, the temporal design of such experiments requires careful consideration. Although many tissues share similarities in the classes of genes altered by CR, as discussed previously, relevant gene expression patterns will change throughout the carcinogenesis process and will vary from tissue to tissue. The challenge is to recognize those causal pathways and their upstream regulators that can serve as targets for cancer prevention. This process will involve validating microarray findings, tying gene expression findings to protein expression findings, and testing hypotheses about perturbations in causal pathways both in vitro and in vivo. Comparisons of energy balance perturbations in normal versus preneoplastic versus tumor tissue across a time course may also provide important insights and facilitate the identification of the most relevant pathways in the midst of a large number of individual gene expression changes. A better understanding of the physiological interactions between IGF-1 and other pathways involved in processes associated with energy balance and carcinogenesis (including new pathways that emerge from genomic and proteomic studies) will be very important in the translation of mechanism-based studies to disease prevention. Examples discussed in this chapter include important interactions among IGF-1, insulin, and other factors involved in glucose metabolism and insulin sensitivity, estrogens and other hormones linking energy balance and reproductive processes, as well as leptin and several other hormones and cytokines that bridge energy balance and inflammation. Although much of the excitement and attention centers on cytokines such as IL-6, TNF-α, monocyte chemoattractant protein-1, and several others, some of the classic hormones, like the adrenal steroids (which are not discussed in detail here) also

likely play a role. Glucocorticoids such as corticosterone are known to affect the cell cycle, induce apoptosis, and reduce inflammation in a variety of cells and tissues and are an integral component of glucose metabolism and mobilization. Several laboratories have shown that CR increases plasma levels and urinary excretion of corticosterone and its metabolites in rodents (Birt et al., 1999; Berrigan et al., 2002; Mai et al., 2003). Therefore, the increase in corticosterone levels with CR could interact with alterations in IGF1 signaling to significantly contribute to the antiaging and anticancer effects of CR. There is also a host of emerging energy balance–related factors, including adiponectin, resistin, gherelin, glucagon-like peptide-1, C-reactive protein, and several others. Many of these are produced in adipose tissue or in the gut, and at least some will likely turn out to play an important role in energy balance and carcinogenesis. Little will be gained by addressing these factors individually; the central goal has to be deciphering how these pathways interact to link energy balance and the carcinogenesis process. The insights from basic and applied research concerning the benefits of CR, exercise, and other antiobesity interventions on healthy aging and cancer prevention require translation into medical interventions and public health recommendations. Unfortunately, the direct translation of CR to humans may never be broadly accepted because major dietary and lifestyle changes, such as a 20–40% decrease in calorie intake, are very difficult for most people (McGuire et al., 1999). Exercise regimens may have broader appeal, although again not everyone can successfully make this change in lifestyle. New approaches to translation are, therefore, needed to fulfill the promise of the health benefits of CR and exercise. In this section, we propose areas relevant to translation that merit further basic and applied research. As described earlier, the IGF-1 pathway has emerged as a potential key mediator of CR’s anticancer and antiaging effects. Agents or interventions that reduce IGF-1 without requiring drastic dietary changes may provide an effective physiological or pharmacological mimetic of those effects, which could be readily adopted by a large proportion of the population, particularly those at high risk for cancer or other chronic diseases associated with high IGF-1 levels. Smallmolecule inhibitors of IGF-1 (Vogt et al., 1998) or IGF/IGFBP (Chen et al., 2001), as well as antisense IGF-1 inhibitor approaches (Scotlandi et al., 2002) and anti-IGF-1 antibody therapies (Granerus and Engstrom, 2001), are under development. It is important to note that any interventions involving IGF-1 modulation must be viewed cautiously. IGF-1 levels decrease with age, and increased IGF-1, directly or indirectly through growth hormone supplementation, in aged rodents and humans has been associated with decreased muscle wasting and improved mobility (Rudman et al., 1990). IGF-1 also plays a key role in bone mineralization

79

References

(Rosen and Donahue, 1998), and studies in rodents have pointed out that CR and other interventions associated with decreased IGF-1 levels reduce bone mineral density (Ferguson et al., 1999). Such findings have prompted proposals that growth hormone supplementation to elevate IGF-1 levels in the elderly would increase bone density and muscle tone (Rudman, 1990). However, three studies have found that higher bone mineral density is associated with increased risk of breast cancer in postmenopausal women (Cauley et al., 1996; Zhang et al., 1997; Buist et al., 2001), consistent with the evidence discussed earlier that links higher IGF-1 levels with higher cancer risk. Taken together, these findings suggest that lowering IGF-1 levels pharmacologically or through lifestyle changes would involve tradeoffs, having positive effects on cancer risk and some aspects of aging but also negative effects such as increased risk of fracture through osteoporosis and decreased muscle tone. Therefore, interventions targeting IGF-1 should proceed with caution. Combination approaches, such as diet or pharmacological modulation of IGF-1 combined with an exercise regimen, may increase the effectiveness of the IGF modulation while protecting the bones and improving muscle tone and insulin sensitivity. In addition, progress in the characterization of the molecular mediators underlying both the desired and the undesired effects of reduced IGF-1 levels will greatly facilitate the translation of CR research into safe and effective interventions. Another strategy for developing pharmacological mimetics of CR is to identify agents that can emulate the modulation of energy metabolism and glucoregulation observed with CR. 2-Deoxyglucose (2DG), a glucose analogue, is the first proposed candidate compound of this type. 2DG is a strong competitor of the enzyme phosphohexose isomerase, which converts glucose 6 phosphate to fructose 6-phosphate. Administration of 2DG greatly reduces the metabolism of glucose. Low-dose regimens (0.3–0.4% of diet) of 2DG had no effect on food intake or body weight but did result in several physiological/metabolic changes observed with CR, such as reduced insulin levels, reduced body temperature, and lower rates of tumor development (Zhu et al., 2005). IGF-1 levels were not reported. These preliminary findings suggest that metabolic mimetics of CR may provide a promising approach. Again, successful translation of this aspect of CR-related research will require attention to the entire biosystem to maximize the benefits while minimizing adverse effects. In conclusion, this chapter, and much of our own research, focuses on the molecular and hormonal mechanisms linking energy balance and cancer. In the postgenomic era, we have access to powerful new research tools that can further elucidate the pathogenesis of human carcinogenesis and the mechanisms underlying effective anticancer interventions such as CR and physical activity. There is tremendous optimism surrounding the application of these new

tools in cancer research, including energy balance–related studies as discussed previously, to hopefully result in the development of important new targets and strategies for both treatment and prevention of human cancer. However, we think a transdisciplinary approach that integrates molecular/cellular/biochemical tools and information with diet and other lifestyle variables in future basic, epidemiological, and clinical studies (as illustrated in Figure 1) is essential to the successful translation of the cancer-preventing effects of energy balance–related interventions such as CR and physical activity into human disease prevention strategies.

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6 Nutritional Epidemiology MARJORIE MCCULLOUGH AND EDWARD GIOVANNUCCI

INTRODUCTION

Evidence from a variety of study designs is required to establish a definitive relationship between diet and disease. Basic biochemistry and physiology, cell culture experiments, laboratory animal studies, and human metabolic studies provide pertinent mechanistic data to implicate a role for a specific dietary factor in carcinogenesis. However, these studies cannot prove that a particular dietary factor will cause or prevent a cancer in humans. Proof can only be established in human studies, preferably through randomized intervention trials. However, such trials are not always feasible. One challenge is the high cost of long-term studies, given that cancer takes years to develop. In addition, it is ethically implausible to test the relationship between a potentially harmful exposure and cancer in humans. For these reasons, the bulk of the available evidence for a particular diet–cancer relationship is garnered from observational nutritional epidemiological studies. This chapter provides an overview of the nutritional epidemiology of cancer, with particular emphasis on study design, dietary assessment, and analysis, as well as interpretation of findings.

Nutritional epidemiology examines dietary or nutritional factors in relation to disease occurrence in populations. Findings from nutritional epidemiology often contribute toward the evidence used in guiding dietary recommendations for prevention of cancer and other diseases (Byers, 1999). Epidemiological methods have been used to study the relationship between diet and disease for centuries, originally to identify nutritional deficiencies and target foods to ameliorate them (Cervantes-Laurean et al., 1999; Jacob, 1999). A classic example is scurvy, a condition observed among sailors of the sixteenth to the eighteenth century manifested by bleeding gums, swollen and inflamed joints, and eventual death. The observation that vegetables and fruits could cure scurvy led Lind to compare the efficacy of various types of these foods. He discovered that lemons and oranges had the most sudden influence on course of the disease. The molecular structure of vitamin C, the nutritional component responsible for the positive effect, was not discovered until centuries later in 1933 (Jacob, 1999). In contrast to studies of nutritional deficiency syndromes, modern nutritional epidemiology focuses on the etiology of chronic diseases such as cardiovascular disease and cancer, the two leading causes of death today (National Center for Health Statistics, 2003). Similar to the early studies of nutrition deficiency, associations between dietary factors and chronic disease may be observed long before a specific etiologic factor can be identified (Jacob, 1999). However, the path from observing to curing disease is more complicated for chronic diseases than deficiency diseases, because chronic disease etiology is multifactorial and diseases take many years to develop or manifest.


STUDY DESIGNS Epidemiologists use a variety of population-based approaches to study the relationship between diet and cancer (Table 1). The two major classifications are observational and intervention studies. Most studies are observational, in which cancer rates are observed among individuals or groups with variable levels of dietary exposure. Observational studies can be further categorized into ecologic and analytical epidemiology studies. Ecologic studies, including

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Chapter 6/Nutritional Epidemiology

TABLE 1 Study design Observational Ecologic: Correlational

Population

Study Designs in Nutritional Epidemiology

Exposure assessment

Outcome assessment

Advantages

Country or region

Population-based, e.g., food balance sheets

Population rates of cancer incidence or death

Examine wide range of exposure; population dietary estimates may be more stable than individual diets

Migrant study

Immigrants

Variety of diet assessment tools measured crosssectionally or over time

Cancer rates in immigrants to new country or region

Examine individuals or ethnic group in transition

Time-trend study

Same population, measured over time (usually as cross-sections of the population)

Population-based methods or varied

Rates of cancer within a population over time

Differences in rates less likely to be influenced by genetics; similar covariates

Cases with cancer (or proxies) vs control

Food frequency questionnaire (FFQ) or other; validate FFQ with daily records or recall or other method

Verified cancer outcome, identified in clinic or hospital

Used for rare cancers; low cost; can tailor exposure assessment to the study

Analytical Case–control:

Hospital-based controls

Population-based controls

Prospective cohort

Nested case–control

Intervention study

Identify population and follow over time Cases and controls within a larger study, e.g., prospective cohort Randomize participants to treatment

FFQ, multiple daily records or recalls or biomarkers; method should be validated FFQ, multiple daily records or recalls or biomarker (collected before disease development) FFQ or daily measures if required to assess baseline diet and changes over time; otherwise, exposure is assigned

Controls admitted to the same hospital for other disease(s) Controls sampled from the population from which the cases arose Cancer incidence or mortality

Cancer incidence

Development of cancer or intermediate endpoint

Special Considerations

Potential for uncontrolled confounding, e.g., unmeasured or uncontrolled aspects of lifestyle related to diet and risk of cancer; potential for biased food estimates because of wastage Other lifestyle factors are likely to change from country to country and over time in both immigrant and time-trend studies Changes in screening procedures may influence rates

Recall bias or misinformation from proxies difficult to avoid

Dietary factor must be unrelated to the other control disease

Volunteers may be more health conscious; keep participation rates high to avoid this bias Recall bias avoided

Potential for confounding; repeat exposure assessments ideal

Saves money for data analysis, e.g., biomarker or genetic analysis, or more detailed analysis of diet Randomization avoids confounding

Choose controls of same age, alive at time of case diagnosis

Long time required to study cancer; questionable utility of intermediate outcomes; expensive; test limited number of doses; compliance may be difficult; subject may not be blinded to status

Study Designs

correlational, migrant, and time-trend studies, are based on population rates of cancer and population estimates of dietary intake, whereas analytical epidemiology studies are based on individual disease outcomes and individual estimates of dietary intake. In contrast to observational studies, intervention studies randomize individuals to a particular exposure and then follow participants for disease outcome.

Observational Studies Ecologic Studies Correlational studies: Correlational studies compare population disease rates with population-based estimates of dietary exposure, most often across countries or regions. Dietary intake on a population basis can be estimated from food disappearance data, which are calculated from governmental food balance sheets as the sum of food produced and imported, and excluding food that is exported, fed to animals, or unavailable for human consumption. Correlation coefficients range from 0, signifying no correlation of per capita dietary intake with cancer rates, to −1.0 or +1.0, signifying a perfect inverse or positive correlation, respectively. Ecologic investigations of diet and cancer in the mid to late 1900s generated several hypotheses for a role of diet in cancer. In a classic ecologic analysis, Armstrong and Doll (1975) noted, among other findings, a positive correlation between estimated meat intake (disappearance) and colon cancer incidence rates of 0.85 for men and 0.89 for women across 23 countries. Migrant studies: Migrant populations provide the opportunity to study environmental risk factors and their interaction with genetic susceptibility. Rates of some cancers vary by up to 100-fold by geographical location (International Agency for Research on Cancer, 2002), and migrating populations have been observed to acquire disease rates of the host country. For example, rates of colorectal cancer in Japan have traditionally been among the lowest in the world, but among Japanese immigrants to Hawaii, these rates have surpassed that of Caucasians. That this phenomenon occurred within one generation further supports an environmental role in carcinogenesis (Le Marchand, 1999). Comparative studies in Hawaii and Japan suggest the discrepancy in disease rates may be attributed to the consumption of Western foods by the Japanese immigrants (Stemmermann et al., 1979). One hypothesis is that certain genotypes for carcinogen metabolism common among Japanese may increase risk among individuals who consume well-done red meat (Le Marchand, 1999). Time-trend studies: These studies examine disease patterns over time within defined populations and geographic locations. From 1963 to 1992, rates of colon cancer in Japan increased fivefold (International Agency for

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Research on Cancer, 2002). The reasons are unclear, but hypotheses include rapid changes in diet. Ecologic studies typically cannot precisely identify causal factors for changing cancer rates, but they provide compelling evidence for an important role of environmental factors in carcinogenesis. Diagnostic techniques and screening practices may change over time and differ across populations and partially explain sudden changes in disease rates (Potosky et al., 1990). Interpretation of Results from Ecologic Studies Ecologic studies have unquestionably provided important hypotheses regarding diet and lifestyle patterns with cancer risk. Their major strength is that contrasts in dietary intake may be substantial. For example, sodium excretion (a highly correlated marker for sodium consumption) across 32 countries varies from 0.2 to 242.0 mEq/day (Intersalt Cooperative Research Group, 1988). Within populations, ranges are narrower, for example 150–170 mEq sodium/day among hypertensive adults in the United States (Intersalt Cooperative Research Group, 1988; Anonymous, 1992). An additional advantage of ecologic studies is that the average diet of individuals within a country is more stable than an individual’s diet, which may fluctuate more dramatically. The primary disadvantage of ecologic studies is that potential determinants of disease other than those under consideration may also vary markedly among populations and regions with extreme incidences of disease. When populations differ by variables other than the main exposure being studied, these other variables could in part explain the exposure–disease relation. For example, smoking, alcohol consumption, physical activity, genetic factors, solar radiation (proxy for vitamin D production and status), selenium intake (based on soil content), and reproductive patterns vary widely across populations. To further illustrate this point, consider an ecologic study of fat intake and cancer among adults in rural 1980s China and current United States. Dietary fat consumption is low among the Chinese and high in U.S. populations. However, the diet–cancer relation may be confounded by differences in physical activity, where physical activity among the Chinese agrarian population exceeds that of most U.S. adults. Even if physical activity were measured perfectly, statistical control for the confounding effects of physical activity may not be feasible if substantial overlap of physical activity level does not exist across both populations. In addition, international comparisons can also be problematic if heterogeneous subgroups living within the same country are considered to represent one entity. Another limitation is the use of food disappearance data in most ecologic studies, which do not account for discarded food and selective avoidance of fat or meat. Higher disappearance of food and nutrients such as meat, fat, and calories for the United States compared with most countries may

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relate in part to a greater amount of wasted food, which correlates with socioeconomic factors. Disappearance data suggest an increase in per capita food availability of fat over time (Enns et al., 1997), whereas evidence based on actual dietary intake from a compilation of 171 studies suggests per capita dietary fat intake has decreased in the United States since 1950 (Stephen and Wald, 1990). Thus, analyses could provide spurious or even opposite conclusions, depending on which data source is used. Analytical Epidemiology Studies In analytical epidemiology, information on diet and disease occurrence is collected on an individual basis, and risk or rates of disease among individuals with a particular exposure are compared with those with no or low levels of exposure. The most common analytical epidemiology studies are case-control, prospective cohort, and nested casecontrol studies, although additional designs exist (Rothman and Greenland, 1998). Case-Control Studies In case-control studies, individuals who have been diagnosed with cancer (or family members, if the individual has already deceased) are interviewed with respect to past diet and other risk factors and compared with individuals without cancer, usually matched on age and other pertinent factors. These are often termed retrospective studies because information about diet is collected after cancer has occurred. Because diet before diagnosis is of interest, reference is usually made to a time before diagnosis. For example, if vegetable intake were hypothesized to lower risk of the disease, one would expect that cases had a lower previous intake of vegetables than control subjects. Statistical models would then control for age and other known risk factors for cancer that are also related to fruit and vegetable intakes to determine whether the observation may have been due to another related risk factor. Results from case-control studies are usually presented as “odds ratios” or the odds of developing disease given a certain dietary exposure, compared with the odds of developing disease with no or very low levels of that dietary factor (Hennekens and Buring, 1987). Data for case-control studies can be collected more rapidly compared with other study designs (described later in this chapter) because there are typically fewer participants and no follow-up is necessary. Dietary questionnaires can be tailored to the specific research question; for example, a study of phytoestrogen intake and breast cancer would include major food sources of phytoestrogens in the questionnaires (Pillow et al., 1999). For relatively rare cancers, case-control studies may be the only feasible option because other designs require cancer cases to accumulate over time.

Two major drawbacks of case-control studies include recall bias and selection bias. Recall bias occurs because dietary information is obtained after the diagnosis and individuals aware of their disease status may systematically overreport or underreport consumption of a food relative to controls. Cases may be more conscious than disease-free controls about past diet, especially if they are aware of a potential diet–disease hypothesis. To test whether this potential problem influenced epidemiological studies of fat and breast cancer, Giovannucci et al. (1993) mailed dietary questionnaires to nurses in a prospective cohort who recently developed breast cancer. More recent self-reports of past diet were compared with information that the same nurses completed before knowledge of cancer diagnosis. Positive associations were observed between total and saturated fat and breast cancer risk when using diet reports completed after diagnosis but not with earlier prediagnostic diet reports. In this case, the relation between fat and breast cancer was overestimated using the case-control approach. Study interviewers not blinded to a participant’s disease status could also contribute to recall bias by inadvertent prompting. The second major concern, selection bias, pertains to the appropriate selection of a control group for the case-control study. Conceptually, controls should be selected from individuals who, had they developed the cancer, would have been identified as a case in that study. Case-control studies can be categorized as “population based,” in which the comparison group is identified from the population from which the cases arise, and “hospital based,” in which the comparison group is identified from other patients hospitalized at the institutions where the cases were diagnosed. In hospitalbased case-control studies, an important assumption is that the exposure under study is unrelated to the condition of the control group. For example, when studying dietary fat and cancer, one should avoid designating patients with gallstones or heart disease as controls. Because diet may affect many diseases, it is often difficult to identify hospitalized patients with conditions that are definitely unrelated to the aspect of diet under investigation. Investigators may choose different sets of controls hospitalized for several diverse conditions; the consistency of results comparing different sets of controls supports the absence of selection bias. In population-based case-control studies, population lists may be used to randomly select a control group from individuals of a particular geographic region. Selection bias may occur in population-based approaches because diet is typically associated with the level of general health consciousness, and the diets of volunteers may differ substantially from those of nonvolunteers (Maclure and Hankinson, 1990). To minimize selection bias, it is essential for case-control studies to have high participation response rates. This may be a challenge for studies in large cities, where participation rates are often low (Hartge et al., 1984). In general, there is a greater likelihood that validity is compromised in studies

Study Designs

with lower participation rates. Acceptable ranges for response rates have yet to be established. Several features of study design can minimize the influence of potential biases. In case-control studies, the collection of information from cases and controls should occur concurrently and in similar settings. Ideally the study interviewer should be unaware of the case status of the interviewee and of the specific study hypotheses. It is critical that response rates be maximized and similar among cases and controls. Prospective Cohort Study The second major type of analytical epidemiology study design is the prospective cohort study. Conceptually, prospective cohort studies are more appealing than casecontrol studies because dietary exposures are measured before the development of disease, thus avoiding recall bias. Typically tens of thousands of individuals are enrolled. Participants complete questionnaires on diet, lifestyle factors, and medical history and are then followed over many years until adequate cases of cancer(s) develop. Follow-up questionnaires may be mailed periodically to inquire about new disease diagnosis and update exposure information. The rate of disease development over time among individuals with different dietary exposures is then compared using Cox proportional hazards modeling (Cox, 1972) or other longitudinal statistical methods while adjusting for potential confounders. For example, after adjusting for age, smoking, and other risk factors, the rate of developing a new colon cancer in individuals whose diet is relatively high in processed meat is compared with the rate of those who consume relatively low amounts of processed meat. The results are usually expressed as a “hazard rate” or “relative risk,” which reflects the rate of disease occurrence in one group divided by the rate of disease occurrence in a specified comparison or “reference” group. A relative risk of 2.0 would imply, for example, that individuals with high consumption of processed meat had twice the incidence of disease as the reference group of individuals with low consumption of processed meat. Repeated assessments of diet may also be obtained over time for several purposes. First, new hypothesized exposures such as heterocyclic amines, phytoestrogens, and acrylamide can be added to questionnaires during follow-up. Second, multiple assessments of diet reduce measurement error and provide the opportunity to study major diet change in relation to disease risk. Finally, multiple assessments of exposure may help identify potentially important exposure periods. Prospective cohort studies avoid most potential sources of methodological bias associated with case-control studies because information is collected from individuals before disease onset. However, as in case-control studies, high follow-up response rates in cohort studies are also important, as losses to follow-up that vary by level of dietary

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factors can result in distorted associations. Also, examining study results that exclude the early follow-up period (e.g., first 2 years) is desirable, as they could be influenced by underlying undiagnosed disease at the beginning of the study, known as “reverse causality.” For example, a prediagnostic cancer-induced weight loss would distort the results between body mass and risk of cancer in the short term. Although biases are largely minimized in prospective cohort studies, unmeasured or inadequate control for confounding remains a concern for both case-control and prospective studies and is reviewed in detail later. Approximately 60 prospective cancer cohort studies with > 10,000 participants each exist worldwide. Most have extensive data on dietary intake, although many have just begun publishing findings. Over the past decade, several prospective cohort studies have reported weaker or null associations between diet and cancer compared with those reported previously from case-control studies. For example, findings from prospective studies of fruit, vegetables, and gastrointestinal cancers are generally weaker than those of case-control studies (International Agency for Research on Cancer, 2003). One potential explanation is that the relationships were overestimated in case-control studies if individuals with cancer systematically recalled their prediagnosis diet erroneously, either because they had a preconception about how diet might influence disease or their current diet changed because of cancer. If this is the primary explanation, it suggests fruit and vegetable consumption is not as strongly associated with gastrointestinal cancers as previously thought. Other methodological issues such as limited ranges of exposure or dietary assessment (discussed later) are unlikely to explain these discrepancies because both study designs are equally susceptible to these limitations. Finally, more potential confounders have been extensively measured and controlled for in prospective studies, suggesting that some associations in case-control studies were confounded. Two large consortium projects may help resolve these questions: the Pooling Project of Diet and Cancer Cohorts (Smith-Warner et al., In press) is a collaborative effort of researchers from 16 cohorts in six countries and includes more than one million study participants. The European Prospective Investigation of Cancer (EPIC) (Riboli and Kaaks, 1997) is a multicenter cohort study of ~500,000 participants representing 22 European centers. These studies enable epidemiologists to evaluate wider ranges of dietary intake and have greater statistical power to examine differences in diet effects among subsets of the participants. Nested Case-Control Study The nested case-control study includes individuals with and without cancer typically sampled from an existing study population such as a prospective cohort. Cases are individuals who develop a cancer during the follow-up; controls

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are individuals from the same cohort who were cancer free at the time of diagnosis of the case and usually matched on age, time period of case diagnosis, and other factors. This design has been popular when biological markers of exposure or disease status are analyzed, to help save on analytical costs and to preserve finite biological resources collected on an entire cohort. A critical advantage of the nested casecontrol design over the standard case-control design is that the exposure is measured before disease development. This advantage may be especially pertinent in studies of biological markers of dietary exposure, which may change in response to underlying disease. Special Considerations in Observational Studies As discussed, bias and confounding represent two important potential problems that should be addressed in observational studies. Bias refers to a distortion in the way data were collected that may produce spurious results. Confounding is a distinct concept that may occur when another factor that is related to risk of disease is correlated with the factor of interest. Generally, bias must be avoided or minimized, whereas confounding represents a characteristic of the specific population that cannot be avoided but must be dealt with statistically or in the study design stage. The major inherent limitation of both ecologic and analytical observational studies is that the exposure, diet, is not randomized. Confounding, from the Latin word “cunfundere,” to mix together, results when the apparent effect of one exposure on risk is brought about by the association with other factors that can influence the outcome (Last, 1983). There are a few key concepts about confounding. First, for a factor to be a confounder, it must be a causal risk factor for the specific disease and be correlated with the exposure of interest. Second, if a study excludes individuals affected by the confounder, then that factor is no longer considered a confounder. For example, smoking would not be considered a confounder if the study excludes tobacco users. Third, the same factor can be a confounder in one population but not another, depending on how it correlates with the factor of interest. In Western populations individuals with healthier diets tend to smoke less, exercise more, weigh less, and take more vitamin supplements, so all of these would be considered potential confounders. In populations where these factors are not all interrelated, these unrelated factors would not be confounders. Overall, confounding is an especially important issue in nutritional epidemiology because dietary factors are often highly correlated. For example, saturated fat and total fat are positively associated, and dietary fiber and total fat intake are inversely associated. In analytical studies several strategies are used to minimize the influence of confounding. In the study design, cases and controls can be matched and individuals restricted on a potential confounder. In dietary-based studies, confounding

is usually dealt with by statistical analysis. An “adjusted relative risk” is a weighted, pooled average of the relative risk across different categories or levels of the confounder. By controlling for smoking, the adjusted relative risk will represent some average of the relative risks between diet and cancer among nonsmokers, past smokers, and current smokers. The ability to control for a confounder statistically is maximized when detailed data are available. “Residual confounding,” or incomplete control for confounding, occurs when information on a confounding factor is measured crudely. For example, if “ever” versus “never” smoking were the only assessment of smoking status, this categorization would fail to distinguish duration of smoking among current and past smokers. Because comprehensive knowledge of disease risk factors is rare, the possibility for uncontrolled confounding persists. The goal of the epidemiologist is to assess the plausibility of uncontrolled confounding based on available information. For a factor to be an important confounder, it must have a strong relationship both with the disease endpoint and with the dietary factor of interest. If research findings vary by category of another variable, results within each stratum should be presented. For example, a dietary factor may influence cancer risk in men but not women or in smokers but not nonsmokers. When associations are different across subgroups, this is referred to as “effect modification” or “interaction.” In addition to concerns for selection and recall bias outlined in the section Case-Control Studies, earlier in this chapter, differential detection rates of cancer by exposure status can distort study results from both case-control and cohort studies. For example, this could arise if dietary intake were related to the likelihood that an individual undergoes a cancer screening test. An implicit assumption in cancer epidemiology is that given the aggressive nature of cancer, individuals with disease would eventually be diagnosed regardless of exposure status. This assumption is reasonable for lung cancer but not for prevalent prostate cancer in the United States because screening by the prostate-specific antigen (PSA) test detects many innocuous cancers (Platz et al., 2004). In addition, individuals who follow breast and colorectal screening recommendations may have healthier lifestyles. Studies should, therefore, examine medical and screening background by diet status when possible. The validity of the relative risk estimate does not require that every cancer be detected but that detection rates are equal across levels of dietary intake.

Randomized Intervention Trials Randomized intervention studies in humans are often considered the gold standard in epidemiological study design because random assignment of treatment naturally controls for important confounders. Dietary exposures can

Study Designs

be clearly defined and comparisons among groups maximized, making this the most definitive test of a diet–cancer hypothesis. Because cancer may take decades to develop, intervention studies measuring cancer outcomes can be cost prohibitive. Intervention studies of diet and cancer prevention, therefore, often utilize surrogate cancer endpoints, such as adenomatous polyp recurrence or other intermediate markers of carcinogenesis, including neoplastic changes or molecular markers (Bostick, 1997; Schatzkin and Gail, 2002). Whether diet influences cancer initiation or progression similarly is not often feasible to study because once a cancer or intermediate risk factor such as a polyp is detected, it is typically removed. Individuals with a history of cancer have also been enrolled in intervention studies, for example, to determine whether high fruit and vegetable/low-fat diets influence breast cancer recurrence or survival (Pierce et al., 2002; Winters et al., 2004). Both dietary supplements and food-based interventions have been tested. Supplementation trials that investigate the biological effects associated with nutrients individually or in combination with others are easier and less costly to conduct than interventions involving foods or whole diet patterns. An additional advantage of providing supplements is that observed effects can be attributed to the specific nutrient(s) under study, because unlike in dietary interventions, nutrient composition of the diet does not change. Furthermore, nutrients can be formulated into pills, which can be counted at follow-up visits to assess compliance. A major limitation of supplement interventions is that the ideal dose and form of interventional supplement are not always known. For example, observational studies that suggested diets high in β-carotene (average U.S. intake < 5 mg/ day [Institute of Medicine, 2000]) were associated with lower risk of lung cancer inspired intervention trials to study lung cancer risk. However, two trials using 20–30 mg of β-carotene supplements reported increased risk of lung cancer in smokers (Institute of Medicine, 1997), whereas in a third trial, 50 mg every other day did not influence risk of malignant neoplasms (Hennekens et al., 1996). These trials led many to believe that β-carotene causes cancer or the hasty conclusion made by the press and public: “Diet is unrelated to cancer risk.” It remains possible that different levels of β-carotene intake reduce the risk of lung cancer, suggesting a nonlinear association, but it is also possible that the earlier studies attributed benefits of foods high in βcarotene to β-carotene alone (Marshall, 1999). Unfortunately, testing various doses and sources of β-carotene is costly. In contrast to β-carotene, intervention trials of 1200 mg and 2000 mg elemental calcium daily (slightly higher than normal intake levels [Institute of Medicine, 1997]) found consistent modest reductions in recurrence of colorectal adenomas (Baron et al., 1999; Bonithon-Kopp et al., 2000); however, whether lower doses have the same effect is still unknown. Underlying interactions may also

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obscure the full effect of a particular intervention. For example, the inverse association with calcium in one colorectal adenoma trial (Baron et al., 1999) was most notable among individuals with adequate serum 25(OH) vitamin D levels (a measure of vitamin D status) (Grau et al., 2003). Follow-up studies are evaluating the effect of calcium and vitamin D on polyp recurrence using a 2 × 2 factorial design (Bostick, personal communication). Another consideration for supplementation trials is whether individuals change their diet during the intervention. A trial of fiber supplementation and colorectal polyp recurrence documented that all participants increased their micronutrient intake and decreased their fat intake; however, few differences in the diets of intervention and control groups were significant (Jacobs et al., 2004). Dietary intervention studies involving changes in food intakes or dietary patterns more closely reflect realistic differences in nutrient intake levels. However, this approach is more labor intensive for both investigators and participants and more costly than supplementation trials, especially when all food is provided to participants. Compliance with dietary regimens in free-living populations is a major concern with food-based intervention trials but has been feasible, as documented by biomarkers, anthropometrics, and self-reports (Bingham, 1987; Windhauser et al., 1999). For dietary intervention studies in which participants are taught how to follow a research diet on their own, studies also suggest that with regular support, participants can learn and sustain major dietary changes (Lanza et al., 2001; Pierce et al., 2004; Rock et al., 2004). Nevertheless, the differences in intervention and control diets may narrow as the study progresses if diet in the control group improves, motivation in the intervention group wanes, or some combination thereof. Although providing food may better test a hypothesis, especially when major dietary differences are required, the diet instruction method addresses long-term feasibility; thus, both approaches are valuable. Advantages and Limitations The major theoretical advantage of randomized intervention studies over observational studies is they naturally control for known and unknown confounders. Additional practical advantages include the ability to test specific doses, durations, and endpoints. Because these studies usually involve several in-person clinic visits, anthropometric measures and other relevant biological parameters can easily be assessed. Despite the superior design of intervention studies, however, theoretical, ethical, and scientific limitations exist. First, null findings may simply reflect that the outcome measure or the duration, dose, or form of diet under investigation was not appropriate to test the research question. Second, how definitive a marker the surrogate endpoint is for cancer development has direct bearing on whether the

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relationship between a dietary factor and surrogate endpoint mirrors the relationship between the dietary factor and cancer itself (Schatzkin and Gail, 2002). Third, for intervention studies of dietary factors, it is difficult to blind participants to the intervention unless micronutrients are formulated into pills. Fourth, these studies take years to evaluate one or two doses of a potential chemopreventive agent. Fifth, whether nutrients supplied in pill form have the same biological effect as those found in food is unclear because food sources contain numerous other dietary factors that may act additively or synergistically to influence carcinogenesis. Pills may also contain doses that exceed amounts found in the diet or forms that do not occur naturally in the diet. Dietary fiber interventions in the form of a high-fiber diet (Schatzkin et al., 2000) or cereal fiber supplements (Alberts et al., 2000) yielded null results for colorectal adenoma recurrence (Alberts et al., 2000; Schatzkin et al., 2000), but the fiber supplement ispaghula husk was associated with increased polyp recurrence (Bonithon-Kopp et al., 2000). Although “fiber” was tested in each trial, the source, form, and vehicle for fiber varied in each study. Sixth, for ethical reasons intervention trials cannot evaluate dietary factors hypothesized to increase cancer risk. Many dietary factors that have hypothesized benefits for various cancers also have potential benefits for other chronic diseases, particularly cardiovascular disease. If a benefit is established for cardiovascular disease, it may be unethical to seek consent for randomization that would lead to longterm deprivation of a factor among those randomized to the placebo group. Seventh, an important scientific limitation relates to generalizability of findings from intervention studies. Populations are heterogeneous with regard to both physiological and lifestyle risk factors, and results in one population may not correspond to those of others. Furthermore, individuals who volunteer to participate in trials may often be quite different from the general population, restricting generalizability of results. For all of these reasons, knowledge of cancer risk in humans is usually based on observational studies and the totality of evidence from several study designs.

SPECIAL ISSUES IN NUTRITIONAL EPIDEMIOLOGY A number of specific issues in nutritional epidemiology may critically influence the quality of a particular study. These include issues regarding the nature of diet and its measurement, statistical analysis, and the induction period required to observe a diet–cancer association. Although not all concepts reviewed in the following section are unique to nutritional epidemiology, each is important to consider when interpreting the quality of epidemiological studies of diet and cancer.

Nutrients, Foods, and Diet Patterns Nutritional epidemiology research examines individual nutrients, foods, or diet patterns. Human diets are complex, and foods contain a wide range of vitamins, minerals, phytochemicals, estrogenic compounds, chemical and natural pesticides, microbial toxins, and chemicals formed during cooking. A single dietary component is typically the focus when it is supported by a hypothesis and is measurable from the questionnaire and its corresponding nutrient database. Nutrients are usually spread out among many different food sources, and a biological association may be overlooked if that factor is not measured across all dietary sources. Because some nutrients are highly intercorrelated because of shared food sources, known as “collinearity,” it is often difficult to attribute an association to a single factor. Usually one of the first steps in nutrition analyses is to examine the correlational structure of various nutrients of interest in the study population. Supplements generally contain much higher nutrient levels than foods; thus, supplement users are usually in the highest of several nutrient intake categories. One approach to evaluate potential confounding or effect modification by supplements is to stratify the results by supplement use patterns. Finally, foods may also be the focus of study when the exact component responsible for a particular health effect is not known (e.g., tomatoes) or when evaluating the effects of adherence to a specific recommendation (e.g., “reduce meat intake”). Researchers have also begun to assess dietary patterns in epidemiology because individuals eat foods in various combinations, and manipulation of a single nutrient (e.g., fiber) usually affects several dietary exposures. Further, many components in a total diet may have additive or interactive effects on health. Diet patterns can be tested in nutritional epidemiology using a variety of approaches (Kant, 1996; Hu, 2002), including empirically based methods such as principal component or factor analysis, the diet-score method reflecting a hypothetically protective diet or dietary guidance (McCullough et al., 2002), or some combination of both (Schulze et al., 2003). The diet-patterns approach characterizes foods that are consumed together (as in factor and cluster analysis) or foods that have hypothesized additive or interactive beneficial effects when consumed together (a priori diet scores). Dietary patterns offer one way to test the effects of dietary combinations on disease prevention, but the specific mechanisms for any observed associations in studies of diet patterns are less transparent. Another issue is that nutrients, food, and diet-pattern studies differ among populations. For example, a study of vegetable consumption and stomach cancer in the United States may not be comparable to a similar study in China because the vegetables consumed and preparation methods differ (not to mention differences in proportion of gastric cancer subtypes by country). That studies of empirically

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Gene-Diet Interactions

derived food patterns have not always been replicated across countries (Slattery et al., 1998; Terry et al., 2001) is not surprising given differences in tradition and food availability.

Ranges of Intake The range of dietary intake should be considered when interpreting study results. For example, the range of fat intake within many Western populations studied is somewhat limited, ranging from ~30–40% of total energy. These studies can be informative across this range, but evidence from populations with a different dietary pattern is required to assess any influence at lower levels of intake. Studies that span multiple populations can usually address wider ranges of exposure (Riboli and Kaaks, 1997; Smith-Warner et al., In press), although confounding and dietary differences should still be considered. Consistency of a finding across populations offers support for a given hypothesis.

Energy Adjustment Because many nutrients are correlated with energy intake, study results can be seriously confounded by other dietary factors that track with higher calorie consumption if not controlled for energy. Generally, changes in nutrient composition of the diet are more relevant than changes in absolute intake. For example, by controlling for energy, a statistical model for the association of fat with disease would be interpreted as the effect of increasing fat intake while reducing calories from protein and/or carbohydrates. This is relevant because most people do not simply add more fat or other sources of calories to the diet (unless they are on a path to weight gain); they usually make substitutions. In the case where energy consumption is also a risk factor for the disease under study, energy may be an independent confounder. Control for energy also reduces measurement error due to overreporting or underreporting total food intake. Several methods are available to control for energy (Willett and Stampfer, 1986; Willett, 1998).

Measurement Error The measurement of many exposures in epidemiology is subject to error, and epidemiologists try to minimize and correct for them when possible. Random or nonsystematic measurement error in dietary studies tends to obscure or weaken any true association with disease. For example, if high intake of a factor has a true relative risk of 2.5 with a specific cancer, error in measurement may attenuate the relative risk to, say, 1.8. Thus, when a significant positive or inverse association is observed in a study, random error in dietary measurement alone cannot account for this, and the true relationship is probably stronger. When an association with a dietary factor exists, potential for bias and con-

founding should be the major consideration. For a null association, it would be important to evaluate whether the degree of error in dietary measurement was so large that it could have obscured a true relationship entirely. Specific statistical techniques have been formulated to quantify the influence of measurement error on relative risks, including the correction of relative risks for measurement error using data from a validation study (Rosner et al., 1990; Spiegelman et al., 2001). Specific concerns have arisen regarding correction for measurement error if the validation study used a comparison method with errors that correlated with the primary method under study (Kipnis et al., 2003). Validation studies should ideally use methods with independent error structures including biomarkers when feasible. Measurement error in several dietary factors and nondietary covariates may produce spurious and erroneous findings (Marshall et al., 1999). It would theoretically be ideal to validate and correct for measurement errors in the assessment of the main exposure and all other confounders. Unfortunately, this is not practical for most studies. Further, not all confounders (e.g., physical activity) have a readily available standard of comparison. It was shown that proper adjustment for energy may partly ameliorate these problems in nutritional analyses (Michels et al., 2004).

Relevant Exposure Periods The long induction period between exposure and cancer diagnosis for most cancers may influence dietary measurement. A typical assessment of diet for an epidemiological study spans a year. Individuals’ diets tend to track over time, but the extent varies across populations and is difficult to assess directly. In response, some cohort studies have repeated assessments of diet. For example, diet has been assessed seven times over 22 years in the Nurses’ Health Study of women (in 1980, 1984, 1986, 1990, 1994, 1998, and 2002) and five times over 16 years in the male Health Professionals Follow-up Study (in 1986, 1990, 1994, 1998, and 2002). Repeated measures provide powerful data to evaluate long-term dietary patterns on cancer risk and enable epidemiologists to examine specific temporal relationships, including latency periods and the effect of changes in diet on disease risk. Repeated measures also reduce measurement error over the long term.

GENE–DIET INTERACTIONS Nutritional factors may influence cancer risk differently according to genotype, haplotype, or molecular characteristic of the tumor. Genetic studies additionally provide a greater understanding of the biological role of certain nutrients in carcinogenesis. Past inconsistencies in nutritional epidemiology literature may be explained in part by

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differences in the distribution of polymorphisms for important cancer-related genes across studies. Ultimately, genetic studies may lead to improved understanding of biological processes and offer potential for targeted interventions for prevention or treatment of cancer. However, cancer genes involve complex pathways with multiple potentially important allelic variants (Vogelstein and Kinzler, 2004). Statistical methods used to test the relationship between multiple genetic polymorphisms, either within a metabolic pathway or between pathways, need further development (Hoh et al., 2001; Kooperberg et al., 2001; Devlin et al., 2003; Ritchie et al., 2003).

INTERPRETATION OF EPIDEMIOLOGICAL STUDIES Because epidemiological studies are correlative in nature, statistical associations do not necessarily represent causeand-effect relationships. When interpreting results from individual studies, the potential for bias and confounding should always be considered, as should the appropriateness of dietary assessment methods, ranges of intake, potential for measurement error, and control for energy and latency period. Ultimately, epidemiologists are interested in whether observed associations represent a true causal relationship and whether modifying the specific dietary factor influences the frequency of disease. Hill (1965) summarized several criteria to establish causality, including the strength of association, the consistency of a finding in various studies and populations, the presence of a dose–response gradient, the appropriate temporal relationship, the biological plausibility, and the coherence of existing data. Though helpful, none can be considered requirements and satisfaction of these cannot be considered proof of a causal relationship. As discussed previously, the greater the magnitude of an association, the less likely the association is due to bias or confounding. The dose–response gradient may be useful, but linear dose–response relationships could also be generated by bias and confounding. Further, nonlinear causal associations in nutrition are certainly plausible. An understanding of underlying mechanisms may enhance the plausibility of a finding but is not required for assessing a diet–disease relationship. For example, obesity is strongly associated with death from many cancers (Calle et al., 2003), although the exact reason for these associations is not clearly understood. One of the most important criterion to consider in observational studies is consistency of results across studies. For epidemiologists, “replication” of findings has a somewhat different connotation than for the experimental scientist. In the laboratory, it is important to replicate findings by creating the circumstances of the experiment as closely as possible. If results cannot be replicated, the question of validity

of the initial experiment may be raised. By the nature of epidemiology, settings can never be replicated precisely, so one typically cannot expect identical results across different studies. Results may vary for methodological and biological reasons. Because findings from epidemiological studies contribute to dietary recommendations for chronic disease reduction (Byers, 1999), several important criteria have been outlined for reviewing the literature in relation to dietary guidance (Freudenheim, 1999; Potischman and Weed, 1999). Evaluation of dietary behaviors that are likely to reduce chronic disease risk requires the consideration of all available epidemiological studies and other evidence bearing on the association. To draw conclusions from a single or even a few epidemiological studies is not desirable; hopefully, numerous well-designed studies possess relevant information. The World Cancer Research Fund and the American Institute for Cancer Research are conducting systematic literature reviews on the epidemiology of dietary factors and several cancers to update their earlier review (World Cancer Research Fund, 1997); this is due out in 2006.

SUMMARY Many types of studies contribute to our understanding of the association between dietary factors and various cancers. Epidemiological data contribute an important part of the total evidence. Data from epidemiological studies must be carefully evaluated using various criteria to determine whether a relationship is likely to be causal, and if so, what factors may modify the relationship. Although randomized trials may eventually provide definitive answers to some of these questions, our knowledge of many of these relationships will depend largely on data from observational studies. Advancements in epidemiological methods, assessment of dietary exposures, and gene–diet interaction studies will improve our ability to identify important dietary factors in the etiology of cancer. Findings can then be applied to reduce the risk of cancer development and recurrence.

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7 Fruit and Vegetable Consumption and Cancer STEPHANIE A. SMITH-WARNER, JEANINE GENKINGER, AND EDWARD GIOVANNUCCI

The association between fruit and vegetable consumption and cancer risk has been reported in hundreds of epidemiological studies (Block et al., 1992; Steinmetz and Potter, 1996; World Cancer Research Fund et al., 1997; The Working Group on Diet and Cancer of the Committee on Medical Aspects of Food and Nutrition Policy, 1998; International Agency for Research on Cancer [IARC], 2003). These studies have been conducted in numerous countries with diverse diets, have evaluated many types of cancer, and have used different dietary assessment methods to assess fruit and vegetable intake. Various aspects of fruit and vegetable consumption have been examined. Studies have evaluated broad subgroups such as total fruits and total vegetables, finer categorization of these groups such as botanically defined groups or groups based on phytochemical content, and individual fruits and vegetables. It has been hypothesized that the wide variety of nutrients and bioactive compounds in fruits and vegetables can inhibit carcinogenesis during the initiation, promotion, and progression stages. The nutrients and bioactive compounds present in fruits and vegetables have been shown to inhibit the deactivation of procarcinogens, induce detoxification pathways, affect the cell cycle by regulating cell cycle progression, influence cell-to-cell communication, quench free radicals, stimulate the immune system, modulate hormone metabolism, and dilute and bind carcinogens, all of which should help to prevent the development of cancer (Table 1). For example, sulforaphanes and isothiocyanates, present in cruciferous vegetables, induce phase I and phase II enzymes to detoxify and eliminate carcinogens (Dragsted et al., 1993; Fahey et al., 1997; Verhoeven et al., 1997a; Belanger, 1998; Crowell, 1999; Hecht, 1999a; van Poppel et al., 1999; Sato


and Miyata, 2000; Greenwald et al., 2001; Lamm and Riggs, 2001; IARC, 2003), regulate cell cycle progression (Bianchini and Vainio, 2001; Greenwald et al., 2001; Pinto et al., 2001), and induce apoptosis (Crowell, 1999; Greenwald et al., 2001; Pinto et al., 2001). The vitamin C found in many fruits and vegetables and organosulfur compounds found in onions, leeks, and garlic may additionally stimulate the immune system (Sato and Miyata, 2000; Venket Rao and Agarwal, 2000; Greenwald et al., 2001; Lee et al., 2003a; Pinto et al., 2001). By promoting the proliferation of lymphocytes, stimulating macrophage phagocytosis, and enhancing the activity of natural killer cells, these nutrients may protect cells against microbes, bacteria, and viral and fungal agents (Head, 1998; Lamm and Riggs, 2001; Lee et al., 2003a). Fruits and vegetables are also rich sources of antioxidants (Cao et al., 1996; Wang et al., 1996), which help to quench exogenous and endogenous free radicals. If the free radicals remain in an oxidative state, they may cause oxidative damage to nucleotides, proteins, and cell membranes and result in the initiation of carcinogenesis. Because randomized clinical trials of β-carotene, vitamin C, and vitamin E have shown no or questionable benefits on cancer outcomes (Hennekens et al., 1996; Lee et al., 1999; Malila et al., 1999; Bjelakovic et al., 2004) and in some cases, a rise in cancer outcomes (The Alpha-Tocopherol and Beta Carotene Cancer Prevention Study Group, 1994; Omenn et al., 1996a), synergism of the variety of phytochemicals present in fruits and vegetables may be more important than consuming high doses of a single nutrient or phytochemical, because of complementary and overlapping mechanisms of action (La Vecchia and Tavani, 1998; Lampe, 1999).

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Chapter 7/Fruit and Vegetable Consumption and Cancer

TABLE 1 Potential anticarcinogenic mechanisms • Enzyme induction of phase I enzymes Inhibits carcinogen uptake and formation (Ariyoshi et al., 1975; Austin et al., 1988; Belanger, 1998; Crowell, 1999; Crowell et al., 1992; Dragsted et al., 1993; Greenwald et al., 2001; Hecht, 1999b; IARC, 2003; Lamm and Riggs, 2001; Maltzman et al., 1991; van Poppel et al., 1999; Verhoeven et al., 1997a) P450, CYP 2B1, CYP 2C, epoxide hydratase

• Inhibition of phase I enzymes P450 (Bianchini and Vainio, 2003; Gusman et al., 2001) • Enzyme induction of phase II enzymes Inactivates carcinogens (Crowell, 1999; Dragsted et al., 1993; Fahey et al., 1997; Greenwald et al., 2001; Hecht, 1999b; IARC, 2003; Kris-Etherton et al., 2002; Pinto et al., 2001; Sato and Miyata, 2000; Shapiro et al., 2001; Talalay and Fahey, 2001; van Poppel et al., 1999)

Examples of phytochemicals

Examples of food sources

Carotenoids Organosulfur compounds Allyl sulfides (allicin) Allyl disulfides Sulforaphanes Glucosinolates Indoles Isothiocyanates Terpenes/D-limonene Carveol Carvone Geranoil Resveratrol

Yellow-orange vegetables Liliaceas (e.g., chives, garlic, leeks, onions)

Carotenoids Flavonoids/Flavonols Quercetin Kaempferol Myricetin Isorhamnetin Organosulfur compounds Allyl sulfides (allicin) Allyl disulfides Resveratrol

Yellow-orange vegetables Apple, blueberry, broccoli, brussels sprout, grape (red), lemon, onion, orange, pear (red/green), pepper (green/red), strawberry Liliaceae (e.g., chives, garlic, leeks, onions)

Sulforaphanes Glucosinolates Indoles Isothiocyanates Terpenes/D-limonene Carveol Carvone Geranoil Sulforaphanes Glucosinolates Indoles Isothiocyantes Organosulfur compounds Allyl sulfides (allicin) Allyl disulfides Vitamin E

Cruciferous (e.g., broccoli, cabbage, cauliflower, greens)

Cruciferous (e.g., broccoli, cabbage, cauliflower, greens)

Citrus fruits, cherries, lemons, oranges, grapefruit, spearmint, tomatoes Vitaceae (e.g., grapes, currants)

GST, Quinine reductase (Jang et al., 1997), UDP-glucuronyl transferase (Belanger, 1998; Crowell, 1999; Elegbede et al., 1993)

May affect cell cycle/DNA through one or more of the following mechanisms • May regulate cell cycle progression (Pinto et al., 2001; Bianchini and Vainio, 2001; Greenwald et al., 2001) Inhibition of tumor cell prolifeation (Belanger, 1998; Crowell, 1999; Morse and Stoner, 1993) Induction of tumor cell differentiation (Belanger, 1998; Crowell, 1999; Greenwald et al., 2001; Haag and Gould, 1994; Haag et al., 1992; Morse and Stoner, 1993) Growth control (Agarwal and Rao, 2000; Sengupta and Das, 1999) Induce cell cycle arrest (Greenwald et al., 2001) Ability to block G2/M phase (Knowles and Milner, 2001) Induce apoptosis (Crowell, 1999; Greenwald et al., 2001; Mills et al., 1995; Morse and Stoner, 1993; Pinto et al., 2001) • Role in DNA metabolism (Choi and Mason, 2000; Krishnaswamy and Madhavan Nair, 2001; Terry et al., 2001c) (Pinto et al., 2001) • Increasing growth factor receptors (Belanger, 1998)

Beta-Carotene Lycopene

Beta-cryptoxanthin

Folate

D-limonene

Vitaceae (e.g., grapes, currants)

Citrus fruits, cherries, lemons, oranges, grapefruit, spearmint, tomatoes Cruciferous (e.g., broccoli, cabbage, cauliflower, greens)

Liliaceae (e.g., chives, garlic, leeks, onions) Avocado; apples; blackberries; bananas; broccoli; dark, green, leafy vegetables; kiwifruit; spinach Yellow-orange fruits and vegetables Grapefruit (red/pink), guava, tomato, watermelon Apple, avocado, broccoli, cantaloupe, corn, grapefruit, kiwi, mango, orange, peach, pepper (red), plum, spinach, tomato, winter squash Citrus fruits, dark green leafy vegetables Cherries, lemons, oranges, grapefruit, peppermint, grasses, tomatoes

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Fruit and Vegetable Consumption and Cancer

TABLE 1 (Continued) Potential anticarcinogenic mechanisms • Inhibits platelet aggregation and adhesion (KrisEtherton et al., 2002) • DNA/lipoproteins Shields sensitive structures such as DNA (Dragsted et al., 1993; Kris-Etherton et al., 2002) Inhibits LDL oxidation Reduced DNA adduct formation (Bianchini and Vainio, 2001; Verhoeven et al., 1997a) Maintains DNA integrity and repair (Choi and Mason, 2000; Dragsted et al., 1993; Kris-Etherton et al., 2002; La Vecchia and Tavani, 1998; Schorah, 1999; Terry et al., 2001c) • May enhance cell to cell communication (Greenwald et al., 2001; Paiva and Russell, 1999) Upregulate gap junction communication (Sengupta and Das, 1999) • Metabolic pathways regulations (Agarwal and Rao, 2000; Venket Rao, 2000) May modulate enzyme activities (Bianchini and Vainio, 2001) • Provitamin A activity (Paiva and Russell, 1999; Agarwal and Rao, 2000; Venket Rao and Agarwal, 2000) • Stimulate immune system (Agarwal and Rao, 2000; Greenwald et al., 2001; Lee et al., 2003a; Pinto et al., 2001; Sato and Miyata, 2000; Venket Rao and Agarwal, 2000) Regulate nuclear factors and inflammation (Pinto et al., 2001) Antibacterial effect, particularly with H. pylori (Fleischauer and Arab, 2001; Sato and Miyata, 2000) Antimicrobial (Sato and Miyata, 2000) Antifungal (Sato and Miyata, 2000) Stimulates proliferation of lymphocytes and macrophage phagocytosis, interleukin-2, tumor necrosis factor-α and



Flavonoids/flavonols Quercetin Kaempferol Myricetin Isorhamnetin

Apple, blueberry, broccoli, Brussels sprout, grape (red), lemon, onion, orange, pear (red/green), pepper (green/red), strawberry

Organosulfur compounds Allyl sulfides (allicin) Allyl disulfides Vitamin C

Liliaceae (e.g., chives, garlic, leeks, onions)

Rutaceae (citrus fruits) Cruciferous vegetables (broccoli, Brussels sprouts, cabbage)

Solanaceae (tomato, peppers, potatoes)

Vitamin E

interferon-γ (Lamm and Riggs, 2001) Stimulates release of enhanced natural killer cell and lymphokine-activated killer cell activity (Lamm and Riggs, 2001) • Antioxidant properties (Bianchini and Vainio, 2003; Greenwald et al., 2001; Gusman et al., 2001; Jang et al., 1997; Kris-Etherton et al., 2002; Lee et al., 2003a; Paiva and Russell, 1999) Quenches exogenous and endogenous oxidants (free radicals, ROS/RNS/RCS) in aqueous solutions, blood, membranes, and within cells (Evans and Halliwell, 2001; IARC, 2003; Schorah, 1999)

Beta-carotene

Avocado; apples; blackberries; bananas; broccoli; dark, green, leafy vegetables; kiwifruit; spinach Yellow-orange fruits and vegetables

Lycopene Resveratrol Vitamin C

Grapefruit (red/pink), guava, tomato, watermelon Vitaceae (red grapes) Rutaceae (citrus fruits) Cruciferous vegetables (broccoli, Brussels sprouts, cabbage)

Nitrosamines (Dragsted et al., 1993; Lee et al., 2003a) lipid carbon-centered hydroperoxyl, lipid peroxyl radicals O2−, H2O2, singlet oxygen, hypochlorite (Evans and Halliwell, 2001), nitrogen dioxide, thiyl, sulphonyl radical (Kris-Etherton et al., 2002)

Vitamin E

b-Carotene

Lycopene

Avocado; apples; blackberries; bananas; broccoli; dark, green, leafy vegetables; kiwifruit; spinach Apricot, broccoli, Brussels, sprouts, cantaloupe, grapefruit (red/pink), mustard greens, peach, pepper (red), romaine, spinach, sweet potato, tomato, turnip greens, winter squash Grapefruit (red/pink), guava, tomato, watermelon (continues)

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TABLE 1 (Continued) Potential anticarcinogenic mechanisms

Singlet oxygen and radical (Agarwal and Rao, 2000; Dragsted et al., 1993; Giovannucci, 1999; Nishino et al., 2000; Paiva and Russell, 1999; Pool-Zobel et al., 1997; Sengupta and Das, 1999; Venket Rao and Agarwal, 2000)

Alkoxyl and peroxyl radicals (Greenwald et al., 2001; Paiva and Russell, 1999) Ultraviolet light and cigarette smoke (Kris-Etherton et al., 2002) Activated mutagens and carcinogens (Dragsted et al., 1993; Kris-Etherton et al., 2002) May inhibit free radical generating reactions (carbon-centered radicals) (Kris-Etherton et al., 2002; Paiva and Russell, 1999) Can regenerate other antioxidants such as vitamin E (Evans and Halliwell, 2001) May chelate prooxidant metal ions (Kris-Etherton et al., 2002) Protects lipids, DNA, lipoproteins (Agarwal and Rao, 2000; Kris-Etherton et al., 2002; Sengupta and Das, 1999) • Increase fecal bulk (Greenwald et al., 2001; IARC, 2003; La Vecchia, 2001) Decrease concentration of bile acid and other carcinogens (Havas, 1997) • Dilution and binding of carcinogens (IARC, 2003) Secondary bile acids and hydrophobic carcinogens (Havas, 1997) Binding of steroid hormones



Flavonoids/flavonols Quercetin Kaempferol Myricetin Isorhamnetin

Apple, blueberry, broccoli, Brussels sprout, cherry, cranberry, grape (red), grapefruit, lemon, orange, onion, pear (red/green), pepper (green/red), plum, raspberry, strawberry, tangerine

Fiber

Apple, apricot, asparagus, bamboo shoots, bananas, bean sprout, beets, blueberries, broccoli, Brussels sprouts, cabbage, cantaloupe, carrots, cauliflower, celery, cherries, corn, cucumber, grapefruit, grapes, green beans, mushrooms, nectarine, onion, orange, pear, pepper (green), pineapple, plum, potato, pumpkin, radish, squash, strawberry, sweet potato, tangerine, tomatoes, turnip greens, watermelon (Marlett, 1992)

• Increase transit time Decrease time that colon is exposed to carcinogens (Havas, 1997) • Produce short-chain fatty acids, which may affect pH • Increase availability of butyrate (Greenwald et al., 2001; IARC, 2003; La Vecchia, 2001) Promotes growth arrest Promotes differentiation Promotes apoptosis • May affect enterohepatic circulation of estrogens and the actions of fiber associated phytoestrogens (Rose, 1990; Rose et al., 1991)

PUBLISHED REVIEWS Several panels have reviewed the results from observational studies on fruit and vegetable consumption and specific cancer sites. In 1997, a panel sponsored by the World Cancer Research Fund and American Institute of Cancer Research reviewed 14 ecologic, 37 cohort, and 196 casecontrol studies that had examined the relationship between fruits and vegetables and cancer risk (World Cancer

Research Fund et al., 1997). The most extensively studied cancers were stomach, colon and rectum, esophagus, mouth, oral cavity, and pharynx. Of the case-control studies, 78% reported a statistically significant inverse association between a specific cancer site and intake of at least one fruit and/or vegetable item or group. The panel determined that the evidence for a protective relationship for fruits and vegetables was convincing for cancers of the mouth and pharynx, esophagus, lung, and stomach; probable for cancers

101

Individual Cancer Sites

of the larynx, pancreas, breast, and bladder; and possible for cancers of the cervix, ovary, endometrium, and thyroid. Associations were examined separately for vegetable consumption in 74 case-control and cohort studies. In these studies, 80% showed an inverse association for at least one vegetable group or item and 12% found positive associations. Among the specific vegetable subgroups that were reviewed, an inverse association was most consistently observed for raw vegetables. For the 56 case-control and cohort studies that examined associations for fruit intake alone, the corresponding numbers were 64% and 9%, respectively. Null findings were more common for fruit compared with vegetable intakes (27% vs 8%, respectively). For vegetables, the evidence was determined to be convincing for cancers of the colon and rectum and possible for cancers of the liver, prostate, and kidney. No consensus statement was made regarding fruit consumption alone. Moreover, the evidence did not suggest that fruits and vegetables increased the risk of any of the cancer sites evaluated (World Cancer Research Fund et al., 1997). The conclusions from a 1998 review of epidemiological and experimental studies on fruits and vegetables and cancer risk by the Working Group on Diet and Cancer of the Committee on Medical Aspects (COMA) of Food and Nutrition Policy made more conservative conclusions than the World Cancer Research Fund and American Institute for Cancer Research panel, even though the two panels reviewed essentially the same studies (The Working Group on Diet and Cancer of the Committee on Medical Aspects of Food and Nutrition Policy, 1998). The COMA Working Group concluded that (1) there is moderate evidence that higher intakes of fruits and vegetables would reduce the risk of gastric cancer, (2) there is moderate evidence that higher intakes of vegetables would reduce the risk of colorectal cancer, and (3) there is weak evidence that higher intakes of fruits and vegetables would reduce the risk of breast cancer. Based on the lack of data showing that fruits and vegetables increase the risk of cancer and the suggestion of a graded decrease in risk with increasing fruit and vegetable consumption, the panel concluded that the “overall picture, therefore, is consistent [with] and supports the hypothesis that the consumption of fruits and vegetables protects against the development of some cancers,” although the evidence is insufficient to define an optimal level of fruit and vegetable consumption to reduce cancer risk. A meta-analysis summarized the association between total fruit and total vegetable intakes and the risk of several cancers (Riboli and Norat, 2003) (Table 2). To reduce the heterogeneity in the exposures examined across studies, the authors limited their meta-analysis to only categories representing total fruit and total vegetable intake. The studyspecific relative risks were extracted from each publication and then reexpressed based on an increase in 100 g/day of intake. For fruit, statistically significant decreases in risk

were observed among case-control studies for cancers of the mouth and pharynx, larynx, esophagus, stomach, colon and rectum, colon, bladder, and lung; a nonsignificant inverse association was observed only for breast cancer. Vegetable consumption was associated with a statistically significant decrease in the risk of cancers of the esophagus, stomach, colon and rectum, bladder, and lung. The magnitude of the association for vegetables generally was stronger than that for fruits only for colorectal and breast cancers. Associations among cohort studies generally were weaker than those observed among case-control studies, with statistically significant decreases in risk being observed only for cancers of the colon (for vegetables only), rectum (for fruit only), bladder (for fruit only), and lung (for fruit only). There was significant heterogeneity in the study-specific results for most cancer sites overall and when case-control studies and cohort studies were examined separately. However, for some cancer sites (i.e., esophagus and stomach), the heterogeneity occurred because the magnitude of the inverse association differed across studies, not because of the directionality of the association. In 2003, a working group sponsored by the IARC reviewed the results on total fruit consumption and total vegetable consumption and the risk of specific cancers (IARC, 2003). Although both positive and inverse associations were observed for the risk of specific cancers with fruit and vegetable consumption, the mean of the study-specific relative risks suggested that higher compared with lower intakes of total fruit intake and total vegetable intake were each associated with a lower risk of most cancers. In general, the results from cohort studies were weaker than those observed in case-control studies. The working group concluded that the evidence indicated that higher fruit consumption probably reduces the risk of esophageal, stomach, and lung cancer and that higher vegetable consumption probably reduces the risk of esophageal and colorectal cancer. For several cancer sites, no conclusion was made because the data were too limited or the results were inconclusive.

INDIVIDUAL CANCER SITES In this chapter, the results for lung, breast, colorectal, stomach, and prostate cancers are summarized in more detail. We chose these five sites because they represent the five most common incident cancers in the world (Ferlay et al., 2004). The discussion follows the progression from broad to specific fruit and vegetable exposures to examine whether specific combinations of fruits and vegetables are more strongly associated with cancer risk compared with overall measures of fruit and vegetable consumption. For each cancer site, associations are summarized for total fruits, citrus fruits, total vegetables, cruciferous vegetables, and green vegetables.

102 TABLE 2


Summary of Meta-Analysis of Case-Control and Cohort Studies Examining Fruit and Vegetable Consumption and Cancer Risk (Adapted from Riboli and Norat, 2003) Fruit

Vegetables

Cancer site

Study design

n

RR (95% CI)

Oral and pharynx

All

9 9 0 5 5 0 13 12

0.72 (0.62–0.83) 0.72 (0.62–0.83)

125 drink-years vs never drank daily

Contrast

(Continued)

Alcohol variable

TABLE 4A

2.07 (1.29, 3.32)

1.1 (0.3, 3.6)

2.56 (1.03, 6.4)

1.85 (0.95, 3.61)

2.5 (1.3, 4.6)

1.5 (0.8, 2.7)

3 (1.6, 5.6)

1.6 (0.9, 2.9)

0.8 (0.4, 1.8)

1.4 (0.5, 3.9)

1.3 (0.7, 2.1)

OR/RR (95% CL)

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Age

Y (cont.)

Y (?)

Y (cont.)

Y (cont.)

Y (?)

Y (?)

Y (?)

Y (?)

Y (cont.)

Y (cont.)

Y (cont.)

Smokinga

Y

Y

Y

Y

N

N

N

N

Y

Y

Y

BMI/ weight

Y

Y

N

N

N

N

N

N

Y

Y

Y

Total energy intake

Covariate adjustment

Y

N

N

N

N

N

N

N

Y

Y

Y

Physical activity

242 Chapter 11/Alcohol and Cancer

105

Japan

411

Denmark

500

179

29,132

3,500

500

176

217

Canada

Sharpe et al., 2002

414 men and women

276

189 424 men and women

236

992

489,979

13,392

90,004

29,132

41,836

163

The Netherlands

Washington State, USA

Meyer and White, 1993

Cohort studies Goldbohm et al., 1994 Pedersen et al., 2003

Sweden

Gerhardsson de Verdier et al., 1993

251

3,291

299

Japan

US (Pooling Project)d

411

598

USA (Iowa Women’s Health Study) Denmark

Beer Case-control studies Longnecker, Various 1990 locations, USA

Cho et al., 2004

Pedersen et al., 2003 Otani et al., 2003 Shimizu et al., 2003

Harnack et al., 2002c

23–95

55–69

35–79

35–70

30–62

>31

>35

40–69

23–95

55–69

Males and femalesb

Males and femalesb

Males, distal colon

Males, proximal colon

Recent intake

Recent intake

Recent intake

Recent intake Recent intake

Females

Males


Consumption 5 years ago Consumption 20 years ago Recent intake

Recent intake

Recent intake Regular drinking Recent intake

Recent intake

Females

Males

Males

Males and females

Males

Males and femalesb Males

Females

≥14 vs 0 drinks/week

≥5 g/day vs none

≥30 g/day vs none Daily drinker of ≥5 drinks/day vs never drank daily Daily drinker of ≥5 drinks/day vs never drank daily

≥10 vs 0– 9.9 g/day ≥30 g/day vs none

Continuous (5 drinks/ day) Continuous (5 drinks/ day) ≥10 vs 0– 9.9 g/day

≥41 vs 300 g/wk vs none >36.7 g/day vs none ≥45 g/day vs none

≥20 vs 31

Age range years (mean)

Males and femalesb Males and females

Males and femalesb

Males, distal colon



Recent intake

Recent intake


Females

Males



Recent intake

Females

Males

Males

Males and females

Subgroup

≥14 vs 31


Males and femalesb

Males, distal colon



Recent intake

Recent intake


Females

Males



Recent intake

Females

Males

Males

30–74

≥14 vs 40 83 drinkyears vs none >29 drinkyears vs none

High vs low quartile High vs low quartile >5 drinks/day vs none >5 drinks/day vs none High vs Low quartile High vs Low tertile ≥11 drinks/wk vs none Daily drinking of ≥50 ml/day vs never ≥500 liters/ lifetime vs nondrinker ≥30 vs 0– 9.9 g/day

Contrast

1.6 (0.6, 4.5)

1 (0.6, 1.8)

0.9 (0.4, 1.9)

1.2 (0.5, 2.7)

0.9 (0.4, 2.2)

0.6 (0.3, 1.3)

1.88 (1.02, 3.45)

1.88 (0.98, 3.58)

1.8 (1.12, 2.89)

Y

1.9 (1.2, 3)

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y Y

Y

Age

1.54 (not shown, p > 0.05) 0.93 (not shown, p > 0.05) 1.5 (0.9, 2.5)

OR/RR (95% CL)

Y (cont.)

Y (cont.)

Y (?)

Y (?)

N

N

N

N

N

Y (?)

Y (?)

N

N

Smokinga

Y

Y

Y

Y

N

N

Y

N

N

N

N

N

N

BMI/ weight

Y

Y

Y

Y

N

N

N

N

N

N

N

N

N

Total energy intake


Population-Based Case-Control and Cohort Studies Examining Alcohol Consumption and Rectal Cancer

186

Cases n

TABLE 4B

Y

Y

Y

Y

N

N

N

N

N

N

N

N

N

Physical activity


USA (Pooling Project)d

Canada

Sharpe et al., 2002

The Netherlands

Sweden


Cohort studies Goldbohm et al., 1994

Western NY, US

Freudenheim et al., 1990

3,500

276 500

110 230

113

236

145

145 107

277

992

489,979

90,004

277

393

3,291

148

Japan

29,132

41,836

123

202

3,500

500

113

230

The Netherlands US (Iowa Women’s Health Study) Denmark

Canada

Beer Case-control studies Longnecker, Various 1990 locations, USA

Pedersen et al., 2003 Otani et al., 2003 Cho et al., 2004

Cohort studies Goldbohm et al., 1994 Harnack 2002

Sharpe et al., 2002c

Females

>40

55–69

35–70

Males

>40

Males and femalesb

Females Males

Males

Men

Males and females

Males and femalesb Males

Males and femalesb Females

Males

>31

40–69

23–95

55–69

55–69

35–70

Recent intake


Lifetime intake Recent intake

Consumption 5 years ago Consumption 20 years ago Lifetime intake

Recent intake Regular drinking Recent intake


Lifetime intake

Recent intake

≥5 g/day vs none

≥10 vs 0–9.9 g/day Daily drinker of ≥5 drinks/day vs never drank daily

Continuous (5 drinks/ day) Continuous (5 drinks/ day) High vs low quartile High vs low tertile ≥10 vs 0– 9.9 g/day

≥41 vs 300 g/wk vs none ≥45 g/day vs none

≥30 g/day vs none ≥20 g/day vs 125 drink-years vs never drank daily

1.7 (0.6, 4.8)

0.9 (0.5, 1.6) 1.5 (0.8, 3)

1.4 (0.6, 3.1)

1.25 (0.69, 2.26)

Y

Y Y

Y

Y

Y

Y

1.5 (1.1, 2.1)

1.86 (1.13, 3.06)

Y

Y

Y

Y

Y

Y

Y

Y

1.8 (1.2, 2.8)

1.49 (1.04, 2.12)

2.4 (1.5, 4)

2.2 (1, 4.6)

0.91 (0.39, 2.1)

2 (0.4, 9.6)

2.1 (1.2, 3.7)

2 (1.1, 3.6)

Y (?)

Y(?) Y (?)

Y (?)

N

N

Y (?)

Y (?)

Y (cat.)

Y (status)

Y (status)

Y (cat.)

Y (?)

Y (?)

Y (?)

Y

Y N

Y

N

N

N

N

Y

Y

Y

Y

Y

N

N

Y

Y N

Y

N

N

N

N

Y

N

N

Y

Y

N

N

(continues)

N

Y N

Y

N

N

N

N

Y

Y

N

N

N

N

N

Summary of the Epidemiological Evidence

247

3,291


3,291


393

202

992

489,979

29,132

3,500

276

110

113

236

145

145 107

277

992

489,979

29,132

277

Denmark

Liquor Case-control studies Longnecker, Various 1990 locations, USA

Pedersen et al., 2003 Cho et al., 2004

The Netherlands

Sweden


Cohort studies Goldbohm et al., 1994

Western NY, USA


393

202

Cases n

Denmark

Study location

Wine Case-control studies Longnecker, Various 1990 locations, USA

Pedersen et al., 2003 Cho et al., 2004

Author, publication year

Controls/ cohort size n

Females

>40

>31

23–95

55–69

Males

>40

Men


Males and femalesb

Females

Males

Men


Subgroup

>31

23–95

Age range Years (mean)

Consumption 5 years ago Consumption 20 years


Recent intake

Recent intake


Consumption 5 years ago Consumption 20 years ago Lifetime intake


Alcohol variable

Continuous (5 drinks/ day) Continuous (5 drinks/

≥14 vs 0 drinks/wk ≥15 g/day vs none

≥5 g/day vs none

≥10 vs 0– 9.9 g/day

Continuous (5 drinks/ day) Continuous (5 drinks/ day) High vs low quartile High vs low tertile ≥10 vs 0– 9.9 g/day

≥14 vs 0 drinks/wk ≥15 g/day vs none

Contrast

TABLE 4B (Continued)

Y

Y

0.9 (0.6, 1.5)

Y

Y

Y

Y

Y

Y

1 (0.6, 1.7)

1.55 (1.11, 2.17)

0.9 (0.7, 2.9)

1.4 (0.4, 9.6)

1 (0.6, 1.6)

1 (0.2, 4.2)

0.94 (0.58, 1.67)

Y

Y

1.1 (0.4, 3.3)

0.87 (0.55, 1.38)

Y

Y

Y

Age

1.4 (0.4, 5.1)

1.59 (1.12, 2.25)

1.4 (0.8, 2.4)

OR/RR (95% CL)

Y (?)

Y (?)

Y (cat.)

Y (status)

Y (?)

Y (?)

Y (?)

N

N

Y (?)

Y (?)

Y (cat.)

Y (status)

Smokinga

N

N

Y

Y

Y

Y

Y

N

N

N

N

Y

Y

BMI/ weight

N

N

Y

N

Y

Y

Y

N

N

N

N

Y

N

Total energy intake


N

N

Y

N

N

Y

Y

N

N

N

N

Y

N

Physical activity


Canada

Sharpe et al., 2002

489,979

29,132

202 3,291

3500

113

500

276

110 230

236

145

145 107

277

277

23–95

55–69

35–70

Females

>40


Males

Males

Females

Males

Males

>40

Recent intake Recent intake Recent intake



ago Lifetime intake

≥7.5 g/day vs none ≥14 vs 0 drinks/wk ≥15 g/day vs none

≥10 vs 0– 9.9 g/day Daily drinker of ≥5 drinks/day vs never drank daily

day) High vs low quartile High vs low tertile ≥10 vs 0– 9.9 g/day

1.1 (0.85, 1.41)

1.3 (0.6, 3)

2.7 (1, 7.5)

1.9 (0.9, 3.9)

0.7 (0.2, 3.7)

1.1 (0.5, 2.1)

1.33 (0.76, 2.35)

1.23 (0.78, 1.94)

Y

Y

Y

Y

Y

Y

Y

Y

Y (cat.)

Y (status)

Y (?)

Y (?)

Y (?)

Y (?)

N

N

Y

Y

Y

N

Y

Y

N

N

Y

N

Y

N

Y

Y

N

N

Y

N

N

N

Y

Y

N

N

a (status), adjusted for smoking status; (cont.), adjusted for cigarette smoking in pack-years or cigarettes/day as a continuous variable; (cat.), Adjusted for cigarette smoking in categories of packyears or cigarettes per day; (?), adjustment method not specified. b Adjusted for gender. c Analyses on wine are also presented in the manuscript, but they were based on very few cases and are not included here. d Includes the Iowa Women’s Health Study, the Health Professionals Follow-up Study, and the Nurses’ Health Study.


The Netherlands Denmark

Sweden


Cohort studies Goldbohm et al., 1994 Pedersen et al., 2003 Cho et al., 2004

Western NY, USA



249

250

Chapter 11/Alcohol and Cancer

a stronger effect for the distal colon than for the proximal colon found in a case-control study (Sharpe et al., 2002) and a pooled analysis of cohort studies (Cho et al., 2004) is intriguing and deserves further exploration. Interestingly, alcohol, and particularly beer, has been found to be more strongly associated with colorectal polyps that overexpress p53 than those that do not, and polyps overexpressing p53 were reported to be more likely to be located in the distal colon and rectum (Terry et al., 2003). Alcohol consumption has been found to exacerbate the elevation in colorectal cancer risk associated with having a family history of the disease at least in two studies (Le Marchand et al., 1999; Fuchs et al., 2002). Because alcohol is known to act as an antagonist of methyl-group metabolism, there has been some interest in exploring its interaction with folate and methionine intakes. In the Health Professionals Follow-up Study, an increased risk of colon cancer was found for those with high alcohol and low folate and methionine intakes, particularly for carcinomas of the distal colon (Giovannucci et al., 1995). In the pooled analysis of eight prospective studies (Cho et al., 2004), the observed relationship with alcohol was limited to those in the lowest tertile of methionine intake, whereas there was no indication of a folate–alcohol interaction. Some studies have examined the possible influence of genetic susceptibility to the effect of alcohol on colorectal cancer. For example, an interaction of alcohol with polymorphisms in methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (Ma et al., 1999), two enzymes involved in the generation of S-adenosylmethionine, the primary methyl donor for DNA methylation, has been reported. The role of polymorphisms in enzymes involved in alcohol metabolism has also been explored. In a nested case-control study within the Physician’s Health Study (Chen et al., 2001), ADH3 polymorphism was unrelated to colorectal cancer, but there was a suggestion of an interaction with alcohol intake. An association and effect modification of the alcohol effect by ALDH2 genotype was reported in two studies conducted in Japan (Murata et al., 1999; Matsuo et al., 2002).

Cancers with a Controversial Association with Alcohol Lung Cancer A relationship between lung cancer and alcohol has been suggested in a number of studies (Bandera et al., 2001), but it has generally been attributed to residual confounding by cigarette smoking. Studying this relationship is indeed a challenge given the strong relationship between cigarette smoking and lung cancer and the fact that smoking and drinking behaviors tend to correlate, at least in some cultures. The 1988 IARC Report concluded that alcohol and lung cancer were “not causally related,” whereas the 1997

WCRF Report concluded that the association was “possible.” A meta-analysis (Bagnardi et al., 2001a) including three case-control studies and three cohort studies found a modest increase in risk of 4% and 8% for approximately four drinks per day and eight drinks per day, respectively. Another more comprehensive meta-analysis (Korte et al., 2002) reported overall pooled smoking-adjusted risk estimates on the basis of highest consumption category in each study of 1.19 (95% CI: 1.11, 1.29) for 11 cohort studies and 1.39 (95% CI: 1.06, 1.83) for 7 case-control studies. This meta-analysis also found that smoking-adjusted risk estimates in hospital-based case-control studies (pooled OR = 1.69, 95% CI: 1.35, 2.12) tended to be of greater magnitude than those reported in population-based case-control studies (pooled OR = 1.09 (95% CI: 0.63, 1.88). This could be explained in part by the fact that two of the hospital-based studies included in these analyses were conducted in Uruguay (De Stefani et al., 1993) and Turkey (Dosemeci et al., 1997) and participants reported much higher levels of intake than in other studies. Population-based studies evaluating the association between alcohol and lung cancer meeting our inclusion criteria are shown in Table 5. Of the 10 studies identified, 6 (Bandera et al., 1992; Potter et al., 1992; Carpenter et al., 1998; Prescott et al., 1999; Djousse et al., 2002; Freudenheim et al., 2003) reported elevated risk for at least one type of alcohol, whereas there was no evidence of an association in other studies (Mayne et al., 1994; Bandera et al., 1997; Woodson et al., 1999; Hu et al., 2002). For example, one study conducted in California (Carpenter et al., 1998) found a relationship only for liquor, whereas in the other studies conducted in western New York (Bandera et al., 1992; Freudenheim et al., 2003) and Iowa (Potter et al., 1992), the association with alcohol was mostly explained by beer consumption. It is uncertain whether there is a true different effect by each alcohol type or an association tends to be found with the most prevalent alcoholic beverage consumed in that population. As illustrated in Table 5, studies examining the effect by alcoholic beverage type have not offered consistent results, but in general they tend to suggest an increased risk with beer, and perhaps liquor, and a decreased lung cancer risk associated with moderate wine consumption. This is of particular interest because wine drinking has been shown to be unrelated to smoking (Prescott et al., 1999). It also points out the need for evaluating the role of the different alcoholic beverages rather than that of total alcohol. To avoid the issue of residual confounding by smoking, studies in never smokers could be particularly informative. However, evaluating the relationship among non/never smokers has been difficult given the low incidence of lung cancer and low prevalence of drinking in this population. In general, studies evaluating the relationship among nonsmokers have presented conflicting results (Bandera et al.,

Study location

New York State, USA

Framingham, US

Bandera et al., 1997

Djousse et al., 2002

Beer Case-control studies Bandera Western et al., 1992 New York, USA Mayne et al., New York 1994 State, USA

Denmark

Prescott et al., 1999

Finland, ATBC study population

Western New York, USA


Cohort studies Woodson et al., 1999

Canada

Hu et al., 2002

Total alcohol Case-control studies Bandera Western et al., 1992 New York, USA Carpenter, California, 1998 USA


564

413

280

413

8969

20,456

130 269

27,544

13,053

194 395

15,107

27,111

1546

480

1,059

111

483

615

261

161

564


F

>20

20–80

35–79

80 28–62

Recent intake “Usual intake”

M and F,b nonsmokers



Recent intake

Lifetime intake

Recent intake

Recent intake

Recent intake

Recent intake

Alcohol variable

M

M and F

F

M

M

>20

80

M, smokers

M and F

F, never smoker

M and F

M, heavy smokers

Subgroup

50–69

35–79

20− >70

40–84

35–79


≥113 drinks/mo vs none High vs low quartiles (cut points not shown)

High vs low drinking quartile (>27.7 g/day vs 41 vs 41 vs 48 drinks/mo vs none High vs low tertile >24 g/day vs none

>82 L vs none

≥22 drinks/ month vs less ≥3 drinks/ day vs 0–3 drinks/mo >1 servings/wk vs none >2.5 L vs none

Contrast

1.18 (CI not presented, includes one)

1.95 (1, 3.7)

1.3 (0.7, 2.4)

1.01 (0.64, 1.58)

1.15 (0.8, 1.65)

0.8 (0.11, 5.79)

1.57 (1.06, 2.33)

1 (0.8, 1.2)

1.13 (0.47, 2.72)

1.35 (0.54, 3.41)

0.8 (0.5, 1.2)

1.07 (0.46, 2.47)

1.6 (1, 2.4)

OR/RR (95% CL)

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Age

Y (cont.)

Y (cont.)

Y (cont.)

Y (cont.)

Y (cont.)

Y (cat.)

Y (cat.)

Y (cont.)

Y (cont.)

Y (cont.)

N

Y (cat.)

Y (cont.)

Smokinga

Y

N

N

N

N

N

N

N

Y

Y

N

N

N

ETS

N

N

N

Y

Y

N

N

Y

Y

Y

N

N

N

Total energy intake


Population-Based Case-Control and Cohort Studies Examining Alcohol Consumption and Lung Cancer

280

Cases n

TABLE 5

(continues)

N

Y

N

N

N

N

N

Y

Y

Y

N

Y

Y

Other dietary factors


251

Canada


Hu et al., 2002


Canada


Hu et al., 2002


111

1,546

483

615

261

161

564

13,053

194

280

15,107

480

Denmark

1,900

27,111

1,546

483

615

109

1,059

111

161

261

Cases n

Iowa, US

Wine Case-control studies Bandera Western et al., 1992 New York, Carpenter California, et al., 1998 USA

Potter et al., 1992c Prescott et al., 1999


California, USA

Carpenter et al., 1998


Study location



F

>20

35–79

20 to >70

40–84

M and F

F, never smoker

M and F

M

M

>20

35–79

W

M, smokers

M and F

F, never smoker

M and F

Subgroup

55–69

50–69

35–79

20 to >70

40–84


Lifetime intake

Recent intake

Recent intake



Recent intake

>19 liters vs none

≥2 drinks/mo vs none ≥1 drinks/day vs 0–3 drinks/mo >0.5 glasses/wk vs none >1 liter vs none

High vs low drinking quartile (>11.6 g/day vs 13 vs 13 vs 62 liters vs none

Lifetime intake

Recent intake


Contrast ≥1 drinks/ day vs 0–3 drinks/mo >0.5 bottles or cans/week vs none >1.6 liters vs none

Alcohol variable

TABLE 5 (Continued)

0.80 (0.51, 1.25)

0.72 (0.4, 1.29)

0.7 (0.4, 1.2)

0.79 (0.34, 1.86)

0.7 (0.5, 1.1)

1.49 (0.7, 3.13)

1.36 (1.02, 1.82)

2 (1.02, 3.8)

0.9 (0.7, 1.1)

1.36 (0.82, 2.27)

1.67 (0.96, 2.92)

0.5 (0.2, 1.1)

0.86 (0.44, 1.75)

OR/RR (95% CL)

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Age

Y (cont.)

Y (cont.)

N

Y (cat.)

Y (cont.)

Y (cat.)

Y (cat.)

Y (cat.)

Y (cont.)

Y (cont.)

Y (cont.)

N

Y (cat.)

Smokinga

Y

Y

N

N

N

N

N

N

N

Y

Y

N

N

ETS

Y

Y

N

N

N

N

N

N

Y

Y

Y

N

N

Total energy intake


Y

Y

N

Y

N

N

N

N

Y

Y

Y

N

Y

Other dietary factors


Finland, ATBC study population Denmark

15,107 13,053

480 194

Denmark

1,900

109

27,111

1,546

483

Iowa, USA

1,059

111



161

615

261

Canada

564

13,053

194

280

15,107

27,111

480

1,059

F

>20

W M F

>20 >20

M, smokers

F, never smoker M and F

M and F

55–69

50–69

20 to >70 35–79

40–84

M

M

>20

35–79

M, smokers

50–69


Recent intake

High vs low drinking quartile (>22.8 g/day vs 13 vs 13 vs 28 liters vs none

Lifetime intake


≥9 drinks/mo vs none

High vs low drinking (>2.1 g/day vs13 vs 13 vs 0.5 shots/wk vs none >1 liter vs none

Recent intake


Recent intake

0.67 (0.21, 2.18)

1.46 (0.99, 2.14)

1.1 (0.6, 2.3)

1.1 (0.9, 1.3)

0.79 (0.52, 1.2)

0.87 (0.51, 1.48)

1.1 (0.6, 2.1)

1.87 (1.02, 3.42)

1.1 (0.7, 1.6)

0.18 (0.03, 1.33)

0.44 (0.22, 0.86)

0.8 (0.6, 1.1)

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y (cat.)

Y (cat.)

Y (cat.)

Y (cont.)

Y (cont.)

Y (cont.)

N

Y (cat.)

Y (cont.)

Y (cat.)

Y (cat.)

Y (cont.)

N

N

N

N

Y

Y

N

N

N

N

N

N

N

N

N

Y

Y

Y

N

N

N

N

N

Y

N

N

N

Y

Y

Y

N

Y

N

N

N

Y

a (status), adjusted for smoking status; (cont.), adjusted for cigarette smoking in pack-years or cigarettes/day as a continuous variable; (cat.), adjusted for cigarette smoking in categories of packyears or cigarettes per day; (?), adjustment method not specified. b Unadjusted by gender. c Nested case-control design. ETS, environmental tobacco smoke; M, males; F, females.

Potter et al., 1992c Prescott et al., 1999


Hu et al., 2002 Freudenheim et al., 2003

Liquor Case-control studies Bandera Western et al., 1992 New York, USA Carpenter California, et al., 1998 US

Prescott et al., 1999



253

254


2001; Korte et al., 2002). The two population-based studies conducted among nonsmokers (Mayne et al., 1994) or never smokers (Hu et al., 2002) did not find an association. If alcohol has an effect in lung carcinogenesis, current drinking rather than lifetime or past drinking appears to be the relevant period (Bandera et al., 2001). The effect by gender and histological type has been evaluated in a number of studies with inconclusive results (Bandera et al., 2001). The few studies that evaluated the effect of alcohol on lung cancer by levels of vitamin A, carotenoids, or vegetable consumption reported with some consistency a stronger association for the lower category of consumption (Bandera et al., 2001). Because one of the proposed mechanisms of action of alcohol is through direct damaging effects of acetaldehyde on the lung tissue, the role of ADH genotype has received some attention. Yang et al. (2002) found that ADH3*1 genotype was associated with higher pulmonary ADH activity and acetaldehyde–DNA adduct levels in Caucasian lung tissue. In contrast, a case-control study (Freudenheim et al., 2003) did not find elevated risk of lung cancer for those with the ADH31-1 genotype, the variant that has been shown to catalyze alcohol to acetaldehyde more rapidly (Bosron and Li, 1986). Furthermore, there was no indication in this study (Freudenheim et al., 2003) of an interaction of alcohol consumption and ADH genotype on lung cancer risk, but these analyses were based on small numbers. Endometrial Cancer The relationship between alcohol and endometrial cancer has been examined in relatively few epidemiological studies. The 1988 IARC Report concluded that there were insufficient data to judge the evidence, and the 1997 WCRF Report did not even mention the possible role of alcohol on endometrial cancer risk. In general, epidemiological studies have not offered much support for the association, although there are some aspects of this potential relationship that warrant further examination (Bandera et al., 2003). Population-based studies examining this relationship (Webster and Weiss, 1989; Gapstur et al., 1993; Swanson et al., 1993; Goodman et al., 1997a,b; Newcomb et al., 1997; Terry et al., 1999; Jain et al., 2000a; Jain et al., 2000b; McCann et al., 2000; Littman et al., 2001; Weiderpass and Baron, 2001), shown in Table 6, have reported risk estimates for total alcohol fluctuating around the null value. However, these studies were confronted with very limited range of alcohol consumption. Hospital-based studies conducted in Italy reporting much higher drinking levels reported an elevated risk (Bandera et al., 2003). A meta-analysis including two cohort studies and four case-control studies reported a 20% increased risk for an alcohol consumption of 100 g/day, whereas for 25 or 50 g/day, there was no association (Bagnardi et al., 2001a). Studies evaluating alcohol type suggested a reduced risk for moderate intake of beer and

wine, whereas liquor intake appeared to increase risk. This may be explained by the beneficial effects of antioxidants and phytoestrogens in beer and wine when consumed at moderate levels (Bandera et al., 2003). Overall, it seems that at moderate levels, alcohol consumption does not increase endometrial cancer risk or it may even be beneficial. The role of heavy alcohol consumption is not clear. Furthermore, important aspects of this relationship that have received limited attention and need to be considered in studies are the possible effect modification by menopausal status and use of estrogen replacement therapy. Ovarian Cancer Compared with other cancer sites, the relationship between alcohol and ovarian cancer has not been widely investigated. The 1988 IARC Report concluded that there was no association between alcohol and ovarian cancer, whereas the relationship was not even mentioned in the 1997 WCRF/AICR Report (WCRF, 1997). A meta-analysis (Bagnardi et al., 2001b) including five case-control studies computed elevated pooled ORs (95% CIs) of 1.11 (1.0–1.24), 1.23 (1.01–1.54), and 1.53 (1.03–2.32) for 25, 50, and 100 g of alcohol per day, respectively (Table 2). Another metaanalysis including seven population-based studies (Webb et al., 2004) computed a pooled risk estimate of 0.72 (95% CI: 0.54–0.97) for the highest level of consumption reported in each study. Results from population-based studies examining the relationship between alcohol and ovarian cancer risk are displayed in Table 7. A decreased risk associated with drinking levels of fewer than three drinks per day was suggested by several case-control studies (Gwinn et al., 1986; Godard et al., 1998; Goodman and Tung, 2003; Webb et al., 2004) and in the Iowa Women’s Health Study (Kushi et al., 1999). The remaining case-control study suggested an elevated risk for drinkers of more than three drinks per day (Kuper et al., 2000). There was no indication of an association among participants in the Netherlands Cohort Study (Schouten et al., 2004). Two case-control studies (Goodman and Tung, 2003; Modugno et al., 2003) have suggested that the effect of alcohol on ovarian cancer risk might vary by histology, perhaps explaining in part the inconsistent results. One of these studies (Modugno et al., 2003) reported an elevated risk only for mucinous tumors, with an OR for those consuming more than 24 g/day of ethanol of 1.93 (95 % CI: 1.02, 3.65). In contrast, another study (Goodman and Tung, 2003) found similar risk estimates for mucinous and nonmucinous tumors but reported a strong protective effect for total alcohol only for invasive epithelial ovarian cancer (OR = 0.36; 95% CI: 0.19, 0.70) and an elevated risk for borderline serous tumors associated with the consumption of spirits (OR = 2.66, 95% CI: 1.46, 4.85). However, a large case-control study found an inverse association between

Study location

Cohort studies Gapstur et al., 1993

2,859

297 2,313

704

400 739

Iowa, USA

167

332

24,848

511

56,837

221

944

679

Canada

639

232

11,659

298

391

133

511

332

24,848

2,313

739

167

2,247

Controls/ Cohort size n

351

Cases n

55–69

18–84

40–59

55–69

40–79

20–74

50–74

45–74

40–85

18–84

40–79

20–54


Recent intake

Recent intake


Intake during adulthood Intake as young adult

Recent intake Recent intake Recent intake Recent intake Recent intake Recent intake Recent intake

Alcohol variable

≥4 vs 0 g/day

>154 vs 0 g of beer/day

≥4 vs 0 g/day >4 vs 0 drinks/wk H vs L quartile

>4 vs 0 drinks/wk ≥14 vs 0 drinks/wk

0 vs ≥150 g/wk ≥14 vs 0 drinks/wk >17.8 vs 0 g/day >8.3 vs 0 g/day >9 vs ≤0.5 g/day ≥1 vs 0 drinks/day >4 vs 0 g/day

Contrast

0.7 (0.3, 1.6)

0.54 (0.18, 1.62)

1 (0.67, 1.5)

1.3 (0.6, 2.8)

1 (0.7, 1.6)

1 (0.58, 1.73)

0.72 (0.39, 1.35)

0.92 (0.7, 1.2)

0.95 (0.66, 1.4)

1 (0.5, 1.8)

0.72 (0.52, 0.99)

0.8

1.27 (0.78, 2.07)

1.83 (1.11, 3.01)

OR/RR (95% CL)

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Age

N

N

Y (status)

N

N

Y (status)

Y (status)

Y (status)

Y (status)

Y (cont.)

Y (status)

N

Y (status)

Y (status)

Smokinga

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

N

BMI/ weight

N

Y

Y

N

N

N

N

N

Y

Y

Y

Y

N

N

Total energy intake


Population-Based Case–Control and Cohort Studies Examining Alcohol Consumption and Endometrial Cancer

Iowa, USA Sweden

Beer Case-control studies Goodman Hawaii, et al., 1997b USA

Cohort studies Gapstur et al., 1993 Terry et al., 1999 Jain et al., 2000b

Total alcohol Case-control studies Webster and USA Weiss, 1989 Newcomb Wisconsin, et al., 1997 USA Goodman Hawaii, et al., 1997a USA Jain et al., Ontario, 2000a Canada McCann Western et al., 2000 NY, USA Littman et al., Washington 2001 State, USA Weiderpass Sweden and Baron, 2001 Swanson USA et al., 1993 Newcomb Wisconsin, et al., 1997 USA


TABLE 6

(continues)

Y

Y

Y

N

Y

Y

N

Y

Y

Y

Y

N

Y

N

HRT/ERT use


255

Study location

Iowa, USA

Iowa, USA

167

332

167

332

Cases n

24,848

511

24,848

511

Controls/ Cohort size n

55–69

18–84

55–69

18–84


Recent intake

Recent intake

Recent intake

Recent intake

Alcohol variable

≥4 vs 0 g/day

>37 vs 0 g of liquor/day

≥4 vs 0 g/day

>3.95 vs 0 g of wine/day

Contrast

1.4 (0.8, 2.4)

2.1

0.8 (0.4, 1.7)

0.7

OR/RR (95% CL)

Y

Y

Y

Y

Age

N

N

N

N

Smokinga

Y

Y

Y

Y

BMI/ weight

N

Y

N

Y

Total energy intake


Y

N

Y

N

HRT/ERT use

a (status), adjusted for smoking status; (cont.), adjusted for cigarette smoking in pack-years or cigarettes/day as a continuous variable; (cat.), Adjusted for cigarette smoking in categories of packyears or cigarettes per day; (?), adjustment method not specified.


Liquor Case-control studies Goodman Hawaii, et al., 1997a USA


Wine Case-control studies Goodman Hawaii, et al., 1997a USA


TABLE 6 (Continued)


Study location

139 214

The Netherlands

696

558

Iowa, USA

Australia

Webb et al., 2004

Cohort studies Kushi et al., 1999 Schouten et al., 2004

Hawaii and Los Angeles, USA

549

Massachusetts and New Hampshire, USA Delaware Valley, USA 761

170

433

Cases n

Canada

Goodman and Tung, 2003

Modugno et al., 2003

Godard et al., 1998 Kuper et al., 2000

Total alcohol Case-control studies Gwinn et al., USA 1986 (multicenter)


TABLE 7

62,573

29,083

786

607

1,352

516

170

2,915


55–69

55–69

18–79

>18

20–69

10 g/day vs 0 >15 g/day vs none

≥2 drinks/day vs none

≥14 drinks/wk vs never ≥25 vs never

≥25 vs never

≥24 g/day vs never ≥14 drinks/wk vs never

≥24 g/day vs never ≥24 g/day vs never

≥24 g/day vs never

≥250 g/wk vs never drank ≥10 drinks/wk vs none >3 drinks/day vs 0

Contrast

0.92 (0.55, 1.54)

0.49 (0.24, 1.01)

0.49 (0.3, 0.81)

0.59 (0.35, 1)

0.36 (0.19, 0.7)

0.88 (0.58, 1.34)

0.84 (0.55, 1.28)

1.29 (0.83, 2)

0.88 (0.57, 1.37)

0.64 (0.22, 1.85)

1.93 (1.02, 3.65)

1.35 (0.8, 2.26)

0.46 (0.13, 1.69)

0.5 (0.2, 0.9)

OR/RR (95% CL)

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Age

Y (status)

Y (cont.)

Y (status)

N

N

N

N

Y (status)

Y (status)

Y (status)

Y (status)

Y (cont.)

N

Y (?)

Smokinga

Y

Y

Y

N

N

N

N

N

N

N

N

Y

N

N

BMI/weight/ WHR


Population-Based Case-Control and Cohort Studies Examining Alcohol Consumption and Ovarian Cancer

Y

N

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

OC use

(continues)

Y

Y

N

N

N

N

N

N

N

N

N

N

N

N

Total energy


257

Study location

Hawaii and Los Angeles, US

Australia


Webb et al., 2004

Delaware Valley, USA




Wine Case-control studies Gwinn et al., USA 1986 (multicenter)



Beer Case-control studies Gwinn et al., USA 1986 (multicenter)


558

761

433

696

558

761

433

Cases n

607

1,352

2,915

786

607

1,352

2,915


>18

20–69

20–54

18–79

>18

20–69

20–54


Nonmucinous tumors (n = 649)

Mucinous tumors (n = 112)



Subgroup

Drinkyears

Current

Former

Current

Former

Current

Recent intake

Drinkyears Recent intake

Current

Former

Current

Former

Current

Recent intake

Alcohol variable

≥25 vs never


0–24 g/day vs never ≥24 g/day vs never

≥150 g/wk vs never drank 0–24 g/day vs never

≥1 drink/day vs none

≥25 vs never



≥150 g/wk vs never drank ≥24 g/day vs never

Contrast

TABLE 7 (Continued)

1.07 (0.71, 1.63)

0.81 (0.52, 1.26)

0.71 (0.25, 2.04)

0.92 (0.38, 2.23)

0.71 (0.29, 1.74)

0.59 (0.28, 1.24)

0.7 (0.3, 1.4)

1.26 (0.65, 2.46)

1.05 (0.62, 1.77)

0.93 (0.53, 1.64)

1.24 (0.63, 2.45)

0.92 (0.39, 2.14)

1.25 (0.4, 3.93)

2.53 (0.86, 7.42)

0.9 (0.5, 1.8)

OR/RR (95% CL)

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Age

N

N

Y (status)

Y (status)

Y (status)

Y (status)

Y (?)

Y (status)

N

N

Y (status)

Y (status)

Y (status)

Y (status)

Y (?)

Smokinga

N

N

N

N

N

N

N

Y

N

N

N

N

N

N

N

BMI/weight/ WHR


N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Total energy

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

OC use


Australia


Australia


Webb et al., 2004

696

558

761

433

214

696

786

607

1,352

2,915

62,573

786

18–79

>18

20–69

20–54

55–69

18–79



Drinkyears Recent intake

Current

Former

Current

Former

Current

Recent intake

Recent intake

Recent intake

≥25 vs never ≥1 drink/day vs none



≥150 g/week vs never drank ≥24 g/day vs never

>15 g/day vs none

≥1 drink/day vs none

1.07 (0.59, 1.95)

0.69 (0.42, 1.14)

1.07 (0.64, 1.78)

1.42 (0.79, 2.57)

1.53 (0.58, 4)

1.07 (0.3, 3.79)

8.83 (2.89, 27.01)

0.6 (0.3, 1.1)

1 (0.57, 1.75)

0.56 (0.33, 0.93)

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y (status)

N

N

Y (status)

Y (status)

Y (status)

Y (status)

Y (?)

Y (status)

Y (status)

Y

N

N

N

N

N

N

N

Y

Y

N

N

N

N

N

N

N

N

Y

N

a (status), adjusted for smoking status; (cont.), adjusted for cigarette smoking in pack-years or cigarettes/day as a continuous variable; (cat.), adjusted for cigarette smoking in categories of packyears or cigarettes per day; (?), adjustment method not specified. BMI, body mass index; WHR, waist-to-hip ratio.



Liquor Case-control studies Gwinn et al., US 1986 (multicenter)

Cohort studies Schouten et al., The 2004 Netherlands

Webb et al., 2004

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y


259

260


alcohol consumption and ovarian cancer, which was of similar magnitude for all histological types (Webb et al., 2004). Although the findings are not conclusive, they suggest that future ovarian cancer studies should examine the effect by histological type. Overall, these results, though inconsistent, tend to indicate that moderate levels of alcohol drinking might be associated with a reduced risk of ovarian cancer. As Table 7 shows, there is no consistent evidence that any particular source of alcohol may be more beneficial or detrimental than another, but in general there is a tendency for studies to show reduced risk for wine consumption and elevated risk estimates for beer and liquor. The high content of antioxidants and resveratrol in wine has been proposed to explain these findings (Webb et al., 2004). If alcohol drinking is related to ovarian cancer, current rather than lifetime drinking seems to be the relevant factor (Goodman and Tung, 2003). Prostate Cancer Both the IARC (1988) and WCRF/AICR (1997) Reports concluded that there was no association between alcohol consumption and prostate cancer risk. A review of the literature reported no association with moderate consumption of alcohol (less than three drinks per day), whereas an elevated risk for drinking more than seven drinks per day was suggested (Dennis and Hayes, 2001). Two independent metaanalyses (Dennis, 2000; Bagnardi et al., 2001b) found an ~20% increased risk associated with drinking levels of about four drinks per day (Table 2). The results of population-based studies examining the role of alcohol on prostate cancer and meeting our inclusion criteria are displayed in Table 8. As shown in the table, results are inconsistent. An increased risk was suggested in several case-control studies (Andersson et al., 1996; Hayes et al., 1996; Breslow et al., 1999; Sharpe and Siemiatycki, 2001) and cohort studies (Breslow et al., 1999; Putnam et al., 2000; Sesso et al., 2001). However, other studies failed to find an association (Hiatt et al., 1994; Le Marchand et al., 1994; Gronberg et al., 1996; Key et al., 1997; Jain et al., 1998; Schuurman et al., 1999; Villeneuve et al., 1999; Ellison, 2000; Albertsen and Gronbaek, 2002). A cohort study (Breslow et al., 1999) reported an inverse association for distant past heavy drinking, but this finding was based on a small number of subjects and has not been confirmed nor evaluated in other investigations to our knowledge. Nevertheless, this aspect of drinking might be important to consider in future studies. The studies evaluating the effect by alcoholic beverage type did not offer consistent results. Unlike other cancer sites, there is no indication that wine, even at moderate levels, may exert a protective effect. The overall evidence does not seem to indicate that recent consumption of alcohol at moderate levels plays a major role in the etiology of prostate cancer.

Cancers Probably Not Associated with Alcohol Consumption Epidemiological studies have generally not shown an association with cancer of the pancreas (IARC, 1988; WCRF, 1997; Longnecker and Tseng, 1999; Nasca, 2001). However, given the high fatality rate for this cancer, most studies used proxy interviews, perhaps leading to considerable random measurement error. If there is a weak association between alcohol and cancer of the pancreas, the relationship may have been missed because of the resulting misclassification. Heavy alcohol consumption increases the risk of chronic pancreatitis, which in turn is a risk factor for pancreatic cancer (Longnecker and Tseng, 1999). Alcohol consumption does not appear to increase the risk of stomach and bladder cancers (IARC, 1988; WCRF, 1997).

ALCOHOL DRINKING AND CANCER SURVIVAL Considering the extensive literature on alcohol and cancer, the relatively small body of literature examining the role of alcohol in cancer survival is surprising. There is a growing interest in this research area, with most research focusing on head and neck and breast cancers.

Alcohol in the Prognosis of Head and Neck Cancers Head and neck cancer patients are at increased risk of developing second primary esophageal squamous cell carcinomas, and chronic use of alcohol and smoking, in particular acetaldehyde, has been implicated in its etiology (Muto et al., 2002). A case-control study found a strong relationship with heavy alcohol consumption but not with cigarette smoking for second primary esophageal cancer among head and neck cancer patients (Tanabe et al., 2001). Confirming the role of acetaldehyde, a case-control study found an interaction between drinking and the ALDH2-2 genotype (coding for the inactive ALDH2) and a weak interaction with ADH3-1 (coding for the more active ADH3) (Muto et al., 2002) on multiple esophageal dysplasia, a precursor for squamous cell esophageal carcinoma, among head and neck cancer patients. In addition to esophageal cancer, head and neck cancer patients who drink and smoke have elevated risk of other second tumors in the aerodigestive tract, and an independent role of alcohol has been suggested in a number of studies (Franco et al., 1991; Day et al., 1994; Barbone et al., 1996). Heavy alcohol drinking has also been associated with decreased survival for head and neck cancer patients. A cohort study found that head and neck cancer patients who drank heavily around the time of diagnosis experienced

Study location

680 101 145

USA (NHANES cohort I) USA (NHANES cohort II)

The Netherlands

Iowa, USA

Canada

Harvard Alumni, US

Denmark

Breslow et al., 1999 Breslow et al., 1999

Schuurman et al., 1999 Putnam et al., 2000 Ellison, 2000

Sesso et al., 2001

Albertsen and Gronbaek, 2002

233

366

3,775

134

12,989

7,612

3,400

1,572

58,279

5,766

43,432

20,316

476

1,623

637

328

252

238

California, USA

399

Canada

Hiatt et al., 1994

1,623

Canada

198

617

Canada

Hawaii, USA

328

252

256

England

1,315

981

Cases n


20–98

(66.6)

50–84

40–86

55–69

25− 65+ 25− 65+

>30

>18

45–70

50–74

(69.8)

41 drinks/wk vs 92 g/wk vs 0

>22 drinks/wk vs none >22 drinks/wk vs none

≥6 drinks/day vs never

≥5261 g vs 0

>96 g/wk vs none 54–61 years vs none >16.6 g/day vs 125 drinkyears vs never drank weekly

≥57 drinks/wk vs never

Contrast

0.66 (0.29, 1.49)

1.33 (0.86, 2.05)

0.93 (0.55, 1.57)

1.5 (0.8, 2.7)

1.1 (0.8, 1.6)

0.23 (0.06, 0.95)

1.42 (0.84, 2.4)

1 (0.4, 2.8)

1.1 (0.7, 1.7)

2.1 (1.3, 3.3)

1.1 (0.9, 1.6)

0.89 (0.64, 1.25)

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

1.2 (0.7, 2.3) 1.04 (0.71, 1.54)

Y

Y

Age

1.5 (0.8, 2.8)

1.9 (1.3, 2.7)

OR/RR (95% CL)

Y (status)

Y (cat.)

N

N

N

N

N

Y (status)

N

Y (?)

Y (cont.)

N

N

N

N

N

Smokinga

Y

Y

N

N

N

N

N

N

N

N

Y

N

N

N

N

N

BMI/ weight

N

N

N

N

N

N

N

N

N

N

N

Y

N

N

N

N

Total energy intake


Population-Based Case-Control and Cohort Studies Examining Alcohol Consumption and Prostate Cancer

Cohort studies Le Marchand et al., 1994

Key et al., 1997 Jain et al., 1998 Villeneuve et al., 1999 Sharpe et al., 2001

Total alcohol Case-control studies Hayes et al., USA (Georgia, 1996 Michigan, New Jersey) Andersson Sweden et al., 1996


TABLE 8

(continues)

Y

Y

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Physical activity

Alcohol Drinking and Cancer Survival

261

Study location

637 1,623

617 1,623

366

Harvard Alumni, US

1,315

1,218 637 1,623

406 617 1,623

12,989

7,612

1,572

58,279

3,775

981

233

101

Iowa, USA

Denmark

680

134

476

1,218

406

399

1,315

981

Cases n

The Netherlands

USA (NHANES cohort II)

Canada

Case-control studies Hayes et al., USA (Georgia, 1996 Michigan, New Jersey) Gronberg Sweden et al., 1996 Jain et al., Canada 1998 Villeneuve Canada et al., 1999

Wine


Schuurman et al., 1999 Putnam et al., 2000 Sesso et al., 2001

Cohort studies Breslow et al., 1999

Sharpe et al., 2001

Beer Case-control studies Hayes et al., USA (Georgia, 1996 Michigan, New Jersey) Gronberg Sweden et al., 1996 Jain et al., Canada 1998 Villeneuve Canada et al., 1999



50–74

(69.8)

47–91

40–79

20–98

(66.6)

40–86

55–69

25− 65+

45–70

50–74

(69.8)

47–91

40–79



“Usual ” consumption

Recent intake


“Usual intake”

Lifetime drinking among daily drinkers


“Usual consumption”

Alcohol variable

Almost daily vs none ≥10 g/day vs none ≥1 drinks/day vs none

>14 drinks/wk vs never

15–21 drinks/wk vs none ≥30 g/day vs none >3 cans/week vs none >3 drinks/day vs almost never >13 drinks/wk vs none

>102 drinkyears vs never drank weekly

Almost daily vs none ≥10 g/day vs none ≥4 drinks/day v none


Contrast

TABLE 8 (Continued)

0.9 (0.7, 1.5)

1.12 (0.8, 1.55)

1.18 (0.23, 5.44)

1.4 (0.9, 2.2)

0.98 (0.63, 1.54)

0.72 (0.18, 2.9)

1.7 (0.9, 3)

0.5 (0.2, 1.3)

0.34 (0.12, 0.92)

1.7 (1, 2.9)

0.5 (0.2, 1)

0.68 (0.49, 0.94)

0.84 (0.56, 1.24)

2.1 (1.4, 3.1)

OR/RR (95% CL)

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Age

Y (cont.)

N

N

N

Y (status)

Y (cat.)

N

N

N

Y (?)

Y (cont.)

N

N

N

Smokinga

Y

N

N

N

Y

Y

N

N

N

N

Y

N

N

N

BMI/ weight

N

Y

N

N

N

N

N

N

N

N

N

Y

N

N

Total energy intake


N

N

N

N

Y

Y

N

N

N

N

N

N

N

N

Physical activity


366

Harvard Alumni, USA

476

399

366

Harvard Alumni, USA 233

101

Iowa, USA

Denmark

680

12,989

7,612

1,572

58,279

3,775

1,623

1,623

134

637

617

The Netherlands


1,218

406

20–98

(66.6)

40–86

55–69

25− 65+

45–70

50–74

(69.8)

47–91

40–79

20–98

(66.6)

40–86

55–69

25− 65+

45–70

Recent intake


“Usual intake”

Recent intake Recent intake Recent intake Lifetime drinking among daily drinkers

“Usual consumption”

Recent intake


“Usual intake”

Lifetime drinking among daily drinkers

15–21 drinks/wk vs none ≥30 g/day vs none >2.5 shots/wk vs none >3 drinks/day vs almost never >13 drinks/week vs none

Almost daily vs none ≥16 g/day vs none ≥4 drinks/day vs none >68 drinkyears vs never drank weekly


15–21 drinks/wk vs none ≥30 g/day vs none >0.9 glasses/ week vs none >3 drinks/day vs almost never >13 drinks/day vs none

>105 drinkyears vs never drank weekly

1.02 (0.52, 2.01)

1.12 (0.64, 1.94)

1.7 (0.9, 3)

1.1 (0.6, 2)

1.01 (0.50, 2.04)

1.7 (0.9, 3.1)

1.8 (0.9, 3.8)

0.86 (0.63, 1.18)

0.78 (0.26, 2.23)

1.9 (1.4, 2.7)

0.92 (0.42, 2.02)

1.05 (0.49, 2.27)

1.9 (0.9, 3.7)

2.3 (1, 5.3)

1.22 (0.38, 3.88)

1 (0.5, 2.1)

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y (status)

Y (cat.)

N

N

N

Y (?)

Y (cont.)

N

N

N

Y (status)

Y (cat.)

N

N

N

Y (?)

Y

Y

N

N

N

N

Y

N

N

N

Y

Y

N

N

N

N

N

N

N

N

N

N

N

Y

N

N

N

N

N

N

N

N

Y

Y

N

N

N

N

N

N

N

N

Y

Y

N

N

N

N

a (status), adjusted for smoking status; (cont.), adjusted for cigarette smoking in pack-years or cigarettes/day as a continuous variable; (cat.), adjusted for cigarette smoking in categories of packyears or cigarettes per day; (?), adjustment method not specified. BMI, body mass index.




1,315

12,989

7,612

1,572

58,279

3,775

476

981

233

101

Iowa, USA

Denmark

680

134

399

The Netherlands


Canada

Liquor Case-control studies Hayes et al., USA (Georgia, 1996 Michigan, New Jersey) Gronberg Sweden et al., 1996 Jain et al., Canada 1998 Villeneuve Canada et al., 1999 Sharpe et al., Canada 2001




Sharpe et al., 2001

Alcohol Drinking and Cancer Survival

263

264


decreased survival after controlling for age, site of cancer, histopathological grade, anatomical stage, antineoplastic treatment, and cigarette smoking (Deleyiannis et al., 1996). Alcohol intake was shown to predict laryngeal cancer survival in one study conducted in Argentina (Pradier et al., 1993), but not in another one conducted in Italy (Boffetta et al., 1997).

sumption was shown in another study to increase the risk of liver metastasis in colorectal cancer patients (Maeda et al., 1998). A Japanese study reported better bladder cancer survival for alcohol drinkers (Wakai et al., 1993), but to our knowledge, this finding has not been replicated in other studies. Possible Biological Mechanisms

Alcohol and Breast Cancer Survival Drinking has been found to be associated with stage of breast cancer at diagnosis, with frequent drinkers in one study (Vaeth and Satariano, 1998) or those drinking ≥14 drinks/wk in another (Weiss et al., 1996) being at increased risk of a regional stage diagnosis. Although these findings could be explained by confounding by other factors affecting survival or detection, such as unhealthy behaviors, access to medical care, or other socioeconomic factors, it is also possible that alcohol affects cancer progression. The few cohort studies that have examined the effect of alcohol drinking on breast cancer survival have offered conflicting results. Overall, most of these studies had limited power due to a small number of events (recurrences or deaths) and/or drinking range, and some of them failed to control for potentially relevant prognostic factors, such as stage at diagnosis, smoking, and treatment. We identified seven cohort studies examining the role of alcohol on breast cancer survival. Of these studies, four (Rohan et al., 1993; Zhang et al., 1995; Holmes et al., 1999; Saxe et al., 1999) did not find an association between drinking and breast cancer survival (death and/or recurrence), whereas one suggested slightly elevated risk of death, though not statistically significant (Ewertz et al., 1991), and two reported elevated risk (Hebert et al., 1998; McDonald et al., 2002). One of these positive studies (Hebert et al., 1998) reported a 41% increased risk of recurrence and a 58% increased risk of death for each additional beer consumed, after adjusting for stage, estrogen receptor status, age, BMI, other dietary factors, and menopausal status. The other positive study conducted among African American women (McDonald et al., 2002) reported an RR of 2.7 (95% CI: 1.3, 5.8) for regular drinkers, after adjusting for stage, radiotherapy, and smoking. Drinking levels were low in these studies, with the highest quartile of intake shown being one drink per day. The effect of heavy alcohol use on breast cancer prognosis has not been evaluated. Overall, there are insufficient data to conclude that drinking has no effect on cancer survival and there is a clear need for research in this area.

Alcohol in the Prognosis of Other Cancers A cohort study of Memorial Sloan-Kettering Cancer Center’s patients found that among nonsmokers, alcohol drinkers with oral and pancreatic cancers experienced poorer prognosis than nondrinkers (Yu et al., 1997). Alcohol con-

A number of causal pathways have been proposed by which alcohol drinking may affect cancer risk. These include direct effects of alcohol on specific organs and tissues, particularly in cancers of the aerodigestive tract, as well as indirect effects by, for example, inducing changes in hormonal levels, interfering with metabolism and absorption of dietary factors, or affecting the metabolism of other carcinogens (WCRF, 1997). Acetaldehyde, the main metabolite of ethanol, is a known carcinogen in experimental animals (IARC, 1988). Elevated DNA adducts of acetaldehyde have been found in peripheral blood cells among alcoholics (Fang and Vaca, 1997). Ethanol has also been shown to generate free radicals and impair DNA repair and immune function (Bandera et al., 2001). Moreover, alcohol drinking can interfere with absorption and/or metabolism of certain dietary factors that have been implicated in cancer etiology, such as retinoids (Wang, 2003) and folate (Bailey, 2003). Although the literature on hormonal effects of alcohol consumption is not consistent, moderate alcohol use has been associated with higher levels of circulating estrogens in premenopausal and postmenopausal women (Gill, 2000). This may explain, at least in part, the increased breast cancer risk associated with alcohol intake. Changes in insulin resistance and the growth hormone–insuline-like growth factor-1 (GH/IGF1) axis have also been implicated (Stoll, 1999). Alcohol may also increase cancer risk by acting as a solvent for tobacco or other carcinogens or affecting the metabolism of carcinogens by reducing their detoxification and/or catalyzing the metabolic activation of procarcinogens (Blot, 1999). These mechanisms may explain the interaction between smoking and alcohol on certain cancers. Besides ethanol, alcoholic beverages contain other substances with carcinogenic (e.g., asbestos filtration products, tannins, N-nitroso compounds, urethan, arsenic, and pesticide residues [Blot, 1999]) and anticarcinogenic potential (e.g., polyphenols, particularly resveratrol in wine [Bianchini and Vainio, 2003]).

CONCLUSIONS AND PUBLIC HEALTH IMPLICATIONS There is convincing evidence that heavy alcohol consumption is strongly related to cancers of the mouth,

265

References

TABLE 9

National and International Guidelines Regarding Alcohol and Health

Source

Year

Alcohol and cancer World Research Fund/American Institute for Cancer Research (WCRF/AICR) National Cancer Institute (NCI) American Cancer Society (ACS)

1997 Current 2002

World Health Organization (WHO)

2003

Alcohol and health National Research Council (NRC)

1989

United States Department of Agriculture (USDA)

2000

Recommendation

“Alcohol consumption is not recommended. If consumed at all, limit alcoholic drinks to less than two drinks a day for men and one for women” “Consume alcoholic beverages in moderation, if at all.” “If you drink alcoholic beverages, limit consumption (no more than two drinks per day for men and one drink for women.)” “Consumption of alcoholic beverages is not recommended: if consumed do not exceed two units (one glass of beer, wine or spirits) per day.” “The committee does not recommend alcohol consumption. For those who drink alcoholic beverages, the committee recommends limiting consumption to the equivalent of less than two cans of beer, two small glasses of wine, or two average cocktails.” “If you drink alcoholic beverages, do so in moderation. Limit intake to one drink per day for women or two per day for men, and take with meals to slow alcohol absorption.”

pharynx, larynx, esophagus, and liver. Alcohol also probably increases the risk of colorectal and breast cancer. For other cancers, such as lung, ovarian, prostate, and endometrial cancers, the relationship remains controversial. Evidence is accumulating that certain populations may be more susceptible to alcohol on the basis of genetic variations on alcohol metabolism. Although the biological mechanisms by which alcohol increases cancer risk are not established, several plausible pathways have been proposed. Issuing recommendations regarding alcohol intake is difficult given the potential beneficial and detrimental effects for both the general public (Anonymous, 2000) and cancer patients (Brown et al., 2003). National and international guidelines regarding alcohol and health are listed in Table 9. Most scientific panels do not recommend drinking for health or cancer prevention, but for those who elect to drink, moderate consumption is recommended (i.e., less than one drink per day for women and two per day for men). Although moderate wine drinking has been shown to be beneficial for certain cancers, there is insufficient evidence to recommend one alcoholic beverage type over another.

Acknowledgments We thank Nirupa Ghai, MPH, and Dina Considine, MPH, for their assistance in conducting searches and in acquiring hundreds of manuscripts.

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12 Environmental Toxins, Nutrition, and Cancer RAMUNE RELIENE AND ROBERT H. SCHIESTL

INTRODUCTION

other most prevalent smoke-related disorders include chronic obstructive pulmonary disease (COPD) and cardiovascular diseases. Inhalation of environmental tobacco smoke (ETS) by nonsmokers is associated with many of the same deleterious health effects as active smoking (Cal-EPA, 1997; Witschi et al., 1997; Collier and Pritsos, 2003). For example, nonsmokers living with smokers have 20–50% access risk of coronary heart disease and a 1.2–2 times elevated risk of developing lung cancer. The U.S. Environmental Protection Agency (EPA) estimates that annually 38,000 heart disease deaths, 3000 lung cancer deaths, and 12,000 other cancer deaths can be attributed to ETS exposure (Cal-EPA, 1997). ETS is particularly hazardous for the developing fetus or young children.

A wide range of environmental toxins have been associated with carcinogenesis of the respiratory system and other organs. Cigarette smoke, particulate matter (PM), and its component diesel exhaust particles (DEPs), as well as arsenic and chromium, are among common environmental toxins that cause a number of adverse health effects predominantly affecting inhabitants of highly populated or industrialized areas. These pollutants have been identified as causative factors for lung cancer, and some are associated with carcinogenesis at other sites. These agents induce oxidative stress and genetic damage, which seem to be the major biological mechanisms responsible for adverse health effects, particularly carcinogenesis. Genetic damage (genotoxicity) includes various aspects of DNA damage ranging from oxidative DNA base lesions and point mutations to chromosomal alterations. Oxidative stress can cause genetic damage. Research data are accumulating that oxidative stress can be alleviated by higher intake of antioxidant vitamins or other antioxidants. In this chapter, we review DNA-damaging effects of cigarette smoke, DEP, arsenic, and chromium and discuss the possible beneficial role of antioxidant dietary supplementation under circumstances of heightened oxidant stress.

Environmental Tobacco Smoke Exposure and Children’s Health More than 150 studies have described effects of ETS on respiratory diseases in children (Jinot and Bayard, 1996). Exposure to ETS increases the prevalence of asthma, wheeze, cough, bronchitis, bronchiolitis, pneumonia, and impaired pulmonary function. Other health effects including ear infections, sudden infant death syndrome, behavioral problems, and neurocognitive deficits are also linked to ETS and prenatal maternal smoking (DiFranza et al., 2004). A very comprehensive study on maternal smoking during pregnancy and childhood ETS exposure in almost 6000 children showed an approximate twofold to threefold significantly elevated risk of asthma and wheezing of different subcategories (Gilliland et al., 2001). The study showed that in utero exposure to maternal smoking without subsequent postnatal ETS exposure had a more pronounced effect on

TOBACCO SMOKE Adverse Health Effects of Tobacco Smoke Cigarette smoking is a major risk factor for lung, oral, and pharyngeal cancer (Bergen and Caporaso, 1999). The


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prevalence of asthma and wheezing than current and previous postnatal ETS exposure. In utero exposure was associated with increased prevalence of asthma and wheezing, while current and previous ETS exposure was not linked to higher prevalence of asthma, although it was associated with wheezing. Other studies showed similarly that effects of in utero smoke exposure on asthma and wheezing are stronger than effects of postnatal ETS exposure (Ehrlich et al., 1996; Strachan and Cook, 1998; Gold et al., 1999). Asthma is a risk factor for lung cancer even in nonsmokers (Boffett et al., 2002; Santillan et al., 2003), and therefore, asthma provoked by ETS at young age poses a risk of developing lung cancer after several decades. A meta-analysis of 30 studies provides evidence that parental smoking is associated with increased risk of childhood cancers (Boffetta et al., 2000).

Genotoxicity of Tobacco Smoke It is generally accepted that cancer develops as a result of multiple genetic events, where gene mutations and particularly large genome rearrangements (deletions, translocations, duplications) are a prerequisite in the process of carcinogenesis. Tobacco smoke contains >4700 chemical compounds, and hundreds of them are mutagenic and carcinogenic (Church and Pryor, 1985). Polycyclic aromatic hydrocarbons (PAHs), heterocyclic nitrosamines, redox cycling quinones, reactive oxygen species (ROS), reactive nitrogen species (RNS), and volatile saturated and unsaturated aldehydes are among the reactive ingredients of tobacco smoke. Many of these structurally diverse chemicals have their own mechanism of action. Perhaps the most common mechanism of tobacco smoke constituents involves oxidative damage of macromolecules because free radicals and other oxidants comprise a high fraction of cigarette smoke (Bluhm et al., 1971; Church and Pryor, 1985; Pryor and Stone, 1993). It has been estimated that each puff of cigarette smoke contains ~1016 oxidant molecules (Church and Pryor, 1985). Oxidative DNA lesions have been found in human smokers and ETS-exposed individuals, indicated by elevated levels of 8-OH deoxyguanosine (8-OHdG), indicating oxidative damage to DNA (Fraga et al., 1996; Howard et al., 1998). Although DNA repair systems evolved to replace damaged DNA bases, 8-OHdG frequently mispairs with an adenosine (A) base (Cheng et al., 1992), causing point mutations (GC → TA transversions) and thereby increasing the gene mutation rate (Ames et al., 1993; Cerutti, 1994). Another prevalent DNA lesion induced by cigarette smoke is formation of covalent DNA adducts. DNA adducts have been detected in various organs of lung cancer patients with a history of smoking (Randerath and Randerath, 1993), “healthy” smokers (Tang et al., 2001), placenta of smoking women (Everson et al., 1986; Hansen et al., 1993), and experimental animals exposed to cigarette

smoke (Balansky et al., 1996). DNA adducts, if not eliminated in a timely manner, may cause miscoding resulting in gene mutations or cause DNA breaks leading to genome rearrangements. For example, specific deletions in the short arm of chromosomes 3, 9, and 17 appear to be associated with lung tumorigenesis (Hung et al., 1995; Kishimoto et al., 1995; Virmani, 1995). The fetus and young child are especially vulnerable to toxic effects of environmental pollutants (Perera et al., 1999; Whyatt et al., 2001), and several experimental and human studies suggest that the fetus clears toxicants less efficiently than the adult (Lu et al., 1986; Lu and Wang, 1990; Whyatt et al., 2001). Some cancer-associated genotoxicity markers detected in newborn infants are particularly concerning. A significant difference in the HPRT mutational spectrum was found in in utero smoke-exposed newborn infants born to mothers exposed to passive cigarette smoke, where the most notable change was an increase in DNA deletions mediated by V(D)J recombinase, a recombination event associated with childhood hematopoietic malignancies (Finette et al., 1998). The micronucleus assay revealed that the frequency of micronuclei, a marker of chromosomal fragmentation, was significantly higher in children of smoking parents and was slightly higher in children with both parents smoking than those with one parent smoking (Baier et al., 2002). Other genetic alterations, such as sister chromatid exchange, have been found in infants born to smoking mothers (Sardas et al., 1995). Interestingly, an animal study has shown a significant increase in the frequency of DNA deletions in the offspring of pregnant mice exposed for only 4 hours to a cigarette smoke concentration that caused the same concentration of blood nicotine as that found in smokers after smoking one cigarette (Jalili et al., 1998).

Antioxidant Dietary Supplementation against Tobacco Smoke Oxidative stress has been implicated in the pathogenesis of tobacco-related diseases, such as COPD, lung cancer, and cardiovascular illness. Numerous studies suggest that a fruitand vegetable-rich diet can reduce the risk of cancer (Block et al., 1992). Smokers are exposed to higher level of oxidants and have lower dietary intake of fruits and vegetables (Traber et al., 2000). It is estimated that vitamin C metabolic turnover of smokers is approximately double that of nonsmokers (Lykkesfeldt et al., 2000). Consequently, plasma levels of major antioxidant vitamins C and E are significantly reduced in smokers, rendering them more susceptible to oxidative damage than nonsmokers (Traber et al., 2000). Besides oxidants derived from cigarette smoke, more oxidants are produced in the organism of a smoker because they have higher numbers of circulating phagocytes primed to produce ROS and RNS as part of the inflammatory immune response against environmental pathogens and

Tobacco Smoke

xenobiotics (Cross et al., 1999). ETS causes qualitatively similar oxidative stress as active smoking. Passive smokers exhibit intermediate vitamin C concentration in their plasma between smokers and nonsmokers who were not exposed to ETS, despite similar intake of vitamin C (Tribble et al., 1993). Thus, it is expected that higher antioxidant intake would minimize some of the smoke-induced effects, and results of the following studies support that. Dietary supplementation of antioxidant vitamins (vitamin C, vitamin E, β-carotene) reduces oxidative DNA damage and singlestrand DNA breaks in smokers (Duthie et al., 1996; Lee et al., 1998). A study on healthy human volunteers exposed to ETS showed that prior ingestion of vitamin C prevented smoke-induced decrease in antioxidant capacity (assessed by the TRAP assay [total peroxyl radical trapping potential of the serum]) and the increase in lipid peroxidation that otherwise were observed after 1.5 hours of passive smoking (Valkonen and Kuusi, 2000). Vitamin C or a combination of vitamin C and E suppressed formation of lipid peroxidation products in chronic smokers (Reilly et al., 1996). Supplementary vitamin E (≥100 IU/day) significantly lowered progression of early preintrusive atherosclerosis compared with non–vitamin E users (Azen et al., 1996). Vitamins C and E seem to ameliorate endothelial dysfunction, an early indicator of atherosclerotic process, in smokers (Traber et al., 2000). An epidemiological study suggested that vitamin E may inhibit lung cancer development by as much as 20% (Woodson et al., 1999). In some studies, however, adverse effects of β-carotene supplements were observed. In human intervention trials, β-carotene supplements actually increased the risk of acquiring lung cancer when administered to heavy smokers of more advanced preclinical stages of carcinogenesis (Greenberg et al., 1996; Omenn et al., 1996). Most of the lung cancers appear in former smokers, and little information is available on the chemosuppression of these tumors. In any case, recommendations on taking antioxidant supplements have to be made with precaution considering compound’s action mechanism, dosage, possible side effects, and the stage of disease.

Antismoke Effects of N-Acetylcysteine N-acetylcysteine (NAC) is a thiol antioxidant that is nontoxic at high doses, tasteless, odorless, and well established in clinical practice (Kelly, 1998). NAC, a synthetic precursor of intracellular cysteine and glutathione (GSH), has been used as a mucolytic drug for more than 4 decades. NAC lowers the viscosity of the mucus and thereby alleviates symptoms of bronchitis and other bronchopulmonary diseases. NAC is an efficient antidote against acute acetaminophen (paracetamol) poisoning (Mitchell et al., 1974; Smilkstein et al., 1988). NAC mitigates acetaminopheninduced hepatorenal toxicity because of its ability to block reactive metabolites of acetaminophen and replenish GSH

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stores that are rapidly depleted by reactive metabolites of xenobiotics. NAC appears to be efficient in inactivating reactive electrophiles and free radical reactions and appears more effective for clinical application than GSH or its precursor cysteine. This is because GSH does not easily cross cell membranes and cysteine is toxic at higher concentrations. Further studies showed that NAC is protective against carcinogenesis-associated end-points (De Flora et al., 1985, 1986, 2001). For example, NAC blocks metabolites of promutagens; inhibits formation of carcinogenic DNA adducts and oxidative DNA damage; and is able to inhibit tumor promotion, progression, and metastases in experimental models. Substantial research on the effects of NAC on DNA damage following cigarette smoke exposure were carried out in Silvio De Flora’s laboratory (De Flora et al., 2001, 2003). Studies showed that dietary NAC administration results in reduction or inhibition of many smoke-induced DNA-damaging effects in experimental animals. This includes decreased levels of DNA adducts in nuclear and mitochondrial DNA, inhibition of oxidative DNA damage, and attenuation of micronucleus formation. All these data indicate that NAC might be protective against smoke-related lung cancer. Unfortunately, this can hardly be tested in animal studies because it has been extremely difficult to induce lung tumors by cigarette smoke in rodent animals (Coggins, 1998). The ability of NAC to modulate smokeinduced biomarkers has been assessed in humans. Oral NAC given at 600–800 mg/day reduced urinary excretion of mutagens in smokers starting on the first day of administration, which was reversed when treatment was withdrawn (De Flora et al., 1996). A battery of biomarkers was examined in healthy smoking volunteers given NAC (2 × 600-mg tablets/day) or placebo in a randomized double-blind phase II chemoprevention trial (Van Schooten et al., 2002). In the placebo group, there was no variation in any of the biomarkers studied, whereas in the NAC group, a significant decrease occurred in some of the markers, including lipophilic DNA adducts and oxidative DNA damage in bronchoalveolar lavage cells. Effect of NAC was studied in a clinical phase III trial in cancer patients. Supplementation with NAC (600 mg/day) and/or retinyl palmitate for 2 years had no beneficial effect on tumor recurrences in the upper or lower airways in the large study composed of almost 2600 patients who have previously been treated for head and neck cancer or lung cancer, and most of them were previous or current smokers (van Zandwijk et al., 2000). Those studies provided some evidence that NAC may possibly reduce cancer risk in healthy human smokers, although it may no longer be effective in cancer patients. Those studies suggest, however, that NAC may be beneficial for ETS-exposed individuals who have not yet developed cancer but suffer from similar health problems as active smokers, albeit to a lesser degree.

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PARTICULATE MATTER PM is airborne sphere-shaped particles forming smog. The particles constitute a carbon core and adsorbed to it organic compounds, low amounts of inorganic compounds (sulphate, nitrate), and metals. The main source of PM is the combustion of fossil fuels (gasoline or diesel) in automobiles and in heavy industries. Some PM comes from natural sources, and secondary particles originate from chemical transformation of the emitted particles in the air. The particles vary in size (categorized as ultrafine, fine, and coarse particles) and composition, although humans are exposed to a complex ambient PM mixture, the concentration of which is high in urban and industrial areas. Exposure to high PM levels has been associated with a variety of adverse health effects, including asthma and pneumonia (Hruba et al., 2001), COPD (Ebelt et al., 2000), heart disease, lung and upper respiratory tract disease, cancer (Dockery et al., 1993; Beeson et al., 1998; Schwela, 2000; Pope et al., 2002), and mortality (Lippmann et al., 2000; Schwartz, 2001). Each 10-µg/m3 elevation in fine PM is associated with an increased risk of mortality by 4%, 6%, and 8%, respectively (Pope et al., 2002).

DIESEL EXHAUST PARTICLES Diesel exhaust (DE) is a complex mixture of gases and particles emitted by combustion engines using diesel oil as a fuel. The diesel engines are used mainly in trucks, buses, locomotives, ships, agricultural, and other off-road equipment. Although diesel fuel is more economic and durable than gasoline fuel, diesel engines emit 30–100 times more PM than gasoline engines (McClellan, 1987). Increased usage of diesel-powered engines has contributed to ambient PM concentrations so that DE particles (DEP) became a significant component of ambient PM, amounting to 20–80% (EPA, 2002). Average DEP concentration in the United States is ~1 µg/m3, but it increases dramatically in big cities. For example, DEP concentration is higher than average in the Los Angeles Basin (1.3–3.6 µg/m3), and at a bus stop in Manhattan, levels reached 13–47 µg/m3 (EPA, 2002). Estimated occupational DEP exposures vary from 2 to 27 µg/m3 for truck drivers, 42–155 µg/m3 for railway workers, and 150–500 µg/m3 for miners (Steenland, 1998). Both the gaseous phase and particulate component are associated with adverse health effects of DEP. There are several toxic components in the gaseous phase of DE such as aldehydes, benzene, carbon monoxide, nitrogen oxides, sulfur dioxides, PAHs, and nitro-PAHs. The particulate component of DE is composed of >450 different organic compounds including toxic PAHs, quinones and aldehydes, and traces of heavy metals (Barfknecht et al., 1982; Casellas et

al., 1995). More than 100 of the chemicals in the particles are carcinogenic and mutagenic. Benzo(a)pyrene, indeno (1,2,3-cd)pyrene, dibenz(a,h)anthracene, benz(a,h)anthracene, benzo(b)fluoranthene, and benzo(k)fluorantene, naphthalene, and chrysene are just a few carcinogenic PAHs that are found in DEP. Epidemiological studies suggest that exposure to DE is associated with an increased risk (20–50%) of lung cancer in transportation workers, railroad workers, miners, and operators of heavy construction equipment (McClellan, 1987; Garshick et al., 1988; Stayner et al., 1998; Steenland et al., 1998; Larkin et al., 2000; EPA, 2002). The Multiple Air Toxic Exposure Study (MATES II) conducted by the South Coast Air Quality Management District estimates that DEP contributes >71% of the total lifetime cancer risk from ambient particles (Mates(II)Study, 2000). There is also weak evidence linking exposure to DE and bladder cancer. Noncancer adverse health effects of DE include acute irritation of the eye and nose, respiratory symptoms (cough and phlegm), and allergic reactions (EPA, 2002).

Genotoxic Effects of Diesel Exhaust Particles The genotoxicity of DE is often expressed in relationship to its particulate component. The lung is the major target organ for PM, and the metabolism of PM is similar to that of other insoluble foreign bodies. The particles are taken up by macrophages followed by inflammatory responses accompanied by the formation of ROS. Research studies show that DEP induces oxidative stress and is mutagenic in bacterial and mammalian cell assays (McClellan, 1987; Schins, 2002). DEP exposure by inhalation or intratracheal instillation induces oxidative DNA damage (Tokiwa et al., 1999; Adachi et al., 2000; Iwai et al., 2000; Risom et al., 2003), DNA adduct formation (Bond et al., 1988; Gallagher et al., 1994), and mutations (Sato et al., 2000) in lung cells. Long-term inhalation exposure causes tumors in the respiratory tract in rodent animals (Mauderly et al., 1987; McClellan, 1987; Schins, 2002). The particle effect, however, is not limited to the lung tissue. Some particlebound material enters blood circulation and becomes bioavailable. Experimental animal studies show that labeled PAHs were detected in blood, liver, and kidney after inhalation exposure (Sun et al., 1982). Elevated levels of DNA adducts are found in peripheral blood lymphocytes of bus maintenance workers (Hou et al., 1995). The most concerning issue is that the particle genotoxic effect can be transmitted to the next generations. A research study by Somers et al. (2004) showed that airborne particles induced heritable alterations at noncoding repetitive DNA loci in mouse offspring after exposed male mice were mated with unexposed females.

Arsenic

ARSENIC Chronic exposure of humans to high concentrations of inorganic arsenic is associated with skin lesions, peripheral vascular disease, hypertension, blackfoot disease (vasoocclusive disease, which leads to gangrene of the extremities), and cancer at various sites (Tseng, 1977; International Agency for Research on Cancer [IARC], 1987; EPA, 1988; Engel et al., 1994; Chen et al., 1995; Tondel et al., 1999). An increased mortality from lung cancer has been observed in humans exposed primarily through inhalation. An increased incidence of skin cancer and cancers of other organs (liver, bladder, and kidney) has been found in the general population consuming water contaminated with high arsenic concentration. Arsenic has been used in a variety of industries, including manufacturing of semiconductor, glass, pigments, paints, alloys, pesticide, and herbicide. Arsenic, as an environmental pollutant, arises from burning coal containing high arsenic concentrations, the mining and smelting of metal ores, or improper disposal of industrial byproduct. The major sources of arsenic in drinking water are the presence of arsenic-rich minerals and rocks in aquifers. More than 200 million people in many countries are chronically exposed to arsenic. Intermediate to high levels of arsenic in ground water (2–5000 µg/liter) is found in Asian countries (Taiwan, Thailand, China, India, Mongolia, Vietnam), South America (Chile, Bolivia, Argentina), Bangladesh, Mexico, and the United States (Arizona, California, Nevada). The highest levels of exposure occur in South America, Taiwan, and the Gulf of Bengal. Intermediate levels of arsenic (≤200 µg/liter) are found in some countries in Europe, such as Hungary, Spain, Greece, and Germany. Most of the epidemiological studies have been conduced in areas with high levels of arsenic contamination (typically >200 µg/liter). Even low or intermediate contamination might be a risk factor for bladder cancer. For example, an ecological study showed that exposure to arsenic concentrations at doses 0.1–0.5 and >0.5 µg/liter relative to the doses below 0.1 µg/liter during 2–9 years prior to cancer diagnosis increases the risk of bladder cancer by 1.5-fold and 2.44-fold, respectively (Kurttio et al., 1999).

Action Mechanism of Arsenic Carcinogenicity Inorganic arsenic in the environment exists as pentavalent inorganic arsenate (AsV) and to a lesser extent as the trivalent arsenite (AsIII). In most mammals, ingested arsenate is readily converted to arsenite, and inorganic arsenic is metabolized to organic forms, namely, first to monomethyl arsonic acid (MMA) and then to dimethylarsinic acid (DMA), and in rats further to trimethylarsine oxide (TMAO) (Aposhian, 1997). Humans exposed to

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arsenic excrete 10–20% inorganic arsenic, 10–20% MMA, and 60–80% DMA (Vahter and Concha, 2001). The exact action mechanism of arsenic is not known, although at least several mechanisms are postulated on the basis of experimental data (Hughes, 2002; Kitchin and Ahmad, 2003). The most prevalent theories include oxidative stress, chromosomal alterations, tumor promotion, and cocarcinogenesis. In vitro and in vivo studies indicate that arsenic induces oxidative stress. ROS are formed within 5 minutes after arsenite exposure in human–hamster hybrid cells (Liu et al., 2001). Peroxyl radicals of DMA, a major metabolite of ingested inorganic arsenic, are detected in vitro (Yamanaka et al., 1990). Oxidative damage is observed in lungs of DMA-exposed mice (Yamanaka et al., 1991; Wanibuchi et al., 2004). Arsenite is a potent clastogenic compound capable of inducing chromosome aberrations (chromatid gaps, breaks, fragmentation, endoreduplication, chromosomal breaks), sister chromatid exchange, micronuclei formation, and large deletions in cultured cells and rodent animals (Tinwell et al., 1991; Jha et al., 1992; Das et al., 1993; RoyChoudhury et al., 1996; Rasmussen and Menzel, 1997; Schiestl et al., 1997; Hei et al., 1998). In contrast, arsenic (inorganic As or its organic metabolites) does not cause point mutations in standard assays. Research studies using animal models show that arsenic is involved in tumor promotion and initiation. DMA given in drinking water significantly enhances tumor induction in the urinary bladder, kidney, liver, and thyroid gland after multiorgan tumor initiation with organ-specific carcinogens in rats (Wanibuchi et al., 2004). Arsenite is cocarcinogenic with ultraviolet (UV) irradiation (Rossman et al., 2001). Mice exposed to arsenite in drinking water and UV irradiation had a 2.4-fold higher skin tumor rate as mice exposed to UV alone. A 2-year carcinogenicity study showed that DMA itself causes bladder tumors in rats (Wanibuchi et al., 2004). Mutational analysis of the tumors shows that no mutations were present in p53, K-ras, or β-catenin genes, or low levels (10%) were found in the H-ras gene and no microsatellite instability using 18 microsatellite loci. The level of oxidative DNA damage, however, was increased after DMA administration in drinking water for 2 weeks, suggesting that ROS may play a role in the early phases of DMA carcinogenesis. Fortunately, some arsenic-induced effects can be reduced by antioxidants. Antioxidative enzymes (catalase and superoxide dismutase) reduce arsenite-induced micronuclei formation (Wang and Huang, 1994), and vitamin E reduces cell killing (Lee and Ho, 1994). Free radical scavenger dimethyl sulfoxide lowers ROS concentration to control level (Liu et al., 2001) and the frequency of deletions by about 75% (Hei et al., 1998). Garlic extract given to mice before exposure to arsenite reduces its clastogenic effects (Das et al., 1993; RoyChoudhury et al., 1996). Vitamin C seems not to be

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beneficial. DMA enhanced iron release from ferritin in the presence of vitamin C, which may lead to higher levels of ROS, because ROS can be formed via cellular iron and other transition metals (Ahmad et al., 2000).

CHROMIUM Chromium exists predominantly as compounds of trivalent chromium (CrIII) or hexavalent chromium (CrVI) (EPA, 2000; IARC, 1990; Cohen et al., 1993; Costa, 1997; Barceloux, 1999). CrIII is suggested to be an essential micronutrient element, while CrVI is carcinogenic. CrIII is found at higher concentrations in green beans, broccoli, cereals, and some brands of beer and wine. It is claimed that insufficient dietary intake of chromium leads to impaired glucose tolerance, glycosuria, and elevations of serum insulin, cholesterol, and total triglycerides. CrIII dietary supplements are recommended for diabetes patients because of its proposed beneficial effect on glucose metabolism (Anderson et al., 1997; Vincent, 2004). Chromite ore is the major source of CrIII. CrVI occurs rarely in nature in the mineral crocoite (PbCrO4). Most CrVI compounds (chromates and Cr trioxide) are produced from chromite ores for commercial applications. CrVI compounds are used in a variety of industries including leather tanning, chrome plating, stainless steel production, chrome pigment manufacturing, and as an antirust agent added to water-cooled machinery. Occupational exposure to CrVI has been associated with respiratory distress, asthma, skin rashes, liver damage, duodenal ulcer formation, and gastritis. The first epidemiological study conducted on chromate production workers in the United States in the 1940s showed that cancer of the respiratory system represented 63% of all deaths from cancer in the exposed group and 21.8% in the general population. Numerous epidemiological studies in different countries confirmed that an increased risk of lung cancer is associated with CrVI exposure and CrVI has been classified as a known human lung carcinogen by the inhalation route of exposure (EPA; IARC, 1990). There is suggestive evidence of an increased incidence of other types of cancer (lymphoma, leukemia, bone, stomach, prostate, genital, bladder, renal cancers) in chrome industry workers (Costa, 1997; Barceloux, 1999). Although CrVI primarily affects individuals in occupational settings, industrial waste contamination and emissions of fossil fuel combustion led to elevated levels of CrVI in the atmosphere, drinking water, and soil. It has been reported that the fourfold increase in childhood leukemia in Woburn, Massachusetts, was attributed to consumption of CrVI-contaminated drinking water at levels two times above the state drinking water standard of 50 ppb (50 µg/liter) (Durant et al., 1995). Epidemiological studies are suggestive that oral exposure to CrVI could lead to increased risk of cancer at various sites, although

there is lack of sufficient evidence (Costa, 1997). Experimental studies found that chronic CrVI ingestion via drinking water causes malignant forestomach tumors (Borneff et al., 1968) or enhances susceptibility to UV-induced skin tumors in mice (Davidson et al., 2004). Other rodent studies showed that CrVI exposure via oral route leads to fetal and embryonic toxicity (Kanojia et al., 1998) and teratogenicity (Kanojia et al., 1996).

Mechanism of Chromium Genotoxicity CrIII is poorly absorbed into the body and does not enter a cell easily, whereas CrVI can be absorbed from gastrointestinal and respiratory tracts or through the skin (Costa, 1997; Barceloux, 1999). Chromium distributes throughout the body and concentrates in the liver and kidney. Absorbed CrVI (highly toxic) is readily taken up into a cell via sulphate and phosphate transporters and is reduced by cellular reducing agents (glutathione, cysteine, ascorbic acid, riboflavin, and others) to CrV, CrIV, and ultimately to relatively inert CrIII. CrVI does not interact with DNA, although its reduced form CrV leads to ROS formation, oxidative DNA damage, and DNA strand breaks. An end product of CrVI metabolism, CrIII, forms DNA adducts, mediates DNA interstrand crosslinks, and protein–DNA crosslinks. The outcome of these DNA–Cr interactions has not been studied in depth. Although available data indicate that CrIII can cause chromosomal aberrations and sister chromatid exchanges, there is little evidence on CrIIIinduced point mutations. Thus, chromium induces many different types of genetic lesions. Findings that CrIII (micronutrient element), the end-metabolite of CrVI (classified carcinogen), accumulates in the body (Stearns et al., 1995) and causes DNA damage provokes questions about the safety of CrIII dietary supplements when used in excess dosages or for an extended time. In these supplements, CrIII is bound to its delivery agents (e.g., picolinate) that allow much better CrIII absorption and cell penetration. This is particularly concerning because the lack of toxicity associated with CrIII is commonly attributed to its inability to penetrate the cellular membrane. Oxidative stress appears to be a major event of CrVImediated genotoxicity, suggesting that antioxidant therapy might be protective against CrVI toxicity. Indeed, as an extracellular agent, vitamin C provides some protection (Cohen et al., 1993). The intravenous administration of large doses of vitamin C has been recommended against CrVIinduced acute renal toxicity (Barceloux, 1999). It appears to be effective when administered immediately after chromium poisoning because 1–2 hours after chromium treatment, vitamin C had no effect or increased nephrotoxicity in animal studies. Intracellularly, however, vitamin C and glutathione (GSH) correlates with increased genetic damage (Costa, 1997). For example, increased cellular concentration

Conclusions

of vitamin C led to an increased level of CrVI-induced DNA–protein crosslinks, and an increase in GSH was associated with DNA strand breaks and Cr–DNA adducts. Apparently intracellular CrVI reduction acts as bioactivation rather than protection.

CONCLUSIONS Common environmental pollutants, such as environmental tobacco smoke, PM, DEP, arsenic, and chromium, cause lung cancer, and some are also risk factors for cancer in other organs. The mechanism of carcinogenesis is largely unknown and it might be different for the different agents. Some of the action mechanisms of these agents, however, appear to be common. Namely, they all induce oxidative stress and damage. Additionally and/or as a consequence of oxidative DNA damage, they can cause a wide variety of genetic alterations. Oxidative stress and genetic alterations can act as tumor initiators or promoters. Thus, it seems that maintaining a physiological antioxidant balance may be essential to prevent or reduce genetic damage and possibly the frequency of long-term disease. Under circumstances of elevated oxidant stress, which occurs in individuals exposed to cigarette smoke, PM, or metals, antioxidant balance could potentially be maintained by increasing the intake of dietary antioxidants. Available research data support the notion that dietary antioxidant vitamin intake is beneficial in smokers or ETS-exposed individuals. A thiol antioxidant NAC (a synthetic dietary supplement) seems to be a good candidate to suppress oxidative stress in smokers. For other agents, effects of dietary antioxidant supplementation are yet to be explored.

Acknowledgments We thank Zhanna Kirpnick of our laboratory for helpful suggestions during the writing of this manuscript. Supported by a research grant from the University of California Toxic Substances Research and Teaching Program (UC TSR&TP) and funds from the UCLA Center for Occupational and Environmental Health to RHS and postgraduate research fellowships of the UC TSR&TP and Philip Morris USA, Inc., to R.R.

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13 Endocrine and Paracrine Factors in Carcinogenesis DAVID HEBER AND PINCHAS COHEN

INTRODUCTION

The IGF-1 signaling pathway evolved hundreds of millions of years ago in many different species to regulate cell growth in accord with the availability of nutrients in the environment (Longo and Finch, 2003). This basic function remains in humans because both insulin and IGF-1 have key roles in regulating cell proliferation and apoptosis. The system includes the ligands IGF-1 and IGF-2; the type 1 and type 2 IGF receptors (IGF-1R and IGF-2R); and the IGF-binding proteins (IGF-BPs), in addition to the proteins involved in intracellular signaling triggered by binding of the ligands to IGF-1R, which include members of the insulin-receptor substrate (IRS) family, AKT, target of rapamycin (TOR), and S6 kinase. Most circulating IGFs are produced in the liver. Hepatic IGF-1 production is subject to complex regulation. Growth hormone (GH), which is produced in the pituitary gland under control of the hypothalamic factors GH-releasing hormone (GHRH) and somatostatin, stimulates IGF-1 production in the liver. Various IGF-BPs are also produced in the liver. In those tissues that respond to IGF, the ligands IGF-1 and IGF-2, as well as IGF-BPs, can be delivered through the circulation from the liver as classical hormones or the IGFs, and IGF-BPs can be locally produced through autocrine or paracrine mechanisms. These mechanisms often involve cell–cell interactions between stromal and epithelial cells. Nutritional status also influences circulating IGF-1 levels. Starvation reduces both IGF-1 levels (Thissen et al., 1994) and intracellular signaling secondary to IGF-1R activation at the level of TOR (Houghton and Huang, 2004). The nutritional adaptations in the IGF axis may have evolved under conditions where food supplies were scarce and inconsistently available to minimize cellular energy and protein consumption at times of inadequate nutrition through the reduction in cell proliferation rates. The repeated observation that caloric restriction provides protection against carcinogenesis and that this can be reversed by

Endocrine and paracrine factors are modulated by nutritional factors, including deficiency states and obesity, which influence carcinogenesis at molecular, subcellular, cellular, organ, and systemic levels. The insulin-like growth factor 1 (IGF-1) axis, estrogens, androgens, and certain classes of lipids are affected by changes in nutrition and influence tumor cell biology. Hepatic IGF-1 production is subject to complex regulation, and IGF-1 is also produced in peripheral tissues where this peptide can promote carcinogenesis. Reproductive steroids affect the IGF-1 axis, and in turn, steroid hormone production is affected by nutritional status. At a cellular and subcellular level, peptides, steroids, and lipids in the microenvironment of the tumor can affect cell proliferation. Peptide hormones act through membrane receptors, and both steroid hormones and lipids act through cytoplasmic and nuclear receptors. There is emerging evidence for cross-talk between peptide and steroid signals and evidence that some lipids interact with high-capacity/lowaffinity receptors, which influence gene expression in the nucleus. Intermittent stimulation by xenobiotics activates metabolic systems through a feed-forward loop and the upregulation of metabolic enzymes that are activated by low-affinity/high-capacity receptors, such as the steroid xenobiotic receptor, and by more specific receptors such as the vitamin D receptor. The study of endocrine and paracrine pathways is vital to an understanding of the processes through which nutrition affects tumor biology and the multistep process of carcinogenesis.

THE IGF AXIS AND ENERGY BALANCE The IGF axis involves complex regulatory networks that operate at the whole organism, cellular, and subcellular levels.


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infusing IGF-1 (see later discussion) suggests a significant role for the IGF axis in mediating many of the protective effects of caloric restriction on carcinogenesis. Other studies indicate that high levels of energy or protein intake are associated with modest increases in IGF1 levels (Heaney et al., 1999; Holmes et al., 2002; Giovannucci et al., 2003). In several studies, IGF-1 levels were seen to increase with increasing dairy product intake (Holmes et al., 2002; Giovannucci et al., 2003; Gunnell et al., 2003). The underlying mechanism and significance of this relationship deserves further study, particularly as prostate cancer risk has been shown to be associated with both increased IGF-1 levels (Pollak, 2001) and increased dairy food consumption (Chan et al., 2001). Micronutrients such as retinoids also influence circulating IGF-BP3 levels, and this is modified by a genetic polymorphism of IGF-BP3 (Deal et al., 2001). At a subcellular level, the signaling systems, which translate IGF effects, are highly complex and this brief description should be viewed only as a summary that does not include all the details of complex regulation. The IGF-1R is a tyrosine kinase cell surface receptor that binds either IGF1 or IGF-2. The local bioavailability of ligands is subject to complex physiological regulation and may be increased in many common forms of cancer. Following ligand binding, intracellular signaling pathways that favor proliferation and cell survival are activated. Initial phosphorylation targets for IGF-1R include IRS proteins, and downstream signaling molecules include phosphatidylinositol 3-kinase, AKT, TOR, S6 kinase, and mitogen-activated protein kinase. IGF2R preferentially binds IGF-2 but has no intracellular kinase domain and may not act as a signaling molecule. The IGF-BP family of proteins determines the relative bioavailabilities of IGF-1 and IGF-2 (Firth and Baxter, 2002). At least six IGF-BPs have been found, and their binding affinity for IGF-1 and IGF-2 is similar to that of the IGF-1R. These proteins are present in the circulation and extravascular fluids. IGF-BP3 provides most of the IGF-binding capacity of serum and greatly prolongs the circulating half-life of the IGFs. IGF-BPs in extracellular tissue fluid modulate interactions between IGF ligands and cell surface IGF receptors. IGF-BP3 stimulates apoptosis through direct interaction with nuclear and membranebound receptors for IGF-BP3 in prostate cancer cells. The complex actions of IGF-BP proteins are poorly understood. They may increase the half-life of the different IGFs and they may bind directly to nuclear and membrane receptors. IGF-BPs modulate bioavailability of IGFs in both the circulation and the cellular microenvironment. The simplest model postulates that the major biological effect of IGFBPs is to compete with receptors for ligands. So, under circumstances in which IGF-BP levels are low, free IGF levels are high and there is more proliferative stimulation by IGFs.

Genetic polymorphisms can influence circulating IGF-1 concentration (Harrela et al., 1996). Dozens of proteins are involved in the physiological systems that regulate IGF-1 levels, and polymorphic variation of the genes encoding these has been proposed to influence circulating IGF-1 concentrations. Examples include genes encoding IGF-1 itself; IGF-BPs and their proteases; GH and its receptor; and somatostatin, GHRH, and their receptors. Only a few of these have been studied in the context of their ability to influence IGF-1 levels within the normal range (Deal et al., 2001; LeMarchand et al., 2002; Bonafe et al., 2003; Johnston et al., 2003), but some are mutated in growth disorders associated with abnormal IGF-1 levels (Laron, 1995; Maheshwari et al., 1998). Some (LeMarchand et al., 2002, Wang et al., 2003) but not all (Schernhammer et al., 2003) reports indicate that polymorphic variation within these genes influences cancer risk or prognosis. Common haplotypes might account for much of the variation in circulating levels of IGFs and their binding proteins (Cheng et al., 2004). Hormones, including endogenous and exogenous steroids, have important influences on IGF-1 regulation. Both tamoxifen and diethylstilbestrol suppress IGF-1 levels (Pollak et al., 1990; Helle et al., 2001). It is believed that estrogens, when administered orally in postmenopausal hormone replacements, reduce circulating levels of IGF-1 by suppressing hepatic production of IGF-1, probably as a consequence of its direct delivery to the liver and suppression of hepatic IGF-1 gene expression (Jernstrom et al., 2001). Estrogen delivery by the transdermal route does not lower circulating IGF-1 levels. In several experimental systems, there is cross-talk between estrogens and IGFs, which may act to stimulate breast cancer growth (Yee and Lee, 2000; Song et al., 2004). Polymorphisms that influence the function of IGF-1R itself (Bonafe et al., 2003; Abuzzahab et al., 2003) or in downstream signaling proteins would also be expected to add complexity to the relation between circulating IGF-1 levels and IGF-1R activation. The IGF-1 signaling pathway has important roles in regulating cellular proliferation and apoptosis acting in both an endocrine and a paracrine manner. Energy balance affects circulating levels of IGF-1 and IGF-BPs. Obesity is associated with increased IGF-1 and decreased IGF-BP1. Many other intriguing functions have been uncovered in the past 2 decades including effects of IGF-1 on body size, longevity, and various organ functions (Jones and Clemmons, 1995; Nakae et al., 2001; Giudice, 2002; Baserga et al., 2003).

IGF Axis and Cancer When one considers the aggregate of all knowledge on the relationship between nutrition and cancer, the impact of excess calorie intake on tumorigenesis has been substantiated in more experiments than any other nutritional factor.

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Estrogens and Caner

FIGURE 1 The IGF axis in the prostate.

For example, caloric restriction in the TRAMP transgenic mouse model of prostate cancer results in a reduction in IGF-1 and an increase in IGF-BP. The inhibition of tumor growth in this model resulting from caloric restriction can be reversed through provision of IGF-1 intraperitoneally but not by growth hormone. With caloric restriction, increases in IGF-BP result in reduced free IGF levels, and the serum has been shown to stimulate apoptosis through binding of IGF-BP3 to a membrane receptor (Figure 1). In human volunteers (Figure 2) undergoing a 3-week dietary intervention with a very low fat, high-fiber diet, the ability of their serum, when substituted for fetal calf serum, to stimulate prostate cancer cell growth ex vivo is reduced. Adding IGF-1 back to the serum samples restores the cell proliferation of cancer cells back to the levels observed with control sera. Insights from population studies and experimental studies in animals suggest that high levels of circulating IGF-1 are associated with increased risk of several common forms of cancer. The key challenge to researchers in nutritional oncology is to define and clarify the mechanisms through which nutrition affects the IGF axis and to determine the extent to which IGF-1 can influence carcinogenesis. The role of IGF-1 in cell proliferation is of particular relevance to nutritional oncology (Holly, 1998; Burroughs et al., 1999). IGF-1 stimulates cell proliferation and prolongs cell survival in vitro (Jones and Clemmons, 1995). In animal models proliferation and metastasis of cancer cells is increased by IGF-1R activation, in relation to either higher levels of circulating IGF-1 in the host or autocrine production of IGF ligands by neoplastic cells (Khandwala et al., 2000). An association has been observed between elevated circulating levels of IGF-1 and cancer risk in populations. There is significant variation of circulating levels of IGF-1, IGF-2, and IGF-BP concentrations among normal individuals. Although IGF has been measured clinically by endocrinologists to diagnose both GH deficiency and acromegaly and by pediatricians to assess nutritional status, the obser-

vations of increased risk are within quartiles spanning the normal range. A number of studies reviewed later in this chapter suggest that the risk of common forms of cancer is increased in individuals who have higher circulating levels of IGF-1, compared with those who have levels at the lower end of the normal range. Significant increases in cancer risk with increasing height have been documented in many studies (Gunnell et al., 2001; Engeland et al., 2003; Lawlor et al., 2003; Mellemkjaer, 2003). Height per se is unlikely to be a risk factor, but the hormonal determinants of height might influence cancer risk. IGF-1 levels are, at most, weakly related to adult height but are related to height early in life (Juul et al., 1994). So, height might be weakly related to the risk in part because it is weakly related to IGF-1 exposure over the first decades of life. Birth weight and size have been associated with risk of breast (Stavola et al., 2000; McCormack et al., 2003), colorectal (Sandhu et al., 2002), prostate (Tibblin et al., 1995), and childhood cancers (Von Behren and Reynolds, 1995) and are positively correlated with cord-blood IGF-1 levels (Vatten et al., 2002a). Conversely, preeclampsia is associated with reduced IGF-1 levels and reduced breast cancer risk (Altinkaynak et al., 2003; Vatten et al., 2002b). Cancers can be stimulated by IGF-1 or IGF-2 that is synthesized locally in an autocrine or paracrine manner or by responding to changes in circulating levels of these two ligands. One hypothesis worth further investigation is that tumors may initially depend on endocrine or paracrine stimulation, but as the process of carcinogenesis proceeds, the tumor cells acquire the ability to produce IGF, particularly IGF-2, which can stimulate growth via an autocrine loop.

ESTROGENS AND CANCER Estrogens modulate cellular growth and differentiation (Warner et al., 1999). Its major target organs in females are the mammary gland and uterus, but in both males and females this hormone is essential for maintenance of bone, brain, the cardiovascular system, and the urogenital tract (Ogawa et al., 1998; Simpson, 1998; Wenger, 1999). The biological actions of estrogens are mediated by estrogen binding to one of two specific estrogen receptors ERα and ERβ (Kuiper et al., 1996), which belong to the nuclear receptor superfamily, a family of ligand-regulated transcription factors. ERα and ERβ are products of different genes and exhibit tissue- and cell-type–specific expression. The characterization of mice lacking ERα, ERβ, or both has revealed that both receptor subtypes have overlapping but also unique roles in estrogen-dependent action in vivo. Additionally, ERα and ERβ have different transcriptional activities in certain ligand, cell-type, and promoter contexts. Both receptors, however, are coexpressed in a number of tissues and form functional heterodimers. The biological

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Chapter 13/Endocrine and Paracrine Factors

Serum IGF-I (ng /m L)

400 300

Serum IGFBP-1 (ng / m l)

100

*

75 p60%. The Lobund–Wistar rats developed tumors of dorsolateral prostate rather than in the ventral prostate (Pollard and Lucker, 1986, 1987). Besides N-methyl-N-nitrosourea, N-nitrosobis (2oxopropyl) amine, 3,2′-dimethyl-4-aminobiphenyl, and 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine have also been used to induce prostate carcinomas in rodents, and extensive review has been provided by other investigators (Shirai et al., 2000). Although these chemical carcinogen–induced prostate tumor models have provided a diverse array of animal models for CaP and have contributed significantly to CaP research, they usually develop multiorgan tumors and are not prostate specific. In addition, it usually takes a long time for animals to develop tumors and incidence is usually low. These limitations have prevented application of these animal models for future studies. Orthotopic Prostate Tumor Models Although subcutaneous implantation is commonly used to develop prostate xenografts, it is well accepted that epithelial–mesenchymal interactions play crucial roles in prostate tumor development and progression. The importance of stromal–epithelial interactions in both normal and abnormal growth of prostate is well recognized, although the precise mechanism has yet to be elucidated. It is widely recognized that a variety of growth factors are implicated in epithelial–stromal interactions. Several orthotopically implanted human prostate tumor models are established that represent diverse phenotypes of CaP. These orthotopic tumor models include ones for androgen-sensitive CaP, for androgen-independent CaP, and for progression of androgen-independent CaP by hormonal manipulation. Orthotopic Tumor Models for Androgen-Sensitive and Androgen-Independent CaP Although no animal model provides all of the characteristics of the human disease, androgen-regulated growth and the potential for metastasis are two clinically important characteristics that are captured in orthotopic implant models utilizing the severely combined immune deficient (SCID) mouse and the LNCaP human CaP cell line (Sato et al., 1997; Zhou et al., 2002a). In addition, this model allows the use of serum PSA as a surrogate marker for tumor growth in response to defined dietary and nutritional interventions. Orthotopic implantation of LNCaP cells results in tumor take rate of >85% and lymph nodes and lung metastases of ~50% (Sato et al., 1997; Zhou et al., 2002a). This tumor model has been widely used in our laboratory to evaluate

Organ-Specific Animal Tumor Models

the preventive effects of several dietary components, such as soy components, tea components, and soy and tea combinations, on the growth and metastasis of androgensensitive prostate tumors (Zhou et al., 2002a, 2003). Similar to the development of the androgen-sensitive LNCaP tumor model, androgen-independent human CaP cell lines, such as PC-3 cell line (Waters et al., 1995; Stephenson et al., 1992) and DU 145 cell line (unpublished data in our laboratory), have also been used to develop orthotopic tumor models for androgen-independent CaP. Because PC-3 and DU 145 cells do not produce PSA, other blood markers are necessary to monitor tumor development and growth. One of the markers is interleukin-6 (IL-6). Both cell lines produce IL-6, and our preliminary data indicate that the blood level of IL-6 is correlated with tumor volume and may be used as a marker to monitor the effect of treatment (unpublished data). An Orthotopic Prostate Tumor Animal Model for Progression of Androgen-Independent CaP by Hormonal Manipulation In men, most prostate tumors respond to androgen withdrawal but relapse after the initial response. Few, if any, prostate tumors in humans have been cured by hormonal manipulation. Models for the development of experimental therapy are limited. Based on our SCID-LNCaP orthotopic tumor model (Zhou et al., 2002a), we further developed an in vivo animal model for human CaP progression from androgen-sensitive to androgen-independent phenotype by surgically castrating SCID mice bearing LNCaP tumors (Zhou et al., 2004b). Tumors responded to androgen ablation initially with growth regression, as measured by serum PSA levels. However, tumors started to grow back after a period of time and developed an androgen-independent phenotype. Progression of androgen-independent prostate tumors was associated with alterations of a series of tumor markers, such as tumor cell proliferation and apoptosis and androgen receptor expression (Zhou et al., 2004b). This orthotopic androgen-independent prostate tumor progression model has been used in our laboratory to evaluate the preventive effects of tea components on delaying this progressive process (Zhou et al., 2002b). Transgenic Mouse Models of Multistage Prostate Carcinogenesis Although mice are relatively resistant to the induction of prostatic tumors by chemical carcinogens, several transgenic mouse lines are now available in which prostate carcinomas preferentially occur. Several targeting vectors are successfully used to express heterologous genes to the prostate epithelium of transgenic mice. These include regulatory elements derived from the rat C3 (1) prostate steroid-binding protein gene, rat probasin gene, human PSA gene, and

339

MMTV long terminal repeat. Several transgenic models that are commonly applied and well characterized are discussed. They are the transgenic adenocarcinoma of the mouse prostate (TRAMP) model, the LADY models, and the male C3 (1)/SV40 Tag model. TRAMP Model A transgene carrying the rat probasin gene fused to the SV40 early region has been used to generate an independent transgenic autochthonous model for CaP designated the TRAMP transgenic line of mice (Greenberg, 1995). The TRAMP model is one such model that mimics progressive forms of human disease. The TRAMP mice develop high-grade PIN within 12 weeks of age and extensive hyperplasia and adenocarcinomas by 18 weeks of age; by 24–30 weeks, all TRAMP male mice develop primary prostate tumors, with about half of them displaying well-differentiated prostatic adenocarcinoma and the other half divided between moderately differentiated and poorly differentiated adenocarcinoma. Metastasis is commonly detected by 30 weeks, primarily to the lungs and lymph nodes and less often to bone, kidneys, and adrenal glands (Greenberg et al., 1995; Gingrich et al., 1996, 1997). Androgen depletion by castration at age 12 weeks resulted in decreased tumor incidence to 80% but subsequently resulted in appearance of androgen-independent disease (Gingrich et al., 1997). Castrated mice developed more poorly differentiated primary tumors and twice the incidence of metastatic disease compared to noncastrated TRAMP controls. The TRAMP model develops progressive primary prostate tumors much faster than the C3 (1)/SV40 Tag model, so it can offer a quick in vivo system for evaluating the preventive effects of chemopreventive agents. On the other hand, since all animals develop PIN and carcinoma of the prostate and metastases in a relatively short period, this model may not be a sensitive system for evaluating preventive activities of agents on CaP progression. This animal model has been used to evaluate the effects of a series of dietary components, such as soy isoflavones (MentorMarcel et al., 2001), tea polyphenols (Gupta et al., 2001), and flaxseed (Lin et al., 2002), on CaP prevention. LADY Models Several related transgenic lines have been developed in which a large (12-kb) region of prostate-specific rat probasin promoter drives the expression of SV40 large T antigen to the mouse prostate (Kasper et al., 1998; Masumori et al., 2001; Abate-Shen and Shen, 2002). Because this transgene lacks small t antigen, disease progression is less aggressive than in TRAMP mice; thus, these mice are collectively named the “LADY” model. The LADY transgenic lines display varying but reproducible rates of tumor formation, ranging from 12 weeks to >20 weeks of age. Each line consistently develops multifocal low-grade PINs that progress

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Chapter 17/Animal Models and Nutritional Oncology

to high-grade PINs and early invasive carcinoma, but that generally fail to metastasize (Kasper et al., 1998; Masumori et al., 2001). Furthermore, the remarkable vascularization was accompanied by tumor growth. Similar to the TRAMP model, androgen deprivation of these transgenic mice resulted in temporary regression, followed by the formation of poorly differentiated metastatic carcinomas (Kasper et al., 1998; Masumori et al., 2001). Compared with the TRAMP model, the “LADY” model has less aggressive disease progression and a longer period of high-grade PIN development. These features make the “LADY” model an appropriate animal model for nutritional prevention studies. In addition, the “LADY” model could serve as an appropriate preclinical model for evaluating the preventive agents that target tumor angiogenesis. However, this model has not been adequately applied in nutritional prevention studies. Male C3 (1)/SV40 Tag Model This is a transgenic mouse model for CaP using a recombinant gene expressing the SV40 early-region transforming sequences under the regulatory control of the androgenresponsive rat prostatic steroid binding protein (C3 [1]) gene. Male transgenic mice develop low-grade PIN in the ventral and dorsolateral lobes, which appear identical to those described for the human disease, after ~8 weeks of age (Maroulakou et al., 1994; Shibata et al., 1996). High-grade PIN was found in both lobes by 5 months of age (Shibata et al., 1996). Prostate carcinomas, which appeared to arise from PIN lesions, were found by 7 months of age in the ventral lobe and 11 months of age in the dorsolateral lobe. Metastases were rare (90,000 cancer deaths in the United States each year could potentially be prevented if men and women maintained normal weight. Development of effective interventions to reduce the prevalence of overweight and obesity is essential. Research in experimental carcinogenesis models indicates that a regimen of caloric restriction (usually 20–40% relative to ad libitum controls), which reduces obesity, may be one of the best broad-based interventions to reduce cancer risk (Hursting et al., 2003), although few consistent data exist in humans. Caloric restriction has a beneficial impact on mechanisms regulated by insulin-like growth factor (IGF)-1, including cell proliferation, apoptosis, and cell cycle regulation. To illustrate, caloric restriction increases the rate of apoptosis by reducing the DNA synthesis, which is necessary to increase the number and volume of preneoplastic lesions (Hursting et al., 2003). Achieving a greater understanding of the relationship between obesity and increased cancer risk will require a concerted effort using an interdisciplinary approach of basic and clinical research. As the U.S. population becomes heavier and less active, the challenge for modulating the impact of diet on chronic disease risk should become a national priority. Current trends begun in the past decade for “super-sizing” restaurant portions should be viewed as an impediment to a healthy

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populace. A study of marketplace portion sizes compared actual served portions with recommended federal portion standards and found most marketplace portions are two to eightfold larger than portions used in federal guidelines (Young and Nestle, 2003). A study of trends in portion sizes from national surveys from 1977 to 1996 found that food portion sizes increased both inside and outside the home for all categories except pizza (Nielsen and Popkin, 2003). In addition, energy intake and portion size of salty snacks increased by 93 kcal (60%), soft drinks by 49 kcal (52%), hamburgers by 97 kcal (23%), French fries by 68 kcal (16%), and Mexican food by 133 kcal (27%). A research center study of self-served portions versus larger served portions (double an age-appropriate portion) among children suggests that the larger served portion leads to an increase in entree size by 25% and total energy intakes by 15% (Orlet et al., 2003). A comparison of National Health and Nutrition Examination Survey (NHANES) III (1988–94) data and earlier studies from the 1970s on energy intake among children and adolescents (2–19 years of age) suggests that there has been little increase in energy intake (Troiano et al., 2001). The same study, however, did show that mean percentage of energy from total and saturated fat decreased but remained above recommendations, with overall means of 33.5% of energy from fat and 12.2% of energy from saturated fat. Because overweight and obesity in this age-group has increased over the past decades, some have suggested that decreasing levels of physical activity may account for this finding. Regular physical activity is one of the most important modifiable risk factors for cancer after dietary choices and smoking. A review of evidence for an association between physical activity and cancer found convincing epidemiological evidence that regular physical exercise, comparing highest to lowest levels, reduces the risk of colon cancer by 40–50% and breast cancer by 30–40% (Friedenreich and Orenstein, 2002). For other cancer sites, this review reported that the association was probable for prostate cancer and possible for cancers of the endometrium and lung. Although the underlying mechanisms for the associations have not been established, possible mechanisms have been proposed. For example, mechanisms that may contribute to a beneficial effect of physical activity on colon cancer include changes in gastrointestinal transit time, altered immune function and prostaglandin levels, and changes in insulin levels, IGFs, bile acid secretion, serum cholesterol, and gastrointestinal and pancreatic hormone profiles (Quadrilatero and Hoffman-Goetz, 2003). Possible mechanisms for a physical activity–breast cancer relationship include decreased levels of exposure to estrogen, increases in the production of sex hormone–binding globulin, and reductions in circulating concentrations of insulin and related growth factors (Friedenreich and Orenstein, 2002). Based on accumulating evidence of the health benefits of physical

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Chapter 18/Challenge of Nutrition in Cancer Prevention

activity for cancer and cardiovascular disease, the American Cancer Society (2002) and other national organizations have adopted the recommendation that adults should engage in at least moderate activity for ≥30 minutes on 5 or more days of the week. Children and adolescents should engage in ≥60 minutes/day of moderate-to-vigorous physical activity at least 5 days per week. Medical Approaches Medical approaches to cancer prevention focus on designing and conducting preclinical and clinical studies to better understand the biological basis of the carcinogenic process and how to influence cancer risk. Chemoprevention (a pharmacological approach to intervention that aims to prevent, arrest, or reverse either the initiation phase of carcinogenesis or the progression of premalignant cells) is an important part of the medical approach developed for cancer prevention and intervention. Laboratory and epidemiological studies have provided the scientific rationale for investigating potential chemopreventive agents (Greenwald et al., 1990). For example, epidemiological studies support an inverse relationship between the intake of vegetables and fruits and cancer risk, and clinical studies have identified possible phytochemical components of these foods (as well as interactions among the components) that might contribute to their ability to reduce cancer risk (Chemoprevention Working Group, 1999; Negri et al., 1991). To illustrate, among the hundreds of phytochemicals and micronutrients with potential chemopreventive effects identified from animal and in vitro studies, diallyl sulfide, a phytochemical found in Allium vegetables such as garlic and onion, has been associated with a reduced risk of prostate (Hsing et al., 2002) and colorectal and stomach cancers (Fleischauer et al., 2000). A review by Milner (2001a) found garlic protects against carcinogenesis by blocking N-nitroso compound formation, suppressing bioactivation of several carcinogens, induces apoptosis, alters the cell cycle, and alters several phase I and II enzymes associated with cancer initiation and progression. Understanding the mechanisms of action of dietary constituents such as garlic with confidence for translating this knowledge into prevention strategies remains a significant challenge.

Dietary Choices and Cancer Food choices produce dietary patterns that may increase or decrease the risk of cancer (World Cancer Research Fund, 1997). A wealth of information shows that certain specific diets may offer protection against cancer at many sites. The challenge for nutritional science researchers is determining which BFCs, or combinations, are responsible for cancer protection or increased risk and for which cancer sites. The study of the American (“Western”) diet and cancer risk has

been ongoing for more than 4 decades, with important clues being discovered that suggest research pathways. For example, an analysis of prostate cancer among 3779 men in the NHANES Epidemiological Followup Study Cohort found three distinct dietary patterns in the United States: (1) a “vegetable–fruit” pattern that includes fish and shellfish; (2) a “red meat–starch” pattern that includes salty snacks, cheese, sweets, and desserts; and (3) a “Southern” pattern that includes traditionally Southern foods such as cornbread, grits, sweet potatoes, and okra (Tseng et al., 2004). The only dietary pattern associated with a decreased risk of prostate cancer was the “Southern” pattern (borderline significance), which was seen in both white and black men. Another prospective study of eating patterns and colon cancer found that a diet with high intakes of dietary fiber and folate was protective, especially among older Americans (Slattery et al., 1998). In the same study, a “Western” dietary pattern (high levels of red meat, processed meat, fast food, refined grains, and sugar-containing foods, and low levels of vegetables and fruits) was associated with an increased risk of colon cancer among men and women. Aside from dietary patterns, a growing base of research exists that indicates specific types of foods or food constituents may reduce the risk of cancer. Table 2 lists selected nutrients that may modify cancer risk. The challenge for nutritional science is to confirm these findings in chemoprevention clinical trials and determine how they should fit into a diet that encourages improved health. Various food choices could satisfy the need for the particular BFCs associated with reduced cancer risk. For example, the carotenoid lycopene has been shown in animal and clinical studies to reduce the risk of prostate cancer by various mechanisms, including acting as an antioxidant, interfering with growth factor receptor signaling and cell cycle progression, and upregulating connexin 43, which allows direct intercellular gap junctional communication (Heber and Lu, 2002). A review of tomato products, lycopene, and prostate cancer risk found that eating one serving of lycopene-containing foods per day is associated with lower prostate cancer risk (Miller et al., 2002). In making food choices, lycopene is found in all tomato-based products regardless of processing, grapefruit, watermelon, papaya, and other fruits.

DISCOVERY Discovery is the initial step in developing hypotheses that can be investigated in clinical investigations and intervention studies. Epidemiological and ecological studies provided clues for avenues of research for elucidating the diet–cancer relationship. In past decades, for example, comprehensive reviews of diet and cancer were published by the U.S. National Academy of Sciences (NAS) and the World Cancer Research Fund (WCRF) (NAS, 1982, 1989; WCRF,

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Discovery

TABLE 2

Selected Examples of Bioactive Food Components That May Modify Cancer Risk

Food source Cruciferous vegetables (arugula, Bok choy, broccoli, Brussels sprouts, cauliflower, collard greens, kale, mustard greens, radishes, rutabaga, turnips) Vegetables

Dark green vegetables (spinach, kale) Vegetables, fruits, black tea Onions, garlic, scallions, chives Citrus fruit Citrus fruit (peel), caraway seed oil Berries, tomatoes, potatoes, broad beans, broccoli, squash, onions Radish, horse radish, kale, endive Tea, chocolate Grapes, red wine Tumeric, curry, mustard fruits, coffee beans, soybeans Strawberries, raspberries, blackberries, walnuts, pecans Cereals, pulses (millet, sorghum, soya beans) Orange vegetables and fruit Tomatoes Tea, coffee, cola, cacao (cocoa and chocolate) Dairy products (milk, cheese, yogurt) Red meat

Class of compound

Bioactive food component (s)

Isothiocyanate

Benzyl isothiocyanate, 2-phenethyl isothiocyanate, sulforaphane, allyl isothiocyanate, 3-methylsulfinylpropyl isothiocyanate

Glycosinolate Minerals Flavonoids Vitamins Carotenoids Vitamins Flavonoid Allium compounds (Organosulfur compounds) Flavonoid Terpenoid Monoterpenes Flavonoid

Indole-3-carbinol, 3,3′-diindoylmethane, indole-3-acetonitrile Calcium, zinc, selenium Quercetin, rutin Folic acid, vitamin A, vitamin E, vitamin C Lutein Vitamin A, vitamin C Anthocyanins Diallyl sulfide, allylmethyl trisulfide, allyl mercaptan, S-allylcysteine Tangertin, nobiletin, rutin d-Limonene, perillyl alcohol, geraniol, menthol, carvone Quercetin

Flavonoid Polyphenol Polyphenol Polyphenol

Kaempferol Epigallocatechin gallate, epigallocatechin, epicatechin, catechin Resveratrol, catechin Curcumin, caffeic acid

Polyphenol

Caffeic acid, ferulic acid, ellagic acid

Isoflavone Carotenoid Carotenoid Methylxanthines Vitamins Vitamins

Genistein α- and β-carotene Lycopene Caffeine, theophylline, theobromine Vitamin D, calcium Iron

Source: Adapted from Manson (2003).

1997), among others. Based on substantive epidemiological and experimental evidence, these reviews indicate strong support for a diet–cancer relationship. In general, these reviews recommended increased intake of fiber and a variety of vegetables and fruits, moderate consumption of alcohol and salt, reduced fat intake, and increased physical activity. To illustrate, the WCRF reported that convincing evidence supported the hypothesis that a diet high in vegetables protects against cancers of the colon and rectum, stomach, lung, esophagus, mouth, and pharynx. Further, vegetables may protect against breast, bladder, pancreas, and larynx cancer, but the evidence was less convincing; and limited evidence suggests that vegetables reduced the risk of prostate, ovary, endometrium, cervix, liver, kidney, and thyroid cancers (WCRF, 1997). In addition, the WCRF proposed that dietary fat, excessive calories, obesity, and alcohol may increase the risk of cancer at various sites, whereas fruits, dietary fiber, and certain micronutrients may protect against cancer. Since the publication of the WCRF review, results from several large population-based epidemiological studies have been

reported that provide additional clues to the relationship between nutrition and cancer.

Large-Scale Prospective Studies Health Professionals Followup Study The Health Professionals Followup Study (HPFS), begun in 1986 with follow-up in 1990 and 1994, is a prospective cohort study of 47,882 men in the United States that uses a validated 131-item semiquantitative food frequency questionnaire. HPFS analyses of dietary factors and prostate cancer suggests reduced risk with the intake of fish more than three times per week (Augustsson et al., 2003); equivocal findings for the intake of cruciferous vegetables, except for reduced risk among men younger than 65 years and those who reported higher intakes over the 10 years before baseline (Giovannucci et al., 2003a); reduced risk with higher intakes of fructose (>5 vs 250 dietdependent compounds could be identified in plasma, which may allow them to be used as biomarkers for the identification of metabolomic genotypes and phenotypes associated with health or disease (Watkins and German, 2002). Proteomic technology has been used to investigate potential prostate cancer biomarkers. Using surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectometry, Zheng et al. (2003) found a protein (PCa-24) present in 16 of 17 prostate carcinoma specimens that may be a potential biomarker for this condition; PCa-24 was not expressed in any of the 12 benign prostatic hyperplasia specimens studied. With the human genome completely sequenced and our improved understanding of the proteins and metabolites involved in gene–nutrient interactions, the challenge for nutrition researchers is to assimilate knowledge from all fields to identify and validate biomarkers that signify changes from good health to clinical cancer.

DEVELOPMENT Development of nutritional interventions within the 3-D approach to cancer prevention is based on the evaluation of findings from discovery that show promise for reducing the cancer burden (von Eschenbach, 2003). Nutritional components have been under investigation at the NCI for more than 2 decades. Table 3 presents information on selected nutritional components being investigated in NCI chemoprevention trials. Phase I clinical trials are designed to determine the dose-related safety and toxicity of the proposed chemopreventive agent. Phase II clinical trials evaluate agent

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TABLE 3 Cancer site Breast Colon Lung

Prostate

Selected NCI-Sponsored Phase I: II: and III Cancer Prevention Trials of Nutritional Factors Phase I Soy isoflavones Indole-3-carbinolb Curcumin lSelenomethionine/vitamin E Lycopene (3 trials) Soy isoflavones Genisteinb

Cervix Bladder Anogenital warts + HPV/HIV Skin

Phase II

Phase III

EGCG/polyphenon E (green tea extract) Folic acida (2 trials) Vitamin Da/calcium Selenized yeast 13-cis-retinoic acidb Selenized yeast Soy (dietary) Soy isoflavones Vitamin D analogue Seleniuma 9-cis-Retinoic acid β-caroteneb

Selenomethionine Selenium/vitamin E a Diet low in fat and high in soy, fruits, vegetables, green tea, vitamin E, and fiber Folic acida High-dose multivitaminsa

EGCG/polyphenon E (Green tea extract)

Indole-3-carbinol Retinol,a Retinyl palmitate EGCGa/polyphenon E (Green tea extract)

Head and Neck

β-carotenea 13-cis-retinoic acida (2 trials)

a

Accrual completed; study closed to new participants. Completed. EGCG, epigallocatechin gallate (polyphenon E). b

efficacy in a larger group of participants at high risk for specific cancers and can provide data that characterize dose, safety, and toxicity in the selected population. Phase III clinical trials are randomized, double-blinded, placebocontrolled trials conducted in a large population of participants. Phase III trials have well-defined primary, and often secondary, endpoints that allow investigators to determine the agent’s usefulness as a prevention or treatment strategy for a specific cancer type. Development with phase III clinical trials also includes large-scale dietary modification trials that investigate the effect of selected BFCs or groups of BFCs on cancer risk. Modification trials generally have endpoints that address changes in lifestyle, reducing the levels of some dietary factors or increasing others. These trials also offer the opportunity to investigate the overall diet for its effect on biomarkers of exposure and susceptibility.

Large-Scale Phase III Chemoprevention Trials Selenium and Vitamin E Cancer Prevention Trial (SELECT) Selenium has been extensively studied in experimental models and has been found to reduce cancer risk through numerous mechanisms, including antioxidant effects, enhancement of immune function, induction of apoptosis,

inhibition of cell proliferation, alteration of carcinogen metabolism, cytotoxicity of metabolites, and influence on testosterone production (reviewed in Klein, 2004). SELECT was designed to further clarify findings from previous population-based trials that reported on the possible benefits of selenium and vitamin E. For example, a population-based clinical trial, the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study (ATBC Study) found in a secondary analysis that men receiving vitamin E had a decrease in prostate cancer mortality (41%) and incidence (36%) (Heinonen et al., 1998). In addition, secondary analysis of the HPFS found that daily use of vitamin E (100 µg/day) decreased the risk of metastatic or fatal prostate cancer 44% compared with nonusers (Chan et al., 1999). Secondary endpoint analyses from a multicenter, double-blind, randomized, placebocontrolled cancer prevention trial indicated that supplemental dietary selenium (200 µg/day) significantly reduced the risk of total cancer mortality by 50% (Clark et al., 1996) and prostate cancer incidence by 63% (Clark et al., 1998). In addition, the Nutrition Intervention Trial in Linxian, China, in a region of low selenium levels in the soil and food, found significant inverse associations between baseline serum selenium and death from esophageal (17% reduction) and gastric cancers (25% reduction) (Wei et al., 2004). Given these encouraging results, the NCI sponsored SELECT, a randomized, prospective, double-blind study, to

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Development

determine whether daily supplementation of selenium and vitamin E will decrease the risk of prostate cancer in healthy men (Klein et al., 2001). SELECT is a four-arm intervention trial comparing vitamin E alone (400 mg of racemic αtocopherol), selenium alone (200 µg of 1-selenomethionine), combined vitamin E and selenium, and placebo. The trial is scheduled to provide a 7- to 12-year regimen that includes an optional multivitamin that does not contain selenium or vitamin E. Routine clinical evaluations will include a yearly digital rectal examination and prostate-specific antigen test. SELECT is the largest prostate prevention trial ever conducted, and as of January 2004, ~90% of the targeted goal of 32,400 men had been enrolled. The primary endpoint is diagnosed prostate cancer; secondary endpoints will be the incidence of and survival from lung and colon cancers. An important role for SELECT in the development of selenium as a chemopreventive agent is the inclusion of a biomarker study within the trial. A nested case-control study within SELECT will assess genetic polymorphisms of four genes, androgen receptor (AR), 5α-reductase type II (SRD5A2), cytochrome P450c 17α (CYP17), and β-hydroxysteroid dehydrogenase (HSD3β2), on prostate cancer incidence (Hoque et al., 2001). Substantial discovery efforts involving epidemiological and experimental studies suggest that these biomarkers of risk may affect susceptibility to prostate cancer (Haiman et al., 2001). For example, experimental studies have shown that selenium induces growth inhibition in human prostate cancer cell lines, but only if the cells have a functioning AR (Venkateswaran et al., 2002). Knowing whether the mechanisms of selenium action are dependent on specific AR polymorphisms could assist researchers in developing more specific preventive strategies for populations affected by the relevant AR polymorphisms. In addition, polymorphisms in CYP17 A1/A1 genotype may confer a significantly higher serum androgen level, which is associated with higher risk of prostate cancer than found in men with either the A1/A2 or A2/A2 genotype (Hoque et al., 2001).

prostate cancer and β-carotene on prostate and total cancer; in addition, it is the only primary prevention trial in healthy men testing multivitamins or any single antioxidant vitamin, alone or in combination, on cancer and CVD (Christen et al., 2000). Follow-up is scheduled to begin after 5 years. Trials of b-Carotene The Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study (ATBC Study) and the Beta-Carotene and Retinol Efficacy Trial (CARET) have been controversial for the surprising finding that β-carotene was associated with an increased risk of lung cancer among smokers (ATBC Group, 1994; Albanes et al., 1996; Omenn et al., 1996). Both trials were conducted in cigarette smokers, with a 16% increase in lung cancer in the β-carotene group of the ATBC Study and a 28% higher incidence of lung cancer in participants receiving the β-carotene/retinyl palmitate combination in CARET. The ensuing international controversy surrounding these findings has been reviewed by Greenwald (2003); potential issues included dose, timing of the dose, interference by β-carotene in absorption of other carotenoids or antioxidants, and the duration of the studies. Subsequent investigations and reviews have added important information to this controversy. A postintervention follow-up of the ATBC Study found that the beneficial effects of vitamin E (α-tocopherol) and the negative effects of β-carotene disappeared after 4 years postintervention (Virtamo et al., 2003). The authors, representing the ATBC Study Group, continued their recommendation that smokers avoid β-carotene. The Pooling Project of Prospective Studies of Diet and Cancer analyzed data from seven cohort studies (~400,000 participants and 3150 cases) of dietary carotenoids and lung cancer (including the ATBC Study) and found that intakes of β-carotene, α-tocopherol, lutein/zeaxanthin, and lycopene were not associated with lung cancer risk (Männistö et al., 2004). Of the carotenoids studied, only β-cryptoxanthin was significantly inversely associated with lung cancer risk.

Physicians’ Health Study-II The Physicians’ Health Study-II (PHS-II) was designed after the end of PHS-I in 1995, which did not support either benefit or harm from 12 years of β-carotene supplementation on the primary prevention of cancer and cardiovascular disease; the aspirin component of PHS-I was stopped early because of the benefit of aspirin on the risk of a first heart attack (Hennekens et al., 1996). PHS-II is a randomized, double-blind, placebo-controlled trial to investigate the role of vitamin C, vitamin E, β-carotene, and a multivitamin for the primary prevention of total cancer, prostate cancer, and cardiovascular disease (Christen et al., 2000). The trial uses a 2 × 2 × 2 × 2 factorial design and is the only trial testing the potential benefits of vitamin E in the prevention of

Large-Scale Dietary Modification Trials Polyp Prevention Trial The Polyp Prevention Trial (PPT) is a multicenter, randomized, controlled dietary intervention trial that is examining the effect of a low-fat (20% of calories from fat), high-fiber (18 g/1000 calories), high-vegetable and -fruit (five to eight daily servings, combined) dietary pattern on the recurrence of adenomatous colorectal polyps (APC) (Lanza et al., 1996; Schatzkin et al., 1996). Participants received extensive dietary and behavioral counseling on how to meet dietary goals. Results reported by Shatzkin et al. (2000) indicated that the PPT dietary intervention did not

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influence the risk of recurrence of APC. A subsequent analysis, however, did show that study participants in the intervention arm of the PPT made sustained significant changes in all PPT goals: reduced fat intake and increases in fiber and fruits and vegetables (Lanza et al., 2001). Intervention participants also reported significantly higher serum carotenoid concentrations and lower body weights than the control group. This finding is of particular importance to cancer prevention researchers as further preventive dietary interventions are designed. Women’s Health Initiative The Women’s Health Initiative (WHI), which began in Fall 1993, is a 15-year, multidisciplinary trial that includes both dietary and chemopreventive interventions. The nutritional components of the WHI include the Low-Fat Dietary Modification Trial (20% of calories from fat) and the Calcium/Vitamin D Supplementation Trial (calcium and vitamin D supplementation) for prevention of cancer, cardiovascular disease, and osteoporosis. A separate WHI initiative on hormone replacement therapy (estrogen plus progestin) was stopped in 2002 because of results indicating an increase in invasive breast cancer (Rossouw et al., 2002). Although disease endpoints are not complete for the nutritional components of the WHI, observational studies suggest that behavioral interventions designed for this trial have resulted in significant dietary changes, especially regarding reduced fat intake (Patterson et al., 2003). Women’s Healthy Eating and Living Study The Women’s Healthy Eating and Living (WHEL) Study, which began in 1996, is a multicenter, randomized dietary intervention trial among breast cancer survivors. The study is investigating the effectiveness of a high-vegetable, lowfat diet in reducing additional breast cancer events and early death in women within 4 years of diagnosis of early-stage invasive breast cancer (Pierce et al., 2002). An important aspect of the study is to investigate the impact of raising circulating carotenoid concentrations through changes in diet. Preliminary results have assessed the methods used in WHEL (FFQ, 24-hour dietary recall, intensive telephone counseling, cooking classes, and print materials) and found that the use of a multimodal, multimethod intervention is beneficial for promoting dietary change (Thomson et al., 2003). The study is scheduled for completion in 2006.

DELIVERY Delivery is the process of disseminating, facilitating, and promoting evidence-based prevention, detection, diagnosis, and treatment practices and policies to reduce the burden of

cancer in all segments of the population (von Eschenbach, 2003). A primary focus of these efforts is to develop strategies for those populations who bear the greatest burden of disease. Delivery works most efficiently when it is part of the processes of “Discovery” and “Development.” SELECT is an excellent example of an integrated 3-D approach. Coordinated by the Southwest Oncology Group (SWOG), SELECT includes >400 study sites throughout the United States, Puerto Rico, and Canada. SWOG and many of the other study sites belong to the NCI’s Community Clinical Oncology Program (CCOP), which is a creative mechanism designed to improve the accrual of patients to NCI phase III clinical trials while encouraging community-based oncologists to participate in clinical research. In addition, CCOP is one of the most practical means to disseminate new information on state-of-the-art cancer treatment outside the traditional cancer centers and research-oriented medical centers (Kaluzny et al., 1989). Clinicians and the public will receive immediate access to the prevention and treatment strategies that are most relevant to their communities because local researchers and facilities will be developing and participating in research translation efforts at the community level. For example, African American men and those in lower socioeconomic strata (SES) have the highest rates of prostate cancer, with race and SES being independent predictors of stage at diagnosis (Schwartz et al., 2003). Prevention and treatment strategies in SELECT can be immediately integrated and delivered in those CCOP communities that include populations that may benefit the most from intervention. Understanding the most efficient and successful nutritional strategies to support cancer prevention, screening, and treatment for those individuals or groups that will benefit the most is a significant challenge for cancer researchers. The small and large hospitals, private practices, and groups of organizations or private practices that compose the CCOP network have been invaluable in creating the environment for research translation to health professionals and the public. CCOP includes 51 centers in 34 states, the District of Columbia, and Puerto Rico, as well as 11 Minority-Based CCOP Programs (MB-CCOP) that serve a large population of minorities. The network provides access to cancer clinical trials in 403 community-based hospitals, with >4000 community physicians participating in NCI clinical trials through this network (CCOP web site, 2003). Many of these clinical trials, such as SELECT, are investigating chemoprevention agents that include natural or synthetic nutritional components and contain programs for dissemination.

Challenges in Delivery for Nutritional Oncology A significant challenge in delivery is determining the benefits within a population of dietary changes and whether

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lifestyle changes per se offer a greater benefit than treatment, screening, or chemoprevention. The analysis of worldwide cancer incidence and mortality rates mentioned previously confirms that diet influences cancer (Young and LeLeu, 2002), although changes in lifestyle generally take many years to accrue benefits compared with the shorter-term benefits of using treatment or chemoprevention approaches. Risks of treatment or chemoprevention, which are higher than dietary interventions, also must be considered when deciding whether lifestyle approaches should be implemented, especially as nutritional oncology appears to be in a transition period emphasizing the integration of lifestyle and medical approaches to cancer prevention. These issues must be weighted carefully in recommendations for lifestyle or medical approaches. Medical Education Delivery of evidence-based practices for the benefit of those most at risk for cancer will depend on improving nutrition education for clinicians and application of proven interventions and programs at the community level. Assessments indicate a lack of time spent on nutrition in our medical training institutions. A survey of medical schools in the United States found that nutrition medical education was required in only ~20% of the programs (Touger-Decker, 2004). A survey of hours of nutrition education in medical schools found that medical schools have an average of only 18 hours of instruction over a 4-year program (Torti et al., 2001). Improving nutrition medical education can encourage delivery of diet-related research results and help integrate delivery into the new nutrition paradigm. There have been calls to provide an integrated nutrition education message within every aspect of medical education so that graduates enter practice with an understanding of the integral role of nutrition in health and disease (Kushner, 2002). Nutrition Policy The awareness of the role of nutrition in cancer prevention should be integrated into all policies at the national, state, and local levels. In the past decade, with the maturity of electronic communications systems such as the Internet, cable TV, and home personal computing, information is becoming increasingly more available at every stratum of the population. The same media that bring information to the consumer, however, also bring conflicting information on the role of nutrition and specific diets in maintaining health. The USDA and the U.S. Department of Health and Human Services have the primary role for providing nutrition education and advice at the national level. Development of the Food Pyramid, and subsequent revisions, has provided consumers with science-based information on appropriate food choices. An interactive USDA web site

(http://www.mypyramidtracker.gov) allows individuals to assess their diets in context of the amount of physical activity they perform and to set goals for maintaining or losing weight. This type of service adds to the knowledge of those who choose to participate. The movement of policymakers at the national level toward evidence-based national dietary guidelines is promising (Cooper and Zlotkin, 2003). Application of nutrition-based policy is exemplified by the 5 A Day For Better Health Program (5 A Day), which was begun by the NCI in 1991 but was transferred to the Centers for Disease Control and Prevention. The 5 A Day Program is a cooperative initiative between the federal government and the vegetable and fruit industry to increase the intake of vegetables and fruit to reduce cancer risk. An evaluation of the 5 A Day program indicated that implementing a media campaign, point-of-purchase initiatives, such as use of the “5 A Day” logo on products, and community-level interventions have significantly increased intake from 1991 to 1997 (Stables et al., 2002). The 5 A Day program evaluation report and more about the program can be found by visiting their web site at http://www.5aday.gov/.

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INTRODUCTION

exposures before cancer induction. Because cancer tends to develop very slowly over a 20–30 year period, memories of subjects’ diets long ago are often inaccurate. Recall bias can occur for many reasons. One example of recall bias is reporting of dietary intake by obese subjects, which is different from reporting of dietary habits by lean subjects. Many obese subjects omit mentioning those foods they know they should not be eating. Patients suffering from cancer malnutrition may be weakened, or even cachectic, so their ability to recall foods may be impaired in comparison to healthy controls. Controls reporting higher consumption with a particular food relative to cases that are cachectic may distort associations toward a preventive or risk-enhancing direction, leading to false conclusions.

Over the past 25 years, dietary assessment methodology has made significant progress in identifying associations between patterns of dietary consumption and cancer risk. Although it is not yet possible to quantify specific cancer risks related to specific foods or dietary patterns, there is considerable evidence that certain foods are associated with increased cancer risk. For example, epidemiological studies have identified a clear association of increased red meat intake with an increased risk for colorectal cancer (Chao et al., 2005; Willett, 2005). The choice of optimal dietary assessment methods depends on the research question being asked, the cancer site under study, the research study design, and the metabolic/ biochemical measurements available for characterizing dietary intake. Moreover, factors intrinsic to the dietary assessment instruments have been shown to affect the results obtained. These include the quality of nutrient and food composition databases, the sensitivity of specific assessment instruments to differences in nutrient intake, and the flexibility of the assessment instrument for diverse dietary intakes. A number of these methodological constraints that can lead to inconsistent results on the association of diet and cancer risk have appeared in published studies from different groups of investigators.

Prospective Longitudinal Study Designs The major advantage of conducting a prospective cohort study is measurement of dietary intake before disease onset, which can provide important insight into causal relationships between dietary patterns and cancer. However, determination of the relevant etiological time period for dietary assessment can be challenging (Tarasuk and Brooker, 1997). Major disadvantages for conducting a prospective cohort study include lengthy time of study, large sample sizes required to detect associations with rare cancer outcomes, the higher costs associated with subject recruitment and measurement over time, and specialized composition of cohorts that limit generalizability.

STUDY DESIGN AND ASSESSMENT Dietary Pattern Assessment

Nutrigenetics and Nutrigenomics

Measurements of dietary patterns are generally conducted using recall methods (Tarasuk and Brooker, 1997). Recall of subjects’ dietary histories are meant to approximate dietary


Genetic and molecular epidemiological study designs such as case–case and gene-association studies are

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beginning to evaluate interactions between dietary intake and genetic polymorphisms, as well as interactions between dietary intake and gene expression. Genetic polymorphisms are a function of inheritance and can be considered fixed exposures over the lifetime, whereas gene expression and epigenetic changes vary over the lifetime. Both acquired genetic changes and inherited susceptibility, coupled with environmental exposures, are responsible for development of cancer. The technology for measuring gene expression and epigenetic changes such as DNA methylation in relationship to nutritional intake and cancer has only been developed recently. A study of DNA methylation of gastric tumors was conducted among 58 male patients who completed food frequency questionnaires (FFQs) and lifestyle questionnaires. Tumors were categorized according to methylation status, and dietary factors were compared in relationship to methylation. The study found an association between CDX2 gastric tumor methylation and decreased intake of green tea and cruciferous vegetables (Yuasa et al., 2005). Gene-association studies examine relationships between polymorphisms of inherited genes, environmental exposures, and risk of disease. Dietary intake and gene association studies in relationship to cancer risk can be conducted prospectively or retrospectively, using cross-sectional, case-control, or prospective cohort designs. One example is a report of serum biomarkers of carotenoids that were studied in relationship to DNA repair gene (XRCC1) haplotypes and breast cancer risk in the Nurses’ Health Study (Han et al., 2003). The study found a marginally significant reduction in breast cancer risk among women who carried at least one 194Trp allele compared wtih noncarriers. One of the haplotypes of the XRCC1 gene, Arg194Trp, modified the inverse associations of plasma α-carotene level and plasma βcarotene level with breast cancer risk (Han et al., 2003).

METHODS OF DIETARY ASSESSMENT Dietary assessment is used to identify and monitor types and amounts of foods and beverages consumed by individuals and groups of individuals. Results of dietary assessments, in turn, shape public health policy and programs. Identifying dietary patterns or food constituents that clearly contribute to or prevent development of cancer is a primary goal and challenge of diet and cancer research. Accurate estimation of nutritional intake is fundamental to studies of diet and cancer. Various methods exist for assessment of dietary intake in free-living individuals. The three main approaches are 24-hour recalls, diet records, and FFQs. Both 24-hour recalls and diet records allow for freeform open-ended recording of food intake. FFQs have preassigned lists of foods and set categories for amount of food consumed.

An ideal dietary assessment instrument should provide an accurate, objective, unbiased, and quantitative measure of long-term exposure to dietary constituents. The accuracy of dietary assessment depends, in part, on the accuracy of available food composition data; the need for expanded composition data for commonly eaten foods is widely recognized. Epidemiological surveys, controlled feeding, metabolic studies, and clinical trials of dietary modification all use dietary assessment methods. The general-purpose instruments used to estimate nutrient intake in populations are not well suited to assessing the modest dietary changes that might result from a dietary intervention. Even within an intervention, different instruments might be used for assessment at baseline versus after intervention because of significant changes in consumption and food preparation techniques. Controlled feeding and metabolic studies, in which all foods are provided, permit close monitoring of food and nutrient intake, as well as biological and metabolic responses that result from dietary change. Such studies are important for development of validated standardized nutritional biomarkers, which may be useful adjuncts to traditional dietary assessment instruments. Selection of the dietary survey methodology, among other things, is dependent on the study design and timing of dietary exposure measurement (Barrett-Connor, 1991; Sempos et al., 1999).

Twenty-four Hour Recall Twenty-four hour recalls ask individuals to describe, in either an in-person or telephone interview with a nutritionist or trained interviewer, what they have eaten during the previous 24 hours (Willett et al., 1985; World Cancer Research Fund [WCRF], 1997). This method has the advantages of requiring minimal effort on the part of the study participant (Willett, 1987). Although single 24-hour recalls are highly accurate and reliable measures of recent intake, they do not account for dayto-day variability in an individual’s diet and generally are not recommended for use in longitudinal large-scale cohort studies (Willett et al., 1985; Willett, 1987; WCRF, 1997). On the other hand, 24-hour recall methods have better reliability for assessing current and immediate dietary patterns for validation of biomarkers that characterize the intake of particular nutrients, as well as assessing dietary compliance in dietary intervention studies. Using data from a carefully designed validation study conducted by Willett et al. (1985), Byar and Freedman (1989) argue that one reason the precision of subjective dietary assessment methods such as FFQs and dietary records is limited or compromised is that individuals are not aware of what they eat. Willett’s validation study included 173 female nurses who were asked to record everything they

Methods of Dietary Assessment

ate for 7 days on four separate occasions ~3 months apart. The same subjects also completed a semiquantitative FFQ on two separate occasions. In this case, the dietary food records served as the “gold standard” against which the questionnaire data were validated. Quintiles of intake from the FFQ were then crossclassified with quintiles from the 7-day food records. If the two methods were measuring the same intakes with no measurement error, a one-to-one direct correlation (a correlation of 1) between the same quintiles of intake would result. Interestingly, however, only 53% of individuals in the first quintile for the questionnaire data also were in the first quintile for the food record data. Correlations were even lower for the other quintile comparisons. Such data provide insight into how well, or poorly, one assessment method compares with another. Whether participants actually provide different dietary profiles in the different instruments, or whether questionnaires and recall methods do not adequately match items described in personal food diaries, warrants further investigation. The new multiple-pass method for collecting 24-hour dietary recalls maintains related systems and databases and is designed to engage the respondent more completely to provide more accurate recalls than earlier methods (Dwyer et al., 2001). These types of 24-hour recalls have been consistently used to characterize food intakes of large population groups such as those surveyed in the National Health and Nutrition Examination Survey (NHANES) (U.S. Department of Health and Human Services, 2005). The Continuing Survey of Food Intakes by Individuals (CSFII) and the NHANES have become integrated (Dwyer et al., 2001). The integrated survey has been administered as part of the NHANES that began in 2002. Another well-respected automated 24-hour recall is the Nutrition Data System for Research (NDS-R), developed by the Nutrition Coordinating Center from the University of Minnesota (Nutrition Coordinating Center, 2005). The computer-based application allows entry of dietary data in a standardized fashion and uses a multiple-pass approach for dietary data collection that prompts for complete food descriptions, food preparation methods, and diverse amount descriptions. The Nutrition Data System links to an extensive database that contains values for 136 nutrients, nutrient ratios, and other food components and includes >18,000 foods, with many ethnic foods and >8000 brand-name products (Nutrition Coordinating Center, 2005).

Food Records Food records or diaries are detailed descriptions of types and amounts of foods and beverages consumed over a prescribed period, usually 3–7 days. The record or diary may be a special form or booklet that contains prompts or suggested categories of foods for each day. In some applica-

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tions, subjects measure food with scales using specific procedures (Willett, 1990). Maintaining a food diary requires meticulous record keeping; sustaining these activities over even a relatively short period requires highly motivated literate individuals. Recording daily intakes often increases subjects’ awareness of what they are eating, which in turn can lead to immediate alterations in the diet. Because of this reactive effect of the participant changing his or her dietary intake by omitting foods to simplify record-keeping, dietary records often do not represent actual intake (Willett, 1987; Bingham and Paul, 1998). In dietary intervention studies, a heightened awareness can be instilled in subjects to enhance the accuracy of diet records, and diet records become a teaching device within the intervention (Willett, 1990). In general, dietary record keeping is expensive and inappropriate for studies focused on past intake. Unlike most other methods, however, food diaries do not depend heavily on memory and thus are relatively free from recall bias, compared with other methods. Food records also reduce dayto-day variation in diet by averaging intake over a number of days and can control for differential intakes between weekdays and weekends. Furthermore, record-keeping methods that require foods and beverages to be weighed or measured dramatically reduce errors associated with estimating portion size (Willett, 1987). Food records can be invaluable tools to monitor group compliance in dietary intervention trials; however, the possibility that compliance may be good during the period of recall and poor otherwise may promote biases in reporting. Diet records are one of the several methods for validating FFQs (Willett, 1990). Food records are being used in large-scale prospective studies to measure diet at various intervals over time. The Data Into Nutrients for Epidemiological Research (DINER) system program is a computerized software system for entry of 7-day diaries in the EPIC-Norfolk cohort (Welch et al., 2001). A food list of 9000 food items and values for 24,000 portion sizes have been incorporated into the database. Daily food intake is recorded in a food diary booklet that is entered into the DINER system using a series of pull-down menus that record time of day and meal, food item, amount of food, method of preparation, and so forth. Food items are then merged to an extensive database that converts the food items to nutrients and food groups (Welch et al., 2001). There are possible drawbacks to recording and inferring usual intake from 7-day food records (Gersovitz et al., 1978; Willett, 1990; Flegal, 1999). Seven-day food records require a high degree of respondent cooperation that may be difficult to achieve in a large study (Flegal, 1999). Moreover, food records tend to become less accurate after the first 4 days of record keeping (Gersovitz et al., 1978). Other limitations to food records include inability to fully represent usual dietary intake and the requirement that the respondent population be motivated, literate, and cooperative (Willett,

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1990). Food records collected over specified time intervals in a prospective design can, however, be a relatively accurate and unbiased method for assessing dietary intake and risk of disease (Barrett-Connor, 1991).

weight, height, menopausal status, parity, hormone replacement therapy, and nonfat energy (Bingham et al., 2003).

NUTRITIONAL BIOMARKERS Food Frequency Questionnaires FFQs are designed to assess frequencies with which food items are consumed during a specified time (Tarasuk and Brooker, 1997). FFQs generally provide a listing of foods and include categories to mark, indicating how often a food item is consumed on a monthly, weekly, or daily basis. The underlying principle of the food frequency approach is that average long-term diet, for example, consumption over months or years, is the conceptually important exposure rather than intake on a few specific days, such as what is measured in 24 hours (Willett, 1990). Although recall of diet >20 years ago is impractical, the food frequency approach may approximate crude underlying dietary patterns that have been present over the long term. Two well-known FFQs, among others, have been used extensively in epidemiological studies of diet and cancer (Willett et al., 1985; Block et al., 1986). In a comparability study of nutrient estimation among subjects who completed both the Willett Food Frequency Questionnaire (Willett et al., 1985) and the Block Food Frequency Questionnaire (Block et al., 1986), both questionnaires were found to be, on the average, interchangeable with respect to estimates of nutrients in the overall diet (McCann et al., 1999). FFQ data may be affected by the educational level of research subjects. Completion of FFQs requires cognitive ability to distinguish how often foods are consumed, remembering which foods are consumed, and recording in such a way that responses reflect usual intake (Flegal, 1999). Portion size estimation also requires that respondents modify and adjust their frequency responses according to, in some cases, prespecified portion sizes (Willett, 1987). Being able to specify portion size is another demanding cognitive task. The challenges of completing FFQs may lead to inaccurate reporting of usual intake. The dietary assessment method selected may impact the associations of diet and cancer risk observed. In one example, diet was assessed with both an FFQ and a detailed 7-day food diary. This study was conducted in the EPICNorfolk cohort among 13,070 women (Bingham et al., 2003). Total fat measured by the 7-day food diaries was positively associated with increased breast cancer risk (p for trend = 0.05), whereas total fat measured by the FFQ was not associated with breast cancer risk (p for trend = 0.14). An even stronger difference in measurement methodology was seen for saturated fat intake, where saturated fat measured by 7-day diaries was strongly associated with breast cancer risk (p for trend = 0.005) and saturated fat measured by the FFQ was not (p for trend = 0.23). Models were adjusted for

Nutritional biomarkers are biological consequences of dietary intake or dietary patterns resulting in the presence of components or metabolic byproducts (Consensus Group for Biomarkers in Cancer Chemoprevention, 2001; Potischman and Freudenheim, 2003). Exposure biomarkers may include endogenous or exogenous agents and their metabolites or adducts in tissues or body products, whether in physiological or pathological amounts. Structural changes in the cell or organism that reflect exposure are also included (Consensus Group for Biomarkers in Cancer Chemoprevention, 2001). Biomarkers have the capacity to improve risk estimation and define mechanisms of exposure–disease linkages. Furthermore, utility of biomarkers in population and prevention studies is continuous; that is, some markers are more informative than others, depending on how they are being used, with a gradation from extremely useful to artifactual (Groopman, 2005). Biomarkers can be sorted into functional use categories (Consensus Group for Biomarkers in Cancer Chemoprevention, 2001), which include the following: 1. Validation of dietary instruments 2. Surrogate indicator of dietary intake 3. Integrated measure of nutritional status for a particular nutrient 4. Measurement of nutrient metabolism or interaction metabolic byproduct with other factors

Biomarkers and Dietary Assessment Biomarkers as Validation Tools for Dietary Assessments Biomarker measurement can provide complementary information to help assess performance of different dietary assessment methods. The objectivity of biomarkers makes reliance on subjects’ memory or cooperativeness less important (Consensus Group for Biomarkers in Cancer Chemoprevention, 2001). The use of randomized controlled feeding trials is one method for validating dietary assessments. Controlled feeding studies in healthy humans have been used to establish quantitative requirements and confirm functional levels of nutrients. These studies rely on small sample sizes with intensive control, including restriction of calories or feeding of specific nutrients with determination of biological availability in specific tissues (Lampe, 2004). Smallscale intensive feeding studies can be enormously useful in describing hypothesized mechanisms observed in large population-based studies.

Nutritional Biomarkers

An example of validation by feeding trial results is a study that measured the effect of vegetable intake on plasma carotenoid concentrations (Martini et al., 1995). High vegetable diets consisted of a control diet plus either carrots and spinach (carotenoid diet), broccoli and cauliflower (cruciferous diet), or tofu and a textured vegetable protein product (soy diet). The control diet consisted of commonly consumed foods and was essentially carotenoid free. Participants consumed each of the experimental diets for 9 days, with at least a 10-day washout period between diets, and were instructed to consume no other foods or beverages. Carotenoid intakes of all diets were calculated using the updated carotenoid food composition database developed by Mangels et al. (1993). When compared with the control diet, mean plasma concentration for α-carotene, β-carotene, and lutein were 5.2, 3.3, and 2.2 times higher on the carotenoid diet, respectively; plasma lutein concentrations were 2.1 times higher on the cruciferous diet. Plasma concentrations of β-cryptoxanthin and lycopene did not differ among diets. Carotenoids are widespread in vegetables and fruits. These data indicate that plasma carotenoid concentrations may be useful exposure markers for total vegetable and fruit intake in a free-living population and might be useful compliance markers in dietary interventions that emphasize vegetable and fruit intake (Martini et al., 1995). Another example of the application of controlled feeding studies involves a comprehensive investigation that compared results of 12 dietary intervention studies using 7-day food records with data from controlled feeding studies for the same individuals (Mertz et al., 1991). Free-living participants were trained by dietitians to keep 7-day food records. Subsequently, participants received diets of conventional foods for 45 days; these diets were adjusted so that each individual maintained his or her body weight. Participants reported intakes using 7-day food records; the estimated energy intake from the food records was compared with the actual intake determined to maintain weight. These comparisons found that 81% of the total subject population reported usual energy intakes that were ~700 kcal below the intake subsequently determined to maintain body weight; 8% reported a higher intake (by ~400 kcal); and 11% reported and calculated intakes that were within 100 kcal of each other. The mean difference between recorded and determined intakes was equally underreported (by ~18%) for both men and women; age had no effect on reporting (Mertz et al., 1991). Use of the doubly labeled water technique, a validated reference method that permits a precise measure of energy expenditure in free-living populations, confirmed the aforementioned finding that subjects recording diet records may underestimate their usual intake substantially (Black et al., 1993; Hebert et al., 1995; Martin et al., 1996; Sawaya et al., 1996). A review of studies in randomly recruited men and

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women that compared dietary energy intake, as assessed from weighed diet records, with total energy expenditure, as measured by doubly labeled water, reported that energy intake was only 82% (men) and 81% (women) of energy expenditure (Black et al., 1993). A comparison of food records with data from the doubly labeled water method found a similar underreporting of ~20% in a subset of women participating in the Canadian Diet and Breast Cancer Prevention Trial (Black et al., 1993). Such analyses suggest that a large proportion of individuals consistently underestimate caloric intake, even those who have received training in keeping accurate food records. The exact reasons for this inaccuracy have not been determined, although the tendency of an individual to convey an image in keeping with social norms (e.g., low-fat vs high-fat diets) and to avoid criticism in a testing situation could bias self-reported dietary intake (Hebert et al., 1995). In any event, caution should be used when interpreting epidemiological survey data and the results of other epidemiological studies that use self-reported dietary assessment instruments to collect food data and estimate nutrient intake, even those that use multiple-day food records. Other validation studies using doubly labeled water found that both men and women underreported energy and protein intakes on 24-hour recalls and FFQs (Subar et al., 2003). In a partnered publication, attenuation of relative risk estimates by FFQs was lessened by adjusting for energy intake using nutrient density or nutrient residuals (Kipnis et al., 2003), suggesting that caution is warranted in interpreting results from epidemiological studies that rely on FFQs to estimate the effects of dietary intake. However, energy adjustment can correct to some degree the imprecision in estimating the true underlying association between intake and cancer risk. In a commentary on both studies, it was noted that the doubly labeled water technique itself is error prone, suggesting that validation biomarkers carry their own level of imprecision (Willett, 2003). Most importantly doubly labeled water sampling needs to be done over several time points to account for within-subject variation. FFQs are designed to assess usual intake, whereas 24-hour recalls and daily food records are designed to assess proximate and immediate intake. It is assumed that individuals’ food intake varies over time, and that it is important to reliably estimate the effect of foods during the 20–30 year cancer induction period.

Biomarkers Used in Combination with Dietary Assessment Despite ongoing improvements in dietary assessment methods, errors inherent in these tools persist. The search for more objective measures of intake is leading researchers

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to utilize biomarkers that reflect dietary intake in combination with those that predict disease outcome or status. Such biomarkers ideally should (see Pearce et al., 1995; Consensus Group for Biomarkers in Cancer Chemoprevention, 2001)

2002; IARC Working Group on the Evaluation of CancerPreventive Strategies, 2003). Well-designed studies of dietary intake and cancer risk ought to include both dietary recall methods and specific biomarkers of intake.

1. be inexpensive to collect and analyze, 2. be present in small amounts of a biological specimen that can be obtained using a minimally invasive collection method, 3. persist for an extended period and reflect all routes of exposure, 4. be specific and highly predictive of the exposure of interest, 5. be measurable using a sensitive, specific, and reliable assay, and 6. be present in low concentrations in unexposed populations at baseline.

Biomarkers as Measures of Dietary Exposure

Although it is difficult to meet all of these criteria, there are several promising biomarkers of intake. For example, tissue and serum long-chain n-3 and n-6 polyunsaturated fatty acids have been reported to be reflective of dietary intake of fish, n-3, and n-6 fatty acids (Lands, 1995; Marckmann et al., 1995; Andersen et al., 1996; Connor, 1996; Bagga et al., 1997; Kohlmeier, 1997) and may be indicative of risk for breast (Kohlmeier, 1997) and prostate (Godley et al., 1996) cancers. Serum levels of β-carotene have been positively associated with intake of carotenoid-rich fruits and vegetables (Mangels et al., 1993; Campbell et al., 1994; Drewnowski et al., 1997). Several studies also corroborate the use of vitamin E (α-tocopherol) concentrations in serum and adipose tissue as measures of external intake (both dietary and supplemental) of that nutrient (Riemersma et al., 1991; Rimm et al., 1993). A convincing body of evidence similarly suggests a direct relationship between consumption of a variety of soy-based products, lignans, isoflavones, isoflavonoid phytoestrogens, and plasma and urinary concentrations (Adlercreutz et al., 1993; Morton et al., 1994; Hutchins et al., 1995a,b; Kelly et al., 1995; Gross et al., 1996). Wu et al. (2004) showed a direct correlation between self-reported soy isoflavone intake from a FFQ and plasma isoflavone levels drawn from a subset of both cases and controls in a population-based study of breast cancer among Asian American women living in Los Angeles County. These findings suggested that, in this instance, breast cancer cases and controls were reliably able to recall their usual soy intake without selective recall biases (Wu et al., 2004). Although biomarkers appear to be a promising method that could replace food frequency methods, it is important to understand that not all foods have biomarkers of intake. Moreover, several studies have shown a weak association between dietary intake and biological markers of intake (Polsinelli et al., 1998; Crews et al., 2001; El-Sohemy et al.,

In instances in which specific biomarkers of intake exist such as serum lycopene level as a marker of tomato product intake, accuracy of dietary exposure measurement can be improved by use of biomarkers. Results of epidemiological studies of fruit, vegetables, and breast cancer risk have been inconsistent and serve as a good example for how biomarkers can potentially increase the accuracy of dietary exposure measurement. Fruits and vegetables contain numerous compounds that have demonstrated anticarcinogenic effects, including carotenoids (Sato et al., 2002), flavonoids (Le, 2002), and isothiocyanates (ITCs) (Fowke et al., 2003). Self-reported dietary intake results from population-based case-control studies of breast cancer all demonstrate a protective association at the highest category of fruit and vegetable consumption (Hirose et al., 2003; Malin et al., 2003; Shannon et al., 2003), whereas eight prospective cohort studies, when summarized together, report a null association (SmithWarner et al., 2001). Most epidemiological studies rely on FFQs to measure dietary intake. The increased variability introduced by self-reported intake has been suggested by several reviews (WCRF, 1997; Smith-Warner et al., 2001; Riboli and Norat, 2003) as one of the reasons for inconsistent results of fruits and vegetables and breast cancer risk observed across studies. Inverse associations observed for biomarkers of fruit and vegetable intake have strengthened biological evidence for fruit and vegetable consumption and reduction of breast cancer risk. Two nested case-control studies found protective associations between serum biomarkers of carotenoids and breast cancer risk (Toniolo et al., 2001; Sato et al., 2002). A protective association was observed for urinary ITCs and breast cancer risk in a case-control study of Shanghai Chinese (Fowke et al., 2003). Dietary intake of soy (flavonoid family) that was later validated by plasma isoflavone was found to be protective for breast cancer risk in a case-control study of Asian Americans conducted in Los Angeles County (Wu et al., 2002, 2004). Protective associations between biomarkers of fruit and vegetable intake and other diseases such as lung cancer have also been found. Observational studies have consistently shown that elevated intake of vegetables reduces the risk of lung cancer (Ziegler et al., 1984, 1986, 1992; Fontham et al., 1988; Le et al., 1989; Dorgan et al., 1993; AlphaTocopherol, 1994; Mayne et al., 1994; Omenn et al., 1996; Speizer et al., 1999). A report of fruit and vegetable consumption in two prospective cohorts found the strongest pro-

References

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SUMMARY As research of diet and cancer moves forward, it is clear that no one method of dietary assessment is ideal. The type of nutrients under investigation, the study design, the study hypothesis, and cancer site under investigation will help determine the optimal assessment instrument and whether biomarkers of intake should be included. If study design permits, use of biomarkers and dietary assessment together appears to be the most accurate and comprehensive method for measurement of diet in relationship to cancer risk.

Acknowledgment This research was supported by the National Cancer Institute (1 P01 CA 42710), California Breast Cancer Research Program (9PB-0117), and the UCLA Center for Human Nutrition.

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20 Prostate Cancer HOWARD PARNES, ASHRAFUL HOQUE, DEMETRIUS ALBANES, PHILIP TAYLOR, AND SCOTT LIPPMAN

INTRODUCTION

et al., 1997). Accordingly, some screening guidelines recommend that African American men begin annual PSA and digital rectal examination (DRE) screening at age 45 years rather than at age 50 years. The risk for Hispanic men is not as clear. Surveillance, epidemiology, and end results (SEER) data from 11 geographic areas in 1992–1996 show a lower incidence of prostate cancers among Hispanic men compared with white men (Canto and Chu, 2000). Results from the Prostate Cancer Outcomes Study, a population-based longitudinal study initiated in 1994 by the National Cancer Institute (NCI) using SEER data from six cancer registries, demonstrated that Hispanic men were more likely than white men to present with clinically advanced stage disease (Hoffman et al., 2001), suggesting that Hispanic men might be at greater risk for more advanced disease. However, the difference between the groups was no longer significant after adjustment for socioeconomic factors. Studies of other ethnic groups have also shown that environmental factors can affect risk profiles: One study found that duration of residence in North America independently increased prostate cancer incidence in Asian-born men living in the United States and Canada (Whittemore et al., 1995), while a second found that mortality from prostate cancer in Japanese-born men living in the United States tends to match the higher rates seen in the overall U.S. population rather than the lower rates seen in the overall Japanese population (Locke and King, 1980). Although the precise mechanisms remain unclear, these data clearly indicate that the effects of diet, lifestyle, and other factors on the development of prostate cancer cannot be overlooked. The third primary risk factor, family history, has also been shown to affect the risk of prostate cancer development.

Prostate cancer is the most common noncutaneous malignancy in U.S. men and remains the second leading cause of cancer death despite advances in screening, early detection, and treatment (Jemal et al., 2005). Prostate cancer represents 33% of all newly diagnosed cancers, with 230,000 new cases and 30,000 deaths expected in 2004 (Jemal et al., 2004). Although most newly diagnosed patients present with nonmetastatic disease, the primary treatments available such as radical prostatectomy and radiation therapy continue to have a significant 30–50% failure rate (Trapasso et al., 1994; Khuntia et al., 2004). Further, treatment of localized disease may adversely affect quality of life and there are no known cures for advanced epithelial cancers, regardless of primary site. The probability of developing prostate cancer increases with age, such that only 1 in nearly 13,000 men younger than 40 years will be diagnosed with prostate cancer, versus 1 in 44 in men 40–59 years of age, 1 in 7 in men 60–79 years of age, and finally 1 in 6 in men older than 70 years. Accordingly, >70% of prostate cancers are diagnosed in men older than 65 years; it is the third leading cause of cancer death in men aged 60–79 years and the second leading cause of cancer death in men 80 years or older (Jemal et al., 2004). Race or ethnicity as a predictor for prostate cancer is most clearly demonstrated in African American men. The incidence of prostate cancer in African American men is ~1.5 times higher than among white men and is 2.7 times higher than among Asian/Pacific Islander men (U.S. Cancer Statistics Working Group, 2003). In addition, a higher prevalence of more aggressive disease and higher prostate-specific antigen (PSA) levels at diagnosis have been noted in African American men versus white men (Moul et al., 1995; Presti


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Hereditary prostate cancer that is attributable to highpenetrance gene mutations and is reflected in obvious family clustering accounts for ~5%–10% of cases in the entire population and for up to 40% in men younger than 55 years (Hsieh and Albertsen, 2003). Data from a population-based case–control study of 563 men younger than 70 years with prostate cancer and 703 age-matched controls with no history of disease indicate that the risk of prostate cancer varies depending on the number of first-degree relatives affected and on the relationship of the affected family members to the case (Lesko et al., 1996). Men with a single relative with a history of prostate cancer had a 2.2-fold risk of cancer, whereas those with two or more relatives with a history of prostate cancer had a 3.9-fold risk of cancer; the risk was higher if the brother had prostate cancer than if the father had prostate cancer (Lesko et al., 1996). Of note, early age at onset of prostate cancer in the affected family members increased the risk, with the highest risk seen in those with family members diagnosed at younger than 60 years (Lesko et al., 1996). Because of this increased risk, the American Cancer Society guidelines recommend that men with a strong family history of prostate cancer initiate regular screening 5 years earlier than the general population, at age 45 years (Smith et al., 2004). As with other epithelial cell cancers, somatic genetic alterations underlie prostate cancer development. A wide variety of alterations have been identified in patients with prostate cancer, but the heterogeneity of these mutations has made study of the genetic underpinnings of prostate cancer challenging. In addition, genetic alterations have been shown to accumulate over time, suggesting that environmental factors not only affect risk profiles and disease incidence rates but can also induce further somatic genetic abnormalities (DeMarzo et al., 2004). The link between prostate cancer and gene–nutrient interaction remains unconfirmed (see discussion later in this chapter), but data clearly suggest that both genetic and nutritional factors play a role in prostate cancer development and progression. Although epidemiological studies strongly suggest that environmental factors contribute to prostate cancer risk, the major risk factors for this disease (age, race, and family history) are not modifiable. Thus, efforts directed at prostate cancer prevention would appear to hold the greatest promise for reducing death and suffering from this disease in the future. The study of finasteride, a drug that inhibits 5αreductase, while demonstrating an overall beneficial effect, resulted in significant side effects both positive and negative. There are observations suggesting that obesity, a Western dietary pattern, and certain types of fat in the diet may influence prostate cancer incidence and mortality. Nutrition researchers have focused on a number of individual vitamins and a number of antioxidant nutrients in an attempt to identify specific dietary elements that can be used to delay

disease development and/or progression. The most work in this area has been conducted with tomato-based products and lycopene, vitamin E, selenium, and vitamin D. The NCIfunded Selenium and Vitamin E Cancer Prevention Trial (SELECT) is the largest trial ever launched in prostate cancer chemoprevention and will provide answers related to the impact of selenium and vitamin E in primary prevention. Future research on gene–nutrient interaction using specific transgenic animal models and translational research promise to advance the field of prostate cancer prevention through nutrition.

INTERNATIONAL EPIDEMIOLOGICAL OBSERVATIONS Although latent or clinically insignificant prostate cancer occurs at equal rates in autopsy studies among men in Asia and the United States (~30% of men older than 50 years), the incidence of clinically significant prostate cancer is 15fold higher in the United States than in Asian countries (Muir et al., 1991). Chinese and Japanese men who immigrate to the United States have a higher incidence of and mortality from prostate cancer than Chinese and Japanese men in their native country (Shimizu et al., 1991; Whittemore et al., 1995). The incidence of prostate cancer in Japan has also been increasing at a time when Western diets and lifestyles are being adopted into that country (Wynder et al., 1991). These data suggest that a significant proportion of prostate cancers may be caused by and, conversely, prevented by changes in the environment. Substantial data suggest that obesity, secondary to dietary patterns and the sedentary lifestyle in Western developed countries, may play an important role in the development, progression, and mortality from prostate cancer.

PROSTATE ANATOMY, PHYSIOLOGY, AND PATHOGENESIS OF PROSTATE CANCER The prostate is a walnut-sized gland located in front of the rectum and underneath the urinary bladder. It contains gland cells that produce some of the seminal fluid, which protects and nourishes sperm cells in semen. Just behind the prostate gland are the seminal vesicles that produce most of the fluid for semen. The prostate surrounds the first part of the urethra, the tube that carries urine from the bladder and semen out of the body through the penis. Male hormones stimulate the prostate gland to develop in the fetus. The prostate continues to grow as a man reaches adulthood and is maintained after it reaches normal size as long as male hormones are produced. If male hormone levels are low, the prostate gland will not fully develop. In older men, the part

Hormones, Obesity, and Prostate Cancer

of the prostate around the urethra often continues to grow, a condition called benign prostatic hypertrophy or benign prostatic hyperplasia. This can cause problems with urinating. Prostate cancer, however, develops in the dorsal prostate epithelium. Pathological and molecular biology studies (Nelson et al., 2004) have led to the hypothesis that chronic or recurrent prostate inflammation may initiate and promote prostate cancer development. The case for prostate inflammation as a cause of prostate cancer is compelling. Epidemiological data have correlated prostatitis and sexually transmitted infections with increased prostate cancer risk and intake of anti-inflammatory drugs and antioxidants with decreased prostate cancer risk.

GENE–NUTRIENT INTERACTION IN PROSTATE CANCER Genetic studies have identified susceptibility genes for familial prostate cancer, which encode an interferoninducible ribonuclease and subunits of the macrophage scavenger receptor. Somatic silencing of a glutathione Stransferase (GSTP1), capable of defending against oxidant cell and genome damage, has been found in almost all prostate cancer cases. Proliferative inflammatory atrophy (PIA) lesions are found adjacent to prostatic intraepithelial neoplasia and carcinoma in situ. These PIA lesions contain activated inflammatory cells, and proliferating epithelial cells appear likely to be precursors to prostatic intraepithelial neoplasia lesions and prostatic carcinomas. Emerging hints that prostate inflammation may contribute to prostatic carcinogenesis provide additional research directions for examining the effects of diet and exercise, altering dietary patterns, or adding antioxidant nutrients to the diet.

Nutritional Factors Prostate cancer appears to be associated with certain nutritional risk factors on the basis of evidence from epidemiological data (variation of prostate cancer incidence according to various regions of the world, modification of these incidences in relation to migratory flows of certain populations) and eating habit studies. These findings are the basis for a number of studies designed to determine the ideal diet to prevent and, if possible, contribute to, the treatment of prostate cancer. Obesity, fat intake, type of fat, vitamins, trace elements, and antioxidants (lycopene, vitamin E) may all constitute nutritional factors involved in prostatic carcinogenesis. However, although some populations appear to be more effectively protected than others by their cultural consumption of particular substances, the link with efficacy, in terms of prevention, of a diet based on this substance administered to another population has yet to be demon-

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strated. Although the ideal diet is unknown, some of these ingredients appear to be good candidates.

Obesity and Prostate Cancer The association of obesity with prostate cancer is somewhat controversial, most likely due to misassignment of body mass index (BMI) and prostate cancer. In one study among men in China, it was found that men in the highest quartile of waist-to-hip ratio (WHR) had an almost threefold increased risk for developing prostate cancer, suggesting a relationship to metabolic syndrome and insulin resistance, as discussed later in this chapter (Hsing et al., 2000). Although the relationship between obesity and prostate cancer risk is unclear, the relationship between obesity and progression and mortality from prostate cancer is well established (Snowdon et al., 1984; Andersson et al., 1996; Rodriguez et al., 2001, 2003). One the basis of this evidence, obese men are more likely to have higher grade disease, and obesity is likely to be an independent predictor of prostate cancer recurrence following radical prostatectomy. In 1959 and 1982, the American Cancer Society enrolled a cohort of patients for longitudinal studies on cancer, known as the Cancer Prevention Study (CPS) I and II, respectively. Men were then followed for 13 years in CPSI and 14 years in CPS-II. Together these studies followed 816,268 men, during which time there were 5212 prostate cancer deaths. Both CPS-I and CPS-II reported that obese men (BMI > 30 kg/m2) were significantly more likely to die from prostate cancer with a 27% increased risk of prostate cancer death from CPS-I and a 21% increased risk of death from CPS-II (Rodriguez et al., 2005). More details regarding CPS-II were published, which showed that severely obese men (BMI > 35 kg/m2) were at even greater risk of dying from prostate cancer, with a 34% higher risk of prostate cancer death relative to normal weight men (Abu-Abid et al., 2002). The data linking obesity with prostate cancer progression and mortality support the urgent need for more research in this area.

HORMONES, OBESITY, AND PROSTATE CANCER Studies regarding obesity and prostate cancer are complicated by the fact that obesity is associated not only with excess body fat, but also with altered serum levels of numerous hormones including testosterone, estrogen, insulin, insulin-like growth factor-1 (IGF-1), and leptin, all of which have, to some degree, been linked to prostate cancer. Androgens act in the prostate by binding and activating the androgen receptor, resulting in enhanced transcription of genes involved in cellular proliferation such as the

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mitogenic growth factors epidermal growth factor (EGF) and IGF-1 (Ho et al., 2004). Studies in animal models of prostate cancer further support the role for androgens in the development of prostate cancer (McCormick et al., 1998). Of interest, obese men are known to have decreased free testosterone levels (Vermeulen, 1996; Niskanen et al., 2004). As discussed later, this fact may explain why obese men have higher-grade prostate cancers. An important advance in the prevention of prostate cancer was the observation from the Prostate Cancer Prevention Trial (PCPT) that finasteride, an inhibitor of 5αreductase (the enzyme that converts testosterone to the major intraprostatic androgen dihydrotestosterone), reduced the 7-year period prevalence of prostate cancer by 24.8% compared with a placebo control group (18.4% vs 24.4%, p < .001). However, this highly beneficial effect was accompanied by a small but significant increase in the period prevalence of high-grade disease among the men randomized to receive finasteride (6.4% vs 5.1%, p = .005). Although it is not clear whether this represents a true increase in high-grade disease or is a consequence of detection bias (finasteride reduces prostate gland size by 25%, thus potentially enhancing detection of high-grade disease), there is clearly a need for additional approaches to prevention of this common and often fatal disease. The men exposed to finasteride had more poorly differentiated tumors than the control group, possibly because of lower prostate dihydrotestosterone levels (Thompson et al., 2003). Data from retrospective studies suggest that testosterone may exert a differentiating effect on prostate cancer and decreased serum testosterone levels have been associated with more advanced and poorly differentiated tumors at presentation (Massengill et al., 2003; Schatzl et al., 2003). Thus, the lower free testosterone levels found in obese men may predispose them to developing more poorly differentiated advanced prostate cancers and explain the higher mortality of prostate cancer among obese men (Freedland and Aronson, 2005). Obese men are known to have increased serum estradiol levels because of peripheral conversion in adipocytes of testosterone to estradiol by aromatase. In fact, 80% of circulating estradiol in men is derived from the peripheral conversion of androgens to estrogens in fat tissue, whereas only 20% is due to intratesticular conversion of testosterone to estradiol. The exact role of estrogen in prostate cancer development and progression is unclear, but animal and experimental studies suggest that elevated estradiol levels may play an important role in testosterone-induced carcinogenesis. Studies in mice with genetically altered aromatase or estrogen receptor expression found that estradiol combined with testosterone plays an important role in regulating proliferation and apoptosis of prostate cells (McPherson et al., 2001). Leav et al. (1989) demonstrated that testosterone needed to be combined with estrogen to develop prolifera-

tive lesions in mouse prostate tissue. In addition, other investigators demonstrated that chronic combined administration of testosterone with estrogens to the Noble rat model resulted in a high incidence of prostate tumors after 52 weeks of treatment, with precancerous lesions seen as early as 16 weeks (Drago, 1984; Bosland et al., 1995). On the basis of these experimental data, it is possible that elevated levels of estradiol in obese men may enhance the growth-promoting effect of testosterone on prostate cancer. Of importance, a short-term low-fat diet and exercise weight-loss program has previously been shown to reduce serum estradiol in men by 48% (Rosenthal et al., 1985).

Metabolic Syndrome and the IGF Axis Obesity is associated with insulin resistance and non–insulin-dependent diabetes mellitus. Increasing evidence suggests that adipose tissue not only stores excess fat but also can function as an endocrine organ. Adipocytes produce multiple polypeptide hormones, of which leptin is the best characterized. Metabolic syndrome is associated with increased intraabdominal fat and a genetic predisposition to diabetes, hypertension, and hyperlipidemia. This syndrome was initially described as an aggregation of risk factors for the development of coronary artery disease with insulin resistance and compensatory hyperinsulinemia and elevated levels of fatty acids and adipocyte-derived peptides as pathogenic factors. IGF-1 levels are increased in the metabolic syndrome associated with abdominal obesity in men. IGF-1 is also a potent mitogen for the growth of androgen-responsive and androgen-independent human prostate cancer cell lines (Iwamura et al., 1993; Yu and Rohan, 2000). Epidemiological investigations have found a positive correlation between elevated serum IGF-1 levels and the risk of developing prostate cancer (Mantzoros et al., 1997; Chan et al., 1998; Wolk et al., 1998; Stattin et al., 2000). Tissue levels of IGF-1 appear to be a critically important factor during initiation and progression of prostate cancer (Kaplan et al., 1999). Animal studies have demonstrated that caloric restriction decreases growth of prostate tumors and decreases serum IGF-1 levels (Dunn et al., 1997; Mukherjee et al., 1999). The activity of IGF-1 is modulated by high-affinity IGFbinding proteins (IGF-BPs) (IGF-BPs 1–6) (Ferry et al., 1999). Circulating levels of IGF-BP1 and IGF-BP2 vary in response to nutritional status and in response to changes in energy metabolism (Yu and Rohan, 2000). In the energyrestricted state, circulating levels of IGFBP1 and IGF-BP2 are increased (Thissen et al., 1994; Donaghy and Baxter, 1996). Overeating and obesity, on the other hand, lead to hyperinsulinemia and decreased levels of IGFBP1, although the effect of insulin on serum IGF-BP2 levels in humans is less clear. Research is necessary to clarify the role of energy


balance through decreased caloric intake and increased physical activity on the IGF axis, prostate cancer risk, and progression.

Red Meat and Type of Dietary Fat Epidemiological research has demonstrated that residence in North America increases the risk of prostate cancer incidence in Asian-born men within a single generation (Whittemore et al., 1995). One hypothesis to explain this observation is that a “Western-style” diet (i.e., high in dietary fats and red meat) may be a contributing factor. Cooked meats contain aromatic hydrocarbons, which can promote prostate carcinogenesis, but red meats are also rich sources of dietary fats and may contribute to obesity and hormonal imbalance (see previous discussion) (Freedland et al., 2004). Prospective data from the National Health and Nutrition Examination Survey Epidemiological Follow-up Study (NHANES) cohort of 3779 men followed for 10 years showed that α-linolenic acid, which is present in some vegetable oils and nuts, leafy vegetables, and animal fats, is an independent risk factor for advanced prostate cancer (Giovannucci et al., 1993). Specifically, after adjusting for potentially confounding variables, the relative risk of advanced prostate cancer conferred by α-linolenic acid from meat, dairy, and nonanimal sources was 1.92; the relative risk went up to 2.12 when evaluating the effect of α-linolenic acid on death from prostate cancer (Leitzmann et al., 2004). On the other hand, increased consumption of fish oils, specifically eicosapentaenoic acid and docosahexaenoic acid, was inversely correlated with the risk of prostate cancer and advanced prostate cancer, such that each additional daily intake of 0.5 g of fish oils from food was associated with a 24% decreased risk of metastatic prostate cancer. Data from the same cohort after 4 years of follow-up demonstrated that total fat intake, and particularly animal fat intake, directly correlated with increased risk of advanced prostate cancer. When analyzed by food group, red meat intake had the strongest association, whereas fat from dairy foods showed no association. At 10 years of follow-up, both red meat and dairy foods showed an association with advanced disease, but after controlling for known risk factors such as calcium and α-linolenic acid, much of the risk was attenuated (Michaud et al., 2001). Two other large epidemiological studies found little to no association between the different types of fat and prostate cancer. A review of data from The Netherlands Cohort Study of 58,279 men after 6.3 years of follow-up showed no associations between prostate cancer and intake of total fat, total saturated fatty acids, or total trans-unsaturated fatty acids (Schuurman et al., 1999). Nonsignificant associations were seen with oleic acid and linolenic acid, whereas no associations were seen with eicosapentaenoic acid or docosa-

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hexaenoic acid. Similarly, in a study of 25,708 Norwegian men followed for 15 years, no associations were found between prostate cancer and energy-adjusted intake of total fat, saturated fat, monounsaturated fat, or polyunsaturated fat (Veierod et al., 1997). At this point, it seems probable that dietary fat intake, particularly that from animal sources, does play some role in prostate cancer development. Whether the effect is strictly due to the already identified components or to other components remains to be explored.

Tomato-Based Foods and Lycopene Data from the Health Professionals Follow-Up Study, a prospective cohort study of 51,529 male health professionals aged 40–75 years in 1986, demonstrated that increased consumption of lycopene-rich tomato-based foods conferred a 35% reduction in the risk of prostate cancer and a 54% reduction in the risk of advanced disease on the basis of data culled from a questionnaire at baseline (Giovannucci et al., 1995). Updated results after 6 years showed that the reduction in prostate cancer risk was durable, but decreased to 17%, with increased consumption of lycopene-rich tomato-based products; after 12 years, although the reduction remained significant, it decreased to 16% (Giovannucci et al., 2002). Specifically, following previous research suggesting that the benefits of tomato-based foods result from the increased bioavailability of lycopene and other compounds when the foods are heated (Giovannucci et al., 1995), the researchers noted that increased consumption of tomato sauce was associated with a 44% reduction in the risk of prostate cancer at baseline and remained significant after 12 years of followup, with a 23% reduction noted in men who consumed at least two servings per week (Giovannucci et al., 2002). Of note, after 12 years of follow-up, men who consumed at least two servings of tomato sauce per week demonstrated a 36% lower risk of metastatic disease and a 35% lower risk in locally advanced disease. A prospective analysis of plasma lycopene in patients enrolled in the Physicians’ Health Study, a randomized placebo-controlled trial of aspirin and β-carotene in 22,071 men, demonstrated an ~40% reduction in the risk of prostate cancer in men with the highest levels of plasma lycopene among those assigned to placebo rather than to β-carotene. In contrast, a possible benefit of β-carotene with regard to prostate cancer risk was only evident among men with the lowest lycopene levels. These results suggest that there is a ceiling to the benefit that can be achieved from the carotenoids, and that this maximum benefit can be achieved by either food intake or supplementation (Gann et al., 1999). Tomatoes contain not only lycopene, which was measured in the blood in the aforementioned study, but a mixture of related compounds including phytoene, phytofluene, vitamin C, and vitamin E. Newer studies suggest that these other

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components of the tomato may also have a preventive benefit. Carcinogen and hormone-treated rats fed whole tomato powder showed a significant decrease in the risk of developing prostate cancer, but those fed a crystalline-pure lycopene supplement did not (Boileau et al., 2003). These results reinforce the notion that a family of related compounds is more effective than a single isolated compound. There may well be complex interactions among the multiple elements and compounds found in foods, and because processing of foods can alter their absorption, it might not be possible to achieve the risk reduction seen with foods by supplementation of purified single compounds alone. Supplements based on extraction of whole tomatoes, juice supplements, and other food-based strategies might be necessary for an effective preventive approach to prostate cancer (Gann and Khachik, 2003). Further research into the benefits of supplementation relative to food-based intake will help better define the optimal nutritional preventive strategy.

Vitamin D On the basis of laboratory and epidemiological data, it has been proposed that 1,25-dihydroxyvitamin D3 (1,25[OH]2 D3), the active metabolite of vitamin D, inhibits prostate carcinogenesis, and that by lowering levels of this metabolite, calcium, in turn, minimizes its protective effects (Giovannucci, 1998). Substantial experimental evidence indicates that the hormonal form of vitamin D promotes the differentiation and inhibits the proliferation, invasiveness, and metastasis of human prostatic cancer cells. Vitamin D receptor (VDR), a member of the steroid/thyroid hormone nuclear receptor family, is bound by the steroid hormone 1,25-dihydroxyvitamin D3, which is thought to play a role in the etiology and progression of prostate cancer. Polymorphisms in the VDR gene have been associated with prostate cancer risk. Results from epidemiological studies of vitamin D status and/or VDR polymorphisms and prostate cancer risk have been mixed. John et al. (2005) conducted a population-based case–control study of advanced prostate cancer among men aged 40–79 years from the San Francisco Bay area. Interview data on lifetime sun exposure and other risk factors were collected for 905 non-Hispanic White men (450 cases and 455 controls). Using a reflectometer, constitutive skin pigmentation was measured on the upper underarm (a sunprotected site) and facultative pigmentation on the forehead (a sun-exposed site) and a sun exposure index was calculated from these measurements. Biospecimens were collected for 426 cases and 440 controls. Genotyping was done for VDR polymorphisms in the 5′ regulatory region (Cdx-2), exon 2 (FokI), and the 3′ region (TaqI and BglI). Reduced risk of advanced prostate cancer was associated with high sun exposure determined by reflectometry (odds ratio [OR], 0.51; 95% confidence interval [CI], 0.33–0.80)

and high occupational outdoor activity (OR, 0.73; 95% CI, 0.48–1.11). Significant risk reductions with the highactivity alleles FokI FF or Ff, TaqI tt, and BglI BB genotypes and a nonsignificant reduction with Cdx-2 AG or AA genotype were observed in the presence of high sun exposure, with ORs ranging from 0.46 to 0.67. These findings support the hypothesis that sun exposure and VDR polymorphisms together play important roles in the etiology of prostate cancer most likely via production and subsequent actions of vitamin D.

Vitamin E The tocopherols and tocotrienols encompass vitamin E and are a class of naturally occurring and synthetic lipidsoluble compounds essential for normal mammalian reproduction (Evans, 1922). There are eight forms of vitamin E (four tocopherols and four tocotrienols) with varying levels of biological activity (Brigelius-Flohe et al., 1999). The richest sources of vitamin E in the U.S. diet are vegetable oils and foods made from vegetable oils, such as salad dressings and mayonnaise, with smaller amounts in grains, nuts, and meats (Eitenmiller, 1995). Estimated average daily dietary intake among U.S. women and men is between 8 and 10 mg, respectively (NHANES, 1999–2000), which is substantially lower than the current Dietary Reference Intake of 15 mg recommended for both men and women (Food and Nutrition Board, 2000). The strongest evidence in support of a preventive effect of vitamin E supplementation comes from a large intervention trial conducted in Finland, the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study. This randomized, double-blind, placebo-controlled trial of synthetic all rac-α-tocopheryl acetate (50 mg daily) and β-carotene (20 mg daily) among 29,133 male smokers 50–69 years old suggested that chronic supplementation with vitamin E can reduce prostate cancer incidence and mortality (Heinonen et al., 1998). During the 5–8 years of intervention and followup, there were 246 new cases of and 64 deaths from prostate cancer. Among those assigned to the α-tocopherol (AT) supplementation arm of the trial (n = 14,564), 99 incident prostate cancers were diagnosed, compared with 147 cases among those assigned to the non-AT arm (n = 14,569), representing a statistically significant 32% reduction in prostate cancer incidence (95% CI, 12–47%; p = .002) (Heinonen et al., 1998). The observed preventive effect appeared stronger in clinically evident prostate cancer cases (i.e., stages B–D) for which the incidence was decreased 40% in subjects receiving vitamin E (95% CI, 20–55%). Prostate cancer mortality was 41% lower in the vitamin E group (95% CI, 1–64%); however, vitamin E supplementation did not affect survival time after diagnosis. Although prostate cancer was prespecified only as a secondary endpoint in this trial, these findings suggested a potentially substantial impact of AT in


reducing prostate cancer incidence and mortality, thus offering an important lead that warranted further evaluation. AT may influence the development of cancer through several plausible mechanisms. AT is a potent antioxidant of highly reactive and genotoxic electrophiles (e.g., hydroxyl, superoxide, lipid peroxyl and hydroperoxyl, and nitrogen radicals) that prevents the propagation of free radical damage in biological membranes and decreases mutagenesis and carcinogenesis (Burton and Ingold, 1981). Vitamin E also blocks nitrosamine formation. AT inhibits protein kinase C activity (Boscoboinik et al., 1991a; Tasinato et al., 1995) and the proliferation of smooth muscle cells (Boscoboinik et al., 1991b; Tasinato et al., 1995) and melanoma cells (Ottino and Duncan, 1997), thus possibly affecting tumor growth or aggressiveness. Vitamin E also induces the detoxification enzyme NADPH:quinone reductase in cancer cell lines (Wang and Higuchi, 1995) and inhibits arachidonic acid and prostaglandin metabolism (Rimbach et al., 2002). Vitamin E effects on hormones that can increase cellular oxidative stress and proliferative activity and on cell-mediated immunity have also been reported (Rimbach et al., 2002). Reductions of serum testosterone and androstenedione were observed in the ATBC Study in response to daily supplementation with AT, 50 mg daily, supporting a highly relevant mechanism of action on prostate carcinogenesis in this study (Hartman et al., 2001). The most biologically active form of vitamin E is AT, which also is the predominant form in human tissues, including the prostate (Brigelius-Flohe and Traber, 1999; Freeman et al., 2000). AT in capsules must be esterified for stability, but esters are hydrolyzed in the intestinal lumen and absorption does not vary among different esters (Traber and Wuller, 1993). AT is also available in the pure RRR configuration, but absorption does not differ between pure RRR and all racemic forms of vitamin E, and once dose is adjusted to account for the 50% of all racemic AT that is biologically active, the formulations are biologically equivalent (Food and Nutrition Board, 2000). In vitro and in vivo experimental data suggest that vitamin E may inhibit carcinogenesis by a variety of mechanisms. Vitamin E can inhibit the growth of human cancer cell lines, including prostate, lung, melanoma, oral carcinoma, and breast (Dieber-Rotheneder et al., 1991; Traber, 1997; Traber and Arai, 1999), and animal experiments show prevention of various chemically induced tumors, including hormonally mediated tumors. In addition, vitamin E has been shown to slow the growth of prostate tumors in rats receiving various doses of chemotherapeutic agents. Other trial data supporting the evaluation of vitamin E and AT in cancer prevention include lower colorectal cancer incidence in the AT arm of the ATBC Study (relative risk 0.78, 95% CI, 0.55–1.09) (Albanes et al., 2000), a finding consistent with some prior epidemiological research of vitamin E supplement use (described later in this chapter).

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Additionally, although only a negligible 1% decrease in lung cancer incidence occurred overall during this study within the vitamin E group (compared with the non–vitamin E group), a supplementation duration analysis showed an increased reduction of lung cancer with longer trial participation (e.g., >3 years), culminating in a 10–15% reduction in risk between the fifth and eighth years in the AT group (Albanes et al., 1996). Further support of the potential effectiveness of vitamin E supplementation in nonprostatic cancers was reported from the first Nutrition Intervention Trial conducted in China. This trial tested a combination of vitamin E, β-carotene, and selenium and found a 21% lower mortality from stomach cancer and a 13% reduction in total cancer mortality in a population of >29,000 individuals, ages 40–69 years, who were at high risk of developing esophageal/gastric cardia cancer (Blot et al., 1993). The study did not specifically examine prostate cancer, and the specific efficacy of vitamin E or any other compound (or combination) could not be determined because the agents were not tested separately. Observational data also suggest that vitamin E may also protect against lung (Comstock et al., 1992; Shibata et al., 1992; Knekt et al., 1991; Yong et al., 1997) and colorectal cancer (Longnecker et al., 1992). In contrast to lung cancer, no association with colorectal cancer risk has been seen for dietary intake levels of vitamin E, although a lower risk of colorectal cancer has been observed among those taking vitamin E supplements at >200 mg daily (Bostick et al., 1993; Ferraroni et al., 1994). Nevertheless, such observational data should be considered hypothesis generating, rather than definitive, given the possibility of measurement error (particularly for dietary vitamin E intake), selection and recall biases, and unrecognized confounding factors. Lung cancer studies have reported lower prediagnostic serum vitamin E (AT) levels among cancer cases compared with noncases (Knekt et al., 1991; Comstock et al., 1992; Shibata, 1992; Yong et al., 1997) or a weak protective association for supplemental vitamin E (Shibata et al., 1992). Five prospective studies showed an inverse association between serum AT and colorectal cancer, with a pooled estimate of a 40% lower relative risk for the highest compared with the lowest categories (Longnecker et al., 1992). Although no association with colorectal cancer risk has been seen for dietary intake levels of vitamin E, a lower risk was observed for vitamin E supplement use (i.e., ≥200 IU daily) (Bostick et al., 1993; Ferraroni et al., 1994). Inherent to such observational studies are problems with measurement error (particularly for dietary vitamin E intake), selection and recall biases, and confounding factors. The epidemiological data supporting a beneficial association between vitamin E and prostate and other cancers are based on assessments of the relationship between cancer development and estimated dietary and supplemental vitamin E intake and AT blood levels. Some prospective

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studies reported lower serum or plasma vitamin E concentrations among prostate cancer cases years before diagnosis, compared with these concentrations among noncases, although dose–response relationships were not observed (Willett et al., 1984; Knekt et al., 1988; Hsing et al., 1990; Eichholzer et al., 1999; Helzlsouer et al., 2000). One study of 3000 subjects over a 17-year follow-up found that lowserum AT concentrations were associated with a higher prostate cancer risk (Eichholzer, 1999), and a trial-based cohort analysis suggested a protective effect from prostate cancer for total vitamin E intake among men who received AT supplementation (Hartman et al., 1998). A study of 117 prostate cancer cases and 223 matched control subjects conducted in Washington county, Maryland, showed a stronger inverse association for γ-tocopherol then for α-tocopherol (Helzlsouer et al., 2000). However, an analysis of 100 cases and 200 matched controls from the ATBC Study cohort revealed a slightly stronger inverse association for αtocopherol than for γ-tocopherol (Weinstein et al., 2005). A long history of research supports the safety AT supplementation in humans (NIH/ODS, 2005). The tolerable upper limit of vitamin E intake has been set at 1000 mg/day, and the dose required to kill 50% of test animals (LD50) for AT acetate is in excess of 2000 mg/kg for rats, mice, and rabbits (Food and Nutrition Board, 2000). No significant adverse effects were reported, with the exception of elevated risk of hemorrhagic stroke among male smokers in the ATBC Study (Alpha Tocopherol Beta Carotene CPSG, 1994), a finding that was subsequently isolated to the subgroup of men with uncontrolled hypertension (Leppala et al., 2000). Thus, poorly controlled hypertension is an exclusion criterion for the SELECT trial (see later discussion), as is the use of anticoagulant medication, with the exception of cardioprotective doses of aspirin.

Selenium Selenium is an essential trace element whose principal dietary sources include seafood, meats, and grain products (El-Bayoumy, 1991). Most dietary selenium is in organic form, primarily selenomethionine and selenocysteine (Combs, 1984). Typical dietary intake of selenium in the United States is in the range of 80–165 µg/day (Schrauzer, 2000), and the Recommended Dietary Allowance is 55 µg/day for adult North Americans (Food and Nutrition Board, 2000). The safe upper limit of intake is considered 400 µg/day (Food and Nutrition Board, 2000). There are extensive in vitro animal experimental and human epidemiological data supporting an association of selenium with reduced prostate cancer risk. In vitro, selenium has antiproliferative and proapoptotic effects (Redman et al., 1997, 1998; Menter, 2000), can alter carcinogen metabolism (Shimada et al., 1997), and can influence several aspects of immune function (Taylor, 1995; Field et al., 2002). More than 60 published animal studies in 10 organ

systems have demonstrated that selenium inhibits tumor formation in chemical, viral, and transplantable tumor models (Medina and Morrison, 1988; Combs, 1991; El-Bayoumy, 1991; Shibata et al., 1992; NCI, 1996). Observational studies in humans, conducted throughout the United States and in many other countries, have also generally found inverse associations between selenium status and cancer risk (Combs, 1997, 2001). Some of this research, however, is weak. Any study based on dietary selenium intake rather than serum or toenail selenium concentration is suspect because selenium intake cannot be measured using standard dietary assessment instruments. Further, case-control studies of serum selenium are difficult to interpret because sequestration of selenium by tumor cells and poor nutritional status may contribute to reduced selenium levels among patients with advanced cancer (Willett, 1986). Thus, the best epidemiological evidence on selenium and prostate cancer is currently from nine prospective cohort studies (Willett et al., 1983; Coates et al., 1988; Knekt et al., 1990; Yoshizawa et al., 1998; Helzlsouer et al., 2000; Nomura et al., 2000; Brooks et al., 2001; Goodman et al., 2001; Steiner et al., 2002). Early studies, published from 1983 to 1991, hinted at a benefit for higher selenium levels but had such small sample sizes (cases from all four studies totaled only 81) that they were largely uninformative. Subsequently, five larger studies with 53–249 cases each (and a total of 834 cases) have provided substantially more information. Although one study reported no association between plasma selenium concentration and prostate cancer risk (Brooks et al., 2002), four studies reported selenium levels that were 3–15% lower in cases than controls, as well as relative risk reductions of 50–76% contrasting the high versus low quantile of serum or toenail selenium. The most compelling evidence for selenium as a potential chemopreventive agent comes from the secondary findings of the Nutritional Prevention of Cancer Study (Clark et al., 1996). In this double-blind clinical trial completed between 1983 and 1991, 1312 patients with prior basal or squamous cell skin cancer were randomized to receive 200 µg/day of selenium as high-selenium yeast versus placebo, with a mean time on study of 4.5 years. The primary endpoint of the trial was the incidence of nonmelanoma skin cancer, and secondary endpoints, added in 1990, included all-cause mortality, total cancer mortality, total cancer incidence, and the incidences of lung, prostate, and colon cancer. Although the incidence of skin cancer was modestly increased among participants randomized to the selenium arm (hazard ratio [HR] = 1.17; 95% CI, 1.02–1.34), primarily because of an elevated risk of squamous cell cancer (HR = 1.25; 95% CI, 1.03–1.51) (Duffield-Lillico et al., 2003), those who received selenium through 1993 had a significant reduction in total (HR = 0.75; 95% CI, 0.58–0.97) and prostate cancer incidence (HR = 0.48; 95% CI, 0.28–0.80) (Duffield-Lillico et al., 2002). The magnitude of reduced prostate cancer risk


depended on baseline selenium levels and was largest among men with low baseline selenium and absent among those with high baseline selenium. Two other large randomized trials have reported findings supporting a protective role of selenium supplementation, but in both, selenium was given in combination with other nutrients, thus precluding evaluation of selenium as an independent agent, and the number of prostate cancers was too small to be separately evaluated (Blot et al., 1993; Li et al., 1993; Mark et al., 2000). There are a variety of potential mechanisms whereby selenium could reduce cancer risk. The best defined biological role for selenium is as an essential constituent of selenoenzymes such as the glutathione peroxidases, where it is incorporated into the active site of the enzyme as the substituted amino acid selenocysteine. This may not account for the antitumor effects of selenium because glutathione peroxidase activity is maximal at physiological selenium levels and the function of this enzyme does not correlate well with tissue selenium levels at cancer-inhibitory doses (NCI, 1996). However, because the glutathione peroxidases are highly polymorphic, selenium supplements may affect enzyme activity in subgroups of men with genetic variants in selenoproteins. Selenium likely has other activity independent of its incorporation into proteins. Both selenite and selenomethionine are metabolized via different pathways to selenide, which is then converted to the highly antitumorigenic monomethylated form, methylselenol. Ip et al. (2000) showed that the antitumor activity of selenocompounds is directly related to their ability to generate methylselenol. In December 1998, a panel of nationally recognized selenium experts was consulted to provide advice on the dose and form of selenium to be used in SELECT. The panel members were unanimous in recommending 200 µg/day as the optimal dose based on the available efficacy and safety data. However, identifying the optimal formulation was more controversial. Although there was considerable interest in several of the newer selenium compounds due to their rapid conversion to the putative active moiety methylselenol, the absence of phase I data in humans with these forms of selenium effectively excluded them from further consideration. Consideration was also given to the inorganic forms, selenite and selenate; however, this option was rejected because of concerns regarding stability, bioavailability, and potential for genotoxicity. Thus, the choice was essentially limited to the two available organic forms of selenium, selenomethionine and high-selenium yeast. Most panel members favored selenomethionine; however, the Data and Safety Monitoring Committee asked that this recommendation be reconsidered given the fact that the strongest data in favor of a prostate cancer preventive effect came from a study (the NPC trial) using the yeast formulation. Therefore, the preliminary decision to use selenomethionine was reexamined by an expanded panel of selenium experts, and reaffirmed in July and October 2000.

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The SELECT Trial SELECT is a randomized, placebo-controlled, phase III trial of selenium (200 µg/day of selenomethionine) and/or vitamin E (400 IU/day of dl-α-tocopheryl acetate) for prostate cancer prevention (Lippman et al., 2005). Eligibility requirements included the following: age older than 55 years (50 years or older for African Americans due to the earlier age at onset in this group), nonsuspicious DRE results, and a PSA level of ≤4 ng/ml within 1 year of entry. Participants were randomized to one of four study arms: selenium + vitamin E placebo, vitamin E + selenium placebo, selenium + vitamin E, or two placebos and are expected to remain on their assigned treatment for 7–12 years. The primary study endpoint is the clinical incidence of prostate cancer detected by routine clinical practice. Between July 25, 2001, and June 24, 2004, 35,534 men from >400 sites in the United States, Puerto Rico, and Canada were accrued to this trial, making SELECT the largest cancer chemoprevention study ever conducted. A panel of experts considered selenomethionine a wellcharacterized chemically discrete compound that would facilitate future in vitro mechanistic and pharmacokinetic analyses. In addition, selenomethionine had demonstrated activity in cell lines (Redman et al., 1997, 1998) and some experimental animal models (Yan et al., 1999). The relative inefficiency with which selenomethionine is metabolized to methylselenol was considered unlikely to be consequential in well-nourished individuals who received prolonged supplementation. In contrast, the primary advantage of selenium yeast is that it would provide the most direct comparison with the Clark trial, which was the major impetus for studying selenium in a large-scale, phase III clinical trial. In addition, selenium yeast contains unidentified selenium metabolites that could potentially generate methylselenol directly, leading to an earlier reduction in cancer risk than would be anticipated for selenomethionine. However, the uniformity of the selenium yeast used throughout the Clark trial and the degree of similarity between that yeast product and currently available selenium yeast formulations was unknown. To address these issues, speciation analyses were undertaken to evaluate four representative yeast tablet lots from the Clark trial in conjunction with a currently available commercial selenium yeast product. Analysis of the currently available selenium yeast product showed good batch-to-batch uniformity and indicated that 69% of its selenium content was selenomethionine. However, archived sample tablets of the selenium yeast used in the Clark trial analyzed by the same procedure had substantial sample-to-sample differences in both the presence and the relative levels of specific organoselenium compounds, some of which seemed to involve oxidation products of selenomethionine. These differences were not readily attributable to

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the time of manufacture or duration of storage. In some cases, released selenomethionine content was as low as 7% and substantial percentages of other organoselenium compounds were found, as yet incompletely characterized (Block et al., 2004; Pter Uden, personal communication). The panel also considered data produced by Menter et al. (2000) on the effects of both inorganic (sodium selenite) and organic (selenomethionine) selenium on monolayer and anchorage-independent growth of normal prostate and prostate cancer cell lines. Differential dose-dependent growth inhibition, apoptosis, and cell cycle arrest were observed in prostate cancer cells versus normal cells. These changes were most pronounced in androgen-sensitive cells and were observed with in vitro concentrations of selenium within the range achieved with pharmacological selenium use. The in vitro selenium results have subsequently been confirmed by other investigators (Venkateswaran et al., 2002; Wan et al., 2003). Given these considerations, the panel recommended selenomethionine over selenium yeast for use in the SELECT by a vote of 6 to 1. This decision was supported by data in men randomized to selenomethionine or placebo during the 4–6 week preprostatectomy period, indicating significant accumulation of selenium in the prostate gland and preferential accumulation in the prostate gland versus seminal vesicles (Sabichi et al., 2002). The formulation of vitamin E being used in SELECT, all rac-α-tocopheryl acetate, encompasses the eight possible stereoisomers (RRR, RRS, RSR . . . SSS) of the AT molecule resulting from methyl group positioning at the 2′, 4′, and 8′ asymmetrical carbon atoms of the chromanol ring. Naturally occurring AT is exclusively in the RRR configuration, and only the four 2-R sterisomers in synthetic AT are incorporated into lipoproteins and thus considered biologically active. This form was used in the ATBC study and is also the most common formulation used in dietary supplements. There are alternative formulations of AT supplements, but for various reasons, these were not relevant when designing SELECT. A report from the Heart Outcomes Prevention Evaluation (HOPE) trial, a randomized, double-blind, placebocontrolled trial of AT, 400 mg a day, in patients at high cardiac risk, revealed no significant increase in the primary endpoint of major cardiovascular events (Lonn et al., 2005). However, an increase in heart failure (RR = 1.13; 95% CI, 1.01–1.26; p = .03) and hospitalizations for heart failure (RR = 1.21; 95% CI, 1.00–1.47; p = .045) among participants randomized to the AT arm was noted on secondary analyses (Lonn et al., 2005). A meta-analysis of 19 randomized controlled studies of vitamin E, reported shortly after SELECT had reached its accrual goal, suggested that high-dose vitamin E, up to 2000 mg/day, may be associated with an increase in all-cause mortality (Miller et al., 2005). These new data on vitamin E were submitted to the SELECT Data

and Safety Monitoring Committee (as had been the increased risk of nonmelanoma skin cancer observed on the selenium arm of the NPC trial, discussed earlier [DuffieldLillico et al., 2003]), and the appropriate measures have been taken to inform participants of the potential risks and to ensure their safety through careful monitoring of adverse events. Although there was general support among the SELECT investigators for using the all rac-α-tocopheryl formulation of vitamin E, the optimal choice of vitamin E dosage was less clear. Some investigators supported using 50 mg to specifically confirm the results of the ATBC, which also used 50 mg. This low dose was rejected, however, in deference to AT’s favorable safety profile and the possibility that higher doses might be required to obtain other health benefits, most notably protection against cardiac disease (Rimm et al., 1993), Alzheimer’s disease (Sano et al., 1997), and macular degeneration (Age-Related Eye Disease Research Group, 2001). In addition, data from the ATBC showed greater benefit for AT supplementation with regard to the reduction in prostate and lung cancer incidence among subjects with higher baseline and thus, overall, AT levels. Secondary Endpoints Prespecified secondary endpoints were a major design consideration of SELECT. A trial of this size allows the inclusion of numerous, hypothesis-generating secondary endpoints. The effects of selenium and/or vitamin E on lung cancer incidence and survival and colon cancer incidence and survival are especially prominent secondary endpoints of the trial. Other prespecified endpoints chosen for prospective secondary evaluation are prostate cancer–specific survival, overall survival, all-cancer incidence and survival, and cardiovascular deaths. In addition, several substudies with clearly specified entry criteria and requiring separate informed consent have been nested within the parent trial. These substudies will explore the potential role of selenium and vitamin E, either alone or in combination, for the prevention of Alzheimer’s disease, macular degeneration, chronic obstructive lung disease, and colonic polyps. Translational Ancillary Endpoints SELECT is a unique opportunity not only to evaluate prespecified secondary endpoints but also to incorporate prespecified translational ancillary studies previously unknown to very large-scale phase III trials. The SELECT biorepository of prospectively collected prostate biopsy tissue, white blood cells, red blood cells, and plasma for correlative mechanistic study also is unique to trials of the SELECT’s scale. The trial will permit studies of diet/nutrient levels, molecular epidemiology, other molecular/cellular biomarkers, and quality of life, using these valuable specimens and/or prospectively collected clinical and questionnaire data.


Prospective collection of blood for research is the key to examining genetic/nutrient background, factors that may affect prostate cancer incidence in various intervention groups. Blood studies will include measurement of micronutrients and hormones in plasma, as well as hemoglobin adducts in red blood cells. Particular emphasis will be placed on the examination of genetic polymorphisms in DNA extracted from lymphocytes. This will allow the conduct of nested case-control studies to determine the relative contribution of genetic polymorphisms to the risk of prostate cancer with the ultimate goal of developing molecular risk profiles for prostate cancer in general and for aggressive prostate cancer in particular. SELECT biospecimens will be available for the best future science to identify highest risk men for prevention and screening and to identify novel molecular targets. Nutrition-Related Ancillary Studies A comprehensive set of nutrition-related measures were added to SELECT to support ancillary studies of the associations between prostate cancer risk and diet and dietary supplement use. There are three primary motives for these ancillary studies. First, we are motivated by the basic nutrient character of both agents, which occur naturally in foods, as added fortification in some foods, and in multivitamins and single supplements. The SELECT will allow a comprehensive understanding of the results of selenium and AT supplementation based on analyses that examine total and long-term exposure to these agents. Second, other nutritional exposures, from supplements or foods, could modify the effects of supplemental selenium and AT. Third, this is a large study of a common and important cancer, and there are many questions about associations of dietary patterns with prostate cancer risk that can be addressed within this trial. Two specific aims will examine whether supplement use or dietary patterns modify the effects of the experimental agents. Specifically, we will evaluate the effects of the duration and dose of prior supplemental AT and selenium, as well as the usual intake of nutrients and foods potentially associated with prostate cancer risk (e.g., fat, lycopene, soy, red meat, and vegetables). Three additional specific aims will examine questions about the relationship of nutrient-related exposures with prostate cancer risk. These will test whether prostate cancer risk is associated with use of dietary supplements other than selenium and AT, nutrient intake (in particular dietary fat, specific fatty acids and lycopene), and specific dietary patterns (consumption of red meat, soy products, and fruits and vegetables). Nutrient-related measures will include (1) at baseline, a 10-year history of dietary supplement use using a selfadministered questionnaire; usual nutrient intake over the past year using a food frequency questionnaire; and nutrient concentrations from blood and toenail samples; and (2)

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at each annual visit, use of dietary supplements over the previous year. Planned biochemical analyses include tissue, serum and toenail selenium and zinc, phospholipid fatty acids, and serum folate, carotenoids, and tocopherols. Results from these studies can be used to better understand the study’s primary outcomes and will be important in formulating public health recommendations for prostate cancer prevention. Molecular Epidemiology One of the most exciting tertiary objectives of SELECT is to conduct timely molecular epidemiological investigations on the basis of blood specimens collected at baseline from all participants and from a subsample annually. The SELECT molecular epidemiology design was reviewed in detail previously (Hoque et al., 2001) and is outlined more briefly here. These studies will generate unprecedented data with the potential to have a substantial impact on generations to come. These studies will permit the examination of the main effects of the interventions on individuals with particular genetic backgrounds and may help to identify those men who may benefit the most (or the least) from the study interventions. In addition to assessing the effects of these supplements on prostate cancer incidence among men with a specific genotype, for example, short androgen receptor (CAG)n repeat length, variations in risk according to the presence or absence of other risk factors, such as high-fat diet, can also be examined. Thus, the molecular epidemiological studies will be critical to fully assessing the potential benefit of SELECT interventions. Identification of any subgroups of men for whom these interventions significantly reduce prostate cancer incidence may enhance our understanding of the underlying molecular defects and environmental exposures that may affect prostate cancer risk. This knowledge may be important for the development of new cancer prevention strategies. The assembly of this large cohort also provides an excellent opportunity to evaluate molecular/genetic markers that contribute to the development of prostate cancer. Nested case-control studies will be performed, with incident cancer patients being matched to participants who are free of disease at the time of the cases’ diagnoses. Because all SELECT enrollees will have blood drawn at baseline and be on the study for up to 12 years, the nested case-control design is highly efficient and affords the advantage of providing sample-based risk estimates that are as stable as those generated from analysis of the entire cohort. Approximately 1500–2000 prostate cancers are expected to occur in this population during the 12-year study. The certainty that new hypotheses regarding prostate carcinogenesis will evolve over time; the identification of new candidate genes and other exposures that may influence susceptibility; and biotechnological improvements in the efficiency of

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analysis, sensitivity, and reproducibility of assays argue in favor of a flexible analytical plan for these investigations. Planned analysis include evaluation of polymorphisms in androgen metabolism genes (e.g., AR, CYP17, HSD3B2, SRD5A1 and 2); genes coding for growth factors (e.g., IGF1 and IGFBP3); and carcinogen metabolism genes (e.g., CYP1A1, GSTM1, and GSTP1); DNA repair (e.g., hMLH1 and 2, OGG1, XRCC1, and XRCC3); and oxidative stress genes (e.g., MPO, ecNOS, MnSOD, GPX1). The role of environmental exposures (e.g., chemicals, dietary intake) can be explored by measuring carcinogen–DNA or carcinogen–protein adducts, as well as plasma nutrients. Tissuebased studies of tumor markers will also be conducted in SELECT. With its activation on July 25, 2001, the SELECT joined an historical march of large-scale phase III cancer chemoprevention trials, beginning in the 1980s. Although most completed phase III trials were negative, chemoprevention finally reached its stride in 1998, when the Breast Cancer Prevention Trial produced a dramatic positive result leading to the U.S. Food and Drug Administration approval of tamoxifen for breast cancer risk reduction in high-risk healthy women. A hallmark of phase III trials is to generate provocative secondary and tertiary translational data that in turn generate new hypotheses for future trials and biological studies. Whatever its primary outcome, the SELECT undoubtedly will spawn new trials, just as the ATBC and NPC studies spawned SELECT. The SELECT was designed prospectively to maximize provocative translational study opportunities of large-scale interventions, in contrast with most (if not all) other large trials, which implemented post hoc methods for biological study. An invaluable biorepository of SELECT blood samples and toenails and prostate biopsy tissue samples will be collected. All these samples will be used in future correlative and other molecular/cellular biological studies of prostate cancer and other disease processes. The SELECT promises to contribute to our understanding of cancer risk and development for generations to come.

ation of previously unavailable models for prostate cancer prevention research. Although animal models have existed for some time for the study of prostate cancer prevention (primarily in the rat), it is uncertain whether the mechanisms that drive prostate carcinogenesis in these models are relevant to those in human prostate cancer. Cell culture studies are of limited usefulness because the conditions are inherently artificial. Factors such as relevant physiological concentrations and metabolism of putative chemoprevention compounds are difficult to model in an in vitro system. These studies also preclude the types of interactions known to occur between multiple cell types in vivo. In addition, all prostate cancer cell lines are already highly progressed and are not representative of the type of cells to which most preventive strategies would be targeted. Due to the advent of transgenic mouse (TgM) models, there are now models of prostate cancer that are dependent on molecular mechanisms already implicated in human prostate carcinogenesis. With these models, it is possible to perform a variety of experiments that could previously only be done in cell culture or in prostate cancer cell line xenografts. These experiments can specify pathways that examine nutrient–nutrient interactions and take into account the metabolism of bioactive compounds into active metabolites. This work will require a combination of expertise from multiple disciplines coordinated around specific issues to advance the field. Then it will be possible using the insights gained from the SELECT to power future studies appropriately through the cooperative group mechanism to ultimately determine optimal nutritional strategies for the prevention of prostate cancer and the prevention of prostate cancer recurrence.

Acknowledgments The SELECT trial is supported by a grant from the NCI (U 10 CA 37429) to the Southwest Oncology Group. Heber and Aronson’s work is supported by the UCLA Clinical Nutrition Research Unit (NCI Grant No 1 P30 CA42710) and the UCLA Prostate Cancer SPORE Nutrition Project (NCI Grant No. 1P50 CA92131).

References CONCLUSION AND FUTURE DIRECTIONS OF RESEARCH Increased research on prostate cancer over the past decade has led to many new insights into the role of nutrition in the pathogenesis and prevention of prostate cancer. At this time, our understanding of how the various phytochemicals and micronutrients interact is incomplete. At a basic science level, there remains much work to be done on gene–nutrient interaction and the specification of where bioactive compounds have their impact. The ability to modify the expression of specific genes in the mouse through genetic engineering technologies allows the gener-

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21 Breast Cancer DAVID HEBER AND GEORGE BLACKBURN

INTRODUCTION

consider the etiology of breast cancer by describing the growth, differentiation, and involution of breast tissue at menopause and the critical role of the breast ductal cell microenvironment.

Breast cancer is the most common form of cancer in women and is the second most common cause of cancerrelated deaths among women. In fact, breast cancer accounts for 22% of all female cancers and 15% of cancer deaths among women (Edwards et al., 2002; Stewart et al., 2004). In the United States, which currently accounts for ~20% of the one million cases occurring worldwide, there has been an increase in breast cancer incidence over the past 20 years with an overall slight decrease in mortality. Therefore, although treatment and early diagnosis has made some modest inroads, much more needs to be done in primary breast cancer prevention and prevention of breast cancer recurrence in the breast cancer survivor. Gene–nutrient interaction plays a key role in breast cancer, and the factors that influence susceptibility to breast cancer from an environmental and genetic standpoint remain poorly understood. There is an urgency to this research because there is a global trend in developing countries of an increase in breast cancer incidence as Western diets and lifestyles are spreading around the world. The etiology of breast cancer has been studied extensively using several different but complementary approaches. Our understanding of the possible etiological factors has been obtained by considering information derived from epidemiological studies, including genetic epidemiology studies and information derived over 7 decades on the basic physiology, anatomy, and cellular signaling within the cellular matrix of breast tissue. Intraductal epithelial cells account for the vast majority of breast cancer (Russo et al., 2001). Physiological studies in animals and humans have provided a valuable framework in which to


BREAST ANATOMY AND PHYSIOLOGY The breast is a mass of glandular, fatty, and fibrous tissues positioned over the pectoral muscles of the chest wall and attached to the chest wall by fibrous strands called Cooper’s ligaments. A layer of fatty tissue surrounds the breast glands and extends throughout the breast. The fatty tissue gives the breast a soft consistency. The breast functions to produce milk and is composed of 15–20 sections called lobes, with each lobe ending in many smaller lobules also known as the terminal ductal lobular units (TDLUs). The lobules terminate in many tiny bulbs that produce milk during lactation. The lobes, lobules, and bulbs are all linked by the ducts. The breast tissues consist of connective or stromal tissues surrounding the complex ductal system opening into about nine orifices on each breast nipple. These openings in the areolae are usually blocked with dried secretions except during lactation (Figure 1). The breast tissue forms in women at about age 8–10 years in a process called thelarche. This process is influenced by hormones related to the onset of puberty and occurs earlier in societies with a Western diet and a higher incidence of obesity (Sales et al., 2003). The stage of thelarche in humans corresponds to the short period of ~7 days around the time of terminal end-bud formation in rodents when the administration of a carcinogen such as DMBA will result in tumor formation in 100% of animals. In order for tumor formation

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FIGURE 1 Breast anatomy. (A) Ducts; (B) lobules; (C) dilated section of duct to hold milk; (D) nipple; (E) fat; (F) pectoralis major muscle; (G) chest wall/ribcage. Enlargement: (A) normal duct cells; (B) basement membrane; (C) lumen (center of duct). www.breastcancer.org.

to occur, the DMBA must be administered at 55 days of age. If administered before 48 days of age or after 62 days of age, carcinogenesis is not initiated. Histological studies have shown high rates of DNA synthesis in the terminal end buds at about 55 days of age, suggesting that the cells are dividing and vulnerable to carcinogens in the environment including diet at this stage of breast development (Klurfeld et al., 1989a). Following initiation in these animal models, the effects of diet on breast tumor growth and angiogenesis are well documented. A high-fat diet promotes the growth of the initiated breast cancers so that they appear at an earlier time and with greater frequency by comparison to animals fed a low-fat diet (Klurfeld et al., 1989b). A number of other dietary factors have been examined, but energy balance and obesity can be demonstrated to affect the incidence of breast cancer in these animal models (Klurfeld et al., 1991). Animals trained to exercise have a reduced rate of growth of tumors compared with sedentary animals, and genetically obese animals have an inherently increased incidence of

breast cancer compared with wild-type animals. In humans, exercise in adolescence reduces breast cancer, whereas adult weight gain and obesity are associated with an increased incidence of breast cancer, as discussed in more detail in Chapter 9. Breast cancer forms in the epithelial ductal cells, which are shed into ductal fluid, which is an apocrine secretion containing a number of peptide and steroid hormones, as well as lipids and proteins. Three major proteins present in breast gross cystic disease fluid (Selim et al., 2001) and expressed by the cyst lining apocrine epithelium are gross cystic disease fluid protein-15 (GCDFP-15), apolipoproteinD (APO-D; GCDFP-24) and zinc α2-glycoprotein (ZnGP; GCDFP-44). These proteins can be identified in breast ductal fluid expressed from the nonlactating breast nipple by simple negative pressure aspiration using a suction pump. APO-D can bind with low affinity to a number of steroids and lipids including a number of reproductive hormones, but its function in the ductal fluid microenvironment is not understood. Ductal fluid production has been proposed as an additional risk factor for breast cancer and is related genetically to the ability to produce liquid ear secretions (Tice et al., 2005). Asians have reduced breast ductal fluid production and dry earwax secretions. Breast ductal fluid production is also reduced after menopause but can be obtained for study by aspiration after cleaning the breast nipple surface. Furthermore, these ducts can be cannulated and infused with contrast material and exfoliated breast ductal cells can be obtained by ductal lavage. Atypia of these cells correlates with breast pathology. Pregnancy results in a differentiation of breast ducts to enable the secretion of milk proteins, and this is driven by reproductive hormones including progesterone and estrogens (Pike et al., 1993). As discussed later, pregnancy is protective by comparison to nulliparity.

EPIDEMIOLOGICAL STUDIES There are five- to ten-fold differences in the incidence and mortality of breast cancer between low-risk and highrisk countries internationally (Ziegler et al., 1993). The developing economies of the world in Asia, Africa, and South America, when compared with developed economies of North America and Northern Europe, demonstrated a difference that was linearly related to dietary fat as a percentage of total dietary intake based on crude food disappearance data (Carroll, 1992). These classical observations first made >30 years ago are striking but likely do not indicate a direct cause-and-effect relationship. In fact, the associations observed may have been due to more than simply dietary fat but to differences in dietary patterns, physical activity, and lifestyle. The large variation of breast cancer incidence among or within different regions of the world

Endocrinology of Breast Cancer

may also be attributed to genetic differences among populations that interact with differences in lifestyle, including diet and environmental exposures (McPherson et al., 2000; Robert et al., 2004). Studies of individuals who migrate from a country with low breast cancer incidence (e.g., Japan) to one with higher breast cancer incidence (the United States) have demonstrated an increased risk of breast cancer within a single generation (Ziegler et al., 1993). Environmental and lifestyle factors may largely explain the observed differences in breast cancer risk in different countries, but this observation may not mean that the effects are entirely independent of the genetic influences on breast cancer risk (McPherson et al., 2000). Environmental influences can be passed on via in utero effects on genetic programming to subsequent generations, which assume the breast cancer risk of the new country for several generations, sometimes to an increased extent compared wtih the first generation of immigrants. Interestingly, women with a higher socioeconomic status (SES) or from urban communities are at slightly increased risk for breast cancer than women who have a low SES or come from rural communities, respectively (relative risks [RR] are 1.20 and 1.17 in that order) (Robert et al., 2004). These differences may relate to differences in physical activity and diet, considered elsewhere.

ENDOCRINOLOGY OF BREAST CANCER Mammary epithelial cell proliferation is correlated with serum ovarian hormonal levels. Proliferation rates are low in the follicular phase of the menstrual cycle, when estradiol and progesterone levels are low as well, whereas during the luteal phase, proliferation rates are twofold higher and correlate with the significantly increased ovarian hormone levels (Feigelson and Henderson, 1996). Increased rates of breast epithelial cellular proliferation have been proposed to expose the DNA in the breast epithelial cell to increased risk of mutagenesis via the action of endogenous oxidants, carcinogens, and exogenous carcinogens (Berkey et al., 1999). Metabolism of endogenous androgens and estrogens, interaction with sex hormone–binding globulin (SHBG), insulin, and insulin-like growth factors (IGFs) all influence the net stimuli to the breast epithelial cell signaling such events as cellular proliferation, differentiation, and apoptosis. Prolonged exposure of breast epithelium to estrogens and progesterone due to earlier regular ovulatory menstrual cycles (Kelsey et al., 1993) has been proposed to increase the lifetime risk of breast cancer. The age at which menarche occurs, the age at first fullterm pregnancy, number of pregnancies, and age at menopause all determine lifetime exposure to estrogens (Brinton et al., 1988; Titus-Ernstoff et al., 1998; Bernstein, 2002; Michels and Willett, 2004). Increase in lifetime expo-

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sure to estrogens increases breast cancer risk, as evidenced by the risk increase noted with onset of menarche at younger than 12 years in comparison with onset after 14 years of age. The earlier age at onset of menarche has been associated with an increase in breast cancer risk of ~10–20% (TitusErnstoff et al., 1998). Circulating levels of estradiol are higher in women with early menarche during their adolescence. These women have higher follicular, but not luteal, phase estradiol levels and lower SHBG levels as well. Lower SHBG levels are associated with a greater fraction of total estradiol being available to breast cells (Titus-Ernstoff et al., 1998). Later onset of menopause results in a greater lifetime number of ovulatory cycles and has been proposed to lead to an increased breast cancer risk. It has been estimated that breast cancer risk is increased by 3% for every 1-year delay beyond average in the onset of menopause (Titus-Ernstoff et al., 1998). Surgically induced menopause (ovariectomy or hysterectomy) before the age of 35 years results in a 40% decreased risk by comparison with women experiencing natural menopause (Kreiger et al., 1999). Lower ovarian hormone levels are associated with a reduced risk of breast cancer, as demonstrated by the fact that unilateral ovariectomy before the age of 45 years is associated with a reduced risk of breast cancer (Kreiger et al., 1999). After menopause, ovarian hormone is reduced and the androgen-to-estrogen ratio changes with a concomitant substantial decrease in mammary epithelial cell proliferation (Russo et al., 2000). Numerous prospective epidemiological studies provide strong evidence for the role of circulating estrogens, which can be derived from fat cell aromatization of circulating androgens as a key mechanism for increasing breast cancer risk in this age group. Postmenopausal women who develop breast cancer have, on average, 15% higher levels of circulating estradiol than postmenopausal women who do not develop breast cancer (Pathak et al., 2000). Because of the effects of pregnancy on breast ductal cell differentiation, early pregnancy has a protective effect against breast cancer (Lambe et al., 1994; Kreiger et al., 1999; Lipworth et al., 2000; Russo et al., 2000; Pathak et al., 2000; Beral, 2002; Bernstein et al., 2002; Helewa et al., 2002). Women who have their first full-term pregnancy before age 20 years have a lower risk of breast cancer compared with women with their first full-term pregnancy after 30 years of age, as do women with an early second pregnancy (Titus-Ernstoff et al., 1998). Women with multiple pregnancies have half the risk of breast cancer of nulliparous women. Women with their first birth after age 35 years are at higher risk than nulliparous women (TitusErnstoff et al., 1998) for reasons that are not clear. Exposure to high levels of estrogens during pregnancy, as evidenced by serious nausea and vomiting during pregnancy (TitusErnstoff et al., 1998), are at higher risk for developing breast cancer.

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Prolonged lactation has been demonstrated to be protective as well (Lambe et al., 1994). There is a 4.3% decrease in the RR of breast cancer for every 12 months of breast feeding, in addition to a decrease of 7.0% for each birth (Helewa et al., 2002). Some of the mechanisms explaining the protective effect of pregnancy have been explored in animal models of breast cancer. One mechanism may involve a markedly reduced susceptibility of the fully differentiated mammary gland to carcinogens due to, at least in part, a decrease in proliferative activity of parous epithelium. Another possibility is that the decrease of the risk is due to the altered hormonal environment during pregnancy (specific molecular changes induced by estrogen and progesterone, decrease in circulating growth hormone, etc.) (Lipworth et al., 2000). The decrease of breast cancer risk due to prolonged lactation may be explained in part by the reduction of total number of ovulatory menstrual cycles and consequently cumulative ovarian hormone exposure. The radiographic density of the breast ducts and associated connective tissues is increased in women taking hormone replacement therapy (HRT) during menopause compared with women who do not. It has been shown that HRT users are more than twice as likely to have high-risk increased breast density patterns on mammography in comparison with nonusers (Sala et al., 2000). In addition, any hormones that stimulate breast cell proliferation can stimulate mammographic density. Mammographic density is a well-established risk factor for breast cancer in both premenopausal and postmenopausal women. The Breast Cancer Detection Demonstration Project (Brinton et al., 1995) and the Canadian National Breast Screening Study (Boyd et al., 1995) have shown that women with >75% increased breast density on the mammography have an approximately fivefold increase in the risk of developing breast cancer by comparison to women with 80% for breast cancer, and for ovarian cancer it is 40% for BRCA1 and 65% for BRCA2 carriers. The New York Breast Cancer Study Group selected 1008 cases of breast cancer regardless of whether other family members were affected. They found that with this method the overall lifetime risk of breast cancer in the entire cohort was 82% regardless of whether other family members were affected. Risk of breast cancer in relatives of individuals with BRCA1 or BRCA2 mutations was 20% by age 40, 55% by age 60, and 80% by age 80 (Levy-Lahad and Plon, 2003). Nongenetic factors were also able to greatly affect risk even in the presence of this high penetrance gene for breast cancer. Two modifiable risk factors were significantly associated with delayed onset of breast cancer among women with BRCA1 or BRCA2 mutations. First, there was lower risk if women were physically active as adolescents, as evidenced by a history of regular exercise in sports, dance, or casual exercise as compared with those who were not physically active. Second, a normal body weight at menarche

rather than overweight and lighter weight at age 21 years were associated with older age at onset of breast cancer among women with BRCA1 and BRCA 2 mutations. Therefore, nongenetic factors including diet and lifestyle can affect the penetrance of even these high penetrance mutations. Although breast cancer risk was high for women with these mutations born before 1940, it was even greater for women born after 1940. These effects of birth cohort were much larger than those of any reported modifier gene. The etiology of these nongenetic effects is not known but may include the dietary, hormonal, reproductive, and environmental differences over time between these two cohorts. For example, early age at menarche is more common in recent generations. Nongenetic effects on the expression of susceptibility genotypes. Germline mutations in highpenetrance breast cancer susceptibility genes such as BRCA1, BRCA2, p53, ATM, or PTEN confer a high individual risk for developing hereditary breast cancer. However, these mutations have been shown to account for up to only 5–10% of all breast cancers, probably because of their low frequencies in the population (Easton et al., 1993; Oesterreich and Fuqua, 1999). Genetic damage can also increase the risk of breast cancer. For example, exposure of the mammary gland to high-dose ionizing radiation, as occurs in survivors of atomic bomb explosions, individuals treated with radiation therapy to the chest, and individuals treated with chemotherapy for Hodgkin’s disease, have increased risks of developing breast cancer. The risk of radiation exposure is dose dependent and decreases gradually over time. The study of inherited genes in populations that affect breast cancer risk has uncovered both high-penetrance and low-penetrance breast cancer susceptibility genes. These genes interact with the environment and a number of the aforementioned factors in a complex way. Relatively common low-penetrance cancer susceptibility genes, acting together with endogenous and lifestyle risk factors, are likely to account for most of the sporadic breast cancers, which comprise the majority of all breast cancers (JohnsonThompson and Guthrie, 2000). Inherited breast cancers usually arise earlier in life and are frequently bilateral, whereas sporadic breast cancers generally occur first in one breast and have a later age at onset (Rebbeck, 1999). As with other common forms of cancer, gene–nutrient interactions have to be studied in order to develop and evaluate new biomarkers of the overall risk of breast tumorigenesis. Necessarily, this will involve nutritional genetics, nutritional genomics, and phenotyping of individual women in order to optimize the nutritional approach to prevention and treatment. Although mutations in BRCA1 and BRCA2 are responsible for ~80–90% of all hereditary breast cancers, these mutations are not at all common in sporadic breast cancers (deJong et al., 2002). Interestingly, the normal expression of

Genetics, Nongenetic Factors, and Gene-Nutrient Interaction in Breast Cancer

the wild-type BRCA1 gene is reduced in most sporadic breast cancers, further implicating this gene in breast carcinogenesis (Deng and Brodie, 2000). The p53 gene was the first tumor suppressor gene linked to hereditary breast cancer and is localized on chromosome 17p13. p53 is one of the most commonly mutated genes, being mutated in ~50% of all human cancers (Malkin, 1994). Germline p53 mutations have been identified in patients with Li-Fraumeni cancer susceptibility syndrome, an autosomal dominant disorder characterized by a markedly increased risk of breast cancer with early onset, among other types of cancers (sarcomas, leukemias, brain tumors, adrenocortical carcinomas, etc.) (Malkin, 1994). Affected women have an 18-fold higher risk for developing breast cancer before age 45 years as compared with the general population, and the risk declines with age (maximum is before age 20 years) (Garber et al., 1991). PTEN germline mutations are present in 80% of patients with Cowden syndrome, a rare hereditary breast and thyroid cancer predisposition syndrome associated with a 25–50% lifetime breast cancer risk (de Jong et al., 2002) (general population has an 8–10% lifetime risk). Ataxia telangiectasia (AT) is an autosomal recessive genetic disease caused by mutations in the ATM gene. AT carriers, who are heterozygous for ATM mutations, appear to be at an increased risk of developing breast cancer (deJong et al., 2002), estimated at 11% by the age of 50 years and 30% by the age of 70 years (Easton, 1994). Germline missense mutations (resulting in a stable but functionally abnormal protein that acts in a dominant negative fashion and inhibits the normal ATM protein), rather than truncating mutations (resulting in an unstable, abnormal ATM protein), confer the high breast cancer risk found in AT carriers (deJong et al., 2002). Polymorphisms in breast cancer susceptibility genes with low penetrance (but present in a high percentage of individuals) have a greater contribution to breast tumorigenesis in combination with exogenous (e.g., diet, pollution) and endogenous (e.g., hormones) exposures (Rothman et al., 2001). Low-penetrance susceptibility genes can be identified by studying the biochemical or physiological pathways that are postulated to be involved in breast carcinogenesis. Candidate polymorphic genes include those encoding for enzymes implicated in the metabolism of estrogen or various carcinogens, detoxification of reactive oxygen species emerging from these reactions, alcohol, and one-carbon metabolism pathways, or proteins that play a role in DNA repair or cell signaling processes. Enzymes from different metabolic pathways can be divided into phase I enzymes that metabolically activate carcinogens (e.g., the CYP family proteins) and phase II enzymes that metabolically inactivate carcinogens (e.g., NAT and GST family proteins). Polymorphisms in both phase I and II enzyme genes involved in xenobiotic and

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endobiotic metabolism, therefore, may modulate the RR of breast cancer for an individual (Okobia and Bunker, 2003). CYP 1A1 encodes aryl hydrocarbon hydroxylase (AHH), an enzyme that activates cigarette smoke constituents and polycyclic aromatic hydrocarbons (PAHs) leading to electrophilic carcinogenic molecules (Bartsch et al., 2000). In addition, it also catalyzes the 2-hydroxylation of estradiol in several extrahepatic tissues, including the breast (Hellmold et al., 1998). Members of the glutathione-S-transferase (GST) superfamily catalyze the conjugation of glutathione (GSH) to a variety of electrophiles, increasing their water solubility and excretability (Strange and Fryer, 1999). Polymorphisms leading to the absence of different GST isoenzymes affect the tolerance of the organism to chemical challenges and may influence cancer susceptibility. A pooled analysis of studies on GSTM1 null genotype (homozygous deletion) has found a small and only marginally significant association with increased breast cancer risk. GSTP1 is expressed consistently in both normal and tumor breast tissues (Albin et al., 1993). A metaanalysis study found that an isoleucine to valine substitution at codon 105, which may reduce the conjugating activity of the enzyme (Gudmundsdottir et al., 2001), has been associated with a moderately increased breast cancer risk in homozygous carriers. Polymorphisms in the rate-limiting enzyme involved in alcohol oxidation, alcohol dehydrogenase (ADH), may modulate breast cancer risk, as alcohol is a well-documented risk factor. Premenopausal women with homozygous for ADH1C*1 allele have been found to be at a 1.8 times higher risk for breast cancer than women with the other two possible genotypes (Freudenheim et al., 1999). Folate metabolism is directly related to methylation of genes, which can lead to silencing of genes or gene activation. Two polymorphisms in MTHFR gene, C677T and A1298C, which have decreased enzyme activity in carriers of these genetic variations, are associated with an increased risk of developing breast cancer (Campbell et al., 2002; Ergul et al., 2003). DNA repair genes constitute another low-penetrance cancer susceptibility gene group. Polymorphisms in these genes leading to attenuated DNA repair capacities, especially after the exposure to endogenous and exogenous genotoxic agents, may contribute to breast cancer risk (Duell et al., 2001; Smith et al., 2003; Goode et al., 2002). Breast cancer is a complex disease, and a variety of risk factors are involved in the etiology and development of breast cancer. Mutations in high-penetrance cancer susceptibility genes, such as BRCA1 and BRCA2, p53, PTEN, and ATM, are responsible for a high proportion of the hereditary breast cancers. Various polymorphisms in low-penetrance genes like CYP 1A1, CYP 2D6, CYP 19, GSTM1 and GSTP1, and MTHFR have been associated with either an increased or a decreased breast cancer risk.

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Chemopreventive agents such as tamoxifen, aromatase inhibitors, or COX-2 inhibitors can be used in women at high risk of developing breast cancer to reduce their risk and have effects in combination with a healthy diet and energy balance that could favorably influence breast cancer risk. There is a clear need for further research on how nutrition affects the identification of phenotypic and genetic traits that account for the increase in breast cancer seen in the past century. An understanding of gene–nutrient interaction in breast cancer can help to enhance these efforts.

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22 Skin Cancer HOMER S. BLACK

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exposure to a subthreshold dose of a carcinogenic agent, whereas promotion has been described as “the process whereby an initiated tissue or organ develops focal proliferations, one or more which may act as precursors for subsequent steps in the carcinogenic process” (Farber, 1982). This factitious segmentation of the carcinogenic continuum has been invaluable in allowing dissection and definition of the biochemical steps in the cancer process, and confirmation of the two-stage theory with the so-called “reverse” experiment of Berenblum and Haran (1955) demonstrated that initiation and promotion stages were actually descriptive of the process occurring in mouse skin. It was from these early studies that the concept of a multistage carcinogenic process developed. Even though the perception prevails that interest in environmental agents as causal factors in human cancer represents a more recent shift in emphasis (Higginson and Muir, 1976; Weisburger et al., 1977), it should be clear from the foregoing that the study of cancer and the carcinogenic process has been driven by early recognition that environmental and occupational factors played a paramount role in the occurrence of cancer (Haagensen, 1931). Foremost among those factors are chemicals, viruses, radiation, and diet. Although there are >200 types of skin cancer, and much of our knowledge gleaned of the carcinogenic process has been obtained from studies with carcinogenic chemicals, exposure to ultraviolet (UV) radiation accounts for ~90% of nonmelanoma skin cancer incidence (Elmets and Mukhtar, 1996). This chapter deals primarily with two of the major extrinsic factors that can influence the development of skin cancer, namely, UV radiation, the primary causal agent, and diet, a potential modifier. Also provided are: (1) a brief historical

The skin represents one of the largest organ systems of the human body, constituting about one-twelfth of total body weight. Situated at the interface between the body and its environment, and acting as a barrier to the harmful effects of an expansive array of extrinsic agents, the skin consequently provides the foremost target for environmental insult (Thody and Friedmann, 1986). Consequently, skin cancer is the most frequently occurring malignant neoplasm in the United States, accounting for an estimated 900,000–1,200,000 new cases annually (Miller and Weinstock, 1994). Using age-adjusted incidence data based on the 2000 population standard of the United States, the American Cancer Society projected that more than a million new cases of skin cancer will have been diagnosed in 2004. Major milestones in the study of cancer development have been achieved through investigations of the carcinogenic potential of various agents on skin (Berenblum, 1954, 1979; Boutwell, 1964) because this target tissue provides a model with obvious advantage for cancer studies. Although Sir Percival Pott (1775) was the first to associate excessive exposure to an external agent (soot) and the unusually high skin cancer incidence of chimney sweeps, it was not until 1915 that Yamagiwa and Ichikawa (1918) first successfully produced cancer (skin) in experimental animals with coal tar—a study that helped launch the investigative era of carcinogenesis. Insight into the carcinogenic process was advanced with development of the two-stage theory, operationally defined as initiation and promotion (Berenblum, 1941; Rous and Kidd, 1941). Tumor initiation is generally regarded as a permanent alteration of the cell genotype that is brought about by a single or sequential


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preface and an overview of the status of our current understanding of the relationship of how diet may influence skin cancer from both an experimental and clinical prospect; (2) suggestions for future research directions; and (3) general dietary guidelines, based on our current knowledge, proposed for the prevention and/or management of skin cancer. The chapter is not intended to be a complete bibliographic reference source of skin cancer. Many major contributions have been made to our understanding of the carcinogenic process in skin, as already noted, through studies with an array of chemicals and even combined with some dietary factors. Only from an historical perspective are some of the early contributions recognized. But we focus on the carcinogenic agent, UV, with the knowledge that many of the chemical studies are irrelevant to most human skin cancers. For example, UV involves no activation or detoxification of the presumed carcinogenic species; no competitive chemical inhibition, no binding to target molecules, and no transport to respective target sites. Changing the chemical milieu through dietary modification could have an impact on any of these activities. Thus, it is fortuitous, in this respect, that the primary causal agent of skin cancer (UV) is a complete physical carcinogen, allowing examination of the underlying mechanisms of dietary modification of the skin cancer process.

THE NATURE OF ULTRAVIOLET RADIATION Solar radiant energy includes a broad region of the electromagnetic spectrum containing UV, visible (light), and infrared radiation (International Agency for Research on Cancer [IARC], 1992). UV radiation is generally considered to include wavelengths between 10 and 400 nm, with the extreme UV extending from 10 to 100 nm; far UV from 100 to 180 nm; middle UV from 180 to 300 nm; and near UV from 300 to 400 nm. Those wavelengths reaching the earth’s surface are usually limited to 290 nm and greater, as shorter wavelengths are absorbed by stratospheric ozone. The photobiological designations (UV radiation of biological importance) of the Commission Internationale de l’Eclairage (CIE, International Commission on Illumination, Vienna, Austria) are reflected in Figure 1, with the exceptions that 320 nm, rather than 315 nm, has been used to define the upper limit of UVB, and UVA has been further segmented into UVA1 and UVA2 based on the recommendations of a task force impanelled at the first conference on the biological effects of UVA (Harber, 1986). Thus, UVC is defined as 100–280 nm; UVB as 280–320 nm; and UVA as 320–400 nm. Although electromagnetic radiation is propagated in the form of waves, radiation may alternatively be considered composed of a very large number of small packets of energy called quanta or photons (Tarrant, 1989). The energy content

Increasing Wavelength Increasing Energy Cosmic, gamma, and X-rays 0.0001

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Middle 180

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WAVELENGTH (nanometers) FIGURE 1 The electromagnetic spectrum with expanded scale of biologically relevant ultraviolet wavelengths.

of a photon is inversely proportional to the wavelength. Photons of the UVB band, at 280–320 nm, exhibit energies from 3.9 to 4.4 eV, whereas those of the longer UVA band range from 3.1 to 3.9 eV (Grossweiner, 1989). In general, photon energy, or wavelength, determines the nature of the photochemical/photobiological process initiated, whereas the “exposure dose” (the energy falling upon a unit surface area of an object and expressed as joules per square meter) limits the rate at which the process takes place. Implicit in this is the requirement that photons are absorbed by a suitable target molecule (chromophore) before the process or response can occur—known as the First Law of Photochemistry. Thus, a photobiological response, per unit exposure of UV radiation, varies with the wavelength of radiation and is dependent on the efficacy of interaction between target molecules and the incident photons. A quantitative plot of this spectral variation is known as an “action spectrum.” When radiation strikes the skin, part of it may be reflected, part may be absorbed in outer layers of the skin, and part may be transmitted inward to deeper layers where the energy of the photon is absorbed (Morison, 1991). Depth of penetration is wavelength dependent. Many chromophores, such as nucleic acids and proteins, absorb the shorter wavelengths, and scattering of shorter wavelengths is normally more pronounced. In a fair-skinned individual, only ~15% of the UVB radiation reaches the dermis, whereas ~50 % of the longer UVA wavelengths penetrate to the dermis. The more energetic UVB band is also the most biologically active portion of the solar spectrum, ~1000 times more so than UVA with regard to erythema (sunburn). With regard to carcinogenesis, Forbes (1984) concluded that wavelengths >330 nm had an average relative efficiency for carcinogenesis in mice of 340 nm may contribute, in no small part, to the carcinogenic action of UV radiation. Thus, the risks from additional UVA exposure, such as from tanning parlors or excessively prolonged sun exposure of individuals protected with effective UVB sunscreens, cannot be ignored, although the latter concern may be unfounded (Urbach, 1992–1993).

THE ETIOLOGICAL ROLE OF UV RADIATION IN SKIN CANCER More than 100 years after Potter (1962) had associated excessive exposure to soot with skin cancer occurrence,

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Unna (1894) associated the severe degenerative changes of the sun-exposed areas of the skin of sailors with the development of skin cancer, “Carcinome der Seemannshaut” (Urbach, 1997). Shortly thereafter, Dubreuilh (1896) confirmed the association of “la lumiere solaire” (sunlight) exposure with keratoses and skin cancer exhibited by vineyard workers in southern France. The predisposition to skin cancer of light-skinned individuals, especially of Celtic origin, and living in geographical areas of high insolation, was also observed by Shield (1899). Experimental proof of the causal role of UV irradiation in skin cancer was provided by Findlay (1928) when he demonstrated that UV radiation delivered daily from a quartz mercury-vapor lamp produced skin cancers in mice. Roffo (1939) demonstrated that skin cancer in rats could be induced by radiation from both a mercury arc lamp and natural sunlight. In addition, he showed that the principal offending UV wavelengths were excluded by clear window glass, thus setting an approximate limit of effectiveness in producing skin cancer to those wavelengths of ≤320 nm. Between 1941 and 1975, Blum and his collaborators (1941, 1975; Blum, 1959) conducted important and extensive quantitative studies on the carcinogenic effects of UV radiation. They concluded that (1) repeated, but not single, doses of UV are required to produce tumors within the lifetime of an animal, and (2) tumor growth is accelerated by successive doses of UV radiation. Development time, a measure of tumor induction, was defined as the time from the application of the first dose of UV to the appearance of a tumor of defined volume. They also found that (3) differences in dose, intensity, or interval between doses did not alter the shape or the slope of the dose–time response but only moved the relative position of the response curve along the dose axis, and (4) reciprocity of the carcinogenic response held until doses of UV became too small to result in tumors during the lifetime of the animal. Other aspects of Blum’s experimental model of UV carcinogenesis have been summarized (Blum, 1948; Urbach et al., 1976; Black and Chan, 1977). Roffo (1939) was the first to conduct an epidemiological study of skin cancer in humans, a study in which skin cancer occurrence was analyzed with respect to anatomical site, gender, nationality, and occupation. Whereas the question of an etiological role of sunlight in human skin cancer does not lend itself to direct experimentation, it is nevertheless firmly based on extensive observation and supported with animal experimentation. The evidence has been summarized as follows (Urbach, 1969; Emmett, 1973; Urbach et al., 1974): • Skin cancers occur most frequently on the head, neck, arms, and hand—those parts of the body habitually exposed to sunlight. • Members of pigmented races who sunburn less readily than light-skinned individuals have much less skin cancer.

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When skin cancer does occur in pigmented races, it affects those areas exposed most frequently to sunlight (Mulay, 1963; Quisenberry, 1963; Segi, 1963). There appears to be much greater incidence of skin cancer among those Caucasians who spend more time outdoors than those who work predominantly indoors (Gellin et al., 1966). Skin cancer is more common in light-skinned people living in areas of greatest insolation (Silverstone and Searle, 1970; Mason et al., 1975). An exceptionally high risk of skin cancer exists among those individuals with genetic diseases characterized by intolerance to sunlight (Cleaver, 1968). Skin cancer is readily produced in mice and rats upon repeated exposure to UV radiation (Roffo, 1939, Blum et al., 1941; Winkelmann et al., 1963).

EXPERIMENTAL DATA ON NUTRITIONAL EFFECTS Background to Nutritional Aspects of Experimental Cancer The fact that diet, and particularly food quantity, could influence cancer was recognized by early physicians who advocated “fames cura,” or a starvation diet, as treatment for cancer. It was surmised that this disease, as others to which humans were subjected, proceeded from the food they ate (Garrison, 1929). Thus, many of the earlier experimental studies examined the growth-inhibiting effect of undernourishment observed on both development of neoplasms and the effects on growth of existing tumors (Stern and Willheim, 1943). This growth-inhibiting effect was widely observed for numerous animal tumor transplants. For example, Bischoff et al. (1935) found that marked caloric restriction (50 %), a caloric intake adequate for maintenance of the animals’ general health, resulted in a 10-fold retardation in tumor growth of transplanted mouse sarcoma 180, whereas a 20% reduction resulted in no significant effect. In a carefully controlled experiment employing semipurified diets, Visscher et al. (1942) found the incidence of spontaneous mammary carcinoma dropped from 67% in control animals fed ad libitum to zero in animals fed a diet adequate in protein, vitamins, and minerals but in which carbohydrates and fats were each reduced to approximately one third of the total calories ingested. Two important observations were noted around this period. First, animals on a restricted caloric diet exhibited greater longevity (Tannenbaum, 1940b), and second, short-term starvation was much less effective in restricting tumor growth than chronic undernourishment—a result that paralleled the clinical experience and undoubtedly tended to relegate dietary treatment of cancer as ineffective. Thus, the practical significance of

caloric restriction appeared to lie in prevention or at least a delay in time of appearance of neoplasms. Tannenbaum (1942a) demonstrated that underfeeding, again a 33–50% caloric restriction, resulted in both a marked delay in appearance of 3,4-benzpyrene–induced skin cancers and a decrease in total number of cancers. He also observed that caloric restriction exerted its main effect on the developmental (postinitiation) stage of carcinogenesis (Tannenbaum, 1944a). This influence of caloric restriction on induced primary tumor formation set the stage for a more analytical approach to the evaluation of the role of dietary factors in carcinogenesis. There seems little doubt that caloric restriction could play an important role in prevention of a wide range of human cancers (Albanes, 1987; Hocman, 1988; Weindruch et al., 1991). Early studies to determine the influence of dietary fat on cancer also began with observations on the development of transplanted tumors (Sugiura and Benedict, 1930). Mixed results were obtained, as exemplified by the finding that with Flexnor-Jobling rat carcinoma, the percentage of positive tumor inoculations (takes) and tumor growth rates were diminished, whereas the number of tumor regressions increased when the host animals were fed high-fat (butter fat) diets. However, the indictment of lipid as a dietary constituent that potentiated carcinogenesis resulted from the studies of Watson and Mellanby (1930) in which dietary fat (12.5–25.0% butterfat) was shown to enhance coal tar–induced skin tumors in mice. This observation was followed with intense investigation of the effect of dietary fat on carcinogenesis in the 1940s and 1950s (Tannenbaum, 1953, 1959). Lavik and Baumann (1941) made several intriguing observations that provided early insight into the nature of the fat effect. First, the presence of lipid peroxides did not alter tumor-promoting power of fat. Both oxygenated and UV-irradiated samples, although exhibiting a high peroxide number, were relatively inactive in their influence on carcinogenesis. In their studies, heated (300°C for 1 hour) fat samples exhibiting lower peroxide numbers were most effective in promoting carcinogenesis. In this regard, it is interesting to note that Haven (1936) found the growth rate of rat carcinoma 256 was lower in animals receiving a diet containing cod liver oil (Iodine number 145–180) than in those fed coconut oil (Iodine number 8–9.5). This was determined to be related to the presence of longer chain fatty acids in cod liver oil, no doubt the long-chain n-3 fatty acids that are now known to exhibit anticarcinogenic effects. The second noteworthy observation in their studies was that mice seldom survived diets with ≥25% fat. A fat level of 15% (lard, butter, or vegetable oils) was adequate to demonstrate an effect on tumor formation, and 10% gave a measurable response. Finally, they observed that the most effective period for enhancing skin carcinogenesis by feeding high fat was 1.5–3.0 months after the beginning of application of methyl chloranthane (MCA). These observations suggest

UV-Induced Skin Cancer and Dietary Modifiction

that the degree of saturation of the dietary lipid was less important than the level and/or fatty acid composition of the dietary lipid source; that the percentage change in level of fat required to elicit a response was considerably less than the percentage of caloric restriction required to produce a similar response; and that the fat effect occurred in the postinitiation or promotion stage of carcinogenesis. Indeed, Tannenbaum (1944b) clearly demonstrated that the dietary fat effect occurred during the postinitiation stage of chemical-induced carcinogenesis. Contemporary studies of that time (Tannenbaum, 1942b; Lavik and Baumann, 1943; Rusch et al., 1945) suggested that although most of the accelerating action of fat on tumor formation could be explained on the basis of an increased calorie intake, fat, per se, increased the rate of tumor formation, particularly when the total intake of calories was restricted. Despite the many methodological problems suffered by the early experimental studies, they clearly pointed to caloric restriction and dietary fat reduction as two important dietary avenues to prevent or moderate the course of cancer for several organ sites and established fertile lines for future investigation. It is interesting to note that nearly 65 years after Tannenbaum (1940a) observed that “persons of average weight or less are not so likely to develop the disease [cancer] as those who are overweight,” Calle et al. (2003), in a large prospective study, found that “increased body weight was associated with increased death rates for all cancers combined and for cancers at multiple sites.” Tannenbaum had suggested “that a caloric restricted and low-fat diet may aid in the prevention of human cancer, or at least delay its onset.”

UV-INDUCED SKIN CANCER AND DIETARY MODIFICATION Roffo (1929), who had provided early evidence for the role of UV irradiation in the etiology of skin cancer, had also recognized the importance of lipids in cancer development. He had hypothesized that cholesterol was an “heliotropic” substance that migrated and accumulated at anatomical sites routinely exposed to UV rays of the sun. Subsequent degradation of cholesterol resulted in substances with carcinogenic activity (Roffo, 1933). This presented an attractive theory because cholesterol is naturally occurring and widely distributed in biological tissues and, thus, could provide a basis for the biogenic origin of cancer. Indeed, a flurry of investigative activity continued through the 1950s in search of carcinogenic cholesterol derivatives and/or evidence of cholesterol involvement in cancer development (Fieser, 1954). It was just such an investigation that resulted in the observation that dietary fat had an influence on UV carcinogenesis (Baumann and Rusch, 1939). These investigators, in an effort to examine the purported role of cholesterol in the carcinogenic process, used a fat-enhanced (5% hydro-

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genated cottonseed oil) ration to ensure sterol absorption. In addition, a second group of animals were fed a high-fat ration containing 30% cottonseed oil. Tumor latency of animals receiving cholesterol-supplemented diets was found to be no different from that of animals receiving a stock ration, whereas latency was shortened by about 4 weeks for animals receiving the high-fat diet. Thus, although failing to find supporting evidence for the role of cholesterol in UVinduced skin cancer, and despite that their experimental design failed to control the usual nutritional variables, as did many of the early investigations, they were the first to demonstrate the potential influence of dietary lipid on UVinduced skin cancer. Pursuant to the potential involvement of cholesterol in UV-induced carcinogenesis, a putative carcinogen, 5α,6αcholesterol epoxide, was identified in UV-irradiated skin from among several photooxidation products of the parent sterol (Black and Lo, 1971). Using the hairless mouse model (Black, 1983), cholesterol epoxide formation was shown to be UV dose dependent, and levels of this compound increased eightfold in chronically irradiated animals (Black and Douglas, 1972, 1973). This increase preceded the appearance of squamous cell carcinomas. Although suggestive, no definitive evidence for the causal involvement of this compound in UV carcinogenesis was forthcoming. In addition, cholesterol feeding studies did not indicate a role for this sterol in UV carcinogenesis, although a slight but statistically significant protective effect was observed (Black et al., 1979). As the formation of cholesterol epoxide was the result of photooxidative reactions, it seemed reasonable that these reactions might be inhibited by antioxidants. This concept was explored by feeding mice a closed formula ration containing a 2% (w/w) antioxidant mixture composed of 1.2% ascorbic acid, 0.5% butylated hydroxytoluene (BHT), 0.2% dl-α-tocopherol (acetate), and 0.1% reduced glutathione. At various intervals, cutaneous antioxidant levels were determined. When skin at the various feeding intervals was irradiated with UV and cholesterol epoxide levels measured, an inverse relationship with antioxidant content was observed (Lo and Black, 1973). Thus, antioxidants, known to impede lipid peroxidation, were shown to inhibit the photochemical conversion of skin cholesterol to its epoxide and provided evidence for peroxidative involvement in carcinogenesis. These findings suggested possible prophylactic effects of systemic antioxidants not only on the formation of this putative carcinogen but also on the subsequent pathological conditions that might result from, or concurrently with, its formation (Black, 1987). Indeed, when fed the same antioxidant mixture and chronically irradiated, animals demonstrated significantly fewer actinic lesions and tumors than control animals (Black, 1974; Black and Chan, 1975). Pauling et al. (1982) corroborated the observations concerning the inhibitory effect of the antioxidant mixture on

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UV carcinogenesis. Subsequent studies demonstrated that BHT, at concentrations in the antioxidant mixture, was the most active principal (Black et al., 1978), although a second study from Pauling’s group (Dunham et al., 1982) found that higher levels of ascorbic acid, alone, could significantly inhibit UV carcinogenesis. Other antioxidants and singlet oxygen quenchers have been shown to provide significant protection against UV carcinogenesis. β-Carotene, a carotenoid that is widely distributed in nature, was shown to provide significant protection (Epstein, 1977; Mathews-Roth, 1982; Mathews-Roth and Krinsky, 1985). The protective effect was thought to be related to quenching of specific reactive oxygen species. βCarotene also acts as an antioxidant (Krinsky, 1987). However, under certain conditions β-carotene may exhibit autocatalytic, prooxidant effects (Burton and Ingold, 1984). Indeed, β-carotene supplementation has been shown to exacerbate UV carcinogenic expression, causing an increase in tumor multiplicity and a shortened tumor latent period (Black, 1998). The exacerbative response was found to be dependent on the type of dietary ration administered (i.e., closed-formula vs semidefined rations) (Black et al., 2000). On the basis of the redox potential of interacting antioxidants, a mechanism was proposed by which β-carotene participated with vitamins E and C to repair oxyradicals (Edge et al., 1998; Edge and Truscott, 2000). The β-carotene radical cation, itself a strong oxidizing agent, was an intermediate in the redox schema and, if left unrepaired, could be responsible for the exacerbative effect of β-carotene. According to the schema, repair of the carotenoid radical cation was dependent on vitamin C. However, in subsequent studies in which experimental animals were fed βcarotene–supplemented semidefined diets with varying levels of vitamin C, no effect on UV carcinogenic expression was observed (Black and Gerguis, 2003). It is suspected that the noninjurious or protective effect of β-carotene found in the previous studies employing closed-formula rations might be dependent on interaction with other dietary factors that are absent in the semidefined diet. At present, βcarotene use as a dietary supplement for photoprotection should be approached cautiously (Black, 2004). Nevertheless, strong indirect evidence for reactive oxygen species and radical involvement comes from studies in which UV carcinogenesis has been inhibited by a wide range of natural and synthetic agents exhibiting antioxidant properties (Black, 1974; Epstein, 1977; Dunham et al., 1982; Bissett et al., 1991; Wang et al., 1991; Burke et al., 1992; Gerrish and Gensler, 1993). As a corollary to antioxidant-inhibited UV carcinogenesis, any condition that limits the level of radical susceptible targets should, likewise, modulate the UV carcinogenic process. Unsaturated fatty acids are a prime center for free radical attack and, therefore, prime candidates for manipulation of radical susceptible targets. Thus, it was nearly 45

FIGURE 3 Relationship of ultraviolet (UV)–carcinogenic parameters, tumor latency, and tumor multiplicity to lipid level intake (corn oil). Regression lines are derived from 15 observations from six experiments evaluated in an incomplete block design. Solid line and circles indicate the tumor latency period; dashed line and open squares indicate tumor multiplicity. Tumor latency period decreases and tumor multiplicity increases as the level of corn oil intake increases. (Reprinted with permission from Black, 1993.)

years after Baumann and Rusch (1939) first reported that dietary lipid could influence UV carcinogenesis that this seminal observation was to again receive attention, albeit from a somewhat different perspective (Black et al., 1983). Indeed, an approximate linear relationship between polyunsaturated lipid (corn or soybean oil) intake and UV carcinogenic expression was observed (Black et al., 1985), with the lowest lipid level resulting in a significantly longer tumor latent period. In addition, with increased lipid intake, the number of tumors per animal (tumor multiplicity) increased. The relationship between level of dietary lipid intake and carcinogenic parameters is reflected in Figure 3 (Black, 1993). Interestingly, dietary antioxidants produced an inhibitory effect almost equal to the degree of exacerbation of UV carcinogenesis evoked by increasing dietary lipid levels. Reeve et al. (1988) found that feeding a diet supplying totally saturated sunflower oil (catalytically hydrogenated) completely abolished the UV carcinogenic response, whereas those animals fed polyunsaturated sunflower oil exhibited 100% tumor incidence. When the diet of the animals receiving hydrogenated fat was reconstituted to a normal mixed fat diet, large numbers of skin tumors rapidly appeared, suggesting that tumor initiation had not been prevented by lack of polyunsaturated fat but that an essential fatty acid deficiency held the tumors in abeyance, that is, at the promotion stage. It was subsequently shown that the principal effect of high dietary fat (corn oil) occurs at the promotion stage of UV carcinogenesis and that by replacing

Possible Mode of Action of Dietary Fatty Acids in Modulation of UV Carcinogenesis

a high-fat diet with one of low fat immediately after UV initiation, the exacerbating effect of high fat could be negated (Black et al., 1992). Further support for a polyunsaturated fat requirement for UV carcinogenic expression was obtained when animals were fed constant levels of fat with graded proportions of polyunsaturated sunflower oil mixed with hydrogenated cottonseed oil (Reeve et al., 1996). The UV carcinogenic response was of increasing severity as the polyunsaturated content of the mixed dietary fat was increased. That degree of dietary fatty acid saturation was an important determinant of carcinogenesis was suggested at an earlier period in nutritional carcinogenesis studies when Miller et al. (1944) found that diets composed almost entirely of saturated fatty acids would retard chemically induced hepatomas in rats. Similarly, Carroll and Kohr (1971) and Carroll and Hopkins (1979) found that rats fed unsaturated lipids developed more mammary tumors than those fed the same levels of saturated lipid. The polyunsaturated fatty acid, linoleic acid, appears to be a requirement for mammary carcinogenesis (Ip et al., 1985). In an earlier study of UV carcinogenesis, partially hydrogenated (60%) corn oil was found to inhibit UV carcinogenesis compared with animals fed 4% or 12% corn oil diets (Black et al., 1983). On careful inspection, those data suggest that a more complex relationship exists between degree of lipid saturation and UV carcinogenesis. The 60% hydrogenated corn oil would have had approximately the same level of linoleic acid (18:2), the principal polyunsaturated fatty acid in corn oil, as that of the 4% corn oil diet, yet, by comparison, significantly inhibited UV carcinogenesis. Potential products of catalytic hydrogenation, such as trans fatty acids or conjugated linoleic acid, have not been examined for effect in UV carcinogenesis. Moreover, when animals were fed a diet employing menhaden oil as lipid source, a polyunsaturated lipid rich in eicosapentaenoic and other n-3 fatty acids, UV carcinogenic expression was not enhanced but markedly inhibited (Orengo et al., 1989). These observations would suggest that degree of dietary fatty acid saturation alone is not a determinant in modulation of UV carcinogenic expression.

POSSIBLE MODE OF ACTION OF DIETARY FATTY ACIDS IN MODULATION OF UV CARCINOGENESIS From the foregoing, it should be apparent that both essential n-6 (particularly linoleic acid) and n-3 fatty acids are important modulators of the carcinogenic process. Both types of fatty acids are important determinants of prostanoid metabolism as well. Prostaglandins (PG), particularly of the 2-series, are recognized as important participants in the inflammatory response to UV, and indeed, it has been

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demonstrated that omega-3 fatty acid supplementation in humans not only results in a small but significant rise in the sunburn threshold (the inflammatory response to UV irradiation) (Orengo et al., 1992; Rhodes et al., 1994, 2000) but also reduces the basal and UVB-induced PGE2 levels in skin (Rhodes et al., 1995). Prostaglandins also act as physiological immunoregulators (Plescia and Racis, 1988). With respect to the latter, some studies suggest that the promotion stage of carcinogenesis, the point at which certain dietary lipids exert maximal influence, may be modulated immunologically (Vitale and Broitman, 1981). Indeed, it has been shown that suppressor T-cell function is PGE2 dependent (Chung et al., 1986). These investigators have demonstrated that UV-induced immunosuppression is abrogated by treatment with an inhibitor (indomethacin) of PG synthesis. Indomethacin treatment has also been reported to convey a protective effect to UV carcinogenesis (Reeve et al., 1995). In addition, celecoxib, a rather specific inhibitor of cyclooxygenase-2, one of the isozymes involved in PG synthesis, has been shown to be an effective inhibitor of UV carcinogenesis (Fischer et al, 1999; Pentland et al., 1999; Orengo et al., 2002). UV not only is immunosuppressive but also is known to activate epidermal enzymes responsible for facilitating PG synthesis from arachidonic acid (AA) through the cyclooxygenase pathway. Omega-3 fatty acids compete for reactive sites on the cyclooxygenase enzyme and may shunt PG precursors through the lipoxygenase path, in effect reducing proinflammatory PG levels. PG synthesis may also be suppressed by reducing the level of hydroperoxide activator, possibly achieved by free radical scavengers or antioxidants that impede lipid peroxidation (Lands et al., 1982). Thus, n3 fatty acids not only act competitively with cyclooxygenase substrates to reduce PG levels, but n-3 fatty acids exhibit a high requirement for hydroperoxide activator, an in vivo level that may normally be insufficient to promote rapid PG synthesis. Indeed, Henderson et al. (1989) have shown that diets containing menhaden oil dramatically suppress plasma and cutaneous PGE2 levels. Furthermore, plasma PGE2 levels exhibit a near linear relationship to the log of corn oil intake, with lower levels of plasma PGE2 present in animals receiving low-fat diets (Fischer and Black, 1991). Epidermal capacity to metabolize AA via the cyclooxygenase pathway is also potentiated by n-6 dietary fatty acid intake and is drastically inhibited in animals receiving dietary n-3 fatty acids. It has been shown that dietary fat can suppress the Tcell–mediated immune status in UV-irradiated mice, both with respect to contact hypersensitivity (CH) and delayedtype hypersensitivity (DTH) (Black et al., 1995a; Reeve et al., 1996). Two questions arise with respect to the relevance of these findings to carcinogenesis. First, is the timing of these influences on specific immune responses compatible with the time (i.e., postinitiation) at which high levels of

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dietary fat (n-6 fatty acids) are known to exacerbate UV carcinogenesis? Second, does dietary fat affect these specific immune responses via T-cell–mediated immunological pathways common with those related to carcinogenesis? Some evidence suggests that they do not, at least for DTH. High dietary fat intake results in DTH suppression, even before UV exposure (Black et al., 1995a). UV irradiation hastens the complete suppression of this response in animals receiving high-fat diets. However, the DTH response rebounds by the time tumors appear in chronically irradiated animals. Thus, the temporal profile of DTH response does not conform to the time at which dietary fat exerts its principal influence on UV carcinogenesis. In contrast, when tumor transplantation studies were undertaken, with animals receiving various periods of UV radiation, tumor rejection was significantly greater in animals fed low-fat diets—but only at a time when the complete tumor-initiating UV dose had been delivered. This, of course, is the time (postinitiation) when dietary fat does exert its influence on carcinogenesis. In conclusion, it is clear that high levels of dietary fat exert profound influence (suppression) over specific immune responses, some of which occur when high dietary fat exacerbates UV carcinogenic expression. High dietary fat has been shown to elevate PGE2 levels, the latter known to act as an immunoregulator of T-cell function and to modulate UV carcinogenesis. These observations make a strong circumstantial case that high dietary fat, especially those rich in essential fatty acids, potentiates UV carcinogenesis via regulation of prostanoid metabolism in a manner that consequently suppresses immune responses that control the outgrowth of UV-induced tumors.

CLINICAL STUDIES OF NUTRITIONAL EFFECTS Nonmelanoma Skin Cancer The magnitude of the skin cancer problem is readily apparent when comparing its occurrence with that of other forms of cancer. As noted earlier, Miller and Weinstock (1994) estimated that the most common nonmelanoma skin cancers—basal and squamous cell carcinomas—account for 900,000–1,200,000 new cases annually in the United States. An estimate of new cases in the United States of all forms of cancer (exclusive of nonmelanoma skin cancer, which is usually not recorded in population-based registries) for 1997 is 1,400,000 (Parker et al., 1997). Thus, the incidence of nonmelanoma skin cancer is approximately equal to the combined incidence of all other cancers. Alarmingly, population-based studies indicate that there has been a steady increase in incidence of nonmelanoma skin cancer over the past 2 decades (Glass and Hoover, 1989; Weinstock, 1989;

Gallagher et al., 1990). This increase occurs in regions of both high and low insolation for both basal and squamous cell carcinomas and appears to affect all age-groups. Epidemiological studies indicate that at least 90% of basal and squamous cell carcinomas can be attributed to UV exposure (Mason et al., 1975; Committee on Chemistry and Physics of Ozone Depletion, 1982; Scotto et al., 1983). Koh et al. (1995) have updated and summarized the evidence linking solar exposure to nonmelanoma skin cancer. It is estimated that >50% of the total lifetime dose of solar UV is received in childhood and adolescence (Marks et al., 1990). In accord with this, it has also been estimated that the regular use of an SPF-15 sunscreen during the first 18 years of life would reduce the lifetime incidence of basal and squamous cell carcinomas by 78% (Stem et al., 1986). About 95% of basal cell carcinomas in men occur after the age of 40 years, whereas squamous cell carcinoma primarily (75–80%) affects men older than 60 years (Scotto et al., 1983). These studies point to the relatively long latent period between time of exposure to solar UV adequate to induce nonmelanoma skin cancer and its actual appearance. Further, when prevention measures are ineffective or fail, it points to a need to develop intervention strategies, a potential role to be filled by dietary modification. Role of Diet in Nonmelanoma Skin Cancer: Epidemiological and Clinical Studies Analytical epidemiological studies have provided the principal evidence associating dietary factors with cancer (Armstrong and Doll, 1975). These associations, even when supported by experimental animal studies or clinical observations, have not always proved to be clinically pertinent (Rackett et al., 1993), as noted from the examples in the following subsections. b-Carotene On the basis of existing epidemiological data, it was suggested that individuals with an above average intake of βcarotene might experience a lower incidence of cancer (Peto et al., 1981). A case-control study found that the incidence of skin cancer was inversely related to the level of serum βcarotene (Kune et al., 1992). In addition, as previously noted, β-carotene had been shown to inhibit UV-induced skin cancer incidence in experimental animals (MathewsRoth, 1982). Thus, β-carotene was examined as a skin cancer preventative agent in a controlled clinical trial. A total of 1805 patients who were diagnosed with a recent nonmelanoma skin cancer were given either 50 mg of oral β-carotene daily or placebo (Greenberg et al., 1990). Adherence to the prescribed treatment was good, determined by annual plasma β-carotene levels. In fact, β-carotene supplementation resulted in about a 10-fold increase of the plasma carotenoid level. However, after 5 years, there was no significant difference between treatment and control

Clinical Studies of Nutritional Effects

groups in any of the predefined primary endpoints (i.e., the mean number of new nonmelanoma skin cancers per patient or with time delay before new tumor occurrence). Under the conditions of the clinical trial, the investigators concluded that β-carotene supplementation was inefficacious with respect to reducing the occurrence of nonmelanoma skin cancer. Subsequent evaluations from nested case-control studies indicated that β-carotene supplementation had no effect in any of the controlled patient subgroups, that is, numbers of previous skin cancers, age, gender, smoking, skin type, or baseline β-carotene levels (Greenberg et al., 1996; Karagas et al., 1997). A clinical study has confirmed that β-carotene supplementation has no effect on risk of monmelanoma skin cancer among men with low baseline plasma β-carotene (Schaumberg et al., 2004). Interestingly, it was found, in the Greenberg and Karagas studies, that those persons in the study who were in the highest quartile of the initial plasma β-carotene level had a lower risk of death from all causes, although β-carotene supplementation did not affect mortality. A second randomized trial, the Nambour Skin Cancer Prevention Trial, examined the influence of daily (30 mg/day) βcarotene supplementation, over a 4-year period, on the incidence of basal and squamous cell carcinomas (Green et al., 1999). A small (1508 vs 1146 per 100,000) but statistically insignificant increase in the incidence of squamous cell carcinoma was indicated with β-carotene supplementation when compared with placebo. The investigators concluded that there was no beneficial or harmful effect on skin cancer rates as a result of β-carotene supplementation. More disturbing than finding no beneficial effect of βcarotene supplementation on cancer occurrence, and despite overwhelming epidemiological evidence for a cancerpreventive effect of the carotenoid, were results from the 8year intervention trial of the α-tocopherol, β-Carotene Cancer Prevention Study Group (1996) in which an 18% increase in lung cancer incidence occurred among βcarotene–supplemented (20 mg/day) smokers. The IARC working group (1998), after extensive review of the epidemiological and intervention trials, concluded: “Until further insight is gained, β-carotene should not be recommended for use in cancer prevention in the general population and it should not be assumed that β-carotene is responsible for the cancer protecting effects of diets rich in carotenoid-containing fruits and vegetables.” Isotretinoin Despite reports of positive responses to treatment of basal and squamous cell carcinomas with oral retinoids, these agents have not proved to be efficacious in prevention of nonmelanoma skin cancer. A total of 981 patients with two or more previously confirmed basal cell carcinomas were randomly assigned to receive either 10 mg of isotretinoin or a placebo daily. After 3 years of treatment, no statistically

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significant difference in either cumulative percent of patients with an occurrence of basal cell carcinoma or annual rate of basal cell carcinoma formation was observed between treatment and placebo groups (Tangrea et al., 1992). There was, however, significant toxicity associated with the low-dose regimen of retinoid. Another randomized, doubleblind, controlled trial of oral retinol (25,000 units) or isotretinoin (5–10 mg) also found no differences in the incidence of nonmelanoma skin cancer in high-risk patients between either treatment or placebo groups (Levine et al., 1997). Noncompliance in a large percentage of patients enrolled in retinol chemoprevention studies was due to symptoms consistent with vitamin A ingestion (Cartmel et al., 2000). Therapeutically, oral treatment of acquired immunodeficiency syndrome (AIDS)–related Kaposi sarcoma (KS) with 9-cis-retinoic acid has shown moderate activity with durable responses, but, again, substantial toxicity limits its use as an anti-KS therapy (Aboulafia et al., 2003). Selenium Selenium is another dietary factor that has been studied as a nonmelanoma skin cancer preventative. Clark et al. (1984), in a case-control study, examined the association between plasma selenium level and nonmelanoma skin cancer. Plasma selenium levels were significantly lower in the skin cancer patients. In a subsequent phase III randomized study of 1300 skin cancer patients, supplemental yeastbased selenium was administered for up to 10 years. Although there was a significant reduction in new cases of colon cancer in the patients randomized to the seleniumsupplemented group, there were no significant effects on the occurrence of nonmelanoma skin cancer (Clark et al., 1996). A report that summarizes the entire blinded treatment period that ended in 1996, in which associations between treatment and time to first nonmelanoma skin cancer diagnosis and time to multiple skin tumors overall were analyzed, continued to show no statistically significant association of selenium supplementation with the risk of basal cell carcinoma. However, selenium supplementation was significantly associated with an elevated risk of squamous cell carcinoma. Overall, the results from the National Prevention of Cancer Trial demonstrate that selenium supplementation is ineffective at preventing basal cell carcinoma and that it increases the risk of squamous cell carcinoma and total nonmelanoma skin cancer (Duffield-Lillico et al., 2003). Tea A number of experimental studies have shown that tea, both green and black teas, contains several constituents that are effective inhibitors of UV carcinogenesis (Wang et al., 1991; Bickers and Athar, 2000). These constituents, monomeric and polymeric polyphenols, exhibit strong antioxidant properties and are capable of inhibiting UV-

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induced erythema in human skin (Katiyar et al., 1999). Tea polyphenols are absorbed and enter the circulation quickly after ingestion, significantly increasing the plasma antioxidant capacity (Benzie et al., 1999). Epidemiological studies have generally failed to provide convincing evidence that the consumption of tea polyphenols contribute to a reduction in human neoplastic disease, although these agents may exert a site-specific effect with respect to skin cancer (Linden et al., 1988; LaVecchia et al., 1992; Goldbohm, et al., 1996; Black and Rhodes, 2001). Indeed, in a populationbased case-control study, Hakim et al. (2000c) found no association between general tea consumption and skin squamous cell carcinoma. However, after adjusting for brewing time, the association between skin squamous cell carcinoma and hot black tea consumption indicated a significantly lower risk in consumers of hot tea compared with nonconsumers. Fat Results from epidemiological and experimental studies regarding the influence of dietary fat on skin cancer have often been in conflict. Whereas experimental studies previously discussed clearly demonstrate a strong influence of dietary fat upon UV-induced skin cancer expression, both case-control and prospective cohort studies have failed to find a relationship of skin cancer incidence with dietary fat intake or specific vitamin supplementation (Graham, 1983; Hunter et al., 1992; van Dam et al., 2000; Davies et al., 2002). The larger prospective study involved a cohort of 73,366 women during a 4-year follow-up. Thind (1986), in an international study, found a positive association of dietary fat with skin cancer incidence but was unaware of a biological basis for these findings and cautioned against the pitfalls of international databases and broad correlation studies. Indeed, these types of studies are fraught with methodological difficulties because of (1) the complexity of the human diet in a free-living population; (2) the difficulties in measuring food intake and analyzing dietary information; in particular, dietary history questionnaires and surveys, although availing epidemiologists with large sample sizes, lack validation procedures that would demonstrate that the method measures what it is intended to measure; (3) the nutritionist seeks methods that accurately reflect current food intake, as opposed to the epidemiologist who requires assessment of dietary patterns that are stable over long periods, usually years if cancer induction is under study (Lyon et al., 1992). Some of the limitations of observational studies of diet and cancer can be circumvented by randomized intervention designs whereby direct answers to the question of dietary impact upon cancer incidence can be obtained (Henderson, 1992). The rationale for undertaking a dietary intervention to modify nonmelanoma skin cancer occurrence rests upon several factors: (1) First, experimental animal studies had

shown that high dietary fat intake exacerbated UV carcinogenesis, principally during the postinitiation period. (2) Further, changing from a high-fat to a low-fat diet, after a cancer-causing dose of UV had been administered, negated the exacerbating effect of high-fat intake (Black et al., 1992). This suggested that dietary modification, even after one had been exposed to skin cancer–inducing doses of UV, could represent a potentially important intervention strategy. Furthermore, (3) the high prevalence of skin cancer and the identification of the relative risks of skin cancer patients developing subsequent skin cancers within 2 years (28% cumulative rate; Karagas, 1994) made it practical to make significant comparisons within a relatively short-term study; and, in addition, (4) an intervention design creates a dietary difference, which, followed with frequent dietary assessment, averts many of the problems associated with epidemiological studies and allows direct comparisons of dietary fat exposure and disease status. Such an intervention trial has been undertaken by Black et al. (1994). Of 133 skin cancer patients (basal or squamous cell carcinomas) recruited for the 2-year clinical intervention trial, 115 successfully completed the study. Fifty-eight patients were randomly assigned to the control arm in which no dietary changes were introduced. The 57 patients assigned to the intervention arm learned how to adopt lowfat eating habits to their food preferences and lifestyles, each patient given a “fat gram goal” that defined the grams of fat that would provide 20% of calories from fat. Baseline and follow-up dietary data were compiled from 7-day food records, from which 4 days were selected for analysis. Food records were verified for types of foods, amounts, and methods of preparation. Nutrient analyses were performed using the Minnesota Nutrition Data System. As the study was specifically designed to examine the influence of dietary fat on nonmelanoma skin cancer, stability of body weight and calorie intake was required to prevent any possible confounding effect due to these variables. Thus, patients in the intervention arm consumed higher levels of complex carbohydrate to compensate for the reduction in fat. The success of the dietary intervention protocol, with respect to meeting the goal of 20% of calories from fat, is reflected in Figure 4. Patients in the intervention group had reduced their percentage of calories from fat from 39% to 21%, a level maintained during the remainder of the 2-year study. The potential for a low-fat intervention became apparent early in the study after only 76 patients had completed it. A clear and significant difference in number of actinic keratoses (premalignant lesions) between groups occurred after 8–12 months, with patients in the control group diagnosed with new keratoses four times as often as those in the lowfat group. Based on diet alone, patients in the control group consuming high levels of fat were found to be at 4.7 times greater risk of having one or more actinic keratoses during the 2-year period than similar patients in the low-fat inter-

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Clinical Studies of Nutritional Effects

FIGURE 4 Dietary fat intake (fat as percentage of calories) of skin cancer patients in control group (solid circles) and in a low-fat intervention group (open circles). (Reprinted with permission from Jaax et al., 1997.)

vention group. The predisposing factors for actinic keratosis are similar to those for basal and squamous cell carcinomas (Marks et al., 1988). Indeed, the influence of reduction in the percentage of calories as fat on nonmelanoma skin cancer occurrence (basal and squamous cell carcinomas) was observed after 101 patients had completed the study (Black et al., 1995b) and became even stronger after all 115 patients had completed it (Table 1). When skin cancer occurrence was examined in 8-month intervals across the 2-year study, occurrence in the control group did not change significantly from the baseline period. However, cancer occurrence in the intervention group was significantly lower (p < .02) in the last 8-month evaluation period. The cumulative rate of occurrence of nonmelanoma skin cancer (cumulative skin cancers/patient/time period) was 0.21 and 0.19 during the first 8-month period of the study and 0.26 and 0.02 during the last 8-month period for control and intervention groups, respectively (Figure 5). With regard to diet, there were no significant differences in total caloric intake between the two groups, but there was a 47% reduction in the percentage of calories consumed as fat in the intervention group. To compensate the total caloric intake of the intervention group, there was a 36% increase in carbohydrates compared with the control group. A small but significant increase in percentage of calories from protein was observed. No difference in percentage of calories from alcohol was observed between groups (Jaax et al., 1997). Dietary parameters are shown in Table 2.

FIGURE 5 Influence of dietary fat intake on nonmelanoma skin cancer occurrence in control (solid circles) and low-fat dietary intervention (open circles) groups. Data points reflect cumulative numbers of nonmelanoma skin cancers per patient at each 4-month follow-up period. (Reprinted with permission from Jaax et al., 1997.)

TABLE 1 Effect of Low-Fat Diet on Incidence and Occurrence of Nonmelanoma Skin Cancer (NMSK)a Treatment

N

NMSK/Patient

Patients with NMSK

Control

58

Intervention

57

0.26 p < 0.01 0.02

9 p < 0.02 1

Improvement 9 → 9 NS 9 → 1 p < 0.05

a

Numbers of new confirmed skin cancers (basal and squamous cell carcinomas) per patient were totaled in 8-month intervals of a 2-year study period. Each of the last two 8-month periods, within each dietary group, was compared with their respective initial 8-month period. In addition, the corresponding 8-month periods of the two dietary groups were compared. Cancer occurrence (nonmelanoma skin cancer [NMSK]/patient) in the dietary intervention group declined by the last 8-month period compared with the control group (0.02 vs 0.26 cancers/patient). The incidence (numbers of patients with NMSK) in the intervention group was less in the last 8-month period than in the control group. This is reflected as a significant improvement in those patients in the intervention group (i.e., the control and intervention groups each had nine patients with NMSK in the first 8-month period). The control group also had nine patients with NMSK in the last 8-month period, with only one patient with NMSK in the intervention group for this period.

Overall, the methods for controlling fat intake permitted considerable flexibility in food choices because foods with little or no fat were emphasized, although higher-fat foods could be included as long as the fat gram goal was not exceeded. Thus, a singular strategy of reducing fat intake,

416 TABLE 2


Major Dietary Variables of Control and Low-Fat Dietary Intervention Groupsa

Dietary variable Total calories (kcal) Control Intervention % of calories from: FAT Control Intervention CARBOHYDRATE Control Intervention PROTEIN Control Intervention ALCOHOL Control Intervention

Baseline

Within study

p

2265 ± 659 2400 ± 629

2196 ± 615 1995 ± 564

NS

39.9 ± 4.4 38.9 ± 3.4

37.8 ± 4.1 20.7 ± 5.5

0.0001

42.7 ± 8.1 44.2 ± 5.4

44.6 ± 6.9 60.3 ± 6.3

0.0001

15.5 ± 2.9 15.6 ± 2.3

15.7 ± 2.4 17.7 ± 2.2

0.0001

3.2 ± 4.5 2.8 ± 3.8

3.2 ± 3.9 3.2 ± 3.4

NS

found a consistent tendency for a lower risk of squamous cell carcinoma with higher intakes of omega-3 fatty acids. Their data also suggested a tendency toward a decreased risk of squamous cell carcinoma with increased intake of diets with high omega-3/omega-6 fatty acid ratios. The preceding discussion suggests that human dietary manipulation, with respect to dietary lipid, offers a safe intervention approach for protection against UV-induced effects that lead to skin cancer. Specific strategies such as a reduction in total fat and omega-3 fatty acid supplementation both show promise in protecting against nonmelanoma skin cancer.

Melanoma

a Baseline values represent values ±SD of diets at the time of randomization into the study. Within the study, values are the mean values from 4 months through 24 months. There were no statistically significant differences in any of the parameters at the time of randomization. p values are shown for the within-study differences between groups. Source: Data taken from Jaax et al., 1997, with permission.

with the goal of 20% of calories from fat, could be an effective aid in the management and prophylaxis of nonmelanoma skin cancer. Moreover, the influence of caloric restriction on cancer development should not be ignored. A diet in which the level of fat intake has been reduced, as in the study described, but in which no effort is made to maintain initial caloric intake or body weight, might be expected to convey even greater protection to nonmelanoma skin cancer and certainly to provide collateral health benefits as well. Not only level, but also composition, of dietary fat must be considered as a potentiator of skin cancer. A considerable body of evidence has previously been discussed on the influence of polyunsaturated fatty acids on UV carcinogenesis. However, it is now clear that a distinction must be made between omega-6 and omega-3 fatty acids with regard to their influence on carcinogenesis. Studies discussed previously have clearly demonstrated that omega-3 fatty acids can elevate the erythema threshold in humans, and it is reasoned that this is the result of the anti-inflammatory properties of these fatty acids (Jackson et al., 2002). An assessment of early genotoxic markers in humans indicates that omega3 fatty acid protects against UV-induced genotoxicity and suggests that longer-term supplementation might reduce skin cancer occurrence (Rhodes et al., 2003). Hakim et al. (2000a), in a population-based case-control study, have

It would be remiss not to consider melanoma in a discussion of skin cancer, although the etiology of this type of skin cancer is not clearly understood. In addition, not until recently have there been animal models to study the role of UV in the etiology of melanoma (Ley et al., 1989; Setlow et al., 1993; Noonan et al., 2001), only one of which lends itself easily to dietary manipulation. Cutaneous melanoma is a cancer of the pigment producing cells (melanocytes) that reside primarily in the basal layer of the epidermis (Koh, 1991). The incidence rate of malignant melanoma has nearly tripled in the past 40 years (Rigel et al., 1987; Glass and Hoover, 1989; Grin-Jorgensen et al., 1992) and currently ranks as the eighth most common cancer among whites in the United States (Koh et al., 1995), where it is the most common cancer among whites between the ages of 25–29 years, although the median age at occurrence is 53 years. In 1993, ~32,000 new cases of melanoma were diagnosed in the United States (Boring et al., 1993). That number was projected to rise to 40,300 new cases in 1997 (Parker et al., 1997), consistent with the 5% increase per annum observed earlier (Glass and Hoover, 1989). It was estimated that by year 2000, malignant melanoma will have afflicted 1 in 90 white Americans (Rigel et al., 1987). The American Cancer Society estimated that >55,000 new cases will have been diagnosed in 2004. Unlike nonmelanocytic skin cancer, the precise causal agent(s) of melanoma is unknown. Nevertheless, examination of worldwide latitude gradients, in association with melanoma rates, provided the first evidence that sunlight might be a causal agent of melanoma (Grin-Jorgensen et al., 1992). Although cumulative UV exposure does not seem to explain the occurrence of melanoma as it does for nonmelanoma skin cancer, there is some evidence that frequent intermittent UV exposure is involved. In addition, anatomical site of occurrence does not conform to those areas of the body habitually exposed to UV, although patients with malignant melanoma tend to have lighter complexions and spend more time outdoors (Gellin et al., 1969). Koh et al. (1990) have summarized the evidence for UV involvement,

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as well as that which disputes such a role. Other potential etiological factors, including an immunological role, have been discussed (Lee and Merrill, 1970; Longstreth et al., 1992). To date, little evidence supports a role for diet in development of melanoma (Koh et al., 1995; Lew et al., l995), although a number of factors have been examined. Retinol, Vitamins E and C, Carotenoids, and Alcohol In a clinic-based case-control study of nutritional factors and risk of malignant melanoma involving 204 patients and 248 controls, little or no evidence was found for a protective effect of increased plasma levels of retinol, αtocopherol (vitamin E), or carotenoids (Stryker et al., 1990). Alcohol consumption (>10 g/day) exhibited a moderate trend of increasing risk. Even though vitamin E supplementation provided no association with decreased risk, there was a consistent increased risk in persons with low vitamin E intake. Millen et al. (2004) also found that high alcohol consumption was associated with increased risk for melanoma but that high carotenoid levels were associated with reduced risk. In a large cohort of U.S. women, vitamins A, C, and E were found to have no association with lower risk of melanoma (Feskanich et al., 2003). Interestingly, higher risks of melanoma were associated with greater intakes of vitamin C from food, as well as a significant positive response with frequency of orange juice consumption. Contrary to this finding with melanoma, Hakim et al. (2000b), in a case-control study, found no association between overall consumption of citrus juices and squamous cell carcinoma. They did, however, observe a dose–response relationship between higher citrus peel intake and degree of risk lowering for squamous cell carcinoma. Citrus peel is a major source of limonene. Vitamin D Speculation regarding the potential preventative effects of vitamin D on melanoma grew from laboratory studies that demonstrated that this vitamin inhibited the growth of cultured melanoma cells. Weinstock et al. (1992) examined the relationship between vitamin D and melanoma risk in a case-control study. Vitamin D intake was assessed by foodfrequency questionnaires in 165 melanoma patients and 209 controls. They found no association of melanoma risk with total vitamin D intake, calorie-adjusted vitamin D intake, vitamin D intake from foods, or consumption of milk or vitamin D supplements. Thus, no evidence was found to support the contention that vitamin D protects against melanoma. Millen et al. (2004), on the other hand, concluded that diets consisting of foods rich in vitamin D may be associated with a reduction in risk for melanoma. Obviously, the relationship of vitamin D and melanoma remains uncertain.

Dietary Fat In 1974, Mackie observed an unusual increase in incidence of melanoma (five patients over a 12-week period) in his clinical practice in Sydney. After an interview with a dietitian, it was determined that all five patients had altered their diets 10 months or more before onset of their melanoma and had enthusiastically replaced saturated fat sources, such as butter, with polyunsaturated fat substitutes. Mackie suggested that the increased intake of polyunsaturated fat may have predisposed patients to the development of melanoma. In a subsequent study involving 142 melanoma patients and 82 controls, dietary questionnaires were administered to assess dietary habits, and fatty acid analysis of subcutaneous adipose tissue was employed as a marker for the percentage of linoleic acid ingestion in the preceding 3 years. No significant differences in linoleic acid intake, use of cooking oils, frequency of cooking habit, meals out, or intake of antioxidants were found (Mackie et al., 1980). This study failed to control for racial differences in which dietary habits might have been influential, and a second study was undertaken with 100 melanoma patients and 100 matched controls. The polyunsaturated fat content of adipose tissue was significantly higher in melanoma patients than in controls and there were significantly more controls than patients who had a low percentage of linoleic acid in the triglyceride fraction of subcutaneous adipose tissue (Mackie et al., 1987). Although the critical studies have not yet been undertaken, these preliminary findings suggest a potential role for diet in the prevention of melanoma (Mackie and Mackie, 1990). A retrospective study compared 5-year melanoma survival rates of patients receiving Gerson’s diet therapy with survival rates in the medical literature (Hildenbrand et al., 1995). The alternative Gerson’s therapy includes a lactovegetarian diet that is low in fat. The 5-year survival rates for patients on this therapy were considerably higher than those reported elsewhere. It would be enlightening to know whether a low-fat diet, such as that used in the nonmelanoma study, would be a beneficial adjunct (in terms of survival rates) to conventional therapies employed in the treatment of melanoma.

CONCLUSIONS • Experimental animal data clearly demonstrate that dietary lipid and certain antioxidants can have significant influence on UV-induced carcinogenic expression. • Degree of saturation, per se, appears not to have as important an influence on carcinogenic response in skin as level of the dietary fat and its fatty acid composition. • A clinical dietary intervention trial indicates that a decrease in percentage of calories consumed as fat reduces

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the occurrence of premalignant actinic keratosis and nonmelanoma skin cancer. Reduction in skin cancer occurrence was observed after 1 year of the low-fat dietary intervention. Reduction in dietary fat reduces the occurrence of basal cell carcinomas in humans and of squamous cell carcinomas in animals. Results from the clinical trial validate the relevancy of the hairless mouse/UV dietary model. In toto, these data suggest that implementation of a lowfat diet and omega-3 fatty acid supplementation show the greatest promise as dietary strategies for the management and prevention of the highly prevalent nonmelanoma skin cancer. Single antioxidant supplementation, in contrast, should be approached with caution.

Acknowledgments The author’s research has been sponsored by grants from the National Cancer Institute; the National Institutes of Health; the American Institute for Cancer Research; and medical research funds from the Department of Veterans Affairs.

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23 Colon Cancer LEO TREYZON, GORDON OHNING, AND DAVID HEBER

INTRODUCTION

preventive measures that can detect and diagnose colonic polyps, which can then be removed, thus preventing the development of a cancer. Although distal colon polyps are poor predictors of subsequent polyps and cancer in the upper colon, the finding of a polyp should motivate changes in lifestyle and diet. In 1981, two investigators (Doll and Peto, 1981), using international comparisons of exposure prevalences and disease rates, estimated that up to 90% of colon cancers may be related to diet and lifestyle. A number of hypotheses attempting to explain these patterns continue to be evaluated in observational and intervention studies. As with other common forms of cancer, the primary risk factor for CRC is age. More than 90% of diagnosed cases are in individuals older than 50 years (ACS, 2005). Risk is increased by a personal or family history of colon cancer and/or polyps or a personal history of long-standing inflammatory bowel disease. The main nutritional factors believed to influence the risk of this disease include obesity, red meat intake, calcium, vitamin D, folic acid, alcohol, fiber, phytonutrients, and the dietary fatty acids. Interactions of dietary factors that can neutralize mutagenic secondary bile acids have revealed interesting nutrient–nutrient interactions. Epidemiological studies have also uncovered interesting interactions among nutrients, such as alcohol and folic acid, which affect colon cancer risk. The development of the Vogelstein model for multistep carcinogenesis is based on the progression of adenomatous colon polyps to cancer, providing insights into the epigenetic changes that occur in this process and providing molecular targets for cancer prevention strategies. The adenoma– carcinoma sequence of colorectal carcinogenesis consists of a progressive loss of differentiation and normal morphology

Colorectal cancer (CRC) is the third most common cancer in both men and women. It is the second leading cause of cancer death in the United States and is usually lethal when diagnosed at later stages of progression. In essentially all economically developed countries, the incidence of CRC is high. Both the number of new cases and the death rates are approximately equal for men and women. In the United States in 2005, 56,290 CRC deaths were projected to occur (American Cancer Society [ACS], 2005). It accounts for 10% of all new cases of cancer and 10% of all cases of cancer death. There were projected to be 145,300 new cases of CRC diagnosed in 2005. Of these cancers, 72% (104,950) occur in the colon and 28% (40,340) in the rectum. Survival from CRC remains poor because precancerous polyps and early cancers are often asymptomatic, resulting in many cases being diagnosed at an advanced stage. This cancer is a highly preventable disease if precancerous lesions are diagnosed early because CRCs develop from adenomas, which are readily identified by colonoscopy. Surveillance research shows that the age-adjusted death rates have been declining for cancers of the colon and rectum since the late 1940s (ACS, 2005). The existing strategies for CRC prevention include dietary prevention, chemoprevention, and endoscopic prevention. Beginning at age 50 years, men and women who are at average risk for developing CRC are advised to begin screening. Fecal occult blood testing has demonstrated the ability to detect CRC, whereas virtual colonoscopy and fecal DNA analysis have yet to achieve adequate sensitivity to become primary screening tools. Flexible sigmoidoscopy and colonoscopy are recommended


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in a growing lesion in association with the acquisition of somatic mutations and of aberrant methylation of CpG islands, leading to gene silencing. These molecular events are accompanied by functional changes, including increased mitosis and loss of apoptosis. Studies of families at increased risk of colon cancer have led to the discovery of inherited abnormalities of DNA mismatch repair enzymes and even to common genetic polymorphisms of metabolic enzymes such as GSTM1, which may affect individual responses to preventive phytochemicals. These nutrigenetics models in combination with the emerging evidence on nutrigenomics of phytochemicals in the colonic epithelium promise to provide important new insights helpful in designing strategies for colon cancer prevention through changes in diet and lifestyle. Diet and lifestyle are most likely related to colon cancer etiology through overconsumption of energy, coupled with inadequate intakes of protective substances, including micronutrients, dietary fiber, and a variety of phytochemicals. The latter are biologically active secondary plant metabolites, many of which modify cell proliferation and induce apoptosis in vitro. There is growing evidence that such effects also occur in vivo and that they can suppress the progress of neoplasia. The risk of carcinomas of the colon appears to be reduced by diets rich in fruits and vegetables, but no definitive evidence has been provided by intervention trials. In fact, the largest such trial (see later discussion) did not demonstrate a reduction in polyp number with a global intervention based on reducing fat, increasing fiber, and increasing fruits and vegetables. Nonetheless, plant foods contain a variety of components including micronutrients, polyunsaturated fatty acids, and secondary metabolites such as glucosinolates and flavonoids, many of which can inhibit cell proliferation and induce apoptosis, and which may well act synergistically when combined in the human diet. The future challenge is to fully characterize and evaluate these effects at the cellular and molecular level, to exploit their full potential as protective mechanisms for the population as a whole. The power of early diagnosis via colonoscopy and the preventive potential of healthy diets and lifestyles should lead to further decreases in the incidence of colon cancer.

THE MULTISTEP PROCESS OF COLORECTAL CARCINOGENESIS Colorectal carcinogenesis is a stepwise process. Through a series of nonlinear genetic alterations, carcinogenesis is characterized by changes from normal mucosa through early and advanced adenomas to invasive carcinoma (Vogelstein et al., 1988). Fearon and Vogelstein (1991) have proposed a phenomenological model of colorectal carcinogenesis correlating specific genetic events with evolving tissue

morphology. This conceptual approach describes a system that proceeds linearly from normal mucosa to a small polyp to a large polyp to an invasive cancer, with each step driven by well-defined alterations in the genome. Some investigators regard this progression as “the adenoma–carcinoma sequence” (Renehan et al., 2002). Formation of a malignant tumor requires mutation of a cascade of genes (Figure 1). The first event is inactivation of the adenomatous polyposis coli (APC) gene in both chromosomes. APC, a tumorsuppressor gene, is inactivated by mutation and the mutagenesis cascade is initiated. Then, mutation in the oncogene k-ras and further mutation of other tumor-suppressing genes SMAD4 and TP53 occurs (Fodde et al., 2001). Other genetic events become important as well, for example, modulation of DNA methylation in CpG sequences of the promoter regions of tumor-suppressor and DNA-repair genes, leading to inactivation, or DNA amplification as a mechanism of oncogene activation (Kinsler and Vogelstein, 1996). These genetic insults are associated with the development of pre-neoplastic lesions such as aberrant crypt foci, polyps, and adenomas (Takayama et al., 1998). Up to 10% of all sporadic cancer types feature an additional pattern that is characterized by development of deficient DNA repair; this leads to genetic instability and, therefore, to an increased rate of mutation (Hawkins and Ward, 2001). This type of tumor often has k-ras mutations. Increased cell proliferation occurs within the colonic crypt as one of the earliest events in the neoplastic process, as first suggested by Dukes (1932). An increased colonic epithelial proliferation rate and an expansion of the cryptal proliferative zone have been the object of extensive research as potential markers of increased susceptibility to colon cancer. An immunohistochemical method using 5-bromodeoxyuridine (BrdU) can measure the proliferation rate of colonic mucosa. Fresh endoscopic colonic biopsy specimens are incubated with BrdU and then processed for immunohistochemistry (IHC) using a monoclonal antibody. The proliferation rate is then expressed as the labeling index (LI). The mean LI in healthy controls has been found to be significantly lower than in patients with colonic polyps and in those with colon cancer. Often, controls are those referred for colonoscopy in whom no lesions are found. Although this method was promising for screening persons at risk for colon cancer and was thought to be of great potential in performing dietary intervention studies in high-risk subjects, results with dietary interventions have been disappointing. Since then, many investigators have found increased cellular proliferation in colonic crypts representing the earliest step in this sequence (Jass et al., 1997). However, many newer studies, using newer techniques, have failed to substantiate the initial literature (Jass et al., 1997). It is now appreciated that the process of apoptosis is vital for normal crypt homeostasis. Its impairment may be an early event in the neoplastic process and this event is marked

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Inherited and Spontaneous Colon Cancer

Molecular Pathways Hypomethylation APC

Normal Epithelium

K-ras

Dysplastic ACF

DCC

Early Adenoma

p53

Advanced Adenoma

NM23

Invasive Carcinoma

Cell Proliferation

Cellular Events

Apoptosis Loss of crypt compartmentalization FIGURE 1 Aberrant crypt focus: adenoma–carcinoma sequence.

by the occurrence of aberrant crypt foci (ACFs) (Renehan et al., 2002). Two ACF types are identifiable: hypercellular and dysplastic. Hypercellular ACFs are more common and are never dysplastic. Increased proliferative activity may be seen in both. The dysplastic entity is most relevant to carcinogenesis. Both animal and human studies support the notion that ACFs grow by crypt fission, leading to formation of microadenoma (Renehan et al., 2002). Adenomas are monoclonal expansions of a disrupted cell, but very early lesions may be polyclonal. Distinction between hyperplastic and dysplastic aberrant crypts has important consequences for the use of biomarkers.

INHERITED AND SPONTANEOUS COLON CANCER Inheritance of genetic alterations accounts for up to 20% of colorectal tumors (Berlau et al., 2004). The best-described genetic contributions include the syndrome familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC, or Lynch syndrome). HNPCC is a dominantly inherited syndrome characterized by the development of CRC, endometrial cancer, and other cancers, as well as the presence of microsatellite instability (MSI) in

tumors. The Bethesda guidelines have been proposed for the identification of families suspected of having HNPCC that require further molecular analysis. The loss of mismatch repair (MMR) protein detected by IHC of CRC and endometrial cancer correlates with the presence of MSI and/or an MMR gene mutation. The Bethesda criteria, with a few modifications, are appropriate to identify families eligible for genetic testing. In addition, MSI and IHC analysis of CRC using antibodies against MLH1, MSH2, MSH6, and PMS2 proteins is equally effective for identifying carriers of the known MMR gene defects. Because other putative MMR genes in hereditary CRC have not been elucidated, IHC analysis cannot completely replace MSI. MSI analysis is still the first step in families suspected of HNPCC. In contrast, in families in which the probability of detecting a mutation is relatively high, IHC is the first diagnostic step because the result might predict the specific underlying MMR gene mutation (Vasen et al., 2004). The identification of HNPCC is important because it makes it possible to target effective preventative measures. MSI and IHC analysis of CRC and endometrial cancer are reliable, cost-effective tools that can be used to identify patients with HNPCC. Such identification may make it more feasible to conduct dietary intervention trials in smaller numbers of subjects and to increase the likelihood

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of uncovering preventive interventions than when low-risk and high-risk individuals are randomly assigned to control and intervention groups. Colorectal tumors are also associated with predisposing factors, namely the inflammatory bowel diseases (Crohn’s disease and ulcerative colitis). The great percentage of colorectal tumors develop sporadically or are caused by exposure to environmental factors. Diet and lifestyle factors (discussed later in this chapter) may modulate genetic and biochemical processes that culminate in initiation, promotion, and progression of carcinogenesis. The evidence from epidemiological and limited intervention trials is reviewed in the following section.

Physical Activity Whereas obesity increases risk, physical activity is associated with a decrease in risk. Although physical activity is difficult to measure in epidemiological studies, >50 studies in diverse populations show that more physically active individuals are at lower risk for colon cancer, though not for rectal cancer (Giovannucci, 2002). Although physical activity is often associated with other lifestyle factors that may be related to colon cancer risk (Giovannucci et al., 1995), the inverse association between colon cancer risk and physical activity appears to be independent of confounders.

Hyperinsulinemia and IGF-1 NUTRITIONAL FACTORS IN COLON CANCER Our knowledge of causative factors in colon carcinogenesis is supported by several epidemiological observations. Direct-acting food components could contribute to the occurrence of cancer because there is a direct interaction between food constituents and the mucosa. Physiological traits, such as obesity, decreased physical activity level, and increased body mass index (BMI), have been reported to be involved in the promotion of colorectal tumorigenesis (Reddy, 1981). Physical inactivity might be associated with diminished mechanical stimulation of the colon, leading to less rapid defection. This increases the time that the mucosa is exposed to potential toxic metabolites. In addition to direct mechanisms, metabolites might also act indirectly to enhance lipid peroxidation (Berlau et al., 2004).

Energy Balance and Obesity As previously mentioned, colon cancer rates are highly correlated with economic development or “Westernization.” Although several factors related to the Western lifestyle may contribute to colon cancer, a large and growing body of evidence implicates energy balance. Many studies, including prospective studies, have found that obesity, usually assessed by BMI, is associated with an increased risk of colon cancer (Wu et al., 1987; Giovannucci et al., 1995; Martinez et al., 1997). The association appears to be stronger for men than for women, possibly because the relationship becomes weaker in women postmenopause (Terry et al., 2001). Evidence also suggests that a tendency for the central distribution of adipose (visceral adiposity) increases risk independently of BMI. For example, in a study of men (Giovannucci et al., 1995), when comparing upper to lower quintiles, the relative risk for colon cancer was about 3.5 in men with a high waist-to-hip ratio compared with those with a low ratio.

The consistent findings for obesity, central obesity, and physical inactivity as risk factors for colon cancer have led to the hypothesis that insulin resistance and resulting hyperinsulinemia is the underlying risk factor (Giovannucci, 1995). This hypothesis has been supported by studies that have found type 2 diabetes mellitus (Hu et al., 1999) and hyperinsulinemia to be directly associated with colon cancer risk many years before colon cancer is diagnosed (Schoen et al., 1999). In addition to hyperinsulinemia, high concentrations of insulin-like growth factors (IGFs) also appear to increase risk of colorectal neoplasia. For example, acromegaly, a condition characterized by chronically elevated growth hormone levels resulting from a pituitary adenoma and IGF-1 hypersecretion, is associated with increased colonic epithelial cell proliferation (Cats et al., 1996) and elevated risk of benign and malignant colon tumors (Jenkins et al., 1997). Prospective studies have found high but normal circulating levels of IGF-1 related to elevated risk of colorectal neoplasia (Ma et al., 1999; Giovannucci et al., 2000).

Fruits, Vegetables, and Fiber Most of the case-control studies (>15 studies) show an inverse association between intake of vegetables, and possibly fruits, and colon cancer risk (Giovannucci, 2002). Trock et al. (1990) conducted a metaanalysis of six casecontrol studies and found that a high intake of vegetables was associated with approximately half the risk for colon cancer, and high fiber was associated with an approximately 40% reduction in risk (Trock et al., 1990). Some studies fail to show a relationship for total fruits and vegetables (Michels et al., 2000). A randomized intervention study in which total servings of fruits and vegetables were increased without regard to the type of fruit or vegetable being eaten and using recurrent adenomas as the endpoint did not find a preventive effect of increasing fruit and vegetable intake (Schatzkin et al., 2000).

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Nutritionl Factors in Colon Cancer

Dietary fiber has been the focus of numerous studies since it was first introduced as a potentially protective dietary component by Burkitt (1971). He observed that diseases of the bowel, including colon cancer, were rare in Africa where a high-fiber diet was consumed. Many correlational and case-control epidemiological studies support this hypothesis (Freudenheim et al., 1990; Martinez et al., 1997; Negri et al., 1998); however, several prospective studies have yielded equivocal results (Platz et al., 1997; Fuchs et al., 1999). For the most part, relationships observed in welldesigned observational studies have not been supported by interventional trials. The results of two randomized clinical trials failed to show a protective effect of increased fiber and/or decreased fat in the diet on the risk of adenomatous polyp recurrence (Alberts et al., 2000; Jacobs et al., 2002). In the Polyp Prevention Trial, there was no reduction in risk of colorectal adenoma recurrence with consumption of a low-fat, high-fiber diet (Jacobs et al., 2002). In one trial there was no difference in the rate of recurrent adenomatous polyps between those randomized to consume a high-fiber supplement as compared with those in the low-fiber group (Alberts et al., 2000). The additional studies that do not point to a cancer-preventive role of fiber include the Finnish ATBC study with >27,000 male volunteers older than 8 years (Pietinen et al., 1999), a cohort study with >45,000 female volunteers (Mai et al., 2003), and intervention trials with patients with existing colonic adenomas and prevention of recurrence by means of increased fiber intake (McKeownEyssen et al., 1994; Bonithon-Kopp et al., 2000; Schatzkin et al., 2000; Jacobs et al., 2002). A Cochrane metaanalysis by Asano and McLeod (2002) found no evidence that increased dietary fiber reduces either incidence or recurrence of adenomatous polyps within a 2–4 year period. However, the largest single study performed (European EPIC study—performed with >500,000 volunteers in 22 European centers) revealed an inverse relationship between fiber intake and risk of developing colon cancer (Bingham et al., 2003). High fiber intake (>30 g/day) was associated with a 25% reduction in colon cancer risk (Bingham et al., 2003). Another study showed that for subjects with colonic adenoma fiber intake was lower, particularly from grains, cereals, and fruit (Peters et al., 2003). It is clear that these data are conflicting. Explanations of these contradictory results might lie in the definition of low and high fiber intake. Compared with the last two studies, the fiber intake in the other studies was not exceptionally high. Moreover, it might not be the fiber itself that has the protective effect, but the whole pattern of a diet that provides less saturated and animal fat and more antioxidants, trace minerals, and phytoestrogens, which might be the real protective elements (Ferguson and Harris, 2003).

Red Meat Early prospective cohort studies have been inconsistent with regard to implicating red meat intake with an increased risk of colon cancer risk (Bjelke, 1980; Phillips and Snowdon, 1984; Stemmermann et al., 1984). A number of prospective studies have shown statistically significant or suggestive positive associations for intake of processed meats and the risk of colon cancer (Bostick et al., 1994; Giovannucci et al., 1994; Goldbohm et al., 1994). Data from the prospective Health Professionals Follow-up Study (Giovannucci et al., 1994) and the Nurses’ Health Study (Willett et al., 1990) showed a direct association between red meat consumption and risk of colon cancer, but no association was observed with other sources of fat or meats such as chicken or fish. The cytotoxic effect of dietary heme has been proposed as a potential mechanism by which red meat increases CRC risk because of higher heme content in red meat compared with poultry and fish (Sesink et al., 1999; Cross et al., 2003). Heme damages the colonic mucosa and stimulates epithelial proliferation in animal studies (Sesink et al., 1999). Both ingestion of red meat and heme iron supplementation have been shown to increase fecal concentrations of N-nitroso compounds (Cross et al., 2003) and DNA adducts in human colonocytes (Hughes et al., 2001, 2002). Non–red meat sources of animal protein—including low-fat dairy products, fish, and poultry—have typically been associated with a lower risk of colon cancer or adenoma (Bostick et al., 1994; Giovannucci et al., 1994). The mechanism for this potential benefit is unknown, however. Diets high in these sources of protein, in contrast to red meat, may also be associated with other healthful behaviors. Regardless of the mechanism, the studies in general tend to support substitution of other protein sources for red meat. Few studies have examined risk in relation to long-term meat intake or the association of red meat with rectal cancer. In 2005, Chao et al. examined 185,000 patients in the CPS-II cohort and found that a high intake of red and processed meats was associated with a higher risk of colon cancer after adjusting for age and energy intake but not after further adjustment for BMI, cigarette smoking, and other covariates. Results of some studies suggest that risk of colon cancer may be increased among meat eaters who consume meat with a heavily browned surface, but not among those who consume meat with a medium or lightly browned surface (Chen et al., 1998; Singh and Fraser, 1998). When meat undergoes prolonged frying, grilling, or broiling at high temperatures, mutagenic heterocyclic aromatic amines are formed from creatinine reacting with amino acids (Hsing et al., 1998). A metaanalysis of the epidemiological literature implicated processed meats as opposed to fresh meats (Thun et al., 1992). Ongoing and future investigations

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should substantiate whether levels of heterocyclic amines caused by cooking or nitrate compounds in processed meats present carcinogenic risks in humans.

Dietary Fats and Fatty Acids Both the type and the amount of dietary fats consumed have been implicated in colon cancer etiology. Studies have demonstrated that n-3 polyunsaturated fatty acids (PUFAs), commonly found in fish oil, could prevent colon cancer development. Evidence shows that n-3 PUFAs act at different stages of cancer development and through several mechanisms including the modulation of arachidonic acid– derived prostaglandin synthesis, and Ras protein and protein kinase C expression and activity. As a result, n-3 PUFAs limit tumor cell proliferation, increase apoptotic potential along the crypt axis, promote cell differentiation, and possibly limit angiogenesis. The modulatory actions of n-3 PUFAs on the immune system and their anti-inflammatory effects might also play a role in reducing colon carcinogenesis. There remains, nevertheless, some ambiguity over the safety of n-3 PUFAs with respect to secondary tumor formation. Epidemiological studies revealed a significantly lower incidence of CRC in Greenland Eskimo populations eating their traditional diet compared with reference populations in the West (Byers, 1996). This population consumed substantial amounts (>10.0 g/day) of long-chain n-3 PUFAs (EPA, DHA, and docosapentaenoic acid). In contrast, the Western diet contains approximately only 1–2 g/day of n-3 PUFAs, mostly as ALNA with long-chain n-3 PUFAs contributing 50% reduction in CRC incidence was shown for a selenium (in the form of brewer’s yeast) intervention in the Nutritional Prevention of Skin Cancer (Clark et al., 1996). Because these results were based on secondary endpoint data among a population of selenium-deficient areas in the United States, it is believed that a large trial is necessary to confirm this provocative finding. In 2005, there was insufficient evidence to support recommendations that individuals obtain supplementary sources of antioxidants to reduce their risk of colon cancer. One possible way to achieve a well-balanced diet is to increase the proportion of plant foods, fruits, and vegetables (Heber and Bowerman, 2001). Phytonutrients are substances that plants make for their own benefit but, when consumed by humans, lead to apparent reductions in incidence of chronic diseases, including CRC. Protective factors from plants, fruits, and vegetables have physical effects such as higher stool volume, decreased transit time, and absorption of mutagens to the fiber itself. They can also affect the release of certain beneficial fermentation products by the gut flora and plant substances with antioxidative potential and other chemoprotective properties (Priebe et al., 2002). One of the defenses of the colonic epithelium is its mucinous layer. This has been reported to have capacity to detoxify reactive substances (Takayama et al., 1989). Despite the protective mucinous layer, colonocytes are still susceptible to damage from toxins. Compared with hepatocytes,

colonocytes express relatively smaller amounts of phase II enzymes such as GST, which inactivate many genotoxic carcinogens (Takahashi et al., 1991). Plant substances or fermentation products (butyrate) from the gut can, however, induce these protective enzyme systems (Ochiai, 1991). Thus, it might be possible to maintain this mechanism of chemoprevention on a continuous optimal level by means of health-promoting nutrition. Results from animal experiments show that phytoprotectants and chemoprotectants such as ethoxyquin, butylated hydroxyanisole, butylated hydroxytoluene, oltipraz and indole-3-carbinol (Langouet et al., 2002), and high-amylose starch, which alters the fermentation profile and the composition of the gut microflora (Takahashi et al., 1991), induce GST-α and GST-π in non-transformed rat colon cells. These studies support the hypothesis that nutrition, by affecting the gut flora, can induce this potentially protective and important class of phase II enzymes in important tumor-target cells. This induction is supposed to provide increased protection from genotoxic carcinogens, as has been demonstrated experimentally for the liver (Pool-Zobel and Leucht, 1997).

CONCLUSION Colon cancer is a diet-related cancer. Most incident colon cancer is preventable through modulation of nutritional choices over a lifetime. As far as this disease is the second leading cause of cancer death in the United States, with >56,000 projected deaths in 2005, it represents a high-priority area for further investigation. Survival remains poor because most cases are diagnosed at an advanced stage. The existing strategies for CRC prevention include dietary prevention, chemoprevention, and endoscopic prevention. Although distal colon polyps are poor predictors of subsequent polyp and cancer in the upper colon, the finding of a polyp should motivate changes in lifestyle and diet. The main nutritional factors believed to influence the risk of this disease include obesity, increased red meat intake, calcium, vitamin D, folic acid, alcohol, fiber, phytonutrients, and dietary fatty acids. Epidemiological studies have also uncovered interesting interactions among nutrients such as alcohol and folic acid, which affect colon cancer risk. Although the relative contributions of these individual dietary factors on colon carcinogenesis are still in question, it remains clear that healthy nutritional choices constitute the main prescription for colon cancer prevention at the societal level. Insofar as the prescription for avoidance of these harmful constituents and consumption of the beneficial ones is associated with prevention of other chronic diseases such as atherosclerotic heart disease, it can be assumed that there are health benefits that this prescription carries beyond colon cancer prevention.

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The Vogelstein model for multistep colon carcinogenesis has provided insights into the epigenetic changes that occur in the process and has provided molecular targets for cancer prevention strategies. Nutritional factors have been shown to be effective at ameliorating transition at each of these steps. Studies of families at increased risk of colon cancer have led to the discovery of inherited abnormalities of DNA mismatch repair enzymes and even to common genetic polymorphisms of metabolic enzymes such as GSTM1, which may affect individual responses to preventive phytochemicals. These nutrigenetic models in combination with the emerging evidence on nutrigenomics of phytochemicals in the colonic epithelium promise to provide important new insights helpful in designing strategies for colon cancer prevention through changes in diet and lifestyle. Carcinomas of the colon appear to be partially preventable by diets rich in fruits and vegetables. Plant foods contain a variety of components including micronutrients, PUFAs, and secondary metabolites such as glucosinolates and flavonoids, many of which can inhibit cell proliferation and induce apoptosis and may act synergistically when combined in the human diet. The challenge is to fully characterize and evaluate these effects at the cellular and molecular level to exploit their full potential as protective mechanisms for the population as a whole. The power of early diagnosis via colonoscopy, fecal DNA analysis, and the preventive potential of healthy diets and lifestyles should lead to further decreases in incident colon cancer.

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24 Gastric Cancer NAI-CHIEH YUKO YOU AND ZUO-FENG ZHANG

INTRODUCTION

et al., 2001a); however, it ranks second in cancer incidence for men, only behind lung cancer (Parkin et al., 2001a). The estimated number of new cases in 2000 was 876,300 worldwide, including 558,500 men and 317,900 women (Parkin et al., 2001a). It is the second leading cause of death from cancer. The estimated number of deaths in the year 2000 was 646,600 (Parkin et al., 2001a), accounting for >10% of all cancer deaths in the world. There is a great geographic variation observed in gastric cancer case distribution. Almost two-thirds of the cases occur in developing countries (Parkin et al., 2001b). High-risk areas include Japan, Central and South America, and Eastern Asia; however, significant differences are observed even within these areas. Incidence rates are low in Southern Asia, North and East Africa, and North America (Parkin et al., 2001b). In the United States, gastric cancer is the fourteenth most common cancer in terms of incidence and the thirteenth most common cause of death from cancer (Jemal et al., 2003). The estimated numbers of new cases in 2003 were 13,400 men and 9000 women. The estimated number of deaths from gastric cancer was 12,100 (Jemal et al., 2003). The survival rate for gastric cancer is moderately good only in Japan (52%) and ranges from 10 to 25% elsewhere (Parkin, 2001). The reason that Japan has the highest gastric cancer survival rate is that mass screening by photofluoroscopy has been practiced in the population since the 1960s (Parkin, 2001). The incidence and mortality rates of gastric cancer have steadily declined in most countries (Parkin et al., 2001b). The estimated incidence rates in 2000 were ~11% lower than those for 1990 (Parkin, 2001), and there has been a 4–5% decrease in age-adjusted risk (Parkin et al., 1999). The decrease may be due to the improvements in preservation

Gastric cancer is the fourth leading cancer incidence in the world. It ranks second in cancer incidence for men, only behind lung cancer, and kills more than half a million people a year according to Globocan 2000. The incidence and mortality rates of gastric cancer have steadily declined worldwide in the past few decades. The reasons for the decline in incidence and mortality rates are not fully understood; however, dietary and lifestyle factors may be associated with the phenomena, including better refrigeration, reduced consumption of preserved foods, increased intake of fruits and vegetables, improved living standards, and a decline in the prevalence of Helicobacter pylori infection. A multifactorial model of human gastric carcinogenesis is currently suggested in which different dietary and nondietary factors, including genetic susceptibility of the host and H. pylori infection, are involved at different stages in the cancer process. Twin studies and immigrant studies have also suggested that dietary factors play an important role in the development of gastric cancer. Dietary modifications may potentially reduce the risk of gastric cancer. In this chapter, we review the epidemiological and animal experimental evidence regarding specific dietary factors in the etiology and prevention of gastric cancer. We discuss the major findings of chemoprevention trials for gastric cancer and the future directions for gastric cancer prevention and control research.

BACKGROUND Gastric cancer ranks fourth in cancer incidence in the world for both sexes according to Globocan 2000 (Parkin


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and storage of foods and the changes in the prevalence of H. pylori by birth cohort (Parkin, 2001). In contrast to the overall decreasing trend, there has been an increasing trend in cancers localized to the cardia of the stomach observed in several populations (Parkin, 2001; Parkin et al., 2001b). The reason is still not clear; however, these changes parallel the increased prevalence of Barrett’s esophagus and adenocarcinoma of the lower third of the esophagus (Parkin, 2001; Parkin et al., 2001b). The incidence rate of gastric cancer rises progressively with age (Lam, 1999; Kelley and Duggan, 2003). The age at which gastric cancer starts to appear is lower in Eastern Asians (~30 years of age) compared with the Western population (~50 years of age) (Lam, 1999). The risk of gastric cancer is higher in men than women (Parkin et al., 2001a; Kelley and Duggan, 2003). Gastric cancer prevalence varies with ethnic distribution, even for those living in the same region (Kelley and Duggan, 2003). Migrants from high-risk to low-risk regions tend to maintain the high-risk patterns of the population of origin (Kono and Hirohata, 1996; Parkin et al., 2001b). Low socioeconomic status is associated with a general increase in gastric cancer risk (Kono and Hirohata, 1996; Kelley and Duggan, 2003). First-degree relatives of patients with gastric cancer histories are at increased risk of about two to threefold (Kelley and Duggan, 2003). This may indicate that familial genetic abnormalities may alter individual susceptibility to gastric cancer carcinogens (Lam, 1999). Body mass index (BMI) above the lowest quartile is responsible for the 19.2% (95% confidence interval [CI], 4.9–52.0%) of gastric cardia adenocarcinoma (Engel et al., 2003). Infection with H. pylori is considered an important risk factor for noncardia gastric cancers (Mayne and Navarro, 2002). The International Agency for Research on Cancer (IARC) accepted H. pylori infection as being carcinogenic for humans in 1994 (Parkin, 1998; Parkin et al., 2001b; Gonzalez, 2002). Meta-analyses have found that the infection increases the risk of noncardia gastric cancer by two to sixfold compared with noninfected control populations (Huang et al., 1998; Eslick et al., 1999; Xue et al., 2001). H. pylori may be responsible for causing ~40% of all gastric cancer cases worldwide (Parkin et al., 2001b). In a twin study (Lichtenstein, 2000; O’Brien, 2000), model fitting found that inherited genes contributed 28% (95% CI, 0–51%), shared environmental factors 10% (95% CI, 0–34%), and environmental factors 62% (95% CI, 0–76%) to the etiology of gastric cancer. The results provide evidence that environmental and lifestyle patterns play major roles in gastric cancer. The constant decline in stomach cancer has been attributed to improved food preservation practices and better nutrition (Reed, 1993; Parkin et al., 2001b). The wide availability of refrigeration has changed the way of preserving food (Reed, 1993; Kono and Hirohata, 1996; Gonzalez, 2002; Kelley and Duggan, 2003); specifi-

cally, refrigeration may reduce the use of salt as a food preservative and decrease the likelihood of mold growth in food (Reed, 1993; Parkin et al., 2001b; Kelley and Duggan, 2003). Smoking weakly increased the risk of gastric cancer (Kono and Hirohata, 1996; Ogimoto et al., 2000; Parkin et al., 2001b; Mayne and Navarro, 2002; Kelley and Duggan, 2003). Other factors that may be associated with gastric cancer risk include blood type A, gastric surgery, gastric polyps, peptic ulcer disease, Epstein–Barr virus (EBV) infection, pernicious anemia (Kelley and Duggan, 2003), obesity (Mayne and Navarro, 2002), chronic reflux (Mayne and Navarro, 2002), use of table salt (Boeing et al., 1991b), the frequency of eating hot meals (Boeing et al., 1991b), irregular eating patterns (Boeing et al., 1991b), ionizing radiation, and asbestos exposure (Kelley and Duggan, 2003). Studies provide evidence that some dietary factors and micronutrients may protect against gastric cancer. In this chapter, we review the epidemiological evidence regarding specific dietary factors in the etiology of gastric cancer. Furthermore, we also discuss the findings of previous and ongoing chemoprevention trials for gastric cancer.

DIETARY FACTORS AND GASTRIC CANCER Fruit and Vegetable Consumption The dominant dietary hypothesis is that fresh fruits and vegetables, or contained micronutrients and antioxidants, are protective against gastric cancer (Kelley and Duggan, 2003). Epidemiological studies have consistently found a decreased risk of gastric cancer associated with frequent consumption of fresh fruits and vegetables independent of other dietary factors. Most of the cohort studies showed a relative risk (RR) of 0.5–0.9, and case–control studies showed an odds ratio (OR) of 0.1–0.8 (Lam, 1999). The results are still remarkable even after adjustment for total caloric intake (Kono and Hirohata, 1996). The World Cancer Research Fund/American Institute of Cancer Research (WCRF/AICR) 1997 report and the COMA 1998 report concluded that there was moderate consistent evidence of the protective effect of vegetable and fruit consumption on gastric cancer (Ogimoto et al., 2000; Gonzalez, 2002). They also considered that some of those findings might be confounded by H. pylori infection. However, the strength of association, dose–response relationship, and consistency of results from those studies went against their doubts (Gonzalez, 2002). Furthermore, WCRF/AICR suggests that ~66–75% of gastric cancer risk could be reduced with high intake of fruits and vegetables in the past decade (Ogimoto et al., 2000; Gonzalez, 2002). The meta-analysis suggests that 40–50% of gastric cancer incidence could be prevented with high intake of fruits and vegetables (Steinmetz and Potter, 1991; Norat and Riboli, 2002).

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Several studies consistently found that high vegetable intake, especially when consumed raw, may decrease gastric cancer risk (Jedrychowski et al., 1986; Kono et al., 1988; Gonzalez et al., 1991; Kono and Hirohata, 1996; Gao et al., 1999; Gonzalez, 2002). A Hawaiian study (Haenszel et al., 1972) observed a 40–50% decrease in gastric cancer risk among those who consumed high levels of each of the following Western-style vegetables: tomatoes, celery, corn, lettuce, and onions. A Polish study (Boeing et al., 1991b) found that increased consumption of vegetables and fruits, especially onions and radishes, decreased risk. For leeks, a Belgian study showed a protective effect against gastric cancer (Tuyns et al., 1992). Finally, a Chinese study suggested that tomato consumption may decrease risk (Gao et al., 1999). Animal studies and in vitro studies that have suggested that garlic and onion extracts, compounds or their synthetically prepared analogues, may inhibit the development of several tumors (Dorant et al., 1993) provide evidence of the anticarcinogenic potential of several bioactive compounds in allium vegetables (Fleischauer and Arab, 2001). Interest has grown in the protective roles and mechanisms that allium vegetables may have in the etiology of stomach cancer. The allium genus of vegetables includes garlic, onions, leeks, scallions, chives, and shallots. These vegetables are characterized by a composition that is high in flavonols and organosulfur compounds (Fleischauer and Arab, 2001). A study in China (You et al., 1989) found that persons in the highest quartile of intake of total consumption of all allium vegetables (garlic, garlic stalks, leeks, Chinese chives and onions) experienced only 40% risk of those in the lowest quartile. Another Chinese study found that in a high-incidence area for gastric cancer, frequent intake of allium vegetables (including garlic, onion, Welsh onion, and Chinese chives) was inversely associated with the risk of gastric cancer (Gao et al., 1999). Garlic is the most studied allium vegetable. In a meta-analysis of the protective effect of garlic on the risk of gastric cancer, the RR estimate was 0.53 (95% CI, 0.31–0.92) (Fleischauer et al., 2000). However, when it comes to garlic products, low study power and lack of product variety limit the reliability of conclusions (Fleischauer et al., 2000). Onions are also a popular allium vegetable; however, the findings are inconsistent (Kono and Hirohata, 1996). A possible explanation for this inconsistency is that the preparation and serving methods of onions vary in different countries, and the effective contents of the vegetable may decrease depending on the cooking method.

raised the possibility that starchy foods may be a risk factor for gastric cancer. Cereals, rice, wheat, noodles, pastas, breads, potatoes, and millets are classified as high-starch foods. One hypothesis is that high-starch, low-protein diets may favor acid-catalyzed nitrosation in the stomach as a result of the poor buffering capacity of such diets (Kono and Hirohata, 1996). Low-protein diets may decrease gastric mucous production and enhance carcinogen absorption (Kono and Hirohata, 1996). They may also cause mechanical damage in the gastric mucosa (Kono and Hirohata, 1996). Another hypothesis is that monotonous diets high in starchy food pose an increased risk, probably because they are deficient in protective dietary constituents (Parkin et al., 2001b). For example, there is an inverse colinearity association observed between dairy product consumption and starchy food consumption (Kono and Hirohata, 1996). It is not easy to compare the studies among different countries or regions because of the huge variation in type and amount of starchy food consumed. Starchy food consumption is higher and less varied in most Asian countries compared with Western countries (Kono and Hirohata, 1996). Therefore, the findings of studies of high-starch food intake on gastric cancer risk are not as consistent as those of fresh fruits and vegetables or salty food intake (Kono and Hirohata, 1996). Most studies found that high consumption of carbohydrates, starch, or cereals increases the risk of gastric cancer (Bjelke, 1974; Modan et al., 1974; Risch et al., 1985; Trichopoulos et al., 1985; Graham et al., 1990; Buiatti et al., 1990; Tuyns et al., 1992; Ramon et al., 1993; Ji et al., 1996; Mathew et al., 2000), whereas other studies found opposite results (Correa et al., 1985; Gonzalez et al., 1991; Memik et al., 1992; Hansson et al., 1994). In the WCRF/AICR 1997 report, for instance, Ogimoto et al. (2000) suggested that whole-grain intake may be a protective factor for gastric cancer. For individual foods, pasta was found to increase the risk of cancer in Greece (Trichopoulos et al., 1985) and Italy (La Vecchia et al., 1987; Munoz et al., 1997). Rice consumption may increase gastric cancer risk of cancer in India (Mathew et al., 2000), Hawaii (among those of Japanese descent) (Haenszel et al., 1972), and Portugal (Azevedo et al., 1999), but not in Japan (Masaki et al., 2003) and China (You et al., 1988). For bread, several studies found that white bread consumption may increase risk (Wu-Williams et al., 1990; Tuyns et al., 1992; Memik et al., 1992; Ji et al., 1996; Munoz et al., 1997), while whole meal bread or nonwhite bread was found to reduce risk (Jedrychowski et al., 1986; Boeing et al., 1991a,b; Hansson et al., 1994).

Starchy Food

Salt and Salty Food

High gastric cancer risk regions (Japan, China, and Korea) also have been characterized as “high starchy food intake areas” (Mettlin, 1986); therefore, researchers have

The hypothesis that excess salt intake could be involved in the etiology of stomach cancer was first presented in 1965. It was postulated that the continuous use of high doses

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of salt would result in early atrophic gastritis, thereby increasing the later risk of stomach cancer (Joossens et al., 1996; Kelley and Duggan, 2003). Although salt is not a carcinogen by itself, it was hypothesized that salt may serve as a co-initiator and promoter in gastric carcinogenesis (Kono and Hirohata, 1996). The mechanism by which salt may be a co-initiator is by irritating gastric mucosa and initial lesions and, thereby, introducing superficial gastritis and atrophic gastritis. The mechanism of the promoting effect is by damaging stomach mucosa and thereby increasing DNA synthesis and cell proliferation, and thus increasing the possibility of endogenous mutation. These mechanisms were demonstrated by animal models (Kono and Hirohata, 1996). Most of the studies estimated salt intake by measuring intake of salted fish, cured meat, and salted vegetables; only a few studies quantitatively estimated total salt intake (Kono and Hirohata, 1996; Ogimoto et al., 2000). Of the studies exploring the association between total salt intake and gastric cancer risk, six of them found positive associations (La Vecchia et al., 1987; Graham et al., 1990; Ramon et al., 1993; Nazario et al., 1993; Sriamporn et al., 2002; van den Brandt et al., 2003), and only one did not (Risch et al., 1985). The results suggest that heavy use of salt is compatible with a 50% increase in gastric cancer risk (Reed, 1993). For studies of salted fish, cured meat, and salted vegetable intake, almost all of them found an increased risk of gastric cancer (Kono and Hirohata, 1996). However, because of the huge variety of these preserved foods, the results may be confounded by other components of the foods.

Nitrite, Nitrate, and N-nitroso Compounds N-nitroso compounds are known animal carcinogens, including N-nitrosodimethylamine and N-nitrosodiethylamine, which are classified as group 2A carcinogens. There is extensive evidence that they are carcinogenic to the stomach and other organs in animals (Mirvish, 1983; Lam, 1999). Such compounds may be formed in the human stomach from dietary nitrites or nitrates (Kelley and Duggan, 2003). Nitrate reacts with amines, amides, and other proteins and may be reduced bacterially to nitrites with the subsequent formation of N-nitroso compounds (Forman, 1989; Kono and Hirohata, 1996; Lam, 1999). The reduction of nitrates to highly reactive nitrites occurs through the action of nitrate-reducing bacteria in the saliva and hypoacidic stomach (Reed, 1993). However, endogenous N-nitroso compounds may also occur under normal gastric conditions (Reed, 1993). The major sources of nitrites and nitrates exposure for humans are vegetables, cured meat, and drinking water (Kono and Hirohata, 1996). However, the evidence from epidemiological studies on dietary nitrates and gastric cancer is relatively weak. Of 15 ecological studies of nitrate exposure and gastric cancer risk

in 10 countries, one-third showed increased risks, one-third showed decreased risks, and another third showed no association (Forman, 1989). Similar results were found in case-control studies. Seven found that nitrate may increase gastric cancer risk, but the associations were relatively weak (Risch et al., 1985; Correa et al., 1985; Buiatti et al., 1990; Boeing et al., 1991a; Palli et al., 1992; Gonzalez et al., 1994; Mayne et al., 2001; Kim et al., 2002), whereas others observed an inverse association (Hansson et al., 1994; Palli et al., 2001). There are some factors that may confound the association between nitrates and gastric cancer. Concomitant intake of vitamin C and α-tocopherol in vegetables may counteract the increased risk of nitrates in vegetables (Lam, 1999). Many studies have evaluated nitrate exposure by evaluating cured meat or nitrate-cured food consumption. However, the results also may be due to other preservation compounds contained in those foods. Another issue is that nitrate intake may be an index of vegetable intake (Kono and Hirohata, 1996). Since vegetable intake is considered a protective factor for gastric cancer, the effect of nitrates may be obscured. In conclusion, more research and better dose evaluation methods are necessary to address this relation.

Soybean Products Interest in soybean products and their role in gastric cancer prevention has increased among researchers in East Asian countries. Soybeans are an abundant source of isoflavones, which are antioxidants and possess other antitumor activities, including inhibition of angiogenesis, topoisomerase, and tyrosine kinase (Wu et al., 2000). There are two main categories of traditional soy foods: nonfermented and fermented. The main nonfermented soy foods include soy milk, tofu (bean curd), soybeans, and soy nuts. The main fermented soy foods include soy paste (miso in Japan) and fermented soybeans (natto in Japan) (Wu et al., 2000). The gastric cancer estimates for nonfermented soy foods were 0.61 (95% CI, 0.38–0.98) for the cohort studies and 0.73 (95% CI, 0.63–0.83) for the case-control studies (Wu et al., 2000). The meta-analysis estimates for fermented soy foods and gastric cancer risk were 1.13 (95% CI, 0.85–1.49) for cohort studies and 1.30 (95% CI, 1.13–1.50) for case-control studies (Wu et al., 2000). These results suggest that nonfermented soy food consumption may decrease risk of gastric cancer, whereas fermented soy foods or other compounds may increase risk.

Green Tea The relation between tea consumption and gastric cancer has been addressed in many studies. The active components in green tea are polyphenols and mainly epigallocatechin gallate. The tea leaf polyphenol oxidase mediates

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oxidation to oolong and black tea, yielding other polyphenols, theaflavin and thearubigins (Weisburger and Chung, 2002). Experimental studies reveal that many polyphenolic compounds have demonstrated anticarcinogenic activities in animal models (Yang et al., 1997). Black tea was also effective, although the activity was weaker than green tea in some experiments. Decaffeinated tea preparations were also active in many model systems (Yang et al., 1997). The possible protective mechanisms of green tea on gastric cancer are (1) its action as an antioxidant, (2) the specific induction of detoxifying enzymes (including cytochrome P450 1A1, 1A2 and 2B1, and glucuronosyl transferase), (3) its molecular regulatory functions on cellular growth, development, and apoptosis (Hayakawa et al., 2001), and (4) a selective improvement in the function of the intestinal bacterial flora (Weisburger and Chung, 2002). There is no conclusive result on gastric cancer when combining the observations on black tea and green tea (Lam, 1999). Looking at green tea only, six case-control studies (Kono et al., 1988; Yu et al., 1995; Ji et al., 1996; Inoue et al., 1998; Setiawan et al., 2001; Mu et al., 2003) observed a reduced risk of gastric cancer with a high level of green tea consumption, whereas prospective studies did not (Galanis et al., 1998; Tsubono et al., 2001; Hoshiyama et al., 2002). For black tea, most of the studies found no association with the risk of gastric cancer. The difference between green tea and black tea is the length of the fermentation process (Weisburger and Chung, 2002). Black tea is fermented for a longer time than green tea; therefore, in black tea some active components may be reduced or changed by the additional process.

Alcohol Although drinking alcohol is an established cancer risk factor, ethanol per se is not a carcinogen. Although several possible mechanisms have been proposed (Blot, 1992), the mechanism by which alcohol induces cancer is not clear. The main components of all alcoholic beverages are ethanol and water; beer also contains substantial amounts of carbohydrates (IARC, 1988). The hypothesized mechanism is that ethanol could act as a syncarcinogen or a cocarcinogen. High concentrations of ethanol may cause local irritation in the upper gastrointestinal tract in humans after consumption of alcoholic beverages. Furthermore, ethanol will be oxidized to acetaldehyde, resulting in increased levels of acetaldehyde in the liver and blood. Acetaldehyde is a recognized animal carcinogen (IARC, 1985) and is suspected to be the key substance in alcohol-related cancer (IARC, 1988). A 1994 review by Franceschi and La Vecchia of the experimental, descriptive, and analytical evidence relating to alcohol and gastric cancer found little to support an association. However, a meta-analysis found that alcohol consumption may elevate gastric cancer risk: ORs were 1.07

(95% CI, 1.04–1.10), 1.15 (95% CI, 1.09–1.22), and 1.32 (95% CI, 1.18–1.49) with alcohol intake of >25 g/ day, 50 g/day, and 100 g/day, respectively (Bagnardi et al., 2001a,b). The results suggest that alcohol may be an important risk factor for gastric cancer.

Dietary Fat and Animal Protein There is limited evidence that dietary fat and animal protein is associated with gastric cancer risk. Most of these studies found that fat and animal protein consumption may increase risk of gastric cancer (Zhang et al., 1997b; LopezCarrillo et al., 1999; De Stefani et al., 2001; Mayne et al., 2001; Palli et al., 2001; Chen et al., 2002; Thomson et al., 2003), whereas other studies failed to observe similar results (Reed, 1993; Ogimoto et al., 2000). Animal protein and fat consumption may induce reflux symptoms (Terry et al., 2000) and well-done meat may contain carcinogens (de Meester and Gerber, 1995). Japanese studies report a significantly positive association between grilled fish consumption and gastric cancer risk and suggest that grilled fish intake may be a risk factor for Japanese gastric cancer (Ogimoto et al., 2000).

Other Dietary Factors Several studies have found that dietary fiber consumption was inversely associated with risk of gastric cancer (Tzonou et al., 1996; Zhang et al., 1997b; Terry et al., 2001; Chen et al., 2002). A Korean study (Kim et al., 2002) found that kimchi (prepared with salted Chinese cabbage and red pepper, etc.) consumption may protect against gastric cancer. Two case-control studies conducted in Mexico (Lopez-Carrillo et al., 1994, 2003) reported that chili pepper consumption may increase the risk of gastric cancer. Some studies suggest that milk consumption may be related to increased gastric cancer risk (Kneller et al., 1991; Memik et al., 1992; Hansson et al., 1993). Conversely, Boeing et al. (1991b) found that consumption of cheese products was associated with decreased gastric cancer risk. Self-reported use of supplemental calcium/Tums was found to be associated with a significant increase in the risk of gastric cancer because it may be associated with gastroesophageal reflux disease (GERD) (Mayne et al., 2001). A Swedish population-based case-control study (Akre et al., 2001) observed that aspirin users had a moderately reduced risk of stomach cancer (RR = 0.7), however, there was a significant inverse trend in risk with frequency of use.

MICRONUTRIENTS With consistent observations that fresh fruit and vegetable intake may protect against gastric cancer, researchers

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hypothesize that the effect may be due to the micronutrients they contain. Vegetables and fruits are rich in vitamin C and carotenoids, and vegetables are also major sources of vitamin E and selenium (Kono and Hirohata, 1996). All of these micronutrients have antioxidative properties with different mechanisms. To further understand the protective mechanisms of fruits and vegetables, many studies have evaluated the effect of individual micronutrients in several populations.

Vitamin C The current U.S. recommended daily allowance (RDA) for vitamin C for adults (>19 years) is 90 mg/day for men and 75 mg/day for women (Frei and Trabe, 2001). Vitamin C is widely distributed in fresh fruits and vegetables. It is present in fruits like oranges, lemons, grapefruits, watermelons, papayas, strawberries, cantaloupe, mangoes, pineapples, raspberries, and cherries. It is also found in green leafy vegetables, tomatoes, broccoli, green and red peppers, cauliflower, and cabbage (Naidu, 2003). Most prospective and case-control studies have found a significant protective effect of dietary vitamin C intake on gastric cancer (Risch et al., 1985; Correa et al., 1985; La Vecchia et al., 1987; You et al., 1988; Buiatti et al., 1990; Boeing et al., 1991a; Ramon et al., 1993; Hansson et al., 1994; Gonzalez et al., 1994; De Stefani et al., 2001; Mayne et al., 2001; Palli et al., 2001). Vitamin C is an important dietary antioxidant; it significantly decreases the adverse effect of free radical species, which can cause oxidative damage to macromolecules such as lipids, DNA, and proteins (Naidu, 2003). Oxidative damage is implicated in chronic diseases including cardiovascular disease, stroke, cancer, neurodegenerative diseases, and cataractogenesis (Naidu, 2003). Another potential mechanism of vitamin C protection against gastric cancer is its function as a free radical scavenger and inhibitor of the formation of potentially carcinogenic N-nitroso compounds in the stomach (Naidu, 2003).

Vitamin E The term vitamin E covers eight forms that are produced by plants alone: α-, β-, γ-, and δ-tocopherol and α-, β-, γ-, and δ-tocotrienol (Brigelius-Flohe et al., 2002). Tocotrienols have an unsaturated side chain, whereas tocopherols contain a phytyl tail with three chiral centers that naturally occur in the RRR configuration (Brigelius-Flohe et al., 2002). The mechanism of vitamin E on gastric cancer is the same as that for vitamin C. It acts as an antioxidant and may also inhibit the formation of N-nitroso compounds in the stomach. Vitamin C reduces N-nitroso compounds in the aqua phase, whereas vitamin E reduces N-nitroso compounds in the lipid phase.

The association between vitamin E and gastric cancer requires further research, as only three case-control studies (Buiatti et al., 1990; Hansson et al., 1994; Nomura et al., 2003) show significant inverse associations between vitamin E and gastric cancer.

Selenium The antioxidative effect of selenium is linked with the activity of glutathione peroxidase, which acts against oxidative tissue damage (Kono and Hirohata, 1996). In animal studies, selenium can inhibit the development of several types of tumors (Kono and Hirohata, 1996). Dietary assessment of selenium intake is difficult because selenium content within food depends on the selenium content in soil (Kono and Hirohata, 1996). Vegetables and fruits contain selenium as well; therefore, it may influence the result of intervention trials. The selenium-rich fruits and vegetables include bananas, kiwi, strawberry, lima beans, peas, mushrooms, kale, and nuts. Epidemiological studies using serum selenium levels to estimate previous exposure show that low serum selenium levels are associated with increased gastric cancer risk (Knekt et al., 1990; Pawlowicz et al., 1991; Kabuto et al., 1994; Mark et al., 2000).

β-Carotene, Vitamin A, and Retinol β-Carotene, like vitamins C and E, is a well-known free radical–trapping agent (Ferguson, 1999). β-Carotene, a violet to yellow plant pigment, can be converted to vitamin A by enzymes in the intestinal wall and liver. Retinol is an alcohol chemical form of vitamin A (Ferguson, 1999). Retinol is converted to retinal by metabolic oxidation and is biologically essential for light perception in the retina. Retinol is further transformed into retinoic acid by strictly controlled metabolic oxidation (Kakizoe, 2003). Vitamin A and its analogues play important roles in cellular processes related to carcinogenesis (Ferguson, 1999). The important biological function of vitamin A is mainly through retinoic acid, which has a close relationship with important cellular functions such as morphogenesis, cellular proliferation, and cellular differentiation (Kakizoe, 2003) and may prevent the malignant transformation of cells (Kono and Hirohata, 1996). Most case-control studies support the protective role of β-carotene on the risk of gastric cancer (Risch et al., 1985; La Vecchia et al., 1987; You et al., 1988; Buiatti et al., 1990; Ramon et al., 1993; Hansson et al., 1994; Kaaks et al., 1998; Azevedo et al., 1999; Ekstrom et al., 2000; Mayne et al., 2001; Jedrychowski et al., 2001; Nomura et al., 2003). There are also studies that found that retinol (Risch et al., 1985; Hansson et al., 1994; Jedrychowski et al., 2001), vitamin A (Azevedo et al., 1999; De Stefani et al., 2000),

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and β-carotene (De Stefani et al., 2000) intake have inverse associations with gastric cancer risk.

Other Micronutrients Several studies have examined the association between other micronutrients and the risk of gastric cancer. The micronutrients that may have a protective effect are folate (Mayne et al., 2001; Nomura et al., 2003), vitamin B6 (Kaaks et al., 1998; Mayne et al., 2001), vitamins B1 and B3 (Kaaks et al., 1998), and lycopene (De Stefani et al., 2000). Those that may increase gastric cancer risk are vitamins B12 (Mayne et al., 2001) and B2 (Kaaks et al., 1998).

PREVENTION TRIALS FOR GASTRIC CANCER Several chemoprevention trials have been done or are currently ongoing for gastric cancer prevention in the general population. Chemoprevention is defined as the use of specific agents to suppress or reverse carcinogenesis and thereby to prevent the development of cancers (Greenwald and Kelloff, 1996). Those trials are all focused on evaluating the effect of micronutrients that may reduce gastric cancer risk in several high-risk populations. The results from those trials may provide more insight into how these micronutrients prevent gastric cancer.

Nutrition Intervention Trials in Linxian, China Linxian, China, has one of the highest gastric cancer rates in the world. In 1985, 29,584 healthy adults aged 40–69 years were recruited from four Linxian communes for a trial in which they received daily vitamin and mineral supplements. Mortality and cancer incidence were ascertained from March 1986 to May 1991 (Blot et al., 1993). This design enabled testing for the effects of four combinations of nutrients: (1) retinol and zinc, (2) riboflavin and niacin, (3) vitamin C and molybdenum, and (4) β-carotene, vitamin E, and selenium. Doses ranged from one to two times the U.S. RDAs. Total mortality was 13% lower among those receiving supplementation with β-carotene, vitamin E, and selenium (Blot et al., 1993). Gastric cancer incidence was 16% lower, and mortality was 21% lower. The prevalence of gastric cancer among participants receiving retinol and zinc was 62% lower than among those not receiving those supplements (Taylor et al., 1994). The pretrial serum micronutrient level analysis showed that the highest quartile of selenium was strongly associated with a reduced risk of gastric cancer (RR = 0.47; 95% CI, 0.33–0.65). The αtocopherol level was also associated with reduced gastric cancer, but was not significant. The serum β-carotene and γtocopherol levels were not associated with the incidence of gastric cancer (Taylor et al., 2003).

The Alpha-Tocopherol, Beta-Carotene Study in Finland In the Alpha-Tocopherol, Beta-Carotene (ATBC) study, 29,133 male smokers aged 50–69 years received either αtocopherol (50 mg), β-carotene (20 mg), both agents, or a placebo daily for 5–8 years (The ATBC Cancer Prevention Study Group, 1994). Ascertainment of cancer cases during the intervention phase was via the Finnish Cancer Registry, which provides almost 100% case coverage. Neither αtocopherol nor β-carotene supplementation had any association with end-of-trial prevalence of gastric cancer (Varis et al., 1998). However, the incidence of gastric cancer was slightly higher among men receiving β-carotene than among those not receiving it. Subgroup analyses by histological type suggested an increased risk of intestinal-type cancers with β-carotene. There were no differences across anatomical locations (cardia/noncardia) in the effects of α-tocopherol or β-carotene supplementation (Malila et al., 2002).

Randomized β-Carotene Trial of Male Physicians in the United States A randomized, double-blind, placebo-controlled trial of β-carotene (50 mg on alternate days) recruited 22,071 male physicians, 40–84 years of age, in the United States (Hennekens et al., 1996). The trial began in 1982 to December 31, 1995. Less than 1% had been lost to follow-up, and compliance was 78% in the group that received β-carotene. Among 11,036 physicians randomly assigned to receive βcarotene and 11,035 assigned to receive placebo, virtually no difference was observed in the incidence of gastric cancer.

Other Trials Several prevention trials are still ongoing. Hong initiated the first clinical trials of green tea extract with humans in the United States, in a population that did not previously consume green tea regularly. In 1997, the U.S. Food and Drug Administration (FDA) approved a phase I clinical trial with green tea capsules (Kakizoe, 2003). Another randomized trial is in progress to assess the effects of diet supplementation with β-carotene and vitamin C on the development of gastric cancer for high-risk persons with chronic atrophic gastritis in Akita, Japan, the region with the highest mortality from gastric cancer in Japan (Kakizoe, 2003). The development of gastric cancer progresses from chronic gastritis to gastric atrophy, intestinal metaplasia, dysplasia, and finally invasive cancer (Correa, 1992). Several chemoprevention trials focus on a gastric premalignant lesion instead of gastric cancer. A chemoprevention trial in Columbia (Correa et al., 2000) with 1219 volunteers

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was assigned to receive anti–H. pylori triple therapy and/or dietary supplementation with ascorbic acid, β-carotene, or their corresponding placebos. The results found that all three interventions resulted in a significantly protective effect by increasing the rate of regression of cancer precursor lesions in the gastric mucosa. The results from those trials may provide more clues regarding gastric cancer prevention.

CONCLUSION AND FUTURE PERSPECTIVES In conclusion, many present epidemiological studies provide evidence for the role of nutrition and diet on the risk of gastric cancer. However, the limitations of different study designs need to be considered. Although animal studies provide important evidence regarding the mechanisms involved, evidence regarding humans is still based primarily on case-control studies, which are susceptible to recall and selection bias. Prospective studies may provide better evidence, but such studies require long-term follow-up. Some prospective and case-control studies use serum levels to estimate current micronutrient consumption. The metabolism and half-life of these metabolites must be considered. Furthermore, any retrospective studies of gastric cancer must consider the health consequences of gastric cancer. Nutrient absorption may decrease as a result of gastric cancer. Gupta and Ihmaidat (2003) found an average of 15% weight loss, reduced indices of body fat, body protein, serum albumin, and total iron-binding capacity in gastric cancer patients. Another issue pertaining to nutrition factors and gastric cancer research is the role of H. pylori infection. Not only is it an important risk factor, but it may interact with several micronutrients. H. pylori may be associated with various micronutrient deficiencies, including vitamins A, E, C, and B12 (Lacy and Rosemore, 2001; Yakoob et al., 2003) in both children and adults. Moreover, in vitro studies show that vitamin C may inhibit the growth of H. pylori in vitro and in vivo (Zhang et al., 1997a; Wang et al., 2000). Interest in how dietary and micronutrient factors play preventive roles in gastric cancer is still growing. More welldesigned epidemiological studies and clinical trials are necessary to clarify the mechanisms involved. Future perspectives of gastric cancer studies may focus on the study of molecular markers and gene–nutrient interaction epidemiology studies. The results may help to improve our understanding of genetic aspects of nutrition and gastric cancer and to aid in the future planning and designing of an appropriate intervention study to help establish prevention strategies for both high-risk and general populations.

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25 Pancreatic Cancer DIANE M. HARRIS, MANISH C. CHAMPANERIA, AND VAY LIANG W. GO

INTRODUCTION

BACKGROUND

Pancreatic cancer is one of the most devastating of all malignancies. It is the fourth leading cause of cancer death in both males and females in the United States, with the lowest survivability rates of any cancer (American Cancer Society [ACS], 2004). The poor prognosis is in large part due to the fact that most pancreatic cancer patients are diagnosed with advanced disease. However, in spite of its lethality, pancreatic cancer has been insufficiently studied at the basic and clinical levels. In 2001, the National Cancer Institute (NCI) convened an expert Progress Review Group that set an agenda for research priorities in pan-creatic cancer (see the report at http://prg.nci.nih.gov/pancreatic/ pancreatic.pdf) (Kern et al., 2001). Among their conclusions was that a critical component in battling pancreatic cancer is understanding environmental risk factors and gene– environmental interactions to identify and treat premalignant lesions and high-risk candidates for prevention. A subsequent workshop held at the National Institutes of Health (NIH) in 2002 was entitled “Nutritional Links to Plausible Mechanisms Underlying Pancreatic Cancer” (Hine et al., 2003). Among the dietary factors discussed were vegetable and fruit intake, as well as related health and lifestyle factors such as alcohol intake, long-standing diabetes, and body mass index (BMI). This chapter outlines the current understanding of the pathogenesis of pancreatic cancer, particularly the identification of the preneoplastic lesion PanIN and some of the most common genetic alterations that have been described in this process. The known and postulated dietary and related lifestyle risk factors are reviewed, and where possible, their relevance to particular molecular targets in the carcinogenesis process is described.

In the United States, ~30,000 cases of pancreatic cancer are diagnosed yearly, with an equal number of deaths (ACS, 2004). In 2004, the expected incidence rates of pancreatic cancer ranked it ninth and tenth among the cancer sites in women and men, respectively. However, the expected rate of deaths from pancreatic cancer place it fourth among men and fifth in women because of the very low survival rates of untreated patients. For all stages combined, the 1-year survival rate is 19% and the 5-year survival rate is 4% (Yeo et al., 2002). This grave prognosis is a consequence of the late development of clinical symptoms in the progression of the disease, so 80% of cases are metastatic at the time of diagnosis. Around 15–20% of patients have potentially resectable pancreatic cancer (including no evidence of extrapancreatic involvement of the tumor, demonstration of fully patent superior mesenteric/portal veins, and no evidence of tumor encroachment on the arterial celiac axis or the superior mesenteric artery); however, even in optimally staged patients, the 5-year survival rate is only 20% (Li et al., 2004a). Therefore, most patients die of pancreatic cancer a short time after diagnosis, making this truly a devastating disease. The majority (80–90%) of pancreatic cancers are ductal adenocarcinomas, although other rarer pancreatic neoplasms are found, including acinar cell carcinoma, pseudopapillary neoplasm, pancreatoblastoma, serous cystadenoma, mucinous cystadenocarcinoma, intraductal papillary-mucinous neoplasm, and undifferentiated carcinoma with osteoclastlike giant cells (Hansel et al., 2003). There is extensive interaction between the endocrine and exocrine portions of the pancreas during pancreatic carcinogenesis; however, malignancies rarely derive from the acinar cells that form the bulk


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Chapter 25/Pancreatic Cancer

of the pancreas, and islet cell carcinomas account for 85%) of K-ras mutations among all human cancers (Li et al., 2004a). Most of the mutations change codon 12 from glycine (wild type) to aspartic acid

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or valine. Other mutations, such as in codons 13 and 61, occur less frequently (Bardeesy and DePinho, 2002). Mutant K-ras genes shed from PanINs have been identified in bile, stool, pancreatic brushings, duodenal aspirate, and pancreatic juice samples (Caldas, 1999; O’Mahony and Sreedharan, 2001). Ras mutations have been associated with cigarette smoking, as well as alcohol and coffee consumption (Li and Jiao, 2003; Li et al., 2004a). However, because K-ras mutations are also found in a significant minority of patients with chronic pancreatitis—the main differential diagnosis for pancreatic cancer—the utility of this approach can be questioned (O’Mahony and Sreedharan, 2001). Ras proteins are involved in cell growth and differentiation and serve as connectors between signals generated at the plasma membrane and nuclear effectors, which eventually affect the production and regulation of other key proteins. Specifically, K-ras mutations impair guanosine triphosphatase (GTPase) activity, thus inhibiting the hydrolysis of guanosine triphosphate (GTP), which in turn keeps the protein constitutively active. The constant ras signaling function activates downstream targets, thus mimicking steady mitogenic stimulus. Waf1/p21 also seems to be coordinately induced with the onset of K-ras mutations, perhaps because of activation of the mitogen-activated protein kinase (MAPK) pathway (Bardeesy and DePinho, 2002). Activation of ras family oncogenes leads to induction of proliferation, survival, and invasion processes through stimulation of a number of downstream pathways. One mechanism includes stimulation of autocrine epidermal growth factor (EGF) signaling. Overexpression of EGF family ligands and receptors is seen in low-grade PanINs, indicating that upregulation of EGF signaling may be an early event in the carcinogenesis pathway (Bardeesy and DePinho, 2002). Oncogenic ras also induces gastrin gene expression via activation of the Raf-MEK-ERK signal transduction pathway (Hine et al., 2003).

Tumor Suppressors Loss of tumor suppressor genes occurs at later stages in the PanIN classification system and facilitates rapid growth of the transformed clone. The commonly silenced tumor suppressor genes include p16, p21, p53, MADH4, and BRCA2. 1. p16: The most commonly mutated tumor suppressor gene in pancreatic cancer is p16. It is located on chromosome 9p and is inactivated in 95% of pancreatic ductal cell carcinomas. The p16 gene is a member of the cyclindependent kinase (CDK) inhibitor family and functions to prevent the phosphorylation of retinoblastoma protein (Rb-1) by CDKs 4 and 6, leading to failure of cell cycle control and unchecked proliferation (Hansel et al., 2003). Loss of p16 in pancreatic ductal adenocarcinomas occurs

through a variable set of alterations, including homozygous deletion, single allelic loss coupled with an intragenic mutation in the second allele, and promoter hypermethylation (Hansel et al., 2003). 2. p53: The p53 tumor suppressor gene is mutated, primarily by missense alterations in the DNA-binding domain, in >50% of pancreatic adenocarcinomas (Bardeesy and DePinho, 2002). Mutations in the p53 gene arise in later stage PanINs that have acquired significant dysplastic characteristics, indicating that loss of p53 promotes malignant progression. The p53 gene, located on chromosome 17p, encodes a transcription factor protein that participates in the upregulation of cell cycle arrest in G1 and G2 following DNA damage or apoptosis pathways. Under genotoxic stress, p53 forms a tetramer, binds to DNA, and upregulates genes involved with cell cycle arrest or apoptosis pathways (Hansel et al., 2003). Loss of p53 allows cell cycle progression in the presence of DNA damage and undoubtedly promotes the rampant genetic instability that characterizes pancreatic adenocarcinoma; pancreatic tumors exhibit aneuploidy and complex cytogenetic rearrangements, as well as cytological heterogeneity within tumors, consistent with ongoing genomic rearrangements (Bardeesy and DePinho, 2002). This mechanism may involve dysfunction of telomeres, as pancreatic tumors have shortened telomere length and activation of telomerase is a late event (Bardeesy and DePinho, 2002). 3. TGF-β/DPC4: The Deleted in Pancreatic Cancer 4 (DPC4; also called SMAD4 or MADH4) gene is a tumor suppressor gene that maps to chromosome 18q21 and is mutated or homozygously deleted in ~55% of pancreatic ductal cancers (Hansel et al., 2003). DPC4 seems to be a progression allele for pancreatic adenocarcinoma, as its loss occurs only in later stage PanINs (Bardeesy and DePinho, 2002). Although patients that inherit a germline DPC4 mutation do not have a strong predisposition to pancreatic adenocarcinoma, loss of DPC4 is a predictor of decreased survival in spontaneous adenocarcinoma (Bardeesy and DePinho, 2002). Mothers Against Decapentaplegic homolog (SMAD) proteins are responsible for the signaling pathways of the transforming growth factor-β (TGF-β) superfamily. In a normal epithelial cell, exposure to TGF-β signaling stops cell proliferation by blocking the G1/S cell cycle transition and promotes apoptosis (Bardeesy and DePinho, 2002). Loss of SMAD4 interferes with intracellular signaling cascades downstream from TGF-β and activin, resulting in decreased growth inhibition via loss of proapoptotic signaling or inappropriate G1/S transition (Hansel et al., 2003). 4. BRCA2: Family-associated pancreatic cancer accounts for some 10–15% of pancreatic cancers. A number of different genes have been implicated in familial

Genes Altered in Sporadic Ductal Adenocarcinomas

syndromes of pancreatic cancer. For example, pancreatic cancer is seen in some breast cancer families carrying mutations in breast cancer genes BRCA1 and BRCA2 (Ghadirian et al., 2003). The cumulative risk of pancreatic cancer to age 75 years among BRCA2 gene carriers in the Ashkenazi Jewish population is reported to be ~7%, compared with 85% risk of breast cancer among carriers of BRCA1 and BRCA2 mutations (Ozcelik et al., 1997). Inflammation and Eicosanoid Pathways Inflammation is a significant factor in the development of a number of solid tumor malignancies. The risk of pancreatic cancer development is enhanced in patients with hereditary and sporadic forms of pancreatitis (Farrow and Evers, 2002). Modulators of the inflammatory response include prostaglandins and other hormone-like members of the eicosanoid pathway, which are thought to induce carcinogenesis through action on nuclear transcription sites and downstream gene products important in the control of cell proliferation. Eicosanoids are locally acting hormone-like compounds derived predominantly from arachidonic acid in tissue cells and tumor-infiltrating leukocytes (Wargovich et al., 2001). The best known eicosanoids, the prostaglandins, are produced by the action of the cyclooxygenases (COX), but the lipoxygenase group of enzymes produce the leukotrienes and hydroperoxyeicosatetraenoic acids, which also have important proinflammatory effects (Wallace, 2002). Substantial evidence from animal studies and human epidemiological and clinical trials shows that nonsteroidal anti-inflammatory drugs (NSAIDs), which are inhibitors of COX, are associated with reduced risk of a number of cancers, including those of the colon-rectum, esophagus, stomach, breast, lung, prostate, bladder, brain, and cervix (Wallace, 2002). At least six epidemiological studies have investigated aspirin and other NSAID use in relation to pancreatic cancer risk, and although the epidemiological data are not consistent, experimental data suggest that NSAIDs inhibit pancreatic cancer cell proliferation (Michaud, 2004). COX-2 has been shown to contribute to the malignant phenotype via various activities, including suppressing the host immune system, conferring resistance to apoptosis, stimulating cancer cell growth and invasion, and recruiting new blood vessels in angiogenesis (Eibl et al., 2004). COX2 expression is regulated in part by the transcription factor nuclear factor-κB (NF-κB), a ubiquitously expressed transcription factor that regulates the expression of a number of inflammatory, apoptotic, and oncogenic genes (Farrow and Evers, 2002). COX-2 in normal pancreas is only detectable in pancreatic islets but is found in preinvasive PanINs and is overexpressed in most invasive pancreatic cancers in the cancer cells themselves and not in the tumor stroma (Eibl et al., 2004). Selective COX-2 inhibitors (termed coxibs)

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used in vivo suppress pancreatic cancer growth in an orthotopic (Tseng et al., 2002) and a chemically induced cancer model (Schuller et al., 2002; Furukawa et al., 2003). However, chronic NSAID use in patients leads to toxicities, including gastric ulceration, perforation, or obstruction, which limit their therapeutic use (Wallace, 2002). Newer selective coxibs have been postulated to be safer, but the voluntary worldwide recall of several of these drugs, including Merck’s product rofecoxib (Vioxx), illustrates that the long-term safety of these drugs has yet to be established (U.S. Food and Drug Administration [FDA], 2005). Therefore, research has focused on identifying and understanding the bioactivity of a number of natural nontoxic agents to control inflammatory eicosanoids. Those agents that have been studied in various cancers include the omega-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) such as from fish oils, vitamin A, vitamin E, and a number of botanically derived anti-inflammatory agents, including boswellic acids, bromelain, curcumin, resveratrol, quercetin, and EGCG (Wallace, 2002). One mechanism of action of these compounds is via inhibition of NFκB, which is activated by proinflammatory signals and upregulates COX-2 expression (Bremner and Heinrich, 2002). Eibl et al. (2004) proposed that the COX and the peroxisome proliferator-activated receptor (PPAR) pathways can interact in pancreatic cancer, and they provide evidence that coxibs have effects on COX-independent pathways, particularly the PPAR system, which either augment or even reverse the expected action of the COX inhibitors (Eibl et al., 2004). Although the exact role of PPARγ, the best characterized member of the PPAR family, in tumorigenesis is ambiguous, it has been shown that PPARγ is overexpressed in pancreatic tumors (Motomura et al., 2000; Itami et al., 2001). In addition, PPARγ ligands induce cell cycle arrest with terminal differentiation (Hashimoto et al., 2002; Kawa et al., 2002; Ohta et al., 2002; Toyota et al., 2002; Tsujie et al., 2003a) and apoptosis (Eibl et al., 2001; Hashimoto et al., 2002) and decrease cellular invasion (Hashimoto et al., 2002; Farrow et al., 2003; Motomura et al., 2004). Coxibs may either increase or decrease PPARγ transcription through different mechanisms, and the model of Eibl et al. (2004) postulates that the overall effect of coxibs on PPARγ activity is a balance between positive and negative influences (Eibl et al., 2004). This also implies that the effect of coxibs on PPARγ activation is likely to depend on COX-2 status of the pancreatic cancer cell. Stimulation of PPAR activity by coxibs (with hypothetically protumorigenic results) in COX-2–positive pancreatic cancers may be offset by inhibition of the production of protumorigenic prostaglandins. However, 10–40% of human pancreatic ductal adenocarcinomas are COX-2 negative, and in these cancers, the PPARγ-activating effects of coxibs may dominate. This conflicting mechanism implies that further study

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of pharmaceutical and natural COX-2 inhibitors in cancer prevention needs to include the possibility of PPARγ activation and enhanced tumor growth in COX-2–negative cancers, making genotyping of tumors prior to initiation of treatment necessary. The other arm of the eicosanoid pathway consists of the lipoxygenases (LOX), termed 5-, 12-, and 15-LOX in humans. Expression of 5-LOX is shown to also be upregulated in human pancreatic cancer tissue, as confirmed by immunohistochemistry (Hennig et al., 2002). The staining of the 5-LOX protein was especially evident in the ductal components of more differentiated tumors, but not present in ductal cells from normal pancreatic tissues. Also, hepatic metastases had higher levels of 5-LOX expression than primary tumors. Use of pharmacological inhibitors of the 5-LOX pathway inhibits growth of pancreatic tumor xenografts in athymic nude mice via induction of apoptosis (Tong et al., 2002). In cell culture, the general LOX inhibitor nordihydroguaiaretic acid (NDGA), the specific 5-LOX inhibitor Rev5901, and the 12-LOX inhibitor baicalein all induced apoptosis in pancreatic cancer lines, as well as increased intracellular carbonic anhydrase activity, a marker of cell differentiation (Ding et al., 1999). Also, the 5-LOX pathway inhibitor MK886 diminished pancreatic carcinogenesis induced by transplacental exposure to ethanol and the tobacco carcinogen NNK in hamsters (Schuller et al., 2002). Further studies to validate the role in pancreatic cancer of other LOX enzymes, including 15-LOX, are required. A number of natural compounds found in foods and spices have anti-LOX activities, and some have dual anti-COX and anti-LOX activities, including quercetin, curcumin, and resveratrol, making them potentially attractive anti-inflammatory and anticancer agents (Wallace, 2002; Schneider and Bucar, 2005). Oxidative Stress Cells are normally in a balance between oxidant stress, as evidenced through the generation of reactive oxygen species (ROS, also called oxygen radicals) and other electrophilic species, and antioxidant mechanisms, which deactivate the reactive species. Generation of ROS occurs during acute and chronic pancreatitis, and increased cellular ROS have been associated with carcinogenesis (Sanfey et al., 1984). ROS, including superoxide and hydroxyl radicals and hydrogen peroxide, are products of cellular metabolism and respiration that at high concentrations cause oxidative damage to cellular DNA, protein, and lipids (such as in membranes). Antioxidant systems include the enzymes superoxide dismutase, catalase, and peroxidase, of which glutathione peroxidase is the most prominent (Benzie, 2000). In addition, a number of nutrients, such as the vitamins C and E and selenium, as well as the dietary constituents β-carotene, quercetin, resveratrol, and genistein

and a host of other phytochemicals, have antioxidant properties (Valko et al., 2004). Expression of the antioxidant enzymes has been shown to progressively decrease in pancreatic cells from normal pancreas to chronic pancreatitis to pancreatic cancer in human histology samples, showing that capacity to disable ROS decreases as cells undergo oncogenic transformation (Cullen et al., 2003a). Overexpression of one antioxidant enzyme, MnSOD, attenuated the growth of human pancreatic cancer cells (Cullen et al., 2003b). Furthermore, growth factor stimulation (by serum, insulin-like growth factor-1 [IGF-1], or fibroblast growth factor-2) of pancreatic cancer cells generated ROS via nonmitochondrial NAD(P)H oxidase (Vaquero et al., 2004). This ROS protects cells from apoptosis, demonstrating that ROS are prosurvival antiapoptotic factors in pancreatic cancer cells and suggesting a mechanism for the general resistance to induction of apoptosis by chemotherapy and radiotherapy of pancreatic malignancy. However, increased intake of antioxidant nutrients can diminish the ROS pool and presumably carcinogenesis. High intake of selenium has been shown to reduce the numbers of histologically diagnosed cancerous pancreatic lesions in a hamster model (Kise et al., 1990). Adenomas and carcinomas of the pancreas were significantly reduced in azaserine-treated rats with diets supplemented with β-carotene, vitamin C, or selenium, but not vitamin E (Woutersen et al., 1999). This effect was most prominent during the promotion phase of the carcinogenic process (Appel and Woutersen, 1996b). Similar studies in BOPinduced pancreatic tumors in hamsters were contradictory. In one, vitamin A and C diminished incidence of pancreatic cancer and decreased levels of superoxide dismutase (Wenger et al., 2001), whereas in another study of longer duration, vitamin C did diminish the number of advanced ductular lesions, but vitamin E, β-carotene, or selenium had no effect on tumor development (Appel et al., 1996). In pancreatic cell lines, treatment with vitamins A and D, but not E and K, decreased cell number (Ohlsson et al., 2004). In humans, however, a supplementation trial determining the effects of β-carotene or α-tocopherol on cancer incidence in male smokers showed no effect on the rate of incidence of pancreatic carcinoma or the rate of mortality caused by the disease; this negative result is perhaps explainable by the lack of statistical power because of the small number of pancreatic cancer cases in this cohort (Rautalahti et al., 1999). DNA Methylation A key role of nutrient action is epigenetic, referring to changes in the phenotype that are not due to changes in the genotype, or in other words, changes in gene expression that are transmissible through mitosis but do not involve mutations of the primary DNA sequence itself (Jaenisch and Bird,

Genes Altered in Sporadic Ductal Adenocarcinomas

2003). A critical mechanism for epigenetic gene regulation involves alterations in patterns of DNA methylation. DNA methylation, or the covalent addition of a methyl group to the 5-position of cytosine within CpG dinucleotides, is particularly important in epigenetic control by nutrients. Tumors commonly exhibit widespread global DNA hypomethylation, region-specific hypermethylation, and increased activity of Dnmt enzymes, which catalyze the transfer of methyl groups from S-adenosylmethionine (SAM) to cytosine residues in DNA. Global genomic hypomethylation is linked to induction of chromosomal instability, whereas hypermethylation is associated with inactivation of most pathways of carcinogenesis, including DNA repair, cell cycle regulation, and apoptosis (Ross, 2003). In pancreatic cancer, a number of genes have been shown to be hypermethylated in promoter regions, including p16, RAS association domain family 1A gene (RASSF1A), heparin sulfate D-glucosaminyl 3-O-sulfotransferase-2 (3-OST-2), cyclin D2, suppressor of cytokine signaling-1 (SOCS-1), retinoic acid receptor-β (RARβ), and adenomatous polyposis coli (APC) genes. Promoter hypermethylation in serum DNA may be used as a marker for the early detection of pancreatic cancer cells, and demethylating agents have potential as therapeutic agents (Kuroki et al., 2004). Dietary factors may influence DNA methylation patterns in several ways. First, nutrient inadequacies will influence the supply of methyl groups for the formation of SAM. Dietary factors that are involved in one-carbon metabolism that influence the availability of SAM include folate, vitamin B12 (cobalamin), vitamin B6 (pyroxidine), vitamin B2 (riboflavin), methionine, and choline; excess alcohol intake is known to deplete these nutrients (Lieber, 2000). Other nutrients including zinc, selenium, and retinoic acid will affect global DNA hypomethylation (Ross, 2003). A second way diet alters methylation is by regulating the use of methyl groups, including modifying DNA methyltransferase activity via Dnmt enzymes. Higher Dnmt activity has been observed in tumor cells compared with normal cells, and activity of these enzymes is upregulated with chronic methyl deficiency in an apparent attempt to compensate for diminished SAM supply. In addition, the DNA demethylation process, previously assumed to be passive, may be a regulated activity (Ross, 2003). The subsequent changes in methylation patterns can influence activity of specific genes; this likely occurs through modifying transcription factor–gene interactions through methyl DNA–binding proteins (Ross, 2003). DNA hypomethylation was associated with overexpression of a number of genes known to be upregulated in pancreatic adenocarcinoma, including claudin 4, lipocalin 2, 14-3-3 sigma, trefoil factor 2, S100A4, mesothelin, and prostate stem cell antigen, as measured in pancreatic cancer cell lines and primary carcinomas (Sato et al., 2003).

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The effects of manipulating dietary levels of several methyl donors have been tested in animal models. Methionine enhanced atrophic change of pancreatic acinar cells in hamsters given BOP, indicating that the inhibitory effects on the post-initiation stage of BOP-induced pancreatic carcinogenesis in hamsters could be generally linked to suppression of growth (Furukawa et al., 2000). The effects of choline-deficient diets on carcinogenesis in azaserinetreated rats are inconsistent. In one study an increased incidence of neoplasms in the choline-deficient diet was attributed to the high fat content (20%) of the experimental diet compared with the control diet (5%) (Roebuck et al., 1981). In a separate study, the incidence of azaserineinduced focal hyperplasia was lower in a choline-deficient diet than in a control choline-sufficient group in a study at the same level of fat (14%) (Shinozuka et al., 1978). In another study, results showed an increased incidence of azaserine-induced focal hyperplasia in rats fed a cholinedeficient diet compared with a group fed a choline-deficient diet against a high-fat (30%) background in a short-term study (Andry et al., 1990); however, in contrasting results from another study in rats fed choline-deficient or cholinesufficient high-fat diets, the incidence of spontaneously occurring acinar cell neoplasms was similar in the two groups (Longnecker et al., 1991). These experimental studies suggest that perturbations in DNA methylation can alter cellular differentiation in the pancreas and contribute to toxic injury that predisposes to pancreatic carcinogenesis. As discussed earlier, folate metabolism is integral to epigenetic regulation of genes important in the carcinogenesis pathway. Therefore, the contribution of folate and other methyl donor nutrients is of interest in interpreting the role of diet in pancreatic cancer risk, but few epidemiological studies have explored this relationship. In the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study cohort, the adjusted hazards ratio for pancreatic cancer comparing the highest with the lowest quintile of dietary folate intake was 0.52, demonstrating the importance of folate intake on pancreatic cancer risk. Dietary methionine, alcohol intake, and smoking history did not modify this relation, and no significant associations were observed between dietary methionine, vitamins B6 and B12, or alcohol intake and pancreatic cancer risk (StolzenbergSolomon et al., 1999). However, results from two large prospective cohorts did not support a strong association between energy-adjusted folate intake and risk of pancreatic cancer (Skinner et al., 2004). Because of the presence of well-defined polymorphisms in the enzymes involved in folate metabolism, study populations probably need to be stratified by genotype. For example, there is an interaction of genotype of 5,10-methylenetetrahydrofolate reductase (MTHFR) that catalyzes the conversion of 5,10methylenetetrahydrofolate into 5-methyltetrahydrofolate (the major circulating form of folate), with pancreatic cancer

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risk; those with the least frequent TT genotype displayed increased risk of pancreatic cancer, and a positive interaction between the TT genotype and heavy smoking or heavy alcohol consumption was detected (Li et al., 2005).

Genetic Susceptibility to Xenobiotic Exposure Metabolic activation is required for the effect of many carcinogens, including those of dietary sources, and substantial interindividual variation exists in the metabolic capacity of carcinogen activation and inactivation. Polymorphisms are present in a number of metabolic genes, including cytochrome P450 (CYP isoforms 1A1, 1B1, and 2E1), N-acetyltransferase (NAT1 and NAT2), and glutathione S-transferase (GST M1 and GST T1); no significant main effect of these genes on pancreatic cancer risk is observed. However, a possible gene–environment interaction among individuals with specific environmental exposures (e.g. dietary heterocyclic amine intake and smoking) was observed (Li and Jiao, 2003). In addition to carcinogen-metabolizing enzymes, DNA repair capability after genotoxic insult has a critical interaction with cancer susceptibility. X-ray repair cross-complementing group 1 (XRCC1) is a base excision repair protein that plays a central role in repair of DNA strand breaks and base damage from various exogenous and endogenous agents including oxidants and DNA alkylating agents (Li and Jiao, 2003). The Arg399Gln polymorphism of XRCC1 is significantly associated with the risk of pancreatic cancer among smokers (Duell et al., 2002). Other DNA repair genes, O6-alkylguanine DNA transferase (AGT) and 8oxoguanine DNA glycosylase/DNA-AP lyase (hOGG1), are being studied in the context of pancreatic cancer (Li and Jiao, 2003). Polymorphisms in the genes involved with metabolic activation, detoxification, and repair have all been shown to confer variable susceptibility to a number of cancers. For example, hOGG1 polymorphisms are associated with increased risk for cancers of lung, prostate, and esophagus (Weiss et al., 2005). We are just beginning to understand the interaction of these polymorphisms with dietary factors. In colon cancer, broccoli consumption in combination with the GST M1 null genotype is associated with a lower prevalence of colorectal adenomas because of higher isothiocyanate levels (Lin et al., 1998). Furthermore, potential interactions of the DNA repair system with flavonoids are being explored (Ferguson, 2001). These types of studies linking dietary factors with interindividual variation in the function of gene products, termed gene–nutrient interactions, are critical to understanding individual susceptibility to cancer and potential individualized recommendations for pancreatic disease prevention and management.

THE IMPORTANCE OF DIET The strongest risk factor for pancreatic cancer is age at diagnosis; >80% of cases are diagnosed in individuals between 60 and 80 years of age (Ghadirian et al., 2003). There are racial/ethnic/gender disparities within the U.S. population in incidence and mortality rates of pancreatic cancer, with generally higher rates among males relative to females, African Americans relative to white Americans, and people of Jewish heritage (Hine et al., 2003). The other established probable risk factors for pancreatic cancer include (1) cigarette smoking; (2) long-standing diabetes; (3) chronic and hereditary pancreatitis; and (4) family history of pancreatic cancer. Possible risk factors include (1) noncigarette tobacco use; (2) other medical conditions (gallbladder disease/cholecystectomy, gastrectomy/peptic ulcer); (3) occupational exposures (e.g., organochlorine compounds [DDT, PCBs] and chlorinated hydrocarbon solvents); (4) low socioeconomic status; (5) “heavy” alcohol consumption; (6) dietary factors (e.g., infrequent intake of vegetables, high intake of grilled or charred meats, positive energy balance, and high BMI); and (7) high caloric intake and frequent meals per day (Hine et al., 2003; Li et al., 2004a). Undoubtedly, many of the listed risk factors are interrelated. Disorders of the exocrine pancreas, such as chronic and acute pancreatitis and pancreatic adenocarcinoma, can induce endocrine pancreatic disorders such as diabetes mellitus and islet cancer. In turn, diabetes and glucose intolerance are often associated with exocrine pancreatic dysfunction and may participate in pancreatic carcinogenesis. Epidemiological studies confirm that glucose intolerance is a risk factor for pancreatic cancer, therefore the association is unlikely due to an adverse impact of early pancreatic cancer on β-cell function and insulin may act as a promoter for pancreatic carcinogenesis (Go and Wang, 2005). In fact, insulin resistance may be the unifying factor linking hyperglycemia, diabetes, obesity, and nutrition in pancreatic cancer development (Michaud et al., 2002; Hine et al., 2003). Relative to other cancer sites, fewer studies have been undertaken to explore associations between particular dietary factors and risk for pancreatic cancer. Given the intimate role of the pancreas in digestion and nutrient absorption, however, one can predict that diet may have a large role in pancreatic cancer development and prevention. Unlike other organs of the gastrointestinal tract, the pancreas is not directly exposed to ingested or absorbed nutrients. Therefore, the effects of diet on carcinogenesis are via changes in the metabolic environment of the pancreas and/or exposure to bloodborne agents (World Cancer Research Fund and American Institute for Cancer Research, 1997). In the 1997 review of diet and cancer prepared by the American Institute for Cancer Research/World Cancer Research Fund, the contribution of dietary factors to development of pancreatic

The Importance of Diet

cancer is estimated to be 35%. For most dietary constituents, the findings at the time of the committee’s review were equivocal, with insufficient data to determine association (World Cancer Research Fund and American Institute for Cancer Research, 1997). However, newer data are being examined and publication of new judgments by the committee is anticipated in 2007. Several limitations in conducting research in pancreatic cancer have slowed progress in understanding risk factors in development of this disease. First, there are few relevant preclinical models of pancreatic cancer. The ideal model should provide morphological, clinical, and genetic alterations comparable with those found in human cases (Ulrich et al., 2002). However, some models exhibit tumor formation in cell types other than ductal epithelial cells or lack mutations in genes associated with human cancers. As mentioned previously, one of the most widely studied animal models for pancreatic duct carcinoma is the use of the carcinogen N-nitrosobis(2-oxopropyl)amine (BOP) in Syrian golden hamsters (Mesocricetus auratus), although other carcinogens, such as azaserine, have also been used to induce pancreatic cancer in hamsters and rats. In the BOP/hamster model, invasive pancreatic adenocarcinomas develop in 80–100% of treated animals, with a tumor latency as short as 8 weeks after a single subcutaneous injection. The most common tumors in this model are ductal adenocarcinomas, which display perineural lymphatic invasion and a pronounced desmoplastic reaction similar to the human disease (Hotz et al., 2000). In addition, hamsters with pancreatic cancer exhibit weight loss, diarrhea, jaundice, ascites, vascular thrombosis, and abnormal glucose metabolism, signs that are similar to those displayed clinically in human pancreatic cancer patients. However, certain human cancer biomarkers, such as carcinoembryonic antigen, pancreatic oncofetal antigen, α-fetoprotein, and DU-PAN-2, are infrequently or not expressed by hamster pancreatic cancer cells (Standop et al., 2001). An alternative approach to generation of animal models involves use of implanted human tumor cell xenografts in immunodeficient mice, including nude mice and severe combined immunodeficient (SCID) mice. These methods of tumor induction include direct injection of human pancreatic cancer cells into the pancreas (orthotopic implant), transplantation of tumor fragments from tumors grown in donor animals (ectopic implant), and transplantation of tumor fragments obtained from resected human tumors (Hotz et al., 2000). All of these techniques have shortcomings; for example, direct injection of tumor cells into the pancreas may result in cell loss into the peritoneal cavity, where abdominal organs may be seeded with tumor cells (Hotz et al., 2000). Cell lines have variable characteristics and can exhibit genetic instability in grafts, and the tumors produced generally are necrotic toward the center of the graft. A refined procedure involves implantation of small

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tumor fragments into surgically prepared tissue pockets in the pancreatic parenchyma (Hotz et al., 2003). The limitation of all immunodeficient mouse models is that the impact of the host immune system is negated; however, they do provide a reliable and reasonably reproducible model for evaluating treatment strategies. Few transgenic mice models of spontaneous pancreatic ductal adenocarcinoma are available, but more are in development (Leach, 2004). Transgenic mice with the elastase promoter SV40 early antigen construct (Ela-1-SV40 T) and variations on this construct develop focal acinar cell lesions that develop into carcinomas with a high incidence after 3–6 months (Standop et al., 2001). The mechanism of T-antigen transformation involves inactivation of the tumor suppressor genes p53 and Rb. However, the tumors formed do not carry K-ras mutations. A mouse model of PanIN has been developed through targeted mutation of KRASG12D in progenitor cells of mouse pancreas (Hingorani et al., 2003). In this model ductal lesions are induced to recapitulate the full spectrum of human PanIN and progress to fully metastatic disease. These models hold much promise for study of gene–nutrient interactions in pancreatic carcinogenesis. In vitro and animal studies are very useful in exploring mechanistic aspects, but the ability to generalize findings from preclinical studies to human populations needs to be evaluated specifically in epidemiological and dietary intervention studies (Gold, 1995). In human studies, attempts to link pancreatic cancer to diet are hampered by the fact that the clinical course of the disease is rapid. Most studies of risk factors for pancreatic cancer are case-control studies, with fewer prospective cohort diet studies, which record diet and food intake in healthy patients before disease development (Hine et al., 2003). At the time of determination of an affected individual, many patients are either at a late stage of the disease, too late for interventions, or even too late to provide accurate detailed answers on dietary questionnaires; proxy respondents, such as close relatives, are often used but are notoriously inaccurate (Howe and Burch, 1996). In addition, there is a lack of biomarkers for preclinical and clinical pancreatic cancer, making early detection and evaluation of the effectiveness of intervention regimens difficult. The following sections review the data for the relationship between selected dietary and lifestyle factors and pancreatic cancer risk in epidemiological studies and nutrient modulation of pancreatic carcinogenesis in preclinical models. Although smoking is a clearly identified and very important risk factor for pancreatic cancer, the contribution of this lifestyle factor has been reviewed previously (Schuller, 2002) and is not discussed here.

Obesity, Physical Activity, and Diabetes Several prospective cohort studies have reported elevated risks of pancreatic cancer among overweight and obese

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individuals. In the ACS study of a very large cohort of >900,000 U.S. adults monitored over 16 years, death rates from pancreatic cancer, as well as for a number of other cancers, were increased as BMI increased; relative risks for individuals with BMIs >30 kg/m2 were 1.49 for men and 2.76 for women (Calle et al., 2003). Further analysis showed that after adjusting for BMI, risk of pancreatic cancer was independently increased among men and women who reported a tendency for central weight gain compared with men and women reporting a tendency for peripheral weight gain; accumulation of intraabdominal fat is particularly associated with development of insulin resistance (Patel et al., 2005). In another prospective analysis of two large cohort studies of 163,689 men and women showed that tall height and greater BMI were independently and positively associated with pancreatic cancer (Michaud et al., 2001). In a Swedish cohort study, a 46% increase in risk was observed among individuals who had gained ≥12 kg as adults, compared with those who gained 2–5 kg (Isaksson et al., 2002). However, several case-control studies have also shown no correlations between BMI or weight and pancreatic cancer; a meta-analysis of these studies illustrates the heterogeneity among results of different studies, which may be linked to such factors as use of proxy respondents, self-reporting of anthropometric measures, and lack of adjustment for potential confounding factors of diabetes or smoking (Berrington de Gonzalez et al., 2003). Physiological mechanisms accounting for the relationship between excess adiposity and enhancement of pancreatic cancer growth are unclear. The mature adipocyte is an active endocrine and metabolic organ known to secrete a number of peptide hormones, such as leptin, tumor necrosis factor-α (TNF-α), resistin, and adiponectin—collectively termed adipocytokines—as well as nonesterified fatty acids (NEFAs), which in numerous cancer cell types stimulate proliferation. In vitro studies, however, demonstrate that leptin inhibits, rather than promotes, proliferation of MIA-PaCa and PANC-1 ductal cancer cells; the mechanism and significance of this effect is unknown (Somasundar et al., 2003). Increased release of NEFA, resistin, and TNF-α, as well as reduced release of adiponectin, gives rise to insulin resistance and compensatory hyperinsulinemia (Calle and Kaaks, 2004). Chronic hyperinsulinemia is associated with increased risk of pancreatic cancer (Weiderpass et al., 1998; Silverman, 2001). Elevated insulin stimulates IGF-1 production and activity (McCarty, 2001). High serum levels of IGF-1 and one of its binding proteins IGF binding protein-3 (IGF-BP3) have been associated with increased risk of pancreatic cancer in some studies (Lin et al., 2004), and in vitro exposure to insulin and IGF-1 stimulates pancreatic cancer cell proliferation and inhibits apoptosis (Ohmura et al., 1990; Takeda and Escribano, 1991; Bergmann et al., 1995; Flossmann-Kast et al., 1998; Nair et al., 2001; Yao et al., 2002).

The association between diabetes mellitus and pancreatic cancer is not entirely clear, as clinical diabetes can be one of the early manifestations of pancreatic cancer. A meta-analysis published in 1995 suggested that diabetics have about a twofold increased risk of pancreatic cancer (Everhart and Wright, 1995). A subsequent population-based case-control study of pancreatic cancer in three U.S. cancer registries showed that a diagnosis of diabetes at least 10 years before diagnosis conferred 50% increased risk for pancreatic cancer (Silverman et al., 1999). This association held within each level of BMI. In a prospective cohort study, a group of >35,000 men and women in the Chicago area were monitored for 25 years (Gapstur et al., 2000). Insulin sensitivity was measured at baseline and at incidence of pancreatic cancer during the 25 years of follow-up. They found that risk for pancreatic cancer increased in proportion to the 2-hour serum glucose values measured during the glucose tolerance test. Furthermore, a meta-analysis of 17 casecontrol and 19 cohort or nested case-control studies showed that the age and sex-adjusted odds ratio for pancreatic cancer was increased by 50% in individuals who had type II diabetes diagnosed within 4 years of pancreatic cancer diagnosis (Huxley et al., 2005). These findings suggest that development of pancreatic cancer is associated with abnormality of islet cell function and glucose intolerance precedes the onset of pancreatic cancer. As discussed earlier in this chapter, plasma insulin and postload plasma glucose may be associated with risk of pancreatic cancer. One measure of the ability of individual foods to raise postprandial glycemia and hence blood insulin levels is the glycemic index (GI). This measure classifies the carbohydrate content of individual foods according to their postfeeding glycemic effects relative to a standard glucose load. Consumption of high GI diets is associated with hyperinsulinemia (Augustin et al., 2002). A perhaps more accurate portrayal is glycemic load, which multiplies the GI by the carbohydrate content of an individual food to take into account carbohydrate density of foods. The relationship between GI, glycemic load, and intake of fructose with pancreatic cancer risk was most apparent among those women participating in the U.S. Nurses’ Health Study with elevated BMI or with low physical activity, suggesting a diet high in glycemic load may increase pancreatic cancer risk in women who already have an underlying degree of insulin resistance (Michaud et al., 2002). However, GI and glycemic load, as well as total sugar and total carbohydrate intake, were not associated with pancreatic cancer risk in a Canadian population (Silvera et al., 2005). Overall, the association of diabetes in pancreatic cancer remains unclear, and the fact that 20–30% of pancreatic cancer patients do not develop a glucose metabolic abnormality suggests multiple mechanisms of diabetes in the development of human pancreatic cancer (Saruc and Pour, 2003).


In animal models, decreasing insulin levels seem to inhibit pancreatic carcinogenesis. For example, pretreatment of hamsters with streptozotocin (a pancreatic β-cell toxin) reduces the incidence of carcinogen-induced pancreatic cancer (Bell et al., 1988, 1989; Fisher et al., 1996). Also, caloric restriction, which decreases daily insulin secretion, inhibits pancreatic cancer induction in hamsters (Roebuck et al., 1993). Conversely, high-fat–fed hamsters have increased insulin levels but normal glucose levels and subsequently increased islet cell proliferation and pancreatic carcinogenesis. Administration of metformin, an oral antihyperglycemic drug, normalized insulin levels and the rate of islet cell turnover (Schneider et al., 2001). Physical activity is associated with a decreased risk of pancreatic cancer, especially among overweight people. The inverse relationship was observed for moderate physical activity, specifically for walking or hiking outdoors for ≥4 hours/wk, but not for vigorous activity (Michaud et al., 2001). In another study, mild obesity (BMI 25–30), adult weight gain, sedentary work, and low physical activity during leisure time were all associated with increased risk of pancreatic cancer (Isaksson et al., 2002). Among Canadian men, men in the highest quartile of the composite moderate and strenuous physical activity index were at a reduced risk of pancreatic cancer, with a trend toward the same relationship in women (Hanley et al., 2001). However, in a study of 32,687 subjects with data on physical activity and BMI collected serially over time, neither physical activity or BMI significantly predicted pancreatic cancer mortality (Lee et al., 2003), nor was there any difference in pancreatic cancer incidence rates between men and women who were most active at baseline compared with those who reported no recreational activity in the ACS Cancer Prevention Cohort (Patel et al., 2005). The mechanisms for any observed effect are unclear, except that exercise helps to maintain normal body weight, again illustrating the interaction between insulin resistance, overweight, and lack of physical activity in cancer risk.

Fruit and Vegetable Intake A growing body of evidence suggests that diets high in fruits and vegetables may be protective against pancreatic cancer. Fruits and vegetables contain a multitude of anticancer agents such as fiber, carotenoids, vitamins C and E, selenium, flavonoids, and plant sterols (Hart, 1999; International Agency for Research on Cancer [IARC] Working Group on the Evaluation of Cancer-Preventive Strategies, 2003). Several reviews have summarized the epidemiological evidence on fruit and vegetables intake and pancreatic cancer prevention (Block et al., 1992; Howe and Burch, 1996; World Cancer Research Fund and American Institute for Cancer Research, 1997). Overall, the pattern is very

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consistent, showing decreasing risk with increasing consumption of fruits and vegetables. However, there is quite a bit of variation in the specific food items showing protective relationships, suggesting perhaps that there are multiple active food components that contribute to this apparent protective effect. Another more recent case-control study differentiated among three dietary patterns in subjects using food frequency questionnaires: Western (characterized by high intake of processed meats, sweets and desserts, refined grains, and potatoes), drinker (high consumption of liquor, wine, and beer), and the potentially beneficial high fruit and vegetable intake pattern (characterized by high intake of fresh fruits and cruciferous vegetables). After adjustment for various anthropometric and demographic factors (age, BMI, smoking, physical activity, socioeconomic factors, etc.), the high fruit and vegetable intake pattern was associated with a 49% reduction in pancreatic cancer risk among men when comparing the highest and lowest quartile of dietary pattern scores (Nkondjock et al., 2005b). Specific phytochemicals have been implicated in the anticancer action of vegetable intake. Studies have shown that genistein, a phytoestrogen and the predominant isoflavonoid of the soy plant, inhibits cell growth in pancreatic cancer cells in vitro and in vivo (Boros et al., 2001; Mouria et al., 2002; Buchler et al., 2003). In an orthotopic nude mouse model, genistein significantly improved survival, almost completely inhibited metastasis, and increased apoptosis (Buchler et al., 2003). When genistein was combined with the tumor necrosis factor–related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo2L), the combination of the two agents decreased cell proliferation in vitro and tumor volume in vivo and increased the number of apoptotic cells to a greater extent than with either agent alone (Nozawa et al., 2004). The constitutively activated signal transducer and activator of transcription-3 protein (STAT3), a mediator of several hormone and growth factors, was modulated by genistein, as well as indole-3-carbinol (I3C; a breakdown product of glucosinolates, which are found primarily in cruciferous vegetables, as described later in this chapter), in pancreatic tumor cell lines (Panc-1 and MIA PaCa-2) (Lian et al., 2004). STAT3 constitutive activation is inhibited at 10 µM of genistein or I3C. Induction of apoptosis by I3C was also shown. Boros et al. (2001) reported that genistein regulates cell tumor proliferation by regulating glucose oxidation and inhibiting synthesis of nucleic acid ribose from glucose through nonoxidative steps of the pentose cycle, which is necessary for nucleic acid synthesis and salvage pathways of purine and pyrimidine bases. Biochanin A, the 4′-methyl ether of genistein found in various legumes, has been reported to inhibit metastasis in breast cancer and colon cancer (Peterson and Barnes, 1996; Wang et al., 1998). A study of two pancreatic adenocarcinoma cell lines, one derived from a male and the other a

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female patient, found that biochanin A inhibited growth in both male and female tumor cells (Lyn-Cook et al., 1999b). However, two other phytoestrogens, equol and coumestrol, which are found in alfalfa sprouts, clover sprouts, and beans, were found to inhibit cancer cell proliferation only in the female cell lines, primarily through downregulation of Kras expression (Lyn-Cook et al., 1999b). The effects of several food-derived polyphenols were compared in in vivo and in vitro models of pancreatic cancer (Mouria et al., 2002). Quercetin (a flavonoid with widespread distribution in vegetables and fruits, including onions, apples, and teas) decreased primary tumor growth, increased apoptosis, and prevented metastasis in nude mice implanted with human pancreatic cells. In human MIA PACa-2 and rat BSp73AS pancreatic cancer cell lines, quercetin and trans-resveratrol (enriched in red wine and grapes) markedly enhanced apoptosis, causing mitochondrial depolarization and cytochrome c release followed by caspase-3 activation; however, the flavonol glycoside of quercetin, rutin, did not. In addition, the effect of a combination of quercetin and trans-resveratrol on mitochondrial cytochrome c release and caspase-3 activity was greater than the expected additive response. The inhibition of mitochondrial permeability transition prevented cytochrome c release, caspase-3 activation, and apoptosis caused by polyphenols. The activity of NFκB was inhibited by quercetin and transresveratrol, but not genistein, indicating that this transcription factor is not the only mediator of the polyphenols’ effects on apoptosis. These results suggest that food-derived polyphenols inhibit pancreatic cancer growth and prevent metastasis by inducing mitochondrial dysfunction, resulting in cytochrome c release, caspase activation, and apoptosis. An additional study confirmed the antiproliferative and proapoptotic effect of resveratrol on human pancreatic cancer cell lines PANC-1 and AsPC-1 (Ding and Adrian, 2002). Vegetables of the Brassica oleracea species (e.g., cabbage, broccoli, cauliflower, Brussels sprouts, kohlrabi, and kale; also called cruciferous vegetables) and many other genera that include a variety of food plants (e.g., arugula, radish, daikon, watercress, horseradish, and wasabi) are known to be rich in glucosinolates (β-thioglycoside-Nhydroxysulfates). These compounds are hydrolyzed by myrosinase, a plant enzyme released when plants are cut, ground, or chewed, releasing the biologically active isothiocyanates (ITCs). Some naturally occurring forms of this phytochemical include 2-phenethyl isothiocyanate (PEITC), benzyl isothiocyanate, and sulforaphanes (Kris-Etherton et al., 2002). ITCs are known to induce expression of phase I and phase II enzymes and, to a lesser extent, directly inhibit the P450s; the effect is dependent on the individual ITC. PEITC has been shown to inhibit pancreatic carcinogenesis in terms of incidence of atypical hyperplasias and multiplicity of pancreatic proliferative lesions including adeno-

carcinomas in BOP-treated hamsters given PEITC during the initiation phase (i.e., administered concomitantly with the carcinogen) (Nishikawa et al., 1996) but not in the post-initiation phase (i.e., PEITC administered after BOP) (Nishikawa et al., 1999). The mechanisms of PEITC on cell kinetics were further investigated in BOP-treated hamsters (Nishikawa et al., 1997). PEITC seems to exert its chemopreventive activity against BOP initiation of carcinogenesis by decreasing cell turnover and DNA methylation in the target organs, and by influencing hepatic xenobioticmetabolizing phase I enzymes (Nishikawa et al., 1997, 2004). Similar studies with other synthetic ITCs with longer alkyl chains showed that 3-phenylpropyl ITC (PPITC) is not effective in BOP-induced pancreatic cancers, in contrast to lung cancers (Nishikawa et al., 1996), whereas 4-phenylbutyl ITC (PBITC) did inhibit the development of pancreatic atypical hyperplasias and adenocarcinomas of ductal origin (Son et al., 2000). In cell culture benzyl ITC (BITC), also derived from cruciferous vegetables, inhibits growth of BxPC-3 pancreatic cancer cells in a concentration-dependent manner with an IC50 of ~8 µM, a concentration achievable in plasma with regular dietary intake of cruciferous vegetables (Srivastava and Singh, 2004). The mechanism of this action involves G2/M cell cycle arrest and induction of apoptosis, which is associated with inhibition of NFκB activation. Sulforaphane, another ITC, also induced G2/M arrest and apoptosis, and cellular toxicity was correlated with a decrease in cellular glutathione levels in vitro; treatment of a xenograft mouse model of pancreatic cancer with sulforaphane resulted in a decrease in mean tumor volume by 40% relative to controls (Pham et al., 2004). Curcumin, a yellow pigment chemical present in the spice turmeric (Curcuma longa), has been shown to inhibit pancreatic cancer growth in vitro (Hidaka et al., 2002; Li et al., 2004b). Furthermore, curcumin ameliorates ethanoland non-ethanol–induced experimental pancreatitis, which may be a precursor condition for pancreatic cancer development (Gukovsky et al., 2003). The mechanism of curcumin’s inhibition of inflammation and cell growth, as well as stimulation of apoptosis, involves downregulation of NFκB, which is constitutively activated in pancreatic cells, and growth control molecules (e.g., prostaglandin E2 [PGE2]) induced by NFκB in human pancreatic cells (Gukovsky et al., 2003; Li et al., 2004b). Furthermore, production of cytokines (and their receptors) such as interleukin-6 (IL-6) and TNF-α, as well as the chemokines IL-8 and KC (a rodent analog of IL-8/GROa) and inducible nitric oxide synthase (iNOS) in pancreas, are diminished by curcumin (Gukovsky et al., 2003; Hidaka et al., 2002). The effectiveness of curcumin in inhibiting pancreatic carcinogenesis in animal models has apparently not been tested. Carotenoids have various cancer preventive activities, including antioxidant activity, enhancement of immune function, stimulation of gap junctional intercellular


communication, induction of detoxifying enzymes, and inhibition of cellular proliferation (Heber and Lu, 2002). Rich sources of carotenoids (such as α- and β-carotenes, lutein, and lycopene) include tomatoes, carrots, cantaloupes, sweet potatoes, and spinach, as well as many leafy greens and yellow-red fruits and vegetables. A case-control study in a Canadian population explored the relationship between dietary carotenoid intake and pancreatic cancer risk (Nkondjock et al., 2005a). It was found that (after adjusting for age, Canadian province of residence, BMI, smoking educational attainment, dietary folate, and total energy intake), lycopene provided primarily by tomatoes was associated with a 31% reduction in pancreatic cancer risk among men.

Meats and Fats Numerous case-control and international mortality and food pattern studies have shown a positive correlation between the average amount of oil and fat consumption and cancer of the pancreas (Baghurst et al., 1991; Ghadirian et al., 1991a,b; Howe et al., 1992; Stolzenberg-Solomon et al., 2002). Similarly, a number of studies have associated consumption of meat, eggs, and milk with an increased risk of pancreatic cancer (reviewed in World Cancer Research Fund and American Institute for Cancer Research, 1997; Howe and Burch, 1996; Michaud, 2004). This trend is illustrated in a Japanese case-control study that showed that intake of meats and animal viscera increased pancreatic cancer risk, whereas vegetables and the traditional Japanese foods (e.g., tofu and raw fish) reduced the risk (Ohba et al., 1996). The responsible component in dietary patterns with high meat intake is unknown but may include total fat or saturated fat, cholesterol, protein, and N-nitroso or heterocyclic aromatic compounds produced in meats by high-temperature cooking (Howe and Burch, 1996; World Cancer Research Fund and American Institute for Cancer Research, 1997). A number of studies in rats and hamsters have demonstrated potentiation of carcinogen-induced carcinogenesis by high-fat diets, particularly those high in lard (a saturated fat) or corn oil (high in PUFAs, especially linoleic acid—an omega-6 fatty acid) (reviewed in Roebuck, 1992; Zhang and Go, 1996). Furthermore, an experimental “Western diet”— high in fat and protein and low in calcium—increases pancreatic epithelial cell hyperproliferation in mice (Xue et al., 1996, 1999). Conversely, dietary restriction does not inhibit pancreatic carcinogenesis induced by BOP in the hamster, at least in one study (Birt et al., 1997), and voluntary physical exercise does not ameliorate the effects of a high-fat diet in promoting pancreatic carcinogenesis in the BOP-treated hamster (Kazakoff et al., 1996). A potential mechanism of the carcinogenesis-promoting effect of PUFA involves the production of various reactive intermediates. ROS are generated during lipid peroxidation and can arise directly from linoleic acid hydroperoxide

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decomposition (Zhang and Go, 1996). Consequently these oxygen free radicals can damage DNA and produce mutations that are associated with the initiation and progression of human cancer. Z’graggen et al. (2001) showed that a high-fat, high-protein diet promoted tumor development in 7,12-dimethylbenzanthracene (DMBA)–induced ductal pancreatic cancer, possibly through faulty gene repair mechanisms or delayed natural regression of early lesions. The authors postulated that a high-fat diet may cause K-ras gene mutations by increasing COX-2 and ras-p21 expression, as well as membrane localization of ras-p21, which is essential for proper ras protein function, phenomena that have been demonstrated in colon cancer (Z’graggen et al., 2001). Another mechanism linking meat intake to pancreatic cancer risk may involve carcinogens produced in grilled meat products. Heterocyclic amines (HCAs) and polycyclic aromatic hydrocarbons are carcinogens formed during cooking of meat in a time- and temperature-dependent manner. A case-control study found that grilled red meat intake specifically was a risk factor for pancreatic cancer, and that method of meat preparation in addition to total intake is important in assessing the effects of meat consumption (Anderson et al., 2002). One study in an animal model of pancreatic cancer has confirmed the carcinogenicity of certain HCAs. Several HCAs were tested in a “rapidproduction” model of pancreatic carcinogenesis in hamsters, whereby carcinogenesis was initiated with BOP, then augmentation pressure was exerted through treatment with dlethionine and methionine and feeding of a choline-deficient diet (Yoshimoto et al., 1999). Out of eight HCAs tested, two compounds, 3-amino-1,4-dimethyl-5H-pyrido[4,3b]indole (Trp-P-1) and 2-amino-3,4,8-trimethylimidazo [4,5-f ]quinoxaline (4,8-DiMeIQx), enhanced pancreatic carcinogenesis in this model. As described earlier in this chapter, diets rich in PUFAs stimulate pancreatic cancer development. In contrast, supplements of long-chain omega-3 PUFA, such as DHA and EPA, which are enriched in fish oils, exert suppressive effects in several cancer models, particularly breast cancer (Terry et al., 2003). A fish oil–enriched nutritional supplement reverses weight loss in patients with pancreatic cancer cachexia (Barber et al., 1999) by diminishing production of IL-6, increasing serum insulin, and decreasing the cortisolto-insulin ratio and the proportion of patients excreting proteolysis-inducing factor (associated with cancer-associated cachexia) (Barber et al., 2001). The first experiments investigating the effect of omega3 fatty acids in animal models showed that a 20% menhaden fish oil diet fed for 4 months produced a significant decrease in the development of both the size and the number of preneoplastic lesions when compared with a 20% corn oil diet rich in omega-6 fatty acids in male azaserine-treated rats (O’Connor et al., 1985). Follow-up studies showed that as the ratio of dietary omega-3 to omega-6 fatty acids increased

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in a diet totaling 20% by weight of fat, the development of preneoplastic atypical acinar cell nodules (AACN) decreased significantly (O’Connor et al., 1989). In addition, serum levels of PGE2, thromboxane B2 (TXB2), and 6keto-prostaglandin F1α (6-keto-PGF1α) decreased significantly. Intervention of the omega-6 fatty acid–rich diet with the omega-3 fatty acid–rich diet significantly decreased focal development when given after carcinogen exposure. However, in a 6-month study using the MaxEPA product, which contains 9.4% fish oil, in a high-linoleic acid background diet did not protect against the increased number and size of pancreatic atypical acinar cell foci stimulated by the high-fat diet in azaserine-treated rats, although cell proliferation in atypical acinar cell foci and prostaglandin levels was decreased (Appel and Woutersen, 1994). In a follow-up 12-month study of MaxEPA feeding, a dose-dependent increase of preneoplastic AACNs was seen with an induction of eicosanoids PGE2, PGF2α, and TXB2 (Appel and Woutersen, 1996a). In BOP-treated hamsters, increasing doses of MaxEPA in a high-fat diet had little effect, although pancreatic levels of PGE2, 6-keto-PGF1α, and PGF2α decreased significantly with increasing dietary MaxEPA (Appel and Woutersen, 1995). The reason for these anomalous results is unclear but may involve the linoleic acid content of the high-fat diets, which could overwhelm any protective effect of the omega-3 PUFA. However, linoleic acid in the BOP-induced pancreatic cancer did not enhance pancreatic tumor growth, even though levels of lipid peroxidation were increased and activity of glutathione peroxidase decreased in pancreatic intratumoral tissue (Kilian et al., 2002; Kilian et al., 2003).

Tea and Coffee An early study from 1981 found that coffee drinkers consuming three or more cups of coffee per day had a relative risk of pancreatic cancer of 2.7 compared with noncoffee drinkers (MacMahon et al., 1981), stimulating interest in a potential link between coffee consumption and pancreatic cancer. A cohort study of >110,000 Japanese subjects reported that heavy coffee consumption (>4 cups/day) may increase risk of pancreatic cancer (Lin et al., 2002). However, a number of other studies have concluded no association (Heuch et al., 1983; Mack et al., 1986; Howe et al., 1992; Gold, 1995; Isaksson et al., 2002). One confounder in these studies could be cigarette smoking, as not all studies control for this factor or self-reporting in subjects is inaccurate (Porta et al., 2000). Interestingly, though, in the Spanish PANKRAS II casecontrol study, an association between coffee consumption and K-ras mutations was identified. Among 121 cases, Kras mutations were significantly more common in tumors of regular coffee drinkers than in nonregular coffee drinkers. The odds of a mutated tumor increased in approximately

linear manner with increasing level of coffee consumption (Porta et al., 1999). It is not clear whether caffeine, other coffee compounds, or other factors with which coffee drinking is associated may modulate K-ras activation. Potentially, coffee components can induce or inhibit relevant metabolic pathways that are involved with activation or inactivation of carcinogenic molecules. Alternatively, these compounds can inhibit DNA repair mechanisms. However, another study conducted in California did not show a significant association between K-ras mutational pattern or p53 staining and coffee drinking (caffeinated coffee, decaffeinated coffee, and total coffee drinking) (Slebos et al., 2000). One limitation of this study may be that the questionnaire used evaluated average total coffee consumption habits during the year before diagnosis, which may not capture the etiologically relevant exposure period. Although the results of these studies are inconsistent and the mechanisms underlying the associations between K-ras mutation and diet are not understood, these studies illustrate the potential for identifying important gene–nutrient interactions in pancreatic cancer. Inhibition of tumorigenesis in animal models by green or black tea preparations has been demonstrated for a number of organ sites, such as skin, lung, oral cavity, esophagus, forestomach, stomach, small intestine, colon, mammary gland, and pancreas. Epidemiological studies, however, are not as clear concerning the protective effects of tea consumption against cancer formation in humans (Yang et al., 2002). Very limited epidemiological evidence from several studies suggests an inverse association between tea consumption and pancreatic cancer in humans (Bushman, 1998). A population-based case-control study in Shanghai indicated a reduction in risk of colorectal and pancreatic cancer in both men and women with consistent green tea consumption (Ji et al., 1997). However, in the Iowa Women’s Study, a prospective cohort study of some 34,000 postmenopausal women, no relationship between tea intake and pancreatic cancer incidence was demonstrated (Harnack et al., 1997). In vitro data support a cancer-inhibiting effect of EGCG, the major polyphenol component of green tea. EGCG inhibits growth of three pancreatic carcinoma cell lines (PANC-1, MIA PaCa-2, and BxPC-3) in a dose-dependent manner (Takada et al., 2002). Further study included black and green tea extracts and components of these extracts to determine their effect on tumor cell growth in vitro. Fractions studied included a mixture of polyphenols from green tea (GTP), mixtures of polyphenols (BTP) and of theaflavins (MF) from black tea, and the purified components epicatechin-3-gallate (ECG) and EGCG. Results showed inhibition (~90%) of cell growth in HPAC pancreatic adenocarcinoma cells by black and green tea extracts (0.02%). GTP (10 µg/ml) and MF (100 µg/ml) significantly inhibited growth (~90%); ECG and EGCG inhibited growth as well (~95%).

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Black and green tea extracts, GTP, and EGCG decreased the expression of the K-ras gene. Green and black tea extracts decreased the multidrug-resistant gene (mdr-1), although GTP and EGCG increased expression (Lyn-Cook et al., 1999a). Other studies explored the molecular mechanism of EGCG action in pancreatic cancer cells in vitro. Takada et al. (2002) showed that EGCG treatment resulted in significant suppression of the invasive ability of these lines but did not affect the cell cycle protein cyclin D1. Qanungo et al. (2005) showed that the antiproliferative action of EGCG is mediated through induction of apoptosis, invoked by oligomerization of Bcl-2–associated X (Bax) protein, depolarization of mitochondrial membranes to facilitate cytochrome c release into the cytosol and downregulation of X Chromosome-Linked Inhibitor of Apoptosis Protein (XIAP). In addition, EGCG elicited production of intracellular ROS, as well as activation of c-Jun N-terminal kinase (JNK). Blocking these pathways with an inhibitor of JNK signaling and the antioxidant N-acetyl-L-cysteine (NAC) blocked EGCG-induced apoptosis, implicating the involvement of ROS-mediated JNK activation in the apoptotic response. Pancreatic carcinogenesis and tumor promotion were inhibited in two hamster models with feeding of green tea extract, demonstrating an in vivo effect (Hiura et al., 1997). In addition, when green tea polyphenols were combined with palm carotene, which consists of 60% β-carotene, 30% α-carotene, 3% γ-carotene, and 4% lycopene, the same effect was seen in diminishing pancreatic preneoplastic lesions and duct epithelial hyperplasia and atypical hyperplasia (Majima et al., 1998). Therefore, compelling preclinical evidence with tea extracts and isolated tea components suggests that tea intake may inhibit pancreatic carcinogenesis, although much further research is warranted.

Alcohol The results of numerous prospective cohort and casecontrol studies for alcohol consumption and pancreatic cancer risk have been inconsistent, with many confounding variables present in various studies. However, heavy alcohol consumption has been known to be a major cause of acute and chronic pancreatitis; in the United States and other developed countries, 60–90% of cases of chronic pancreatitis are linked to alcohol consumption (Durbec and Sarles, 1978; Dufour and Adamson, 2003; Swaroop et al., 2004). The chronic inflammation of pancreatitis accelerates the pancreatic oncogenic processes, and chronic pancreatitis has been linked to pancreatic cancer (Gordis and Gold, 1984; Lowenfels et al., 1993). It is thought that chronic heavy alcohol consumption potentiates other risk factors such as smoking in pancreatic and other cancers. In fact, according to the Eleventh Annual Report on Carcinogens by the U.S. National Toxicology Program (http://ntp.niehs.nih.gov/ntp/

roc/toc11.html), alcoholic beverage consumption has been classified as a human carcinogen since 2000 (U.S. Department of Health and Human Services, 2005). An important mediator of the deleterious effects of ethanol consumption is acetaldehyde, which is produced in the pancreas through oxidative metabolism of ethanol via alcohol dehydrogenase, as well as CYP 2E1. Acetaldehyde is mutagenic and carcinogenic in vitro and in vivo (Poschl and Seitz, 2004). The molecular mechanisms by which alcohol and its metabolites such as acetaldehyde and fatty acid ethyl esters induce inflammation and carcinogenesis have been studied and are reviewed by Go et al. (2005b). In summary, these mechanisms include (1) premature activation of zymogens; (2) induction of the inflammatory response through activation of nuclear transcription factors (including NFκB) and activation protein 1 (AP-1); (3) increased production of ROS resulting in oxidative DNA damage and altered effect of dietary antioxidants; (4) activation of pancreatic stellate cells that lead to fibrosis; (5) gene mutation in enzymes related to CYP, glutathione Stransferase, aldehyde dehydrogenase, cationic trypsinogen, and pancreatic secretory trypsin inhibitor (PST1); (6) effects on metabolism of tobacco carcinogen NNK; and (7) dysregulation of proliferation and apoptosis. In addition to direct effects of ethanol, a number of factors including genetics, intestinal infection, and dietary factors may make the pancreas more susceptible to damage induced by alcohol consumption. Dietary fat appears to potentiate the development of alcoholic pancreatitis by modulating oxidative stress. For example, unsaturated fat (corn oil) potentiated development of alcoholic pancreatitis in female Wistar rats fed high levels of ethanol. This effect was mediated through oxidative stress as measured by radical adducts in pancreatic secretions and levels of lipid peroxidation in the pancreas (Kono et al., 2001). However, saturated fat (medium-chain triglycerides) attenuated the injury and blunted oxidative stress.

Isoprenoids Various other phytochemicals found in plants also have antitumor or anticarcinogenic effects. Isoprenoids are nonnutritive dietary phytochemicals found in the essential oils of citrus fruits, cherry, spearmint, dill, caraway, and other plants. Bioactive isoprenoids include limonene, perillyl alcohol, geraniol, and farnesol. The important mechanistic activities of this class of compounds are as regulators of the mevalonate pathway, which produces essential sterols, ubiquinones, retinoids, and isoprenoids, which are essential to cell survival. Several of these compounds are required intermediates for the post-translational modification of small G proteins, including ras, nuclear lamins, and growth factor receptors. Suppressors of activities providing and transferring mevalonate-derived substrates for the modifications of

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these growth-associated proteins diminish cellular growth (Mo and Elson, 2004). A number of pharmacological inhibitors of the mevalonate pathway show promising activities in inhibiting pancreatic carcinogenesis in preclinical studies. For example, lovastatin, a competitive inhibitor of 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase activity, inhibited the proliferation of human and hamster pancreatic tumor cells in culture (Sumi et al., 1994; Muller et al., 1998) and of pancreatic tumor xenografts implanted in nude mice (Sumi et al., 1992). However, these types of nondiscriminate inhibitors of the mevalonate pathway may have doselimiting toxicities. Mevalonate-derived isoprenoids from natural sources post-transcriptionally downregulate HMGCoA reductase activity with specificity for tumors and may have greater potential in pancreatic tumor chemoprevention (Mo and Elson, 2004). Significant inhibition of human pancreatic tumor cells in vitro (60–90%) is attained by administration of farnesol, geranyl geraniol, perillyl amine, geraniol, and perillyl alcohol (Burke et al., 1997). In the same report, hamsters fed geraniol or farnesol at 20 g/kg diet exhibited complete inhibition of BOP-induced pancreatic tumor growth. Both farnesol and geraniol were more potent than perillyl alcohol, which inhibited tumor growth by 50% at 40 g/kg diet with no effects on plasma cholesterol levels (Burke et al., 1997). In vitro, farnesol, geraniol, and perillyl alcohol increased apoptosis and proapoptotic Bak levels (Stayrook et al., 1997; Burke et al., 2002). Similarly, in vivo treatment during the promotion/progression phase of BOP-induced pancreatic carcinogenesis with perillyl alcohol or farnesol resulted in hyperplastic pancreatic ductal neoplasms with higher apoptotic rates, increased Bak protein expression, diminished expression of the antiapoptotic protein Bcl-XL, and lowered rates of DNA synthesis (Burke et al., 2002). Interestingly, farnesylation of H-ras, but not K-ras, was inhibited by perillyl alcohol in pancreatic tumor cells transformed with either ras isoform; similarly, the downstream effector MAP kinase phosphorylation was inhibited in H-ras tumor cells only (Stayrook et al., 1998). Farnesol mimetics S-trans, trans-farnesylthiosalicylic acid (FTS) inhibited the proliferation of Panc-1 cells and the growth of Panc-1 xenograft in nude mice (Weisz et al., 1999). Geranyl geraniol and menaquinone-4 (vitamin K2; a mixed isoprenoid with a geranyl geraniol side chain) inhibited the proliferation of MIA PaCa-2 cells and induced apoptosis (Shibayama-Imazu et al., 2003).

Retinoids and Vitamin D: Differentiation Agents The vitamin A metabolite, retinoic acid, has been shown to have chemopreventive and therapeutic activity against several cancers. These effects are the results of induction of differentiation and growth arrest, which are mediated

through retinoid nuclear receptors termed retinoic acid receptors (RARs) and retinoic X receptors (RXRs) (Niles, 2004). In cell culture, treatment with several retinoids, including all-trans-retinoic acid, 9-cis-retinoic acid, and 13cis-retinoic acid, results in a time- and dose-dependent growth inhibition in ductal but not acinar pancreatic tumor cells, although results for individual ligands are not consistent among studies (Rosewicz et al., 1995; Bold et al., 1996; Vickers et al., 1997; Albrechtsson et al., 2002). One report (El-Metwally et al., 2005a) showed that natural all-trans-retinoic acid inhibited proliferation in 10 human pancreatic adenocarcinoma cell lines with varying degrees of differentiation, some of which were characterized previously as retinoid resistant. Retinoid treatment in vitro induces a differentiated phenotype, as evidenced by cellular morphology and expression of differentiation-specific genes such as carbonic anhydrase II (Rosewicz et al., 1995; ElMetwally et al., 2005b). In addition, 9-cis retinoic acid enhanced the G1 cell cycle arrest induced by troglitazone, a PPARγ ligand, implicating the action of the RXRα/PPARγ pathway in growth inhibition (Tsujie et al., 2003b). Retinoids also induce apoptosis and decrease the Bcl-2/Bax ratio in pancreatic adenocarcinoma cells, activity that requires RARα (Pettersson et al., 2002; El-Metwally et al., 2005a). Additional targets for retinoic acid action identified in pancreatic cancer cells include protein kinase C (Rosewicz et al., 1996), stromelysin 3 (an MMP) (von Marschall Z. et al., 1998), kinesin-related protein HsEg5 (important for spindle assembly and function during mitosis) (Kaiser et al., 1999), α6,β1-integrin receptor (modulates cell adhesion to laminin) (Rosewicz et al., 1997), MUC4 (mucin; high molecular weight glycoprotein overexpressed in pancreatic tumors) (Choudhury et al., 2000), cMet (hepatocyte growth factor receptor) (Leelawat et al., 2005), and TGF-β protein and EGF receptor (autocrine growth factor pathways) (El-Metwally et al., 2005b). On the basis of promising in vitro data, a number of studies evaluated retinoic acid action in animal models. In azaserine-initiated pancreatic carcinogenesis in rats, longterm treatment with various synthetically derived retinoids inhibited progression of pancreatic carcinomas (Longnecker et al., 1982, 1983); similar results were born out in shortterm studies looking at number and size of acidophilic foci, believed to be precancerous lesions (preceding the definition of PanIN) (Roebuck et al., 1984). The inhibitory effect of low-dose retinoids could be enhanced to a small degree by addition of selenium to the diet (Curphey et al., 1988). Results of retinoid action in BOP-induced carcinogenesis are not as clear. In this model there was either little to no effect (Longnecker et al., 1983, 1986) or a small enhancing effect of retinoids on BOP-induced carcinogenesis (Birt et al., 1981, 1983). Based on these findings, clinical trials have been initiated, but with disappointing results. Phase II studies have

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Future Directions

been conducted on retinoic acid co-administered with interferon-α (IFN-α) in patients with advanced unresectable pancreatic carcinoma, without impressive results (Moore et al., 1995; Brembeck et al., 1998). In another study, retinoids were added to chemotherapy plus IFN-β treatment in patients with metastatic pancreatic cancer. However, there was not a large effect on survival, and moreover the incidence of toxicities was high (Recchia et al., 1998). Therefore, the prospect of retinoic acid as a therapeutic agent is not great; however, there still may be applications in prevention. Another differentiation agent, vitamin D and analogs thereof, shows growth inhibitory effects in pancreatic cancer cells (Kawa et al., 1996; Zugmaier et al., 1996; Colston et al., 1997; Pettersson et al., 2000; Albrechtsson et al., 2003), and these effects interact with those of retinoids (Zugmaier et al., 1996; Pettersson et al., 2000). Most pancreatic cancer cells express the vitamin D receptor (Kawa et al., 1996; Colston et al., 1997; Albrechtsson et al., 2003), as well as the enzyme 25-hydroxyvitamin D-1α-hydroxylase that converts the vitamin prohormone to the most active form (Schwartz et al., 2004). Vitamin D analogs block the G1/S transition by upregulating p21 and p27 proteins (Kawa et al., 1997; Schwartz et al., 2004). A Western-style diet (high fat, calcium, and vitamin D) fed to mice induces hyperproliferation of epithelial cells of the exocrine pancreas, but the effect is ameliorated by replacement of dietary calcium and vitamin D (Xue et al., 1996, 1999). A phase II trial of a non-hypercalcemic vitamin D analog Seocalcitol (EB1089) has been conducted in patients with inoperable pancreatic cancer; the drug was well tolerated, but no cytostatic activity was seen in this trial (Evans et al., 2002). Further research is warranted to determine the therapeutic potential of vitamin D analogs in less advanced pancreatic cancer and in prevention of pancreatic carcinogenesis.

METABOLIC HYPOTHESIS FOR PANCREATIC CANCER Pancreatic tumor cells are characterized by poor differentiation and a high glucose-utilizing phenotype. Boros et al. (2002) have hypothesized that a specific metabolic phenotype is associated with tumor cells relative to normal cells. Tumor cells assume their unique characteristics according to their diverse genetic aberrations. Their invasive and proliferative characteristics, however, are limited by the availability of substrates, nutrients, and metabolic pathway enzyme activities. On the basis of these factors, tumor cells exhibit distinct metabolic phenotypes determining the rate of proliferation, apoptosis, cell cycle arrest, and differentiation. Hormones, signaling pathways, environmental factors, and nutritional habits have a strong influence on these metabolic phenotypes. Cell transformation and tumor growth are

associated with the activation of metabolic enzymes that increase glucose carbon utilization for nucleic acid synthesis, whereas enzymes of the lipid and amino acid synthesis pathways are activated in tumor growth inhibition. Furthermore, phosphorylation and allosteric and transcriptional regulation of intermediary metabolic enzymes and their substrate availability together mediate and sustain cell transformation from one condition to another. Metabolic profiling of cancer cells using labeled tracers have shown opposite changes in metabolic phenotypes induced by TGF-β, which is a cell-transforming agent (Boros et al., 2000), and tumor growth–inhibiting phytochemicals such as genistein, as outlined earlier in this chapter (Boros et al., 2001). The authors illustrate that understanding adaptive metabolic changes in glycolysis and anabolic reactions in response to tumor growth–modulating agents is fundamental to the understanding of tumor pathophysiology in the pancreas. The proposed metabolic hypothesis of tumor cell growth and death permits a wide range of basic and clinical studies in developing new strategies to revert tumor-specific metabolic changes. Complex metabolic networks of key regulatory metabolic enzymes offer a large number of targets for direct intervention. This new class of metabolic regulators offers a potentially effective alternative to current gene therapeutics, chemotherapeutics, and signal pathway regulators to achieve the same endpoint effect of reducing cell proliferation through limiting glucose carbon use for nucleic acid synthesis (Boros et al., 2002).

FUTURE DIRECTIONS Progress in the understanding of the genome has been paralleled by development in the understanding of the process of carcinogenesis. As outlined in this chapter, cancer is now considered a genetic disease; tumor cells result from multiple genetic defects caused by exposure to environmental and infectious agents, as well as dietary and various lifestyle factors (Go et al., 2003). Knowledge of the genetic signatures and molecular markers of tumorigenesis provides us with the opportunity to use approaches such as dietary intervention to prevent cancer development. High-dimensional technologies to assay genetic and metabolic changes within the cell on a global basis are new weapons in the scientific arsenal that allow nutrition scientists to move forward beyond the reductionist method of investigating single nutrient effects on an isolated biological pathway toward a more holistic approach of exploring the molecular details of food and nutrient effects on an entire biological organism. As these concepts are evolving, so has the very definition of a nutrient. The classic definition of a nutrient is a constituent of food necessary for normal physiological function and essential nutrients are those required for optimal health (Go et al., 2003). With our expanding working knowledge

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of the role of nutrients in gene expression and cellular response to changes in nutrient availability, the pursuit of a definitive definition of the term nutrient is a work in progress. Young (2002) defined the term nutrient in the postgenomic era as a “fully characterized (physical, chemical, physiological) constituent of a diet, natural or designed, that serves as a significant energy yielding substrate or a precursor for the synthesis of macromolecules or of other components needed for normal cell differentiation, growth, renewal, repair, defense and/or maintenance or a required signaling molecule, cofactor or determinant of normal molecular structure/function and/or promoter of cell and organ integrity.” In addition, nutrients can catalyze reactions and promote the assembly of mechanistic structures. Nutrient–genome interactions may differ according to the life cycle of the organism and have a profound influence on health maintenance and disease (cancer) prevention. Within this mechanistic definition of nutrients, one must consider that the requirement range of a particular nutrient is contingent upon the functionality of the cell and organism, that the required amount may vary depending on whether the nutrient is necessary for normal cell growth or cancer prevention, and that certain nutrients may also be harmful in supernormal doses. The new definition of nutrients can provide the appropriate mode of gene–nutrient analysis needed at the genome, transcriptome, proteome, metabolome, physiome/phenome, and populome level to generate appropriate biomarkers (Figure 3). With the development of novel technologies and the advent of nutritional genomics, proteomics, metabolomics, and other so-called “-omics” sciences, there is renewed interest in dietary components that affect global gene expression and the integrative physiological and metabolic functions of an organism. Nutrition science has thus evolved into a multidisciplinary field that applies molecular biochemistry and integration of individual health to epidemiological investigation and population health (German et al., 2004). Therefore, there exists ample justification for creating an innovative research model to further explore the role of diet in health promotion and disease prevention, including pancreatic cancer. The implications of our broadening knowledge of the impact of nutrient intake on cancer biology are that we can hope to utilize nutritional interventions to slow the progression of tumor development in the intraepithelial hyperplasia phase before tumor size becomes large enough for diagnosis and probability of metastasis increases (Figure 2). Opportunity exists to stretch this prevention phase so that symptom-free life of the future cancer patient is prolonged. Because the median age at cancer diagnosis in the United Sates is 70 years, and the average life expectancies are 74 years for men and 79 years for women, cancer delay may result in total prevention for many people (Lippman and Hong, 2002).

FIGURE 3 Nutritional genomics and biomarker discovery. The steps involved in gene expression (center); the stages at which diet, represented by nutrients, can modulate these processes from cell to population (left); and the functional genomics techniques used to analyze each stage, with appropriate biomarkers (reprinted with permission from Go et al., 2003.)

The results of new approaches will be to facilitate molecular analysis of nutrient action and identification of appropriate biomarkers that target individuals who are at risk and predisposed to pancreatic cancer. Ever-increasing evidence, including that presented in this volume, substantiates the beneficial effects of certain nutrients and interactions between nutrients in the carcinogenesis pathway, paving the way for modification of nutritional requirements as a cancer prevention strategy. In the future, diet, nutrition, and cancer prevention will be included in public health programs that target cancer risk management in the population at large, as well as on individual programs that focus on particular cancer risk profiles. Concomitantly, agricultural sciences will continue to develop improved plants through both traditional breeding techniques and genetic modification, and food industries will provide functional foods enriched with beneficial nutrients (Milner et al., 2001). Targeting pancreatic cancer prevention with specific foods and/or bioactive components is a relatively nontoxic and cost-effective strategy for reducing cancer burden. However, the complexity of the human diet coupled with individual variation in the carcinogenesis process will make continued research in the area of diet and pancreatic cancer prevention mandatory (Milner, 2002). Toward this end, we would like to highlight the following research priorities for identifying gene–nutrient interactions in pancreatic cancer (Kern et al., 2001; Hine et al., 2003): • Strengthening the evidence base linking dietary and lifestyle factors with cancer risk via epidemiological studies, especially where associations between specific genetic and metabolic alterations and dietary factors can be examined

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• Identification of physiologically relevant polymorphisms in genes related to metabolism and detoxification of foodderived agents, both anticarcinogens and procarcinogens, and relate these individual variations to pancreatic cancer risk • Refinement of relevant biomarkers and/or imaging systems for pancreatic cancer, especially present in the early PanIN stages, to identify affected individuals and to track impact of nutritional intervention on carcinogenesis • Further development of animal models of pancreatic ductal adenocarcinoma, especially of transgenic models that recapitulate the human disease • Validate use of high-dimensional analysis systems, including genomics, proteomics, and metabolomics, as biomarkers of pancreatic disease and to discriminate among subtypes to develop most effective intervention regimens.

Acknowledgment Funding provided by the National Cancer Institute (NCI)/UCLA Clinical Nutrition Research Unit (CA42710) and NCI/National Center for Complementary and Alternative Medicine (AT1535). The authors recognize Yu Wang, Raymond Gonzales, Debra Wong, and Anita Stein for their expert editorial and technical assistance and Drs. Guido Eibl and Huanbiao Mo for critical review of the manuscript.

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26 Bladder Cancer ALLAN J. PANTUCK, RON LIEBERMAN, KELLY KAWAOKA, OLEG SHVARTS, AND DONALD LAMM

BACKGROUND

disease recurrence. Additionally, genetic changes may occur and induce the development of higher-grade papillary lesions that result in the transformation of low-grade lesions to higher-grade, more aggressive lesions. Between 50 and 70% of patients with superficial tumors will develop new superficial TCC, often within 12 months of diagnosis, and 10–20% progress to infiltrate muscle (Heney et al., 1982). There is increasing evidence to support the concept of histopathological and molecular biochemical field disease occurring years in advance of overt malignancy in bladder cancer (Rao et al., 1999), offering an ample latency period and opportunity for cancer preventive strategies. The development of biomarkers of susceptibility, exposure, and effect establishes a powerful paradigm for individual risk assessment. Although primary prevention is ideal, the premalignant field serves as a useful target for chemoprevention by targeting functional pathways relevant to apoptosis, proliferation, differentiation, and cytotoxicity. Drugs, biologics, or nutrients that halt, slow, or reverse the carcinogenic process are likely to be effective when administered before the emergence of genetic instability.

Bladder cancer, which currently ranks the fourth most common cancer site in men and the eighth most leading site in women (Jemal et al., 2003), represents an important health problem in the United States. An estimated 57,400 new cases of bladder cancer will be diagnosed in the United States, with ~12,500 estimated cancer deaths (Jemal et al., 2003). The highest incidence of new cases in the United States is estimated to be in California (5300 cases, or nearly 10%). The expected male-to-female ratio is 2.6 : 1.0, the disease is more common in whites than blacks, and the average age at diagnosis is 65 years. There has been a slow rise in the total number of new cases in the past 20 years. Approximately 90% of bladder tumors arise from the urothelial lining, and transitional cell carcinoma (TCC) is the most common histological type in cases arising in the United States. Exogenous risk factors appear to play a pivotal role in the development of bladder cancer. Cigarette smoking represents the single most significant preventable cause of bladder cancer in the United States. The process of bladder tumor carcinogenesis results from the interaction of environmental exposures and genetic susceptibility. The molecular pathology of bladder cancer begins not with the appearance of visually aberrant lesions, but with altered biochemical and genetic processes. Clinically and pathologically, one can separate bladder tumors into two entities: low-grade papillary lesions and high-grade in situ or invasive malignancies. Most bladder tumors (70–80%) are classified as superficial. Papillary tumors that are confined to the mucosa can be cured by surgical resection; however, there may be changes elsewhere in the bladder that are not clinically detectable that can lead to


SMOKING AND BLADDER CANCER TCCs of the bladder are clearly related to occupational, chemical, and environmental exposures, such as aniline dyes, arsenic, nitrates, and certain analgesics. In the past, occupational exposures have been estimated to account for 20% of the cases of bladder cancer in the United States with long latency periods of 30–50 years (Cole et al., 1972). Occupations that have been associated with exposures to bladder carcinogens include autoworkers, painters, truck

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Chapter 26/Bladder Cancer

drivers, drill-press operators, leather workers, metalworkers, machinists, dry cleaners, paper manufacturers, rope and twine makers, dental technicians, barbers and beauticians, plumbers, apparel manufacturers, and physicians (Morrison, 1984; Silverman et al., 1989). Today, tobacco smoke represents the most important exogenous risk factor for bladder cancer. Smokers are at least two to three times more at risk for developing TCC of the bladder than nonsmokers (Hoover and Cole, 1971), with heavy smokers being at five times the risk (Wynder and Goldsmith, 1977). Furthermore, it has been estimated that up to 50% of all bladder tumors are directly attributable to cigarette smoking (Wynder and Goldsmith, 1977). A dose–effect relationship appears to be present because increasing the number of packs smoked per day increases the risk ratio of the individual (Morrison et al., 1984). It has been estimated that smoking two or more packs of cigarettes can increase the risk to about seven times that for nonsmokers (Morrison et al., 1984). More than 3000 chemicals are present in tobacco smoke, including at least 60 known carcinogens such as nitrosamines and polycyclic aromatic hydrocarbons (PAHs). Tobacco contains many urothelial carcinogens, including 4-amino-biphenyl, acrolein, and oxygen free radicals (Bartsch et al., 1993a). The exact mechanism of bladder carcinogenicity remains unknown, but data support the arylamines, including the amino-biphenyls, as the major carcinogen in smokinginduced bladder carcinogenesis (Zang and Wynder, 1996). Mutations in the p53 tumor suppressor gene have also been associated with smoking (Curigliano et al., 1996). Furthermore, Thompson et al. (1987) demonstrated a positive correlation between smoking history and histological grade, stage, number, and size of bladder tumors. All three variables were increased in smokers. Other studies suggest that bladder cancers that develop in association with cigarette smoking lead to higher mortality with survival data, suggesting that 40% of smokers versus 27% of nonsmokers died of their disease (Raitanen et al., 1995). Estimates suggest that ~50% of adults are former smokers, including 44 million in the United States (U.S. Department of Health and Human Services, 1993). From the current smoking trends, former smokers will account for a growing percentage of all bladder cancer cases. Conflicting data exist on the prognostic value of smoking cessation (Thompsin et al., 1987; Carpenter, 1989; Castelao et al., 2001). The risk of developing bladder cancer is not precipitously reduced by smoking cessation, and former smokers still have a higher bladder cancer risk than nonsmokers. A decrease in smoking-related incidence of bladder cancer to a rate equal to that of nonsmokers may not occur until after 15 years of abstinence (Wynder and Goldsmith, 1977). A dose response between smoking and DNA adduct formation has been confirmed, and the slow and fast acetylation NAT2 genes relate to adduct formation in cigarette exposure, but do not necessarily result in a biological effect (Vineis et al., 1990).

An understanding of the procarcinogens related to bladder cancer development permits a better understanding of the genetic polymorphisms in phase I and phase II enzymes, which may play an important role in activating mutagens, as well as repair of DNA damage and detoxification and excretion of mutagens. Furthermore, this knowledge opens avenues of exploration for the identification of nutritional factors that may modulate the balance between detoxification and cancer formation.

INTRINSIC GENETIC SUSCEPTIBILITY FACTORS IN BLADDER CANCER No inherited syndromes have pointed to specific genes involved in bladder carcinogenesis in the way that familial adenopolyposis has identified a sequence for colon cancer (Kinzler and Vogelstein, 1996). However, Lynch syndrome involving multiple inherited cancer, which manifests as hereditary nonpolyposis colon cancer (HNPCC), involves bladder cancer frequently and identifies the importance of DNA repair genes in the process of carcinogenesis (Schulte, 1988). There are multiple genes that could potentially contribute to the network of signaling pathways related directly or indirectly to DNA repair in addition to the specific enzyme pathways, which have been defined and concisely summarized by Larminat et al. (1995). Enzymes related to the activation and inactivation of xenobiotics also may function as biomarkers of genetic susceptibility. These may be assayed by defining the genetic polymorphisms or from phenotypic functional assays. In most circumstances, the two approaches provide similar data, but the genetic analysis does not reflect the influence of subtle endogenous and exogenous factors influencing the metabolism of the xenobiotic.

GSTM1 Glutathione S-transferase (GST) M1 belongs to a family of phase II detoxification enzymes (Awasthi et al., 1994). GSTM1 detoxifies reactive chemical species, by catalyzing their conjugation to glutathione (Bell et al., 1993a). Deficiency in enzyme activity is caused by homozygous deletion of the GSTM1 gene. The frequency of homozygous deletion of the GSTM1 (i.e., GSTM1 null or 0/0 genotype) gene is about 50% in white populations and 30–70% in other racial/ethnic groups. Bell et al. (1993) studied 229 patients with TCCs of the bladder and 211 control subjects and reported that GSTM1 0/0 genotype conferred a 70% increased risk of bladder cancer (95% confidence interval [CI], 1.2–2.5), compared with individuals with at least one copy of the gene. Subsequent studies have confirmed this finding. A study in Spain by Lafuente et al. (1993) reported a twofold increased risk of bladder cancer in smokers for

Chemoprevention of Bladder Cancer

this polymorphic alteration. Daly et al. (1993) reported that odds ratios for bladder cancer associated with homozygous deletion of GSTM1 were 1.4 and 3.8, compared with hospitalized and healthy controls, respectively. Brockmoller et al. (1994) reported an odds ratio of 1.4 (95% CI, 1.0–1.9) for GSTM1 0/0 genotype and reported an etiological fraction of 17% for bladder cancer in Germany. In a pilot study, 175 patients with TCC and 162 cancer-free controls were assessed for GSTM1 genotype. The odds ratio was 1.7 for homozygous deletion of the GSTM1 gene.

GSTP1 GSTP1 belongs to the Pi class of the GST family. GSTP11 is the only member of the Pi class of GSTs expressed in humans. GSTP1 catalyzes the detoxification of polycyclic aromatic hydrocarbons (Strange et al., 1999). GSTP1 demonstrated an A-G polymorphism at codon 104, which has been indicated in bladder, testicular, and prostate cancer. Harries et al. (1997) reported that two variant GST cDNAs have been described at the GSTP1 locus, which differ by a single base pair (A-G) substitution at nucleotide 313 of the GSTP1 cDNA (GSTP1a and GSTP1b). This results in an amino acid substitution, which alters the function of the enzyme. The Pi-class GST has been associated with preneoplastic and neoplastic changes (Esteller et al., 1998).

N-Acetyltransferase N-Acetyltransferase (NAT) activity in humans is coded by two distinct genes named NAT1 and NAT2. NAT2 displayed polymorphism in humans, which results in the detection of individuals with slow and rapid N-acetylating phenotypes (Bartsch et al., 1993b). Slow acetylators have been found to be associated with higher incidence of bladder cancer, but fast acetylators are associated with higher incidence of colon cancer, which suggests that acetyltransferase polymorphism is related to both metabolic activation and deactivation of carcinogenic arylamines (Hein et al., 1993). The N-hydroxy metabolite of arylamine interacts with DNA and initiates tumorigenesis. The detoxification and elimination of carcinogens at this stage is through N-acetylation by N-acetyltransferase (Vineis et al., 1990). It has been estimated that slow acetylators are 55% of Caucasian populations and 41% in African American population (Bell et al., 1993b).

CHEMOPREVENTION OF BLADDER CANCER Bladder cancer is an ideal organ system for testing chemoprevention strategies. The urinary bladder is easily accessible and can be monitored by various noninvasive or minimally invasive surveillance techniques. The entire

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mucosal surface at risk can be visually examined by cystoscopy, and if necessary, biopsy specimens can be taken. The accessibility of the voided urine for noninvasive longitudinal sampling is suitable for mass screening studies, and the accessibility of the bladder for cystoscopic observation and pathological sampling provides tissue and cells for longitudinal follow-up and allows the process of bladder carcinogenesis to be followed. Thus, the bladder can be evaluated easily for evidence of early or premalignant lesions. Furthermore, the mean time from the initial carcinogen exposure to the development of overt TCC is close to 20 years, a lengthy period that provides ample opportunity to apply chemopreventive measures. The past 10 years have revealed the potential of chemoprevention in the reduction of mortality associated with common epithelial cancers (Hong and Sporn, 1997). Despite this fact, few large-scale trials have been performed for bladder cancer prevention. Currently, there are very few bladder cancer chemoprevention trials actively enrolling patients for study. From a clinical standpoint, chemopreventive agents should be easy to administer, have minimal side effects to encourage compliance, should be specific to tumor tissue, and have no deleterious effects on normal tissue. Thus, chemoprevention measures should target people who are generally healthy, at high risk for bladder cancer, have precancerous lesions, or other cancer histories but have not yet developed the disease.

Dietary Aspects for the Chemoprevention of Bladder Cancer There is an urgent need for research on dietary phytochemicals, which may prevent cancer through antioxidation and via gene–nutrient interactions, or which may be useful adjuncts in the prevention of cancer recurrence among successfully treated cancer survivors where prevention of cancer recurrence is vital. The role of nutrition in the development and course of bladder cancer and other malignancies is expanding rapidly but was recognized by early ancestors. Most research in bladder cancer has evaluated specific substances such as the various vitamins or specific foods such as green tea or garlic, but there is a vast starry-sky array of micronutrients in fruits and vegetables that are only beginning to be characterized. A meta-analysis of 38 articles on six dietary variables shows that the risk of bladder cancer is increased by diets low in fruits and vegetables or high in fat with an estimated relative risk (RR) of 0.7 for high vegetable and 0.8 for high fruit in the diet (Steinmaus et al., 2000). Future reviews will undoubtedly extol the enhanced benefits of micronutrients that are not appreciated today. Caloric Intake Numerous studies have investigated the impact of diet upon the incidence of various malignancies. One aspect of

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TABLE 1

Bladder cancer prevention clinical trials

Agent

Group/location

Study

DFMO Pyridoxine

ILEX Oncology, University of Wisconsin Byar and Blackard, 1977

Vitamin C

Shibata et al., 1992

High-dose combination multivitamins Celecoxib

Lamm et al., 1994

450 patients randomized, double-blind DFMO or placebo 121 patients in a multicenter trial comparing pyridoxine or thiotepa with placebo Cohort study of 11,590 patients over 9 years with diets high in vitamin C 65 patients given BCG and multivitamins

Tarceva and green tea polyphenol extract

M.D. Anderson; University of Texas UCLA and Mayo Clinics

Patients receive BCG and are randomized to either celecoxib or placebo 330 high-risk former smokers randomized to three arms

these dietary studies has been the role of caloric intake. Animal studies with mice have demonstrated that a reduction in total calories consumed decreased the development of numerous cancers including prostate and breast carcinoma (Cohen et al., 1988; Weindruch, 1992). The growth of bladder tumors was also demonstrated to decrease through caloric restriction in a mouse model (Dunn et al., 1992). A human study also demonstrated that higher caloric intake was associated with a higher incidence of bladder cancer in American men younger than 65 years (Vena et al., 1993). The effect of caloric intake on tumorigenesis appears to be related to insulin-like growth factor (IGF). Animal studies have demonstrated that the decrease in the growth of bladder tumors seen in calorically restricted mice was reversed when these mice were given IGF-1 (Dunn et al., 1992). Similarly, a human study also demonstrated that people with the highest quartile of plasma IGF-1 were three times more likely to develop bladder cancer (Zhao et al., 2003). Fluid Intake Increased fluid intake has also been isolated as a potential dietary method of decreasing the risk of bladder cancer. Studies focused on fluid intake as a result of the theory that the development of bladder cancer was related to the duration of time during which the bladder urothelium is exposed to carcinogens, as well as the concentration of the carcinogens within the urine (Oyasu and Hupp, 1974). Most bladder carcinogens are thought to exert their effect by direct contact with the urothelium through excretion in the urine. Increased fluid intake, in theory, should shorten this exposure time and decrease the risk of subsequent development of bladder cancer by diluting metabolites and increasing the frequency of voiding. The largest cohort study to date assessing this question was the Health Professionals’ Follow-up Study, which followed 48,000 men for 10 years (Michaud et al.,

Result In progress; accrual completed No significant difference 0.59 relative risk for bladder cancer patients Decreased recurrence compared with MDR In progress In progress

1999a). The study demonstrated that fluid consumption was inversely correlated with the risk of bladder cancer. Those men meeting the criteria for the highest quintile of fluid consumption (2531 ml/day) demonstrated approximately half the risk of developing bladder cancer as those men whose fluid consumption placed them in the lowest quintile (1290 ml/day) (Michaud et al., 1999a). Water consumption, in particular, resulted in a significantly lower RR of 0.49 for those men drinking more than six cups a day as compared with those drinking only a single cup per day (Michaud et al., 1999a). The consumption of all other fluids also demonstrated an inverse, though not statistically significant, relationship with the development of bladder cancer in this population. Other case-control studies have, in contrast, demonstrated a positive correlation between the consumption of some beverages and the incidence of bladder cancer. A pooled analysis of case-control studies, for example, reported a significant increase in bladder cancer in those people drinking large amounts of coffee (≥10 cups/day) (Sala et al., 2000). Other studies have demonstrated that increased intake of any fluids, including tap water, led to an increased incidence of bladder cancer, whereas still others have argued that fluid intake has no impact on bladder cancer, either positive or negative (Kunze et al., 1986; Geoffroy-Perez et al., 2001). Overall, the evidence on fluid intake and bladder cancer risk remains controversial, with results ranging from increased risk, to inverse risk, to no association. The inconsistencies in these studies may result from the difficulty in measuring total fluid intake. Furthermore, the source and purity of the water may also be important, with metals such as arsenic or disinfectants such as chlorine adversely affecting the overall protective effect of increased fluid intake. More data are necessary before definite recommendations regarding this potential preventative strategy can be made.


Fat Consumption Restriction of dietary fat has also been proposed as a method of bladder cancer prevention. Numerous studies have correlated dietary fat intake with carcinogenesis through such mechanisms as oxidative stress and the formation of free radicals (Fleshner and Klotz, 1998–1999). Studies have demonstrated an increased incidence of bladder cancer in people with high-fat diets. A multicenter casecontrol study from Spain demonstrated that men with the highest intake of saturated fat had a significantly increased risk of bladder cancer, with an RR of 2.25 (Riboli et al., 1991). In addition, Steineck et al. (1990) demonstrated a dose–response relationship between fat consumption and bladder cancer incidence. Other case-control studies have verified these results, correlating an increased risk of bladder cancer with greater ingestion of saturated fats and fried foods (Bruemmer et al., 1996). Still other studies have demonstrated that the ingestion of meat, in particular, is associated with an increased risk of bladder cancer (Mills et al., 1991). Whether the increased risk demonstrated by these studies is attributable to increased intake of fat rather than to merely increased calories is yet to be determined. Nonetheless, reduction of dietary fat should be advised as a potential method of reducing the risk of bladder cancer and other malignancies.

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tective effect against bladder cancer than fruits. The Health Professionals’ Follow-up Study reported that only the consumption of cruciferous vegetables had a significant impact on the risk of developing bladder cancer when comparing study participants with the greatest versus the least consumption of these vegetables (Michaud et al., 1999b). Also, whereas all cruciferous vegetables in the diet were associated with a decreased risk of bladder cancer, only broccoli and cabbage were found to have a statistically significant impact when comparing people with intakes less than once a week to those with intake of at least once per week. The study by Nagano et al. (2000) also reported the greatest effect from green and yellow vegetables. Interestingly, while tomatoes and tomato products have been demonstrated to have a preventative role in prostate cancer secondary to their lycopene contents, no such association has been found in relation to bladder cancer (Michaud et al., 1999b). Also, studies have reported that the risk reduction provided by vegetables seems have the greatest impact on nonsmokers (Michaud et al., 1999b). Nonetheless, dietary fruit consumption, particularly that of green vegetables, has definitely been demonstrated to serve some benefit in preventing bladder cancer and deserves mention during counseling of at-risk patients. Garlic

Consumption of Fruits and Vegetables In 1997, an international review panel concluded that there was convincing evidence that high intake of vegetables decreased the risk of cancers of the mouth, pharynx, esophagus lung, stomach, colon, and rectum, and that it probably decreases the risk of cancers of the larynx, pancreas, breast, and bladder (World Cancer Research Fund, 1997). Numerous studies have evaluated the effect of fruit and vegetable consumption on the risk of developing bladder cancer, and most of these studies have reported a lower risk of bladder cancer in subjects with high consumption (Negri and La Vecchia, 2001). A meta-analysis of 38 articles on six dietary variables shows that the risk of bladder cancer is increased by diets low in fruits and vegetables or high in fat, with an estimated RR of 0.7 for high vegetable and 0.8 for high fruit in the diet (Steinmaus et al., 2000). A large prospective study from Japan of 39,000 atomic bomb survivors in Japan (Nagano et al., 2000) demonstrated that regular fruit and vegetable consumption led to up to a 50% decreased risk of the future development of bladder cancer. Similarly, the Health Professionals’ Follow-up Study, based on 252 cases of bladder cancer, reported an inverse, though not significant, relationship between fruit and vegetable consumption and bladder cancer (Michaud et al., 1999). Further analysis from these studies demonstrated that vegetables appeared to have a greater pro-

Garlic, in use before recorded history, was known to have medicinal properties by ancient health professionals, including Hippocrates (Rivlin, 2001). Epidemiological studies have found reduced risk of stomach cancer (You et al., 1989) and prostate cancer (Hsing et al., 2002). Scientific study dates from the 1950s when Weisberger and Pensky (1958) demonstrated in vitro and in vivo that thiosulfinate extracts of garlic inhibited the growth of malignant cells and prevented growth of sarcoma 180 ascites tumor. Since that time, garlic has been shown in animal models to have antitumor activity in sarcoma, mammary carcinoma, hepatoma, colon cancer, and squamous cell carcinoma of the skin and esophagus (Lau et al., 1990). The activity of garlic in reducing the growth of bladder cancer in animals is quite striking. In 1986, Lau et al. compared intralesional and intraperitoneal garlic extract therapy with effective immunotherapies for bladder cancer: BCG, Corynebacterium parvum (CP, now termed Propionibacter acnes), and keyhole limpet hemocyanin (KLH) in the transplantable murine bladder tumor model MBT2. Intralesional garlic significantly inhibited the growth of transplanted bladder carcinoma. This efficacy was subsequently confirmed when Marsh (1987) reported intralesional garlic to be more effective and less toxic than BCG. Lamm, who has championed BCG therapy, was skeptical of these results and found that in his hands BCG could provide superior protection, but both intralesional and oral garlic significantly inhibited the growth of murine bladder

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cancer and prolonged survival. Clinical trials of garlic have not been reported (Lamm and Riggs, 2001). Green Tea Polyphenols Flavonoids found in green tea and available as botanical dietary supplements may play a role in bladder cancer protection and have been shown to have potent antioxidant and antitumor effects. Direct effects on cells and effects on enzyme systems, which inactivate carcinogens or act as part of the antioxidant response of cells, may mediate the effects of green tea. Tea is the most widely consumed beverage in the world and is a complex mixture of many substances including caffeine (2–4%), amino acids (4%), lignin (6.5%), organic acids (1.5%), protein (15%), chlorophyll (0.5%), and polyphenols (8–12%). Green tea contains 35–52% catechins and flavonols combined. Catechins belong to the general natural product class of flavonoids. The four major catechins in green tea are (−)-epicatechin, (−)-epicatechin3-gallate, (−)-epigallocatechin, and (−)-epigallocatechin-3gallate (EGCG). These catechins, which are believed to be the key active constituents in green tea polyphenol preparations, are potent antioxidants, which have been shown to reduce tumor growth, metastatic capacity, and angiogenesis in several studies. EGCG is the primary component, accounting for 40% of the total polyphenolic mixture, but it is not clear that this is the only component important in the action of green tea. One cup of green tea usually contains ~300–400 mg of polyphenols, and commercial preparations are now available that have reduced caffeine content and are enriched to contain 60–80% or more dry weight polyphenols. Numerous in vitro, human, and animal studies have identified these antioxidant polyphenols, in particular EGCG, as cancer-chemoprevention agents (Komori et al., 1993; Mukhtar et al., 1994). Preliminary work suggests that these polyphenols protect DNA to a much greater extent than either vitamin E or β-carotene in the retro-Ames assay. EGCG has been predicted to guard against carcinogenesis by blocking cell membrane receptors, repressing the catalytic activities of several cytochrome P450 (CYP) enzymes including CYP 1A and CYP 2B1, and enhancing cancerdetoxification enzymes. In in vitro studies, green tea polyphenols blocked nitrosamines and suppressed carcinogenic activity in lung, breast, colon, melanoma (Khan et al., 1992; Chung et al., 1993), and bladder cancers (Sato, 1999). Human studies suggest that green tea polyphenols may have anticarcinogenic effects on nitrosation (Xu et al., 1993) and chromosome damage (Shim et al., 1995). In humans (Xu et al., 1993), the urinary excretion of N-nitrosoproline is decreased with consumption of green tea extract, showing evidence of decreased nitrosation in vivo. Blood samples, cultures, and chromosome spreads have been examined and have shown an inverse incidence with tea intake (Shim et al., 1995). Moreover, urinary excretion of polyphenols can

be measured and may be useful in quantifying tea ingestion and polyphenol exposure in humans (Lee et al., 1995). There is increasing evidence of an inverse association with various cancers, in particular pancreatic (Ji et al., 1997), colon, stomach, and urinary bladder cancers. Of note, the incidence of bladder cancer in Japan and elsewhere in Asia is very low compared with the incidence in the United States and western Europe. The occurrence of bladder cancer in Japanese families emigrating from Japan to the United States beyond the second generation is twice that seen in natives remaining in Japan (Heuper, 1969). A number of epidemiological and human studies have shown an inverse association between green tea consumption and the risk of bladder cancer (Bushman, 1998; Bianchi, 2000). In preclinical studies, green tea has been shown to dosedependently inhibit bladder tumor growth in animal models, including the induction of bladder tumors by N-butyl-N-(4hydroxybutyl)-nitrosamine in rats (Sato, 1999). In addition to its antioxidant and antiangiogenic effects, in vivo studies have shown that green tea polyphenols are capable of inhibiting the enzymatic activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in the pathway of mammalian polyamine synthesis (Steele et al., 2000) and an enzyme that appears to play an important role in the process of tumor promotion. Polyamines affect DNA, RNA, and protein synthesis. For these reasons, ODC activity is said to be closely associated with tumor promotion. Green tea polyphenols have been demonstrated to inhibit ODC induction caused by tumor promoters in a number of tissues (Gupta et al., 1999). The inhibition of ODC results in a decrease in polyamine synthesis and cell growth. The biological properties of bladder cancer may render it susceptible to the effects of ODC blockade. Although normal and malignant human bladder epithelial cells have similar baseline ODC activities, activity of this enzyme is preferentially inducible by epidermal growth factor (EGF) in TCC (Messing et al., 1987). Also, ODC activity is significantly higher in TCC than in normal urothelium (Messing et al., 1995). Furthermore, human bladder cancer cell lines have been shown to be quite sensitive to the specific inhibitor of ornithine decarboxylase, α-difluoromethylornithine (DFMO) (Messing et al., 1988). DFMO has already been identified by the National Cancer Institute (NCI) as a promising bladder cancer chemopreventive agent for these capabilities (Malone et al., 1987). A phase II study of a green tea polyphenol extract is under way at the David Geffen School of Medicine at UCLA. Soy Products Soy products have been found to be beneficial in protection from numerous disease processes. Studies have demonstrated an association between higher intake of soy products and a lower risk of malignancies of the breast, colon, and


prostate (Messina and Barnes, 1991). Experimental evidence has also demonstrated that soy products may affect the growth of bladder cancer cells in vitro and in vivo. The mechanism behind this effect appears to be cell cycle arrest, induction of apoptosis, and reduction of angiogenesis by means of interaction of various isoflavones such as genistein (Zhou et al., 1998; Su et al., 2000). Given that isoflavones are excreted in the urine with ingestion of soy products, the direct contact of these compounds with the bladder urothelium may prevent the development of bladder tumors (Seow et al., 1998). Very few epidemiological and clinical studies have investigated the role of soy in bladder cancer prevention. A small study of 40 patients by Lu et al. (1999) reported a small, insignificant decrease in bladder cancer risk in people who regularly consumed soy juice. This benefit was further reduced when adjustments were made for confounding variables. Similarly, Garcia et al. (1999) also failed to demonstrate a relationship between flavonoid intake and reduction of bladder cancer risk. Vitamins Vitamins that have been implicated by chemical, epidemiological, physiological, immunological, or clinical trial evidence to be potentially beneficial in bladder cancer include vitamins A, B6, C, D, and E. Each of these are briefly discussed as they related to bladder cancer, but it important to emphasize that the key to nutritional therapy in bladder cancer is balance rather than bullet: Combinations of beneficial nutrients working together will likely produce superior results to an approach based on single factors. Vitamin A As early as 1972 clinical evidence suggested that vitamin A was effective in bladder cancer. Evard and Bolag (1972) gave an oral dose of 100 mg of vitamin A to 15 patients and reported 26.7% complete and 46.7% partial response in the treatment of existing TCCs in the bladder. Vitamin A and its analogs, especially the retinoids, are among the best-studied chemopreventive agents in bladder cancer. Vitamin A is important for cell differentiation and has been found to enhance the differentiation of normal and neoplastic cells in tissue culture (Lotan, 1980). The reported biological actions of vitamin A analogs include antioxidant activity and immunoenhancement. In addition to their effects on singlet oxygen, carotenoids are also thought to suppress oxygen free radicals. All-trans-retinoic acid causes acute promyelocytic leukemia blasts to differentiate into polymorphonuclear leukocytes in vitro and has been observed to significantly improve response in controlled trials when used in combination with chemotherapy in acute promyelocytic leukemia (Chomienne et al., 1991). Epidemiological data regarding vitamin A remain conflicting. Studies that favor a protective role include that of

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Mettlin and Graham (1979), who compared retrospective data from 569 bladder cancer patients and 1025 age-matched an increase in sex-adjusted RR for bladder cancer with lower levels of vitamin A intake. In another study of 2974 men followed for 17 years, low mean plasma levels of carotene were associated with increased overall mortality from cancer (Eichholzer et al., 1996). Both serum carotene and serum retinol levels are significantly reduced in patients with bladder cancer when compared with controls (Hicks, 1983). However, a meta-analytical review of epidemiological studies linking six dietary factors to bladder cancer demonstrated no increased risks for diets low in retinol (RR = 1.01, 95% CI, 0.83, 1.23) or β-carotene (RR = 1.10, 95% CI, 0.93, 1.30) intake (Steinmaus et al., 2000). The effect of low vitamin A serum levels is often lost when corrected for smoking. Retinoids are potent suppressors of neoplastic transformation induced by various carcinogens, including viral, chemical, and radioactive agents, and antitumor activity against carcinogen-induced cancer of the skin, mammary gland, urinary bladder, esophagus, cervix, and liver has been demonstrated in animal models (Sporn and Newton, 1979). Like most chemopreventive agents, retinoids are most effective when given before or shortly after the inciting carcinogen, but efficacy in preventing bladder cancer is reported even when treatment is delayed (Becci et al., 1979). Hypervitaminosis A inhibits keratinization and squamous metaplasia in bladder lesions induced by the carcinogen Nbutyl-N-4-hydroxybutyl nitrosamine (BBN) and reduces incidence of transitional cell carcinoma and papilloma of the urinary bladder (p < .02) (Miyata et al., 1978). Becci et al. (1978) further showed that 13-cis-retinoic acid can reduce the incidence, average number, and grade of TCCs, as well as hyperplasia and cellular atypia in BBN-exposed mice urinary bladders even when the treatment was delayed by 9 weeks (Becci et al., 1978). Similar results have been reported with β-carotene. Mice supplemented with βcarotene for 5 weeks before receiving the carcinogen and maintained on β-carotene for an additional 26 weeks developed significantly fewer tumors than did control mice (Mathews-Roth et al., 1991). Using DNA flow cytometry as an intermediate endpoint, Decensi et al. (1992) demonstrated a reversion to normal cytology with the administration of 4-HPR to patients with previously suspicious or positive DNA flow cytometry. Although 4-HPR and other retinoids have been touted as “superior chemopreventive agents,” clinical trials such as the CARROT and Finnish study suggest that a combination of retinoids, as well as other vitamins, may be preferable. Studies combining retinoids with BCG have shown improved efficacy and suggest that the activity of retinoids may be potentiated by nonspecific immune stimulation. Treatment of mice with a combination of the retinoid Ro 10-

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9359 and BCG results in an 83.3% incidence of complete tumor regression within 80 days (Pang and Morales, 1983). Similar findings have also been noted when combining retinoids with interferons (IFNs). Furthermore, it has been shown that human tumor-induced inhibition of IFN in vitro can be reversed by the β-carotene (Rhodes et al., 1983). Further support for immune-mediated response is demonstrated by the reduction of insulin-like growth factor 2 (IGFII) (an important factor in IGFs in the pathogenesis of different solid tumors) by 16% with fenretinide treatment (Torrisi et al., 2000). Clinical trials of retinoids in bladder cancer have been generally remarkably successful but largely ignored. In one of the earliest human trials Evard and Bolag in 1972 gave 15 patients 100 mg of vitamin A orally and found complete response in 26.7% and partial response in 46.7% (Evard and Bolag, 1972). A controlled clinical trial by the National Bladder Cancer Collaborative Group A of 13-cis-retinoic acid in patients with rapidly recurring bladder cancer refractory to other treatments closed early and failed to show any benefit (Prout and Barton, 1992). However, this study in patients who had failed multiple prior treatments would not be expected to show benefit of chemoprevention. Another form of vitamin A, etretinate, was reported in two independent studies to be an effective chemopreventive for superficial bladder tumors. Thirty patients were enrolled in a double-blind, placebo-controlled study using etretinate in grade 1–2 Ta or T1 lesions. With duration of treatment ranging from 10 to 26 months, the recurrences were reduced from 87% to 60% (p < .01) in patients given etretinate (Alfthan et al., 1983). This report stimulated further larger studies with conforming results. Studer et al. (1995) studied 79 eligible patients with superficial (Ta, T1) papillary bladder tumor in a prospective double-blind multicenter trial. These patients were randomized to receive either 25 mg/day of etretinate orally or a placebo. Although the time to first recurrence was similar in both groups (13.5 and 13.6 months in the placebo and etretinate groups, respectively), the mean interval to subsequent tumor recurrence was 20.3 months in the etretinate group as compared with 12.7 months in the placebo arm ( p = .006). A longer time to recurrence resulted in a decrease in the number of transurethral resections per patient-year from 2.1 in the control arm to 0.95 in the etretinate arm (p < .001). The side effects of etretinate were acceptable to most patients. Vitamin B Vitamin B6 (pyridoxine, pyridoxal, and pyridoxamine) and its metabolites are coenzymes for multiple pathways including tryptophan metabolism. Metabolites of tryptophan have been observed to have carcinogenic properties, and studies of human families suggest a clustering of abnormal tryptophan metabolism in patients with bladder cancer. Kynurenine, 3-hydroxy-anthranilic acid, and other tryptophan breakdown products can be elevated in the urine of

patients with bladder cancer, and in animal models, these substances induce bladder cancer (Brown et al., 1960). Pyridoxine shifts the metabolism of tryptophan away from the carcinogenic derivatives and, therefore, prevents recurrences of superficial bladder cancers. The benefit observed with vitamin B6 may also be related to its effect on augmentation of immunity because elevated levels of vitamin B6 stimulate lymphocyte response in the geriatric population, suppress tumor growth, and boost anti-tumor immunity in mice (Gridley et al., 1987; Talbott et al., 1997). Two large human trials have been instituted with conflicting results. In 1977, Byar et al. (1977) studied 118 patients with stage I bladder cancer randomized to placebo, pyridoxine 25 mg orally, or intravesical thiotepa. Tumor recurrence rate was 60% of the controls, 46% in the pyridoxine group, and 47% in the thiotepa group. If events during the first 10 months were excluded from analysis (allowing for time to action of pyridoxine), pyridoxine was as effective as thiotepa and significantly better than placebo (p = .03) (Byar et al., 1977). Another trial by the EORTC GU group failed to show an advantage for pyridoxine therapy. In this double-blind randomized trial comparing 20 mg/day of pyridoxine with placebo in 291 patients, no difference in time to first recurrence or the recurrence rate was observed. Adjusting for prognostic factors such as the recurrence rate or number of tumors before entry, the tumor grade or the levels of tryptophan metabolites did not change the results (Newling et al., 1995). Vitamin C Vitamin C is a major circulating water-soluble antioxidant and free radical scavenger. In vitro studies show it to decrease chromosome damage in lymphocytes induced by exposure to bleomycin (Pohl and Reidy, 1989). Diets high in vitamin C are associated with lower risk for oral, gastrointestinal, and lung cancers. For gastric and esophageal cancer, there is evidence that this association is due to an inhibition of in vivo N-nitroso compound formation (Mirvish, 1986). Vitamin C reduces the in vivo formation of N-nitroso compounds, including 3-hydroxanthranilic acid and nitrosamines, which are implicated as potential causative agents in bladder cancers (Schlegel, 1975; Kakizoe et al., 1988). Bladder cancer decreases with increasing levels of vitamin C intake (p = .03). Nomura et al. (1991) reported that the odds ratio for the highest quartile of vitamin C intake compared with the lowest quartile was 0.4. In a similar study, Shibata et al. (1992) followed 11,580 residents of a retirement community who were initially free from cancer. With 8 years of follow-up, 1335 cancer cases were diagnosed. After adjusting for age and smoking, there was an inverse association between vitamin C supplementation and bladder cancer risk (Shibata et al., 1992). Some studies have hinted that sodium l-ascorbate and lascorbic acid might promote urinary bladder carcinogenesis

The Future of Bladder Cancer Chemoprevention

in a dose-related manner (Birt, 1986), but studies are again inconsistent (Fukushima et al., 1987). The cytotoxic effects of ascorbic acid on the sensitive cell lines are time and dosage dependent (Kao and Meyer, 1993). It has been suggested that there is a threshold dose beyond which vitamin C has carcinogen-promoting effect (Fukushima et al., 1983) and that cancer cells may exist in two forms, one sensitive to inhibition by ascorbic acid and the other resistant to its anticarcinogenic effect. It may be best to limit the dose of vitamin C to moderate doses of 2000 mg daily until further data are available. Vitamin D In a study of ultraviolet B (UVB) radiation and U.S. cancer mortality rates, decreased sunlight was associated with increased risk of cancer of the bladder and other tumors including breast, colon, ovary, prostate, kidney, and lung (Grant, 2002). Vitamin D inhibits the proliferation of various tumor cells in tissue culture, including human bladder cancer cell lines, and in vivo inhibits the growth of N-methyl nitrosourea–induced bladder cancer in rats (Konety et al., 2001). Vitamin E Vitamin E is a major lipid-soluble antioxidant and free radical scavenger and acts as an antioxidant, protecting unsaturated lipids in cell membranes and metabolic enzymes from free radical oxidation damage. Diets high in vitamin E are associated with a lower risk of cancer (Byers and Guerrero, 1995). Nitrosamines, occurring in tobacco products, certain industrial chemicals, and nitrite-cured meat and other foods, have been implicated in cancers of the stomach, esophagus, nasopharynx, urinary bladder, and colon (Mirvish, 1995). In a cohort study of 991,522 U.S. adults in which 1289 bladder cancer deaths occurred, long-term vitamin E consumption, but not vitamin C consumption, significantly reduced mortality from bladder cancer (Jacobs et al., 2002). Vitamin E inhibits N-nitroso compound formation and produces 30–60% inhibition of induced carcinogenesis in most animal experiments (Mirvish et al., 1986). It is also possible that vitamin E provides protection from cancer by stimulating antitumor immunity. Supplementation of vitamin E enhances resistance to infection, reticuloendothelial system activity, and delayed cutaneous hypersensitivity response and antibody production (Beisel et al., 1981). Studies of thymidine incorporation have shown that vitamin E supplementation reduces DNA synthesis and causes fragmentation leading to apoptosis and, thus, inhibits cancer cell growth in a dose-dependent fashion. Cells vary in sensitivity to this effect, with breast and prostate cancer cells being most sensitive (Sigounas et al., 1997). Selenium Conflicting data exist as to the potential role of selenium in preventing bladder cancer. This essential trace mineral has

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been extensively investigated by numerous studies through the measurement and comparison of serum and toenail selenium levels of bladder cancer patients and controls. Helzlsouer et al. (1989) reported on a population of 25,802 people followed for 12 years and found to have 35 cases of bladder cancer. In this population, serum selenium was found to be significantly lower in patients with bladder cancer than in controls. In contrast, in the Nurses’ Health Study, selenium levels were measured from toenail clippings of more than 62,500 women, and women with bladder cancer were actually found to have higher selenium levels than controls (Garland et al., 1995). Also, an overall higher cancer risk was also found in those women taking selenium supplements. In another large trial of selenium supplementation, a nonsignificant association was reported between selenium supplementation and bladder cancer risk (Clark et al., 1996). More study is necessary before dietary or supplemental selenium can be recommended for the prevention of bladder cancer. Combination Vitamins Lamm et al. (1994) studied the effect of a megadose vitamin combination in a double-blind randomized trial. Sixty-five patients with biopsy-confirmed TCC of the bladder were randomized to receive either the RDA of multiple vitamins or the RDA plus 40,000 IU vitamin A, 100 mg vitamin B6, 2000 mg vitamin C, and 400 IU vitamin E and 90 mg zinc. As might be expected from a chemoprevention trial, curves for time to recurrence for the two groups were identical for the first 10 months but diverged significantly thereafter. Five-year estimates of tumor recurrence were 91% in the RDA arm and 41% in the high-dose vitamin arm (p = .0014). Overall recurrence was 80% (25/30) in the RDA arm and 40% (14/35) in the high-dose vitamin arm ( p = .0011). Stratifying the patients according to tumor stage showed a statistically significant (p = .003) benefit of a 42% reduction in tumor recurrence in those with stage Ta or T1 TCC. Stratifying the patients according to tumor grade also showed a statistically significant ( p = .007) benefit of 53% in those with low grade (G1, G2) TCC. The high-dose vitamins were generally well tolerated, with mild nausea being the most common side effect.

THE FUTURE OF BLADDER CANCER CHEMOPREVENTION: A RATIONAL STRATEGY FOR THE IDENTIFICATION AND TESTING OF NEW AGENTS Development of newer or more effective preventive strategies for bladder cancer is of critical importance. Chemoprevention lies at an interesting intersection between disease management and health promotion, and competing values play a role in determining the nature and magnitude of the risks and benefits of chemoprevention of cancer.

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Ethical questions related to these trials concern the enrollment of healthy or “at-risk” individuals rather than cancer patients, and one has to balance competing benefits and toxicities of treatment. Many authorities question the validity of current recommendations for nutritional chemoprevention against bladder cancer. The reason for this skepticism revolves around the wide variations reported in epidemiological studies that are in the nature of observational studies. Observation dietary studies have been limited in their conclusions because the protection afforded by the consumption of a particular nutrient may be multifactorial, with different components of the food exerting potential chemopreventive effects. Furthermore, measuring levels of nutrients in the food intake of populations is confounded by factors that might affect these levels and the incidence of cancer. In addition, chemoprevention studies using dietary strategies may be expected to have mild effects, and large studies may be required to confirm statistical significance. Therefore, prospective randomized trials with a large sample size, longer follow-up, an extended duration of treatment, and validated biomarkers of risk, effect, and prognosis are necessary to clarify the association between micronutrients and cancer protection. The final section of this chapter explores key issues related to the prospective testing of new agents for bladder cancer prevention.

A Strategy for Developing Interventions for Superficial TCC: The ABCD Matrix The design and conduct of investigational studies for the prevention and management of superficial bladder cancer involves the rational integration of several key factors including (1) agents (pharmaceuticals, biologics, and natural products); (2) biomarkers (intermediate endpoints that reflect biological activity and cancer risk reduction); (3) cohorts (well-defined individuals with high risk for recurrence and progression); (4) designs (efficient randomized controlled trials linked to the phase of Food and Drug Administration–guided drug development); and (5) endpoints (meaningful clinical outcomes such as reduction of cancer recurrence and progression). Because patients with superficial TCC typically present with early-stage disease (Ta, T1, CIS) that frequently recurs and is readily accessible and monitored through serial cystoscopies and urine cytology, superficial bladder cancer can serve as a prototypical clinical model for conducting prevention and adjuvant treatment trials. Clinical Models and Trial Designs for Evaluating Nutritional, Biological, and Chemopreventive Agents Prevention of Recurrence of Superficial TCC The design most frequently employed to demonstrate clinical and biological activity is to administer the experi-

mental agent(s) after the initial or recurrent lesion is surgically removed (TURBT) as adjuvant therapy. Because recurrences can represent a new second primary tumor or can reflect recurrence of the original clonal outgrowth, interventions in this setting are also considered to be secondary prevention. The cohort is typically a patient with low to intermediate grade Ta or T1 lesions. However, with the FDA approval of intravesical BCG immunotherapy initially for the treatment of carcinoma in situ (CIS) and more recently for an expanded indication to include adjunct treatment of resected stage Ta or T1 tumors, an increasing number of patients with high grade (grade 2 or 3) Ta and T1 lesions that are multifocal and larger than 3 cm are also managed after TURBT with BCG induction and/or maintenance therapy. Based on current recommendations of the FDA, definitive clinical response would be assessed by the 2-year recurrence rate and longer term follow-up of disease progression (Hirsch, personal communication, 2004). Neoadjuvant Treatment Prior to Definitive TURBT A practical design that has gained popularity for several cancers (e.g., prostate and breast) is to administer the experimental agent(s) in the window of opportunity between the diagnostic or surveillance positive cystoscopy and definitive treatment (TURBT for superficial or cystectomy for muscle invasive disease). An advantage of this approach is the clinical histological response of the index lesion(s) and the effect on the field changes (dysplasia), and intermediate endpoint biomarkers in the bladder and urine can be concurrently assessed over a relatively shorter interval (3 weeks to 3 months). This approach should allow for more rapid screening of promising agents. Intravesical Administration Another strategy that is feasible is the intralesional or intravesical administration of experimental agents including immunological vaccines, chemopreventive, chemotherapeutic, and gene therapy. This approach would appear to be well suited for the evaluation of the prevention of low-grade recurrent disease implantation, as well as adjuvant treatment of CIS in combination with BCG or as salvage treatment of BCG failures (e.g., valrubicin). Intermediate Endpoint Biomarkers: The Search for Surrogate Endpoints Validation of Surrogate Endpoints and Accelerated Drug Development A major goal of chemoprevention is the validation of surrogate endpoints (SEs) for bladder cancer incidence reduction. The rationale for identifying SEs to evaluate efficacy relates to the improved efficiency and predictability by reduced sample size, decreased duration to conduct clinical trials, and more cost-effective drug development for phar-

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The Future of Bladder Cancer Chemoprevention

maceutical and biotechnology sponsors of phase 3 clinical trials organized to gain FDA approval for marketing. Validation can be achieved during phase 3 randomized controlled trials by incorporating candidate SEs and correlating changes in the SE with the primary clinical endpoint of cancer incidence reduction. In this context, information should be elucidated regarding the relationship between the SE and the causal pathway of bladder carcinogenesis. Once validated, an SE of bladder cancer carcinogenesis can accelerate new agent development because it may now be used as the primary endpoint versus the clinical endpoint of bladder cancer recurrence in phase 2b/3 trials under current FDA clinical guidelines. However, no SE has been prospectively validated as a substitute for cancer incidence/recurrence in bladder cancer.

DNA damage

Procarcinogens

Cancer

Mutagens

P450 Enzymes

GSTs,MEH NQO1,UGTs

Detoxified mutagens

Excretion

Legend Promote

Soy protein, green tea, Vit A, B6, C, D, E.

Isoflavones, Phytoestrogens, Catechins

Inhibit

FIGURE 1 Gene–environment interactions: Diet may modify enzyme metabolism and subsequent risk of cancer development.

Categories of Intermediate Endpoint Biomarkers Currently, several types of intermediate endpoint biomarkers (IEBs) are under active evaluation or have been proposed as candidate SEs. These include (1) histological markers such as IEN and nuclear histomorphometric indices in the urothelium at risk; these histological-based markers can be quantitated by nuclear morphometric analysis; (2) tissue and cellular markers such as apoptosis, proliferation, angiogenesis, inflammation, and differentiation; (3) genetic markers such as specific mutations, aneuploidy, loss of heterozygosity, microsatellite analysis, and cDNA gene expression microarrays; (4) markers associated with signal transduction pathways such as overexpression or loss of function of oncogenes and tumor suppressor genes; (5) markers associated with regulatory and cell cycle control; (6) markers of inflammation and oxidative stress; (7) markers associated with proteomics patterns and protein products in the serum, urine, and bladder washings; (8) markers based on epigenetics (methylated DNA) in the serum and urine; and (9) markers associated with functional imaging. Although the FDA has approved five bladder cancer markers including NMP-22, BLCA-4, and BTA, the clinical gold standard for confirming bladder cancer incidence and recurrence remains cystoscopic surveillance with examination of bladder washings and urine cytology. Several promising urine markers include MSA, survivin, telomerase, and methylated genes.

Evidence-Based Leads for Identifying New Agents Molecular Epidemiology, Pharmacogenomics, and Nutritional Science Epidemiology and nutritional science continue to provide promising leads for identifying populations at risk and novel interventions. This would include subjects with polymor-

phisms in known antioxidant/anticarcinogen defense mechanisms related to cytochrome P450 metabolism (i.e., GSTM1 null variants), acetylator phenotypes (i.e., NAT2 slow acetylator), DNA repair enzymes (i.e., hMSH2), and gene–environment interactions with carcinogens in tobacco, common drugs such as nonsteroidal anti-inflammatory drugs (NSAIDs), and dietary exposures to micronutrient antioxidants (i.e., carotenoids, selenium, vitamin E, and phytochemicals such as soy isoflavones, green tea polyphenols, and garlic). Adjuvant Trials Successful adjuvant and secondary prevention trials using intravesical administration as noted earlier have been the gold standard to identify agents such as BCG and KLH that could be applied earlier in superficial bladder cancer. Furthermore, the realization of significant limitations in the ability to prevent recurrences and especially progression to muscle invasive disease in CIS and high-grade Ta and T1 lesions with these intravesical agents alone underscores the need for other modes of administration including chronic systemic approaches. In this regard, the successful experience with systemic adjuvant treatment regimens for other epithelial cancers such as breast and colon cancer is relevant. This strategy to use chronically administered oral agents is being pursued through several collaborations among the NCI, pharmaceutical/nutriceutical industry, and academia. One innovative design is a three-arm randomized controlled trial using two promising oral agents (i.e., extracts derived from green tea [polyphenon E] and the selective EGF receptor [EGFR] inhibitor [erlotinib]). This trial is actually two parallel randomized trials that use a common control group (Belldegrun, personal communication).

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Secondary Analyses of Large Prevention Trials Historically, post hoc analyses of large prevention trials have proven informative in hypothesis generation, which can be tested in prospective randomized controlled trials. Two antioxidant nutrients for prostate cancer prevention (i.e., vitamin E and selenium) were identified in this way and are now undergoing clinical evaluation in a large primary chemoprevention trial for prostate cancer known as SELECT. Thus, even negative phase 3 trials for bladder cancer chemoprevention (e.g., DFMO) could provide new leads thru secondary analyses. In Vitro and In Vivo Experimental Models of Bladder Carcinogenesis Chemopreventive activity has been identified for several classes of agents including NSAIDs and their derivatives, synthetic and natural retinoids, phase 2 enzyme inducers, antioxidant nutrients, phytochemical natural products, immune modulators, and molecularly targeted agents such as EGFR inhibitors. Experimental model systems include in vitro human bladder cell lines, human xenografted rodent models, carcinogen (OH-BBN) rodent models of bladder carcinogenesis, and new transgenic murine models of bladder cancer using p53 heterozygotes. These models have yielded positive results and have led to randomized controlled trials (e.g., DFMO, 4-HPR, celecoxib, green tea extracts, and EGFR inhibitors).

CONCLUSIONS Preclinical and limited clinical data demonstrate that bladder cancer is responsive to primary and secondary preventive efforts. Furthermore, epidemiological studies imply that natural products, such as vitamins and herbal compounds, may provide preventive benefit, but these agents have not been prospectively studied. Bladder cancer is an attractive target for prevention efforts because renal excretion of agents enables prolonged exposure to high concentrations of drugs in the urine. Additionally, the urothelium is easily evaluated and monitored with strategies that are highly successful and relatively noninvasive. The frequent recurrence of superficial bladder cancer with low occurrence of muscle-invasive disease permits the safe and efficient assessment of strategies to prevent secondary bladder cancers with a modest number of patients and short followup. Although improved understanding of the pathways of bladder carcinogenesis presents new targets for prevention, the lack of identifiable high-risk populations and SEBs (for both malignancies) limits our ability to implement prevention studies. The development of validated surrogate endpoints will facilitate the future evaluation of novel chemopreventive agents and strategies.

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Torrisi, R., Mezzetti, M., Johansson, H., Barreca, A., Pigatto, F., Robertson, C., and Decensi, A. 2000. Time course of fenretinideinduced modulation of circulating insulin-like growth factor (IGF)-I, IGF-II and IGFBP-3 in a bladder cancer chemoprevention trial. Int J Cancer 87(4): 601–605. Vena, J., Graham, S., Freudenheim, J., Marshall, J., Zielezny, M., Swanson, M., et al. 1993. Diet in the epidemiology of bladder cancer in western New York. Nutr Cancer 48: 191. Vineis, P., Caporaso, N., Tannenbaum, S.R., Skipper, P.L., Glogowski, J., Bartsch, H., et al. 1990. Acetylation phenotype, carcinogenhemoglobin adducts, and cigarette smoking. Cancer Res 50: 3002– 3004. Weindruch, R. 1992. Effect of caloric restriction on age associated cancers. Exp Gerontol 27: 575. Weisberger, A.S., and Pensky, J. 1958. Tumor inhibition by a sulfhydrylblocking agent related to an active principle of garlic (Allium sativum). Cancer Res 18: 1301–1308. World Cancer Research Fund. 1997. “Food, Nutrition and the Prevention of Cancer: A Global Perspective.” American Institute for Cancer Research, Washington, D.C. Wynder, E.L., and Goldsmith, R. 1977. The epidemiology of bladder cancer: a second look. Cancer 40: 1246–1268. Xu, G.P., Song, P.J., and Reed, P.I. 1993. Effects of fruit juices, processed vegetable juice, orange peel and green tea on endogenous formation of N-nitrosoproline in subjects from a high-risk area for gastric cancer in Moping County, China. Eur J Cancer Prev 2: 327–335. You, W.C., Blot, W.J., Chang, Y.S., Ershow, A., Yang, Z.T., An, Q., Henderson, B.E., Fraumeni, J.F., Jr., and Wang, T.G. 1989. Allium vegetables and reduced risk of stomach cancer. J Natl Cancer Inst 81: 162–164. Zang, E.A., and Wynder, E.L. 1996. Differences in lung cancer risk between men and women: examination of the evidence. J Natl Cancer Inst 88: 183–192. Zhao, H., Grossman, H., Spitz, M., Lerner, S., Zhang, K, and Wu, X. 2003. Plasma levels of insulin-like-growth-factor 1 and binding protein 3 and their association with bladder cancer risk. J Urol 169: 714. Zhou, J., Mukherjee, P., and Gugger, E., et al. 1998. Inhibition of murine bladder tumorigenesis by soy isoflavones via alterations in the cell cycle, apoptosis, and angiogenesis. Cancer Res 58: 5231.

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27 Differentiation Induction in Leukemia and Lymphoma SVEN DE VOS AND H. PHILLIP KOEFFLER

INTRODUCTION

diagnosed AML. The best outcomes occur in patients with acute promyelocytic leukemia (APL) harboring the t(15;17) translocation. Overall, long-term survival or cure rates vary between 20 and 40%. Myelodysplastic syndrome (MDS) is characterized by peripheral blood cytopenias, and a bone marrow (BM) that shows ineffective hematopoiesis. Karyotypic and genetic marker data indicate that MDS results from a preneoplastic transformation at a pluripotent stem cell level (Koeffler, 1996). The MDS clone has a selective growth advantage over the normal stem cells compartment, perhaps in part mediated by a maturational block. The mechanism of this maturational block is unknown. Treatment of MDS has long been frustrating for the physician. Although it represents a preleukemic state in some patients, therapy with standard antileukemic chemotherapy is excessively toxic. Cytokines, transfusions, and antibiotics for infections have been the mainstays of patient management. Theoretically the use of differentiation therapy to induce normal maturation of the aberrant clone of cells is an interesting approach (Koeffler 1983). The goal of this therapy is not to eliminate the dysplastic clone of cells but to induce the defective hematopoietic progenitors to acquire more normal maturational and functional characteristics. Differentiation therapy attempts to improve the ability of the abnormal progenitors to produce functional blood cells. The WHO classifies lymphomas as either B-cell neoplasms, T/NK-cell neoplasms, or Hodgkin’s lymphoma. The classification and further subdivisions are based on the integration of morphological, immunophenotypic, genetic, and clinical features. Low-grade lymphomas are frequently responsive to a variety of treatments but remain fairly resistant to cure. Still, patients usually achieve long survival

Leukemia can be divided into four major subtypes: lymphoid and myeloid, each of which can be either chronic or acute. The median age at diagnosis of patients with chronic lymphocytic leukemia (CLL) is 60–65 years, with a steadily increasing incidence with age. Individuals with this disease often survive in excess of 7–10 years. CLL is characterized by a clonal expansion of mature, long-lived, and functionally defective lymphocytes. Acute lymphocytic leukemia (ALL) is the most common leukemia in children. With aggressive chemotherapy, long-term disease-free survival (DFS) rates of 70–75% are possible. In contrast, ALL is less common and less curable in adults. Only 20% of all adult acute leukemias are ALL, and the 5-year survival rates reach only 25–35%. Chronic myelocytic leukemia (CML) is a clonal stem cell disorder characterized by a balanced translocation between the long arms of chromosome 9 and 22 (t[9;22], known as Philadelphia chromosome). CML is very rare in children; the median age at presentation is 50–60 years. The medium survival is 5–7 years. Initially the myeloid cells are expanded in number but mature correctly. However, toward the end of the disease, additional chromosomal changes besides the Philadelphia chromosome occur and the leukemia evolves into blast crisis, which is nearly impossible to cure. Acute myelogenous leukemias (AMLs) occur at all ages. The World Health Organization (WHO) classification (WHO) distinguishes (a) AMLs with recurrent cytogenetic translocations, (b) AMLs with multilineage dysplasia, (c) therapy-related AML, (d) AMLs not otherwise characterized, and (e) biphenotypic leukemias that display features of ALL and AML. Chromosomal abnormalities are the most important prognostic factors in patients with newly


491


492

Chapter 27/Differentiation Induction in Leukemia and Lymphoma

times. Patients with high-grade lymphomas are potentially curable with aggressive chemotherapy regimens including high-dose chemotherapy with peripheral stem cell transplantation. Today, most patients with Hodgkin’s lymphoma can expect to be cured with combination chemotherapy regimen. The current emphasis in clinical research is to maintain this treatment success with less long-term toxicities. No clear association exists between nutrition and the development of leukemias and lymphomas. Occasionally, some nutritional deficiencies can be confused with either preleukemia or leukemia. For example, individuals with severe vitamin B12 or folate deficiency display BM abnormalities that resemble a preleukemia or erythroleukemia. These individuals respond dramatically to replacement therapy with vitamin B12 or folate and do not have an increased incidence of leukemias or lymphomas. However, evidence points to a possible role of folate deficiency in the development of childhood or adult ALL. Considerable epidemiological evidence suggests that a low folate intake confers a higher risk of several types of cancers (Ames and Wakimoto, 2002). A polymorphism (C677T) in the gene encoding methylene-THF reductase (MTHFR) reduces its enzymatic function resulting in decreased uracil incorporation and increased occurrence of chromosome breaks (Ames and Wakimoto, 2002). Both adult (Skibola et al., 1999) and childhood ALL (Wiemels et al., 2001) have an inverse relationship with the C677T homozygous MTHFR-TT genotype, suggesting that folate deficiency might promote ALL. Pan et al. (2004), in line with earlier publications, has observed an increased risk of cancer associated with obesity, including non-Hodgkin’s lymphoma (Wolk et al., 2001), leukemia (Moller et al., 1994), and multiple myeloma (Friedman et al., 1994). The mechanistic link between obesity and these hematological malignancies remains elusive, although speculations point to impaired immune responses (Stallone, 1994; Chandra, 1997) and lower intake of antioxidants and nutrients (Hotzel, 1986).

RETINOIDS Mechanism of Action Retinoids are a family of molecules important for many biological processes such as embryonal morphogenesis, epidermal cell growth, and hematopoiesis in vivo and in vitro. The natural retinoids, 9-cis-retinoic acid (RA), 13-cis-RA (cRA), and their genomic isomer, all-trans-RA (ATRA), constitute a group of structurally similar compounds with diverse profiles of pharmacokinetics, therapeutic, and adverse effects. Retinoids affect the transcription of various genes critical to cellular proliferation and differentiation. Upon enter-

ing the cell, retinoids must be transported to the nucleus to exert their effect. The cellular responses to RA are mediated by two families of transcription factors, which include the RA receptors (RARs) and the retinoid X receptors (RXRs). ATRA crosses the cell membrane by passive diffusion. It can be bound by cellular RA-binding protein (CRABP) and degraded by cytochrome P450 (CYP) enzymes in the endoplasmic reticulum. Only unbound ATRA or its isomers enter the cell nucleus and bind to RARs or to RXRs. After dimerization (homodimer or heterodimer), these RA-activated receptors bind with high affinity to specific DNA segments (RA response elements) and effect the transcription of messenger RNA of target genes. Binding of retinoids to these receptors increases the DNA-binding affinity of other nuclear receptors including vitamin D receptor (VDR), thyroid hormone receptor (THR), and peroxisome proliferation-activating receptor (PPAR). Each of these nuclear receptors has its own specific ligand (Mangelsdorf et al., 1990). Each retinoid receptor superfamily consists of several receptor isoforms: RARα (Petkovich et al., 1987), RARβ (Brand et al., 1988), RARγ (Krust et al., 1989), and RXRα,β,γ (Mangelsdorf et al., 1992). RARs and RXRs act as transacting enhancer factors and modulate transcription of target genes by binding as either heterodimers (RAR/RXR) or homodimers (RAR/RAR and RXR/RXR) to RA-responsive elements (RAREs). The consensus DNA sequences recognized by RAR are represented by a tandem repeat of the sequence AGGTCA separated by five nucleotides (Umesono et al., 1991). In contrast, the consensus sequences recognized by RXR consist of the same tandem repeats separated by only one nucleotide (Mangelsdorf et al., 1991).

Retinoids and Hematopoiesis Selective vitamin A deprivation induces anemia in humans and rats that is reversible by readministering retinol (Hodges et al., 1978; Mejia et al., 1979). Progenitor cells capable of giving rise to erythroid colonies in the presence of erythropoietin are termed erythroid burst-forming units (BFUs-E). The addition of 10−8 to 10−7 M of either ATRA, 13-cRA, or 9-cRA to BM cells or peripheral blood cells in methylcellulose cultures containing erythropoietin increases BFU-E formation (Douer and Koeffler, 1982a,b; Sakashita et al., 1993). Retinoids have a similar effect on myeloid colony formation. Bone marrow mononuclear cells contain a population of committed progenitors termed granulocytemonocyte colony-forming units (GM-CFUs), which are capable of producing myeloid colonies in vitro in the presence of colony-stimulating factors (CSFs). Colony formation by GM-CFU in methylcellulose or soft agar containing CFUs is enhanced by either ATRA, 13-cRA, or 9-cRA (10−8 to 10−7 M) (Douer and Koeffler, 1982a,b; Sakashita et al., 1993).

Retinoids

493

Retinoids and Leukemia Cells Cultured in the presence of retinoids, the late myeloidpromyelocytic cell lines HL-60 and NB4 are induced to differentiate down the granulocytic pathway (Breitman et al., 1980; Lanotte et al., 1991). A dose-dependent increase in HL-60 differentiation is observed with exposure to ATRA concentrations between 10−9 and 10−6 M. The monoblastic cell lines THP-1 and U937 are induced to undergo monocytic differentiation by retinoids (Olsson et al., 1984; Hemmi and Breitman, 1985). This process is accompanied by the progressive loss of proliferative capacity. On the other hand, the myeloblastic cell lines KG1 and KG1a and many fresh leukemia samples are growth inhibited but not induced to differentiate when exposed to RA (Douer and Koeffler, 1982a,b). The erythroid cell line HEL undergoes differentiation with RA and is growth inhibited (Suedhoff et al., 1990), whereas the K562 cell line is not affected by retinoids but differentiates down the erythroid pathway when cultured with hemin and butyrate (Miller et al., 1984). Fresh cells from 21 patients with either acute myelocytic leukemia (AML) or chronic myelogenous leukemia (CML) in blast crisis were incubated in the presence of ATRA (10−6 M) for 5–7 days (Breitman et al., 1981). Differentiation was seen only in cells obtained from two patients with APL. A subsequent study confirmed the differentiating activity of RA on cells from APL patients (Honma et al., 1983) (Figure 1). Therefore, retinoid concentrations capable of enhancing the proliferation of normal myeloid progenitors inhibit the clonal growth of myeloid leukemia cell lines. Retinoidinduced leukemia cell differentiation is invariably accompanied by loss of proliferative capacity, whereas growth inhibition need not be attended by morphological or functional changes.

Retinoid Therapy of Acute Promyelocytic Leukemia Mechanism of Action in APL The leukemia cells from almost all APL patients possess the reciprocal translocation t(15;17). The gene on chromosome 17 that is disrupted by this rearrangement encodes RARα (Borrow et al., 1990). The translocation eliminates the promoter region and exon 1 of RARα and fuses the remainder of the gene to the PML gene present on chromosome 15 (de The et al., 1991). The fusion gene encodes a chimeric protein that retains the RARα domain capable of binding the retinoid ligand and the DNA. The resulting PML-RARα fusion protein is present in all APL patients with the reciprocal translocation t(15;17) and likely contributes to the pathogenesis of the disease. This concept is supported by studies showing that artificial expression of the PML-RARα fusion protein in myelocytic or erythrocytic

FIGURE 1 Morphological changes of acute promyelocytic leukemia cells treated with all-trans-RA. Cells were cultured for 7 days in (A) culture medium and (B) culture medium containing 10−7 M all-trans-RA. Control cells (A) demonstrate prominent azurophilic granules, and a fine nuclear chromatin pattern. All-trans-RA–treated cells (B) show loss of primary granules and appearance of secondary granulation, nuclear condensation and segmentation.

leukemic cell lines blocks differentiation induced by vitamin D3 (VD3), VD3 and transforming growth factor-1 (TGF-1) in combination, or hemin (Grignani et al., 1993, 1995). Furthermore, the ability of the PML-RARα product to transform chicken hematopoietic progenitor cells in vitro and to induce acute leukemias further demonstrates its oncogenic potential (Altabef et al., 1996). The PML-RARα protein suppresses transcription of RARE-containing genes to a greater degree than RARα protein in the absence of ligand (Kakizuka et al., 1991). The transcriptional repression by PML-RARα can be attributed to the recruitment of nuclear co-repressors that inhibits transactivation from RARα target genes through recruitment of molecules sin3, Dnmt1, and Dnmt3a (Di Croce et al., 2002), and histone deacetylases (HDs) (Grignani et al., 1998; He et al., 1998; Lin et al., 1998). HDs deacetylate histones and keep the DNA in a state inaccessible for the transcription machinery, effectively shutting down the expression of genes needed for further

494

Chapter 27/Differentiation Induction in Leukemia and Lymphoma No gene expression

NCoR

A)

HDAC

X

RAR PML

NCoR HDAC

B)

Gene expression

ATRA

Myeloid maturation

HAT RAR PML

FIGURE 2 Model for the effect of retinoic acid and PML-RARa on myeloid differentiation. (A) In cells harboring the t(15;17) translocation, the PML-RARα chimeric protein binds to RA response elements and effectively inhibits the expression of genes required for normal myeloid maturation. This is facilitated by the recruitment of nuclear corepressors (NCoRs) and histone deacetylases (HDs). (B) When treating cells with ATRA, ATRA binds to the RARα part of the PML-RARα chimeric protein, which results in the release of the transcriptional repressor complex by inducing a conformational change of RARα and degradation of the PMLRARα protein. RARα now attracts histone acetylases (HATs), which open the chromatin and allow the expression of the myeloid differentiation program.

maturation of promyelocytes. ATRA binds to RARα of the PML-RARα chimeric protein and induces a conformational change that releases the nuclear co-repressor and HDs. Consequently, the differentiation program is expressed again leading to the rapid and simultaneous maturation of the promyelocytes (Figure 2). PML-RARα also plays a role in the induction of differentiation and maturation of leukemic cells by RA. Immunohistochemical studies of PML and PML-RARα show that they co-localize in an APL-specific microparticulate structure, whereas in normal cells, PML displays a punctuate pattern (POD) (Dyck et al., 1994). Treatment with RA reconstitutes the normal POD pattern in APL cells, suggesting that deranged PML function may also play a role in the pathophysiology of APL. These data show that retinoids induce APL cells to mature by reversing the oncogenic properties of PML-RARα. An additional mechanism by which retinoids induce differentiation of APL cells involves retinoid-induced degradation of PML-RARα, and thus releasing the suppressive activity of PML-RARα (Raelson et al., 1996). Clinical Trials APL (M3 in the French-American-British (FAB) classification) (Bennett et al., 1985) is characterized as a prolif-

eration of cells blocked at the promyelocytic differentiation stage and is complicated in its clinical course by disseminated intravascular coagulation (DIC). PML-RARα is the central leukemia-inducing lesion in APL and is directly targeted by ATRA and by arsenic, both compounds able to induce complete remissions by overcoming the differentiation block. In rare cases, the translocation fuses other partners rather than the PML gene with the RARα gene, namely promyelocytic leukemia zinc-finger gene (PLZF), nucleophosmin (NMP), nuclear mitotic apparatus (NUMA), and STAT5b (Melnick and Licht, 1999). Usually, these alternative chimeric–protein expressing leukemias are insensitive to differentiation therapy with ATRA. Unique to APL compared with other AML subtypes is the occurrence of lifethreatening DIC (Barbui and Falanga, 2001) and the achievement of complete remissions (CRs) in ~90% of patients treated with ATRA (Degos and Wang, 2001) or 75–90% of patients treated with low-dose arsenic trioxide (Lengfelder et al., 2003). Although APL is very sensitive to chemotherapy, in early clinical experience, 10–20% of the patients died of fatal hemorrhage resulting from DIC (Bauer and Rosenberg, 1984). Treatment with oral 13-cRA (100 mg/m2/day) for various durations resulted in either clinical improvement (Flynn et al., 1983) or CR in APL patients (Fontana et al., 1986). The first trial of ATRA for APL patients was reported from China (Huang et al., 1988). Twenty-four APL patients were treated with ATRA (45–100 mg/m2/day). Of these, eight cases had been resistant to prior chemotherapy; the other 16 cases were previously untreated. All but one patient achieved a CR with 20–119 days of continuous treatment. During the course of therapy, DIC either disappeared rapidly or was not observed after the onset of treatment. All but six complete responders received various types of consolidation chemotherapy. In spite of this, eight patients relapsed after 2–5 months of remission. Two subsequent studies have confirmed the promising result of the initial Chinese trial. A French center treated 22 APL patients with ATRA (45 mg/m2/day) for 90 days (Castaigne et al., 1990). Fourteen patients (including patients in relapse) had a complete response in 30–90 days after starting therapy, four patients had a transient response, one patient had no response, and three patients died 40 mo Median survival > 43 mo

Historical (29 cases)

CR rate: 76% EFS: 28% at 4 yr Survival: 40% at 4 yr Median CR 14 mo Median survival 17 mo

Historical (80 cases)

APL, acute promyelocytic leukemia; CR, complete remission; EFS, event-free survival.

APL cells, only ATRA is effective at 10−7 M. A review published in 1993 showed that ATRA was able to induce CR in 84% of 565 APL patients (Warrell et al., 1993). These studies demonstrate a dramatic achievement for differentiation therapy. This therapy has now become the first choice in the treatment of APL. Nevertheless, most patients treated solely with the retinoid relapsed shortly after a complete response is attained and remained unresponsive to reinduction with ATRA. The treatment programs using ATRA for induction followed by several cycles of cytotoxic chemotherapy as consolidation yielded superior overall survival compared with chemotherapy alone (Tables 1). Clinical results show that the combination of ATRA and cytosine arabinoside (CA) chemotherapy can increase the CR rate to >90%, and patients presenting with high leukocyte counts seem to benefit particularly from this combination therapy (Fenaux and Degos, 1996). ATRA followed by chemotherapy also reduced the incidence of relapse (particularly early relapse) as compared with chemotherapy alone. In a randomized trial of 93 European centers, APL patients with an initial white blood cell (WBC) count 75% took multivitamins and almost half took vitamin C; among cancer survivors, calcium use was significantly higher among women (34.9%) than men (13.8%), and vitamin A use was higher among men (9.0%) than women (7.6%) 79 individuals reported use of “unconventional medical therapies”; among them, 46% took megavitamins (especially high-dose vitamin C [at >10 g/ day] and vitamin E), 34% took herbal supplements (23 identified), and 16% took other supplements (such as shark cartilage and hydrazine sulfate); admitting use of unconventional therapies increased from 7% to 40% when patients directly queried 139 individuals took multivitamins/ mineral supplements, 205 used individual vitamins (vitamins E and C most frequently), 104 used botanical supplements (typically garlic, ginseng, soy, ginkgo, and echinacea), and 67 used individual minerals (calcium, followed by iron and selenium) Both groups took similar numbers of supplements. Among cancer survivors, 47.3% and 54.5% of men and women, respectively, took a multivitamin, 41.0% and 57.5% had high use (two or more per day) of vitamin and mineral supplements, and 16.0% and 20.7% had high use of herbal and specialty supplements; strongest positive associations were found for cranberry pills with bladder cancer, zinc with ovarian cancer, soy with prostate cancer, melatonin with cervical cancer, and vitamin D with thyroid cancer 54.5% of men and 72.5% of women took any vitamin or mineral supplement beyond a basic multi; 32.6% of men and 42.7% of women took any herbal or other type of supplement; conclusion: men and women “differ considerably” in their use of complementary and alternative medicine, including use of dietary supplements Most commonly used supplements were multivitamins (80.3%) and minerals (40.6%); 10 took herbal supplements. 74% of users reported benefits, including improved health and energy; 38% did not disclose supplement use to their physicians

McDavid et al., 2001

Cross-sectional, nationally representative probability household survey

Entire United States

Male and female cancer survivors (median age 63 years) and individuals with no reported history of cancer (median age 40 years)

33,456 (689 cancer survivors and 32,767 without cancer)

Metz et al., 2001

Prospective evaluation of consecutive patients at a university cancer center

Philadelphia, Pennsylvania

Patients with a malignancy at first visit; median age 61 years

196 (133 men and 63 women)

Kumar et al., 2002

Retrospective chart review of consecutive patients at a university cancer center

Tampa, Florida

Patients ranging in age from younger than 30 years (4% of total) to older than 60 years (56% of total)


Greenlee et al., 2004

Cross-sectional cohort study of participants in the Vitamins and Lifestyle (VITAL) study

Western Washington State

Men and women 50–76 years of age

75,083 (10,857 cancer survivors and 64,226 cancer-free controls)

Hedderson et al., 2004

Telephone survey of randomly selected cancer patients in state surveillance system

Washington State

Individuals 20–70 years of age diagnosed with breast, colon, or prostate cancer


Jazieh et al., 2004

Cross-sectional study of cancer patients at a veterans’ hospital oncology clinic

Cincinnati, Ohio

Military veterans with a malignancy; median age 68 years


510

Chapter 28/Dietary Supplements

TABLE 2 Reference

Prevalence of Dietary Supplement Use by Diagnosis of Breast, Prostate, and Colorectal Cancer Type of study

Location

Population

N

Results On regular basis, 64% used vitamin and mineral supplements, 33% took antioxidants, and 13% used herbs; more than half did not take supplements before diagnosis, and majority discussed supplement use with doctor 84 took vitamins (53 used vitamin E), 52 took a multivitamin, and 29 used an herbal supplement (most commonly lycopene and saw palmetto); many believed supplements helped cure their cancer and helped them to feel better 48% took new supplements after diagnosis (primarily multivitamins, vitamins E and C, calcium, garlic, and echinacea); women were 2.2 times more likely to do this than men; >90% reported that supplement use improved health and well-being At enrollment, 58% took multivitamins, 46% vitamin E (17% of them at intakes ≥500 mg/day), 42% vitamin C (24% at intakes ≥1000 mg/day), 11% vitamin A and carotenoids, and ~10% antioxidant mixtures; trend toward use of multiingredient products containing herbs; supplements commonly used for general health and “to feel better” 50% of the Australians and 47% of the Finns took supplements, primarily vitamins and minerals

Lengacher et al., 2002

Descriptive crosssectional survey

Tampa, Florida

Convenience sample of women with diagnosis of breast cancer; mean age 59 years

105

Hall et al., 2003

Descriptive survey of responses to mailed questionnaire

Charlottesville, Virginia

Men treated for prostate cancer at a medical center

238

Patterson et al., 2003

Telephone survey of randomly selected patients in state surveillance system

Washington State

Adults with breast, prostate, or colorectal cancer

Rock et al., 2004

Multisite, randomized controlled trial

Sites in California, Arizona, and Oregon

Women with history of early-stage breast cancer

356 (126 with breast cancer, 116 with colorectal cancer, and 114 with prostate cancer 3,008

Salminen et al., 2004

Responses to questionnaires administered to patients on site

Melbourne, Australia and Turku, Finland

Women with newly diagnosed breast cancer

cancer. The second such study offering comparative statistics was the Vitamins and Lifestyle (VITAL) study, composed of a self-selected sample of adults in western Washington State, most of whom took at least one vitamin supplement at the start (Greenlee et al., 2004). This study, which focused on supplement use at least five times weekly, showed little difference among cancer patients and those without cancer with respect to both multivitamin, single vitamin or mineral supplement, and herbal supplement use. Differences among prevalence rates reported in these and the remaining four studies in Table 1 reflect variation in

354 (215 from Australia and 139 from Finland)

study design and outcome indicators (Metz et al., 2001; Kumar et al., 2002; Hedderson et al., 2004; Jazieh et al., 2004). The differences in results reported in Table 2 also reflect the variation in study design, sample selection, and outcome variables. Patterson et al. (2003) only report on new supplements taken after a diagnosis of breast, prostate, and colorectal cancer and do not include usual supplements taken before and after diagnosis. Hall et al. (2003) limited their study to men with prostate cancer, and Lengacher et al. (2002) and Salminen et al. (2004) limited their studies to

511

Prevalence of Dietary Supplement Usage

TABLE 3 Reference

Prevalence of Dietary Supplement Use among Pediatric Cancer Patients

Type of study

Location

Population

N

Neuhouser et al., 2001b

Telephone survey of parents of randomly selected patients in state surveillance system

Western Washington State

Pediatric cancer patients 18 years or younger

75

Ball et al., 2005

Convenience sample of parents of chronically ill patients who completed questionnaire at clinic

Salt Lake City, Utah

Children and adolescents with solid-tumor cancer or leukemia (mean age ~9 years)

women with breast cancer. Rock et al. (2004) presented comparative data from a study conducted with breast cancer patients and one with the general population. Reported multivitamin use in women with a history of earlystage breast cancer in the Women’s Healthy Eating and Living (WHEL) Study (n = 3,088) was 58%, with 42% using vitamin C. The two studies of children with cancer (Table 3) also use different outcome measures. Ball et al. (2005) reported on the prevalence of dietary supplement use separately for children with leukemia and solid tumors (42% and 50%, respectively, used vitamins; 10% and 16% used minerals; and 18% and 24% used botanicals). Neuhouser et al. (2001b) did not differentiate by type of cancer and reported 29% use of single vitamin supplements, 15% use of vitamin and mineral mixtures, and 35% use of herbal supplements. Most parents of the children in this study reported perceived improvement from single vitamin use (76.9%) and herbal supplement use (85.7%). Motivators for dietary supplement use included treating side effects or symptoms of cancer or cancer treatment (47.2%), preventing recurrence or spread of cancer (33.3%), preventing or treating noncancer symptoms such as a cold or flu (51.4%), and maintaining general good health (72.2%). Despite the variations in populations, study design, sample size, and outcome variables, it is clear that significant proportions of people who have been diagnosed with cancer are using dietary supplements. This underscores the need for additional information on the efficacy and safety of

100 (50 with each type of cancer)

Results After cancer diagnosis, subjects took miscellaneous supplements (n = 28; including antioxidant mixtures and shark cartilage), herbal supplements (14), single-nutrient supplements (13), vitamin C (7), and echinacea (6); majority used supplements to maintain health or to treat noncancer symptoms like cold and flu 50% took supplements (typically without doctor’s knowledge), primarily vitamins, botanicals, and minerals; most common reasons: improve health, supplement diet, and prevent disease; supplements were discontinued within past year by one third of parents

these supplements for people with different types of cancers, undergoing different types of treatments, and at varying stages of life.

Importance of Evidence-Based Research Any recommendations for supplementation must be based on scientific evidence that the supplements are both effective and safe. Ideally, a rigorous systematic research approach (Table 4) is carried out and the results are evaluated to assess the health benefits of a dietary supplement and whether its use is recommended. The review begins with preclinical (in vitro and in vivo studies) and epidemiological evidence. Although these lines of evidence may provide insight into anticipated outcomes, it is important that research be taken to the next level of clinical trials. Before the conduct of human clinical trials, however, all available evidence must be reviewed thoroughly and objectively to determine whether data on efficacy and safety justify proceeding to clinical trials. Such evidence-based reviews differ from traditional opinion-based narrative reviews in that they systematically attempt to reduce bias by the comprehensiveness and reproducibility of the search for and selection of articles for review. Systematic reviews also assess the methodological quality of the included studies and evaluate the overall strength of the body of evidence (Agency for Healthcare Research and Quality [AHRQ], 2002). When the body of evidence on safety and efficacy justifies proceeding to clinical trials, the trials are usually conducted in three

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TABLE 4

Evaluating Dietary Supplements: A Research Approach

BASIC BIOMEDICAL LABORATORY RESEARCH (In vitro experiments and in vivo animal experiments) HUMAN OBSERVATIONAL EPIDEMIOLOGICAL STUDIES (Identify possible links between dietary supplements and cancer prevention)

DECISION POINT

HYPOTHESIS DEVELOPMENT (Evaluation of existing laboratory and epidemiological evidence on dietary supplement safety and effectiveness: as related to cancer prevention and therapy) DO NOT PROCEED

PROCEED HUMAN SAFETY TRIALS (Identify adverse side effects: safe dosage: and potential interactions) SMALL TRIALS IN DEFINED POPULATIONS (Measure supplement effectiveness at various safe doses) LARGE-SCALE: DOUBLE-BLIND: PLACEBO-CONTROLLED: RANDOMIZED CLINICAL INTERVENTION TRIALS (Test whether supplementation has the hypothesized human health benefit) DEVELOP SUPPLEMENTATION RECOMMENDATION

phases: human safety trials, small efficacy trials (usually in defined target groups), and large-scale trials that are essential in moving from basic and observational science to evidence-based public health recommendations that have human benefits. The large-scale, double-blind, randomized, placebocontrolled clinical trial, which is designed to eliminate all possible bias, is considered the gold standard of scientific intervention research. In such trials, some people receive the substance being tested and some receive an inactive placebo. These trials may not be possible in all circumstances, however, because of ethical issues that make it inappropriate to withhold the substance being tested from any trial participants. For example, after it was observed that low folate intake by pregnant women was linked to neural tube defects, a placebo-controlled intervention trial to test the validity of this association would not have been ethical. In such cases, all available evidence from in vitro laboratory research and in vivo animal studies, as well as epidemiological studies and surveys, must be reviewed systematically and objectively to draw conclusions about the possible effectiveness

and safety of the supplement of interest and to make recommendations for supplementation. Two evidence-based reviews have been conducted on the effect of specific supplements on cancer prevention. The first was a review by the U.S. Preventive Services Task Force (PSTF) on routine vitamin supplementation to prevent cancer and cardiovascular disease. For cancer, the PSTF recommended against the use of β-carotene supplements, either alone or in combination, and concluded that insufficient evidence exists either for or against the use of supplements of vitamins A, C, or E, multivitamins with folic acid, or antioxidant combinations for the prevention of cancer (PSTF, 2003). The second systematic review by the AHRQ on the use of the antioxidant vitamins C and E and CoQ10 supported the PSTF recommendations for vitamins C and E and determined that the literature does not support the use of CoQ10 supplements to help prevent or treat cancer (AHRQ, 2003). The AHRQ recognized that a few individual trials did report benefits in patients with bladder cancer and that other trials reported beneficial intermediate outcomes, such as colonic crypt cell proliferation with vitamin C and E supplementation. Although clinical trials provide a wealth of information, various interactions must be accounted for when interpreting the results and developing public health recommendations. These factors include a person’s stage of life, general health status, genetic makeup, and health and lifestyle behaviors. Each may influence the absorption, usefulness, and need for any particular dietary supplement. For example, the results of a large randomized clinical trial, the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) study conducted in Finland suggested a substantial benefit of vitamin E in reducing prostate cancer (Heinonen et al., 1998). However, almost all the participants were current or past smokers. Another major concern associated with clinical trials designed to evaluate the health effects of dietary supplements is that participants might take additional supplements, which could influence trial outcomes. In the Prostate Cancer Prevention Trial (PCPT) of the drug finasteride, for example, almost half of the participants reported using a multivitamin supplement, about a third used single supplements of either vitamin C or E, and one in five used calcium supplements. Limitations to the study included the lack of control for dosage amount, frequency of intake, type of supplement, and the limited data on micronutrient intake from fortified foods (Neuhouser et al., 2001a). Very little evidence is available about how individual micronutrients might interact with one another to influence health outcomes. The Selenium and Vitamin E Prevention Trial (SELECT) is expected to help clarify the association of dietary supplement use with prostate and other cancers. SELECT is a randomized, prospective, double-blind study designed to determine whether selenium and vitamin E reduce the risk of prostate

Role of Dietary Supplements in Cancer Prevention and During Therapy

cancer in healthy men (Klein et al., 2003). The vitamin E supplement will be a higher dose than that used in the ATBC study (400 vs 50 mg), and final results are expected in 2013. The following section explores the relationship of these interactive factors with respect to the role of dietary supplements and cancer.

ROLE OF DIETARY SUPPLEMENTS IN CANCER PREVENTION AND DURING THERAPY To understand the potential role of dietary supplements in the prevention of cancer, scientists have developed models of molecular mechanisms through which nutrient and nonnutrient supplements might affect metabolic processes that lead to cancer. Potential mechanisms include inhibiting carcinogen uptake, inhibiting the formation or activation of carcinogens, and preventing dietary carcinogen binding to DNA (American Institute for Cancer Research, 2000). Different supplements use different pathways to influence carcinogenesis. For example, antioxidants neutralize free radicals, preventing them from damaging other molecules, which over time may lead to cancer. In addition to the well-known antioxidants (vitamin C, vitamin E, and βcarotene), other substances such as mistletoe extract exhibit antioxidant properties. Calcium inhibits carcinogen uptake most often in conjunction with vitamin D. Folic acid helps to synthesize and repair DNA, potentially preventing cancer development. Phytoestrogens, the most common of which (genistein and daidzein) come from soy products, may inhibit the growth of estrogen receptor (ER)-positive and ER-negative breast cancer cells (Jennings, 1995; Peterson and Barnes, 1996). These are only a few supplement-related mechanisms that have been or are being studied. Researchers have also investigated additional factors that might influence the involvement of dietary supplements in the prevention of cancer. They include the timing of supplement use, the effect of dose and dose–response, the role of specific supplement components, and the impact of interactive factors. Each factor is discussed in the following subsections with examples.

Timing The issue of timing in dietary supplement use with respect to cancer prevention and treatment reflects on the age of the person and the time within the course of the disease that supplements are taken. A better understanding of these issues may help explain some of the conflicting results from epidemiological and clinical studies on dietary supplement use. Calcium and the soy isoflavone genistein provide examples of the importance of timing in supplement use.

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Timing with the Framework of a Day: The Example of Calcium Calcium has been investigated for its role in cancer prevention because it participates in multiple molecular signaling pathways and alterations of gene expression associated with cancer and for its role in many other key biological processes, such as bone formation and proper functioning of the nervous system (NIH Consensus Conference, 1994; Patton et al., 2003). However, the time of supplementation may influence its impact. Ingesting calcium supplements between meals supports calcium bioavailability because some foods contain compounds such as oxalates that reduce calcium absorption (NIH Consensus Conference, 1994). Also, high intakes of calcium from foods or supplements taken with meals may inhibit nonheme iron absorption and negatively affect the redox and antioxidant availability of iron (NIH Consensus Conference, 1994; Whiting, 1995).

Timing with the Lifespan: The Example of Phytoestrogens Another example of timing is exposure during different periods of life. Throughout the lifespan, estrogens increase mammary cell proliferation, but other factors, such as hormonal levels, may influence estrogens’ ability to induce differentiation or affect mammary growth by other means. Thus, estrogens can have a different impact on the breast if the exposure occurs in utero; during childhood, puberty, or pregnancy; premenopausally; or during postmenopause (Hilakivi-Clarke and Clarke, 1998). There is evidence that genistein also has different effects on the breast depending on the timing of exposure. For example, studies in rats have shown that prepubertal exposure to genistein protects against chemically induced mammary tumors, possibly because genistein increases cellular differentiation at early stages of mammary development (Lamartiniere et al., 2002). During the reproductive years, genistein increases mammary gland proliferation, as has been shown in both animal and human studies (Petrakis et al., 1996; Hsieh et al., 1998; McMichael-Phillips et al., 1998). Differences have been noted in the effect of genistein premenopausally and postmenopausally. Although there is no evidence that genistein promotes breast cancer in premenopausal women, animal studies suggest that it may play a role in the growth of cancer cells in postmenopausal women (Hsieh et al., 1998; Trock et al., 2000). It is possible that the different impact of genistein on premenopausal and postmenopausal women reflects the increased likelihood that postmenopausal women already have malignant cells in their breasts and that genistein, acting as an estrogenic agent, proliferates mammary cell growth, be it healthy or malignant cells (Bouker and Hilakivi-Clarke, 2000).

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Dose and Dose–Response Because dietary supplements are ingested to add to or replace dietary factors generally found in food products, issues of dose and bioavailability are important in discussions of their efficacy and safety. Often, the dose of a dietary supplement is greater than the amount normally found in food and may equal or exceed recommended levels of intake. For example, the recommendation for vitamin C is 75 mg/day for adults older than 18 years, but an average dose of a vitamin C supplement is 500 mg. Although dietary recommendations suggest the value of eating large amounts of fruits and vegetables, which contain vitamin C and other antioxidants, research is necessary to determine the levels of specific nutrient or nonnutrient components of these foods, which when used as supplements will have an impact on carcinogenesis.

independent of antioxidant activity. Dose–response studies show that growth-inhibitory doses of tocotrienols are five to six times lower than their corresponding lethal doses, suggesting that different mechanisms control their antiproliferative and cytotoxic effects (Sylvester and Shah, 2005). Folic Acid Epidemiological studies suggest that dose and dose– response are important factors in folate supplementation to reduce cancer risk, especially for colon cancer and colorectal adenoma. A 35–40% risk reduction was observed in those with the highest folate intake compared with those with the lowest intake (Kim, 1999). A randomized study of patients with recurrent polyps reported that supplementation with 2 mg of folate significantly decreased colonic mucosal-cell proliferation in the treatment group compared with controls (no supplementation) (Khosraviani et al., 2002).

Vitamin A and b-Carotene For example, both the α-Tocopherol, β-Carotene Cancer Prevention (ATBC) Study and the β-Carotene and Retinol Efficacy Trial (CARET) reported that the use of β-carotene supplements in smokers may promote lung cancer (ATBC Study Group, 1994; Omenn et al., 1996). Among the explanations for these results is that the dose of β-carotene in the trial was 5–10 times greater than that supplied by a healthy diet; this higher dose may have inhibited the absorption of other antioxidants with cancer-preventive properties (Greenwald, 2003). In addition, tissues of trial participants supplemented with β-carotene showed a 50-fold higher concentration than those of individuals who consumed large amounts of fruits and vegetables (Borrás et al., 2003). Dose–response to vitamin A also is dependent on the vitamin A status of cells. Vitamin A circulates in the body after binding to a retinol-binding protein (RBP), which is accumulated in the liver, and homeostasis results in extra retinol being stored for future use. When cells are deficient in vitamin A, the liver accumulates large amounts of RBP in anticipation of future availability of the vitamin (Russell, 2000). Ingesting vitamin A through the diet or by supplement in a vitamin A–deficient state causes a rapid large rise in serum retinol that is short-lived. Vitamin A ingestion when cells are not deficient results in a slower and smaller rise in serum retinol, with extra amounts being stored for later use (Russell, 2000). Vitamin E and Its Constituents The two subgroups of vitamin E are tocopherols and tocotrienols. Tocotrienols have been shown to have potent anticancer activity at doses that do not appear to affect normal cell growth or function. Their antitumor activity is

Calcium and Vitamin D Calcium, which has the potential to reduce the risk of colon cancer, also has been shown to exhibit a dose–response relationship (Wu et al., 2002). Data from the Nurses’ Health Study (Martínez et al., 1996) and Health Professionals Follow-up Study (Kearney et al., 1996) indicate that higher calcium intake is associated with a reduced risk of distal colon cancer. The incremental benefit of additional calcium intake >700 mg/day was minimal. Interestingly, it has been shown that the relationship between calcium and vitamin D is important in their associations with cancer risk (Milner et al., 2001). Results of the Calcium Polyp Prevention Study show that vitamin D status strongly influenced the impact of calcium supplementation on adenoma recurrence (Grau et al., 2003). Calcium supplements only lowered the risk of adenoma in subjects with 25hydroxyvitamin D levels above the median. Similarly, 25hydroxyvitamin D was associated with reduced risk only among those supplemented with calcium. It was concluded that vitamin D and calcium supplements appear to act together, not separately, on colorectal carcinogenesis.

Interactive Impacts: Environment, Gender, Genetic Differences Environmental, genetic, and other differences may determine whether benefit or harm is derived from the use of dietary supplements in healthy individuals, populations at risk for certain diseases, and patients undergoing disease therapy. Selenium, folate, genistein, and zinc are examples of dietary supplements that have been investigated for their association with environmental, genetic, and hormonal factors and cancer risk.

Role of Dietary Supplements in Cancer Prevention and During Therapy

Environmental Factors Epidemiological studies suggest an increase in colon cancer in areas where selenium levels are low in the soil (Clark et al., 1991). Because the amount of selenium provided by the diet is dependent on the amount found in the soil used to grow food products, the level of intake among populations is varied, especially when most food consumed comes from a single geographic source. Clinical trial results from Linxian, China, an area characterized by epidemic rates of squamous esophageal and adenomatous gastriccardia cancers, indicated a significant inverse association of serum selenium levels with these cancers when the highestto-lowest quartiles of serum selenium were compared (Mark et al., 2000). Selenium supplementation has been associated with a reduction in prostate, lung, and colorectal cancers (Greenwald et al., 2002). Genetic Factors Genetic variability and selenium intake may both play important roles in reducing cancer risk. A large randomized phase III trial, The Selenium and Vitamin E Cancer Prevention Trial (SELECT), is investigating the effect of supplementation with selenium and vitamin E, alone or in combination, on prostate cancer incidence. A nested casecontrol study within SELECT will assess genetic polymorphisms of four genes (androgen receptor [AR], 5α-reductase type II [SRD5A2], cytochrome P450c 17α [CYP17], and β-hydroxysteroid dehydrogenase [HSD3β2]) on prostate cancer incidence (Hoque et al., 2001). CYP17 is of particular interest because previous studies have suggested that the A1/A1 genotype confers a significantly higher serum androgen level than is found in men with either the A1/A2 or the A2/A2 genotype. Folate provides another example of genetic differences that can influence the potential benefits of supplementation. Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme that regulates the metabolism of folate by converting 5,10-methylenetetrahydrofolate (methyleneTHF) to 5-methyltetrahydrofolate (methylTHF), the major form of circulating folate in plasma. A common polymorphism of the MTHFR gene (677C→T) results in an alanine→valine substitution in the enzyme and, subsequently, in significantly decreased activity (Greenwald et al., 2002). This results in increased methyleneTHF, which results in reduced incorporation of uracil in DNA, which leads to fewer chromosome breaks and possibly reduced cancer risk (Greenwald et al., 2001). Studies of data from the Health Professionals Follow-Up Study and the Physician’s Health Study on the 677→6T MTHFR polymorphism and dietary intake of folate in colorectal tumorigenesis found that when the dietary methyl supply was high, individuals with the MTHFR polymor-

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phism were at reduced risk of colorectal cancer. Interestingly, alcohol consumption reversed this association—possibly by depletion of the dietary methyl supply and folate breakdown by acetaldehyde—and suggests that individuals with this genotype may be more susceptible to the carcinogenic effects of alcohol (Greenwald et al., 2001). Epidemiological studies report that zinc deficiency is associated with an increased risk of esophageal squamous cell carcinoma in high incidence areas of China and Iran (Fong et al., 2003). Abnormalities in the p53 tumor suppressor gene, which causes a loss of function leading to increased tumor proliferation and decreased apoptosis, has been studied in zinc-deficient mice exposed to the carcinogen N-nitromethylbenzylamine (NMBA). An investigation of esophageal NMBA-induced tumor proliferation in p53−/− zinc-deficient mice suggests that zinc modulates genetic susceptibility to cancer caused by p53 inactivation (Fong et al., 2003). Hormonal Factors The relationship between genistein and hormones in the lifespan of women was described in the section “Timing with the Lifespan: The Example of Phytoestrogens.” In men, epidemiological and experimental evidence suggests that genistein may inhibit prostate tumor growth through various mechanisms, including cell proliferation and increased apoptosis (Greenwald et al., 2002). In a study in LNCaP prostate cancer cells, genistein completely inhibited expression of prostate-regulated transcript 1 (PART-1), an androgen-induced gene that may represent a novel tumor marker for prostate cancer (Yu et al., 2003). In a small study of patients with prostate cancer, a dietary supplement of redclover isoflavones, including genistein, was administered before surgery. After prostatectomy, apoptosis in cells from treated patients was significantly higher than in cells from controls, specifically in regions of low- to moderate-grade cancer (Jarred et al., 2002).

Therapeutic Interactions Just as there are many mechanisms through which dietary supplements influence the prevention of cancer, so are there a variety of ways in which they influence cancer treatment. The following highlights some of the mechanisms that have an impact on the efficacy of treatment and its side effects. This is not meant to be a complete list of all influential factors, but a highlight of examples of potential interactions. Antioxidants Chemotherapeutic agents include alkylating agents (cyclophosphamides), anthracycline antibiotics (doxoru-

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bicin), platinum compounds (cisplatin), mitotic inhibitors (vincristine), antimetabolites (5-fluorouracil), camptothecin derivatives (topotecan), biological response modifiers (interferon), and hormonal therapies (tamoxifen). Anticancer therapies that may potentially be influenced by antioxidants include: alkylating agents (cyclophosphamide and iphosphamide), platinum compounds (cisplatin), antibiotics (doxorubicin and bleomycin), topoisomerase II inhibitors (etoposide), and radiation (Conklin, 2000). However, the evidence with respect to the impact of antioxidants on chemotherapy or radiation is controversial. Studies show both that antioxidants are safe and effective enhancers of chemotherapy and that they interfere with the oxidative breakdown of cellular DNA and cell membranes needed for the chemotherapy to be effective (Norman et al., 2003). Although most clinical trials have not shown significant impacts of antioxidant supplementation on chemotherapy or radiation, some have reported either the potentiation or the inhibition of these therapies by antioxidants (Weiger et al., 2002). Three clinical trials indicate that melatonin (an antioxidant) enhances the efficacy of radiation therapy and chemotherapy (Lissoni et al., 1996, 1997, 1999). Animal studies indicate that the impact of antioxidants may depend on dosage and timing of administration with respect to radiation (Sakamoto and Sakka, 1973). Phytoestrogens As was discussed in the section “Timing with the Lifespan: The Example of Phytoestrogens,” soy isoflavonoids, particularly genistein and daidzein, have both positive and negative estrogenic effects on breast tissue. In a review of 26 animal studies, soy was found to have a positive effect in most cases (Messina et al., 1994). However, other animal studies suggest the need for concern that soy supplementation in women with breast cancer, particularly with ERpositive tumors, may cause a proliferation of the cancerous cells (Weiger et al., 2002). A review addressed this dichotomous role and concluded that the data are not strong enough on either side to support the use or nonuse of soy supplements (Messina and Loprinzi, 2001). Additional effects of soy on cancer treatments have also been examined. Animal study data indicate that genistein can negate the inhibitory effect of tamoxifen on breast cancer growth (Ju et al., 2002). Given its antioxidant activity, there is also concern with respect to the use of soy supplementation during radiation or chemotherapy (Wiseman, 1996). Other Supplements Supplements that are neither antioxidants nor phytoestrogens also may affect treatment. Blood levels of medications may be influenced by the use of St. John’s wort. This herb is an inducer of the cytochrome P450 enzyme

system and drug-transporting P glycoprotein. Studies have found that St. John’s wort reduces levels of drugs such as cyclosporine and indinavir, as well as levels of the active metabolite of irinotecan, a chemotherapeutic agent (Mathijssen et al., 2002; Weiger et al., 2002).

CONCLUSIONS Despite varied results with respect to specific foods and specific cancers, results of observational, ecological, and clinical studies provide strong evidence that diets high in vegetables, fruits, and plant-based foods and low in animal fats lower the risk for cancer. The specific agents responsible for cancer protection are unknown (World Cancer Research Fund, 1997). At best, the evidence is mixed that dietary supplements taken for health promotion and disease prevention actually provide the benefits expected by consumers and patients. However, given the high rate of dietary supplement use among the general population and those diagnosed with cancer, a better understanding of possible differences between a dietary factor in food and the same factor as a supplement is necessary. As new molecular and technological approaches are developed to study the nutritional sciences, investigations can be designed to elucidate the mechanisms of action of dietary factors in both forms. Experimental and animal models must be developed to help assess the safety and efficacy of the multitude of vitamins, minerals, and botanicals in the marketplace. Also, identification and use of intermediate outcomes as endpoints in future clinical research could provide a more cost-effective method for gauging the efficacy of dietary supplements. Furthermore, attention should be directed toward possible confounding effects of supplement use by participants in clinical trials for cancer prevention and control. From a broader research perspective, there is a need to investigate dietary supplement use in the context of health disparities and cultural, ethnic, and demographic determinants. A better understanding of supplement timing, dose and dose–response, and vulnerability of specific populations is essential for providing scientifically sound information on the use of dietary supplements. Important issues to be addressed in research aimed at determining the effects of dietary supplements on cancer include developing better methods to measure the contribution of dietary supplements for various population groups and to monitor these usage trends over time. Although data are available on the prevalence of dietary supplement use among people with cancer, the data collection is not systematic and the data are not collected both before and after cancer diagnosis. The majority of information is collected on people who have been diagnosed with cancer. This makes inferences to the role of supplements in cancer prevention difficult, if not impossible. Although some of the nationally representative survey data provide comparisons between

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References

cancer patients and individuals not diagnosed with cancer, the number of cancer patients is too small, limiting the strength of the comparative results. Additionally, although some studies collect data on new supplement use (post diagnosis), most do not ask about length of time for which supplements have been used. The systematic collection of prevalence data could include the development of a set of core prevalence indicators that include definitions of cancer patient or survivor, frequency of supplement use (daily, regularly, ever), length of time a supplement has been used, supplement dose (low to high for each supplement), motivators for use, perceived benefits, and other user characteristics. Such systematic prevalence data could assist researchers, healthcare professionals, and policymakers in identifying the supplements most frequently used and the characteristics of those most likely to use them, helping to set an agenda for future research. Along with ensuring that prevalence data on supplements includes botanicals and other nonbotanical nonmicronutrient products, additional work is necessary to understand how best to ask people questions about these supplements, given the different languages and names used in their sale, the multitude of herbal combinations used, and the variety of forms in which people take them (e.g., prepared teas, concentrated drinks, powders, and tablets). Taking dietary supplements is likely to remain a significant strategy used by consumers and patients to improve health and combat diseases such as cancer. The scientific community must respond by providing guidance about the responsible use of these products to the public, medical professionals, and policymakers that is based on sound scientific evidence. Research—experimental, epidemiological, and clinical—on nutrition and cancer is the best way to identify dietary factors that show promise as cancer prevention or control agents. This approach will allow scientists to either confirm or refute the growing amount of consumer information on the benefits and risks of dietary supplement use. Physicians and healthcare providers need to openly discuss the use of dietary supplements with their patients, especially because many supplement users get most of their information about these products from friends, family, the media, and other word-of-mouth sources. Patients need to understand the potential positive and negative impacts of the supplements they may choose to use and need to be informed that supplements should not be used as a substitute for medical therapies. Healthcare providers should encourage patients to enumerate the various supplements they take and provide expert advice about using these products responsibly. The NIH Office of Dietary Supplements, for example, provides helpful materials for this purpose, including a consumer-friendly brochure titled “What Dietary Supplements Are You Taking? Does Your Health Care Provider Know? It Matters and Here’s Why” (Office of Dietary Supplements, 2005).

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Koplan, J.P., Annest, J.L., Layde, P.M., and Rubin, G.L. 1986. Nutrient intake and supplementation in the United States (NHANES II). Am J Public Health 76: 287–289. Kumar, N.B., Hopkins, K., Allen, K., Riccardi, D., Besterman-Dahan, K., and Moyers, S. 2002. Use of complementary/integrative nutritional therapies during cancer treatment: implications in clinical practice. Cancer Control 9: 236–243. Lamartiniere, C.A., Cotroneo, M.S., Fritz, W.A., Wang, J., Mentor-Marcel, R., and Elgavish, A. 2002. Genistein chemoprevention: timing and mechanisms of action in murine mammary and prostate. J Nutr 132: 552S–558S. Lengacher, C.A., Bennett, M.P., Kip, K.E., Keller, R., LaVance, M.S., Smith, L.S., and Cox, C.E. 2002. Frequency of use of complementary and alternative medicine in women with breast cancer. Oncol Nurs Forum 29: 1445–1452. Lissoni, P., Barni, S., Mandala, M., Ardizzoia, A., Paolorossi, F., Vaghi, M., Longarini, R., Malugani, F., and Tancini, G. 1999. Decreased toxicity and increased efficacy of cancer chemotherapy using the pineal hormone melatonin in metastatic solid tumour patients with poor clinical status. Eur J Cancer 35: 1688–1692. Lissoni, P., Meregalli, S., Nosetto, L., Barni, S., Tancini, G., Fossati, V., and Maestroni, G. 1996. Increased survival time in brain glioblastomas by a radioneuroendocrine strategy with radiotherapy plus melatonin compared to radiotherapy alone. Oncology 53: 43–46. Lissoni, P., Paolorossi, F., Ardizzoia, A., Barni, S., Chilelli, M., Mancuso, M., Tancini, G., Conti, A., and Maestroni, G.J. 1997. A randomized study of chemotherapy with cisplatin plus etoposide versus chemoendocrine therapy with cisplatin, etoposide and the pineal hormone melatonin as a first-line treatment of advanced non–small cell lung cancer patients in a poor clinical state. J Pineal Res 23: 15–19. Lyle, B., Mares-Perlman, J., Klein, B., Klein, R., and Greger, J.L. 1998. Supplement users differ from nonusers in demographic, lifestyle, dietary and health characteristics. J Nutr 128: 2855–2862. Mark, S.D., Qiao, Y.L., Dawsey, S.M., Wu, Y.P., Katki, H., Gunter, E.W., Fraumeni, J.F. Jr, Blot, W.J., Dong, Z.W., and Taylor, P.R. 2000. Prospective study of serum selenium levels and incident esophageal and gastric cancers. J Natl Cancer Inst 92: 1753–1763. Martínez, M.E., Giovannucci, E.L., Colditz, G.A., Stampfer, M.J., Hunter, D.J., Speizer, F.E., Wing, A., and Willett, W.C. 1996. Calcium, vitamin D, and the occurrence of colorectal cancer among women. J Natl Cancer Inst 88: 1375–1382. Mathijssen, R.H., Verweij, J., de Bruijn, P., Loos, W.J., and Sparreboom, A. 2002. Effects of St. John’s wort on irinotecan metabolism. J Natl Cancer Inst 94: 1247–1249. McDavid, K., Breslow, R.A., and Radimer, K. 2001. Vitamin/mineral supplementation among cancer survivors: 1987 and 1992 National Health Interview Surveys. Nutr Cancer 41: 29–32. McMichael-Phillips, D.F., Harding, C., Morton, M., Robert, S.A., Howell, A., Potten, C.S., and Bundred, N.J. 1998. Effects of soy-protein supplementation on epithelial proliferation in the histologically normal human breast. Am J Clin Nutr 68: 1431S–1436S. Medeiros, D.M., Bock, M.A., Ortiz, M., Raab, C., Read, M., Schutz, H.G., Sheehan, E.T., and Williams, D.K. 1989. Vitamin and mineral supplementation practices of adults in seven western states. J Am Diet Assoc 89: 383–386. Metz, J.M., Jones, H., Devine, P., Hahn, S., and Glatstein, E. 2001. Cancer patients use unconventional medical therapies far more frequently than standard history and physical examination suggest. Cancer J 7: 149–154. Messina, M. J., and Loprinzi, C. L. 2001. Soy for breast cancer survivors: a critical review of the literature. J Nutr 131: 3095S–3108S. Messina, M. J., Persky, V., Setchell, K. D., and Barnes, S. 1994. Soy intake and cancer risk: a review of the in vitro and in vivo data. Nutr Cancer 21: 113–131.

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29 Dietary Fiber and Carbohydrates MARÍA ELENA MARTÍNEZ AND ELIZABETH T. JACOBS

INTRODUCTION

charides cannot be hydrolyzed to a simpler form, disaccharides may be hydrolyzed to give two molecules of the same or different monosaccharides, and polysaccharides yield >10 units. Carbohydrates are absorbed through the intestinal mucosa as monosaccharides, primarily glucose, with minor quantities of other sugars. All carbohydrates are then carried in the portal blood to the liver, after which they are utilized in one of several ways. The principal function of carbohydrates is to serve as a major source of energy for the body. The body tissues require a constant daily supply of carbohydrates in the form of glucose in all metabolic reactions. Much of the glucose is used for immediate energy needs via oxidation to CO2 and water. Part is stored as glycogen in the liver and muscle tissue, and some is converted to fatty acids and possibly stored as triglycerides in fat tissue. A small amount is converted to other necessary carbohydrates and some becomes the carbon skeletons for the production by the body of the nonessential amino acids. Carbohydrates that escape digestion and absorption in the small intestine and yield short-chain fatty acids in the large intestine are mainly the nonstarch polysaccharides (NSPs) and the resistant starches. Cellulose and other insoluble indigestible carbohydrates aid in normal elimination by stimulating the peristaltic movements of the gastrointestinal (GI) tract and absorbing water to give bulk to the intestinal contents.

Various dietary factors have long been suspected to play a prominent role in the etiology of cancer. A substantial amount of research has been carried out in attempts to identify the specific risk factors. Dietary hypotheses comprise several macronutrients and micronutrients, as well as other dietary constituents. Although little focus has been placed on the direct effect of carbohydrates in general, a great deal of effort has been spent on identifying the specific role of dietary fiber and its food sources as potential protective factors for various cancers. However, the overall contribution of fiber to cancer risk is unclear. This uncertainty is due to a variety of reasons, including the chemical complexity of dietary fiber, the lack of direct evidence regarding the exact mechanism of action specific to each type of fiber, and our limited understanding of the carcinogenic process. This chapter focuses on malignancies for which the effect of carbohydrates and dietary fiber has been investigated. Although the data suggest some consistent patterns for some malignancies, some uncertainties are also evident. An attempt is made to outline plausible new and existing mechanisms of action for these effects. Overall, the complexities inherent in the study of diet and cancer, which involve complex biological mechanisms and human behavior, create an enormous and exciting challenge for future investigations.

DIETARY FIBER

CARBOHYDRATE CLASSIFICATION AND METABOLISM

Definition and Physiological Effects Dietary fiber is defined as the endogenous components of plant materials in the diet that are resistant to digestion by

Carbohydrates are classified as monosaccharides, disaccharides, and polysaccharides (Table 1). Whereas monosac-


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TABLE 1

Classification of Carbohydrates Polysaccharides

Monosaccharides Glucose Galactose Fructose

Disaccharides

Digestible

Sucrose Lactose Maltose

Starch Resistant starch Dextrins Glycogen Inulin Mannosans Raffinose Stachyose Pentosans

Nonresistant starch Cellulose Hemicellulose Pectin Gums Mucilages

the enzymes produced by intestinal flora (Trowell et al., 1985; Cummings and Bingham, 1987; Pilch, 1987; Kritchevsky, 1988). Fibers in foods are complex carbohydrates; however, due to their chemical complexity, they are poorly defined. Fiber consists of a heterogeneous mixture of complex polysaccharides and nonpolysaccharide polymers mostly made up of plant cell wall carbohydrate and NSP. By its definition, dietary fiber reaches the colon intact and is a major source of energy for colonic bacteria. Analytical methods have determined that dietary fiber is composed of at least six general components, which include cellulose, hemicellulose, pectins, gums, mucilages, and lignin (Table 1). According to the definition given by Trowell et al. (1985), dietary fiber is the sum of lignin and the plant polysaccharides that are not digested by the endogenous secretions of the human digestive tract. Thus, lignin, a noncarbohydrate, can be included in this definition. Lignin, a highly polymeric substance occurring in woody plant tissues, is virtually indigestible. Values for lignins (enterolactone and enterodiol) can be obtained from published sources (Thompson et al., 1991). The physiological effects of dietary fiber depend largely on the type of fiber. Thus, these effects differ whether reference is made to foods rich in fiber such as grain products, legumes, fruit, and vegetables or isolated fibers such as cellulose, pectin, or lignin. Although several types of fiber have been identified, for purposes of their mechanistic protective action in carcinogenesis, they can be classified into two general types according to water solubility. Soluble fibers are present in fruit, vegetables, and certain grains, such as oats. These include the gel-forming fibers such as pectins, gums, and mucilages. This type of fiber undergoes metabolism in the small intestine and especially in the large intestine, as bacterial enzymes convert it to products that increase stool size. The function of the pectins is mainly to absorb water, form gel, and increase bulk. Another important func-

tion of soluble fiber is its potential to slow down glucose absorption and lower serum cholesterol. These polysaccharides can prevent the absorption of nutrients such as carbohydrates and lipids from the gut (Jenkins et al., 1978; Blackburn and Johnson, 1981; Sandberg et al., 1983). In most cases, this results from slowing of absorption rather than an inhibition in amount absorbed; this action also involves delayed gastric emptying and slowed small bowel absorption (Jenkins et al., 1978). Enzymes in the intestinal flora do not substantially metabolize insoluble fibers, present in considerable amounts in bran cereals (i.e., wheat, rice). These fibers, which include cellulose, lignin, and some hemicelluloses, are insoluble and nonfermentable. Among these, cellulose is not hydrolyzed, whereas hemicellulose forms bulk and roughage. Such fibers increase stool size substantially through several mechanisms, including water retention. Therefore, these affect intestinal function by retaining water in the stool, thereby increasing fecal bulk and decreasing GI transit time. The larger bulk generated by the action of these fibers dilutes carcinogens, especially tumor promoters such as secondary bile acids, which may result in lower risk of some cancers, such as colon cancer. The fermentable soluble fibers are also capable of decreasing intestinal transit time by stimulating microbial growth in the intestine resulting in higher fecal bacterial mass. The total amount of dietary fiber varies markedly in different foods. Furthermore, each type of fiber is composed of different proportions and combinations of the six basic components and no two are the same in their action in the GI tract. Although the exact action of dietary fiber depends on the type and amount of fiber ingested, most of this is limited to the large intestine. Adding to its complexity, the physiological effects of dietary fiber depend not only on the type of fiber ingested but also on the composition of the rest of the meal or diet and the unique physiology of the individual.

Analysis of Fiber in Foods No method of dietary intake assessment is without flaws. Earlier animal research related to fiber focused on crude fiber and other methods that did not allow complete analysis of fiber in foods for human studies (Goering and van Soest, 1970; van Soest and van Soest, 1973). However, crude fiber consists mainly of lignin and cellulose, whereas dietary fiber contains these plus other components (hemicellulose, gums, pectin, etc.). Subsequently, analysis methods described by Southgate (1969, 1976) and Englyst (1980) evolved and became more applicable to studies in humans. The problems of isolating dietary polysaccharides (which are equivalent to dietary fiber) have been the objective of more recent methods for measuring fiber in food (Asp et al., 1992; Li, 1995). However, controversy regarding the most appropriate method of chemical analysis continues.

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Simple Sugars and Cancer

Dietary fiber, chemically speaking, is a polysaccharide and, in terms of food composition, is best seen as such. Currently, there is no one accepted definition or method of analysis for dietary fiber. Englyst and Cummings (1988) define it as a component of NSP. In this method of analysis, starch is removed enzymatically, after solubilization, and NSP is measured as the sum of constituent sugars released by acid hydrolysis. The Southgate method provides data on total dietary fiber and individual fiber components (cellulose, lignin, etc.); thus, the Englyst method is considered a modified Southgate method. The Association of Analytical Chemists (AOAC) describes fiber as endogenous plant food material from the diet that is resistant to human digestive secretions but that is substantially fermented in the colon (Prosky et al., 1985). This method relies on an enzymatic gravimetric system of analysis and has been suggested to be the most practical and simplest approach to measuring the major components of dietary fiber as a single unit (Dreher, 1987). The AOAC method is the most accepted in the United States, whereas that of Englyst is preferred in European countries. When data on dietary fiber and disease are reported using different analytical techniques, comparison across studies may be difficult. Thus, standardized analytical chemical techniques of fiber content in foods are imperative.

CARBOHYDRATES AND CANCER Relatively few studies have focused solely on the effects of carbohydrate and its role in cancer risk. For colon cancer, results of correlational studies show a strong inverse association between intake of starch and risk of this malignancy (Cassidy et al., 1994). In analytical epidemiological studies, for the most part, investigators have focused on various food sources of carbohydrate, although analyses of total carbohydrate and colorectal adenoma and cancer were conducted in a large cohort of >34,000 women. The results showed no association between total carbohydrate intake and risk of colorectal adenoma (Oh et al., 2004) but did find an increased risk for colorectal cancer with higher carbohydrate intake (Higginbotham et al., 2004). A higher risk of colon cancer has been shown for higher intake of starch in countries such as Japan, where rice is the main source (Wynder et al., 1969); southern Europe, where pasta, rice, bread, polenta, potatoes, and cereals are the major sources (Macquart-Moulin et al., 1986; La Vecchia et al., 1988; Benito et al., 1990; Bidoli, et al., 1992; Centonze et al., 1994); Australia (Steinmetz and Potter, 1993); and Russia (Zaridze et al., 1985). Because of its low incidence rate, investigations of diet and pancreatic cancer are few. Furthermore, most of the evidence relies on data from retrospective studies, many of which use proxy information on the case individuals. The largest data set comes from five case-control studies

conducted simultaneously by the International Agency for Research on Cancer (IARC) SEARCH program (Howe et al., 1992). A main objective for the implementation of these studies was for the combination of the data in a pooled analysis. The data comprise >800 case and 2000 control individuals. For carbohydrate intake, four (Bueno de Mesquita et al., 1990; Howe et al., 1990b; Baghurst et al., 1991; Zatonoski et al., 1991) of the five case-control studies show positive associations with pancreatic cancer, although only one (Baghurst et al., 1991) was statistically significant. A combined analysis of these studies shows a significant positive association (odds ratio [OR] = 1.74; 95% confidence interval [CI], 1.26–2.40). However, data from two case-control studies not included in the SEARCH pooled analysis (Kalapothaki et al., 1993) do not support these findings. Thus, the evidence for the effect of carbohydrate- or starch-rich foods on risk of pancreatic cancer is inconsistent across studies. Furthermore, no biological mechanism has been identified. As in the case of pancreatic cancer, epidemiological data on diet and gastric cancer have mainly derived from casecontrol studies. Although intake of fruit and vegetables is inversely correlated with risk of stomach cancer (Kono and Hirohata, 1996), the results for diets high in carbohydrate are less clear. Various case-control studies (Bjelke, 1974; Modan et al., 1974; Correa et al., 1985; Trichopoulos et al., 1985; Buiatti et al., 1990; Graham et al., 1990; Tuyns et al., 1992; Ramon et al., 1993) and one cohort study (Kneller et al., 1991) support a positive association between carbohydrate intake and risk of stomach cancer, although a small number did not (Correa et al., 1985; Gonzalez et al., 1991; Hansson et al., 1993). Among individual food sources of carbohydrate, pasta (Trichopoulos et al., 1985; La Vecchia et al., 1987) and rice (Haenszel et al., 1972; Nomura et al., 1990) consumption have been shown to increase risk; however, in countries where sufficient interindividual variation may be lacking, such an association is nonexistent (Hirayama, 1971; Haenszel et al., 1976; Hirohata, 1983; You et al., 1988; Kono et al., 1988). Thus, current data are inconclusive as to whether high-carbohydrate diets confer an increased risk for gastric cancer.

SIMPLE SUGARS AND CANCER Investigations on the relationship between sucrose and the simple sugars and cancer have mainly been focused on colorectal malignancies. Biologically plausible mechanisms for an enhanced risk for colorectal cancer with higher intake of sucrose have been published (Bostick et al., 1994). Sucrose increases colonic epithelial cell proliferation and increases microadenoma formation (Corpet et al., 1990; Stamp et al., 1993) in rodents. In humans, a high sucrose diet increases mouth-to-anus transit time despite decreasing

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the mouth-to-cecum time and increases fecal concentration of both total and secondary bile acids (Kruis et al., 1991). At least 11 case-control studies (Phillips, 1975; Manousos et al., 1983; Miller et al., 1983; Pickle et al., 1984; Bristol et al., 1985; Macquart-Moulin et al., 1986; La Vecchia et al., 1988; Tuyns et al., 1988; Benito et al., 1990; Bidoli et al., 1992; Peters et al., 1992) and one cohort study (Bostick et al., 1994) have reported results on the relation of sucrose intake and colon cancer; of these, 10 (Phillips, 1975; Miller et al., 1983; Pickle et al., 1984; Bristol et al., 1985; Macquart-Moulin et al., 1986; La Vecchia et al., 1988; Tuyns et al., 1988; Benito et al., 1990; Bidoli et al., 1992; Bostick et al., 1994) indicate an association in the direction of increased risk with results being significant in three (Bristol et al., 1985; Tuyns et al., 1988; Bostick et al., 1994). In particular, in the Iowa Women’s Health Study (Bostick, et al., 1994), the relative risk (RR) for the upper versus the lower quintile of sucrose-containing foods was 1.74 (95% CI, 1.06–2.87). Few studies have investigated the association of sugar and other malignancies. Results of four case-control studies of pancreatic cancer (Raymond et al., 1987; Bueno de Mesquita et al., 1990; Baghurst et al., 1991; Kalapothaki et al., 1993) showed one null finding, one of a nonsignificant slightly elevated risk, and two of significant positive associations. In a cohort investigation conducted among women in the Nurses’ Health Study, sucrose was not associated with increased risk of pancreatic cancer, although fructose appeared to be associated with increased risk, particularly among overweight and sedentary women (Michaud et al., 2002). However, data on this malignancy continue to be sparse and inconclusive.

GLYCEMIC INDEX AND CANCER Glycemic index and glycemic load in the diet are among the new methodologies to assess the role of carbohydrates in disease etiology. Glycemic index uses a system whereby carbohydrates are ranked according to their acute effects on blood glucose levels. For example, the highest glycemic indexes are assigned to carbohydrates that are quickly broken down, whereas those that are more slowly metabolized are assigned a lower index score. Glycemic load takes into account the amount of carbohydrates by multiplying the grams of carbohydrates in the serving of food by its glycemic index. High intake of highly refined carbohydrates has been recognized as a risk factor for a variety of chronic diseases, including type 2 diabetes, cardiovascular disease, obesity, and some cancers (Liu et al., 2000; Jenkins et al., 2002; Willett et al., 2002). Glycemic index represents a dietary indicator used to classify carbohydrate content of foods. Diets high in glycemic index or glycemic load have been

associated with increased risk of colon cancer in some (Slattery et al., 1997; Franceschi, Dal Maso et al., 2001) but not all (Terry et al., 2003; Michaud et al., 2005) studies. In the Women’s Health Study (Higginbotham et al., 2004), dietary glycemic load was statistically significantly associated with an increased risk of colorectal cancer (adjusted RR = 2.85; 95% CI, 1.40–5.80), comparing high versus low quintiles of dietary glycemic load. High glycemic diets have also been investigated in relation to breast cancer risk. Results of a prospective study (Silvera et al., 2004) showed a positive association between glycemic index and postmenopausal breast cancer (RR = 1.87; 95% CI, 1.18–2.97). However, results of both the Cancer Prevention Study II (Jonas et al., 2003) and the Nurses’ Health Study (Holmes et al., 2004) do not support this association. Other malignancies where glycemic index or load have been shown to be positively associated with risk include prostate cancer (Augustin et al., 2004) and pancreatic cancer (Michaud et al., 2002). Clearly, this active area of research is still in its infancy. As is the case with any observational study, reproducibility of findings from large studies, especially those prospective in nature, will be necessary before firm conclusions can be drawn regarding high glycemic diets and cancer risk.

FIBER AND CANCER The malignancy most closely linked to fiber consumption is that of the colorectum. The observations by Burkitt in 1971 of lower incidence rates of colon cancer in African populations than in Western countries led to the hypothesis of an association of this malignancy with fiber intake. Correlational studies (Schrauzer, 1976; IARC, Intestinal Microecology Group, 1977; Liu et al., 1979) later supported this hypothesis. However, these studies also alluded to the complexity of exposure assessment, as the associations were often attenuated when other nutrients such as fat and cholesterol were taken into account (Greenwald et al., 1987). Correlational data comparing areas with high colon cancer rates and low fiber intake (e.g., New York) and those with low rates and high fiber intake (e.g., Finland) suggest that dietary fiber may account for the differences in the rates, given the similarities in dietary fat consumption (MacLennan et al., 1978). Results of analytical epidemiological studies of a retrospective nature strongly support the fiber–colon cancer hypothesis. The results of a meta-analysis of 13 case-control studies of colon cancer showed a combined OR of 0.58 ( p < 0.001) between the upper and lower quintile of fiber intake (Howe et al., 1992). Giovannucci and Willett (1994a) noted that most case-control studies have found an inverse association between total fiber and risk of colon cancer, although a stronger case can be made for the protective effect of

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fruit and/or vegetable consumption. Results of prospective studies (Heilbrun et al., 1989; Willett et al., 1990; Thun et al., 1992; Giovannucci et al., 1994; Steinmetz et al., 1994), on the other hand, are less convincing. Although an inverse association is shown when comparing highest with lowest categories of intake, the dose–response relationship is nonexistent, except for the American Cancer Society (ACS) study of colon cancer mortality (Thun et al., 1992). In this study, the exposure factor (the combination of vegetables, citrus, and high-fiber grain consumption) was inversely related to risk of fatal colon cancer (RR < 0.76; 95% CI, 0.57–1.02; p for trend < 0.031). Heilbrun et al. (1989) also showed a protective effect for dietary fiber and colon cancer risk; however, assessment of diet was made using a single 24-hour recall. The reasons for the inconsistency in the findings of the retrospective and prospective studies are not clear. It is possible that bias related to the difference in study design may account for the discrepancies. Also, early epidemiological studies focused on foods and food groups high in fiber only or used a measure of crude fiber rather than dietary fiber. Of interest, among the case-control studies reviewed by Giovannucci and Willett (1994a) published prior to 1990, only two of the nine reviewed show a significant reduction by dietary fiber on colon cancer risk, whereas the majority published in or after 1990 show a significant inverse association. Because issues related to the measurement and definition of fiber have only been recently addressed, future studies utilizing standard definitions and approaches should help clarify this field of research. Although many studies appear to support a benefit of fiber, a puzzling observation has been that cereal fiber, generally the major determinant of fecal mass and determinant of transit time (Cummings et al., 1978), has not been related consistently to colon cancer risk in analytical epidemiological studies. Results of case-control studies that have examined the role of grain fiber or cereal fiber generally show intake to be unrelated or positively related to risk of colon cancer (Giovannucci and Willett, 1994a). Furthermore, only one prospective study (Thun et al., 1992) has shown a significant reduction in colon cancer risk associated with intake of high-fiber grains. In fact, vegetables and fruits, important contributors of water-soluble fibers, have historically been more consistently associated with reduced risk. Nonetheless, two large cohort studies of >135,000 men and women from the Nurses’ Health Study and the Health Professionals’ Follow-Up Study failed to find a protective effect of fruit and vegetable consumption on colorectal cancers (Michels et al., 2000). The RR for colon cancer associated with one extra serving of fruit per day was 1.02 (95% CI, 0.98–1.05), whereas for an additional vegetable serving, it was 1.03 (95% CI, 0.97–1.09). Results from the Nurses’ Health Study also failed to show any impact of dietary fiber overall on colorectal cancers or adenomas (Fuchs et al., 1999), as discussed later in this chapter.

TABLE 2

Baseline Characteristics in the Wheat Bran Fiber and Polyp Prevention Trials

Characteristic Age (mean ± sd) Gender (male) Family history of colorectal cancer (yes) Previous adenomas (yes) Aspirin use (yes) Folate (µg/day) Calcium (mg/day) Fiber (g/day) Alcohol (g/day) Energy (kcal/day) Fat (g/day)

TABLE 3

Wheat Bran Fiber Trial

Polyp Prevention Trial

65.9 ± 8.8 874 (67%) 220 (17%)

61.1 ± 9.9 1228 (64%) 479 (25%)

452 (35%) 368 (28.2) 326.5 ± 136.6 892.3 ± 379.2 21.9 ± 9.9 7.2 ± 14.9 1927.2 ± 699.0 69.2 ± 30.5

334 (18%) 438 (23%) 304.4 ± 122.0 843.9 ± 433.9 17.8 ± 7.8 7.1 ± 12.4 1923.1 ± 582.7 76.9 ± 31.2

Results from the Wheat Bran Fiber and Polyp Prevention Trials Recurrence (%)

Wheat Bran Fiber Trial Polyp Prevention Trial

Low fiber

High fiber

Relative risk (95% confidence interval)

51.2 39.5

47.0 39.7

0.88 (0.70–1.11) 1.00 (0.90–1.12)

Further adding to the equivocal results in the literature, two large clinical trials of fiber and colorectal adenoma recurrence showed that fiber appeared to have no protective effect. Table 2 presents selected baseline characteristics of the participants in these trials. The Wheat Bran Fiber Trial (Alberts et al., 2000) tested the effect of a high-fiber cereal supplement (13.5 g/day) and a low-fiber supplement (2.0 g/day) on adenoma recurrence over 3 years of supplementation among patients who had recently had an adenoma removed at colonoscopy (Alberts et al., 2000). No difference was observed between the treatment groups for adenoma recurrence (Table 3). Upon further analysis of this trial, there was a suggestion that, in those who were already consuming relatively high amounts of fiber at baseline, the fiber intervention did result in protection from adenoma recurrence (Jacobs et al., 2002), but these results must be further explored. The Polyp Prevention Trial (PPT) was a multicenter study conducted by the National Cancer Institute to evaluate whether a dietary intervention aimed at decreasing fat intake and increasing consumption of fiber, fruits, and vegetables reduced the rate of adenoma recurrence when compared with a usual diet after 4 years of follow-up (Schatzkin et al., 2000). As shown in Table 3, no

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reduction in risk was observed for adenoma recurrence (Schatzkin et al., 2000). Following the publication of these two clinical trials, two large prospective investigations reported that higher intake of fiber was indeed associated with a significantly decreased risk of colorectal adenoma (Peters et al., 2003) and cancer (Bingham et al., 2003). In the study conducted within the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial, participants in the highest quintile of total dietary fiber intake had a 27% decreased risk of adenoma compared with those in the lowest quintile (p trend = 0.002). The European Prospective Investigation into Cancer and Nutrition (EPIC) study results showed that the highest quintile of dietary fiber intake was associated with a relative risk of 0.75 (95% CI, 0.59–0.95) for colorectal cancer compared with the lowest quintile of intake. Again, the reasons for the discrepant results are unclear. One possibility is related to differences in fiber intake in the populations under study. For example, participants in the highest quartile of the PLCO and EPIC studies, in which a protective effect of fiber was observed, had a median fiber intake of ~31 g/day (Figure 1). In contrast, those in the highest quartile of the Nurses’ Health Study, in which no protection from fiber was reported, were consuming a comparatively lower 25 g/day (Figure 1). These results suggest that the amount of fiber necessary for protection from colorectal neoplasia may be higher than has been attained in clinical trials thus far. Equivocal results between the cohort studies and the clinical intervention trials might relate to limitations inherent in intervention studies of adenoma recurrence (Martinez, 2001); these include short intervention phase as well as the fact that these studies are assessing recurrence of the premalignant lesion rather than cancer. Lifetime intake of a variety of foods high in fiber may be necessary for protection from colorectal neoplasia. Therefore, a negative finding from clinical trials of adenoma recurrence does not necessarily translate into a lack of efficacy of the intervention.

1.1

Relative Risk

1

EPIC PLCO NHS

0.9 0.8 0.7 0.6 EPIC PLCO NHS

Fiber intake (g/day) 12.7 15.4 9.8

33.8 30.6 24.9

FIGURE 1 Association between dietary fiber and colon cancer (European Prospective Investigation into Cancer and Nutrition [EPIC] and Nurses’ Health Study) or colorectal adenoma (Prostate, Lung, Colorectal, and Ovarian [PLCO).

Potential Mechanisms of Action There are several, not mutually exclusive, mechanisms by which dietary fiber may modify colon cancer risk. One important mechanism involves the physiochemical capacity of fiber to bind cytotoxic bile acids, which may act as tumor promoters, thereby making them unavailable for metabolic activity (Jacobs, 1988). Fibers, particularly insoluble fibers, modify the enzyme activities of the intestinal bacterial flora, usually yielding a lower level of such enzymes (Weisburger et al., 1993). Some of the fibers also generate a lower intestinal pH such that the hydrolytic enzymes are not in a medium. An important aspect of intestinal biochemistry stemming from fiber, particularly insoluble fiber, is the lower rate of conversion of primary bile acids produced in the liver and secreted in the bile. Primary bile acids are converted to secondary bile acids through the action of enzymes from the bacterial flora, the activity of which is a function of intestinal fiber content. Most studies that have investigated the effect of wheat bran fiber dietary supplementation on the concentration and excretion of fecal bile acids have shown an increase in total and/or secondary fecal bile acid excretion, as well as a significant decrease in fecal bile acid concentrations (Reddy et al., 1977; Hill, 1991). Results of a published double-blinded phase II trial of wheat bran fiber (2.0 or 13.5 g/day) and calcium carbonate (250 or 1500 mg/day) showed that high-dose wheat bran fiber supplement was associated with a significant reduction in total and secondary fecal bile acid concentrations after 9 months of intervention (Alberts et al., 1996). These results suggest that one important mechanism by which wheat bran fiber may reduce colon cancer risk is through the reduction in concentration of secondary bile acids. Given the international variations in breast cancer rates, it has been suggested that diet and differences in endogenous estrogen metabolism and excretion may play a role in the development of this malignancy (Miller and Bulbrook, 1986; Parkin et al., 1993). Although a modest number of studies have assessed the role of fiber or its components in breast cancer risk, a protective effect is largely supported by results of case-control studies (Lubin et al., 1986; Rohan et al., 1988; Howe et al., 1990a; Van’T Veer et al., 1990; Graham et al., 1991; Lee et al., 1991; Baghurst and Rohan, 1994; Yuan et al., 1995; Freudenheim et al., 1996; De Stefani et al., 1997; Bonilla-Fernandez et al., 2003; Mattisson et al., 2004). Howe et al. (1990a) conducted a combined analysis of 12 case-control studies carried out in Argentina, Australia, Canada, China, Greece, Hawaii, Israel, and Italy. These results also support a modest protective effect for dietary fiber and risk of breast cancer, with an increase of 20 g of fiber per day associated with RRs of 0.89 (p < 0.15) for premenopausal and 0.83 (p < .02) for postmenopausal women. Of some concern, however, are data from the few published cohort studies (Kushi et al., 1992; Willett et al., 1992) less prone to recall and selection bias, which do not

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support an inverse association between dietary fiber and breast cancer. Among the proposed mechanisms for the protective effect of fiber on breast carcinogenesis, the most widely accepted is that of the alteration of estrogen levels by dietary fiber (Adlercreutz, 1990). The exact mechanism responsible for this action is unclear. It is suggested that fiber may affect the enterohepatic circulation of estrogens and the actions of fiber-associated phytoestrogens (Rose, 1990; Rose et al., 1991). Based on this hypothesis, intervention studies have been conducted to assess the indirect effect of various fiber components on mammary tumors. Although the results of these studies have demonstrated the potential for change in estrogen levels by means of interventions consisting of highfiber or low-fat diets, they do not provide clear evidence for these effects (Adlercreutz et al., 1989; Woods et al., 1989, 1996; Rose et al., 1991; Goldin et al., 1994). Overall, data concerning the relationship between dietary fiber and breast cancer indicate that the degree of protection due to fiber intake is unclear, and that the exact mechanism by which fiber may act is unknown. The potential for a protective effect of fiber on breast cancer risk is of great public health importance, given that most recognized risk factors for this disease are not modifiable. Data on the relation between fiber and pancreatic cancer are consistent with a protective effect of this nutrient in all (Bueno de Mesquita et al., 1990, 1991; Howe et al., 1990b; Baghurst et al., 1991; Ji et al., 1995), with one of the studies (Zatonoski et al., 1991) reporting data on fiber. The association was significant in most studies (Bueno de Mesquita et al., 1990; Howe et al., 1990b, 1991). Consequently, the pooled analysis of the five SEARCH studies showed a significant association (OR = 0.42; 95% CI, 0.12–0.63; p for trend < 0.01). Simultaneous adjustment for saturated, monounsaturated, and polyunsaturated fat, protein, carbohydrate, cholesterol, fiber, and vitamin C showed an even stronger effect for fiber where an increase of 26 g/day was associated with an OR of 0.37 (95% CI, 0.25–0.55). From the SEARCH data, it is suggested that dietary fiber, as well as fruit and vegetable consumption, may play an important role in decreasing risk of pancreatic cancer.

Additional Plausible Mechanisms Dietary carbohydrate is heterogeneous in nature, and therefore a simple carbohydrate– or even a fiber–cancer hypothesis may be unjustified. The various existing proposed biological mechanisms for the specific carbohydrates and their constituents have been addressed earlier in this chapter. A strong case can also be made for two additional hypotheses: One is based on existing data based on the correlation of fiber and carbohydrates with other nutrients known to be related to cancer risk, and the second is from a proposal by Giovannucci (1995) dealing with insulin resistance.

Giovannucci (1995) proposed that exposure to elevated blood-insulin levels may promote the growth of colon tumors. Thus, hyperinsulinemia or its determinants may increase the occurrence of colon cancer. If this hypothesis is correct, the determinants of both fasting and postprandial serum insulin levels should have an effect on colon cancer risk. As discussed earlier, epidemiological data suggest that a diet high in refined carbohydrates and low in water-soluble fiber increases risk of colon cancer. These same factors lead to the rapid absorption of glucose into the blood and cause postprandial hyperinsulinemia. Among dietary factors, simple sugars, easily digestible starch, low water-soluble fiber, and extensive food processing, which contribute to a high glycemic load, result in hyperinsulinemia and a subsequent increase in risk of colon cancer. Among all fibers, water-soluble fibers appear to be most effective in leveling off blood glucose response and moderating insulin levels (Riccardi and Riverllese, 1991; Wolever et al., 1991) in response to carbohydrate intake. Therefore, it is plausible that the carcinogenic or protective action of carbohydrates and/or its constituents in colon carcinogenesis might involve the effect of these nutrients or their sources on insulin levels. This mechanism may involve the stimulation of growth of colon tumors by insulin’s action as a growth factor of colonic epithelial cells (Koenuma et al., 1989). Based on present knowledge, it can be further hypothesized that the carcinogenic mechanism of action differs depending on the type of fiber. If focus is placed on insoluble fiber, particularly that derived from wheat bran, its action is known to be mediated through the direct effect on bile acid metabolism and excretion. However, if emphasis is placed on fruit and vegetables, rich sources of soluble fiber, their anticarcinogenic effect may be mediated through their action on insulin levels. It must be emphasized, however, that sufficient direct evidence is lacking to support the various proposed steps. Clearly, much research needs to take place before we can put all the pieces of this complex puzzle together. There are many future directions for elucidating the relationship between fiber and colorectal neoplasia. The effect of fiber may vary by age, gender, and fiber intake throughout life, and each of these factors may in turn have effects on different areas of the colorectum. Further, the actions of fiber related to the development of more advanced neoplasia are unknown. Single trials that have been conducted may not have sufficient statistical power to investigate these issues in depth. Therefore, large studies are under way to combine data from multiple large trials to investigate the role of fiber in colorectal neoplasia in more detail.

SUMMARY AND CONCLUSIONS The role of carbohydrate, per se, in the carcinogenic process is difficult to assess because of its chemical diver-

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sity and the physiological effects specific to each type of carbohydrate. Furthermore, few studies have assessed carbohydrate intake without focusing on individual food sources or specific types of polysaccharides, especially the NSP. Therefore, it is not possible to ascertain any direct contribution of carbohydrate consumption on cancer risk. As a result, public health recommendations (National Academy of Sciences, 1989; World Health Organization, 1990) emphasize a higher consumption of complex carbohydrates, whole-grain foods and cereal products, and dietary fiber and limitation of sugar consumption. Although many studies of dietary fiber and cancer have been conducted, the evidence for its protective effect is not clear. The epidemiological evidence suggests some consistent patterns along with some apparent disparities. Some of the inconsistent findings have been attributed to insufficient variation in fiber intake plus inadequate dietary instruments in analytical studies. In addition, the chemical complexity of fiber, inconsistency in its definition, and our limited understanding of its chemical characteristics make it difficult to define its overall contribution to cancer risk. These contradictions may also reflect our incomplete understanding of the carcinogenic process. A conservative interpretation of the data suggests that some component of plant products, especially those present in fruit and vegetables, may reduce the risk of cancer of the colon. Although a plausible biological mechanism for the action of fiber on breast cancer risk has been identified, the overall evidence is inconsistent. For pancreatic cancer, although there is some consistency in the findings supporting a protective effect of fiber, it is unclear whether this effect is due to the effect of fiber or to the nutrients or dietary components associated with fiber. For all other cancers, there is little or no evidence to support the role of fiber. Further investigations focusing on the proposed relationships with special attention on types and sources of fiber, as well as a full consideration of other factors in fruit and vegetables, are warranted. As large prospective studies continue to mature, their results will be extremely valuable in providing more concise evidence, particularly for rare cancers.

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30 Dietary Lipids HUSEYIN AKTAS, MICHAEL CHOREV, AND J.A. HALPERIN

INTRODUCTION

ogy. Many physiologically relevant fatty acids have linear, even-numbered chains of 18 carbons or longer and are polyunsaturated, presenting several sequential double bonds separated by single methylene units. These double bonds are exclusively in the cis configuration in which the two substituting hydrogens are on one side while the two substituting methylenes are on the opposite side of the double bond (Table 1). Because they lack the enzymes required to introduce double bonds within the last seven carbons proximate to the methyl end of the molecule, mammals can neither synthesize linoleic (18:2 n-6) or linolenic acid (18:3 n-3) nor interconvert one to the other. These two fatty acids must be obtained from the diet and are, therefore, termed essential; they are extremely important constituents of the membrane lipids and indispensable precursors in the biosynthesis of eicosanoids such as prostaglandins, thromboxanes, leukotrienes, and 5-hydroxyeicosatetraenoic acid (Hansen et al., 1958; Collins et al., 1971; Paulsrud et al., 1972; Holman et al., 1982).

A combination of epidemiological, case-control, and cohort studies in the second half of the past century underscored the possibility that high dietary intake of n-3 polyunsaturated fatty acids (n-3 PUFAs) could have a protective effect against cancer. Although the results of those studies were controversial and not uniformly embraced in the field of nutritional epidemiology (Willett, 1997), the notion that n-3 PUFAs may exert anticancer properties was strongly supported by extensive experimental studies documenting an anticancer effect both in cancer cells in vitro and in animal models of experimental cancer. These studies opened the door to more detailed experimental work aimed at elucidating the cellular and molecular mechanism of the putative anticancer properties of n-3 PUFAs. In this chapter, we summarize the sometimes-inconsistent nomenclature of fatty acids, the basic biochemistry needed to understand the principal biological differences between n-3 and n-6 PUFAs, and the mechanisms proposed to explain the putative anticancer properties of n-3 PUFAs. Finally, we summarize our work that led to include n-3 PUFAs among an emerging class of anticancer agents generally known as inhibitors of translation initiation (Clemens and Bommer, 1999; Palakurthi et al., 2000). The chapter focuses only on putative anticancer effects of n-3 PUFAs; other postulated health benefits are acknowledged but are not discussed.

Nomenclature The systematic nomenclature of fatty acids is based on the length of the carbon chain, the number of double bonds, their location relative to the carboxyl end, and the geometric configuration. For example, the systematic name for arachidonic acid, the precursor of many eicosanoid signaling molecules, is cis,cis,cis,cis-5,8,11,14-eicosatetraenoic acid. In addition to the trivial and systematic names, there are two numerical systems that describe attributes of PUFAs. In one of these numerical systems, arachidonic acid is denoted “20:4 n-6,” meaning that it contains 20 carbons, 4

FATTY ACID BIOSYNTHESIS Metabolism of both dietary and de novo synthesized fatty acids generates the fatty acids required for normal physiol-


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Chapter 30/Dietary Lipids

TABLE 1 Fatty acid Saturated fatty acids Caprylic acid Caproic acid Lauric acid Myristic acid Palmitic acid

Fatty Acids Structure and Nomenclature

CO2H CO2H CO2H CO2H CO2H

Stearic acid cis Monounsaturated fatty acids Myristoleic acid

∆-Designationb

n-Designationa

Structure

CO2H

CO2H

Palmitoleic acid

CO2H

8:0

8:0

10:0

10:0

12:0

12:0

14:0

14:0

16:0

16:0

18:0

18:0

14:1 n-5

14:1 ∆9

16:1 n-7

16:1 ∆9

Vaccenic acid

CO2H

18:1 n-7

18:1 ∆11

Oleic acid

CO2H

18:1 n-9

18:1 ∆9

20:1 n-9

20:1 ∆9

22:1 n-9

22:1 ∆9

Eicosanoic acid

CO2H

Erucic acid Primary n-6 polyunsaturated fatty acidsc Linoleic acid γ-Linolenic acid

CO2H

CO2H

18:2 n-6

18:2 ∆9,12

CO2H

18:3 n-6

18:3 ∆6,9,12

Dihomo-γ-linolenic acid

CO2H

20:3 n-6

20:3 ∆8,11,14

Arachidonic acid

CO2H

20:4 n-6

20:4 ∆5,8,11,14

Adrenic acid

CO2H

22:4 n-6

22:4 ∆7,10,13,16

Docosapentaenoic acid

CO2H

22:5 n-6

22:5 ∆4,7,10,13,16

18:3 n-3

18:3 ∆9,12,15

20:5 n-3

20:5 ∆5,8,11,14,17

n-3 polyunsaturated fatty acidsd α-Linolenic acid Eicosapentaenoic acid

CO2H CO2H

Docosapentaenoic acid

CO2H

22:5 n-3

22:5 ∆7,10,13,16,17

Docosahexaenoic acid

CO2H

22:6 n-3

22:6 ∆4,7,10,13,16,19

a

Carbon numbering starts from the methyl terminal useful to link diet with tissue fatty acid metabolism. Carbon numbering starts from the carboxyl terminal useful to describe biochemistry of fatty acid metabolism. c Also called ω6-polyunsaturated fatty acids. d Also called ω3-polyunsaturated fatty acids. b

double bonds, and the first double bond is located between carbons 6 (n-6) and 7 counting from the methyl end of the molecule (Table 1). In the other system, arachidonic acid is denoted “20:4 ∆5,8,11,14,” meaning that it contains 20 carbons with 4 double bonds, and that the first double bond is between carbons 5 (∆) and 6 starting from the carboxyl end, and the other 3 double bonds start at carbons 8, 11, and 14, from the same end. The first numerical system indicates the relatedness to the essential fatty acid from which a given PUFA is derived: n-6 PUFAs are derived from linoleic (18:2 n-6) and n-3 PUFAs from linolenic acid (18:3 n-3). The

second numeric system reflects the biochemistry of a given fatty acid metabolism. Table 1 lists the trivial names, structures, and designations for some of the more abundant series of saturated fatty acids, monosaturated fatty acids, and PUFAs in mammalian biology.

Metabolism of Essential Fatty Acids The 20-carbon or longer polyunsaturated n-6 or n-3 fatty acids are, respectively, derived from linoleic (18:2 n-6) or linolenic (18:3 n-3) acid (Figure 1). Linoleic acid is

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Epidemiological, Prospective, and Experimental Studies CO2H

18:0 S tearic a.

9-desaturase

C O 2H

6-desaturase

C O 2H

C O 2H

18:1 n-9 Oleic a.

6-desaturase

18:2 n-6 Linoleic a.*

6-desaturase

18:3 n-3 -Linolenic a.*

elongase

18:4 n-3 S tearidonic a.

C O 2H

CO2H

C O 2H

C O 2H

12-desaturase

18:1 n-9 Oleic a.

elongase

elongase C O 2H

C O 2H

15-desaturase

18:3 n-6 -Linolenic a.

18:2 n-9

18:2 n-6 Linoleic a.*

5-desaturase

20:2 n-9 5-desaturase

C O 2H

18:3 n-3 -Linolenic a.*

C O 2H

C O 2H

elongase

C O 2H

20:3 n-6 eicos anoids Dihomo- -Linolenic a.

5-desaturase

20:4 n-3 E icos atetraenoic a.

C O 2H

20:3 n-9 Mead a.

elongase

20:4 n-6 Arachidonic a.

elongase

22:4 n-6 Adrenic a.

C O 2H

C O 2H

eicos anoids elongase

20:5 n-3 eicos anoids E icos apentaenoic a. (E P A)

elongase

22:5 n-3 Docos apentaenoic a.

C O 2H

22:3 n-9

C O 2H

C O 2H

C O 2H

6-desaturase peroxisomal -oxydation

6-desaturase peroxisomal -oxydation

24:4 n-6

24:5 n-3

C O 2H

22:5 n-6 Docos apentaenoic a.

P L A NT S

C O 2H

22:6 n-3 Docos ahexaenoic a. (DHA)

A NIMA L S

FIGURE 1 Biosynthetic pathways for polyunsaturated fatty acids (PUFAs) in plants and animals. The fatty acids indicated by an asterisk (*) are the essential PUFAs in the animal diet.

metabolized by a ∆6-desaturase that adds a double bond between carbons 6 and 7 from the carboxyl group generating γ-linolenic acid (18:3 n-6). This is followed by elongase generating the dihomo-γ-linolenic acid (20:3 n-6), which is longer by two carbons added in proximity to the carboxyl group. A subsequent modification by ∆5desaturase adds another double bond between carbons 5 and 6 from the carboxyl group yielding arachidonic acid (20:4 n-6). Arachidonic acid is a substrate for prostaglandin synthases, which produce the two double-bond–containing prostanoids: prostaglandins, prostacyclin, and thromboxanes, and for lipoxygenases, which produce hydroxyeicosatetraenoic acids and the four double bonds containing leukotrienes. α-Linolenic acid (18:3 n-3) is similarly metabolized to eicosapentaenoic acid (20:5 n-3; EPA), the precursor of the three double bonds containing prostanoids (Needleman et al., 1979; Fischer and Weber, 1984) and the 5 double bonds containing leukotrienes such as LTA5, LTB5, and LTC5 (Lee et al., 1984; Prescott, 1984; Strasser et al., 1985). Although the metabolism of both n-6 and n-3 PUFA series utilizes the same elongase and desaturase enzymes, some observations suggest that the desaturase has a higher affinity for n-3 than for n-6 PUFAs (Brenner, 1974). As a result, dietary n-3 PUFAs reduce the synthesis of arachidonic acid and the eicosanoids derived from it (Chen and Nilsson, 1993; Emken, 1994; Sauerwald et al., 1996; Emken et al., 1998, 1999).

Animals cannot interconvert n-3 and n-6 PUFAs and lack the ∆12- and ∆15-desaturases necessary to convert oleic acid (18:1 n-9) to linoleic (18:2 n-6) and α-linolenic (18:3 n-3) acids, the two essential fatty acids. In contrast, algae have the full repertoire of enzymes to synthesize stearic (18:0 n-9) and oleic (18:1 n-9) acids and to convert oleic acid to linoleic acid (18:2 n-6) by ∆12-desaturase and subsequently to α-linolenic acid (18:3 n-3) by ∆15-desaturase (Sayanova and Napier, 2004) (Figure 1). Fish obtain both n-6 and n-3 fatty acids from the food available to them, and the n-6/n-3 fatty acid distribution of fish oils is determined in great part by the availability of n-3 fatty acids producing microalgae in the marine food chain in the geographic location of their habitat. Humans obtain their n-3 fatty acids by dietary intake of fish or fish oils. n-3 PUFAs are more abundant in coldwater fatty fish than in warm-water lean fish. It is also important to highlight that because the n-3 PUFA content of the fish fat is dependent on the fishes’ food source, aquaculture fish fed with processed food tend to have a lower content of n-3 PUFAs as a percentage of the total fat.

EPIDEMIOLOGICAL, PROSPECTIVE, AND EXPERIMENTAL STUDIES The incidence of some cancers in different populations around the world shows dramatic variation. For example, the

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incidence of prostate and breast cancer is much higher in the United States and northern Europe than in Asia. This difference cannot be explained by ethnic or racial differences because one-to-two generations after Asian men or women migrate to the United States, the age-adjusted risk of prostate and breast cancer rises to reach the risk of the white American population (Ziegler et al., 1993; Deapen et al., 2002). Because dietary habits are prominent among the major differences in the lifestyles of Western and Asian societies, the regional distribution of breast and prostate cancers, together with other comparable observations, fueled the speculation that dietary factors, especially the amount of fat consumed and its dietary origin, critically influence the genesis and/or progression of some cancers. A classical example frequently quoted in support of the fat–cancer risk connection is that populations such as Alaskan Eskimos and Greenland populations, whose diets are based almost exclusively on cold-water fish rich in n-3 PUFAs, reportedly have a low incidence of prostate and breast cancer (Bjarnason et al., 1974; Karmali et al., 1987; Bruce et al., 2000a,b). Another example is that in countries historically considered to have low incidence of cancer, cancer rates are increasing coincidentally with the adoption of more Westernized diets, including a higher fat intake (Nielson and Hansen, 1980; Lanier, 1996; Lanier et al., 1996a; Tsuji et al., 1996; You et al., 2002). In Japan, for example, parallel to a progressive increase in the incidence of breast cancer, the average fat consumption increased from 9% of total energy in 1955 to 25% in 1987 (Hirayama, 1978; Karmali et al., 1987; Wynder et al., 1991). Increasing attention has been paid to a connection between cancer risk and the intake of specific fatty acids rather than total fat intake, and notable among these have been marine fatty acids. This is because most of those populations mentioned earlier, with apparent low rates of cancer, derive a very significant part of their diet from fish and marine animals, whose fat contains large amounts of n-3 PUFA, and the progressive increase in cancer risk parallels a decline in the intake of n-3 PUFAs concomitant with an increased intake of n-6 PUFAs (Bjarnason et al., 1974; Armstrong and Doll, 1975; Jansson et al., 1975; Nielson and Hansen, 1980; Kaizer et al., 1989; Sasaki et al., 1993; Lanier et al., 1996b). Although ecological studies of special populations seem to support a negative association between dietary n-3 PUFA intake and cancer risk, it is worth noting that the epidemiological evidence is purely inferential and correlative, and its interpretation is difficult because of other confounding factors. For example, factors such as early age at menarche and at first pregnancy, and prolonged lactation, known to reduce the risk of breast cancer, are also prevalent among most of the populations shown to have low incidence of breast cancer and high consumption of n-3 PUFAs. These behav-

ioral/cultural patterns may also be changing in response to Western influence. In summary, although nutritional epidemiology studies have intrinsic problems related to both methodology and multifactorial confounding factors that are difficult to control, they were useful in bringing to the forefront the notion that dietary fat in general and the relative intake of n-3 marine fatty acids in particular could exert a protective effect against some cancers. Case–control and cohort studies provide another approach to analyze possible correlations between diet and the risk of cancer. In case-control studies, the diet and lifestyle of the cancer patients are compared with those of control subjects, whereas in cohort studies a group of healthy individuals is followed for an extended period and the incidence of cancer and dietary habits among the participants is compared at the end of the study period. In contrast with the frequently quoted ecological studies mentioned earlier, most case-control studies do not support a potential cancer-protective effect of consuming a predominantly fish-based diet (reviewed by Willett, 1997). In an extensive review of epidemiological, case-control, and cohort studies examining the association between consumption of fish and risk of breast cancer, prostate cancer, and other hormone-dependent cancers published by Terry et al. (2003), the authors concluded that “. . . the development and progression of breast and prostate cancer appear to be affected by a process in which eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) play important roles; yet whether the consumption of fish containing marine fatty acids can alter the risk of these or of other cancers is unclear.” Importantly, these authors point out some critical factors that could have clouded the results of those studies. For example, the concentration of EPA and DHA contained in fish varies among species. Relatively high concentrations are found in wild cold-water fatty fish such as salmon, mackerel, sardines, and herring, whereas warm-water lean fish usually have lower concentrations of n-3 PUFAs and sometimes higher content of arachidonic acid (n-6 FA). Thus, case-control or cohort studies that analyzed only total fish consumption in relation to cancer risk regardless of the type of fish may generate conflicting results. Terry et al. (2003) also point out that an analysis of published studies indicates that the duration of the follow-up period may also affect the results of a study because stronger inverse associations between cancer risk and fish/n-3 PUFA consumption were found in studies with the longest follow-up periods. It seems, therefore, that a clearer picture of the potential association between fatty acid intake and cancer risk would emerge from studies and a long follow-up period, with repeated assessment of diet during that period, would provide information on cancers at various stages of growth and progression. This was the case in a report within the Health Professionals’ Follow-up Study that examined prospectively

Epidemiological, Prospective, and Experimental Studies

consumption of fish and marine oil intake in relation to risk of prostate cancer in a cohort of 50,000 men followed for 12 years. The participants responded to a semiquantitative food frequency questionnaire mailed four times during the 12-year period. The food questionnaire was validated measuring actual food intake for a week among a sample of 127 cohort members, and dietary intake of n-3 PUFAs verified by analysis of the relative composition of fatty acids in a subcutaneous fat aspirate taken from a sample of men from this cohort. The results, adjusted for other dietary and nondietary risk factors, showed that men consuming more than three servings of fish per week had almost half the risk of metastatic prostate cancer as compared with rare or nonconsumers of fish (Augustsson et al., 2003). Even more remarkable in that study is the finding that each additional daily intake of 0.5 g of marine oil further reduced the risk of metastatic prostate cancer by 24%. Interestingly, no association was found between fish intake and the overall risk (incidence) of prostate cancer among the cohort, suggesting that the intake of fish or marine oils affects tumor progression and metastatic potential rather than the malignant transformation. Experimental studies have provided more consistent data showing that both EPA and DHA, the main n-3 fatty acids in fish oils, exert anticancer properties in cancer cell lines in vitro and in animal xenograft models of human cancer (Falconer et al., 1994; Grammatikos et al., 1994; Rose et al., 1996; Calviello et al., 1998; Whelan et al., 2002). In our laboratory, daily oral administration of EPA doubled the life expectancy of p53−/− mice, which develop multiple cancers with 100% penetrance and die at ~40 weeks of age (unpublished observation). Taken together, studies in humans, animals, and cells indicate that n-3 PUFAs may exert a protective anticancer effect that deserves further investigation. Critical for such future studies is defining the cellular and molecular mechanism(s) underlying the anticancer effects of n-3 PUFAs, and deriving from this research much-needed mechanismspecific biomarkers to be used in human studies.

Proposed Mechanisms of Anti-cancer Activity of n-3 PUFAs

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channels (Xiao et al., 2001), a diversity of cellular effects believed to contribute to their anticancer properties. Inhibition of Eicosanoid Production from Arachidonic Acid Eicosanoids, including prostaglandins, leukotrienes, and thromboxanes, which are derived from 20-carbon PUFAs, affect a wide variety of cellular processes, including cell proliferation, differentiation, and apoptosis. Arachidonic acid, a 20-carbon fatty acid abundant in cell membrane phospholipids, is the major precursor of two double-bond– containing prostanoids. Cyclooxygenase (COX) catalyzes the first step in the conversion of arachidonic acid to prostaglandins and thromboxanes, and lipoxygenase catalyzes its conversion to four double-bond–containing leukotrienes. Increased expression of COX and overproduction of some eicosanoids have been implicated in both the development of cancers and the promotion of angiogenesis. Prostaglandins derived from arachidonic acid by the COX2 enzyme, notably prostaglandin E2 (PGE2), have been linked to carcinogenesis in studies of the proliferation of breast and prostate cancer cell lines in vitro, in experimental animal models leading to the development of mammary tumors, and in human studies on the effect of fish oil intake on epithelial cell proliferation rates (Rose and Connolly, 1999). Increasing the dietary intake of n-3 PUFAs reduces the production of arachidonic acid (Christiansen et al., 1991) and thereby the generation of arachidonic acid–derived eicosanoids. In addition, both EPA and DHA can displace arachidonic acid in cell membrane phospholipids (Rose et al., 1994) and in diacylglycerols (Madani et al., 2004) and have been shown to inhibit COX-2 (Rose and Connolly, 1999) and lipoxygenase. Thus, replacement and reduced formation of arachidonic acid, as well as inhibition of key enzymes in the eicosanoid synthesis pathways, are proposed mechanisms underlying the anticancer effects of n-3 PUFAs, a view supported in part by the apparent inhibitory effect on cancer cells’ growth of some nonsteroidal anti-inflammatory drugs that inhibit COX activity.

Effect on Membrane Structure and Function

Effect on Estrogen and Testosterone Metabolism

Because n-3 PUFAs incorporate into and are an integral part of membrane phospholipids, they can exert profound effects on membrane physical properties, including permeability, lateral diffusion, lipid packing, and domain formation, and thereby affect the function of membrane proteins intimately involved in intracellular signaling. Consistently, n-3 PUFAs have been shown to influence G protein–coupled receptor and receptor tyrosine kinase signaling pathways (Zhang et al., 1999; Mitchell et al., 2003), as well as ion

17β-Estradiol, the main natural estrogen, stimulates normal mammary development and promotes the neoplastic transformation of breast cells. Estradiol is metabolized along two major pathways, one generating 16-hydroxyestrone and the other 2-hydroxyestrone. 16-Hydroxyestrone is considered more bioactive than the 2-estrone metabolites. 16-Hydroxyestrone produces aberrant hyperproliferation in mammary explants and is considered a mediator of estrogen-induced transformation of breast epithelial cells

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(Chajes et al., 1995); also, clinical studies suggest that an elevated C16-hydroxylation of estradiol may provide a biomarker for breast cancer risk (Telang et al., 1997). Osborne et al. (1988) reported that feeding an n-3 fatty acid–rich fish-oil supplement to women reduced the extent of C16hydroxylation, suggesting that a reduced production of 16-hydroxyestrone and perhaps increased generation of 2hydroxyestrone may contribute to an anticancer effect of n-3 PUFAs in breast cancer. Testosterone promotes proliferation and neoplastic transformation of prostate cells. It has also been shown that both n-3 and n-6 PUFAs interfere with testosterone metabolism by inhibiting the enzyme 5α-reductase and thereby the conversion of testosterone to dihydrotestosterone (Liang and Liao, 1992). However, the anticancer properties of PUFAs in prostate cancer seem to be limited to the n-3 series. Also, finasteride, a specific inhibitor of the 5α-reductase that lowers 5-dihydrotestosterone to the castrate levels and is clinically used for treatment of benign prostate hyperplasia, was shown to significantly increase the risk of highgrade prostate cancers in a large prospective study. This study argues against the 5α-reductase as the primary target of the n-3 PUFAs, which appear to decrease the incidence of aggressive prostate cancer (Augustsson et al., 2003). Effect of Lipid Peroxidation The long-chain PUFAs are highly susceptible to lipid peroxidation, and peroxidation products of the marine fatty acids have been proposed as mediators of their anticancer effects (Gonzalez, 1995; Welsch, 1997). For example, in breast cancer cell lines, DHA increased lipid peroxides and enhanced the toxicity of anthracyclines (agents that generate oxidative stress); both effects were inhibited by the antioxidant vitamin E (Gonzalez et al., 1993). Similar results have been reported in animal models of experimental breast cancer (Gonzalez et al., 1991). However, the mechanisms by which these oxidation products of n-3 PUFAs inhibit cancer cell growth are still uncertain (Welsch, 1995). Thus, although effects on the cell membrane, eicosanoid formation, estrogen and testosterone metabolism, and lipid peroxidation have been proposed as potential mediators of the anticancer effects of marine fish oils, these mechanisms are not embraced because of the lack of stringent and direct evidence for their causative role.

THE TRANSLATION INITIATION CONNECTION Work has identified translation initiation as a molecular target of the anticancer effects of n-3 PUFAs. Translation, the cellular process by which mRNAs are translated into proteins, is operationally divided in three phases: initiation,

elongation, and termination. Translation initiation, a highly regulated process, requires the concerted participation of >20 proteins/cellular factors known as eukaryotic translation initiation factors (eIFs), plays a critical role in the control of growth and division in eukaryotic cells. This is because structural features in the mRNAs’ coding for most proto-oncogenic and cell cycle–regulatory proteins make their translation rather inefficient and critically dependent on the activity of translation initiation factors such as eIF2, eIF4E, eIF4A, and eIF4G. Indeed, experimental evidence indicates that the rate of translation controls the expression of most cell growth–regulatory proteins. For example, early mitogenic signals that turn on the transcription of cell growth–regulatory genes simultaneously activate translation initiation factors such as eIF2 and eIF4E that are rate limiting for translation initiation. In this manner, extracellular signals that stimulate cell proliferation couple transcription with translation, resulting in a dramatic increase in the expression of growth regulatory proteins at the G0–G1 transition and during the G1 phase of the cell cycle. Consistently, we and others have shown experimentally that reducing the rate of translation initiation preferentially inhibits the synthesis and expression of oncogenic proteins and cell growth regulatory proteins such as the G1-cyclins (cyclin D1, cyclin E, and cyclin A), whereas other “housekeeping” proteins are minimally affected (Aktas et al., 1998). The tight translational control of proteins that promote cell proliferation represents a key physiological restraint to cell growth, and uncontrolled translation results in malignant transformation (Lazaris-Karatzas et al., 1990; Koromilas et al., 1992a; Donze et al., 1995).

Translation Initiation and Cancer Cancer cells proliferate disregarding the checkpoints that restrain growth in normal cells. This ability is acquired through mutations that lead to inactivation of growth inhibitory genes such as Rb (retinoblastoma) or of tumor suppressor genes such as p53, and/or to activation of protooncogenes such as cyclin D1, c-myc, or Ras. Products of these genes regulate specific events in cell growth and division. Interestingly, overexpression of proteins that regulate translation initiation causes neoplastic transformation because the consequent increase in the rate of protein synthesis leads to a disproportionately higher translation of oncogenic proteins such as cyclin D1 and c-myc, which are overexpressed in a large number of human cancers (LazarisKaratzas et al., 1990; Duan et al., 1995; Shilatifard et al., 1996). In human cancers, overexpression of the translation initiation factor eIF2α correlates with neoplastic transformation of mammary epithelial cells and with the aggressiveness of non-Hodgkin’s lymphomas (Raught et al., 1996; Wang et al., 1999). Overexpression of eIF4E reportedly is a

The Translation Initiation Connection

prognostic tumor marker for breast cancers (Li et al., 2002), a predictor of recurrence in head and neck tumors (Nathan et al., 1997; Rosenwald et al., 2001), and is abundant in breast (Li et al., 1998), head and neck (Nathan et al., 2002), primary bladder (Crew et al., 2000) and colon carcinomas (Berkel et al., 2001), as well as non-Hodgkin’s lymphomas (Wang et al., 1999). Also, in many human cancers, the translational efficiency of oncogenic proteins and growth factors such as c-myc, vascular endothelial growth factor (VEGF), or transforming growth factor-β (TGF-β) is significantly enhanced through variations that simplify the structure of their mRNA, thus enabling them to escape tight control of translation initiation (Scott et al., 1998). For example, the mRNA for TGF-β has two alternative splicing forms: one with an 1100-nucleotide-long and highly structured 5′UTR and the other with a 230-nucleotide-long and simpler 5′UTR that has sevenfold higher translational efficiency. The shorter, translationally stronger, and upregulated TGF-β mRNA is almost exclusively seen in cancers and is believed to contribute to the metastatic potential of some breast cancers (Arrick et al., 1994). In contrast, inhibition of translation initiation interferes with both cell growth and malignant transformation (Sonenberg, 1994; Graff et al., 1995; Rousseau et al., 1996). For instance, attenuation of translation initiation by overexpression of the inhibitory eIF4E-binding protein or interferons suppresses cancer cell and tumor growth (Rastinejad et al., 1993; Davis and Watson, 1996; Rousseau et al., 1996). The previous paragraphs highlight the critical role played by translation initiation in the physiological control of cell growth, as well as in both malignant transformation and in maintenance of transformed phenotypes. The cellular translation initiation machinery, therefore, represents an attractive target for cancer treatment (Clemens and Bommer, 1999), and translation initiation inhibitors are now recognized as an emerging class of anticancer agents (Dua et al., 2001).

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initiator AUG codon, a process that requires the participation of several translation initiation factors including eIF4E, eIF4G, and the RNA helicase eIF4A. At the AUG codon, the 60S ribosomal subunit is recruited to form the 80S ribosome. Concomitantly, GTP associated with eIF2 is hydrolyzed to GDP, which must be exchanged for GTP to initiate a new cycle of translation. This GDP–GTP exchange is catalyzed by the multisubunit guanine nucleotide exchange factor eIF2B and is inhibited when the alpha subunit of eIF2 (eIF2α) is phosphorylated. The higher affinity of phosphorylated eIF2α for eIF2B sequesters it from the cytosol so it cannot catalyze the GDP-GTP exchange (Pain, 1996). In other words, phosphorylated eIF2α is a competitive inhibitor of eIF2B. Because the stoichiometric ratio of eIF2B to eIF2 in the cytosol is quite low (i.e., molecules of eIF2 are far more abundant than molecules of eIF2B), even partial phosphorylation of eIF2α is sufficient to eliminate the free eIF2B necessary to recycle the eIF2·GDP into the functional eIF2·GTP and reduce the overall rate of translation initiation (Brostrom et al., 1989; Srivastava et al., 1995). Figure 2 summarizes the translation initiation process. The enzymes that phosphorylate eIF2α on its serine 51 residue are known as eIF2α kinases. At least two eIF2α kinases, interferon-inducible double-stranded RNAdependent protein kinase R (PKR) and PKR-like ERresident kinase (PERK), are activated by signals from a “stressed” endoplasmic reticulum (ER) triggering a cascade of events generally termed the ER-stress response. Most proteins synthesized in the cytoplasm are translocated to the ER for folding and post-translational modifications. Increased or accelerated protein synthesis that overwhelms the ER capacity for folding or other perturbations that prevent protein

Inhibition of Translation Initiation Mediates the Anticancer Effect of EPA In the following sections, we briefly describe the translation initiation process and summarize the experimental evidence generated in our laboratories demonstrating that the anticancer effects of EPA are mediated by inhibition of translation initiation. In the initiation phase of mRNA translation, the translation initiation factor eIF2 forms a ternary complex with GTP and the initiating methionyl-tRNA (Met-tRNAi). The eIF2·GTP·Met-tRNAi ternary complex recruits the 40S ribosomal subunit forming the 43S preinitiation complex, which then binds to the mRNA cap with the help of other translation initiation factors. The preinitiation complex scans the 5′ untranslated region (5′UTR) of mRNA for the

FIGURE 2 Schematic representation of translation initiation highlighting the sites of action of the n-3 polyunsaturated fatty acids.

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folding or transport induce ER stress (Kaufman, 1999; Harding et al., 2000b). Another ER stressor is the partial depletion of intracellular calcium stores (Kaufman, 1999; Harding et al., 2000b). Indeed, it is well established that partial depletion of ER Ca2+ stores rapidly activates eIF2α kinases that phosphorylate eIF2α, thus limiting the rate of translation initiation and protein synthesis (Brostrom et al., 1989; Aktas et al., 1998). The exact mechanism by which reduction of intracellular Ca2+ activates eIF2α kinases is not clearly understood (Figure 2 identifies the putative sites for EPA action on the translation initiation process).

(nm)

A

EPA Depletes Intracellular Ca2+ Stores Binding of many physiological agonists of cell processes such as hormones, growth factors, or cytokines to their cognate cell membrane receptors induces a transient rise in cytosolic Ca2+ following its release from intracellular stores. When Ca2+ is released from intracellular stores, Ca2+ channels in the plasma membrane, known as store-operated calcium (SOC) channels, open to refill the intracellular stores by capacitative Ca2+ entry from the extracellular medium, thus reestablishing cellular Ca2+ homeostasis (Berridge, 1995; Putney, 1997). EPA has a dual effect on intracellular Ca2+ homeostasis. On the one hand, it induces Ca2+ release from the intracellular Ca2+ stores, and on the other, it inhibits Ca2+ influx through SOC in the plasma membrane; these cellular effects require peroxidation of EPA because they are blocked by vitamin E (Palakurthi et al., 2000). By releasing Ca2+ from the ER stores while simultaneously closing SOC, EPA partially depletes intracellular Ca2+ stores (Figure 3A). Depletion of the intracellular Ca2+ stores by EPA was confirmed by transfecting cells with ER-targeted “cameleon” proteins that monitor the ER calcium content in real time. Figure 3B shows the ER calcium–depleting effect of EPA. As mentioned earlier, depletion of intracellular Ca2+ stores activates eIF2α kinases and inhibits translation initiation. Inhibition of translation initiation by EPA was demonstrated by sucrose density gradient centrifugation of cell lysates followed by determination of the cell polysome profile. Treatment of cells with EPA shifts the polysome profile from heavy polyribosomal fractions towards light polysomes and free ribosomal subunits (Palakurthi et al., 2000) (Figure 4). This shift of the cell polysome profile toward lighter fractions is recognized as the hallmark of inhibition of translation initiation. Phosphorylation of eIF2α Mediates Inhibition of Translation Initiation by EPA Inhibition of translation initiation by EPA is mediated by activation of eIF2α kinase–dependent phosphorylation of eIF2α. This conclusion is based on the experimental findings in cancer cell lines treated with EPA: (a) EPA causes phosphorylation of eIF2α (inset to Figure 4); (b) it inhibits

B FIGURE 3 Eicosapentaenoic acid (EPA) releases Ca2+ from ER stores and closes store-operated calcium (SOC) channels. Fura-2–loaded cells were treated with EPA in the presence or absence of vitamin E (A) in Ca2+-free media or with thapsigargin (TG) in Ca2+-containing media to open SOC channels and then treated with or without EPA (Palakurthi, 2000). (B) ERtargeted Ca2+-sensitive cameleon-expressing cells were treated with EPA excited at 440 nm and fluorescence resonance energy transfer (FRET) was measured by determining the emission ratio at 530 nm (Yellow Fluorescent Protein) versus 480 nm (Cyan Fluorescent Protein) (C).

translation initiation in wild-type cells but not in cells expressing a dominant-negative mutant of PKR; and (c) cells transfected with a constitutively active but phosphorylation-resistant mutant of eIF2α (eIF2α-S51A) are resistant to the effects of EPA on translation initiation, protein synthesis, and cell growth (Palakurthi et al., 2000). EPA Downregulates G1 Cyclins and Blocks Cell Cycle Progression in the G1 Phase Phosphorylation of eIF2α results in preferential downregulation of oncogenes and G1 cyclins. It is well established that several structural features influence the translational efficiency of individual mRNAs. For example, long and complex 5′UTRs are associated with inefficient translation probably because in the presence of stable secondary structures, ribosomes cannot scan efficiently the entire 5′UTR to reach the AUG initiation codon (Koromilas et al., 1992b; Rousseau et al., 1996b). In contrast, mRNAs with simple, less structured 5′UTRs are translated more efficiently.

The Translation Initiation Connection

FIGURE 4 Eicosapentaenoic acid (EPA) shifts the cell polysome profile from heavy to lighter polysomes. Lysates of exponentially growing cells were processed by sucrose density gradient centrifugation and then read from the bottom at 214 nm. The inset shows phosphorylation of eIF2α by EPA.

Interestingly, the leader sequences of ~90% of vertebrate mRNAs are between 10 and 200 bases long, mostly without a complex secondary structure, and are efficiently translated (“strong” mRNAs). On the other hand, most mRNAs encoding for cell growth regulatory proteins or proto-oncogenes contain atypical 5′UTRs, which are >200 bases long and complex, which restricts their translational efficiency and renders their translation highly dependent on the activity of translation initiation factors (“weak” mRNAs) (Kozak, 1991). This translational inefficiency of proteins that regulate cell proliferation probably plays a crucial role in the maintenance of proper restraints on cell growth; unrestricted translation due to overexpression or dysregulation of translation initiation factors mostly increases the expression of oncogenic proteins and results in malignant transformation. For the reasons summarized earlier, interventions that restrict the rate of translation initiation by targeting translation initiation factors (such as eIF2) preferentially decrease the expression of growth-promoting and oncogenic proteins and can thereby inhibit the growth and metastatic potential of cancers (Graff et al., 1995; Rosenwald, 1996; Willis, 1999). Consistently, EPA-mediated phosphorylation of eIF2α limits the rate of translation initiation and results in a preferential translational downregulation of G1 cyclins. Figure 5A shows that EPA inhibits the synthesis and expression of cyclin D1, cyclin E, and Ras while minimally affecting the synthesis and expression of housekeeping proteins such as β-actin or ubiquitin. Figure 5B shows that cyclin D1 expression is downregulated at the level of translation. In this

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experiment, cells were made quiescent by serum withdrawal for 18 hours and then stimulated with basic fibroblast growth factor (bFGF). The figure shows that in quiescent cells there is no cyclin D1 mRNA. Eight hours after mitogenic stimulation with bFGF, the expression of cyclin D1 mRNA is fully induced, and the cyclin D1 protein is synthesized at a high level. In contrast, cells stimulated with bFGF in the presence of EPA show full expression of cyclin D1 mRNA but reduced synthesis of cyclin D1 protein. This experiment confirms that EPA inhibits cyclin D1 synthesis and expression at the level of translation. Importantly, this experiment also shows that EPA does not inhibit the bFGF-induced mitogenic signal upstream from the transcriptional activation of cyclin D1. Furthermore, EPA also downregulated cyclin D1 expression in the tumors in vivo. Taken together, these data indicate that EPA inhibits preferentially the translation initiation of cell cycle–regulatory but not of housekeeping proteins that may account for the potent anticancer effects of EPA with low toxicity. Downregulation of G1 cyclins by EPA causes cell cycle arrest in the G1 phase, as would have been expected from an agent that inhibits expression of G1 cyclins (Palakurthi et al., 2000). EPA Induces the Translation of ER-stress Genes Including Expression of Proapoptotic Proteins EPA induces the translation of the activating transcription factor-4 (ATF-4)–regulated gene cluster. As previously discussed, treatment of cells with EPA limits the availability of the eIF2·GTP·Met-tRNAi ternary complex and decreases the overall rate of translation initiation. Scarcity of the ternary complex has different consequences for the translation of the various mRNAs. Under conditions of limited ternary complex availability, the translation of mRNAs’ coding for housekeeping proteins such as β-actin and ubiquitin is minimally affected, while the translation of mRNAs’ coding for oncogenic proteins such as cyclin D1 is dramatically reduced. Paradoxically, the translational efficiency of another subset of mRNAs is significantly enhanced when the ternary complex is scarce. Among these is the mRNA encoding for activating ATF-4, which regulates the transcription of the ER-stress response gene cluster (Harding et al., 2000a; Scheuner et al., 2001). The ATF-4 mRNA is more efficiently translated under conditions of limited ternary complex availability because its 5′UTR contains several upstream open reading frames (uORFs) that render its translation highly inefficient when the ternary complex is abundant but significantly more efficient when the ternary complex is scarce. When the 43S preinitiation complex binds to the 5′ end of the ATF-4 mRNA, it scans the 5′UTR and initiates translation at the AUG codon of the first uORF (exemplified in the schematic mRNA represented in Figure 5D). By recognizing the initiation codon of the first uORF, the ribosomal machinery is primed to also recognize its stop codon and dissociate. However, a small fraction of

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A

C

B

D FIGURE 5 Eicosapentaenoic acid (EPA) inhibits synthesis and expression of growth-regulatory proteins at the level of translation initiation. (A) Exponentially growing cells were pulsed with [35S]Met-Cys with or without 30 mM EPA for 1 hour, and equal protein-containing cell lysates immunoprecipitated with anti-cyclin D1, cyclin E, Ras, β-actin, or ubiquitin antibodies. Immunocomplexes were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by PhosphorImager (synthesis) or by Western blot (expression). (B) Quiescent NIH 3T3 cells were stimulated with bFGF (5 ng/ml)/0.1% calf serum for 8 hours with or without 30 mM EPA, and total RNA was extracted and Northern blotted with a cyclin D1–specific probe using 18S mRNA as loading control. Parallel cultures were pulse-labeled with [35 S]Met-Cys, and cell lysates were immunoprecipitated with anti-cyclin D1 antibodies. (C) MCF-7 breast cancer cells treated with EPA increase the expression of BiP and CHOP. (D) Schematic representation of an mRNA with several uORFs that restrict its translational efficacy when the ternary complex is abundant but increase its translation when the ternary complex is scarce.

the 40S ribosomal subunit that remains associated with the mRNA continues scanning toward the 3′ end, restarting initiation and falling off at the subsequent initiation and stop codons, respectively. In summary, the probability of reaching the initiation codon of the ATF-4 bona fide uORF is very low when the ternary complex is abundant. In contrast, when

the ternary complex is scarce, the probability that the ribosomal machinery would translate the uORFs is reduced, and the probability that the 43S subunit would reach and translate the bona fide uORF of ATF4 is enhanced several-fold (Harding et al., 2000a). As a consequence, stimuli like EPA treatment that induce phosphorylation of eIF2α limit the

541

Conclusion

availability of the ternary complex, translationally upregulate the expression of ATF-4, and increase the transcription and expression of ATF-4 target genes such as chaperon binding protein (BiP) and C/EBP-homologous protein (CHOP). Indeed, treatment of cancer cells with EPA induces the expression of both BiP and proapoptotic CHOP (Figure 5C), providing a molecular explanation for the increased apoptotic rate reportedly seen in cancer cells upon prolonged treatment with EPA. Together with downregulation of oncogenic proteins and the blockade of cell cycle progression in the G1 phase, increased apoptosis would also contribute to the anticancer properties of EPA. In summary, we have identified the molecular mechanism of the anticancer activity of n-3 PUFAs such as EPA in vitro and most likely in vivo. By partially depleting ER Ca2+ stores, these natural dietary products inhibit translation initiation and preferentially downregulate the synthesis and expression of oncogenic proteins, thereby blocking the progression of the cell cycle in G1, in association with increased apoptosis, mediated, at least in part, by upregulation of ER-stress response genes. Figure 6 summarizes the proposed mechanism of the anticancer effect of n-3 PUFAs such as EPA.

regulation of genes that control cell proliferation and survival can be conceived as an environmental clue to maintain a correct restraint on physiological cell growth. Evolutionary and cultural changes have shifted the human diet over time toward a lower n-3 to n-6 PUFA ratio to the point that the modern Western diet is overwhelmingly rich in n-6 PUFAs. The observed increase in cancer rates in populations that until recently relied on n-3 PUFA-rich diets may be a reflection of the potential impact of the dietary transition from n-3 to n-6 PUFA–rich diets. Efforts to reintroduce an adequate balance of n-3 to n-6 PUFAs in the modern diet might be hampered by the precipitous decline of marine fish stocks. Indeed, it seems that current marine sources would not be capable of sustaining a progressively increasing demand for dietary n-3 PUFAs by the world population. Fish produced in aquaculture farms do not represent a viable alternative to increase the n-3 PUFA content of the modern human diet because the fish diet contains vegetable oil that is rich in n-6 PUFAs. These fish will be low in n-3 PUFAs, relative to the population in the wild. There may be hope, however, because genes that can convert n-6 to n-3 fatty acids such as fat-1 can be transgenically introduced into both plants and animals, as reported in mice (Kang et al., 2004). Thus, given the magnitude of the potential public health benefits that could derive from increasing n-3 PUFAs in the human diet, and the multifactorial economic, cultural, and practical difficulties implicated in achieving that goal, extensive intervention studies are needed to determine conclusively whether n-3 PUFA–rich diets reduce cancer risk in a manner that justifies the effort. The conduction and success of such human trials critically depends on the availability of biomarkers that will provide clear-cut and efficient endpoints, thus limiting their duration and facilitating the interpretation of their results. The generation of such biomarkers is an important contribution of the research summarized in this chapter. Indeed, preliminary results in human cancer patients indicate that administration of EPA-rich fish oils induces phosphorylation of eIF2α in vivo. Thus, the theoretical and practical tools needed are now available for the design of prospective trials to validate first the translation initiation machinery as the molecular target of the putative effects of n-3 PUFAs in human cancers and, if confirmed, conduct adequate trials to assess the preventive and perhaps therapeutic effects of these nutrients, an approach recently advocated in The Lancet by Professor David Horrobin, a pioneer investigator on the potential therapeutic effects of n3 PUFAs (Horrobin, 2003a, b).

CONCLUSION

References

n-3 PUFAs were readily available nutrients in the primitive food chain. The effect of n-3 PUFAs on the translational

Aktas, H., Fluckiger, R., Acosta, J.A., Savage, J.M., Palakurthi, S.S., and Halperin, J.A. 1998. Depletion of intracellular Ca2+ stores, phosphorylation of eIF2alpha, and sustained inhibition of translation initiation

FIGURE 6 Schematic representation of the effect of eicosapentaenoic acid (EPA) on translation initiation. EPA induces Ca2+ release from ER calcium stores and closes SOC thus reducing the Ca content of the ER stores. Partial depletion of calcium in the ER activates eIF2α kinases, which phosphorylate eIF2α and thereby inhibit translation initiation. Inhibition of translation initiation by eIF2α phosphorylation has a differential effect on the translation of mRNAs that depends in part on the structure of their 5′UTR. Most are minimally affected; oncogenic and cell growth–regulatory proteins such the G1 cyclins are translationally downregulated; proteins such as BiP and the proapoptotic CHOP, which are under the transcriptional control of ATF-4, are upregulated. This results in interruption of the cell cycle in the G1 phase and in activation of proapoptotic pathways.

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31 Calcium and Vitamin D JOELLEN WELSH

OVERVIEW: CALCIUM, VITAMIN D, AND CANCER

calcium availability induces transient hypocalcemia that stimulates secretion of parathyroid hormone (PTH) and enhances metabolic activation of vitamin D. Vitamin D in turn promotes absorption of dietary calcium in enterocytes, release of calcium from bone, and reabsorption of calcium in the kidney, processes mediated by the vitamin D receptor (VDR) (Holick, 2003). Once calcium influx restores normocalcemia, PTH secretion is diminished in a classic endocrine negative feedback loop. Under normal circumstances, therefore, this endocrine system maintains extracellular calcium homeostasis and allows for normal bone mineralization as long as sufficient calcium and vitamin D are available. The two naturally occurring forms of vitamin D are cholecalciferol (vitamin D3, from animal sources) and ergocalciferol (vitamin D2, from plant sources); both forms require metabolism for biological activity. For simplicity, this review focuses on vitamin D3 (Figure 1), but the metabolism and functions of vitamin D2 are similar. Vitamin D3 can be synthesized from a cholesterol derivative (7dehydrocholesterol) in the epidermis, a conversion that requires ultraviolet B (UVB) radiation. Vitamin D3 can also be obtained from natural and fortified foods and supplements (discussed in the section “Diet, Sunlight, and Vitamin D,” later in this chapter) and is absorbed along with other dietary lipids. Regardless of source (endogenous synthesis or diet), the initial step in metabolism of vitamin D3 is hepatic hydroxylation at the 25 position, generating 25hydroxyvitamin D3 (25[OH]D3). 25(OH)D3 is the major circulating form, which is also stored in adipose tissue and is the most accurate biomarker of overall vitamin D3 status. Further metabolism of 25(OH)D3 generates two metabolites: 24,25-dihydroxyvitamin D3 (24,25[OH]2D3) or 1α,25-

Epidemiological, molecular, and cellular studies have implicated vitamin D, a fat-soluble vitamin, in the development or progression of cancer. The activation of vitamin D in the body is intricately linked to dietary calcium, another nutrient that has been associated with cancer risk in epidemiological studies. Although the physiology of calcium and vitamin D are intricately connected, particularly in maintenance of skeletal health, it is now recognized that these nutrients also exert independent effects on cell behavior, including proliferation, differentiation, and apoptosis. This chapter focuses on the cellular and molecular mechanisms whereby calcium and vitamin D might have an impact on cancer risk and briefly discusses supportive data from epidemiological and clinical studies.

VITAMIN D AND CALCIUM METABOLISM: INTERPLAY OF ENDOCRINOLOGY AND NUTRITION Forms, Functions, and Metabolism of Vitamin D The term vitamin D refers to calciferols, steroid compounds originally identified as lipid-soluble compounds that could ameliorate the childhood bone disease rickets. Indeed, the best-characterized role of vitamin D is maintenance of extracellular calcium homeostasis, and rickets results from impaired bone mineralization secondary to insufficient calcium availability to the growing skeleton. Normally, low


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Chapter 31/Calcium and Vitamin D

7-Dehydrocholesterol

Skin

DIETARY VITAMIN D •Natural foods •Fortified products •Supplements

UVB Vitamin D3

Skin

25(OH)D3

Liver PTH , Low Ca++

1,25(OH)2D3 1,25(OH)2D3

Diet, Sunlight, and Vitamin D Deficiency

Kidney

VDR: BONE Ca resorption

VDR: INTESTINE Ca absorption

duction of 1,25(OH)2D3, the biologically active vitamin D3 metabolite, is mediated by the 25(OH)D3 1α-hydroxylase (also termed CYP27B1 or P450C1), an enzyme that is highly expressed in renal proximal tubules (Miller and Portale, 2003). 1,25(OH)2D3, or calcitriol, is the ligand for the VDR and is a potent calcium-elevating hormone. Because the kidney 1α-hydroxylase produces 1,25(OH)2D3 for the systemic circulation, its activity reflects calcium availability. If calcium demand is increased, renal 1αhydroxylase activity is induced and more 1,25(OH)2D3 is generated (it is this activation step that is enhanced by PTH). Elevated circulating 1,25(OH)2D3 subsequently interacts with VDR in target tissues such as kidney, intestine, and bone to mobilize calcium. Conversely, when calcium demands are low, the renal 1α-hydroxylase is suppressed and the 24-hydroxylase is enhanced, leading to formation of 24,25(OH)2D3 and initiation of catabolism. These regulatory concepts of 25(OH)D3 hydroxylation are based on the renal enzymes and control of extracellular calcium homeostasis and are not likely to be applicable to regulation of these enzymes in other tissues. This issue is discussed further in the section “Uptake and Metabolism of Vitamin D Metabolites in Novel Target Tissues,” later in this chapter, in the context of control of epithelial cell turnover by 1,25(OH)2D3.

VDR: KIDNEY Ca re-absorption

FIGURE 1 Vitamin D3: structures and metabolism. Cholecalciferol (vitamin D3) can be endogenously synthesized in the epidermis upon exposure to ultraviolet B (UVB) radiation from sunlight, indoor tanning, or other sources. UVB energy cleaves the B ring in 7-dehydrocholesterol, creating the flexible A ring characteristic of cholecalciferol (D3). Cholecalciferol can also be obtained from the diet, either from foods or from supplements; therefore, this compound was initially labeled a vitamin, and 7-dehydrocholesterol a previtamin. In the liver, cholecalciferol is metabolically converted to 25-hydroxycholecalciferol or 25(OH)D3 (the prohormone form) by the microsomal 25-hydroxylase enzyme. Production of the hormonal form, 1,25-dihydroxycholecalciferol or 1,25(OH)2D3, in the proximal renal tubules is catalyzed by the mitochondrial 1α-hydroxylase, whose activity is tightly controlled by parathyroid hormone (PTH), calcium, and 1,25(OH)2D3 itself. The metabolism of vitamin D2 (ergocalciferol) is similar. In the classic endocrine control of calcium homeostasis, both 1,25(OH)2D3 and 1,25(OH)2D2 act as ligand for the vitamin D receptor (VDR) to mediate calcium influx into the circulation from bone, intestine, and kidney.

dihydroxyvitamin D3 (1,25[OH]2D3). Production of 24,25(OH)2D3 is catalyzed by the 25(OH)D3 24-hydroxylase (also termed CYP24 or P450C24), an enzyme present in the majority of vitamin D target tissues. The 24,25(OH)2D3 metabolite does not readily bind VDR, and its production is considered the first step in degradation of 25(OH)D3. Pro-

Natural foods, with the exception of certain fish, are relatively low in calciferols, and epidermal synthesis is highly variable. For this reason, milk and other products are fortified with vitamin D3 in the United States, Canada, and many other countries (Tangpricha et al., 2003). It should be noted, however, that fortification is voluntary, and the actual vitamin D3 content of fortified milk is often less than the stated 400 IU/quart (Holick et al., 1992). Despite the fortification of vitamin D3 in foods and endogenous synthesis, the prevalence of vitamin D insufficiency, as defined by low circulating 25(OH)D, is surprisingly common, especially in populations living in northern climates and in the elderly (Lips, 2001; Zittermann, 2003). More than 50% of patients admitted to Massachusetts General Hospital in a 1998 sample were diagnosed as vitamin D deficient (Thomas et al., 1998), and >40% of healthy young men in Boston had serum 25(OH)D levels in the insufficient range at the end of winter (Tangpricha et al., 2002). Similar rates of vitamin D insufficiency were reported for middle-aged men in Finland (Ahonen et al., 2000). A study conducted in Boston reported that 24% of healthy adolescents were vitamin D deficient as defined by low serum 25(OH)D levels (Gordon et al., 2004). Factors associated with low vitamin D status include limited epidermal synthesis of cholecalciferol (because of infrequent exposure to sunlight, living in geographic areas with low solar radiation, dark pigmentation, and liberal use of sunscreen), liver or kidney disease, certain medications,

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Cellular Mechanisms of Vitamin D Action

poor diet, and aging. Indeed the current recommended daily allowance for vitamin D increases with age (200 IU for those younger than 50 years, 400 IU for those 51–70 years, and 600 IU for those 71 years or older). The increasing number of reports of vitamin D insufficiency has prompted reevaluation of the recommended dietary allowances, which were designed to prevent rickets. There is fairly compelling evidence that prolonged subclinical vitamin D deficiency, which may not be associated with hypocalcemia but could limit availability of vitamin D metabolites to tissues, contributes to chronic disease in human populations (Zwittermann, 2003). However, relevant biomarkers of vitamin D status that reflect newly identified actions in target tissues such as colon, prostate, and breast (as discussed below) remain to be identified. In most cases, overt vitamin D deficiency can be prevented or cured by dietary adjustments or use of a daily multivitamin supplement (Tangpricha et al., 2002; Zittermann, 2003). There are a number of hereditary defects in humans that impair bioactivation or utilization of vitamin D, and these are not cured by vitamin D itself. These vitamin D–resistant syndromes are rare and have been well characterized at the biochemical and molecular levels (Malloy and Feldman, 2003). Mouse models of hereditary vitamin D–resistance syndromes have become powerful research tools for identification of new functions of vitamin D (Li et al., 1997; Dardenne et al., 2001).

CELLULAR MECHANISMS OF VITAMIN D ACTION Uptake and Metabolism of Vitamin D Metabolites in Novel Target Tissues The identification of the VDR in tissues that do not participate in control of extracellular calcium homeostasis prompted studies of vitamin D metabolism and action in additional cell types (Figure 2). Vitamin D metabolites, including 25(OH)D3 and 1,25(OH)2D3, circulate in the free form, as well as bind to the vitamin D binding protein (DBP), a member of the albumin gene family. Free steroids, in particular 1,25(OH)2D3, which has relatively low affinity for DBP, are presumed to enter cells via diffusion through the plasma membrane. In contrast, 25(OH)D3 bound to DBP enters renal cells via receptor-mediated endocytosis, facilitated by the megalin–cubulin complex (Willnow and Nykjaer, 2002). Though not yet demonstrated experimentally, it is likely that 25(OH)D3 bound to DBP can also be imported into nonrenal cells that express megalin. If so, the metabolic fate of 25(OH)D3 would depend on the relative expression/activity of 25(OH)D3–metabolizing enzymes. In cells with high levels of the 25(OH)D3 24-hydroxylase, catabolism would predominate, whereas in cells with

DBP 25D 1,25D Endocytosis DBP

25D DBP Mitochondria 24-OHASE

1-OHASE

24,25D

1,25D

Catabolism VDR VDR RXR

Nucleus

25D 1,25D 24,25D

25(OH)D

Gene Regulation

VDR Cytoplasm, membrane Signal transduction

1,25(OH)2D 24,25(OH)2D

Growth Arrest, Differentiation, Apoptosis

FIGURE 2 Cellular pathways of vitamin D signaling. Vitamin D metabolites circulate as free steroids and in complex with vitamin D binding protein (DBP). Free steroids, including 1,25(OH)2D3, diffuse through the plasma membrane, whereas DBP containing bound vitamin D metabolites such as 25(OH)D3 is imported via receptor-mediated endocytosis. Inside cells, 25(OH)D3 is released from DBP and transported into mitochondria, likely via intracellular binding proteins that act as chaperones (not shown). Metabolism of 25(OH)D3 to 24,25(OH)2D3 or 1,25(OH)2D3 depends on the relative activities of the 24-hydroxylase (24OHASE) and 1α-hydroxylase (1-OHASE) mitochondrial enzymes. Generation of 24,25(OH)2D3 is considered the first step in a catabolism pathway, whereas 1,25(OH)2D3, whether generated in mitochondria or taken up from the circulation, acts as ligand for the vitamin D receptor (VDR). VDR can mediate transcriptional regulation (as a heterodimer with RXR or other nuclear receptors) or nongenomic effects on membrane-initiated or cytoplasmic signal transduction pathways, leading to growth arrest, differentiation, and apoptosis.

functional 25(OH)D3 1α-hydroxylase, activation to 1,25(OH)2D3 would occur. Examples of nonrenal cell types that express 25(OH)D 1α-hydroxylase include keratinocytes, macrophages, and epithelial cells derived from the prostate, breast, pancreas, and colon (Rizk-Rabin et al., 1994; Chen et al., 2003; Cross et al., 2003; Hewison et al., 2003; Welsh et al., 2003; Schwartz et al., 2004). The presence of 1α-hydroxylase in extrarenal tissues suggests that concentrations of 1,25(OH)2D3 sufficient to elicit effects on cell proliferation could be generated from 25(OH)D3. In support of this possibility, some epithelial cells that express 1α-hydroxylase are growth inhibited by physiological concentrations of 25(OH)D3 in vitro, presumably due to its bioactivation to 1,25(OH)2D3 (Hsu et al., 2001; Welsh et al.,

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2003). This new concept of tissue-specific vitamin D metabolism and action indicates that the optimal serum levels of 25(OH)D3 will need to be redefined in terms of maintenance of local 1,25(OH)2D3 generation. These observations have thus identified two distinct pathways of vitamin D biosynthesis and action: an endocrine pathway geared toward maintenance of calcemia via circulating 1,25(OH)2D3 (Figure 1) and an autocrine pathway (Figure 2), which mediates tissue-specific cell-regulatory effects via local generation of 1,25(OH)2D3. The implication of the autocrine pathway is that cellular production of 1,25(OH)2D3 would likely be regulated in a tissue-specific fashion independently of systemic calcium homeostasis. Similarly, the actions of locally produced 1,25(OH)2D3 would be confined to the immediate cellular environment and would not necessarily affect body calcium homeostasis. Existence of the autocrine pathway implies that circulating 25(OH)D3 becomes the critical determinant of cellular vitamin D activity.

The Vitamin D Receptor Whether generated in cells from 25(OH)D3 or taken up from the circulation, 1,25(OH)2D3 binds to the VDR, a member of the steroid receptor family of ligand-dependent transcription factors that modulate gene expression in a tissue-specific manner (Carlberg, 2003; Christakos et al., 2003). Gene regulation by the liganded VDR requires dimerization, most often with the retinoid X receptor (RXR) family, and binding to specific DNA sequences in target gene promoters (Figure 2). Although a variety of structurally distinct vitamin D–responsive elements have been identified, the best characterized is a hexanucleotide direct repeat separated by three variable base pairs (DR3) to which VDR:RXR heterodimers bind. However, the recognition that VDR also functions as a homodimer or as a heterodimer with partners other than RXR and can bind diverse DNA sequences suggests enormous flexibility to the genomic pathways regulated by vitamin D. VDR can also influence gene expression via interactions with other transcription factors such as Sp1. In addition, the VDR is subject to posttranslational modifications, including phosphorylation, which affect its transcriptional activity. In addition to genomic signaling, 1,25(OH)2D3 can exert rapid effects on signal transduction pathways, leading to biological responses at the plasma membrane or in the cytoplasm (Figure 2). Identification of an alternative binding pocket in the VDR for ligands that mediate rapid effects (Mizwicki et al., 2004) suggests that the VDR mediates some of these nongenomic effects, a suggestion supported by studies with cells from VDR-null mice (Erben et al., 2002; Zanello and Norman, 2004). Localization of the nuclear VDR protein to caveolae, specialized signaling complexes

present in plasma membrane, further supports this concept (Huhtakangas et al., 2004). Examples of nontranscriptional effects of the 1,25(OH)2D3–VDR complex with potential relevance to cancer cell regulation include calcium uptake, protein kinase C activation, interaction with β-catenin, and activation of protein phosphatases PP1c and PP2Ac (Palmer et al., 2001; Bettoun et al., 2002; Norman et al., 2002). The possibility that alternative receptors for vitamin D metabolites, which have been linked to rapid responses (FarachCarson and Nemere, 2003), may contribute to cancer cell regulation by 1,25(OH)2D3 has yet to be thoroughly investigated. Thus, the relative contributions of genomic and nongenomic signaling in mediating the diverse biological effects of 1,25(OH)2D3, particularly in relation to its anticancer properties, remain to be fully clarified.

Newly Identified Vitamin D Target Cells Although originally identified based on its role in bone homeostasis, vitamin D signaling has an impact on many cell types, including leukemic cells, osteoblasts, keratinocytes, and mammary, colon, and prostate epithelial cells. In an early study, 23 of 33 established human cancer cell lines surveyed expressed VDR (Frampton et al., 1982). In most VDR-positive cells, 1,25(OH)2D3 mediates antiproliferative effects, and it may subsequently trigger differentiation or apoptosis. Importantly, studies with cells from VDR-null mice have demonstrated that the VDR is required for the growth-regulatory effects of 1,25(OH)2D3 in transformed epithelial cells (Zinser et al., 2003). Expression profiling of breast, prostate, colon, and squamous carcinoma cells has identified 1,25(OH)2D3–responsive gene clusters involved in regulation of cell cycle, differentiation, cell adhesion, and immune responses, indicating a diverse and broad range of VDR targets potentially involved in cell regulation (Palmer et al., 2001; Lin et al., 2002; Swami et al., 2003; Krishnan et al., 2004). The mechanisms by which 1,25(OH)2D3 mediates growth-regulatory effects are briefly described; more details can be found in several reviews (Mork-Hansen et al., 2001; Lowe et al., 2003; Welsh et al., 2003).

Cell Regulatory Effects of 1,25(OH)2D3 Cell Cycle Regulation The antiproliferative effects of 1,25(OH)2D3 result from alterations in key cell cycle regulators, which culminate in dephosphorylation of the retinoblastoma protein (Liu et al., 1996; Simboli-Campbell et al., 1997; Flanagan et al., 2003) and arrest of cells in G0/G1 (Munker et al., 1986; Zhuang and Burnstein, 1998; Park et al., 2000b). The cyclindependent kinase inhibitors p21 and p27 are genomic targets

Cellular Mechanisms of Vitamin D Action

of the 1,25(OH)2D3–VDR complex in many cell types (Liu et al., 1996; Park et al., 2000b; Scaglione-Sewell et al., 2000; Hager et al., 2001; Liu et al., 2002). In colon cancer cells, liganded VDR activates protein phosphatases PP1c and PP2Ac, leading to dephosphorylation of S6 kinase, which prevents progression from G1 to S phase (Bettoun et al., 2002). In leukemic cells, 1,25(OH)2D3–mediated growth arrest is paralleled by activation of Erk, JNK, and p38 mitogen-activated protein kinase pathways (Pepper et al., 2003). Thus, although the mechanisms vary with cell type, a common feature of vitamin D signaling is to prevent transition of cells from G1 into S phase. In addition to direct regulation of cell cycle modulators, 1,25(OH)2D3 blocks mitogenic signaling, including that of estrogen, epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and KGF (Tong et al., 1999; Xie et al., 1999; Crescioli et al., 2000), and upregulates negative growth factors such as transforming growth factor-β (TGFβ) (Mehta et al., 1997; Jung et al., 1999; Yang et al., 2001). Although the tumor suppressor p53 mediates cell cycle arrest in response to many stimuli, 1,25(OH)2D3 inhibits growth of cells that express mutant p53 (Eisman et al., 1989), indicating that functional p53 is not required for the antiproliferative effects of vitamin D. This notion is consistent with data indicating that 1,25(OH)2D3–mediated activation of the p21 gene promoter is p53 independent (Liu et al, 1996). Induction of Differentiation In many cells, 1,25(OH)2D3 has been shown to induce differentiation markers, suggesting that a major function of 1,25(OH)2D3 in normal cells may be maintenance of the differentiated phenotype. The best studied example of vitamin D–induced differentiation is the keratinocyte (Bikle et al., 1993). In keratinocytes, 1,25(OH)2D3–mediated differentiation is dependent on increases in intracellular calcium and induction of the calcium sensing receptor and phospholipase C (Bikle et al., 2003). The ability of vitamin D compounds to mediate keratinocyte differentiation has become important therapeutically, as vitamin D–based drugs are effective against psoriasis, a hyperproliferative skin disorder. Furthermore, skin from VDR-null mice exhibits abnormal differentiation (Xie et al., 2002) and is highly sensitive to tumor formation in response to chemical carcinogens (Zinser et al., 2002b). Cancer cells that retain the ability to differentiate may be programmed to do so in response to 1,25(OH)2D3. Examples include induction of β-casein in breast cancer cells (Lazzaro et al., 2000; Wang et al., 2001) and modulation of β-catenin signaling in colon cancer cells (Palmer et al., 2001). In leukemic cell lines such as HL-60, NB4, and U937 cells, 1,25(OH)2D3 induces differentiation along the macrophage/

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monocyte pathway (Elstner et al., 1997). Genes identified as targets of 1,25(OH)2D3 in leukemic cells include p21 and the homeobox gene HOXA10 (Liu et al., 1996; Rots et al., 1998). Numerous signaling pathways have been implicated in 1,25(OH)2D3–induced monocytic differentiation, including nuclear factor-κB (NFkB), protein kinase C, ERKs, JNK, and p38 MAP kinases (Pepper et al., 2003). There is evidence that the induction of differentiation by 1,25(OH)2D3 in NB4 leukemic cells is mediated, at least partially, through nongenomic pathways involving protein kinase C, changes in intracellular calcium, calpain activation, and NFkB signaling (Norman et al., 2002). Activation of Apoptotic Cell Death In some transformed cells, 1,25(OH)2D3 induces apoptotic cell death via generation of reactive oxygen species, dissipation of the mitochondrial membrane potential, and cytochrome c release (Welsh et al., 1995; Narvaez and Welsh, 2001; Guzey et al., 2002; Flanagan et al., 2003), features of the intrinsic (mitochondrial) pathway of apoptosis. Cell lines reported to undergo apoptosis in response to 1,25(OH)2D3 or various synthetic analogs include those derived from breast cancer, prostate cancer, colon carcinoma, squamous carcinoma, myeloma, retinoblastoma, neuroblastoma, and glioma (Simboli-Campbell et al., 1996; Diaz et al., 2000; Park et al., 2000a; McGuire et al., 2001; Audo et al., 2003; Elias et al., 2003; Wagner et al., 2003). Furthermore, 1,25(OH)2D3 exerts additive or synergistic effects in combination with other triggers of apoptosis, such as radiation and chemotherapeutic agents (Chaudhry et al., 2001; Posner et al., 2001; Sundaram et al., 2003). Sensitivity to 1,25(OH)2D3–mediated apoptosis reflects the relative expression and/or subcellular localization of the Bcl-2 family of proapoptotic and antiapoptotic proteins, although the specific proteins involved and their modulation vary with cell type. In breast cancer cells, 1,25(OH)2D3 downregulates the antiapoptotic protein Bcl-2 and induces redistribution of the proapoptotic protein Bax from cytosol to mitochondria (Narvaez and Welsh, 2001; Flanagan et al., 2003; Narvaez et al., 2003; Wagner et al., 2003). Furthermore, overexpression of Bcl-2 renders prostate and breast cancer cells resistant to 1,25(OH)2D3–mediated apoptosis (Mathiasen et al., 1999; Blutt et al., 2000; Guzey et al., 2002). The role of caspases and other proteases in 1,25(OH)2D3–mediated cell death appears to vary with cell type. Activation of caspases 3 and 9 occurs during 1,25(OH)2D3–induced apoptosis in some cells, and caspase inhibition can prevent some features of 1,25(OH)2D3–mediated apoptosis (Park et al., 2000a; McGuire et al., 2001; Narvaez and Welsh, 2001; Guzey et al., 2002; Pepper et al., 2003). However, caspase inhibitors do not prevent

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1,25(OH)2D3–mediated death (Mathiasen et al., 1999; Narvaez and Welsh, 2001; Pirianov and Colston, 2001; Guzey et al., 2002). Another protease pathway implicated in 1,25(OH)2D3–mediated apoptosis involves calcium release from the endoplasmic reticulum and activation of µ-calpain (Mathiasen et al., 2002). In this study, 1,25(OH)2D3–mediated apoptosis could be prevented by either calpain inhibitors or calcium-buffering agents such as calbindin D28K, indicating a requirement for both enzyme activation and calcium signaling (Mathiasen et al., 2002). Collectively, these studies indicate that a wide variety of signaling pathways, apoptotic-regulatory proteins, and proteases may contribute to 1,25(OH)2D3–mediated apoptosis depending on the specific cell type or context.

MODULATION OF CELL TURNOVER AND CARCINOGENESIS BY VITAMIN D IN VIVO: ANIMAL STUDIES Additional evidence that vitamin D can have an impact on both prevention and treatment of several common cancers has come from animal feeding studies. Rodents fed Western-style diets (low in vitamin D and calcium, high in saturated fat) developed hyperproliferation and/or enhanced rates of tumor formation in colon, prostate, and mammary gland compared with rats fed adequate calcium and vitamin D (Pence and Buddingh, 1988; Jacobson et al., 1989; Newmark et al., 1990; Xue et al., 1997, 1999). In mammary gland organ culture, 1,25(OH)2D3 inhibits hormone-driven proliferation (Zinser et al., 2002a) and reduces the number of carcinogen-initiated preneoplastic lesions during both the initiation and the promotion stages (Mehta et al., 1997), indicating that vitamin D signaling can exert direct antineoplastic effects at multiple steps in the carcinogenesis process. VDR agonists have also been shown to inhibit angiogenesis, invasion, and metastasis (Hansen et al., 1994; Schwartz et al., 1997; Bernardi et al., 2002; Flanagan et al., 2003), indicating a potential benefit of vitamin D on later stages of cancer progression. In addition to dietary manipulation, the effects of 1,25(OH)2D3 and synthetic forms of vitamin D have been studied in animal models of cancer. In 1987, Eisman et al. presented the first evidence that 1,25(OH)2D3 could inhibit growth of human colon carcinoma xenografts in immunesuppressed mice (Eisman et al., 1987). In most subsequent in vivo studies, synthetic vitamin D compounds have been utilized in attempts to avoid the hypercalcemic effects associated with chronic 1,25(OH)2D3 administration. Pharmaceutical firms have since developed thousands of vitamin D analogs, some of which display enhanced cell-regulatory effects with reduced calcemic activity (Carlberg, 2003; Guyton et al., 2003; Pinette et al., 2003). Although their specific mechanisms of action are not completely understood,

most vitamin D analogs that display antiproliferative effects bind to and activate VDR. Many synthetic analogs of vitamin D delay tumor formation in chemical carcinogenesis models and inhibit growth of established tumors in xenograft models (James et al., 1998; VanWeelden et al., 1998; Polek et al., 2001; Kumagai et al., 2003). Both 1,25(OH)2D3 and a synthetic analog reduced polyp formation and tumor burden in the Apc/min mouse, a spontaneous model of hereditary colon cancer (Huerta et al., 2002). Analysis of regressing tumors indicated that vitamin D analogs induce growth arrest and apoptosis in vivo (James et al., 1998; VanWeelden et al., 1998; Audo et al., 2003), similar to that observed with VDR agonists in vitro. Vitamin D analogs have also been shown to reduce the number of secondary tumors and experimental metastasis in nude mouse models of breast and prostate cancer (Lokeshwar et al., 1999; El Abdaimi et al., 2000; Flanagan et al., 2003). Newer studies with VDR-null mice have demonstrated that VDR status can modulate proliferation and carcinogenesis in normal tissues including mammary gland, colon, and skin (Kallay et al., 2001; Zinser et al., 2002a,b). VDR ablation significantly delays apoptotic remodeling of normal breast tissue during postlactational involution (Zinser and Welsh, 2004), providing the first evidence that vitamin D signaling has an impact on physiological apoptosis in vivo. VDR status alters tumor incidence in a transgenic model of breast cancer (MMTV-neu mice) and in chemically induced skin carcinogenesis (Zinser et al., 2002b; Zinser and Welsh, 2004). Collectively, these and other animal studies (summarized in Tables 1 and 2) have confirmed that the effects of vitamin D signaling observed in vitro translate to effects on cell proliferation, differentiation, and apoptosis in vivo that are of sufficient magnitude to have an impact on the carcinogenic process.

TABLE 1

Vitamin D and Prevention of Cancer

• Inverse associations reported between biomarkers of sunlight exposure, dairy products, and/or dietary vitamin D and risk of colon, prostate, and breast cancer • Low circulating 25(OH)D3 and/or 1,25(OH)2D3 associated with enhanced cancer risk and/or disease activity • Amplification of 25(OH)D3 24-hydroxylase in breast and esophageal cancers • Vitamin D receptor (VDR) polymorphisms linked to cancer risk and/or metastatic progression • VDR and 25(OH)D3 hydroxylases are expressed in normal epithelial tissues • 1,25(OH)2D3 inhibits carcinogen induced preneoplastic lesions in organ culture • VDR-null mice exhibit abnormal cell turnover in colon, mammary gland, and epidermis • VDR status correlates with carcinogenesis susceptibility in animal studies

Epidemiological and Clinical Studies on Vitamin D, Calcium, and Cancer

TABLE 2

Vitamin D in Cancer Therapy

• Vitamin D receptor (VDR) is expressed in transformed cell lines and in human tumor biopsies. • Natural and synthetic VDR agonists induce G1 arrest, apoptosis, and/or differentiation in vivo and in vitro. • 1,25(OH)2D3 inhibits angiogenesis and invasion via effects on tumor cells, endothelial cells, and extracellular matrix proteases. • Synthetic vitamin D analogs inhibit growth of carcinogen-induced tumors and human xenografts in absence of weight loss or hypercalcemia. • Clinical trials indicate vitamin D–based drugs are well tolerated with some antitumor efficacy.

CALCIUM AND CANCER RISK Calcium Intakes and Calcium Status The majority of dietary calcium is derived from dairy products, although the mineral is also present in reasonable concentrations in certain vegetables, nuts, grains, and beans. Because of increased awareness that calcium intakes are frequently low, calcium-fortified foods (such as orange juice) and calcium supplements are encouraged. Although most attention has been paid to the role of calcium in protection against osteoporosis, the mineral has also been linked to protection against cancer of the colon and possibly other tissues (see the next subsection). Because of the tight homeostatic controls exerted by the PTH–vitamin D axis, which allow the body to adapt to a wide range of calcium intakes, calcium deficiency is not easy to define. Adaptation to chronic low dietary calcium is associated with compensatory increases in circulating PTH, which maintains serum calcium at the expense of calcium release from bone. Thus, unless severe or accompanied by vitamin D deficiency, low dietary calcium is not usually associated with changes in serum calcium.

Potential Mechanisms of Cancer Prevention by Calcium In general, animal studies have supported the concept that high dietary calcium can inhibit colon, and possibly breast, cancer (Lipkin and Newmark, 1999; Lamprecht and Lipkin, 2003). Feeding a Western-style diet (low in calcium and vitamin D but high in saturated fat) induces hyperplasia and aberrant cell proliferation in colon and other tissues that is prevented by supplemental calcium (Richter et al., 1995). In the colonic lumen, calcium may bind lipids and/or bile acids and minimize damage to mucosal epithelial cells. In support of this concept, high dietary calcium reduces both incidence and multiplicity of cholic acid–promoted colon tumors in rats, an effect that correlates with inhibition of ornithine decarboxylase (Pence et al., 1995).

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In some studies, interdependent effects of calcium and vitamin D on growth control have been identified. The ability of 1,25(OH)2D3 to inhibit proliferation, induce differentiation, and activate apoptosis in colon cancer cells has been linked to changes in intracellular calcium (Zhao and Feldman, 1993; Vandewalle et al., 1995). In rodents, dietary vitamin D modulated the intracellular calcium gradient normally present along the colonic crypt–villus axis (Brenner et al., 1998). Other evidence supports direct effects of calcium on epithelial cells (Lamprecht and Lipkin, 2003). Low extracellular calcium increases caco-2 cell proliferation via induction of c-myc (Hulla et al., 1995), and high calcium media inhibits proliferation and induces differentiation in many cell types (Lipkin and Newmark, 1999). The calciumsensing receptor, originally identified in cells of the parathyroid gland, has been linked to calcium-mediated growth control. The calcium-sensing receptor is expressed in many cell types, including breast, ovarian, and intestinal epithelial cells and keratinocytes (Rodland, 2004). The calciumsensing receptor is a G protein–coupled receptor in the plasma membrane that responds to physiological changes in extracellular calcium concentration by activating downstream signal transduction pathways involving phospholipase Cβ, inositol triphosphate, and diacylglycerol. Ultimately, these signals provoke calcium release from the endoplasmic reticulum, leading to elevated intracellular calcium and activation of protein kinase C. In colon cancer cells, calcium-sensing receptor signaling induces E-cadherin and suppresses B-catenin signaling (Chakrabarty et al., 2003), and resistance to calcium-mediated growth suppression correlates with loss of the calcium-sensing receptor (Rodland, 2004). The calcium-sensing receptor is also present in normal human breast cells, where it co-localizes with the vitamin D–dependent calcium-binding protein calbindin D28K, suggesting potential crosstalk between 1,25(OH)2D3 and calcium signaling in this signal transduction pathway (Cheng et al., 1998; Parkash et al., 2004). Collectively, these data indicate that the calcium-sensing receptor represents a molecular link among extracellular calcium, intracellular calcium signaling, and growth control.

EPIDEMIOLOGICAL AND CLINICAL STUDIES ON VITAMIN D, CALCIUM, AND CANCER Overview Motivated by the cellular, molecular, and whole animal studies demonstrating antitumor effects of calcium and vitamin D, multiple studies have addressed the impact of these nutrients on cancer risk, particularly for breast, colon, and prostate carcinoma, in human populations. Because most cells that give rise to cancer express functional VDR

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and are sensitive to calcium-mediated signaling, it is envisioned that optimization of these pathways may actually prevent cancer development (Table 1). This concept has not yet been adequately tested in large-scale intervention trials in humans, although the ongoing Women’s Health Initiative is addressing the effect of supplemental calcium and vitamin D on colon cancer development (Jackson et al., 2003). The available evidence linking vitamin D and calcium to breast, prostate, and colon cancers is briefly reviewed in the following subsections, and readers are encouraged to consult the references cited in each section for more specific information. There are many similarities among these three common carcinomas: All are derived from epithelial tissues that normally express VDR and are independently growth inhibited by calcium and 1,25(OH)2D3. All three tissues express 25(OH)D 1α-hydroxylase, highlighting the probability of local 1,25(OH)2D3 production acting in an autocrine fashion to regulate cellular proliferation, differentiation, or apoptosis. Potentially relevant to cancer etiology, transformation may be associated with reduced function of the 25(OH)D 1α-hydroxylase (Ma et al., 2004) and amplification of the 25(OH)D 24-hydroxylase, which can inactivate both 25(OH)D and 1,25(OH)2D3 (Albertson et al., 2000; Mimori et al., 2004). Furthermore, biopsies indicate that the majority of human colonic, breast, and prostate tumors retain expression of the VDR, highlighting the possibility that VDR agonists may have therapeutic value (Table 2).

Breast Cancer Several studies have reported inverse associations between indices of vitamin D status (such as intake, sunlight exposure, solar radiation, and/or latitude) and breast cancer incidence or mortality (Garland et al., 1990; Knekt et al., 1996; John et al., 1999; Grant, 2002). A review of the epidemiological literature concluded that although there is no consistent correlation between dairy product intake and breast cancer risk (Moorman and Terry, 2004), there are inverse correlations between certain forms of dietary calcium and breast cancer (Shin et al., 2002; Boyapati et al., 2003). In a 2004 study, Berube et al. demonstrated that high dietary intakes of calcium and vitamin D are associated with reduced mammographic breast density, a strong risk factor for development of breast cancer. Although no differences in serum 1,25(OH)2D3 were found between breast cancer cases and controls 15 years prior to diagnosis (Hiatt et al., 1998), low levels of 1,25(OH)2D3 were associated with increased breast cancer risk or disease progression in two case-control studies (Mawer et al., 1997; Janowsky et al., 1999). A high percentage of breast cancers express VDR, but there is no significant correlation between VDR expression and estrogen receptor expression, lymph node status, or tumor grade (Freake et al., 1984; Eisman et al., 1986; Berger et al., 1987,

1991). Tumor VDR status is not related to overall survival or to survival after relapse, but women with VDR-negative tumors relapsed earlier than women with VDR-positive tumors (Colston et al., 1989; Berger et al., 1991). A few small clinical trials with vitamin D analog therapy have included patients with advanced breast cancer. Topical treatment with a rapidly metabolized analog (calcipotriol) was assessed in women with locally advanced or cutaneous metastatic disease (Bower et al., 1991), and ∼20% of the participants showed partial responses. Similarly, disease stabilization was observed in some patients with breast cancer who received the low calcemic vitamin D analog EB1089 orally (Gulliford et al., 1998). Although no clear antitumor effects have been observed, these and other trials have demonstrated proof of principle that chronic administration of vitamin D analogs can be tolerated in breast cancer patients.

Prostate Cancer Like breast cancer, prostate cancer risk is inversely correlated with solar radiation (Hanchette and Schwartz, 1992, Grant, 2002). Two studies have found a higher risk for early onset and/or more aggressive prostate cancer in young men with low serum 25(OH)D3 (Ahonen et al., 2000). Interestingly, high consumption of dairy foods or calcium (>2000 mg/day) may enhance prostate cancer incidence and mortality (Chan et al., 2001), an effect postulated to be secondary to inhibition of 1,25(OH)2D3 production. However, no differences in serum 1,25(OH)2D3 or 25(OH)D3 were found between prostate cancer cases and controls in another prospective study (Platz et al., 2004). A number of phase I and II clinical trials with 1,25(OH)2D3 have been conducted in patients with androgen-responsive and androgen-unresponsive prostate cancer (Johnson et al., 2002; Beer et al., 2003; Krishnan et al., 2003). These trials have evaluated toxicity, administration schedules, and changes in prostate-specific antigen (PSA), a serum biomarker of tumor responsiveness. In general, these studies have demonstrated reduction or stabilization of serum PSA level in a subset of patients, with limited toxicity particularly with intermittent 1,25(OH)2D3 administration. Furthermore, combination therapy of 1,25(OH)2D3 with other drugs such as paclitaxel and dexamethasone was associated with less toxicity, suggesting that simultaneously targeting vitamin D signaling and conventional cytotoxic pathways may be a more effective approach clinically.

Colon Cancer Studies that combine multiple sources of vitamin D (intakes, supplements, and solar UV radiation) or measure serum 25(OH)D levels have generally reported inverse associations between vitamin D status and colon cancer risk

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(Peters et al., 2001; Feskanich et al., 2004; Grant and Garland, 2004). In one human trial, calcium in milk products was shown to precipitate bile acids and increase their excretion (Govers et al., 1996). More significantly, supplemental dietary calcium inhibited colonic epithelial cell proliferation in subjects at high risk for colon cancer (Lipkin and Newmark, 1985). Several cohort and case-control studies support the association between dietary calcium and reduction of colon cancer risk (Schatzkin and Peters, 2004). Furthermore, two randomized, placebo-controlled clinical trials have shown that calcium supplements modestly decrease risk of colorectal adenoma recurrence (Baron et al., 1999; Grau et al., 2003). In the Calcium Polyp Prevention Study, 1200 mg elemental calcium reduced recurrence rate 19% within 1 year of intervention and had an even more pronounced effect on advanced colorectal lesions (Baron et al., 1999; Wallace et al., 2004). In studies that have addressed the role of both calcium and vitamin D, data strongly suggest that these nutrients interact to reduce the risk of colon carcinoma (Grau et al., 2003; Peters et al., 2004; Slattery et al., 2004).

VDR Gene Variants May Modify Cancer Risk A number of common variants, or polymorphisms, in the human VDR gene have been studied in relation to cancer risk (Uitterlinden et al., 2004). VDR polymorphisms are distributed throughout the gene, including within the extensive promoter region (Cdx-2 site), the coding sequence (FokI, BsmI, ApaI, and TaqI sites), and the 3’UTR (polyA repeat variants). Although the majority of VDR polymorphisms do not alter the amount, structure, or function of the VDR protein, the FokI variation may have functional significance because individuals carrying the FokI site produce a shorter VDR, which may be less active than the full-length VDR (Jurutka et al., 2000). More than a dozen studies have reported associations between one or more VDR polymorphisms and incidence or progression of breast, prostate, and colon cancer. Not unexpectedly, many studies report that associations between VDR polymorphisms and disease are highly dependent on other factors, including dietary calcium, ethnicity, UV radiation, and tumor histopathology (Habuchi et al., 2000; Kim et al., 2001; Hamasaki et al., 2002; Guy et al., 2003). Although more research is necessary to determine whether VDR genotypes have an impact on cancer risk, these studies highlight the importance of identifying the role of individual genetic variability in the response to dietary factors such as calcium and vitamin D.

SUMMARY In this review, the possible relationship between vitamin D status, calcium intake, and cancer risk has been discussed on the basis of available evidence. Although the data are

predominantly supportive that these nutrients can reduce cancer risk, many questions remain to be answered. For example, to what degree does individual genetic variation in components of the calcium or vitamin D signaling pathways alter the effectiveness of dietary interventions? Are specific subtypes of tumors (i.e., those harboring particular mutations) or patients (those with hereditary cancer syndromes) more or less responsive to dietary factors? Can biomarkers for vitamin D and calcium signaling at the cellular level be identified that can be used to define optimal status and the intakes necessary to achieve such status? Once these questions have been satisfactorily answered, public health measures to increase calcium and vitamin D intake via diet, food fortification, or supplementation might be warranted.

Acknowledgments The author is indebted to her excellent research team at the University of Notre Dame, consisting of Lindsay Barnett, Belinda Byrne, Carly Kemmis, Carmen J. Narvaez, Matthew Rowling, Meggan Valrance, and Glendon Zinser, who contributed significantly to the studies and concepts discussed in this review. Work in the author’s laboratory is supported by the National Cancer Institute, the Susan G. Komen Foundation, and the Department of Defense Breast Cancer Research Program.

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32 Soy Isoflavones STEPHEN BARNES, JEEVAN PRASAIN, TRACY D’ALESSANDRO, CHAO-CHENG WANG, HUANG-GE ZHANG, AND HELEN KIM

INTRODUCTION

HISTORY OF SOY

The value of soy as an important part of the diet has been known for several millennia. Systematic research over the past 30–40 years has established a beneficial role of soy foods and their constituents in several chronic diseases, including cancer. Soy’s presence in foods has risen over the past 10–20 years in the United States and Western Europe, mostly in the form of increasingly purified protein preparations. This is in contrast to the use of soy in Southeast Asia, where it more commonly appears in forms prepared from whole soybeans using fermentation techniques. Several constituents of soy (isoflavones, protease inhibitors, phytosterols, and inositol phosphates) have been shown to have biological activity in models of cancer. The isoflavones have an estrogen-like activity (hence, the term phytoestrogen), but this is not their only mode of action. Unlike many other bioflavonoids, isoflavones are well absorbed, although they undergo substantial metabolism. It is possible that a bacterial metabolite, equol, has unique biochemical properties, giving advantage to those who can form it. A new class of bioactive compounds from soy may include unique peptides that can pass intact into the blood circulation. The link between diet and lowered risk of several chronic diseases is well recognized. It has stimulated groups of investigators to pursue cancer-prevention hypotheses on the basis of the roles of specific foods and compounds derived from them. In this chapter, we examine the connection between soy food consumption and lowered risk of several cancers, as well as one class of the phytochemicals in soybeans, the isoflavones. We explore the historical aspects of soy foods, their modern forms, and apparent influence on cancer risk, and the biochemistry, chemistry, metabolism, and mode of action of the soy isoflavones.

The soybean is a plant (Glycine max) that originated in Southeast Asia. The seeds of the soybean were mentioned as a medicine in the writings of the Chinese Emperor ChengNung in 2838 BC. They were used by monks in Japan in the second through sixth centuries AD to prepare an alternative to salted meat garnishes. This fermented product is soy sauce, now commonly used as a condiment in meals in many countries of the world. Soybeans were brought to Europe and America in the eighteenth century by traders who traveled to and from Southeast Asia. In the years leading up to the American Revolution of 1776, soybeans were cultivated as a crop in South Carolina (Hymowitz, 1990). However, the agricultural use of soybeans did not occur for more than 100 years. In the period from 1890 to 1900, the U.S. Department of Agriculture (USDA) systematically investigated the selection of soybean strains for the different growing conditions in each agricultural state. Since then, soybeans have become an important crop in the United States. Ironically, the United States has become a major provider of soybeans to Japan and other Asian countries, although over the past 25 years it has faced increasing competition from new soybean suppliers in countries such as Brazil.


FOOD MATERIALS DERIVED FROM SOYBEANS The soybean is processed to provide a number of different foods and food products (Figure 1). Soy milk is prepared by grinding soybeans and extracting them with water. Soy milk is an advantage to Asians because many of them lack

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Chapter 32/Soy Isoflavones Soy paste

Tempeh Soy sauce

Miso

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hot water

Soy milk coagulation

Soy sauce

chemical

Hot aq. alcohol

Soy Beans Defatted soy flour

Tofu

mild base

water

Soy protein concentrate

Soy protein concentrate

Solubilized soy protein Acid precipitation, washed and dried

Textured vegetable protein

Isolated soy protein

FIGURE 1 Processing of soybeans to produce soy foods. In the upper part of this diagram, soy foods that are common in the Asian diet are shown. Extraction of proteins from soybeans with water creates a liquid form of soy that takes the place of cow’s milk. This soy milk is converted to a semisolid form, tofu, by coagulation. Miso, soy paste, tempeh, and soy sauce are each fermented products of soybeans. The remaining items represent soy products prepared in the United States, mostly those enriched in soy proteins. There is a transition soy food, soy sauce, prepared by acid hydrolysis.

intestinal lactase and, thereby, cannot digest lactose in cow’s milk (Simoons, 1978). It is also an alternative for those infants who are intolerant to cow’s milk, and it is used in infant milk formula. The sales of soy milk have risen sharply in the past 15–20 years, and it is sold in the United States in full-fat and low-fat varieties. Tofu (or bean curd) is a protein fraction prepared from soy milk by calcium-induced coagulation, although it can also be formed by weak acids in a closed sterile container, thereby giving an extended shelf life. It comes in several forms by reducing the water content by pressing the curd in a filter. Silken tofu can be blended with other ingredients in puddings, whereas firm tofu is often a separate protein dish in a meal. Fermented soy products such as soybean paste are common in Asia. In Korea, soybean paste is combined with peppers to produce a hot sauce. In Japan, the fermentation is carried out slowly with barley and wheat to produce miso. Miso is widely used as a condiment and in soups. In Indonesia, intact soybeans are incubated with microorganisms to produce Tempeh, a fermented food with a meatlike appearance. In the United States, soybeans were initially largely grown as a source of cooking oil, with the residual protein fraction being used as an animal feed. However, there was an early interest in creating novel products from soybeans. Henry Ford explored the use of soybeans to make plastics. In the second half of the twentieth century, food chemists created protein fractions (soy flour, textured soy protein, and isolated soy protein) that have appeared in a wide variety of food products. Defatted soy flour is the protein fraction

(50% of solids) left after the extraction of the oil fraction with hexane. It is prepared in various forms dependent on the extent of heating used to denature the enzymes left after extraction. The roasted form is the one fed to livestock. Treatment of soy flour with hot water or hot aqueous ethanol removes the soluble carbohydrates (some of these, raffinose and stachyose, are poorly hydrolyzed in the gut). The alcoholic wash also removes other low molecular weight compounds such as the isoflavones (see Table 1 for data on the isoflavone content of soy foods). The protein content of the washed soy protein concentrate is ∼70%. Extruded soy protein concentrate is textured soy protein. If the defatted soy flour is treated with a mild alkaline solution, the protein fraction is largely solubilized, leaving behind the insoluble complex carbohydrate fraction. The protein-rich extract is treated with acid to pH 4–5 to precipitate the protein fraction. It is washed and dried to form soy protein isolate, a fraction containing 90–92% protein and 95%) the isoflavone content. The preparation of isolated soy protein leads to a loss of isoflavones, particularly the β-glycosides. Interestingly, the unconjugated isoflavones are increased, suggesting that the process causes hydrolysis of the conjugated isoflavones. Changes in the process of preparing isolated soy protein occurred in the mid-1990s and materials used in many clinical studies contained isoflavone concentrations in excess of 1.0 mg/g dry weight. Miso, soy paste, and tempeh each have total isoflavone concentrations that are comparable to unprocessed soybeans or soy flakes. However, the fermentation processes used in their preparation convert the β-glycosides to the unconjugated isoflavones. Soy foods in which other materials are added (barley, rice, or wheat) have a lower isoflavone content per gram. Soy milk and tofu have a high isoflavone content per gram, mostly as β-glycosides. Thus, it is critical in a preclinical or clinical study to carefully determine the isoflavones composition and concentrations. Because of differences in absorption of each of the isoflavone forms, the isoflavones that enter the systemic circulation, and thus, the tissues, will be a function of the composition of the soy preparation used. A large database of the isoflavone content of a wide variety of foods is available at http://www.nal.usda.gov/fnic/foodcomp/Data/isoflav/isofla v.html. Because soy protein is added to many foods and dietary or energy-producing supplements, investigators should not rely on dietary questionnaires to assess isoflavone intake but instead collect urine samples and perform isoflavone analysis (Horn-Ross et al., 1997). An example of an unusual source of isoflavones is in certain brands of canned tuna in which a protein (soy) broth is added to the fish (Horn-Ross et al., 2000).

When soybeans are extracted with hot water or pressurized steam to produce soy milk and tofu, there is substantial hydrolysis of the malonyl esters to form the simple βglycosides of the isoflavones (Barnes et al., 1994b) (Figure 4). This also occurred when analytical chemists used hot aqueous-organic solvent mixtures to extract isoflavones from plant and food materials (Barnes et al., 1994b). To avoid this analytical complication, extraction should be carried out at 4°C (Coward et al., 1998). Once extracted, the solution should be kept cold. We found that the isoflavone β-glycoside malonyl esters in such extracts when placed on a high-performance liquid chromatography (HPLC) autosampler slowly degrade (M. Smith-Johnson and S. Barnes, unpublished observations). A cooled sample tray prevents this problem. Processing of the soybeans with hexane to recover soy oil does not damage the isoflavone conjugates. However, post-extraction heating leads to the well-known decarboxylation of the malonyl group to an acetyl group (Figure 4). Thus, defatted toasted soy flakes/flour have substantial amounts of 6′-O-acetyl-β-glycoside conjugates of isoflavones (Barnes et al., 1994b). Soy protein concentrates have variable amounts of isoflavones (Table 1). In those protein concentrates derived by water washing of soy flour, the isoflavones are largely

OH

OH O

HO O OH

O

HO

A

O Genistin

OH hot aqueous conditions

OH

OH O

-

OOC.CH2 .COO

Importance of Glycoside Conjugates in Isoflavone Chemistry and Biological Action In the soybean and red clover, isoflavones are converted to their O-glycosides by O-glycosyltransferases and then to 6-O-malonate esters of the glycosides (Kudou et al., 1991). The O-glycoside conjugates are formed by a carbon–oxygen bond between the C-1 carbon of the sugar moiety and (generally) the O-7 oxygen of the isoflavones. The majority of the isoflavones in Kudzu root are unusual C-glycosides (Prasain et al., 2003). These conjugates are formed by a carbon–carbon bond between the C-1 carbon of the sugar moiety and the C-8 carbon of the isoflavones. In the groundnut, A. americana, a 7-O-glycosylglycoside conjugate of genistein is the principal isoflavone in the edible tuber of this plant (N. Barnes and S. Barnes, unpublished observations).

B

O OH HO

O

O 6"-O-malonylgenistin

OH

dry heat; -CO 2 OH

OH O

CH3 COO O OH

O

HO OH

O

C

6"-O-acetylgenistin

FIGURE 4 Chemistry of the glycosides of isoflavones. The principal form of isoflavones O-glycosides in the soy and other plants is the 6′-Omalonyl ester of the sugar moiety (B). The ester rapidly undergoes hydrolysis to form β-glycoside when heated in aqueous solvents, but also slowly even at room temperature. In the absence of water, the malonate loses CO2 to form a 6′-O-acetyl ester (C). It is also hydrolyzed in aqueous solvents.

563

Chemistry and Biosynthesis of Isoflavones

retained (Coward et al., 1998). There is some loss of the more hydrophilic isoflavone forms, daidzein > glycitein > genistein and of the conjugates, malonyl esters of βglycosides > β-glycosides > acetyl esters of β-glycosides > aglycons (Ji Guo-Ping and S. Barnes, unpublished observations). Again, the drying process can lead to more decarboxylation of residual malonyl esters of β-glycosides. Soy protein isolates have the highest protein content (>91% by weight) of soy food products. They have a lower concentration of the isoflavones than soy flour and waterextracted soy protein concentrate (Table 1). However, high isoflavone-containing soy protein isolates have been prepared for investigational research. The first step in their preparation, treatment with mild alkali, would be expected to dissociate the isoflavones from the now soluble proteins. Isoflavones undergo an interesting reaction with alkalis wherein the heterocyclic ring is opened and then closed to form a five-member ring system (A. Franke and S. Barnes, unpublished observations). The precipitation of the solubilized proteins with a weak acid leads to a further loss of isoflavones, as described for water washing of soy flour. This may account for the larger proportion of isoflavone aglycons in the soy protein isolates (Barnes et al., 1994b).

Uptake and Metabolism of Isoflavones Unconjugated isoflavones are readily absorbed passively by the cells of the intestine; in this respect, they behave like many other organic compounds used for therapeutic treatment of disease and minor ailments. The more common forms in soy, the O-glycosides, have poor passive absorption because of their lower hydrophobicity than the aglycons. Isoflavone β-glucosides may also undergo intestinal transport via the sodium-dependent glucose transporter (SGLT). Phlorizin, a β-glucoside of the 5-hydroxyflavonoid, has long been used as an inhibitor of glucose transport (Dimitrakoudis et al., 1992). However, no O-glucosides of isoflavones are found in the blood (Barnes et al., 2001). In contrast, when puerarin, the 8-C-glucoside of daidzein, is administered to rats by gavage, it is the only isoflavone that is detected in the blood and urine for the first 24 hours (Prasain et al., 2004). This suggests that puerarin is transported by SGLT. The lack of isoflavone O-glucosides in blood is due to an enzyme, lactose phlorizin hydrolase (LPH), embedded in the apical membrane of the small intestinal mucosal cells that carries out hydrolysis of the β-glycoside conjugates of isoflavones and thereby allows the isoflavone aglycons so formed to be readily absorbed (Day et al., 2000). In the intestinal cells, the isoflavone aglycons are largely converted to β-glucuronide conjugates by uridine diphosphate glucuronosyl transferase (UDPGT) (Sfakianos et al., 1997). This enzyme activity also is present in the liver so that much of the circulating forms of isoflavones in the blood are β-

glucuronide conjugates. Only at high-dose levels are unconjugated isoflavones a significant part of the circulating forms of isoflavones (Sfakianos et al., 1997). A second conjugating reaction, O-sulfation, is catalyzed by phenol sulfotransferases. The isoflavone sulfates are minor metabolites in female rats but are more common in male rats (Bayer et al., 2001) and in humans (Shelnutt et al., 2002) and by some human breast cancer cell lines (Peterson et al., 1996, 1998).

Enterohepatic Recycling of Isoflavones If 4-14C–labeled genistein is introduced into the duodenum of female rats with an indwelling cannula in the common bile duct, ∼80% of the dose appears in the bile within 3 hours (Sfakianos et al., 1997). If it is introduced into the portal vein, then ∼100% of the dose is transported into the collected bile. Furthermore, if the radioactive form in the bile (shown to be the 7-O-β-glucuronide) is reinfused into the small intestine, radioactivity quickly reappears in the bile (Sfakianos et al., 1997). If genistein 7-O-β-glucuronide is introduced into the duodenum, 25% of the administered dose is recovered in the bile in a 4-hour period; however, if reinfusion occurs in the mid-jejunum, then the recovered dose in the bile rises to 70–80% (Sfakianos et al., 1997). These observations are consistent with genistein undergoing a substantial enterohepatic circulation (Figure 5), with formation bile

Liver

Genistein GlcA Genistein Genistein GlcA

Duodenum - jejunum - ileum

Colon Gen iste in G lcA

Other metabolites

Gen iste in

FIGURE 5 Cartoon of the enterohepatic circulation and pathways of genistein transport and metabolism. Genistein is readily absorbed from the upper small intestine. Its β-glucoside, genistein, undergoes hydrolysis either due to luminal bacterial β-glucosidases or small intestine apical membrane lactase phlorizin hydrolase. Genistein is absorbed into the enterocytes and there most is converted to its β-glucuronide. Although much of this metabolite enters the bloodstream, some of it refluxes back into the intestinal lumen. Genistein and its β-glucuronide that enter the blood circulation are mostly taken up by the liver and excreted into the bile and thereby reenter the small intestine, completing an enterohepatic circuit. Genistein β-glucuronide undergoes hydrolysis by β-glucuronidases in the mid small intestine, releasing genistein that is taken up into the intestine once again. Some of the genistein β-glucuronide enters the large intestine where hydrolysis is completed and genistein is also reabsorbed. However, in this anaerobic environment degradation of genistein to other metabolites can occur.

564

Chapter 32/Soy Isoflavones

of genistein from its β-glucuronide by the action of intestinal β-glucuronidases. Indeed, we have observed genistein βglucuronide in the bile of rats 1 week after being taken off a diet containing soy and its isoflavones (J. Sfakianos and S. Barnes, unpublished observations).

Bacterial Metabolism of Isoflavones Isoflavones undergo additional metabolism when they come into contact with intestinal bacteria in the colon. Several metabolites of daidzein (dihydrodaidzein, Odesmethylangolensin and equol) (Figure 6) are commonly observed in blood and urine from most species, including humans. These, as for the primary isoflavones (daidzein, glycitein, and genistein), undergo β-glucuronidation and sulfation. Very few intact isoflavones are found in feces; the major route of excretion is via the kidney. Up to 60% of orally administered doses of both daidzein and glycitein appears in the urine (Kelly et al., 1995). In contrast, only 15–20% of genistein is excreted in urine. This led some to state that daidzein was more bioavailable than genistein (Xu et al., 1994); however, this statement is misleading because both isoflavones are well absorbed. Instead, daidzein has a higher renal clearance than genistein and therefore actually spends less time in the body. Metabolites of genistein have been less commonly found. These include dihydrogenistein, 6′-hydroxy-O-desmethylangolensin, 2-(4-hydroxyphenyl)propionic acid, and 4-ethyl phenol (Setchell, 1998; Coldham et al., 1999) (Figure 7). The latter accumulates in the prostate of male rats, whereas genistein does not. Interestingly, in elderly Seventh Day Adventists on soy-containing diets,

daidzein and its metabolites, but not genistein, accumulate in prostatic fluid to levels up to 50 times their blood concentrations (Hedlund et al., 2005). Equol, 7,4′-dihydroxyisoflavan, is a major daidzein metabolite in almost all species except in humans. Only onethird of humans have a blood concentration of equol that exceeds 10 nM (the amounts less than this may come from dietary sources such as cow’s milk) (Urban et al., 2001). In rats fed soy, the blood equol levels are five to seven times higher than daidzein or genistein (Kim et al., 2004) and are in the 1–3 µM range. Pharmacokinetic analysis suggests that the accumulation of equol in the blood is due not only to its rate of formation from daidzein, but also to it having a lower renal clearance than the other isoflavones (Kim et al., 2004). This marked difference in isoflavone metabolism between experimental animals (rats, mice, monkeys) and humans is a concern because administration of a soy product produces a very different array of isoflavones and their metabolites to tissues of each species. Even in the one third of humans who can consistently generate equol, the blood equol concentra-

OH

O

HO OH

A OH

O

O

HO

OH

OH O

B

O

OH

B HO

A HO

O

OH

O

HO

OH

O

OH

O

OH

C

O

C HO

OH

O

E

O

OH

OH

OH OH

HO

D

D

FIGURE 6 Major bacterial metabolites of daidzein. Daidzein is either (A) mostly reduced to dihydrodaidzein or (B) undergoes ring opening to form (C) O-desmethylangolensin. In most animals, but only in one third of human subjects, daidzein undergoes a more complex reduction to form the isoflavone (D) equol.

HO

OH

OH

F COOH

FIGURE 7 Metabolism of genistein. Genistein (A) undergoes reduction to form dihydrogenistein (B) or ring opening to form 6′-hydroxy-Odesmethylangolensin (C). Although the pathways are not well defined, genistein is also converted to 2,4,6-trihydroxybenzoic acid (D), pethylphenol (E), and 2-(4-hydroxyphenyl)-propionic acid (F).

Preclinical Experiments Exploring the Soy–Cancer Prevention Hypothesis

tions are substantially lower than in animals (Urban et al., 2001).

Isoflavones and Cytochrome P450 Another relatively unexplored area of metabolism is the hydroxylation of isoflavones by the cytochrome P450 (CYP) system. In experiments in vitro with isolated rat and human microsomes (Kulling et al., 2000; Kulling and Lehmann, 2002), hydroxylation can occur slowly in the 6, 8, and 3′ positions. 6- and 8-hydroxydaidzein and genistein have been detected in miso prepared by fermentation (Esaki et al., 1999). These isoflavones are much stronger antioxidants than their parent isoflavones because they are catechols. This property may have prevented their detection in clinical and preclinical animal experiments, unless the blood and urine specimens are treated with an excess of antioxidants (e.g., vitamin C or a metal chelator) before storage, the 6- and 8hydroxyisoflavones may undergo degradation. We have observed that both 6- and 8-hydroxydaidzein when dissolved in 80% aqueous methanol are unstable and completely disappear when stored for 1 month at 4°C (K. Jones, D.R. Moore, II, and S. Barnes, unpublished observations).

Metabolism of Isoflavones in Inflammatory Cells The inflammatory response consists of the formation of powerful oxidant species such as superoxide anion, O2−, hydrogen peroxide (H2O2), hydroxyl radical (OH), hypochlorous acid (HOCl), and peroxynitrite (ONO2−), as well as cytokines and hydrolytic enzymes (Schwartsburd, 2004). HOCl and ONO2− react with tyrosine residues in proteins to form 3-chloro- and 3-nitrotyrosine (Ischiropoulos et al., 1992; Hazen and Heinecke, 1997). Analogous reactions occur for isoflavones. Genistein and daidzein are nitrated in the B-ring to form 3′-nitro derivatives. Chlorination occurs in both the A- and B-rings to form 6-, 8- and 3′-mono and dichloro derivatives (Boersma et al., 1999). The chloro isoflavones are formed both by differentiated HL-60 cells (Boersma et al., 2003) and by freshly isolated human polymorphonuclear cells (D’Alessandro et al., 2003) following activation with a phorbol ester. The further metabolism of these compounds is not yet defined, but it does not include reaction with glutathione (D’Alessandro et al., 2005).

565

diseases such as atherosclerosis and cancer. It became clear from animal studies that the diet was a variable in models of atherosclerosis and that plant proteins were superior to animal proteins in this context (Hamilton and Carroll, 1976). As a result, a soy protein fraction was used to treat patients with hypercholesterolemia (Sirtori et al., 1977). Over the next 20 years various reports appeared in the literature and led to an important meta-analysis of the effects of soy protein on serum lipids (Anderson et al., 1995). This in turn enabled the establishment in 1999 of a health claim for the use of soy protein to prevent cardiovascular disease (http://vm.cfsan.fda.gov/~lrd/fr991026.html). Suggestions that soy might also have an important role in lowering the risk of cancer came from several angles. First, Doll and Peto (1981) observed that the worldwide variation in the incidence and death from cancer could not be explained solely by ethnic or genetic origins. They concluded that diet was a major factor (35–70% of the risk). Initially, the much higher fat content of the U.S. and Western European diets was seen as the driving force for the higher risk of breast and prostate cancer in these countries as opposed to Asia. Indeed, international comparisons of fat intake and breast cancer risk were supportive of this hypothesis. However, in Americans there was no correlation between fat content in the diet and breast cancer risk (Willett et al., 1987). A plausible alternative explanation was that the Asian diet contained cancer-preventing substances (Barnes et al., 1990). A hint came from an unintended change in 1986 in the design of chemoprevention experiments in rodent models of breast cancer. The National Cancer Institute decided to switch from a laboratory chow diet to a semidefined AIN76A diet. It was intended that the change would lead to a more reproducible model to test chemopreventive agents. A chow diet is an unregulated mixture of soymeal, wheat, and fishmeal, the composition of which was dependent on market prices of its components, whereas the AIN76A diet contained casein as the only protein. Surprisingly, the number of mammary tumors induced by the carcinogen Nmethyl-N-nitrosourea (NMU) on the AIN76A diet rose sharply, resulting in both a higher incidence and a substantial loss of animals during the observation period to their tumor burden (Grubbs et al., 1985). In 1980, Troll et al. had shown that adding soybeans in the diet of rats reduced the number of mammary tumors in an X-ray model of mammary carcinogenesis. However, it was not clear which soybean component was responsible.

SOY AND HUMAN HEALTH As noted earlier, the medical value of the soybean was first appreciated several millennia ago. The modern discovery of the effects of soybeans on human health occurred as medical research in the 1960s and 1970s looked beyond surgical and pharmacological treatments of the major chronic

PRECLINICAL EXPERIMENTS EXPLORING THE SOY–CANCER PREVENTION HYPOTHESIS The introduction of experimental designs where the dietary intake was carefully controlled for caloric and


protein content has demonstrated that diets containing soy prevent experimentally induced cancer in most cases. Barnes et al. (1990, 1994a) and Hawrylewicz et al. (1995) showed that various soy fractions lowered the number of tumors in carcinogen-induced mammary carcinogenesis. The rationale for soy’s effect was proposed at that time as being due to its isoflavone content, although there are other theories. One of these, a small peptide, the Bowman-Birk inhibitor, was studied in detail by Kennedy (1998). An analogous peptide, lunasin, has been identified by a group of investigators at UC-Davis (Galvez et al., 2001). Phytosterols (Awad et al., 2000; Ju et al., 2004) and inositol phosphates (Shamsuddin et al., 1997) have also had their advocates.

8 7

Mammary tumors/rat

566

6 5 4 3 2 1 0 20

60

80

100

Days post MNU

GENISTEIN AND BREAST CANCER MODELS A test of the role of a dietary substance in disease prevention is to examine it in isolation (i.e., to add it to a standard semisynthetic diet and to ensure that the animal’s diet consumption, rate of growth, and final adult weight are unaffected). This is a pharmacological approach to a dietary problem and not necessarily an ideal model to test the effect of a dietary constituent (Barnes and Prasain, 2005). It was fortunate that the initial rodent experiments on the soy isoflavone genistein were carried out in Coral Lamartiniere’s laboratory. Dr. Lamartiniere is a toxicologist with an interest in the effects of synthetic estrogen exposure during the perinatal and prepubertal periods of development. Although at that time genistein was a very expensive phytochemical, nonetheless, he demonstrated that rat pups treated i.p. with genistein (in DMSO as the solvent) on postnatal days 2, 4, and 6 had 50% less mammary tumors induced by the carcinogen DMBA than pups treated with DMSO alone (Lamartiniere et al., 1995a). A study where genistein was administered on postnatal days 14, 16, and 18 produced similar data (Lamartiniere et al., 1995b). Because genistein was administered i.p. in these experiments, the question remained as to whether this was relevant to dietary exposure. Subsequently, Fritz et al. (1998) discovered that delivering genistein to the dam’s diet (and hence to the pups via her milk) also reduced the number of mammary tumors in a dose-dependent manner. However, a study from our laboratory sowed confusion by showing that administering genistein to adult animals in their diet was not chemopreventive (Kim et al., 2004). Lamartiniere et al. (2002) reported a similar negative finding. However, they went on to show that when prepubertal delivery of genistein (as described earlier) is combined with adding it to the adult diet, there is a significant increase in the level of inhibition of mammary tumors (Table 2). Ironically, in the late 1990s, the National Cancer Institute switched back from the AIN76A diet to a laboratory chow diet. When genistein was administered to adult female Sprague–Dawley rats in the laboratory chow

40

FIGURE 8 Effect of dietary genistein on mammary rumors in adult female rats treated with the carcinogen, N-methyl-N-nitrosourea. The control animals (open triangles) were fed a laboratory chow diet. Genistein was added to this diet at two doses (200 mg/kg diet, closed squares; 2000 mg/kg diet, open diamonds).

TABLE 2 The Effect of Dietary Genistein on the Average Tumor Number per Rat in a Carcinogen-Induceda Model of Breast Cancer Experimental group Control Prepubertal Adult Prepubertal + adult

Exposure to genistein None 1–21 days After tumors appear (100 days) From birth to end of study

Mean no. of mammary tumors per rat 8.9 4.3 8.2 2.8

Note: The control AIN76A diet contained no phytoestrogen. The other AIN76A diets contained 250 mg of genistein per kilogram of body weight. a All rats were treated with 80 mg of dimethylbenz[a]anthracene per kilogram of body weight at day 50 postpartum. Source: The data were adapted from Lamartiniere et al. (2002).

background, it had a strong chemopreventive effect (Figure 8). These data strongly suggest that early life exposure to genistein sets the stage for a beneficial sensitivity to genistein in adult life. Hilakivi et al. (1999) showed that exposure to genistein prenatally by its administration to the dams increased the number of mammary tumors induced by DMBA in rats. However, genistein was administered i.v. and thereby bypassed the enzymes responsible for its metabolism (see previous sections on metabolism). In a subsequent experiment when genistein was administered neonatally, Hilakivi et al. (2001) found that it caused a decrease in the number

567

Soy, Isoflavones, and Preexisting Breast Cancer

of mammary tumors, confirming the previous results of Lamartiniere et al. (1995a).

TIMING OF EXPOSURE TO SOY IN HUMANS Analogous results to the preclinical experiments noted earlier have been observed in epidemiological studies. In a study on breast cancer in Shanghai, China, there was a dosedependent–negative association between eating soy in adolescence and breast cancer (Shu et al., 2001) (Table 3). However, there was no correlation with other fresh legumes in the diet. In a second study in Asian-Americans in California, it was found that women who ate tofu more than once a week in adolescence, but little in adult life, were less likely to have breast cancer (adjusted odds ratio [OR] = 0.77) than those who ate tofu less than once a month at any stage during life. Those who ate tofu more than once a week only in adult life (adjusted OR = 0.93) were at a similar risk to low tofu eaters (Wu et al., 2002). In contrast, those who ate tofu at least once a week throughout life had the lowest risk of breast cancer (adjusted OR = 0.53, p trend = 0.001). A report on identical twins found that they do not start menstruation (a measure of estrogen sensitivity) at the same time; typically in 50% of the twins, the difference was at least 1 year (Hamilton and Mack, 2003). Importantly, the twin who had two of the first signs of puberty was 5.4 times more likely to have breast cancer than their sibling.

SOY, ISOFLAVONES, AND PREEXISTING BREAST CANCER There are two quite different views regarding the roles of soy and isoflavones on existing mammary tumors. Hawrylewicz et al. (1995) showed that when female rats treated with DMBA on an AIN76A diet were subjected to TABLE 3 Group interviewed Premenopausalb Postmenopausalc Mothersd

resection of the first mammary tumor and then randomly placed on AIN76A diet or AIN76A diet where the casein was replaced by soy protein isolate (containing isoflavones), the soy-free animals had statistically more mammary tumors than those on a soy diet (Figure 9). These data suggest a chemopreventive effect of soy protein in this model. In contrast, a number of authors (Hsieh et al., 1998; Allred et al., 2001a,b; Ju et al., 2001, 2002) have used an athymic nude mouse model to implant human breast cancer cells into this animal to create a model of recurrent mammary cancer. For this model to work, the mice must be ovariectomized. This maneuver removes the physiological source of estrogens. Under these conditions human MCF-7 cells do not grow. However, adding soy or soy isoflavones to the diet leads to slow but steady growth of the cancer cells (Figure 10). One interpretation of these experimental results is that genistein or soy containing genistein will promote the growth of cancers and therefore should be avoided in women with preexisting breast cancer or at high risk of breast cancer. However, it should be noted that in this animal model, the thymus and hence the source of T cells have been removed. This is the way that the human breast cancer cells can be implanted without rejection by the mouse immune system. Detection of foreign antigens is carried out by the innate immune system (Figure 11). Natural killer (NK) cells respond to foreignness by producing interferon-γ (IFN-γ). This in turn causes recruitment of T cells producing antibodies to nonself antigens and clonal expansion of the T cells. It is this aspect of the immune system that is absent in the nude mouse. It is, therefore, of some interest that a theory of immune tolerance and onset of cancer is emerging (Cheng et al., 2004). There are mouse strains where the onset of breast cancer is age dependent. In a search for a rationale for this, it was noted that the breast cancer cells when grown in culture produce an extracellular fraction that when incubated with natural killer cells or T cells causes an inhibition of

Epidemiological Data Linking Adolescent Soy Intake and Breast Cancer Risk Food

Lowest quintilea

Third quintilea

Fifth quintilea

Trend, p

Soy Fresh legumes Soy Fresh legumes Soy Fresh legumes

1.00 1.00 1.00 1.00 1.00 1.00

0.72 (0.54–96) 1.04 (0.82–1.33) 0.65 (0.44–0.97) 0.92 (0.65–1.30 0.59 (0.32–1.07) 0.88 (0.57–1.38)

0.53 (0.39–0.72) 0.96 (0.72–1.29) 0.49 (0.33–0.74) 0.96 (0.67–1.39) 0.35 (0.21–0.60) 0.67 (0.40–1.12)

0.0001 0.51 0.006 0.82 100 nM

>100 nM >1 µM >1 µM >10 µM

>10 µM

Mechanism Estrogen receptor β expression Estrogen receptor α proliferation Restores interferon-γ production by T cells Blocks production of tumor exosomes Inhibition of TNFα/NFκB activation Inhibition of 17β-hydroxysteroid dehydrogenase Inhibition of steroid and phenol sulfotransferases Alters transforming growth factor β signaling Free radical scavenging Protein tyrosine kinase inhibition DNA topoisomerase II Inhibition of metalloproteinase-9 Cell cycle progression

Reference(s) Kuiper et al., 1998 Wang and Kurzer, 1997; Zava and Duwe, 1998 Barnes et al., 2005 Barnes et al., 2005 Davis et al., 1999 Makela et al., 1995

Kirk et al., 2001 Kim et al., 1998 Wei et al., 1993 Akiyama et al., 1987 Markovits et al., 1989 Shao et al., 1998 Pagliacci et al., 1994

IFN-γ in the immune system (Barnes et al., 2005; Liu et al., 2005), and production of exosomes by tumor cells (Barnes et al., 2005), to effects at the low micromolar level via inhibition of activation of tumor necrosis factor-α (Davis et al., 2001), antioxidant events (Surh, 2003), and tyrosine kinase inhibition (Akiyama and Ogawara, 1991). They are molecules with complex pharmacological effects, and the investigator must be very careful in developing suitable experimental designs that model aspects of human disease. With this in mind, the role of isoflavones, indeed of any preventive or therapeutic agent, on the growth of tumor cells in culture must be viewed very cautiously so as to not draw grandiose conclusions concerning human health and disease from a grossly simplified system.

Acknowledgments Federal support for research on isoflavones has been in the form of grants-in-aid from the National Center for Complementary and Alternative Medicine for the Purdue University-University of Alabama at Birmingham Botanicals Center for Age-related Disease (P50 AT00477; C. Weaver, PI) and from the National Cancer Institute to the University of Alabama at Birmingham Center for Nutrient–Gene Interaction (U54 CA100949; S. Barnes, PI). Funds for the mass spectrometers used in research at UAB were provided by a Shared Instrumentation Award from the National Center for Research Resources (S10 RR06487).

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References Makela, S., Poutanen, M., Lehtimaki, J., Kostian, M.L., Santti, R., and Vihko, R. 1995. Estrogen-specific 17 beta-hydroxysteroid oxidoreductase type 1 (E.C. 1.1.1.62) as a possible target for the action of phytoestrogens. Proc Soc Exp Biol Med 208: 51–59. Markovits, J., Linassier, C., Fosse, P., Couprie, J., Pierre, J., JacqueminSablon, A., Saucier, J.M., Le Pecq, J.B., and Larsen, A.K. 1989. Inhibitory effects of the tyrosine kinase inhibitor genistein on mammalian DNA topoisomerase II. Cancer Res 49: 5111–5117. Pagliacci, M.C., Smacchia, M., Migliorati, G., Grignani, F., Riccardi, C., and Nicoletti, I. 1994. Growth-inhibitory effects of the natural phytooestrogen genistein in MCF-7 human breast cancer cells. Eur J Cancer 30A: 1675–1682. Peterson, T.G., Coward, L., Kirk, M., Falany, C.N., and Barnes, S. 1996. Isoflavones and breast epithelial cell growth: the importance of genistein and biochanin A metabolism in the breast. Carcinogenesis 17: 1861–1869. Peterson, T.G., Ji, G-P., Kirk, M., Coward, L., Falany, C.N., and Barnes, S. 1998. Metabolism of the isoflavones genistein and biochanin A in human breast cancer cell lines. Am J Clin Nutr 68: 1505–1511. Prasain, J.K., Jones, K., Kirk, M., Wilson, L., Smith-Johnson, M., Weaver, C.M., and Barnes, S. 2003. Identification and quantitation of isoflavonoids in Kudzu dietary supplements by HPLC and electrospray ionization tandem mass spectrometry. J Agric Food Chem 51: 4213–4218. Prasain, J.K., Jones, K., Brissie, N., Moore, D.R. II, Wyss, J.M., and Barnes, S. 2004. Identification of puerarin and its metabolites in rats by liquid chromatography-tandem mass spectrometry. J Agric Food Chem 52: 3708–3712. Schultz, G. 1965. Isoflavone glucoside formononetin-7-glucoside and biochanin A-7-glucoside in Trifolium pratense L. Naturwissenschaften 52: 517. Schwartsburd, P.M. 2004. Age-promoted creation of a pro-cancer microenvironment by inflammation: pathogenesis of dyscoordinated feedback control. Mech Age Develop 125: 581–590. Sfakianos, J., Coward, L, Kirk, M., and Barnes, S. 1997. Intestinal uptake and biliary excretion of the isoflavone genistein in the rat. J Nutr 127: 1260–1268. Shamsuddin, A.M., Vucenik, I., and Cole, K.E. 1997. IP6: a novel anticancer agent. Life Sci 61: 343–354. Shao, Z.M., Wu, J., Shen, Z.Z., and Barsky, S.H. 1998. Genistein exerts multiple suppressive effects on human breast carcinoma cells. Cancer Res 58: 4851–4857. Shao, Z.M., Shen, Z.Z., Fontana, J.A., and Barsky, S.H. 2000. Genistein’s “ER-dependent and independent” actions are mediated through ER

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33 Selenium and Cancer Prevention CLEMENT IP, JAMES MARSHALL, YOUNG-MEE PARK, HAITAO ZHANG, YAN DONG, YUE WU, AND ALLEN C. GAO

INTRODUCTION

many cellular processes are sensitive to the integration of redox-sensitive signals. Each reactive thiol-containing protein may act as a unique cellular sensor to monitor the redox status of the microenvironment. Based on this paradigm, selenium may activate or inactivate a circuitry of switches in controlling the fate of cell proliferation, differentiation, or apoptosis. In summary, genomics and proteomics technologies have provided evidence that selenium affects not just a few key targets, but a multitude of targets. A challenge is to validate these discoveries in innovative human intervention trials. This chapter reviews the evidence supporting an association of selenium with common forms of cancer. In some detail, the evidence for the SELECT trial of selenium and vitamin E in prostate cancer is also considered as an illustration of the types of controlled trials necessary to establish the effectiveness of selenium in cancer prevention.

Research over the past 35 years has demonstrated a role for selenium (Se) in cancer prevention. Low blood Se levels were found to be associated with an increased incidence and mortality from various types of cancers. The role of selenium in the prevention of a number of degenerative conditions including cancer, inflammatory diseases, cardiovascular disease, neurological diseases, aging, infertility, and infections has been established by laboratory experiments, clinical trials, and epidemiological data. Most of the effects in these conditions are related to the function of selenium in antioxidant enzyme systems. Replenishing selenium in deficiency conditions appears to have immune-stimulating effects, particularly in patients undergoing chemotherapy. However, increasing the levels of selenoprotein antioxidant enzymes (glutathione peroxidase, thioredoxin reductase, etc.) appears to be only one of many ways in which selenium-based metabolites contribute to normal cellular growth and function. Animal data, epidemiological data, and intervention trials have shown a clear role for selenium compounds in both prevention of specific cancers and antitumorigenic effects in post-initiation phases of cancer. Redox regulation of protein thiols is a fundamental aspect of protein chemistry that is known to have a significant impact on cell biology. Because of its strong nucleophilic nature, methylselenol (an active selenium metabolite) can react avidly with protein thiols and result in either a gain or a loss of these thiol groups. Using a display thiol-proteomics approach, studies have demonstrated that methylselenol is able to cause global thiol modification of proteins. These changes are manifested in distinct quantifiable patterns as a function of time. There is increasing recognition that


BREAST CANCER Selenium is effective in the reduction of cancer incidence when provided to animals at nontoxic doses only 5- to 10-fold above the nutritional requirement (Ip, 1986; El-Bayoumy, 1991). Numerous studies have indicated the efficacy of selenium in the significant reduction of mammary tumor incidence after exposure to carcinogens, including 2acetylaminofluorene, methylnitrosourea, and 7,12-dimethylbenz(a)anthracene, and selenium has also been shown to be effective against the development of spontaneous mammary tumors in the C3H mouse model (El-Bayoumy, 1991). The relationship between selenium status and the incidence of breast cancer remains somewhat controversial, with one

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study indicating a protective role (McConnell et al., 1980) and other studies not (Hunter et al., 1990; Garland et al., 1993, 1995; Ghadirian et al., 2000).

COLON CANCER Progress in chemoprevention research has brought about innovative approaches to the prevention and control of colon cancer (Reddy, 1986; Wattenberg, 1992; Giovannucci and Willett, 1994; Kelloff, 2000). Epidemiological studies have pointed to an inverse association between dietary intake of selenium and colorectal cancer risk in humans (Clark, 1986; El-Bayoumy, 1991). A clinical trial by Clark et al. (1996) demonstrated that administration of selenium-enriched yeast significantly inhibited colon cancer incidence in humans. A series of experiments in laboratory animals also showed that supplementation of sodium selenite inhibits carcinogenesis in the colon (El Bayoumy, 1995). Generally, humans ingest organic forms of selenium, such as SM and SC; however, cancer prevention studies in preclinical models have not revealed any significant differences between the inorganic forms of selenium and those naturally occurring forms of selenium (Nayini et al., 1991; El Bayoumy et al., 1995). A preclinical study indicated that dietary SM, one of the major forms of selenium in the selenium-enriched yeast, lacked chemopreventive efficacy against AOM-induced colon carcinogenesis in F344 rats (Nayini et al., 1991). There are also studies to indicate that chronic feeding of inorganic and certain organic forms of selenium at >5 ppm induces toxic effects (Wilber, 1980; Fan and Kizer, 1990). Therefore, substantial efforts have been made to develop organoselenium compounds with maximal chemopreventive efficacy but low toxicity. Organoselenium compounds hold great promise as chemopreventive agents (Fan and Kizer, 1990; Nayini, 1991) against cancers of the colon, at least, in preclinical models (Reddy et al., 1992, 1997; Rao et al., 2000).

SELENIUM AND PROSTATE CANCER Selenium supplementation showed great promise in lowering prostate cancer risk in a previous controlled intervention trial. The complete 13 years’ worth of results of the Nutritional Prevention of Cancer Trial (Clark et al., 1996; Duffield-Lillico et al., 2003) have been analyzed, confirming that Se supplementation was associated with marked reductions in risks to total (all-site except skin) carcinomas and to cancers of the prostate and colon-rectum. Of the treatment effects, the most robust was for prostate cancer, which was more frequent and was confirmed by serum prostatespecific antigen (PSA) level. Therefore, the role of selenium supplementation is considered in some detail in the following with regard to prostate cancer.

Androgen signaling plays an important role in the development of prostate cancer. Laboratory findings indicate that exposure of androgen-responsive human prostate cancer cells to an active monomethylated selenium metabolite decreases the expression of androgen receptor (AR) and its trans-activating activity. PSA is a gene known to be positively regulated by AR and is a well-accepted biomarker for the diagnosis and prognosis of prostate cancer. As expected, selenium also depresses PSA transcript and protein in a dose response and time schedule similar to that for AR. In addition to PSA, microarray data mining analysis suggests that the expression of many androgen-responsive genes can also be reversed by selenium. This information supports a mechanism-driven role of selenium in prostate cancer chemoprevention.

The Nutritional Prevention of Cancer Trial Results A number of case–control studies have demonstrated an inverse relationship between selenium status and prostate cancer risk (Yoshizawa et al., 1998; Helzlsouer et al., 2000; Nomura et al., 2000; Brooks et al., 2001; Li et al., 2004). The litmus test of the efficacy of a chemopreventive agent is a randomized intervention design involving an appropriate cohort of subjects. One of the more important studies of selenium as a chemopreventive agent is the Nutritional Prevention of Cancer (NPC) study initiated by Larry Clark. Clark et al. (1996) and Duffield-Lillico et al. (2003) reported that supplementation of people with selenized yeast is capable of reducing the overall cancer morbidity by nearly 50%. The study was a randomized, double-blind, placebocontrolled trial involving 1312 patients (mostly men) who were recruited initially because of a history of basal cell or squamous cell carcinoma of the skin. Individuals in the treatment arm were given Se at 200 µg/day (average daily intake in the United States is ∼100 µg) for a mean of 4.5 years. After a total follow-up of 8271 person-years, selenium treatment did not decrease the incidence of these nonmelanoma skin cancers. However, patients receiving the supplement showed a much lower prevalence of developing lung (relative risk [RR] = 0.54), colon (RR = 0.42), or prostate cancer (RR = 0.37). Larry Clark passed away in 2000; his colleague James Marshall (then at the Arizona Cancer Center but since relocated to Roswell Park) assumed responsibility for completing the trial. The original report by Clark et al. (1996) was based on an interim analysis of the data collected from 1983 to 1993, even though the follow-up of all participants actually continued until 1996. There were justifiable reasons behind this rather unusual decision. Although the trial was still blinded, a decrease in overall cancer morbidity and mortality in the selenium-supplemented cohort became apparent. In 1994, the Safety Monitoring and Advisory

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Committee recommended that the trial be unblinded and the results published. After the completion of an audit of the study by the National Cancer Institute (NCI) in 1995, the blinded phase of the trial was terminated early the following year. At this time, all participants were informed of their treatment status, given the opportunity to take selenium supplement, and reconsented to enroll in the open-label phase of the trial. Extending the analysis for an additional 3 years to the end of the blinded phase in 1996, an update by Duffield-Lillico et al. (2002) reaffirmed the significant reduction in prostate cancer incidence by selenium (RR = 0.48, 95% confidence interval [CI] = 0.28–0.80). Because of the randomized and double-blinded nature of the trial design, the NPC results command a high degree of credibility. The one caveat about the NPC trial is that the observed preventive effects of selenium are secondary endpoints that were not hypothesized at the start of the study. They were instead proposed after the investigators had noticed a trend for differential cancer incidence and mortality between the placebo and treatment groups.

The SELECT Trial The fact that the hypothesis for selenium protection of prostate cancer in the NPC was not stated a priori raised suspicion that the findings might represent a statistical artifact. Nonetheless, the NPC prostate cancer data were instrumental in leading to the development of the current Selenium and Vitamin E Chemoprevention Trial (SELECT), which is aimed specifically to determine the efficacy of these two agents, either alone or in combination, in reducing prostate cancer incidence among average-risk men (Klein et al., 2001). In spite of the value of SELECT, it will not be possible to use this trial to gain insight into the mechanism of action of selenium in prostate cancer prevention. The selenium chemoprevention research team at Roswell Park is working on a translational model to address this gap of knowledge. An intriguing piece of information from the NPC prostate cancer results is that baseline plasma selenium is highly correlated with the impact of selenium treatment (DuffieldLillico et al., 2003). For those in the lowest and middle tertile of baseline selenium, treatment decreased the risk of prostate cancer by fivefold and threefold, respectively. However, for those in the highest tertile of baseline selenium, treatment failed to produce any significant protective effect. It is not yet understood whether this suggests that selenium is protective only within a narrow range, that differences in baseline selenium reflect variations in metabolic capacity, or that exposure to other environmental agents might modify the impact of selenium. This issue is revisited in the section “Future Directions.” An analysis of the blood PSA data also turned out to be quite illuminating. PSA is a well-accepted marker for the diagnosis and prognosis of prostate cancer. In brief, the

PSA trajectory over time in placebo- and selenium-treated patients who did not develop prostate cancer is not statistically different from each other. However, among those who developed prostate cancer, the PSA trajectory of the selenium-treated patients is significantly less steep than that of the placebo-treated patients. This divergence suggests that PSA might be a reliable biomarker to monitor the responsiveness to selenium. Statistical analysis of this result is under way. Laboratory studies to elucidate the mechanism underlying the effect of selenium on PSA production are discussed in the section “MSA Interference of Androgen Receptor Signaling in Human Prostate Cancer Cells.”

Methylseleninic Acid In a previous review, Ip et al. (2002) pointed out the need to use a direct- and fast-acting selenium compound to investigate the mechanism of action of selenium. Selenomethionine, the supplement used in SELECT and presumably the major form of selenium in selenized yeast, was studied extensively in the early 1990s. It was originally proposed (Ip and Ganther, 1990; Ip et al., 1991; Ip, 1998) that the metabolism of selenomethionine to methylselenol (CH3SeH) would be important for the expression of chemopreventive efficacy. In the whole animal, liver and kidney are the major organs for selenium metabolism; the conversion of selenomethionine to methylselenol requires five enzymatic steps (Ip, 1998) (Figure 1). Tissues such as breast and prostate have a low capacity to produce the monomethylated selenium metabolite from selenomethionine. For this reason, cultured breast or prostate cells generally are growth inhibited by selenomethionine only when it is present at levels of 100–400 µM in the medium. These concentrations of selenium are much higher than the plasma concentrations of 2– 4 µM attainable by supplementation. The dose of 200 µg/ day in the NPC study increased blood concentrations of selenium-treated subjects from a baseline of 1.4 µM to ∼2.5 µM (Clark et al., 1996). Methylselenol is highly reactive, difficult to prepare, and cannot be tested as is. To obviate this problem, Ip et al. (2000) developed a stable metabolite called methylseleninic acid (CH3SeO2H, abbreviated to MSA) for cell culture studies. Once taken up by cells, MSA is readily reduced to CH3SeH through nonenzymatic reactions involving glutathione or NADPH. Therefore, biochemically, MSA serves as a ready-made reagent to generate CH3SeH endogenously as soon as it enters into cells. We found that mouse and human cancer cells are sensitive to MSA at a concentration as low as 2.5 µM (Ip et al., 2000; Dong et al., 2002). Thus, both selenomethionine and MSA produce the same active metabolite. The notable difference is that whereas selenomethionine has to be added to the culture medium at exceedingly high levels, a physiological concentration of MSA is sufficient to elicit biological responses. As expected, MSA

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Transsulfuration pathway of selenomethionine metabolism demethylation

selenomethionine

+ serine

selenohomocysteine

selenocystathionine cystathionase

methylselenol

methyl transferase

hydrogen selenide

lyase

active Se metabolite

selenocysteine + α-ketobutyric acid

FIGURE 1 Selenium metabolic pathway.

has excellent anticancer activity in vivo; its efficacy is similar to that of selenoamino acids (Ip et al., 2000).

MSA Interference of Androgen Receptor Signaling in Human Prostate Cancer Cells In vitro studies with human prostate cancer cells showed that exposure to MSA results in cell cycle arrest and induction of apoptosis (Jiang et al., 2001, 2002; Wang et al., 2002; Dong et al., 2003, 2004; Zu and Ip, 2003). These events are accompanied by changes in the expression of many key cell cycle– and apoptosis-regulatory molecules, as well as by changes in the activities of a number of survival and death signaling pathways. The clonal expansion of prostate cancer at the early stage is mostly dependent on androgen stimulation. Androgen response is mediated by binding to the AR, which subsequently translocates into the nucleus and interacts with specific androgen-responsive elements (AREs) in the promoters of target genes. The interaction leads to the activation or repression of genes involved in the proliferation and differentiation of the prostate cells (Jenster, 1999). Almost all patients with advanced prostate cancer respond initially to treatments that interfere with the AR signaling process. However, these treatments fail after prolonged use and the growth of the cancer recurs (Koivisto et al., 1998). Recurrent prostate cancer is generally androgen independent, although the expression of AR is maintained regardless of the clinical stage of the disease (Sadi et al., 1991; Hobisch et al., 1996). The fact that PSA continues to be produced by the pathologically advanced cancer suggests that the AR signal transduction pathway is still intact. Several hypotheses have been proposed to explain this phenomenon. Mutations of the AR may enable the cells to be sensitized by very low levels of androgens, perhaps even by non-androgen steroids (Zhao et al., 2000). Alterna-

tively, the receptor may become promiscuous and can be activated by nonsteroidal growth factors and cytokines (Culig et al., 1998). Prostate cancer may also adapt to androgen deprivation by increasing the expression of AR through gene amplification (Visakorpi et al., 1995; Koivisto et al., 1997; Ford et al., 2003). Therefore, an intervention strategy aimed at dampening the intensity of AR signaling would be helpful not only for controlling prostate cancer in high-risk men but also for preventing relapses after endocrine therapy. We reported that MSA is able to markedly depress AR transcript and protein levels in a dose-dependent manner within hours in the androgen-responsive human LNCaP prostate cancer cells (Dong et al., 2004). PSA is a gene known to be positively regulated by AR. As expected, decreases in PSA transcript and protein follow a dose response and time schedule similar to that for AR upon exposure to MSA. The reduction of AR and PSA expression by MSA occurs well before any detectable change in cell number. With the use of a luciferase reporter construct linked to either the PSA promoter or the ARE, we found that MSA inhibits the transactivating activity of AR in cells transfected with the wild-type AR expression vector. MSA also suppresses the binding of AR to the ARE site, as evidenced by electrophoretic mobility shift assay of the AR–ARE complex. However, we cannot distinguish whether this is due to a block in nuclear translocation of the activated AR or to physical interference of AR association with the ARE through modulation of other co-regulators. These various possibilities will be investigated systematically. Incidentally, cellular PSA is inhibited to a greater magnitude by MSA compared with secretory PSA (Cho et al., 2004), suggesting that the turnover of PSA might also be affected by MSA. The half-life of PSA is about 3.6 hours in control untreated cells but is reduced to 1.5 hr in MSAtreated cells, thus confirming that PSA degradation is indeed

Redox Modification of Proteins by MSA

enhanced by MSA. The decrease in the half-life of PSA is reversed by lysosomal inhibitor, but not by proteasomal inhibitor. This finding is consistent with our preliminary data that MSA greatly enhances the transcript level of lysosomal cathepsin B and cathepsin L. There could be two mechanisms by which MSA decreases cellular PSA expression. The first is at the gene transcription level and the second at the level of protein degradation. For this reason, cellular, rather than secretory, PSA may provide a better handle to study mechanisms of selenium intervention. The NPC finding of a decreased PSA trajectory by selenium in prostate cancer patients might reflect an important biological response that is happening in the prostate tissue.

MICROARRAY METHODS SEARCHING FOR POTENTIAL Se TARGETS It has been widely recognized that microarray gene expression analysis offers great promise in unraveling the mechanisms of anticancer agents. Bioinformatics holds the key to unlocking the treasure of data from these array studies. We described a data mining approach to gain further insight into selenium biology utilizing published microarray datasets (Zhang et al., 2004). The paradigm combines laboratory- and bioinformatics-based research to identify molecular targets of prostate cancer intervention by selenium. Previously we had profiled gene expression changes in PC-3 and LNCaP prostate cancer cells treated with MSA, using either the Affymetrix oligonucleotide U95A chip or a custom cDNA array. Zhao et al. (2004) had also performed microarray analysis in MSA-treated LNCaP cells using a high-density cDNA array. Our goal was to use these three selenium datasets and develop a systematic data mining strategy to earmark putative prostate cancer genes that are sensitive to selenium intervention; these genes are derived from three newly published microarray datasets generated by using human surgical prostate tumor specimen. The first is an Affymetrix oligonucleotide array study in 50 normal and 52 prostate cancers reported by Singh et al. (2002). The second, described by Welsh et al. (2001), is similar to the first with the exception that a fewer number of samples were examined (9 normal and 25 prostate cancers). The third is an analysis of 41 normal and 62 prostate cancers by Lapointe et al. (2004), using a 26K-gene cDNA microarray. These three prostate cancer datasets proffer a fountain of information of dysregulated genes implicated in prostate carcinogenesis. First, we devised a method to merge and methodically analyze the three selenium microarray datasets according to standardized criteria. Second, we subjected the prostate cancer datasets to permutation t-test analysis to identify a list of genes consistently dysregulated in prostate carcinogenesis. Then we matched the first analysis with the second analysis to cull a subset of dysregulated prostate cancer

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genes of which expression could be reversed or restored to normal by selenium intervention. With the above procedure, we pared down to 42 genes, which are reciprocally regulated; many of them are involved in controlling cell cycle progression and/or cell death. Selenium modulates their expression in a way that is consistent with cell growth inhibition, cell cycle block, and apoptosis induction. Our bioinformatics exercise also revealed four genes with tumorsuppressing activities (SERPINA5, gelsolin, CYLD, and SSBP2), which can be upregulated by selenium. The finding that selenium decreases the expression and transactivation of AR fueled our interest in applying a similar microarray analysis approach to investigate whether the transcription of AR-regulated genes, in addition to PSA, might be counteracted by selenium. Recent events have made this query possible. In separate studies by DePrimo et al. (2002) and Nelson et al. (2002), LNCaP cells were treated with synthetic androgen and microarray analyses were then performed to identify genes responsive to androgen stimulation. These two androgen datasets are well suited to serve as a tool to mine the selenium datasets for fresh clues. Altogether, 92 genes are commonly modulated by both selenium and androgen, and slightly less than half of them (38 out of 92) are altered in opposite directions. A plausible explanation for this is that genes have multiple regulatory elements, both positive and negative, in their promoter regions. Selenium is known to alter the expression of many transcription factors, co-activators, and co-repressors (Dong et al., 2003). The ARE is but one of many regulatory elements controlling the transcription of androgen-responsive genes. Thus, it is not surprising that selenium could counteract the expression of some, but not all, androgen-regulated genes. Collectively, the above information has provided us with a trove of exciting clues to investigate the mechanism of selenium chemoprevention of prostate cancer. Moreover, these selenium target genes could also serve as biomarkers in clinical trials to gauge the efficacy of selenium intervention.

REDOX MODIFICATION OF PROTEINS BY MSA Redox regulation of protein cysteines is a fundamental aspect of protein chemistry that is known to have a significant impact on cell biology. As discussed earlier, the generation of a monomethylated selenium metabolite is critical for the anticancer activity of selenium. At physiological pH, methylselenol is present in an ionic form (i.e., CH3Se−). Because of its strong nucleophilic nature, methylselenol is expected to react avidly with protein thiols. However, because most protein thiols exhibit a pKa value of 8.0–8.5, few cellular protein thiols would react with micromolar concentrations of methylselenol at physiological pH, even under conditions of oxidative stress. But some protein cysteine

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residues exist as thiolate anion at neutral pH, because their pKa values are lowered as a result of the influence of neighboring nucleophilic groups. More often than not, the thiolate is stabilized by salt bridges to positively charged residues (Polgar and Halasz, 1978; Page and West, 1986). Those thiols with lower pKa values may thus exhibit enhanced reactivity because of the nature of the thiolate, and they are called “reactive thiols.” The interaction of proteins with a methyl selenium metabolite could potentially result in a gain or loss of reactive thiols, depending on the reduction potential. These modifications have never been examined systematically before because of the lack of a reliable methodology to quantify reactive protein thiols globally in cells. We completed a study to investigate the extent of protein thiol modification by MSA in human prostate cancer cells using a biotinylated iodoacetamide (BIAM)–based display proteomics approach (Park et al., 2004). The labeling of reactive thiols with BIAM was adapted from a previously published method (Kim et al., 2000) with minor modifications. PC-3 cells were treated with MSA for different times ranging from 0.5 to 24 hours (no cell growth inhibition was evident during this period), and the protein extract was labeled with BIAM. After two-dimensional (2D) gel electrophoresis, the samples were processed for the detection of reactive thiol-containing proteins by reaction with HRPconjugated streptavidin. The image data were analyzed by the Self Organizing Maps (SOMs) clustering algorithm. Out of 194 reactive thiol-containing protein spots on the 2D gel display, 100 of them (cluster 1) were not sensitive to MSA modulation. The remaining 94 were categorized into three distinct patterns of change. Cluster 2 showed an immediate but sustained loss of reactive thiols for at least 24 hours; cluster 3 showed a transient loss of reactive thiols followed by rapid rebound; and cluster 4 showed a transient gain followed by a rapid return to normal. Because of limitations in the methodology, the present approach is likely to underestimate the number of redox-sensitive proteins amenable to modification by selenium. Our attention is focused on the idea that global protein redox changes induced by selenium represent a form of cellular stress because these changes are likely to lead to protein misfolding, unfolding, or aggregation. The concept of quality control monitoring of newly synthesized proteins in the endoplasmic reticulum (ER) is well described (Kaufman, 2002; Rao et al., 2004). An accumulation of misfolded or unfolded proteins in the ER triggers a defined set of sensor and modulator signals to stop protein synthesis and to refold or degrade the aberrant proteins so that the cells may continue to survive. Our preliminary data showed that exposure of PC-3 cells to MSA induces a number of signature ER stress molecular markers, including phosphoPERK, phospho-eIF2α, and GRP-78, within a few hours. GRP-78 belongs to the family of chaperones; it is a key

protein charged with promoting folding and preventing aggregation of proteins in the ER (Kaufman, 2002). MSA also greatly increases GADD153 and caspase 12 activation. The latter two markers are usually associated with cell cycle exit and/or apoptosis when ER stress due to protein unfolding is too severe and beyond repair. These same cellular changes have been demonstrated by numerous studies to occur in cancer cells (but not necessarily normal untransformed cells) treated with selenium. Based on this information, we are proposing a novel hypothesis that typical ER stress signaling responses governing the balance between survival and death may also be initiated by selenium as a result of damage to mature and newly synthesized proteins. Because metabolic oxidative stress is greater in cancer cells than in normal cells, cancer cells may be more susceptible to redox stress than normal cells (Spitz et al., 2000). This might be one reason that normal untransformed cells are not nearly as sensitive as cancer cells to selenium inhibition of growth (Ip and Medina, 1987; Menter et al., 2000). It has been well documented that the redox status of proteins has a profound effect on their functions/activities. Two examples are cited to illustrate how selenium causes defined redox changes, which are accompanied by either a gain or a loss of function/activity of the protein. First, using a 20kDa carboxyl-terminal fragment of p53 with two cysteines at codons 275 and 277, Seo et al. (2002) reported that a reduction of these two thiols by selenium significantly increases sequence specific DNA binding and transactivation of p53. MSA and selenite are more potent than selenomethionine in inducing p53 redox changes (Smith et al., 2004). The finding is congruent with the interpretation that MSA and selenite are converted to the reactive monomethylated metabolite much more efficiently than selenomethionine. A second example is provided by the work of Gopalakrishna et al. (1997) regarding the redox modification of protein kinase C by selenite. There are two cysteine-rich regions in protein kinase C: The regulatory domain contains 12 cysteine residues, and the catalytic domain contains 6 or 7 cysteine residues. At low concentrations, selenite converts four cysteine residues to two disulfides; and at high concentrations, it converts eight cysteine residues to four disulfides. The former modification is associated with a loss of affinity to ATP, whereas the latter with a lower Vmax of the enzyme. Thus, in addition to causing ER stress, these studies raise the provocative suggestion that selenium might act as a chemical switch to turn a protein on or off without affecting the expression level of the protein.

FUTURE DIRECTIONS Thiol methyltransferase (TMT) is a key enzyme involved in generating the active metabolite, methylselenol, from selenomethionine. TMT is a membrane-bound enzyme and

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References

is extremely labile once separated from the membrane. Therefore, this enzyme is very difficult to purify, and efforts to clone and sequence the TMT gene have failed mainly for this reason. As a result, molecular pharmacogenetic studies on the polymorphism of this gene could not be conducted. However, TMT is expressed on the membrane of human red blood cells, which are easily accessible, and a radiochemical assay has been developed to study its enzymatic activity (Weinshilboum et al., 1979). Using this assay, Price et al. (1989) observed a fivefold variation in TMT activity in a large random sample. Ninety-eight percent of this variation was attributed to heredity. This study suggests the presence of polymorphisms in this genetic locus; at least one allele encoding high TMT activity may be responsible. TMT plays a critical role in selenium metabolism by catalyzing the reaction from hydrogen selenide to methylselenol, the active selenium metabolite (Figure 1). Variations in the enzymatic activity of TMT could conceivably affect an individual’s ability to metabolize selenium and lead to differences in responsiveness to selenium supplementation. As described earlier, the NPC trial showed that among individuals with low and medium levels of baseline plasma selenium, selenium treatment decreased prostate cancer incidence by threefold or more, whereas no protective effect by selenium was observed among those with high levels of baseline selenium. The differences in baseline selenium could be explained by the differences in the enzymatic activity of TMT. In individuals with high TMT activity, selenium can be efficiently metabolized and excreted, resulting in low baseline selenium levels. Therefore, we hypothesize that individuals with higher TMT activity are likely to benefit more from selenium supplementation. The level of methylselenol would be a direct measurement of the ability to generate the active selenium metabolite. However, methylselenol is very difficult to speciate. To the best of our knowledge, no methodology has been developed for quantifying the level of methylselenol in biological fluids. On the other hand, a simple assay for determining TMT activity in red blood cells is available. We plan to examine the activity of TMT in the cohort of prebrachytherapy patients and assess the value of using TMT activity to predict the responsiveness to selenium supplementation. The outcome could have significant clinical implications, as it could provide important information in designing custom-tailored intervention strategy for prostate cancer.

CONCLUSION There is a great deal of evidence from epidemiological studies and basic science that has informed our understanding of the anticancer effects of selenium. The application of genomics and proteomics technologies will open new avenues of research into uncharted frontiers of selenium

action. Based on the microarray data, we have shown that selenium affects not just a few key targets, but a multitude of targets. In doing so, the impact of selenium is amplified. The diversity of the molecular targets also makes it difficult for cancer cells to escape the inhibitory effects of selenium. Through the methodology of display thiol-proteomics, we have, for the first time, provided convincing evidence that selenium is capable of inducing global redox modification of proteins. This finding has significant implications in terms of cellular stress response and activation/inactivation of protein function. A challenge is to design innovative intervention trials that will allow us to validate these discoveries from the laboratory.

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C

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34 Glucosinolates RADHA M. BHEEMREDDY AND ELIZABETH H. JEFFERY

INTRODUCTION

feeds (Price et al., 1993). There has been renewed interest in these compounds because of the bioactivity of their aglucon metabolites that may be responsible for lowered cancer risk in persons eating cruciferous vegetables. Glucosinolates are the thioglucosides of modified amino acids, which are formed as secondary metabolites in a limited number of plant families. There are >120 glucosinolates identified, belonging to 16 families of angiosperms. The Brassicaceae, which include the cruciferous vegetables, are an economically important family because of their role in the human diet (Fahey et al., 2001) (Table 1). Most plant species contain more than one glucosinolate, and whereas concentrations vary considerably across varieties, the same spectrum or profile of glucosinolates usually occurs across all varieties of any given subspecies. Although some plants contain many glucosinolates, typically relatively few (one to four) predominate (Rosa et al., 1997). Interestingly, the distribution of glucosinolates frequently differs both qualitatively and quantitatively among plant parts (roots, leaves, seeds, etc.) of a single plant. Seeds, some of which are used as condiments, contain higher concentrations of glucosinolates than other edible parts (Carlson et al., 1987) (Table 2). Furthermore, the glucosinolate content depends not only on the genetic makeup, but also on the growing environment (Brown et al., 2002). Storage and processing may lead to either enhancement or loss of bioactivity depending on the specific conditions. For these reasons, it is often difficult to estimate intake of bioactive components in a population. For a more detailed discussion of the occurrence, distribution, and concentrations of glucosinolates, the reader is directed to several extensive review articles (Fenwick et al., 1983; Rosa et al., 1997; Fahey et al., 2001; Jeffery and Jarrell, 2001).

Epidemiological studies have associated diets rich in cruciferous vegetables and other glucosinolate-containing plants with reduced risk for a number of cancers. Animal studies have identified several glucosinolate derivatives from cruciferous vegetables that exhibit these chemopreventive properties. A large body of literature shows that glucosinolate derivatives modify many mammalian detoxification enzymes that make up part of our host defense against foreign chemicals, by inhibiting carcinogen activation and increasing carcinogen detoxification, resulting in clearance of carcinogens from the body. For more than 3 decades, these effects have been considered responsible for the reduced cancer initiation or decreased promotion and progression of tumors seen in animal and human studies. These same glucosinolate derivatives have been shown to arrest the cell cycle, slow or stop proliferation, and enhance apoptosis in cancer cell lines. Some of these compounds, in purified form, have either already shown encouraging results or are poised ready for testing in clinical trials of cancer prevention in high-risk groups. In this chapter, we summarize some epidemiological, clinical, animal, and cell culture studies on cancer prevention by dietary crucifers and glucosinolate derivatives and highlight the areas that need further research.

OCCURRENCE AND DISTRIBUTION OF GLUCOSINOLATES IN EDIBLE PLANTS Glucosinolates have been a topic of agricultural research for more than a century, although frequently focused on adverse effects in animals fed concentrated crucifer-based


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Chapter 34/Glucosinolates

TABLE 1

Common name

List of Widely Consumed Edible Plants and Their Prominent Glucosinolates Average Glucosinolate (mg/100 g)

Scientific name

Arugula Broccoli Brussels sprouts

Eruca sativa Brassica oleracea var. italica Brassica olerace var. gemmifera

— 62 237

Cabbage Chinese cabbage Cauliflower Collard greens Drumstick Garden cress Horseradish Kohlrabi

Brassica oleracea var. capitata Brassica oleracea var. pekinensis Brassica oleracea var. botrytis Brassica oleracea var. acephala Moringa oleifera Lepidium sativum Armoracia lapathifolia Brassica oleracea var. gongylodes

Mustard Black

Brassica nigra

4,630

Brown White Water cress Rutabaga

Brassica juncea Sinapis alba Nasturtium officinale Brassica oleracea var. napobrassica

4,660 6,410 95 92

Radish

Raphunus sativa

93

Turnip

Brassica campestris

93

59 21 43 201 20,200 390 160 73

Prominent glucosinolates Glucoerucin, Glucoraphanin Glucoraphanin, Sinigrin, Gluconapin, Glucobrassicin Sinigrin, Gluconapin, Progoitrin, Glucoraphanin, Glucoiberin, Glucobrassicin Sinigrin, Glucoiberin, Progoitrin, Glucobrassicin Sinigrin, Progoitrin, Glucobrassicin, Gluconasturtiin Sinigrin, Glucoraphanin, Glucoiberin, Glucobrassicin Sinigrin, Glucoiberin, Glucobrassicin Glucoconringiin (rhamnopyranosyloxy), benzyl glucosinolate Glucotropaeolin Sinigrin, Gluconapin, Glucoputranjivin, Gluconasturtiin Gluconapin, Glucoerucin, Glucoraphanin, Gluconasturtiin, Glucobrassicin Sinigrin, Gluconapin, Gluconasturtiin, 1-Methylpropyl Glucosinolate Sinigrin, Progoitrin, Gluconapin, Glucobrassicanapin Glucosinalbin, Glucoputranjivin Gluconasturtiin, Glucobrassicin, Glucosiberin, Glucohirsutin Sinigrin, Gluconapin, Progoitrin, Glucoerucin, Glucoraphanin, Gluconasturtiin Sinigrin, Glucoerucin, Glucotropaeolin, Gluconasturtiin, Glucobrassicin Gluconapin, Glucobrassicanapin

Source: Fenwick et al., 1983; Rosa et al., 1997; Kiddle et al., 2001; Bennett et al., 2003; McNaughton and Marks, 2003.

TABLE 2

Glucosinolate Content of Seed and Vegetative Tissue

Cruciferous vegetable

Seed (mmol/g)

Broccoli Brussels sprouts Cauliflower Collard greens Kale Mustard greens Kohlrabi

151.6 135.8 140.2 176.0 186.6 185.0 193.9

S-Glucose

R

Fresh tissue (mmol/g)

N O

1.9 5.5 1.0 4.4 3.2 11.9 0.9

Tissue damage Myrosinas pH, Fe, ESP Temp

Glucose SH N O

SO3-

HSO4-

S

Glucosinolate Structure and Diversity Glucosinolate structure consists of a β-D-thioglucose group linked to a sulfonated aldoxime moiety and a side chain (R group) derived from one of several amino acids (Figure 1A). Based on the amino acid precursors, glucosinolates are grouped as aliphatic, aromatic, or indolyl glucosinolates when derived from methionine, phenylalanine, or tryptophan, respectively (Mithen et al., 2000). Extensive research has identified enzymes that elongate the aliphatic side chain (three to six carbons in length are commonly

A

H2O

R

Source: Carlson et al., 1987.

SO3-

Nitrile

Thiocyanate

Isothiocyanate FIGURE 1 Schematic representation of glucosinolates hydrolysis.

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Occurrence and Distribution of Glucosinolates in Edible Plants

found in dietary crucifers). Other enzymes support desaturation, oxidation, or reduction, steps that follow side-chain elongation and that produce, for example, sulfinyl, sulfonyl, sulfide, and de-sulfo products (Wittstock and Halkier, 2002). Further, in the Moringaceae family (producing the Indian fruit drumstick), there may be an additional sugar moiety (rhamnose or arabinose) glycosylated to the benzene ring of the phenylalanine-derived side chain (Fahey et al., 2001). The abundance and the type of glucosinolate present in each species is dependent on the presence of the enzymes involved in these synthetic pathways and therefore may be genetically modified to breed for vegetables with the desired glucosinolate profile. Broccoli is one vegetable that has already been genetically modified to provide about 80 times the efficacy, when compared with the standard broccoli (Mithen et al., 2003).

Glucosinolate Hydrolysis to Bioactive Derivatives Glucosinolates are not bioactive but must undergo hydrolysis to an aglucon, followed by rearrangement, to form the bioactive glucosinolates derivatives. This hydrolysis may occur either within the plant tissue, catalyzed by the plant enzyme myrosinase, or following ingestion, catalyzed by the gut microflora (Mithen et al., 2000). Mammalian cells do not possess an active thioglucosidase. In intact plants, glucosinolates are physically separated from myrosinase, so glucosinolates remain chemically stable until the plant tissue is disrupted due to food preparation or chewing. When glucosinolates come into contact with myrosinase, the glucose is lost, and an unstable aglucon intermediate remains, which rapidly rearranges nonenzymatically to form an isothiocyanate, nitrile, or thiocyanate (Figure 1). These different products that are formed vary in their potency as anticancer agents. Some isothiocyanates with a hydroxyl group in the 2 position spontaneously cyclize to form oxazolidine2-thiones; aglucons with terminal double bonds form epithionitriles in the presence of a myrosinase cofactor, the epithiospecifier protein (Wittstock and Halkier, 2002; Matusheski et al., 2004). Under acid conditions, in the pres-

TABLE 4 Vegetable

Fresh (g/yr)

Cabbage Broccoli Cauliflower Total

4044 2578 889 7511

ence of millimolar concentrations of iron or in the presence of the epithiospecifier protein and micromolar concentrations of iron, a nitrile can be formed from any aglucon (Matusheski, personal communications). The importance of this is that of all the hydrolysis products, isothiocyanates appear to be the most bioactive. Table 3 lists several bioactive compounds, their parent glucosinolates, and the vegetables in which they are abundant.

Dietary Intake of Glucosinolates Detailed information on dietary glucosinolate or isothiocyanate intake is difficult to obtain. However, rough estimates can be made from available data on consumption of cruciferous vegetables. Estimated per capita consumption of fresh and frozen broccoli, cabbage, and cauliflower within the United States (USDA-NASS Agriculture Statistics, 2003) is presented in Table 4. The total per capita consumption of crucifers for year 2001 was reported at 19.7 lbs: roughly 1/3 cup or 24 g/person/day, based on total commercial production divided by total population. Glucosinolate content of commonly consumed vegetables varies from 500 to 2000 µg/g (Table 1), giving an average daily consumption in the range of ∼13 mg glucosinolates per day per person within the United States. Intake is substantially greater in a number of Asian countries. For example, in a study of 1724 Shanghai women, average cruciferous vegetable consumption was 98 g/day, four times higher than in the United States (Fowke et al., 2003b). Also, different cruciferous vegetables are popular in different parts of the world. The Japanese

TABLE 3

Bioactive Glucosinolates Derivatives

Glucosinolate Sinigrin Glucotropaeolin Gluconaturtiin Glucoraphanin Glucobrassicin Progoitrin

Bioactive compound Allyl isothiocyanate (AITC) Benzyl isothiocyanate (BITC) Phenylethyl isothiocyanate (PEITC) Sulforaphane (SF) Indole-3-carbinol (I3C) Crambene (CHB)

Consumption of Glucosinolate-Containing Vegetables in the United States (2001) GS content (mg/g) 0.59 0.62 0.43

Source: USDA-NASS Agricultural Statistics (2003). GS, glucosinolates; TVC, total vegetable consumption.

Frozen (g/yr)

978 267 1244

GS content (mg/g)

0.51 0.41

TVC (g/yr)

Total GS consumption (mg/yr)

4044 3556 1156 8755

2382 1698 492 4573

586


consume substantial amounts of wasabi, a Japanese horseradish, as a condiment with sushi, and the Koreans consume kimchi, a fermented cabbage or cabbage/radish product with almost every meal, whereas Americans consume mostly cabbage and broccoli (Fenwick et al., 1983). A database of glucosinolate content of common vegetables may soon be available, greatly simplifying calculation of glucosinolates consumption (McNaughton and Marks, 2003).

EPIDEMIOLOGICAL INVESTIGATIONS RELATING GLUCOSINOLATES AND CANCER PREVENTION Individuals with a high intake of fruits and vegetables appear to be at lower risk for developing a number of cancers (Table 5). A cohort study of survivors of ovarian cancer reported a statistically significant survival advantage for women in the highest quartile of intake for all vegetables in general (adjusted hazard ratio [HR] = 0.75, p < .01) and for those in the highest quartile of intake for cruciferous vegetables in particular (HR = 0.75, p < .03) (Nagle et al., 2003). A case-control study investigating the association between renal cell carcinoma incidence and diet identified a strong inverse relationship between renal cell carcinoma and intake of both dark green vegetables (p < .001) and cruciferous vegetables (p < .001); the protective effect of crucifers persisted even when data were adjusted for known nutrients in the crucifers, such as vitamin C and carotenoids (Yuan et al., 1998). The authors concluded that nonnutrients such as glucosinolate derivatives may play a role in protecting against renal cell carcinoma (Yuan et al., 1998). The results of many studies support the possibility that consumption of cruciferous vegetables has a stronger inverse association to cancer risk than the consumption of all vegetables (Jain et al., 1999; Cohen et al., 2000; Kolonel et al., 2000). For example, the Health Professionals’ Followup Study monitored 47,909 men over 10 years and reported 252 cases of bladder cancer. Analysis of food frequency

questionnaires collected three times during that 10-year period revealed a significant reduction in risk (relative risk [RR], 0.49; p = .008) for bladder cancer in individuals ingesting five or more servings of cruciferous vegetables a week, compared wtih those ingesting one or fewer servings per week (Michaud et al., 1999). A similar calculation for consumption of green leafy vegetables or for total fruits and vegetables did not show a significant risk reduction. Data are accumulating to suggest that, as one might expect, the protective response varies with the amount of cruciferous vegetables consumed: Risk is typically decreased most in the highest quartile of intake. For example, one study reported a significant 40% reduction in prostate cancer risk in individuals who consumed three to five servings or more of cruciferous vegetables per week, compared with those consuming only one serving or fewer (Cohen et al., 2000; Kolonel et al., 2000). Similarly, a metaanalysis of 20 studies, controlling for total vegetable intake, found an 8% decrease in risk for colon cancer with every 10-g increase in cruciferous vegetables consumed per day (Kohlmeier and Su, 1997). In the Nurses’ Health Study, a significant reduction in risk (RR, 0.67; p = .03) for nonHodgkin’s lymphoma was seen among women who consumed five or more servings per week in comparison with women who consumed fewer than two servings per week (Zhang et al., 2000). Whereas the number of studies showing an inverse relation between cruciferous vegetable intake and a decrease in risk for cancer is growing, there are a substantial number of well-controlled studies that do not show this effect. A review of 94 studies concluded that consumption of cruciferous vegetables is associated with reduced risk of cancer at several sites (Verhoeven et al., 1996). However, in that review, only 58 of the 87 case-control studies evaluated showed an inverse association between consumption of cruciferous vegetables and cancer risk, and findings were only statistically significant in 39 of these studies. Cohort studies showed the same trend as case-control studies, with five of seven studies showing an inverse effect. Studies continue to

TABLE 5 Epidemiological Studies Showing the Effect of Dietary Cruciferous Vegetables on Risk of Cancer Cancer site

Study type

Intake

Risk

p value

Reference

Bladder Lung Lymphoma Prostrate Prostrate Breast Kidney Ovarian

Prospective Case-control Prospective Case-control Case-control Case-control Case-control Case-control

>5 ser/wk 0.5 ser/day >5 ser/wk >3 ser/wk 5 ser/wk Quartile 4 Quartile 4 >0.83 ser/day

RR 0.49 OR 0.31 RR 0.67 OR 0.59 OR 0.61 OR 0.50 OR 0.53 HR 0.75

0.008 CI (0.1–0.92) 0.03 0.02 0.006 0.01 0.001 0.03

Michaud et al., 1999 Zhao et al., 2001 Zhang et al., 2000 Cohen et al., 2000 Kolonel et al., 2000 Fowke et al., 2003a Yuan et al., 1998 Nagle et al., 2003

CI, confidence interval; HR, hazard ratio; OR, odds ratio; RR, relative risk; ser, serving.

Chemoprevention Studies in Animal Models

show protection in a preponderance of studies, but not in all studies (Kristal and Lampe, 2002). One possible cause for these inconsistent findings may be the genetic variability in the makeup of the individuals. Cancer protective effects of cruciferous vegetables are mostly attributed to their ability to modulate the xenobiotic metabolizing enzymes. A number of studies have uncovered the fact that human genetic polymorphisms in xenobiotic metabolizing enzymes can change both an individual’s risk for cancer and the extent to which cruciferous vegetables can protect that individual (Spitz et al., 2000; Lin et al. 2002). In a study of the relationship between incidence of colon cancer and ingestion of cruciferous vegetables, sensitivity to protection by cruciferous vegetables was found to vary substantially with polymorphism in the family of glutathione S-transferase (GST) enzymes. Thus, a greater protective effect of broccoli specifically, and cruciferous vegetables in general, was seen in individuals who were missing the GSTM1 isoenzyme (GSTM1-null genotype) than in the general study population (Lin et al., 2002). A similar study conducted among lung cancer patients showed that a protective effect of cruciferous vegetables was significant in current smokers who were both GSTM1- and GSTT1-null genotype (Spitz et al., 2000). Whereas Spitz et al. (2000) found no significant protective effect of cruciferous vegetables against lung cancer incidence in former smokers, Zhao et al. (2001) showed a protective effect in nonsmoking Singapore Chinese women who were GSTM1 null. The different results from these two studies may reflect a difference in the range of cruciferous vegetable intake in the United States and Singapore: The latter study reported a mean intake very similar to that reported for the United States (∼50 µmol isothiocyanates/wk), but the range of intake was up to 10-fold greater (Zhao et al., 2001). Most of the cited epidemiological studies calculate dietary intake of cruciferous vegetables using food frequency questionnaires. That method of data collection might, however, cause some inaccuracy because of multiple causes including poor recall, recall bias, errors in identification of cruciferous vegetables, variation in glucosinolate levels due to variety, growing, and storage conditions, as well as difficulty in estimation of serving size. Several studies have used urinary isothiocyanate excretion as a measure of dietary exposure to cruciferous vegetables (Spitz et al., 2000; Zhao et al., 2001; Fowke et al., 2003a). This can give a precise measure of excretion and therefore a relative measure of intake. However, it is limited by the fact that much of a dose is excreted during the first 12 hours, and therefore, it is difficult to interpret period urine samples if intake varies substantially day to day (Shapiro et al., 1998). Albeit, when viewed together, the epidemiological studies strongly support a role for cruciferous vegetables in dietary prevention of cancer.

587

CHEMOPREVENTION STUDIES IN ANIMAL MODELS The capacity for cruciferous vegetables to modulate metabolism of carcinogens and prevent mutagenic and carcinogenic outcomes has been demonstrated in numerous animal models (Verhoeven et al., 1997; Hecht, 2000; Jeffery and Jarrell, 2001; Murillo and Mehta, 2001; Conaway et al., 2002). In addition, studies using animal models of cancer have found that in addition to the whole vegetable, purified dietary isothiocyanates and indoles, bioactive metabolites of glucosinolates, are also effective chemopreventive agents (Table 6). Animal feeding studies that evaluate changes in detoxification enzymes as an endpoint of efficacy have employed a broad range of cruciferous vegetables including broccoli and watercress. In contrast, feeding studies evaluating tumor incidence or tumor size as an endpoint have mostly utilized Brussels sprouts or cabbage. For example, in a study by Kassie et al. (2003), male F344 rats were given the liver and colon carcinogen 2-amino-3-methylimidazoquinoline (IQ) by gavage. In addition, some of these rats received either Brussels sprouts or red cabbage juice (5% v/v) in their drinking water. Dietary Brussels sprouts and red cabbage juice both significantly decreased IQ-induced glutathione-S-transferase-P-positive foci (GST-P+) number (a measure of initiation) and size (a measure of promotion/progression) in the livers of these rats. The Brussels sprouts diet effectively decreased initiation, but not progression in colon, and the red cabbage juice was without effect on initiation or progression in the colon. In this study, cooking did not diminish the cancer protective effects of this vegetable diet. These studies included the feeding of juices from two varieties of each vegetable. Although the glucosinolate content varied substantially among varieties, there appeared little difference in efficacy. The authors speculated that the interactive effect of all glucosinolate derivatives might have more impact than the content of any single glucosinolate. Another possibility is that the percentage yield of bioactive isothiocyanates, relative to the less active nitriles or thiocyanates, was greater from the variety with lower glucosinolate levels, equalizing the bioactive isothiocyanate yield (Keck et al., 2003). The majority of whole animal studies evaluating cancer prevention by cruciferous vegetables or glucosinolate derivatives have focused on prevention of initiation. However, several studies report efficacy of isothiocyanates given either before or after exposure to carcinogens, demonstrating that at least in some cancer models, cruciferous vegetables, glucosinolates, or isothiocyanates may be protective during both initiation and promotion/progression. For example, when rats were given the purified glucosinolate sinigrin, starting 22 hours after administration of the carcinogen 1,2-dimethylhydrazine (DMH), a significant

TABLE 6 Anticarcinogena

Effect of Glucosinolate Derivatives on Chemical-Induced Cancers Carcinogenb

Test system

Type of cancer

Effect

2-Phenethyl isothiocyanate (PEITC)e Before NNK Before BaP Before BaP Before BOP Before DMBA During DMBA Before and during NNK Before, during, and after NMBA Before and after DEN Before and after DMBA After NNK

Mouse Mouse Mouse Hamster Rat Mouse Rat Rat Mouse Rat Mouse

Lung Forestomach Lung Lung Mammary Lung and forestomach Lung Esophageal Liver Mammary Lung

Inhibition Inhibition No effect Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition No effect No effect

PEITC-NAC conjugatesg Before or after

AOM

Rat

Colon

Inhibition

Benzyl isothiocyanate (BITC)e Before Before Before Before Before During Before and after Before and after Before and after After After After

NNK DEN DEN BaP BaP DMBA/BaP NMBA DMBA DEN DMBA DMH NNK

Mouse Mouse Mouse Mouse Mouse Mouse Rat Rat Rat Rat Mouse Mouse

Lung Forestomach Lung Lung and forestomach Skin and forestomach Lung and forestomach Esophageal Mammary Lung Mammary Colon Lung

No effect Inhibition No effect Inhibition No effect Inhibition No effect Inhibition Inhibition Inhibition Inhibition Inhibition

Sulforaphane (SF)g,c,d Before and after Before, during, and after Before or after

DMBA BaP AOM

Rat Mice Rat

Stomach Stomach Colon (ACF)

Inhibition Inhibition Inhibition

Indole-3-carbinol (I3C)e Before After Before, during, and after Before and after After

AFB1 AFB1 DMH MNU/ DMBA DEN and MNU and DBN

Rainbow trout Rainbow trout Rat Rat Rat

Liver Liver Colon Mammary Liver and lung

Inhibition Enhanced Enhanced Inhibition Inhibition

I3C, DIM, I3Ae Before

DMBA/BaP

Rat/Mouse

Mammary/forestomach

Inhibition

DEN/4NQO

Rat

Liver/tongue

Inhibition

DMH

Rat

Colon

Inhibition

MNU AFB1/DMH

Rat Rat/Mouse

Mammary Liver

Inhibition Inhibition

Brussels sproutse,f Before or after During or after

AOM DMBA

Rat Rat

Colon (ACF) Mammary

Inhibition Inhibition

Cabbage Cauliflower Broccolie After

DMBA

Rat

Mammary

Inhibition

Gardencress juicei Before

IQ

Rat

Colon (ACF)

Inhibition

Broccoli sproutsh Before and after

DMBA

Rat

Mammary

Inhibition

e

I3C, Sinigrin Before, during, and after Singirin After

j

Cabbage Before During

a

e

Dosing schedule of potential anticarcinogen, relative to dosing of carcinogen. Carcinogen abbreviations: AFB1 Aflatoxin B1; AOM, azoxymethane; BaP, benz(a)pyrene; BOP, N-nitrosobis(2-oxopropyl)amine; DBN, N,N-dibutyl nitrosamine; DEN, diethylnitrosamine; DMBA, 9,10-dimethyl-1,2-benzanthracene; DMH, 1,2-dimethylhydrazine dihydrochloride; IQ, 2-amino-3methylimidazoquinoline; MNU, N-methyl-n-nitrosourea; 4NQO, 4-nitroquinoline1-oxide; NMBA, N-nitrosomethylbenzylamine; NNK, 4-(N-nitrosomethylamino)-1-(3-pyfldyl)-1-butanone; ACF, aberrant crypt foci; DIM, 3,3′-diindolylmethane; I3A, indole-3-acetonitrile (Zhang et al., 1994; Fahey et al., 1997, 2002; Verhoeven et al., 1997; Rijken et al., 1999; Chung et al., 2000; Kassie et al., 2002; Smith et al., 2003). b

Mechanisms of Cancer Prevention by Glucosinolate Derivatives

inhibition of appearance of colonic aberrant crypt foci was seen (Smith et al., 1998). Also, the purified glucosinolate derivatives sulforaphane (SF) and phenethyl isothiocyanate (PEITC) have been shown to significantly decrease (p < .01) azoxymethane-induced colonic aberrant crypt foci in F344 rats, when given in the diet before or after the carcinogen, suggesting protection against both initiation and promotion/progression (Chung et al., 2000). It is possible that the preponderance of anti-initiation studies in the literature may not reflect a lack of inhibition of proliferation, but that until recently the effect of isothiocyanates on carcinogenesis was frequently limited to anti-initiation studies (Table 6). Two reasons may have been responsible. Because inhibition of proliferation requires feeding of the test substance for a prolonged period, often months, it may have been technically difficult to produce sufficient test substance, when purification and/or synthetic systems were first under development. More significant, hypothesis-driven research demanded that initiation be the target of experiments, since the accepted hypothesis for the mechanism of action of glucosinolate derivatives was improved metabolism and clearance of chemical carcinogens. Studies using cultured cells have identified that isothiocyanates also arrest the cell cycle and cause apoptosis (see later discussion). This has led to a strong rationale for evaluating the effect of isothiocyanates on progression/proliferation. For example, Smith et al. (2003) focused on effects of Brussels sprouts on apoptosis and mitosis in the colonic mucosa of male Wistar rats given DMH. Oral Brussels sprouts juice or dietary freeze-dried uncooked Brussels sprouts increased apoptosis and decreased mitosis in colonic mucosa of DMH-treated rats. Importantly, in rats not receiving the carcinogen, neither juice nor freeze-dried Brussels sprouts had any inhibitory effect on normal mitosis. In contrast to the study described earlier, where cooking Brussels sprouts or red cabbage juice had no effect on efficacy (Kassie et al., 2003), when the Brussels sprouts were blanched before freeze-drying for addition to the diet, the protective effect was lost. Further studies are necessary to determine the cause of the lack of consistency in results from studies evaluating the effect of cooked cruciferous vegetables on cancer prevention. Effects of cruciferous vegetables on cancer prevention are not limited to any one vegetable type, carcinogen, or species of animal tested. The studies compiled in Table 6 are taken from several review articles and research papers to give an overall perspective on the breadth of the cancer-preventive action of glucosinolate derivatives that has been reported in studies using animal models of cancer. Efficacy has been reported for juice, raw and cooked cruciferous vegetables, purified glucosinolates, isothiocyanates, and even the Nacetylcysteine conjugate metabolites of isothiocyanates. Efficacy appears to cross species, type of cancer, carcinogen, treatment protocol, initiation, and promotion/progression, although the extent of protection appears to depend on a

589

number of factors, including the frequency and amount ingested, the stage of carcinogenesis, and the type of cancer (Xu et al., 2001). Even though one cannot extrapolate these findings from animal studies directly to human efficacy, these data do strongly implicate glucosinolate derivatives from cruciferous vegetables in chemoprevention.

MECHANISMS OF CANCER PREVENTION BY GLUCOSINOLATE DERIVATIVES Our understanding of mechanisms involved in cancer prevention by cruciferous vegetables has evolved concurrently with the fields of xenobiotic metabolism and cancer biology. Cruciferous vegetables were first shown to modify the enzymes that metabolize drugs and other xenobiotics in the 1970s, as our knowledge of these enzymes first began to unfold (Pantuck et al., 1979). With this knowledge arose the hypothesis that induction of detoxification enzymes might decrease the activity of carcinogens. In support of this, cancer prevention, decreased formation of DNA adducts, and an increase in elimination of carcinogens were found to occur in parallel with the increase in detoxification enzymes (Verhoeven et al., 1997). Glucosinolate derivatives have been found to trigger signal transduction events leading to cell cycle arrest and apoptosis. It remains to be determined whether these effects are secondary to changes in detoxification enzymes or changes in the antioxidant/redox status of the cell, whether they are primary mechanisms for anticarcinogenesis, or whether these mechanisms work in conjunction with enzyme induction to provide anticarcinogenesis. In addition, glucosinolate metabolites may also have epigenetic effects; by inhibiting histone, deacetylase be able to slow or even reverse tumor formation (Myzak et al., 2004).

Detoxification Enzymes The rate of metabolism and clearance of many environmental procarcinogens and carcinogens is enhanced by a diet rich in cruciferous vegetables or glucosinolate derivatives. Phase I detoxification enzymes, also called bioactivation enzymes, are primarily made up from the family of cytochrome P450 (CYP) isoenzymes. Phase I reactions support oxidation, reduction, and hydrolysis of carcinogens and other xenobiotics, and the products may be more reactive (bioactivated) or less reactive (detoxified). The products of bioactivation, reactive carcinogenic or toxic products, may cause adverse effects on the body such as DNA damage leading to initiation of carcinogenesis, or they may immediately undergo further phase II metabolism to form detoxified conjugation products. Conjugation enhances hydrophilicity of the metabolites, thus facilitating elimination of the carcinogen from the body. The compounds that are detoxified, rather than bioactivated, by phase I metabolism may be excreted directly

590


or undergo further metabolism by phase II conjugation enzymes. Glucosinolate derivatives have been found to induce a few CYP enzymes, inhibit a few more CYP enzymes, and induce several phase II conjugation enzymes. Therefore, if CYP-dependent bioactivation of a carcinogen could be inhibited and detoxification and clearance enhanced, this could slow or prevent the initiation of carcinogenesis by chemical carcinogens, leading to lowered risk of developing cancer. One example of glucosinolates altering metabolism is seen with 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK), a carcinogen present in tobacco smoke. NNK undergoes CYP-dependent bioactivation (carbonyl reduction) to form 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a direct-acting carcinogen that can methylate DNA (Hecht, 1999) (Figure 2). In humans, metabolism of NNAL by phase II conjugation results in detoxification and excretion of NNAL as a glucuronide. Another metabolic route for NNK is through CYP-dependent carbon hydroxylation to form the mutagenic metabolite diazohydroxide, which can cause DNA point mutations (Hecht, 1999). A third route is via CYP-dependent N-oxidation to pyridine N-oxide, which has no carcinogenic effect and can be excreted without further metabolism. The glucosinolate derivative PEITC prevents NNK lung cancer in rats and mice and enhances NNAL and NNAL glucuronide excretion in rats and humans, supporting the theory that PEITC has diverted metabolism away from formation of the carcinogenic diazohydroxide. In humans, bioactivation appears to involve several CYP enzymes, including CYP 1A2, 2A6, and 3A4, whereas detoxification through N-oxidation is supported by CYP 2B1, and possibly CYP 3A4. Thus, induction of glucuronidation and inhibition of the specific CYP enzymes that bioactivate NNK could be responsible for the decreased carcinogenic potential of NNK. The shared role of CYP enzymes in bioactivation and detoxification has led some scientists to suggest that induction of phase II conjugating enzymes and/or inhibition of phase I CYP enzymes may be more reliably anticarcinogenic than induction of phase I enzymes (Talalay and Fahey, 2001). Yet whole vegetables protect against cancer in epidemiological studies even

though they contain indolyl derivatives like indole-3carbinol, known to cause induction of some CYP enzymes. A broad range of glucosinolate derivatives have been studied for the ability to affect both phase I and phase II detoxification enzymes in whole animals and in cell culture systems. Table 7 lists some of the enzymes that have been studied in this context. The mechanism of enzyme induction by these derivatives has been reviewed (Lampe and Peterson, 2002; Thornalley, 2002). In brief, promoter regions of the genes of upregulated enzymes contain one or both of two regulatory regions that respond to glucosinolate derivatives, the xenobiotic response element (XRE) and the antioxidant response element (ARE) (Figure 3). Indolyl isothiocyanate derivatives, such as indole-3-carbinol and its metabolite diindolylmethane, trigger the XRE through binding to a TABLE 7

Partial List of Enzymes Modulated by Glucosinolate Derivatives

Aflatoxin aldehyde reductase Cytochrome P450 1A and 2E Epoxide hydrolases Ferritin Glutathione reductase Glutathione peroxidase Glutathione S-transferases γ-Glutamylcysteine synthetase Heptoglobin Multidrug resistance protein (MRP-1) NAD(P)H: quinone oxidoreductase P-glycoprotein Thioredoxin reductase UDP-glucuronosyltransferases Source: Thimmulappa et al., 2002; Hintze et al., 2003.

ITC

Nrf2

KEAP 1 maf

ARE

Non Carcinogenic N-Oxide

Phase II XRE

QR, GST

XRE

CYP 1A1

Carcinogenic Diazohydroxide

NNK

Phase I ARNT

DNA methylation DNA adducts N-Oxide

I3C

Diazohydroxide

NNAL

ARNT

Nucleus

DNA methylation DNA adducts Glucuronoide

Ah Receptor

Cytosol

FIGURE 3 Transcriptional regulation of detoxification enzymes by gluFIGURE 2 NNK metabolism.

cosinolate derivatives.

Clinical Studies with Clucosinolate Derivatives

cytosolic receptor. This complex of the metabolite and the Ah receptor then translocates to the nucleus, where the complex binds and activates the XRE, leading to the induction of a battery of enzymes, including both phase I (CYP 1A) and phase II enzymes. Other glucosinolate derivatives, such as the isothiocyanates allyl isothiocyanate (AITC) and SF, cause the endogenous cytosolic transcription factor Nrf2 to translocate to the nucleus and activate the ARE, but the isothiocyanates themselves are not bound to the Nrf2 transcription factor. Comparing the potency with which different isothiocyanates upregulate phase II enzymes in cell culture, SF appears very much more potent than other isothiocyanates (Talalay and Fahey, 2001). One possible reason for this is that SF accumulates rapidly in cultured cells, increasing the intracellular concentration by several orders of magnitude over the concentration of SF added to the medium (Zhang and Callaway, 2002). Whether this occurs within the body or only in cell culture remains to be determined because SF does not appear to maintain this potency advantage in animal feeding experiments (Keck et al., 2002; Munday and Munday, 2004)

591

exhibited a significant increase in apoptosis, together with a decrease in mitotic cell number compared with xenografts from mice with no AITC (Srivastava et al. 2003). These findings using the immune-compromised mouse xenograft model are in agreement with the chemical carcinogenesis model in immune-sufficient rats, in which rats given the carcinogen DMH to induce colon carcinogenesis, dietary sinigrin caused enhanced apoptosis of the aberrant crypt cells (Smith et al., 1998). The pathways leading to cell cycle arrest and apoptosis are still under investigation. Phenethyl isothiocyanate has been reported to induce apoptosis through a p53-dependent pathway, in studies using the JB6 cell line (Huang et al., 1998). In another study, activation of ERK, but not JNK, was implicated in PEITC-induced apoptosis of PC-3 cells (Xiao and Singh, 2002). In HeLa, Jurkat, and 293 cells, it was found that PEITC-dependent JNK activation was associated with apoptosis (Xiao and Singh, 2002; Hu et al., 2003). Taken together, these studies suggest that there are multiple pathways by which isothiocyanates trigger apoptosis, and that involvement of any one pathway may be specific to the cell line under study.

Modulation of Signal Transduction, Cell Cycle Arrest, and Apoptosis Studies have established the ability of glucosinolate derivatives to induce cell cycle arrest and apoptosis in cancer cells in culture while having little or no cytotoxic effect on normal noncancerous cells (Xiao et al., 2003). The mechanisms involved in triggering these events are under investigation Chen and Kong, 2004). One study evaluated the effect of four common isothiocyanate derivatives of glucosinolates—benzyl isothiocyanate (BITC), AITC, PEITC, and SF—on cultured cancer cell lines from several major organ sites: liver (HepG2 cells), breast (MCF-7 cells), colon (HT-29 cells), skin (HaCaT cells), and blood (HL60/S and 8226/S cells) (Zhang et al., 2003). Interestingly, both BITC and AITC were more potent in arresting cell growth than either PEITC or SF, even though SF had earlier been shown to be the most potent in induction of phase II enzymes in cultured cells (Zhang and Callaway, 2002). These results suggest that there may be distinct mechanisms involved in the induction of phase II enzymes and the arrest of the cell cycle by these compounds. Because both mechanisms may play a role in prevention of cancer, vegetables that contain a mixture of glucosinolate derivatives acting through different mechanisms may prove synergistic in their action. Studies employing xenografts of tumor cells grown under the dorsal skin of immune-deficient mice show that glucosinolate derivatives can induce apoptosis in this model also. In a mouse xenograft model implanted with PC-3 human prostate cells, 10 µmol AITC given i.p. three times a week significantly inhibited tumor growth (p = .05). Prostate cancer cell xenografts excised from mice receiving AITC

CLINICAL STUDIES WITH GLUCOSINOLATE DERIVATIVES Absorption and Metabolic Effects of Glucosinolate Derivatives Metabolism studies using rodents have shown that ingested isothiocyanates are conjugated to glutathione, further metabolized, and excreted in urine as N-acetylcysteine conjugates. These conjugates can be individually identified and quantified by high-performance liquid chromatography (HPLC)/UV (Duncan et al., 1997). Several clinical studies have used urinary excretion of individual conjugates as biomarkers and a relative measure of absorption of various cruciferous vegetables (Shapiro et al., 1998; Conaway et al., 2000; Fowke et al., 2003a,b). Total urinary isothiocyanates, measured as compounds that react with 1,2-benzenedithiol to form cyclic condensation products, have also been used as a measure of dietary intake (Ye et al., 2002). The cyclic condensation assay has the advantage that a single analysis can be used regardless of the particular vegetable in the diet. However, the measure of specific isothiocyanates is less easily confounded and can be used to evaluate intake of specific crucifers more accurately. Clinical studies have also used urinary products to estimate modification of carcinogen metabolism by diets rich in cruciferous vegetables. A broccoli/Brussels sprouts diet significantly decreased urinary excretion of 2-amino3,8-dimethylimidazo(4,5-f) quinoxaline (MeIQx) and 1-amino-1methyl-6-phenylimidazo(4,5-b)pyridine (PhIP),

592


carcinogenic heterocyclic aromatic amine metabolites from cooked meats (Murray et al., 2001). These data were interpreted to mean that phase I bioactivation to carcinogens had been inhibited. Similarly, a group of individuals given cruciferous vegetables together with fried meat over the 6-week study period excreted approximately twofold more conjugates than a group receiving noncruciferous vegetables with the fried meat (DeMarini et al., 1997). This was interpreted to mean that carcinogenic products formed were twice as likely to be metabolized by phase II enzymes and excreted. Based on rodent studies that show that PEITC enhances detoxification and excretion of the tobacco carcinogen NNK, a clinical trial evaluated the effect of watercress, a rich source of PEITC, on urinary tobacco metabolites in smokers (Hecht, 1995). Similar to results from the rodent studies, an increased excretion of NNAL and its glucuronide was observed in smokers following ingestion of watercress. Because in the rodent studies this change in metabolism correlated with PEITC protection from lung cancer, it has been proposed that PEITC should be developed as prophylactic therapy for addicted smokers (Hecht et al., 1995). Furthermore, a second tobacco carcinogen, benzo(a)pyrene, shows a shift in metabolic products and decreased carcinogenicity in rodents fed BITC (Lin et al., 1993). Therefore, PEITC and BITC may best function together to prevent lung cancer from the mixture of carcinogens present in cigarette smoke (Hecht, 1995). Clinical studies also support the hypothesis that cruciferous vegetables modulate estrogen metabolism in a manner consistent with reduced risk for estrogen-dependent cancers such as breast or endometrial cancers (Kall et al., 1996; Fowke et al., 2000) (Figure 4). Indole-3-carbinol is present in most cruciferous vegetables as its parent compound glucobrassicin and has been identified as the main contributor to the shift in estrogen urinary metabolites seen following ingestion of cruciferous vegetables. The change in urinary metabolites is from production of 16α-hydroxyestrone to the less estrogenic metabolite 2-hydroxyestrone (Figure 4). Women given 250 g of broccoli twice daily for 12 days exhibited this shift, with a 29.5% (p > .05) increase in the ratio of urinary 2 : 16α-hydroxyestrone metabolites (Kall et al., 1996). Similarly, a dietary intervention trial in healthy postmenopausal women found that for each 10-g incremental increase in cruciferous vegetable consumption per day, there was an 0.08% (CI 95%, 0.02–0.15) increase in the urinary 2 : 16α-hydroxyestrone ratio (Fowke et al., 2000). Enzymes associated with formation of 2-hydroxy metabolites are CYP 1A1 and 1B1 in extrahepatic tissue and CYP 3A4/5 and 1A2 in hepatic tissue; CYP 1A and possibly 1B are upregulated by indole 3-carbinol and its metabolites (Sanderson et al., 2001). These promising results have prompted phase I trials with indole-3-carbinol. The collection of urine samples is both minimally invasive and safer than handling blood samples. This methodology will no

Estradiol

Estrone

Active estrogen

Active estrogen

CYP 1A1 CYP 3A4

2-OH Estradiol 2-OH Estrone Non-Estrogenic

2-Methoxy Estradiol 2-Methoxy Estrone

CYP

16α-OH Estradiol 16α-OH Estrone Active estrogen

CYP 1B1

4-OH Estradiol 4-OH Estrone Quinone/DNA binding Carcinogenic

FIGURE 4 Effect of indole-3-carbinol and its metabolites on estrogen metabolism. Alteration of estrogen metabolism results in an increased 2OH/16-OH estrogen ratio (box in figure), which has been found to correlate with reduced risk of female cancers.

doubt be expanded to include measurement of additional metabolites for determination of efficacy of glucosinolate derivatives in slowing or preventing many cancers.

Cancer Prevention Clinical trials of cancer prevention and treatment are still very few and are typically, as a result of clearcut prevention studies in rodents, linked to urinary biomarkers of altered metabolism. As described earlier, indole-3-carbinol and its more potent metabolite diindolylmethane protect against estrogen-dependent breast cancer in rats (Wattenberg and Loub, 1978; Chen et al., 1998), this protection is linked to an increase in the urinary 2 : 16α-hydroxyestrone ratio (Jellinck et al., 1993), and this change in metabolite ratio can be seen in women ingesting cruciferous vegetables. Epidemiological studies have shown clearly that a low urinary 2 : 16α-hydroxyestrone ratio is a risk factor for breast cancer and that a high crucifer diet, indole-3-carbinol, or diindolylmethane can each elevate the ratio (Bradlow and Sepkovic, 2002). Furthermore, tamoxifen and diindolylmethane appear to work by independent means, possibly suggesting a role for indolyl products in addition to tamoxifen in women at high risk for breast cancer (Cover et al., 1999). Patients with laryngeal papillomas require periodic surgery. When patients were given daily indole-3-carbinol for 8 months, there was a cessation in the growth of papillomas in six patients and these patients did not require surgery. Six patients showed reduced papilloma growth, and the other six did not respond to the treatment (Rosen et al., 1998). Another small trial evaluated the effect of indole-3carbinol on women with cervical epithelial neoplasia (CIN). Thirty individuals in stage II or III CIN were randomized to placebo, 200 or 400 mg/day of oral indole-3-carbinol. After 12 weeks of treatments, 50% of those taking 200 mg and

593

Conclusion

45% of those taking 400 mg exhibited complete regression and did not need surgery (Bell et al., 2000). Although a key stumbling block has been the purification of sufficient quantities to perform clinical trials, there is a move toward developing isothiocyanates as prophylactic treatment for individuals at high risk for recurrent adenoma or papilloma. Phase I pharmacokinetic studies have been initiated using PEITC (Conaway et al., 2002), and methods are being developed for the estimation of urinary products of other isothiocyanates. The next few years should see considerable development in the evaluation of glucosinolate derivatives as chemopreventive tools in medicine.

Safety of Glucosinolate Derivatives Before glucosinolate derivatives can be used in supernutritional amounts, they must be evaluated for adverse effects. Early studies on feeding rapeseed meal to livestock can act as pointers to suggest which adverse effects might occur at higher than usual intake levels. Rapeseed meal is a rich source of the nitrile crambene (cyanohydroxybutene), which may be present at 10- or even 50-fold the amount typically found in cruciferous vegetables. Hepatic, renal, and pancreatic lesions, as well as thyroid enlargement, have been associated with these high doses (Papas et al., 1979; Wallig et al., 1992). Feeding a rapeseed meal diet to both poultry and laboratory animals has been reported to cause growth retardation, weight loss, and liver necrosis (Tookey et al., 1980). Such adverse effects have not been reported in individuals consuming a crucifer-rich diet because the content of glucosinolates in a serving of cruciferous vegetables, 500 mg or less, is a fraction of that present in a 40% rapeseed meal diet, 3–5 g/00 g feed (Kloss et al., 1994). Cruciferous vegetables are a rich source of vitamin K, often providing the entire daily requirement in a single serving, causing physicians to advise patients taking coumadin or related anticoagulant drugs to exclude crucifers from their diets, because coumadin functions by inhibiting the recycling of vitamin K. Another concern has been raised that consumption of a diet rich in cruciferous vegetables might cause goiter. The oxzaolidine-2-thione derivatives of glucosinolates have been shown to inhibit incorporation of iodine into precursors of thyroxine and thus to interfere with secretion of thyroxine hormone (Tookey et al., 1980). Even if glucosinolate-containing foods are not the primary cause of goiter, it is possible that they might aggravate goiter formation in individuals with very low iodine intake. However, even taking this into consideration, based on serving size, several studies have concluded that eating cruciferous vegetables is not a public health concern (Tookey et al., 1980). Nevertheless, there may be a need to examine the antinutritional effects of glucosinolate derivatives, because over the past decade, with the growing interest in glucosinolate derivatives for their anticarcinogenic properties, intake has

increased in some sectors of society (Mithen et al., 2000). For example, encapsulated broccoli concentrates are available over the counter as dietary supplements. Manufacturers of dietary supplements are not required to carry out extensive preclinical or clinical safety evaluations on their products. Whereas a small randomized trial evaluating the use of a broccoli supplement reported no adverse effects, no beneficial effects were reported either, suggesting that larger doses may soon be evaluated (Clapper et al., 1997). With the use of extracts or concentrates, the daily intake of glucosinolates has the potential to be increased many fold, highlighting the need for safety studies. In one study, purified PEITC (0.1% of the diet) was fed to rats for 32 weeks and caused weight loss and bladder carcinoma in >50% of the rats over the following 16-week period (Sugiura et al., 2003). Thus, administration of glucosinolate derivatives in supplements and purified powders may cause harm at the elevated doses possible with supplements. Another possibility is that ingestion of purified glucosinolates or derivatives may have very different effects to the whole food, even if taken in similar amounts to those found in the diet. For these reasons, there is a growing need for research to determine the safe upper levels for intake of glucosinolate derivatives, both purified and within cruciferous vegetables. However, it is also necessary to maintain a perspective on the need for concern due to adverse effects that are only reported for extremely high intake levels.

CONCLUSION There is an abundant literature on epidemiological, animal, and mechanistic studies strongly supporting a role for cruciferous vegetables in prevention of cancers. These have led to promising results in a few small clinical trials. They have also encouraged plant breeders and food manufacturers to take up projects to improve the content of glucosinolates and their bioactive derivatives in crucifers and crucifer-containing food products. There are ongoing research efforts to create both vegetable varieties with enhanced glucosinolate levels and a database quantifying glucosinolates in many commonly consumed cruciferous vegetables. Dietary supplements containing glucosinolates or their bioactive derivatives are also commonly available. However, there are still areas that need further research if the full health benefits of dietary crucifers are to be recognized. Further details on absorption, metabolism, and mechanism of action are necessary if we are to determine an effective dietary portion/quota/allocation. Most importantly, safety studies are needed that evaluate both safe upper limits and potential interactions between these bioactive compounds and cancer chemotherapeutics. With the advances that are occurring in the field, the goal of using cruciferous vegetables for dietary cancer prevention may soon become a reality.

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35 Green Tea JANELLE M. LANDAU, JOSHUA D. LAMBERT, AND CHUNG S. YANG

INTRODUCTION

tions that produce the distinctive color and taste of black tea. Oolong tea falls somewhere between green and black teas, in that the polyphenols in leaves are only partially oxidized. Tea is also divided by grades, determined by the leaf size. Herbal teas are not from the C. sinensis plant and, therefore, require completely different categorizing. For the purposes of this chapter, we discuss green tea, whose composition is similar to that of fresh tea leaves. The notion that tea may aid in the prevention of certain diseases and may promote good health is quite popular. Indeed, many animal studies and in vitro experiments have shown beneficial health effects of green tea, including the prevention of cardiovascular disease and cancer. Strong evidence for such beneficial health effects in humans, however, is scarce. This chapter discusses the chemistry, oxidative properties, and bioavailability of green tea polyphenols, as well as their possible cancer prevention activities and mechanisms in animals and humans.

Many studies suggest that green tea consumption may promote good health and prevent cancer. Scientific investigation in cell culture and animal studies continues to show promise in this regard. Ambiguous results from epidemiological studies, however, prevent forming a declarative statement for the cancer preventive effects of tea in humans. Possible explanations for the varied results from human studies could be related to the organ site investigated, the type and amount of tea consumed, or lifestyle confounding factors. In this chapter, we discuss the chemistry, oxidativereduction properties, metabolism, and bioavailability of green tea polyphenols, as well as their possible cancer prevention activities and mechanisms in animals and humans. More than 12 centuries ago, tea became a popular drink in China. When sailors began to bring tea to England from Asia around 1644, tea began to replace ale as the national drink of England. Tea shrubs were introduced in the United States in 1799, and in 1901, Thomas Sullivan developed the first tea bag. Tea is now second only to water as the world’s most consumed beverage. All tea comes from Camellia sinensis, a warm-weather evergreen. Tea is grown in thousands of tea gardens around the world, resulting in thousands of flavorful variations. Leaves of the C. sinensis plant are dried for stability and shelf life. Various processing techniques and the extent of oxygen exposure determine the type of tea that is produced from the fresh tea leaves. In the manufacturing of green tea, the leaves are steamed, rolled, and dried with minimum oxidation of the constituents. In black tea production, the leaves are crushed to allow enzyme-catalyzed oxidation of polyphenolic compounds, leading to polymerization and other chemical reac-


TEA CHEMISTRY AND OXIDATIONREDUCTION PROPERTIES The dried green tea leaves preserve the original constituents in the tea leaves. The characteristic polyphenolic compounds in tea are known as catechins, which include (−)-epigallocatechin-3-gallate (EGCG), (−)-epigallocatechin (EGC), (−)-epicatechin-3-gallate (ECG), and (−)-epicatechin (EC), with EGCG being the major catechin in tea (Figure 1) (Balentine et al., 1997). Polyphenolic structures are characterized by several hydroxyl groups on aromatic rings. These catechins (or flavonols) are made in plants for the purpose of defense against ultraviolet (UV) radiation, herbivores, and

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Chapter 35/Green Tea OH

OH HO

O

OH

OH HO

O

OH

OH

OH

OH

OH (-)-Epigallocatechin

(-)-Epicatechin

OH

OH

OH

OH HO

O

OH

HO

O O

O OH

OH

O

OH

OH

O

OH

OH OH

OH (-)-Epigallocatechin-3-gallate

(-)-Epicatechin-3-gallate

FIGURE 1 Catechin structures.

pathogens. Tea leaves also contain other polyphenols in small quantities such as quercetin, kaempferol, and myricetin, as well as the alkaloids caffeine and theobromine. A typical brewed green tea beverage (e.g., 2.5 g tea leaves in 250 ml of hot water) contains 240–320 mg of catechins and 20–50 mg of caffeine (Balentine et al., 1997). Tea catechins and other tea polyphenols are efficient scavengers of free radicals. Several functional groups in their structures appear to be important in conferring their low reduction potentials. All catechins have two hydroxyl groups in ortho position in the B-ring (Figure 1), which participate in electron delocalization. Both EGC and EGCG have three hydroxyl groups in the B-ring. In ECG and EGCG, the hydroxyl group at the 3 position in the C-ring is esterified with gallic acid, thus providing three more hydroxyl groups. The tri-hydroxyl group in both the B-ring and the gallate moiety have been associated with increased antioxidant activity. There is also some evidence that the Aring of EGC and EGCG may provide an antioxidant site (Zhu et al., 2000). Whereas tea polyphenols have been shown to have strong antioxidant activity in vitro, such activity has been demonstrated only in some in vivo experiments (reviewed by Higdon and Frei, 2003). An intervention trial among smokers found that green tea consumption decreased oxidative DNA damage (Hakim et al., 2003). Urinary 8hydroxydeoxyguanosine (8-OHdG) levels were 31% lower after 4 months of decaffeinated green tea (4 cups/day) consumption. The relevance of this biomarker and the importance of this antioxidative mechanism in the inhibition of carcinogenesis require further investigation. On the other hand, studies have suggested that the cell-killing activity of tea polyphenols, at least in vitro, may be related to their prooxidant activity. For example, we have shown that EGCG-

induced apoptosis in H661 human lung cancer cells and Ras-transformed human bronchial cells is completely or partially blocked by the inclusion in the medium of catalase, which catalyzes the decomposition of H2O2 (Yang et al., 1998, 2000). When EGCG was added to cell culture medium, H2O2 was produced (Hong et al., 2002). Preincubation of cells with EGCG has been shown to block signaling systems induced by the epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). Our results with esophageal squamous cells indicated that this blockage of EGF signaling was associated with the inactivation or degradation of the EGF receptor, and the effect could be abolished by the inclusion of superoxide dismutase, which converts oxygen radicals to H2O2, in the preincubation system (unpublished results). The addition of superoxide dismutase also stabilized EGCG and increased its growth inhibition effect. Both of these observations suggest the involvement of EGCG pro-oxidation in some of the reported activities of EGCG in vitro. It is not known whether such reactions occur in tissues where antioxidative capacity is much higher and oxygen partial pressure is much lower than that in cell culture medium.

INHIBITION OF CARCINOGENESIS IN ANIMAL MODELS Green tea has been shown to inhibit carcinogenesis induced by UV light and chemical carcinogens in rodents, as well as spontaneous tumorigenesis in wild-type and genetically modified mice. The organs for which tea has demonstrated a protective effect include the lung, skin, oral cavity, esophagus, stomach, liver, pancreas, bladder, small intestine, colon, and prostate (Yang and Wang, 1993; Yang et al., 2002; Chung et al., 2003). The lung tumorigenesis model, which exposes A/J mice to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), has demonstrated that green tea can inhibit tumor formation at several stages. In this model, treatment of female A/J mice with a single dose of NNK results in the formation of pulmonary tumors in nearly every mouse with an average of 9.3 tumors/mouse after 16 weeks. Decaffeinated green tea given as the sole source of drinking fluid during the initiation stage, during the post-initiation stage, or after lung adenoma formation inhibited the number of tumors that were formed (Yang et al., 1998). These results suggest that tea can inhibit lung tumorigenesis at the initiation and promotion stages. Inhibition of tumor invasion and metastasis in transplanted and spontaneous metastatic tumor models by intragastric infusion of green tea or EGCG has also been reported (Sazuka et al., 1995; Taniguchi et al., 1992). Tea has also been shown to inhibit spontaneous lung tumorigenesis (e.g., administration of 1% freshly brewed green tea inhibited the spontaneous development of lung adenoma and

Mechanisms of Cancer Prevention

rhabdomyosarcoma in A/J mice [Landau et al., 1997]). In this study, the body weights and retroperitoneal fat-pad weights of mice drinking 1% green tea were 14% and 35% lower, respectively, than those of the control mice. Likewise, the inhibition of skin tumorigenesis by caffeine or tea was shown to be closely correlated to the reduction of body fat (Conney et al., 2002). The relationship between tea consumption, lower body fat, and carcinogenesis requires further investigation. Inhibition of lung tumorigenesis by tea preparations has not, however, been unequivocally demonstrated. For example, Witschi et al. (1998) reported that green tea extract did not reduce lung tumor multiplicity in male A/J mice treated with one dose of NNK or in a cigarette smoke–induced lung tumorigenesis model. Whereas the NNK experiments already described in this chapter used decaffeinated green tea and the cancer preventive activity of EGCG has been demonstrated (Yang et al., 1998), the caffeine in tea is also important in the inhibition of carcinogenesis in some animal models. In the UVBinduced skin carcinogenesis mouse model, caffeine plays a significant role. Orally administered green tea and black tea were effective in reducing the incidence and multiplicity of UVB-induced skin tumors, whereas orally administered decaffeinated teas were much less or not effective (Huang et al., 1997). The addition of caffeine restored the protective activity to the decaffeinated teas. Topical application of caffeine or EGCG to hairless mice that had been pretreated twice weekly for 20 weeks with UVB decreased the multiplicity of skin tumors by 44–72% or 55–66%, respectively (Lu et al., 2002). In addition, both compounds were shown to increase the apoptotic index of the tumors by 56–92%. The bioavailability of tea constituents is apparently a key factor in determining the effectiveness of tea in inhibiting tumor formation. The oral cavity and digestive tract, which have direct contact with orally administered tea, may receive the most benefit from tea consumption. For example, in the 7, 12-dimethylbenz[a]anthracene (DMBA)–induced hamster model of oral carcinogenesis, treatment with 0.6% green tea, as the sole source of drinking fluid, reduced the number of visible tumors by 35% and tumor volume by 57% (Li et al., 2002). Immunohistochemical assays showed that tea increased the apoptotic index (the percent of cells undergoing programmed cell death) of the tumors while it decreased the proliferation index and microvessel density. Tea preparations have been shown to inhibit esophageal, forestomach, and intestinal cancer. Tea preparations have also been shown to inhibit colon carcinogenesis in several studies, although such an effect was not observed in others (reviewed by Yang et al., 2002). The transgenic adenocarcinoma of the mouse prostate (TRAMP) model emulates the progressive forms of human prostate cancer without the need for induction with chemicals or hormones. When these mice were given 0.1% green

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tea extract (which contains the polyphenol levels equivalent to 6 cups of green tea per day) as the source of drinking fluid for 24 weeks, prostate cancer development was inhibited and no metastasis was seen (Gupta et al., 2001). In addition, green tea infusion to TRAMP mice caused inhibition of insulin-like growth factor-1 (IGF-1), restoration of IGFbinding protein-3, and elevation of apoptosis. In a breast cancer model in SCID female mice, IGF-1 was also inhibited by green tea in combination with soy phytochemical concentrate (Zhou et al., 2004). Green tea (1.5%) as the sole source of drinking fluid reduced the mammary tumor weight significantly. A second transplanted tumor model in mice also showed reductions in tumor size with green tea extract consumption (Sartippour et al., 2001). In two other breast cancer models, green tea extract significantly increased the latency period to first tumor formation (Kavanagh et al., 2001; Yanaga et al., 2002). Studies using DMBA to induce mammary tumors in rats, however, showed little or no effect of green tea polyphenols on inhibiting post-initiation, promotion, or progression of mammary carcinogenesis (Hirose et al., 1997; Tanaka et al., 1997).

MECHANISMS OF CANCER PREVENTION Alterations in Intracellular Signaling Cascades Activation of the transcription factors activator protein 1 (AP-1) and nuclear factor-κB (NFκB) is commonly seen in carcinogenesis. These transcription factors promote the uncontrolled growth of cells. EGCG and other tea polyphenols have been shown to inhibit the activation of AP-1 and NFκB. Although the antioxidative mechanisms have been implicated in this activity, the results can be better explained by the direct inhibition of specific protein kinases by these tea polyphenols. Several studies using different cell lines have shown that EGCG inhibits the activity of various kinases (Ahmad et al., 2000; Chung et al., 2001; Yang et al., 2001). These include direct inhibition of MAP kinases, and the results are consistent with previous observations that EGCG (5–20 µM) inhibited the phosphorylation of jun Nterminal kinase, activation of AP-1, and the transformation of mouse epidermal cells, as well as the suppression of AP1 activity by topical application of EGCG to B6D2 transgenic mice. Downregulation of AP-1 by tea polyphenols may contribute to the p53 increase and the stress-induced elevation of apoptosis seen in tea-treated cells (Chung et al., 2001). EGCG has been shown to inhibit the activity of IκB kinase in tumor necrosis factor-α (TNF-α)–stimulated intestinal epithelial cells and lipopolysaccharide (LPS)stimulated murine macrophages (Yang et al., 2001). In both cases, there was diminished IκB degradation and NFκB

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activity in response to stimulation. Likewise, Ahmad et al. (2000) demonstrated that EGCG inhibited the activity of NFκB in TNF-α– and LPS-stimulated human epidermoid carcinoma cells. This effect could also be mediated by inhibition of IκB phosphorylation and degradation. The transcription factor NFκB plays important roles in inflammation and in suppressing apoptosis in cancer cells.

Regulation of Cell Cycle The development of tumors has been associated with the dysfunction of the cell cycle checkpoints and overexpression of growth-promoting cell cycle factors such as cyclin D1 and cyclin-dependent kinases (CDKs) (Diehl, 2002; Semczuk and Jakowicki, 2004). EGCG has been reported to inhibit CDK-2 and -4 (Liang et al., 1999), leading to the inhibition of hyperphosphorylation of the retinoblastoma (Rb) protein and causing G0/G1-phase arrest. This type of cell cycle arrest by EGCG has been demonstrated in several human tumor cell lines, including breast, epidermal, prostate, and head and neck squamous cell cancers. The direct inhibition of CDK by EGCG may be a primary event (Liang et al., 1999).

Induction of Apoptosis Apoptosis is the process by which a damaged cell degrades itself to prevent the proliferation of the genetic damage. Typically, in cancer cells, this cell death is inhibited. EGCG has been reported to induce apoptosis in many cell lines, including leukemia, skin, lung, stomach, and prostate cancer cells (Ahmad et al., 1997; Mukhtar et al., 1999; Yang and Chung, 1999). The H2O2 generated in the cell culture system due to autoxidation of EGCG could account for some of the reported apoptotic activity. It appears that EGCG can induce apoptosis by H2O2-dependent and H2O2-independent pathways. A study by Leone et al. (2003) suggested that certain green tea polyphenols bind to the antiapoptotic proteins Bcl-2 and Bcl-xL and, thus, may prevent inhibition of apoptosis. Using a combination of advanced physical measurements and computational docking studies, these investigators determined that tea polyphenols with a gallate moiety inhibit the aforementioned antiapoptotic proteins at nanomolar levels. If this action does indeed take place in the cells, one would expect to observe the induction of apoptosis with nanomolar amounts of EGCG. However, enhanced apoptosis was usually observed with much higher concentrations of EGCG (20–100 µM) (Yang et al., 2002). Caspase 3 activation has been demonstrated to be required for green tea polyphenol-induced apoptosis. Caspase 3 activity was increased in cells that were treated with green tea polyphenols, and caspase 3–deficient tumor cells did not undergo apoptosis when exposed to polyphenols (Hsu et al., 2003).

Inhibition of Angiogenesis, Invasion, and Metastasis Angiogenesis, the formation of new blood vessels, is important for tumor development. Tumors induce new blood cell growth to provide a pathway for nutrient and waste transport as it grows larger. Cao and Cao (1999) demonstrated the inhibition of endothelial growth and angiogenesis in the chorioallantoic membrane assay by EGCG (20 µM). They also showed that oral administration of 1.25% green tea to mice inhibited new corneal vessel formation stimulated by vascular endothelial growth factor (VEGF). Several investigators have demonstrated the EGCG inhibits the expression of VEGF in head and neck, breast, and colon carcinoma cells (Jung et al., 2001; Masuda et al., 2002; Sartippour et al., 2002). In the TRAMP mouse model using gene array techniques and immunoblot analysis, the expression of VEGF and matrix metalloproteinases (MMP-2 and MMP-9) were shown to be elevated in the transgenic mice, and these gene expressions were inhibited in TRAMP mice consuming green tea extract (Adhami et al., 2003). EGF receptor (EGFR) is frequently overexpressed in neoplastic cells, activating signaling transduction pathways that promote cell proliferation and tumor progression. EGCG inhibited the autophosphorylation of EGFR in head and neck and breast carcinoma cell lines (Masuda et al., 2001, 2002). Overexpression of MMP has been shown to increase the invasive and metastatic potential of tumor cells, whereas the suppression of these zinc-dependent proteases has been shown to inhibit tumor growth and invasion. EGCG inhibited the activity of secreted MMP-2 and MMP9 with fairly low doses (8–13 µM) (Garbisa et al., 2001).

Inhibition of Aberrant Arachidonic Acid Metabolism Arachidonic acid is metabolized by cyclooxygenase (COX) to form prostaglandin E2 and by 5-, 12-, or 15lipoxygenase (LOX) to form leukotrienes. Overexpression of COX-2, 5-LOX, and other LOX enzymes is observed in several types of cancers. EGCG has been shown to inhibit the induction of COX-2 in both in vitro and in vivo systems. EGCG at doses of 100–200 µM reduced COX-2 protein expression and activity in human cartilage cells (Ahmed et al., 2002). Whereas these findings are potentially interesting, the concentrations of EGCG used in the study are 10–100 times greater than those observed in vivo. The results must, therefore, be confirmed in animal model or in humans. Mice consuming EGCG before 12-tetradecanoyl-phorbol-13acetate application to the skin had significantly less COX-2 expression in their skin compared to control mice (Kundu et al., 2003). Inhibition of COX-2 with the ingestion of green tea extract was observed in the colons of rats (Metz et al., 2000) and the skin of mice (Katiyar et al., 1992). Within our

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Bioavailability and Metabolism

laboratory, various catechins were shown to inhibit the formation of COX and LOX-dependent metabolites in human normal and tumor colon microsomes (Hong et al., 2001). ECG and EGCG were the most potent inhibitors.

BIOAVAILABILITY AND METABOLISM Human Data An understanding of the processes involved in the absorption, distribution, and metabolism of tea polyphenols is essential for determining their potential actions in vivo and their overall significance in human disease prevention. Interindividual variation in the plasma and tissue bioavailability of active tea components is substantial and may result, in part, from genetic polymorphisms in the enzymes involved in polyphenol metabolism and individual variation in colonic microflora (Scalbert and Williamson, 2002). Several studies of the systemic bioavailability of orally administered green tea and catechins in human volunteers have been conducted (Yang et al., 1998; Chow et al., 2001; Van Amelsvoort et al., 2001; Lee et al., 2002). Most recently, we have shown that oral administration of 20 mg green tea solids/kg body weight resulted in the maximum concentration (Cmax) of 223, 124, and 77.9 ng/ml in the plasma for EGC, EC, and EGCG, respectively (Lee et al., 2002). Time to reach a maximum concentration (Tmax) was found to range from 1.3 to 1.6 hours with half-lives (t1/2 of 3.4, 1.7, and 2 hours for EGCG, EGC, and EC, respectively). The Tmax increased with greater dose of catechins (Chow et al., 2001). When the catechins (EGCG, EGC, or ECG) were given individually in a single 1.5-mmol dose with breakfast, the plasma kinetics showed a difference in appearance among the catechins (Van Amelsvoort et al., 2001). The peak value of EGCG, EGC, and ECG occurred at 2.9, 1.4, and 4 hours, respectively; the half-lives were 4, 1.7, and 7, respectively. Plasma EC and EGC were present mainly in the conjugated form, whereas 77% of the EGCG was in the free form (Lee et al., 2002). The data support earlier findings that plasma EGC was present as glucuronide (57–71%) and sulfate (23–36%) with only a small fraction of free EGC (Yang et al., 1998; Chow et al., 2001). Likewise, plasma EC was largely in the sulfated form (66%) with less glucuronide (33%). EGC was also methylated (4′-O-methyl-EGC) in humans, and its plasma and urine levels were higher than those of EGC (Lee et al., 2002). Pharmacokinetics and toxicity of EGCG at a dose of 800 mg in pure form or in Polyphenon E (a mixture of the main green tea polyphenols) have been investigated (Chow et al., 2003). These doses were found to be safe and acceptable to humans. However, in a Phase II trial investigating the effect of green tea on patients with prostate carcinomas, a daily dose of 6 g green tea (given in six 1-g doses/day) led to adverse effects in 69%

of the subjects, which included nausea, vomiting, insomnia, fatigue, diarrhea, abdominal pain, and confusion (Jatoi et al., 2003). In contrast to previous studies in rodents employing multiple-dose regimens, Chow et al. (2003) have shown that daily dosing with 800 mg EGCG to human volunteers for 4 weeks resulted in a 60% increase in the systemic exposure to free EGCG. This increased availability may be due to alterations in drug-metabolizing enzymes or the accumulation of EGCG in a nonplasma compartment.

Comparison of Human to Rat and Mouse The pharmacokinetics of EGCG and the other catechins have been investigated in rats, mice, and humans (Yang et al., 2002). Studies of [3H]-EGCG in both rats and mice showed that following a single intragastric (i.g.) dose, radioactivity was distributed throughout the body (Suganuma et al., 1998; Kohri et al., 2001). After 24 hours, 10% of the initial dose of radioactivity was found in the blood with ∼1% in the prostate, heart, lungs, liver, kidneys, and other tissues of rats and mice. The major route of elimination was through the feces in both species. In the rat, 77% of an intravenous (IV) dose of [3H]-EGCG was eliminated in the bile, whereas only 2% was eliminated through the urine. Treatment of rats with a green tea polyphenol preparation (0.6% w/v in distilled water) resulted in increased plasma levels of polyphenols over a 14-day period, with levels of EGC and EC being higher than those of EGCG (Kim et al., 2000). Plasma levels then decreased over the subsequent 14 days, suggesting an adaptive effect. EGCG levels were found to be the highest in the rat esophagus, intestine, and colon, which have direct contact with tea catechins, whereas EGCG levels were lower in the bladder, kidneys, liver, lungs, and prostate, which depend on systemic bioavailability. When the same polyphenol preparation was given to mice, the EGCG levels in the plasma, lungs, and liver were much higher than in rats (Kim et al., 2000). On the basis of the numerous biotransformation studies that have been conducted, it appears that mice are more similar to humans than rats in terms of enzymatic ability to conjugate tea catechins.

Metabolites Formed in the Body Tea polyphenols are extensively metabolized by intestinal and hepatic enzymes and by the intestinal microflora. Knowledge of their bioavailability and metabolism is necessary to evaluate their biological activity within target tissues. The metabolites that are found in blood and target organs may differ from the native substances in terms of biological activity. Our laboratory has proposed a schematic describing the possible factors governing the bioavailability of catechins (Figure 2). This model includes several biotransformations including methylation, glucuronidation,

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Systemic circulation

X

MRP2

EGCG-X EGCG

EGCG EGC

EGCG EGC

EGCG EGC Phase II

EGCG-X EGCG

Tea Consumption

EGCG EGC MM

Phase II

MRP1

EGCG-X EGCG

MRP1

EGCG-X EGCG

MRP2

Microbial Metabolism

EGCG-X EGCG

Bile Hepatocyte Fecal excretion Urinary excretion

Enterocyte

Systemic circulation

Fecal excretion

EGCG-X: EGCG conjugate MM: Microbial Metabolite X: Unknown Metabolite

FIGURE 2 Factors governing bioavailability. Proposed model illustrating the possible factors governing the bioavailability of tea catechins. Multidrug resistance–associated proteins (MRPs) include MRP1, which is expressed on the basolateral membrane of most cell types, and MRP2, which is expressed on the apical side of cells of the liver, kidney, and intestine.

sulfation, and ring-fission metabolism. For example, anaerobic fermentation of EGC, EC, and ECG with human fecal microflora has been shown to result in the production of the ring fission products, 5-(3′,4′,5′-trihydroxyphenyl)-γvalerolactone (M4), 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone (M6), and 5-(3′,5′-dihydroxyphenyl)-γ-valerolactone (M6′) (Figure 3), and these are detectable in urine (Meselhy et al., 1997; Meng et al., 2002). These compounds contain a valerolactone structure while retaining the polyphenolic character of the parent compound and may, therefore, have biological activities. EGCG has also been shown to undergo methylation, forming 4′,4″-di-O-methyl-EGCG (Meng et al., 2002). As another example, EGCG has been observed to rapidly oxidize in intestinal fluid (pH 8.5) with the amount of EGCG decreasing 81.6% in only 5 minutes, whereas a similar incubation in mouse plasma (pH 7.4) resulted in only a 29.3% decrease in EGCG (Yoshino et al., 1999). This oxidation, however, was found to create dimerized products, which had greater superoxide radical scavenging activity than EGCG. Investigation into the biological activities of the metabolites of green tea is an open area of research.

EPIDEMIOLOGICAL STUDIES The plethora of animal studies demonstrating protective effects of tea against tumorigenesis suggests that one would expect to see such an effect in humans. The effect of tea con-

sumption on human cancer has been studied extensively and reviewed in the literature (Yang and Wang, 1993; Blot et al., 1996; Kohlmeier et al., 1997; Buschman, 1998; Yang et al., 2002; Higdon and Frei, 2003). Many case-control studies have shown that subjects who consume large amounts of tea had lower cancer risk; in particular, risk of gastric and esophageal cancers was lower in green tea consumers in Japan and China. However, many other studies did not observe this protective effect. For example, a systematic review of 21 epidemiological investigations of gastrointestinal cancer or precancerous lesions suggested a protective effect of green tea on adenomatous polyps and chronic atrophic gastritis formation, but no clear support for green tea’s preventive role in stomach and intestinal cancer (Borrelli et al., 2004). A population-based case-control study of breast cancer was conducted among women of Asian descent living in Los Angeles to investigate the effect of green and black tea (Wu et al., 2003a). Green tea drinkers had a significantly reduced risk of breast cancer that was maintained even after adjusting for several potential confounding factors including smoking, alcohol, coffee, and black tea intake, family history of breast cancer, physical activity, and intake of soy and dark green vegetables. Among women who carried at least one low-activity catechol-O-methyltransferase (COMT) allele, both green and black tea drinkers showed a significantly reduced risk of breast cancer (Wu et al., 2003b), as opposed to the initial study (Wu et al., 2003a), in which

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Intervention Studies

OCH3

OH

OH

OH OH

HO

Cytosolic COMT

O

O O

OH 5-(3',4'-Dihydroxyphenyl) -γ-valerolactone (M6)

OH Colonic OH Microflora

SAM

HO

O OH

SAH

OH

EC

3'-MeEC OH OCH3

OH OH HO

OH

O

SAM

O

OH OH

SAH

OH

OH

Colonic OH Microflora

O

HO

OH

OH O

Cytosolic COMT

4'-MeEGC

OH

EGC

5-(3',4',5'-Trihydroxyphenyl) -γ-valerolactone (M4)

Catechin and MeCatechin Conjugates

UGT/SULT Conjugation

OH

OH OH

OH

HO A

O C

OH

O

O

O

OH 5-(3',5'-Dihydroxyphenyl) -γ-valerolactone (M6')

OCH3

B

OH

O

Cytosolic HO COMT

OH O

OH

O

SAM

SAH

OH

O

OH

EGCG OH

4',4''-DiMeEGCG OH

OH OCH3

FIGURE 3 Ring fission products. Metabolic fate of tea catechins (COMT, catechol-O-methyltransferase; SAM, sadenosylmethionine; SAH, homocysteine; SULT, sulfotransferase; UGT, uridine 5′-diphosphoglucuronosyltransferase).

no effect of black tea was seen. The authors concluded that individuals with a low-activity COMT allele have a reduced risk of breast cancer because they metabolize tea polyphenols less efficiently and, therefore, had prolonged exposure to these compounds. In two prospective studies in Japan, green tea was not, however, associated with decreased breast cancer risk when women drinking five or more cups of tea per day were compared with women drinking less than one cup (Suzuki et al., 2004). A case-control study of prostate cancer in southeast China found that prostate cancer risk declined with increasing frequency, duration, and quantity of green tea consumed (Jian et al., 2003). One Japanese cohort study found that total cancer risk was significantly lower in women who drank >10 cups of green tea per day, but not men (Imai et al., 1997). One explanation for the difficulty in interpreting epidemiological results is that “tea” is a diverse beverage. The polyphenolic content depends on how the leaves are processed before drying, the geographic location, and

growing conditions. Within a particular tea beverage, the polyphenol concentration varies among the type of tea (e.g., blended, instant, or decaffeinated) and preparation (e.g., amount used, brew time, and temperature). The highest amounts of polyphenols are found in brewed hot tea (541–692 µg/ml), less in instant preparations (90–100 µg/ml), and lower amounts in iced and ready-to-drink tea (Hakim et al., 2000). Despite some early controversy, it is now widely accepted that the addition of milk to tea does not interfere with polyphenol absorption (Hollman et al., 2001). Yet other issues such as tea drinking alone or with meals remain to be investigated further.

INTERVENTION STUDIES Several intervention trials have investigated the effect of tea consumption on various biomarkers in smokers. In one intervention trial, 143 heavy smokers were randomized to

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drink 4 cups/day of green tea, black tea, or water (Hakim et al., 2003). After 4 months of intervention, the green tea group had significantly lower urinary 8-OHdG levels after adjusting for baseline measurement and other potential confounding factors. A double-blind intervention trial in which 64 smokers with oral leukoplakia were given daily capsules containing a mixture of tea polyphenols and topical applied tea mixture was conducted (Li et al., 1999). Oral lesions were significantly reduced in 38% of the experimental patients as compared with 10% of the placebo control patients. The incidence of micronucleated exfoliated oral mucosal cells was significantly lower in the tea-treated group. Patients with androgen-independent prostate carcinoma were given 1 g of green tea six times per day and monitored monthly for changes in prostate-specific antigen (PSA) levels (Jatoi et al., 2003). After 1 month of treatment, no beneficial effects were seen on the PSA levels of these patients, and many patients complained about side effects. Certainly, intervention trials with other high-risk groups are warranted.

CONCLUSIONS Because of the wide consumption of tea beverages worldwide, the biological activity of tea constituents continues to be an important topic for scientific investigation. The biological mechanisms discussed in this chapter suggest that tea possesses cancer-preventive activities. Nevertheless, these activities have not been convincingly demonstrated in humans, in spite of the many epidemiological studies. Studies using a more quantitative assessment of tea consumption (Sun et al., 2002) and analyzing subgroups of the population (Wu et al., 2003b) have begun to demonstrate the expected protective effect. More positive results are expected when methodology in epidemiological studies is further refined. Another consideration is that at the levels of human tea consumption, which are usually lower than those used in animal cancer chemoprevention studies, the amount of the tea polyphenols that reaches the target tissues is a limiting factor. For cancer prevention, one possible approach is to increase the amount of tea intake, but this may be limited by the taste and possible undesirable side effects. At moderate levels of tea consumption, the organ sites that are more accessible to tea polyphenols, such as the oral cavity and intestinal tract, are likely to be protected by tea. This concept could be tested in future epidemiological and intervention human studies.

References Adhami, V.M., Ahmad, N., and Mukhtar, H. 2003. Molecular targets for green tea in prostate cancer prevention. J Nutr 133: 2417S–2424S. Ahmad, N., Feyes, D.K., Nieminen, A.L., Agarwal, R., and Mukhtar, H. 1997. Green tea constituent epigallocatechin-3-gallate and induction of

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36 Garlic JOHN MILNER

INTRODUCTION

characteristics and their apparent health benefits (Rivlin, 2001). The ability of these foods to serve as antimicrobial, antithrombotic, antitumor, hypolipidemic, antiarthritic, and hypoglycemic agents has surely been important to the widespread belief in these vegetables as medicinal foods. Although clinical studies provide some important insights into the potential physiological significance of dietary garlic and individual variability, especially for heart disease (Rahman, 2001; Dhawan and Jain, 2004; Franco et al., 2004; Tattelman, 2005), the most compelling evidence linking garlic and related foods with cancer comes from preclinical studies using cultured cells or animal models (Milner, 2001; Thomson and Ali, 2003). Epidemiological support for the anticancer effects of garlic, admittedly sparse, is suggestive that those with higher intakes have increased protection against some cancer (Fleischauer and Arab, 2001; Hsing et al., 2002; Khanum et al., 2004; Sengupta et al., 2004). Cohort studies provide evidence, though not overly compelling, that an inverse association existed between garlic intake and the incidence of colorectal cancer and possibly the incidence at several other cancer sites (Fleischauer and Arab, 2001). Evidence again suggests that increased garlic intake, as well as related allium foods, was associated with a reduction in prostate cancer risk (Hsing et al., 2002). The apparent protection was independent of body size, intake of other foods, and total calories. Although data relating garlic intake to human cancer risk are tantalizing, it is likely that variation in a variety of genetic and environmental factors may influence the response found among individuals. Thus, it should not be surprising that variability occurs in the literature about the importance of garlic and related allium foods and overall cancer risk and/or tumor behavior. The role of genomics is increasingly being recognized as a key factor in the biological response to foods and their

Mounting evidence continues to raise the possibility that garlic is a protective factor against cancer. Studies using fresh garlic extracts, aged garlic, garlic oil, and a number of specific organosulfur compounds arising from processed garlic provide support for a reduction in cancer risk and a modification in tumor behavior, although there is considerable variability in response. Regardless, the ability of garlic and related allyl sulfur compounds to block models for colon, lung, breast, and liver suggests general mechanisms of action are a real possibility. The anticancer characteristics appear to arise through both a dose- and a temporalrelated change in a number of cellular events involved with the cancer process, including those involving drug metabolism, inflammation, hormonal regulation, immunocompetence, cell cycle regulation, apoptosis, and angiogenesis. When using chemical carcinogens in animal models, there is little difference in the relative efficacy of water- and lipid-soluble allyl sulfur compounds. However, the tumor proliferation/apoptosis response is highly dependent on the species provided. A shift in sulfhydryl groups, alterations in glutathione/oxidized glutathione ratios, and resultant changes in cellular redox status may be involved in some of the phenotypic changes caused by allyl sulfur compounds. Although the anticarcinogenic and antitumorigenic data are intriguing, additional studies are necessary with more reasonable exposures over prolonged periods to verify efficacy in humans. Finally, it is critical that genomic, epigenomic, proteomic, and metabolomic factors are considered so that models can be developed to predict who might benefit most from expanded use of garlic or its allyl sulfur components. Allium vegetables including garlic, onion, leeks, chives, and scallions are used throughout history for their sensory


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components (Davis and Milner, 2004; Fenech, 2005). Collectively, genetic background (nutrigenetic effects), DNA methylation and histone regulation (nutritional epigenomic effects), ability to induce or repress gene expression patterns (nutritional transcriptomics effects), the occurrence and activity of specific proteins (nutriproteomic effects), and/or the dose and temporal changes in cellular small molecular weight compounds (metabolomics effects) can influence the overall response to any food (Hsing et al., 2002). Knowledge about each of these genomic variables will allow for a preemptive approach to those who will benefit most from the ingestion of garlic or other related allium foods (Figure 1). This “omic” information may also provide valuable clues about specificity in response and will assist in the identification of surrogate fluids/tissues and associated biomarkers that can be used for predicting responders or identify anyone who might be placed at risk because of dietary change (Ommen and Groten, 2004). Unraveling the importance of each of the potential sites of regulation within the “omics” is particularly challenging but holds promise to help explain the mounting inconsistencies in the scientific literature about diet and cancer prevention interrelationships, especially those related to garlic and cancer prevention.

BIOACTIVE FOOD COMPONENTS AND THEIR METABOLITES The most important constituent within garlic that accounts for its anticancer properties in a model system remains to be determined. Nevertheless, there is a wealth of evidence suggesting that organosulfur constituents are likely significant contributors. Sulfur in garlic is known to reach concentrations as high as 1% of its dry weight (Amagase et al., 2001). Within the allium family, considerable variability occurs in the content and chemical sulfur species, which can be markedly influenced by the vegetation period examined, as well as plant genetics (Krest et al., 2000; Brandolini et al., 2005). Thus, it is not surprising that considerable variability is observed when food disappearance data for garlic or associated allium foods are correlated with cancer risk and mortality. The complexity of this interrelationship can also be further modified by constituents other than sulfur content and speciation including the rather significant amount of oligosaccharide that occurs. Oligosaccharides have been reported to influence gastrointestinal (GI) flora or GI function, both of which are associated with a cancer risk in some experimental models (VanLoo, 2004). Although garlic has a moderate amount of protein, it is a relatively rich source of the amino acid arginine, which has experimentally been reported to suppress inflammatory processes, which again has been linked to reduced cancer risk (Lind, 2004). The presence of several other factors, including selenium and flavonoids, may also influence several cellular

FIGURE 1 Garlic and the “omics.” Evidence indicates that a number of processes associated with genes and their products are influenced by garlic or one or more of its allyl sulfur components. Thus, genes may influence absorption, metabolism, or the site of action of bioactive components within garlic. Each of these potential gene and gene product modifiers of the action of garlic are briefly discussed in the text.

processes that have been linked experimentally to cancer incidence and tumor behavior (Borek, 2001; Dong et al., 2001; Sengupta et al., 2004). The intracellular metabolite actually accounting for the change in cell signaling due to garlic and thereby leading to a phenotypic change has also yet to be defined. Although considerable information points to the ability of garlic to suppress the incidence and multiplicity of chemically induced tumors, it does not do so by changing the growth of the host. Thus, normal nonneoplastic cells do not appear to be as sensitive to the effects of garlic or its organosulfur constituents as neoplastic cells. In fact, some of the allyl sulfur compounds may be protective to normal functioning cells. Data from Koh et al. (2005) suggest that low concentration of diallyl disulfide (DADS) was neuroprotective possibly by activating PI3K/Akt and by inhibiting GSK-3 activation, cytochrome c release, caspase-3 activation, and PARP cleavage. However, when exposures were raised to excess, cytotoxicity occurred. Likewise, normal liver cells appear to be more resistant to the toxic effects of selected allyl sulfur compounds than neoplastic cells (Sundaram and Milner, 1993; Liu and Yeh, 2000). These inconsistencies indicate that biological responses reflect the uptake and/or the formation of the active intermediate and its rate of removal, which is likely variable across cell types. Differences in each of these variables likely explains the variation in sensitivity of various neoplastic cells to the organosulfur compounds, as well as the lower susceptibility of nonneoplastic cells to growth inhibition by these agents (Sundaram and Milner, 1996).

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It is certainly possible that the active organosulfur intermediate may be a radical. Koh et al. (2005) have provided some of the strongest evidence that enhanced radical formation is likely involved because growth inhibition was found to correlate with the occurrence of free radicals and membrane lipid peroxidation in neuronal cells (nPC12) treated with DADS. Other cellular events started to change when exposures were increased to ~50 µM DADS, including a block in PI3K/Akt and the activation of GSK-3 and caspase-3, the release of cytochrome c, and the cleavage of PARP (Koh et al., 2005). It remains to be determined whether a block in radical formation would prevent these subsequent cellular responses. These observations also raise the possibility that the protective effects of garlic against chemical carcinogenesis might relate to its antioxidant properties (Khanum et al., 2004). However, its ability to alter the growth of an existing tumor may relate to the ability to increase intracellular oxidative damage (Koh et al., 2005). Clearly, additional studies are necessary to resolve this interesting conundrum. Not all allyl sulfur compounds appear equally toxic to cells. Water-soluble compounds found in deodorized garlic, such as S-allyl cysteine (SAC), are far less toxic than the lipid-soluble compounds such as DADS (Liu and Yeh, 2000; Knowles and Milner, 2001; Elango et al., 2004). Generally, the antiproliferative effects of the allyl sulfur compounds increase as the number of sulfur atoms increases (Sundaram and Milner, 1996; Knowles and Milner, 2001; Elango et al., 2004). Although all cells will ultimately become susceptible to the toxic effects of allyl sulfur compounds when the concentration becomes sufficiently high, there are clear differences in response among various cell types. More attention to how uptake, metabolism, and excretion of specific allyl sulfur compounds vary should help explain why these differences in sensitivity exist and help point to which tissue(s) may be most responsive to dietary fortification with garlic.

FREE RADICALS AND GARLIC Reactive oxygen species (ROS) are known to arise from endogenous processes and exogenous exposures. These ROS are believed to cause genetic oxidation/damage to DNA and other macromolecules. Unchecked, this oxidative damage may lead to a host of conditions including cancer. Normally, this process is held in check by elaborate endogenous or exogenous antioxidant processes. Garlic is one of several foods with proposed antioxidant properties (Milner, 2001; Riblin, 2001; Thomson and Ali, 2003). Deodorized garlic preparations also appear to be protective against oxidative damage. Studies by Gedik et al. (2005) revealed that long-term administration of aqueous garlic extract (AGE) alleviated liver fibrosis and oxidative damage, as indicated by reduced myeloperoxidase activity in rats with

biliary obstruction. Using another model, Sener et al. (2003) reported that peeled crushed garlic extracts reversed the decrease in GSH level and the increase in fatty acid oxidation radicals (malondialdehyde) and MPO activity resulting from thermal stress. Although it remains unclear whether a block in oxidation accounts for the plethora of published manuscripts about the anticarcinogenic and properties associated with garlic and its sulfur constituents, there is evidence to the contrary. Because disulfides and trisulfides arising from garlic can deplete intracellular glutathione, oxidative stress might be involved in the upregulation of carcinogen metabolism, especially those involved with phase II detoxifying enzymes. Adding glutathione and Nacetylcysteine to HepG2 cells was discovered to markedly reduce the induction of glutathione S-transferase (GST), presumably by blocking antioxidant-responsive element (ARE) activity, presumably because it helped maintain Nrf2 in a complex form in the cytosol, thus preventing its migration to the nucleus (Chen et al., 2004). Diallyl compounds are also known to form volatile epoxide intermediates by way of unsaturated double-bond oxidation and, thus, may contribute to the induction of phase II enzymes. Support for the idea that oxidative intermediates are involved in the response to diallyl trisulfide (DATS) comes from the observation that addition of epoxide hydrolase will decrease, at least partially, the ability of diallyl compounds to increase ARE activity while also slightly attenuating Nrf2 protein induction (Chen et al., 2004).

EPIGENOMICS AND GARLIC Epigenomics can also influence genetic expression patterns. Several regulatory proteins including DNA methyltransferases, methyl-cytosine guanine dinucleotide binding proteins, histone-modifying enzymes, chromatin remodeling factors, and their multimolecular complexes are involved in controlling epigenomic processes (Ross, 2003; GallouKabani and Junien, 2005). Because several dietary components may influence epigenomic events, these processes represent additional control sites at which bioactive food components in garlic may reduce cancer burden. Although the impact of garlic on DNA methylation has not been adequately investigated, it is known that allyl sulfurs can influence DNA methylation processes indirectly by influencing blocking carcinogen metabolism. Carcinogen bind-ing to DNA is known to influence DNA methylation patterns (Zhang et al., 2005), and this process is recognized to be influenced by dietary fortification with garlic and many of its sulfur constituents (Milner, 2001; Khanum et al., 2004). There is also evidence that the components of garlic can affect histone homeostasis. Druesne et al. (2004) found that DADS and allyl mercaptan were very effective in increasing histone H3 acetylation in Caco-2 and HT-29 cells in

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culture. The reason for the histone H4 hyperacetylation occurring preferentially at the lysine residues 12 and 16 remains to be resolved but appears to relate to reduction in histone deacetylase activity (Druesne et al., 2004). The DADS-induced hyperacetylation was accompanied by an increase in p21 (waf1/cip1) expression, at mRNA and protein levels, again demonstrating that epigenomic events can influence subsequent gene expression patterns. Such intracellular changes may account for the ability of DADS and other allyl sulfur compounds to cause the accumulation of cells in the G2 phase of the cell cycle (Knowles and Milner, 2003). DADS and allyl mercaptan are rather unique in that they join a relatively short list of dietary factors that have been found to alter histone homeostasis (i.e., caloric restriction, butyrate, catechins, and sulforaphane). Studies by Lea and Randolph (2001) indicate that the ability of allyl sulfurs to affect epigenomic events is not consistent across all cell types. Interestingly, their data suggest that normal liver cells are more responsive to histone regulation than other nonneoplastic cells. More comparative studies are necessary to explain why these differences exist among various types of nonneoplastic and neoplastic cells. Regardless, such changes would typically be associated with differentiation of cells and, therefore, may have profound implications in the role of diet in the regulation of stem cells.

GARLIC AND TRANSCRIPTOMICS Data from cDNA array studies reveal that the antiproliferative effects of DADS may relate to changes in the expression of a host of genes including those related to alterations in cellular matrix gene expression (Frantz et al., 2000). Specifically, DADS exposure was shown to downregulate the expression of aggrecan 1, tenascin R, vitronectin, and cadherin 5, whereas it upregulated 40S ribosomal protein SA, platelet-derived growth factor–associated protein, and glia-derived neurite-promoting factor levels. These changes in matrix protein expression may influence cellular adhesion, as sometimes observed in studies using allicin and other allyl sulfides (Ledezma et al., 2004; Sela et al., 2004). Frantz et al. (2000) reported that the increase in HT-29 cell detachment by aqueous garlic extracts related to an increase in epidermal growth factor receptor and integrin-α6 mRNA expressions. Additional studies are necessary to characterize more fully which changes in gene expression patterns explain the likely multiple targets involved with the anticancer and antitumorigenic properties associated with increased intake of garlic and its related sulfur constituents. The GST family of xenobiotic-metabolizing enzymes is involved in the metabolic detoxification of various environmental carcinogens and may influence a number of cellular processes including the clearance of oxidative stress products and the modulation of cell proliferation and apoptosis

signaling pathways (Mahajan and Atkins, 2005). Andorfer et al. (2004) compared in a short-term feeding study the effects of DADS, diallylthiosulfinate (allicin), and butylated hydroxyanisole on GST expression in the GI tract and liver of mice. The effects of DADS and allicin on GST expression were especially prominent in stomach and small intestine, where there were major coordinate changes in GST subunit profiles. In particular, the transcripts of the mGSTM1 and mGSTM4 genes, which share large segments of common 5′-flanking sequences, and their corresponding subunits were selectively induced. Although liver and colon GSTs were also increased, but to a lesser extent, there was no effect on heart, brain, and testis, suggesting gene expression patterns are not equally influenced across all tissues. Their data also indicate these organosulfur compounds may operate on GST transcription through a reversible modification of certain protein sulfhydryl groups, shifts in reduced glutathione/oxidized glutathione ratios, and resultant changes in cellular redox status. Additional studies are necessary to clarify the role of thiol switches as a site of action of organosulfur compounds occurring in garlic and other foods. As mentioned previously, studies by Chen et al. (2004) demonstrate that the ability of DATS to cause ARE gene activation and Nrf2 protein accumulation correlated with phase II gene expression induction. Using transient transfection HepG2 cells, they found that DATS-induced ARE activity was inhibited by dominant-negative Nrf2 Kelch-like ECH-associating protein 1 and constructs. The ability of DATS to influence cellular thiol status may account for this phenomenon. Pretreatment with various upstream protein kinase inhibitors revealed that the protein kinase C pathway was not directly involved in this induction of ARE activity, but that a calcium-dependent signaling pathway was involved (Chen et al., 2004).

GARLIC AND PROTEOMICS The examination of patterns of changes in protein expression and their modifications, or proteomics, presents a formidable challenge to the scientific community (Barnes and Kim, 2004). Undeniably, considerable evidence points to the ability of essential nutrient deficiencies to alter the rate of protein synthesis and degradation. Today, it is clear that both essential and nonessential nutrients can influence not only protein anabolism and catabolism but markedly influence post-translation processes by influencing the degree of phosphorylation, glycosylation, nitration, and ubiquitination. New information generated by a proteomic approach will undeniably have a major impact on understanding how subtle changes in diet can influence protein signals involved with cancer. Understanding proteomics will require a greater attention to not only which food exposure causes a change

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Multiple Targets

but how this response is influenced by the duration of consumption and how compensatory cellular processes influence these changes. The occurrence and post-translation patterns of specific proteins have been observed to be modified by the ingestion of allium vegetables or their associated sulfur constituents. Although in many cases scientists have focused on the impact of allyl sulfur compounds on enzymatic activity, it is clear that activity may reflect a difference in the protein content and the amount occurring in the active state. For example, diallyl sulfide (DAS) was found to induce the expression of the wild-type p53 and to downregulate the expression of mutant (mut) p53 in cells in culture when Western blot analysis and immunohistochemical protein detection were combined with multivariable flow cytometry (Wen et al., 2004). The increase in the expression of the wild-type tumor suppressor gene protein p53 was accompanied by elevation of the levels of cyclin-dependent kinase inhibitor p21/waf1. Changes in activity may also be a result of a combination in the quantity and activity of specific cellular proteins. Exposure of synchronized colonic cells to DADS increased p34(cdc2) hyperphosphorylation by 15% (Knowles and Milner, 2000). Consistent with its ability to slightly increase the quantity of hyperphosphorylated p34 (cdc2), DADS also decreased cdc25C protein expression. These findings suggest that the ability of DADS to inhibit p34(cdc2) kinase activation occurs because of decreased p34(cdc2)/cyclin B1 complex formation and a shift in the p34(cdc2) hyperphosphorylation state (Knowles and Milner, 2000). Similar conclusions were reached by Wu et al. (2004) when the effects of DAS, DADS, and DATS were found to modify the cell cycle of human liver tumor cells (J5) in culture.

MULTIPLE TARGETS There is compelling evidence that several organosulfur compounds in garlic can influence a number of processes involved with cancer. Interestingly, both water-soluble and lipid-soluble allyl sulfurs have been reported to influence these molecular events (Figure 2). Some of the processes modified include the blocking of mutagenesis, blocking carcinogen-DNA adduct formation, serving as a free-radical scavenger, blocking cell proliferation, promoting differentiation, and decreasing angiogenesis. Although there is a large body of evidence supporting each of these plus other mechanisms, there is a need for additional research to determine which change is causally related to a cancer-preventive activity associated with garlic and its allyl sulfur components. The following is a brief account of some of the evidence linking garlic and related sulfur components with some of the processes associated with cancer.

FIGURE 2 Garlic may influence multiple cancer processes. Dietary garlic may influence genetic and epigenetic events associated with several diseased states including cancer. Alterations in each of these processes is highly dependent on the form and quantity of allyl sulfur provided and the duration of exposure.

Carcinogen Bioactivation By far the most compelling evidence about the mechanism by which allyl sulfur compounds can inhibit the cancer process comes from studies demonstrating that they can markedly suppress carcinogen bioactivation and the subsequent formation of DNA adducts. Studies with several chemical carcinogens document the fact that the anticancer properties associated with garlic are not limited to a specific animal model or to a particular tissue. Moreover, these studies demonstrate that both water- and lipid-soluble allyl sulfur compounds are effective in retarding chemical carcinogenesis. Because several types of allyl sulfur compounds offer protection against chemical carcinogenesis, multiple mechanisms are possible (Borek, 2001; Milner, 2001; Yang et al., 2001; Thomson and Ali, 2003; Andorfer et al., 2004; Khanum et al., 2004; Sengupta et al., 2004). Certainly, it does not appear that a single cytochrome P450 (CYP) enzymatic change could account for the observed protection based on the variety of carcinogens examined. Nevertheless, one class of carcinogens that appear to be particularly sensitive to water- and lipid-soluble allyl sulfurs is the nitrosamines (Yang et al., 2001). Much of the evidence in the literature suggests that garlic may promote the formation of nitrosothiols, thereby reducing their ability to form carcinogenic nitrosamines (Dion et al., 1997). The decrease in carcinogenicity observed may also arise from a block in the formation and/or bioactivation of nitrosamines (Gao et al., 2002). A competitive block or autocatalysis of CYP 2E1 may account for part of this inhibition, at least for lipid-soluble organosulfur agents (Gao et al., 2002). DAS is sequentially converted to diallyl sulfoxide (DASO) and diallyl sulfone (DASO2) by CYP 2E1. Studies by Davenport and Wargovich (2005) found that although DADS and AMS

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decreased CYP 2E1, adding dipropyl sulfide, dipropyl disulfide, propyl methyl sulfide, or SAC did not influence the expression of hepatic protein or influence CYP 2E1 mRNA levels. Although polymorphisms in CYP 2E1 might be logically assumed to influence the response to garlic, no such relationship has been observed, at least with the risk of esophageal and stomach cancer (Gao et al., 2002). Nevertheless, several studies have shown that different garlic compounds can reduce CYP 2E1 activity, presumably by serving as competitive inhibitors. Because allyl sulfurs inhibit the actions of several carcinogens not requiring CYP 2E1 activity, it is logical to assume that alterations in other phase I, or also II (Andorfer et al., 2004; Chen et al., 2004), enzymatic targets are involved.

Hormonal Regulation The association between estrogen exposure, either with or without progestin, and breast cancer risk continues to be a topic of immense interest and debate (Creasman, 2005). Although no significant effect of garlic or its constituents on estradiol have been reported, a change in the biological response to diethylstilbestrol (DES), a synthetic estrogen known to increase mammary cancer in animal models, has been observed (Green et al., 2003). Part of the effects of DAS may arise from its ability to block DES metabolism and the subsequent formation of DNA adducts by blocking lipid hydroperoxides in mammary tissue (Green et al., 2003). The androgen dependence of the growth of some tumor types including prostate cancer is well recognized. Adding several allyl sulfur compounds, in particular S-allylmercaptocysteine (SAMC), was found to enhance the rate of testosterone disappearance from cells in culture and may have accounted in part for the antitumorigenic properties observed (Pinto et al., 2000). Adding SAMC created a situation similar to androgen deprivation. Although the mechanism responsible for this effect remains to be determined, it is conceivable that it involves the conversion of testosterone to metabolites that have less affinity for androgen receptors (Pinto et al., 2000). Other hormones may also be involved, including luteinizing hormone, as demonstrated in research by Oi et al. (2001).

Inflammation and Immunocompetence The anticancer properties linked with garlic may also arise from suppression of inflammation and promotion of immunocompetence. Garlic oil derivatives have been found to differentially suppress the production of nitric oxide and prostaglandin E2 (PGE2) in activated macrophages (Chang and Chen, 2005). These studies found that DAS decreased stimulated nitric oxide and PGE2 production by inhibiting inducible synthase and cyclooxygenase-2 expressions. DADS inhibited activated nitric oxide production by

decreasing inducible synthase expression and by directly clearing nitric oxide (Chang and Chen, 2005). Cytokine production has also been reported to be reduced significantly in the presence of garlic extract (Hodge et al., 2002). Studies by Lang et al. (2004) suggest that another organosulfur compound, allicin, exerts its immunomodulatory effect on intestinal epithelial cells by attenuating intestinal inflammation. Allicin was found to markedly inhibit the spontaneous and tumor necrosis factor-α (TNF-α)–induced secretion of IL-1β, IL-8, IP-10, and MIG from the two cell lines in a dose-dependent manner and suppressed the expression of IL-8 and IL-1β mRNA levels. Allicin was also found to suppress the degradation of IκB, which may have mediated these effects. However, the effects are complex because garlic derivatives appear to have both stimulatory and inhibitory properties in lymphocyte proliferation and lipopolysaccharide (LPS)-induced TNF-α generation (Romano et al., 1997; Salman et al., 1999). Whether these variations in response relate to the type of sulfur compound tested, the duration of exposure, or some other modifier remains to be determined. Evidence exists that a nonsteroidal anti-inflammatory drug (NSAID)–activated gene (NAG-1) possesses both proapoptotic and antitumorigenic activities. Data in the literature reveal that this gene is upregulated by anticancer agents such as DADS and NSAIDs (Bottone et al., 2002). Adding DADS in vitro produced a dose-dependent increase in NAG-1. Induction of p53 was found to precede that of NAG-1. DADS did not induce NAG-1 or p53 in a p53 mutant cell line (Bottone et al., 2002).

Antiproliferation and Apoptosis A variety of allyl sulfur compounds have been reported to reduce the growth of neoplastic cells in culture and in vivo (Sundaram and Milner, 1993, 1996; Knowles and Milner, 2001; Wen et al., 2004; Wu et al., 2004). At least part of this reduced growth relates to a blockage in the cell cycle, most frequently in the G2/M phase (Wen et al., 2004; Wu et al., 2004). The transitory nature of this inhibition suggests that the rates of clearance of allyl sulfur are likely an important determinant of the magnitude of the response. Certainly, all cells are not equally susceptible to the antiproliferative effects of these sulfur compounds, and in particular nonneoplastic cells tend to be less responsive. Generally, as the concentration of the allyl sulfur compound increases, there is also a shift from a depression in cell proliferation to a greater involvement of apoptosis. However, insufficient attention has been given to which process is actually influenced first. Considerable evidence points to the dependence of the allyl component and the number of sulfur atoms as determinants of the degree of antiproliferative and apoptotic response. DATS has been reported to be >10 times as effective as DADS in retarding some tumors. As reviewed previously, alterations in several molecular targets may explain

Interaction with Other Food Components

the antiproliferative and apoptotic effects of allyl sulfur compounds. As additional information about the molecular targets for the various organosulfur compounds arises, it will be possible to develop better predictive models for determining which individuals will benefit most from dietary change with allium foods. The use of this “nutritional preemption” approach should allow for the use of specific foods, such as garlic, at critical points that allow for a block in the initiation and progression of a pathway that leads to an unhealthy or lethal phenotype.

INTERACTION WITH OTHER FOOD COMPONENTS It is certainly conceivable that a variety of food components might influence the ability of garlic to suppress the cancer process. It is somewhat logical that variation in the sulfur content of the diet might be one variable (Amagase et al., 1996). Although the amount of evidence is not overwhelming, it appears that variation in dietary sulfur amino acids, unsaturated fats, and selenium can influence the response to allyl sulfur compounds (Amagase et al., 1996). When carcinogen-DNA adducts were used as the biomarker, there was a beneficial effect of combining garlic, selenite, and retinyl acetate beyond that caused by each ingredient alone. The effects of combining garlic with tomatoes were examined for their ability to synergistically inhibit hamster buccal pouch cancer (Bhuvaneswari et al., 2005). Combining garlic and tomatoes suppressed the incidence and mean tumor burden of hamster buccal pouch carcinomas possibly by decreasing phase I enzyme activities and by increasing phase II enzyme activities. The effect of combining bioactive food components on the antitumorigenic properties of allyl sulfur compounds has not been adequately examined. As the era of molecular nutrition unfolds, we will gain a greater understanding about which of the many processes modified by garlic is critical to induce a phenotypic change. This information will be fundamental to the development of tailored strategies for reducing cancer burden. The identification of biomarkers that can be used to predict who will respond is essential for effective intervention to occur.

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37 Berries NAVINDRA P. SEERAM

INTRODUCTION

STRUCTURAL TYPES OF BERRY BIOACTIVES

Studies suggest that consumption of a phytochemicalrich diet, which includes fruits and vegetables, contributes toward reducing the risk of certain types of human cancers. Among fruits, berries such as blackberries, black raspberries, blueberries, cranberries, raspberries, and strawberries are popularly consumed in our diet in fresh and in processed forms such as beverages, yogurts, jellies, and jams. In addition, berry extracts are widely consumed in dietary supplement forms for their potential human health benefits. A wide number of laboratory and animal studies have shown that berries have anticancer properties that are attributed to their high content of a diverse range of bioactive phytochemicals. These compounds include flavonoids (anthocyanins, flavonols, and flavanols), condensed tannins (proanthocyanidins [PAs]), hydrolyzable tannins (ellagitannins [ETs] and gallotannins [GTs]), stilbenoids (e.g., resveratrol), phenolic acids (hydroxybenzoic and hydroxycinnamic acids), and lignans. Berry bioactives may impart anticancer effects through various complementary and overlapping mechanisms of action. These include antioxidant effects as free radical scavengers, as well as acting indirectly through antioxidant actions that protect DNA from damage, the regulation of enzymes important in metabolizing xenobiotics and carcinogens, the modulation of nuclear receptors, gene expression, and subcellular signaling pathways of proliferation, angiogenesis, and apoptosis. This chapter reviews the research progress, advances, future challenges, and impact of berry consumption on cancer prevention.

Over the past few decades, knowledge of the composition of berry fruits in fresh, freeze-dried, and extract forms has rapidly expanded with the advent of highly sensitive analytical methods, allowing researchers to establish phytochemical profiles or “chemical fingerprints” of these fruits. These bioactive phytochemicals have been identified as flavonoids (anthocyanins, flavonols, and flavanols), condensed tannins (PAs), hydrolyzable tannins (ETs and GTs), stilbenoids, phenolic acids (hydroxybenzoic and hydroxycinnamic acids), and lignans (Seeram and Nair, 2002; Manach et al., 2004). The considerable diversity in the skeletal structures of these compounds imparts unique biological properties to each class that affects their absorption, distribution, metabolism, and excretion in humans (Beecher, 2003; Manach et al., 2004, 2005; Williamson and Manach, 2005). The structural diversity of berry bioactives can be observed by the varying types and oxidation levels of their heterocycle ring, their substitution patterns of hydroxylation (bearing an -OH group), the existence of stereoisomers, their glycosylation by various sugars, and/or acylation by organic and phenolic acids, and by conjugation with themselves to form polymers, etc. The main structural classes of berry bioactives are shown in Figure 1 and are discussed in the following subsections.


Anthocyanins and Anthocyanidins Of the numerous phytochemicals found in berries, the anthocyanins are probably the best known. Anthocyanins are

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Chapter 37/Berries R1 R1

R2 OH

O+

HO

HO

O

R3

R2

OH

OH

OH

O

OH

R 1 = R 2 = OH; R 3 = H; Quercetin R 1 = R 3 = H; R 2 = OH; Kaempferol R 1 = R 2 = R 3 = OH; Myricetin

R 1 = R 2 = H; Pelargonidin R 1 = OH; R 2 = H; Cyanidin R 1 = R 2 = OH; Delphinidin Anthocyanidins

Flavonols

R1

R1

OH

R2 HO

HO

O

O

R2

8 3

R3 OH

OH

OH

O

R 1 = R 2 = R 3 = OH; Catechin R 1 = R 2 = OH; R 3 = gallate; Gallocatechin

n

R1= R2 = H, Propelargonidins R1= H, R2= OH, Procyanidins R1, R2 = OH, Prodelphinidins Proanthocyanidins

Flavanols

R1

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O

R2 OH

R2

R3

OH

R 1 = R 2 = OH; R 1 = OH;

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Caffeic acid Coumaric acid

Hydroxycinnamic acid

Hydroxybenzoic acid

HO

CH2OH

HO

CH2OH

HO

OH HO

OCH3 OCH3

Resveratrol

Secoisolariciresinol

Stilbenoids

Lignans

OH HO OH HO O HO HO HO

O C O C O O O HO HO O O O O HO C OH O HO O OH OH HO HO

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O O O

OH O

C

O OH OH OH

OH OH OH

OH OH OH

Sanguiin H-6 Ellagitannin

FIGURE 1 Structural classes of bioactives found in berries: anthocyanins, flavonol, flavanols, proanthocyanidins, ellagitannins, hydroxycinnamic and hydroxybenzoic acids, stilbenoids, and lignans.

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the pigments that impart the attractive red, blue, purple, violet, and intermediate red-purple to berries and many other fruits, vegetables, and grains (Mazza and Miniati, 1993; Seeram et al., 2001a,b; Wu and Prior, 2005). Similar to most other flavonoids, anthocyanins occur naturally in fruits and vegetables as glycosides. The de-glycosylated or aglycone forms of anthocyanins are known as anthocyanidins, which exist as different chemical structures, both colored and uncolored, according to variations in pH (Seeram et al., 2001b). Several hundred anthocyanins are known varying in the basic anthocyanidin skeleton, with the six most common being cyanidin (the most ubiquitous), delphinidin, pelargonidin, malvidin, petunidin, and peonidin (Seeram and Nair, 2002; Seeram et al., 2003) (Figure 1). Apart from variations in the anthocyanidin core, structural diversification is further achieved by the identity and extent and position at which glycosides and/or acyl groups are attached to the skeleton. The most common glycosides encountered on anthocyanins are glucose, galactose, rhamnose, and arabinose usually as 3-glycosides or as 3,5-diglycosides. However, rutinosides (6-O-L-rhamnosyl-d-glucosides), sophorosides (2-O-d-glucosyl-d-glucosides), and sambubiosides (2-O-d-xylosyl-d-glucosides) are also common, as are some 3,7-diglycosides and 3-triosides (Clifford, 2000). Common acylating agents include phenolic acids (caffeic, pcoumaric, ferulic, and sinapic), which may themselves bear glycosidic sugars, and a range of aliphatic acids (e.g., acetic, malic, malonic, oxalic, and succinic).

Flavonols Flavonols are the most ubiquitous flavonoids found in foods and, similar to anthocyanins, also occur naturally in glycosylated forms where the associated sugar moiety is often glucose or rhamnose, although other substituents such as galactose, arabinose, xylose, and glucuronic acid may also be involved. The most common flavonol aglycons are quercetin (the most ubiquitous), kaempferol, and myricetin (Figure 1). Because their biosynthesis is stimulated by light, flavonols accumulate in the outer and aerial tissues of plants (in leaves) and fruits (in skin). However, in berries, they are commonly found in the flesh and achenes of the fruits such as in strawberries (Seeram et al., 2006) and in raspberries (Kaehkoenen et al., 2001).

Flavanols Flavanols are the only class of flavonoids that do not occur naturally as glycosides. Flavanols exist in both monomeric (catechins) and polymeric (PAs; see later discussion) forms (Figure 1). Catechins and epicatechin are the main flavanols found in berry fruits, whereas gallocatechin, epigallocatechin, and epigallocatechin gallate are found in certain seeds of leguminous plants, in grapes, and in tea (Manach et al., 2004).

Condensed Tannins (Proanthocyanidins) Condensed tannins, or PAs, are dimers, oligomers, and polymers of catechins that are bound together by links between C4 and C8 (or C6) (Buelga-Santos and Scalbert, 2000) (Figure 1). However, in addition to C–C linkages, PAs can also have ether linkages between C2–O5 or C2–O7, referred to as A-type linkages. PAs can also be classified based on their constituent units that are produced on acid hydrolysis. In this case, they are referred to by the nomenclature system established for anthocyanidins. The most ubiquitous PAs are procyanidins, consisting of individual (epi)catechin units. Others include the less common propelargonidins, consisting of (epi)afzelechin units, and prodelphinidins, consisting of (epi)gallocatechin units. It is difficult to estimate the PA content of berries and other foods because they have a wide range of structures and molecular weights and their mean degree of polymerization in foods has rarely been determined (Manach et al., 2004; Gu et al., 2004). Among common edible berries, blueberries and cranberries contain high levels of PAs (Gu et al., 2004).

Hydrolyzable Tannins (Ellagitannins and Gallotannins) Hydrolyzable tannins are categorized into GTs and ETs. GTs are esters of gallic acid, whereas ETs are composed of esters of hexahydroxydiphenic acid (HHDP: 6,6′dicarbonyl-2,2′,3,3′,4,4′-hexahydroxybiphenyl moiety). On hydrolysis of ETs, the HHDP moiety spontaneously rearranges to release ellagic acid, hence, their name (Clifford and Scalbert, 2000). The hydrolysis reaction to form ellagic acid is usually employed for the detection and quantification or ETs in berry fruits (Amakura et al., 2000). ET monomers can be further oxidized in plants and form dimers, trimers, and tetramers with molecular weights up to 4000 Da. Among common edible berries, strawberries, raspberries, blackberries, and black raspberries contain high levels of ETs (Maeaettae-Riihinen et al., 2004a,b).

Phenolic Acids Berries contain a wide variety of phenolic acids (Zadernowski et al., 2005), which occur as derivatives of hydroxybenzoic acid (e.g., gallic acid) and hydroxycinnamic acid (e.g., caffeic acid) (Figure 1). The hydroxycinnamic acids are more common than the hydroxybenzoic acids and consist chiefly of p-coumaric, caffeic, ferulic, and sinapic acids. Hydroxycinnamic acids are found in all parts of fruits, although the highest concentrations are seen in the outer parts of ripe fruits. Concentrations generally decrease during the course of ripening, but total quantities increase as the fruit increases in size (Clifford, 2004). Although phenolic acids are found in berries in free forms, they can be considered components of complex polymers such as hydrolyzable

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tannins (GTs or ETs) or PAs. Hence, they can be released from their “parent” compounds in vivo by changes in physiological pH, enzymatic, or gut bacterial action, or in vitro by storage, processing conditions, and so on (see the section “Bioavailability and Metabolism of Berry Bioactives”).

Stilbenoids Stilbenes are phenolic-based compounds, of which the most widely recognized is resveratrol (3,4′,5-trihydroxystilbene) (Figure 1). Resveratrol has attracted immense attention because of its biological properties including its anticancer effects (reviewed in Aggarwal et al., 2004). Resveratrol has a number of closely related analogs (e.g., pterostilbene and piceatannol), and it plays an important role as the parent molecule of oligomers known as the viniferins (Aggarwal et al., 2004). Among edible berries, resveratrol and its analogs have been reported in members of the Vaccinum genus, for example, blueberry, bilberry, lingonberry, and cranberry (Rimando et al., 2004).

genus Rubus (e.g., cloudberry and raspberry), the main phenolics are ETs, and in the genus Fragaria (e.g., strawberry), ETs are the second largest group after anthocyanins (Kaehkoenen et al., 2001). Flavonols based on quercetin and kaempferol aglycons are also reported to be present in substantial quantities in strawberries (Seeram et al., 2006). Phenolic acids are found in high levels in rowanberries (genus Sorbus) and anthocyanins in chokeberries (genus Aronia). In the genus Ribes (currants and gooseberries), anthocyanins predominate, as well as in crowberries (genus Empetrum) (Kaehkoenen et al., 2001). Maeaettae-Riihinen (2004) identified and quantified soluble and insoluble phenolics in 18 species of berries belonging to the families Grossulariaceae, Ericaceae, Rosaceae, Empetraceae, Elaeagnaceae, and Caprifoliaceae. The berry phenolics were identified as conjugated hydroxycinnamic acids, flavonol glycosides, and anthocyanins. The study showed similarities in the distribution of conjugated forms of phenolics among berry species of the same family and differences in the profiles and compositions of anthocyanins among individual types of berries.

Lignans Lignans are formed from two phenylpropane units. Although the richest dietary sources of lignans are flaxseed and linseed, which contain secoisolariciresinol (Figure 1) and low quantities of matairesinol (Scalbert and Williamson, 2000; Clifford, 2004), some cereals, grains, fruits, and certain vegetables also contain traces of lignans. Lignans have been reported in berries such as strawberry, blackberry, raspberry, cloudberry, cranberry, lingonberry, and blueberry (Mazur et al., 2000).

Triterpenes and Sterols Although the predominant bioactive phytochemicals in berries are phenolic compounds (see previous subsections), there have been reports of nonpolar compounds such as ursolic acid, triterpene hydroxycinnamates, and β-sitosterol in members of the Vaccinum species, for example, cranberries (Murphy et al., 2003; Schmandke, 2004).

DISTRIBUTION OF BERRY BIOACTIVES Berries contain a wide range of phytochemicals, the most predominant of which are phenolic (aromatic ring bearing hydroxyl, -OH, group) in nature. Phenolic contents have been reported to vary considerably among different berry genera. For example, in the Vaccinum genus, anthocyanins are reported as the main phenolics in bilberry, bog-whortleberry, and cranberry, but in cowberries, flavanols and procyanidins predominate (Kaehkoenen et al., 2001). In the

Mechanisms of Chemoprevention by Berry Bioactives Epidemiological evidence has shown that the consumption of a phytochemical-rich diet contributes toward reducing the risk of certain types of human cancers (Steinmetz and Potter, 1991; Meyskens and Zabo, 2005). Although the predominant phytochemicals in berry fruits are phenolic compounds such as anthocyanins, its other phytochemicals may also contribute synergistically and/or additively to its anticancer activities (Camire, 2002; Seeram et al., 2004). Hence, berries contain a wide range of phytochemicals that may impart anticancer effects through various complementary and overlapping mechanisms of action (Liu, 2003; Heber, 2004). The individual constituents of berries, as well as total berry extracts, have been shown in in vitro and in vivo studies to exert anticancer properties through different mechanisms. For example, berry bioactives may exert anticancer effects through their antioxidant properties as free radical scavengers while acting indirectly through antioxidant actions that protect DNA from damage, the regulation of enzymes important in metabolizing xenobiotics and carcinogens, the modulation of nuclear receptors, gene expression, and subcellular signaling pathways of proliferation, angiogenesis, and apoptosis. Although an effort has been made to categorize the actions of berry bioactives in the following discussion, the reader will notice overlap between sections, which is due to the multimechanistic and complementary pathways through which berry phytochemicals exert their anticancer effects.

Distribution of Berry Bioactives

Modulation of Signaling Pathways of Proliferation, Apoptosis, and Cell Cycle Arrest Studies have investigated the subcellular signaling and molecular mechanisms through which berry phytochemicals may exert their anticancer properties. These include the ability of berry extracts and their purified bioactives to inhibit cell proliferation and modulate cell cycle arrest, induce DNA repair and signal transduction, and apoptosis in cancer cells while having little or no cytotoxic effect on normal noncancerous cells. Although many of these studies have focused on evaluating whole berry extracts, a significant number of bioassay-guided fractionations, aimed at isolating and identifying the active constituents present in the berry extracts, have been done. Some of the in vitro anticancer studies conducted on berries are discussed in the following paragraphs. Blueberry, black chokeberry, lingonberry, and raspberry extracts were shown to decrease the proliferation of human colon HT-29 and breast MCF-7 cancer cells in a dosedependent manner (Olsson et al., 2004). Similarly, whole cranberry fruit extracts were assayed for tumor growth inhibition using seven tumor cell lines and selective inhibition of K562 leukemia, and HT-29 colon cells were observed from a methanolic extract in the range of 16–125 µg/ml (Yan et al., 2002). Bilberry extract was shown to inhibit the growth of human HL60 leukemia cells and HCT116 colon carcinoma cells and induce apoptotic cell bodies and nucleosomal DNA fragmentation in the HL60 leukemia cells (Katsube et al., 2003). Juranic (2005) compared the antiproliferative action of red raspberries to malignant human colon carcinoma LS174 cells and to normal immune competent cells, with the action of ellagic acid (a bioactive constituent of berries) alone. Results from this study showed that raspberry extracts possess the potential for antiproliferative action against human colon carcinoma cells, which was correlated with its content of ellagic acid. In this study, the cytotoxic activity of the extracts was not pronounced on normal human PBMC colon cells (Juranic, 2005). A bioactivity-guided fractionation of cranberries identified triterpenoid esters that inhibited the growth of MCF-7 breast, ME180 cervical, and PC3 prostate tumor cell lines (Murphy et al., 2003). The major bioactives were identified as the cis- and trans- isomers of 3-O-p-hydroxycinnamoyl ursolic acid. The authors reported that the cis- isomer showed superior antiproliferative activity when compared with its trans counterpart and to quercetin and cyanidin-3galactoside. Phenylboronic acid was also isolated from the cranberry extract, but it did not exhibit significant antitumor activity (Murphy et al., 2003). Ramos (2005) investigated the effects of individual purified berry bioactives, quercetin, chlorogenic acid, and

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epicatechin, as well as whole strawberry fruit extract on the viability and apoptosis of human hepatoma HepG2 cells. Quercetin and the strawberry fruit extract inhibited cell viability in a dose-dependent manner, whereas chlorogenic acid and epicatechin had no prominent effects on the cell death rate. Similarly, quercetin and the strawberry extract, but not chlorogenic acid and epicatechin, induced apoptosis in hepatoma HepG2 cells. In cell cycle progression experiments, quercetin and the strawberry extract were observed to arrest the G1 phase in the cell cycle before apoptosis (Ramos et al., 2005). In a study to identify the chemopreventive phytochemicals in black raspberries, Han et al. (2005) identified two of its bioactives as ferulic acid and β-sitosterol. In the bioassay-guided fractionation experiment, Han (2005) also demonstrated that a purified fraction eluted with ethanol during chromatography of the organic extract of freeze-dried black raspberries inhibited the growth of premalignant and malignant but not normal human oral epithelial cell lines. However, purified ellagic acid alone was found to inhibit the growth of normal, premalignant, and malignant human oral cell lines. Using flow cytometry and Western blotting of cell cycle–regulatory proteins, these workers also investigated molecular mechanisms by which ferulic acid, β-sitosterol, and the berry ethanol fraction could selectively inhibit the growth of premalignant and malignant oral cells. They observed no discernible change in the cell cycle distribution following treatment of cells with the berry ethanol fraction. Premalignant and malignant cells redistributed to the G2/M phase of the cell cycle following incubation with ferulic acid, whereas β-sitosterol–treated premalignant and malignant cells accumulated in the G0/G1 and G2/M phases. The berry ethanol fraction reduced the levels of cyclin A and cell division cycle gene 2 (cdc2) in premalignant cells and cyclin B1, cyclin D1, and cdc2 in the malignant cell lines. The berry ethanol fraction also elevated the levels of p21waf1/cip1 in the malignant cell line. Ferulic acid treatment led to increased levels of cyclin B1 and cdc2 in both cell lines, and p21waf1/cip1 was induced in the malignant cell line; on the other hand, β-sitosterol reduced the levels of cyclin B1 and cdc2 while increasing p21waf1/cip1 in both the premalignant and the malignant cell lines. The authors concluded that the growth-inhibitory effects of black raspberries on premalignant and malignant human oral cells may reside in specific components that target aberrant signaling pathways regulating cell cycle progression (Han et al., 2005). Cell lines of differing origins have been shown to respond with varying degrees of sensitivity in growth toward berry extracts. For example, a cranberry presscake (the material remaining after squeezing the juice from berries) extract inhibited proliferation of eight human tumor cell lines of multiple origins (Ferguson et al., 2004). The androgendependent prostate cell line, LNCaP, was the most sensitive

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of those tested, whereas the estrogen-independent breast line, MDA-MB-435, and the androgen-independent prostate line, DU145, were the least sensitive. Other human tumor lines originating from breast (MCF-7), skin (SK-MEL-5), colon (HT-29), lung (DMS114), and brain (U87) had intermediate sensitivities to the cranberry extract. Using flow cytometric analyses of DNA distribution (for cell cycle) and annexin V positivity (for apoptosis), the authors showed that a purified fraction blocked cell cycle progression and induced cells to undergo apoptosis in a dose-dependent manner in MDA-MB-435 breast cells (Ferguson et al., 2004). In another study, human oral, prostate, and colon cancer cells responded with differing sensitivities to cranberry bioactives (Seeram et al., 2004). In this study, Seeram et al. (2004) showed that although the individual cranberry phytochemicals, such as its flavonols, organic acids, PAs, and anthocyanins, inhibited the growth of the tumor cells, when they were combined, as found in a total cranberry extract, antiproliferative activities were significantly enhanced. The effects of berry extracts on signal transduction pathways have also been reported. For example, Huang (2002) reported on the effects of a black raspberry methanol extract and its purified fractions on transactivation of activated protein-1 (AP-1) and nuclear factor-κB (NFκB) induced by the carcinogen BaP diol-epoxide (BPDE) in mouse epidermal cells. AP-1 and NFκB are transcription factors associated with carcinogenesis (Bode and Dong, 2000). Inhibition of AP-1 activity has been shown to lead to the suppression of cell transformation (Dong et al., 1997). NFκB is also an important regulator in deciding cell fate, such as programmed cell death and proliferation control, and is critical in tumorigenesis (Baldwin, 1996). The inhibitory effects of a purified black raspberry fraction on AP-1 and NFκB were mediated via inhibition of mitogen-activated protein kinase (MAPK) activation and inhibitory subunit κB phosphorylation, respectively. Pretreatment of cells with purified berry fractions did not result in an inhibition of BPDE binding to DNA, which suggested that this was not a mechanism of reduced AP-1 and NFκB activities. In addition, none of the purified fractions were found to affect p53-dependent transcription activity. In view of the important roles of AP-1 and NFκB in tumor promotion and progression, the authors concluded that the ability of black raspberries to inhibit tumor development may be mediated by impairing signal transduction pathways, leading to activation of AP-1 and NFκB (Huang et al., 2002). The inhibitory effects of strawberry on tetradecanoylphorbol-13-acetate (TPA)—or ultraviolet B (UVB)– induced AP-1 and NFκB were recently demonstrated by Wang (2005). TPA and UVB are well-known tumor promoters and can produce reactive oxygen species (ROS) and stimulate AP-1 and NFκB activities by activating MAPK signaling pathways such as the extracellular signal– regulated kinases 1/2 (ERK1/2), c-Jun amino-terminal

kinases (JNKs), and the p38 MAPK (Schulze-Osthoff et al., 1997; Hou et al., 2004). These workers also evaluated strawberry extracts for inhibition of proliferation and transformation of human and mouse cancer cells. The strawberry extracts inhibited the proliferation of human lung epithelial cancer cell line A549 and decreased TPA-induced neoplastic transformation of mouse epidermal cells. In addition, pretreatment of the mouse epidermal cells with strawberry extracts resulted in the inhibition of both UVB- and TPAinduced AP-1 and NFκB transactivation. Furthermore, the strawberry extracts also blocked TPA-induced phosphorylation of ERKs and UVB-induced phosphorylation of ERKs and JNK kinase in the mouse epidermal cell culture. These results suggest that the ability of strawberries to block UVBand TPA-induced AP-1 and NFκB activation might be due to their antioxidant properties and their ability to reduce oxidative stress. The authors concluded that the oxidative events that regulate AP-1 and NFκB transactivation could be important molecular targets for cancer prevention. Therefore, strawberries may be highly effective as chemopreventive agents that act by targeting the downregulation of AP-1 and NFκB activities, blocking MAPK signaling, and suppressing cancer cell proliferation and transformation (Wang et al., 2005). In another study, freeze-dried strawberries and freezedried black raspberries were extracted with methanol, partitioned, and chromatographed into several fractions (Xue et al., 2001). The extracts, along with ellagic acid, were analyzed for anti-transformation activity in a Syrian hamster embryo (SHE) cell transformation model. None of the extracts or ellagic acid alone produced an increase in morphological transformation. For assessment of chemopreventive activity, SHE cells were treated with test samples and benzopyrene for 7 days. Ellagic acid and two of the purified fractions produced a dose-dependent decrease in transformation compared with the benzopyrene treatment only. Ellagic acid and the two purified fractions were further examined using a 24-hour co-treatment with benzopyrene or a 6-day treatment following 24 hours with benzopyrene. Ellagic acid showed inhibitory ability in both protocols. The two purified fractions significantly reduced benzopyreneinduced transformation when co-treated with benzopyrene for 24 hours. The authors concluded that the possible mechanism by which the purified fractions inhibited cell transformation appear to involve interference of uptake, activation, detoxification of benzopyrene, and/or intervention of DNA binding and DNA repair (Xue et al., 2001).

Angiogenesis Antiangiogenic (the ability to reduce unwanted growth of blood vessels) approaches to treat cancer represent a priority area in vascular tumor biology. Angiogenesisinhibiting agents have the potential for inhibiting tumor growth and limiting the dissemination of metastasis, thus

Distribution of Berry Bioactives

keeping cancers in a static growth state for prolonged periods. Extracts of blueberry, bilberry, cranberry, elderberry, raspberry, and strawberry were studied for antioxidant efficacy, cytotoxic potential, cellular uptake, and antiangiogenic properties (Roy et al., 2002; Bagchi et al., 2004). The authors evaluated various combinations of the extracts and showed that a “synergistic extract” significantly inhibited both hydrogen peroxide (H2O2) and tumor necrosis factor-α (TNF-α)–induced vascular endothelial growth factor (VEGF, a key regulator of tumor angiogenesis) expression by human keratinocytes (Roy et al., 2002; Bagchi et al., 2004). The same research group also studied the synergistic extract in an in vivo mice model of angiogenesis and observed that it significantly inhibited basal monocyte chemotactic protein-1 (MCP-1), a protein responsible for facilitating angiogenesis (Atalay et al., 2003). In addition, the synergistic extract significantly inhibited inducible NFκB transcription. Endothelioma cells pretreated with the synergistic berry extract showed a diminished ability to form hemangioma and markedly decreased tumor growth by >50% (Atalay et al., 2003). Liu (2005) reported that black raspberry extract showed antiangiogenic properties in a human tissue–based in vitro fibrin clot angiogenesis assay. Bioassay-guided fractionation of the berry extract resulted in a highly potent antiangiogenic fraction that completely inhibited angiogenic initiation and vessel growth. Further subfractionation of this active fraction revealed the coexistence of multiple antiangiogenic compounds, one of which has been identified as gallic acid (a bioactive constituent of berries). However, the authors concluded that the whole fraction was superior to its subfractions and that the active ingredients may be additive and/or synergistic in their antiangiogenic effects (Liu et al., 2005).

Antimutagenicity The initial step in the formation of cancer is damage to the genome of a somatic cell producing a mutation in an oncogene or a tumor suppressor gene. Strawberry, blueberry, and raspberry juices and extracts were evaluated for their ability to inhibit the production of mutations by the directacting mutagen methyl methanesulfonate and the metabolically activated carcinogen benzopyrene (Hope et al., 2004). The berry juices significantly inhibited mutagenesis caused by both carcinogens. Ethanol extracts from freeze-dried fruits of strawberry and blueberry cultivars were also evaluated, and hydrolyzable tannin-containing fractions from strawberries were found to be most effective at inhibiting mutations.

Induction of Antioxidant Enzymes ROS are formed during the normal endogenous metabolic process and from exogenous factors such as ionizing

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radiation, diet, and xenobiotics (Davis, 1987; Halliwell and Gutteridge, 1989). Oxidative stress arises either from the overproduction of ROS or from the deficiency of antioxidant defense or repair mechanisms, resulting in reversible or irreversible damage to critical cellular macromolecules such as lipids, proteins, and DNA (Davis, 1987). Oxidative stress has been implicated in initiation, promotion, and progression phases of carcinogenesis (Cerutti, 1985), and the resulting unrepaired oxidative damage has been suggested to play a role in other chronic diseases, including cancer (Ames et al., 1993). Aerobic organisms constantly battle the adverse effects of ROS by increasing the production of biochemical antioxidants (such as glutathione and ascorbate) or by inducing endogenous antioxidant enzymes including superoxide dismutase (SOD), catalase, glutathione peroxidase (GPOD), and glutathione reductase (GR). These scavenging antioxidant molecules and the endogenous antioxidant enzymes attenuate the ROS concentration to maintain an intracellular reduction and oxidation (redox) balance. Strawberries have been shown to have antioxidant capacity against ROS such as ROO, O2−, H2O2, OH, and 1O2 (Wang and Lin, 2000; Wang and Zheng, 2001). These workers also demonstrated the activities of antioxidant enzymes including SOD, G-POD, and GR in strawberries (Wang et al., 2005). The activities of antioxidant enzymes in blackberries have also been shown (Jiao and Wang, 2000). The activities of antioxidant enzymes in both berries were shown to be positively correlated with their antioxidant capacity (Jiao and Wang, 2000; Wang et al., 2005). Studies have been designed to investigate correlations between antioxidative potential and antiproliferative activities of berries. For example, Meyers (2003) investigated eight strawberry cultivars to find out if their antioxidant capacities, by the total oxyradical scavenging capacity (TOSC) assay, can be correlated with their antiproliferative activities. Overall, although the proliferation of HepG2 human liver cancer cells was significantly inhibited in a dose-dependent manner after exposure to all strawberry cultivar extracts, these workers found no relationship between antiproliferative activity and antioxidant content. In another study, similar results were observed when extracts of four raspberry cultivars were evaluated for total antioxidant capacity and cancer cell antiproliferative activity (Weber and Liu, 2002). In this study, the antioxidant activity of each cultivar was directly related to the total amount of phenolics, but no significant relationship was found between antiproliferative activity and the total amount of phenolics (Weber and Liu, 2002). Berry extracts and their purified bioactives have been investigated for effects on the production of cytokines such as TNF-α, which mediates a variety of cell functions including stimulation of nitric oxide (NO) production. TNF-α has been related to oxidative stress and diseases such as chronic inflammation (Park et al., 2000). Wang (2002) investigated common purified berry phenolics and anthocyanin-enriched

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blueberry, blackberry, Saskatoon berries, and black currant extracts for their effects on the production of TNF-α in RAW 264.7 macrophages. Gallic acid and catechin showed small but significant effects, whereas chlorogenic acid had no effect on TNF-α production. The flavonol quercetin inhibited TNF-α production, whereas kaempferol and myricetin induced the secretion of TNF-α. The individual anthocyanidins (pelargonidin, cyanidin, delphinidin, peonidin, and malvidin), anthocyanins (malvidin 3-glucoside and malvidin 3,5-diglucosides), and anthocyanin-enriched blueberry extracts induced TNF-α production and acted as modulators of the immune response in the activated macrophages (Wang and Mazza, 2002).

Inhibitors of Detoxification Enzymes Phase I and phase II metabolizing enzymes play an important role in the biotransformation of carcinogens and xenobiotics in the human body. In phase I reactions, these chemicals undergo bioactivation catalyzed by cytochrome, P450 (CYP) isozymes to produce strong electrophiles which are capable of interacting with cellular nucleophiles such as DNA to form adducts eventually culminating in mutagenesis and neoplastic transformation. In normal cells, the reactive intermediates formed by phase I reactions are then conjugated via phase II enzymes with glucuronides, sulfate, or glutathione, facilitating their excretion. Phase II xenobiotic detoxification enzymes include glutathione S-transferase, sulfotran ferases, UDP-glucuronyl transferases, and quinone reductase (QR). Conjugation enhances hydrophilicity of the metabolites, thus facilitating elimination of the carcinogen from the body. Although phase I enzymes increase the carcinogenic potency of a chemical, phase II enzymes serve to detoxify the electrophilic metabolites. An imbalance in phase I and phase II carcinogen-metabolizing enzymes has been documented in a wide range of malignant tumors including breast cancer (Williams and Phillips, 2000). Kansanen et al. (1996) investigated the in vitro effects of some flavonoids and phenolic acids common to berries, as well as extracts of strawberry and black currant, on CYP 1A1 isozyme. These workers found that the flavonoid aglycons and berry extracts were effective inhibitors of CYP 1A1, whereas the flavonoid glycosides and phenolic acids were not (Kansanen et al., 1996). Other studies have shown that flavonoid-rich fractions from Vaccinium species, such as cranberries, induce QR in vitro (Bomser et al., 1996). This study also showed that a cranberry extract inhibited expression of ornithine decarboxylase (ODC), a key enzyme responsible for polyamine biosynthesis (Bomser et al., 1996).

Inhibitors of Metalloproteinase Enzymes Matrix metalloproteinases (MMPs) are enzymes essential for development, and remodeling of tissues and aberrant

overexpression of these enzymes contributes to several pathological conditions. In particular, MMP overexpression in cancer plays a significant role in metastasis by providing a mechanism for invasion and progression. MMPs are involved in proteolysis of the extracellular matrix, which can lead to the progression of tumors (Pupa et al., 2002). Raspberries and blackberries have been shown to inhibit the activities of MMP-2 and MMP-9 (Tate et al., 2004). Quercetin, a typical berry flavonol, has been shown to have a chemoprotective role through complex effects on signal transduction involved in cell proliferation, including increased expression of endogenous tissue inhibitors of MMPs (Morrow et al., 2001). Ursolic acid, which has been reported in berries such as cranberries (Murphy et al., 2003), reduced the expression of MMP-9 in HT-1080 fibrosarcoma cells and consequently inhibited tumor invasion (Cha et al., 1996, 1998). As part of a study to determine the effects of cranberry extracts on prostate tumor proliferation, evaluation of the effects of whole cranberry extract and purified fractions on MMP expression in DU145 prostate cells was conducted (Kondo et al., 2004; Neto et al., 2006). The whole cranberry extract inhibited expression of MMP-2 and MMP-9 in the cells at 100 µg/ml concentrations. A purified PA fraction, at 500 µg/ml, inhibited MMP-2 expression completely and resulted in ~75% inhibition of MMP-9 activity (Kondo et al., 2004; Neto et al., 2006).

CHEMOPREVENTION STUDIES WITH BERRY BIOACTIVES Animal Studies The chemopreventive potential of freeze-dried berries against aerodigestive tract cancers, such as oral cavity and esophageal cancers, has been demonstrated in a number of animal studies (Stoner et al., 1999; Kresty et al., 2001; Aziz et al., 2002; Casto et al., 2002). Studies have suggested that berries, which contain high amounts of ETs and ellagic acid (e.g., strawberries and black raspberries), show better effects against these cancers than those that contain PAs as their predominant tannins (e.g., blueberries) (Aziz et al., 2002). However, although ellagic acid, an abundant component in berries, has been shown to inhibit carcinogenesis both in vitro and in vivo, several studies have reported that other compounds in berries may also contribute to the observed anticancer effects (Stoner et al., 1999). Studies have been designed to investigate the chemopreventive effects of berries during initiation and progression phases of cancer. Lyophilized black raspberries (LBRs) were evaluated against N-nitrosomethylbenzylamine (NMBA)– induced esophageal tumorigenesis in the F344 rat during initiation and post-initiation phases of carcinogenesis

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(Kresty et al., 2001). Anti-initiation studies included a 30week tumorigenicity period, quantification of DNA adducts, and NMBA metabolism study. Feeding 5 and 10% LBRs, for 2 weeks prior to NMBA treatment and throughout a 30week period significantly reduced tumor multiplicity (39 and 49%, respectively). The post-initiation inhibitory potential of berries was evaluated in a second experiment with administration of LBRs after NMBA treatment. In this experiment, animals were sacrificed at 15, 25, and 35 weeks. The LBRs were found to inhibit tumor progression as evidenced by significant reductions in the formation of preneoplastic esophageal lesions, decreased tumor incidence and multiplicity, and reduced cellular proliferation. At 25 weeks, both 5 and 10% LBRs significantly reduced tumor incidence, tumor multiplicity, proliferation rates, and preneoplastic lesion development. At 35 weeks, only the 5% LBRs significantly reduced tumor incidence and multiplicity, proliferation indices, and preneoplastic lesion formation. The authors concluded that dietary administration of LBRs inhibited events associated with the initiation, promotion, and progression stages of carcinogenesis. In another study with LBRs, the hamster cheek pouch (HCP) assay was used to evaluate the ability of the berries to inhibit oral cavity tumors (Casto et al., 2002). Hamsters were fed 5 and 10% LBRs in the diet for 2 weeks prior to treatment with dimethylbenzanthracene (DMBA) and for 10 weeks thereafter. HCPs were painted with the DMBA to induce tumor formation. The animals were sacrificed 12–13 weeks after the beginning of DMBA treatment and the number and volume of tumors were determined. The authors observed a significant difference in tumor number between the LBR-treated and control groups (Casto et al., 2002). Blueberries were evaluated for their ability to inhibit NMBA tumorigenesis in the rat esophagus (Aziz et al., 2002). As previously mentioned, blueberries differ in phytochemical content from strawberries and black raspberries in that their predominant tannins are PAs and not ETs. Two weeks prior to NMBA treatment, animals were placed on a control diet or diets containing 5 and 10% freeze-dried blueberries. At 25 weeks, animals on 5 and 10% blueberries produced no significant differences in tumor incidence, multiplicity, or size when compared with NMBA-treated controls. In addition, blueberries did not reduce the formation of NMBA-induced O6-methylguanine adducts in esophageal DNA when fed at 10% of the diet. The authors concluded that blueberries appear to lack components that inhibit the initiation and progression of NMBA-induced tumori-genesis in the rat esophagus (Aziz et al., 2002). It should be noted that although blueberries did not show anticancer properties in these studies, its consumption has been correlated with other health benefits such as antineurodegenerative properties and so on (Joseph et al., 1998, 2003).

Human Studies A survey of the literature revealed no published human clinical studies examining the anticancer effects of berries. However, data are available on the absorption, distribution, metabolism, and excretion of berry bioactives in humans obtained from foods, beverages, extracts, and as singly purified compounds (see the section “Bioavailability and Metabolism of Berry Bioactives,” later in this chapter). A phase I study sought to examine tolerance for high dietary levels of freeze-dried berries that would be necessary for chemoprevention studies (The James 2002/2003 Annual Report, Comprehensive Cancer Center, Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, Ohio State University). Because the berries were well tolerated, studies are being designed to test the anticancer effects of these berries among individuals with precancerous lesions and at high risk for esophageal, colon, and oral cancers (www.jamesline.com/patientsandpublic/research/ index.cfm). A human study (Moller et al., 2004) investigated the effects of black currant anthocyanins on the steady state level of oxidative DNA damage in mononuclear blood cells of 57 healthy human subjects, determined as strand breaks, as well as endonuclease III and formamidopyrimidine DNA glycosylase (Fpg). The baseline level of oxidative DNA damage was low, and Fpg-sensitive sites increased during the intervention within the black currant anthocyanin group, whereas there were no differences between treatments in any of the DNA damage markers. The authors concluded that even large amounts of berry antioxidants did not decrease the already low steady state levels of oxidative DNA damage in healthy, adequately nourished humans (Moller et al., 2004).

DIETARY INTAKE OF BERRY BIOACTIVES As previously discussed, phenolics are the predominant phytochemicals present in berry fruits. Unfortunately, because of the considerable number of factors that can modify their concentrations, reference food composition tables are not available. Their estimation in foods is also extremely challenging because of their wide structural diversity existing as different conjugated forms and complex polymeric nature, ill-defined structures, and unavailability of commercial standards. As a result, data on their dietary intake, as well as data on their bioavailability and pharmacokinetics in humans, are limited. Only partial data for certain phenolics, such as flavonols, have been published on the basis of direct food analysis or bibliographic compilations (Manach et al., 2004). In the United States, agencies such as the U.S. Department of Agriculture (USDA) have established databases in which the flavonoid contents of

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selected foods, compiled from varying bibliographic sources, are available (USDA web site). Studies have shown a high variability in polyphenol intake based on variations in individual food preferences. A diet consisting of several servings of fruit and vegetables per day can provide up to 1 g of phenolics consisting of the following: 16% flavonols, flavones, and flavanones; 17% anthocyanins; 20% catechins; and 45% PAs, ETs, and other “bioflavonoids” (Kuhnau, 1976). Among the berry bioactives, research has targeted individual data for specific classes of compounds. For example, consumption of flavonols has been estimated at 20–25 mg/day in the United States, Denmark, and Holland (Hertog et al., 1993; Justesen et al., 1997; Sampson et al., 2002). In Italy, consumption ranged from 5 to 125 mg/day, and the mean value was 35 mg/day (Pietta et al., 1996). In Finland, where high amounts of berries are eaten, anthocyanin consumption was found to be 82 mg/day on average, although some intakes exceeded 200 mg/day (Heinonen, 2001). Intake of phenolic acids ranged from 6 to 987 mg/day in Germany (Radtke et al., 1998). Dietary burden, nature, and occurrence of specific classes of berry bioactives have been previously reviewed. These include anthocyanins (Clifford, 2000a); ETs (Clifford and Scalbert, 2000); PAs (Santos-Buelga and Scalbert, 2000); sterols (Piironen, 2000); hydroxybenzoic acid derivatives (Tomás-Barberán and Clifford, 2000); chlorogenic acid and other cinnamates (Clifford, 2000b); lignans and stilbenes (Cassidy et al., 2000); and flavonols, flavones, and flavanols (Hollman and Art, 2000).

BIOAVAILABILITY AND METABOLISM OF BERRY BIOACTIVES Several review articles have been published on the bioavailability and metabolism of phenolics (Scalbert and Williamson, 2000; Rechner et al., 2002; Manach et al., 2004, 2005a; Walle, 2004; Williamson and Manach, 2005). A wide body of studies has shown that although phenolics are the predominant phytochemicals in human diet, they are not necessarily the most active in vivo, either because they are poorly absorbed from the gut, highly metabolized, or rapidly eliminated. In addition, because of digestive and hepatic activities, the bioactivities of phenolic metabolites that are bioavailable in blood and target organs may differ significantly from their native forms. Hence, extensive knowledge of the bioavailability of phenolics is essential if their health effects are to be understood. Because most phenolics are present in food in the form of esters, glycosides, or polymers that cannot be absorbed in their native form, they must be hydrolyzed by intestinal enzymes or by the colonic microflora before they can be absorbed, for example, into aglycons, which can then be

absorbed from the small intestine. When the gut microflora is involved, the efficiency of absorption is often reduced because the flora also degrades the aglycons that it releases and produces various simple phenolic and aromatic acids in the process. During the course of absorption, phenolics are conjugated (usually methylated, sulfated, and glucuronidated) in the small intestine and later in the liver, a metabolic detoxification process that facilitates biliary and urinary elimination. Because the conjugation mechanisms are highly efficient, aglycons are either absent in blood or present in very low concentrations after consumption of nutritional doses. Circulating phenolics are conjugated derivatives that are extensively bound to albumin (Scalbert and Williamson, 2000). Phenolics are able to penetrate tissues, particularly those in which they are metabolized, but reports on their ability to accumulate within specific target tissues are scarce (Manach et al., 2004). Phenolics are secreted via the biliary route into the duodenum, where they are subjected to the action of bacterial enzymes, especially β-glucuronidase, in the distal segments of the intestine, after which they may be reabsorbed. This enterohepatic recycling may lead to a longer presence of phenolics within the body. The metabolism of the major classes of berry bioactives is discussed in the following subsections.

Metabolism of Anthocyanins Among berry bioactives, the metabolism and bioavailability of anthocyanins in both human and animal models have been well studied (Prior, 2002). Human studies with anthocyanins have shown that albeit at low concentrations, they are detectable intact in human plasma (Cao and Prior, 1999; Cao et al., 2001; Milbury et al., 2002; Mazza et al., 2002; Bitsch, 2004). The elimination of plasma anthocyanins appears to follow first-order kinetics, and most anthocyanins were excreted in the urine within 4 hours of feeding (Milbury et al., 2002). Other studies have shown that anthocyanins and their metabolites are detectable in human urine after consumption of boysenberries (Cooney et al., 2004), strawberries (Felgines et al., 2003), and elderberries (Murkovic et al., 2001). The bioavailability of 15 structurally different anthocyanins from blueberry, boysenberry, black raspberry, and black currant in both humans and rats was investigated (McGhie et al., 2003). This study showed that intact and unmetabolized anthocyanins were detected in urine, although the relative concentrations of dosing varied, indicating that differences in bioavailability were due to variations in chemical structure. Anthocyanin metabolites and tissue distribution in digestive organs (stomach, jejunum, liver), kidney, and brain were studied in male Wistar rats fed with blackberry anthocyanins for 15 days (Talavera et al., 2005). Intact blackberry anthocyanins were detected in the stomach, while other organs (jejunum, liver, kidney) contained the anthocyanins in their intact and in

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their methylated and monoglucuronidated forms. Jejunum and blood plasma also contained anthocyanins in their aglycon forms. In the brain, the total anthocyanin content reached 0.25 nmol/g tissue (Talavera et al., 2005). Milbury et al. (2005) also demonstrated that berry anthocyanins cross the blood–brain barrier.

Metabolism of Flavonols Among berry flavonols, quercetin, the most ubiquitous flavonol in plant foods, is probably the most investigated. Hollman (1995, 1996) showed that quercetin is bioavailable in human plasma and demonstrated that glucosides of quercetin were more efficiently absorbed than quercetin itself, whereas the rhamnoglucoside (rutin) was less efficiently and less rapidly absorbed. The bioavailability of quercetin differs among food sources, depending on the type of glycosides they contain. For example, onions, which contain glucosides, are better sources of bioavailable quercetin than apples and tea, which contain rutin and other glycosides. The presence of intact glycosides of quercetin in plasma had been debated, but it is now accepted that such compounds are absent from plasma after nutritional doses (Manach et al., 2005). On metabolism, phenolic acids can also be produced from flavonols by the gut microflora. Quercetin degradation produces mainly 3,4-dihydroxyphenylacetic, 3-methoxy-4-hydroxyphenylacetic (homovanillic acid), and 3-hydroxyphenylacetic acids (Manach et al., 2005).

Metabolism of Tannins (Proanthocyanidins and Ellagitannins) Assessment of the bioavailability and metabolism of tannins (PAs, ETs, and GTs) remains a challenge because of their ill-defined structures, lack of authentic standards, and lack of accurate data on their compositions in foods. Because of these challenges, there are few human studies reporting bioavailability of PAs and ETs (Seeram et al., 2004; Manach et al., 2005). Although the detection of PA dimers B1 and B2 in human plasma has been reported (Holt et al., 2002), studies done both in vitro and in animals have shown that polymerization greatly impairs intestinal absorption (Déprez et al., 2001; Donovan et al., 2002). An ET, punicalagin (MW 1084), was detected intact in rat plasma and was reported as the largest polyphenol observed in vivo (Cerda et al., 2003). Given the poor absorption of these molecules in their intact forms, it is possible that their biological effects may be attributable not only to direct actions of tannins themselves, but to the actions of some of their metabolites that can be more readily absorbed. PAs and ETs may be degraded into various phenolic and aromatic acids and other metabolites by the microflora (Cerda et al.,

2005a,b; Manach et al., 2005). ETs have been shown to release ellagic acid in human plasma (Seeram et al., 2004). Whereas the microbial metabolism of PAs has never been studied in humans after consumption of purified PA polymers (Manach et al., 2005), that of ETs has been reported (Cerda et al., 2004, 2005a,b). Therefore, further investigations into the degradation of PAs into microbial metabolites must be further evaluated in humans.

CONCLUSIONS AND FUTURE DIRECTIONS In conclusion, an overwhelming number of cell culture and animal studies suggest that berries may have immense potential for the prevention and treatment of cancer. Berry bioactives may act individually, additively, and synergistically to exert their chemopreventive properties. Because extrapolations cannot be made between in vitro and animal studies to humans, future clinical trials should be designed to investigate the potential of berries for the prevention and treatment of human cancers. In addition, further details on absorption, distribution, metabolism, and mechanisms of action of berry bioactives in humans are necessary to determine effective dietary portions of berries. Whether bioactivities of berries are made stronger by the interactions of the many substances within a particular fruit, as well as in combination with phytochemicals from other fruits and vegetables, should be investigated. In addition, interactions of berries with prescription drugs and other herbal medicines, through their ability to modulate enzymes or cell receptors, should be investigated in carefully planned and controlled human clinical studies.

References Aggarwal, B.B., Bhardwaj, A., Aggarwal, R.S., Seeram, N.P., Shishodia, S., and Takada, Y. 2004. Role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies. Anticancer Res 24: 2783–2840. Amakura, Y., Okada, M., Tsuji, A., and Tonogai, Y. 2000. High-performance liquid chromatography determination with photodiode array detection of ellagic acid in fresh and processed fruits. J Chromatogr B 896: 87–93. Ames, B.N., Shigena, M.K., and Hagen, T.M. 1993. Oxidants, antioxidants and the degenerative diseases of aging. Proc Natl Acad Sci USA 90: 7915–7922. Atalay, M., Gordillo, G., Roy, S., Rovin, B., Bagchi, D., Bagchi, M., and Sen, C.K. 2003. Antiangiogenic property of edible berry in a model of hemangioma. FEBS Lett 544: 252–257. Aziz, R.M., Nines, R., Rodrigo, K., Harris, K., Hudson, T., Gupta, A., Morse, M., Carlton, P., and Stoner, G.D. 2002. The effect of freezedried blueberries on N-nitrosomethylbenzylamine tumorigenesis in the rat esophagus. Pharm Bio 40: 43–49. Bagchi, D., Sen, C.K., Bagchi, M., and Atalay, M. 2004. Anti-angiogenic, antioxidant, and anticarcinogenic properties of a novel anthocyanin-rich berry extract formula. Biochem Trans Biokhimiya 69: 75–80. Baldwin, A.S. 1996. The NF-kappa B and I kappa B proteins: new discoveries and insights. Annu Rev Immunol 14: 649–683.

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38 Isoprenoids and Novel Inhibitors of Mevalonate Pathway Activities HUANBIAO MO AND CHARLES E. ELSON

INTRODUCTION

Stayrook et al., 1997; Ura et al., 1998; Yaguchi et al., 1998; Ariazi et al., 1999; Elson et al., 1999; Mo and Elson, 1999; Mo et al., 1999; Sahin et al., 1999; McIntyre et al., 2000; Rioja et al., 2000; Crowell and Elson, 2001; Smalley and Eisen, 2002; Beaupre et al., 2003; Clark et al., 2003). Reviews record the characterization of 23,000 isoprenoids. Some of the mevalonate-derived secondary products are “pure” isoprenoids of varying structural complexity (Sacchettini and Poulter, 1997) but consisting only of multiples of the five-carbon isoprene unit (×), for example, monoterpenes (2×), sesquiterpenes (3×), diterpenes (4×), triterpenes (6×), tetraterpenes (8×), and polyterpenes (n×) (Bach, 1995). Prominent among the monocyclic monoterpenes reported to suppress tumor growth are a hydrocarbon (d-limonene), several alcohols (carvacrol, cresol, eugenol, perillyl alcohol, thymol), aldehydes (menthal, perillaldehyde), and a ketone (menthone). Bicyclic monoterpenes of interest include an alcohol (myrentol) and a ketone (verbenone). Acyclic monoterpenes include alcohols (geraniol, linalool) and an aldehyde (geranial). Corresponding acyclic monoterpenes containing a cis bond (nerol, neral) have lower tumorsuppressive potency (Tatman and Mo, 2002). The acyclic sesquiterpenoid alcohol, trans, trans farnesol is reportedly the most potent of the simple (volatile) isoprenoids (Tatman and Mo, 2002). Others are “mixed” isoprenoids: the prenylated coumarins, flavones, flavanols, isoflavones, chalcones, quinones, and chromanols, each with only a part of the molecule geraniol or farnesol being derived via the mevalonate pathway (Barron and Ibrahim, 1996). Among the mixed isoprenoids widely distributed in nature, the farnesylated chromanols δ-d- and γ-d-tocotrienol (He et al., 1997) and the farnesylated quinone menaquinone-3 (Yaguchi et al., 1997,

The mevalonate pathway supports pools of farnesyl diphosphate, geranylgeranyl diphosphate, and dolichol phosphate, products essential for cell survival and proliferation (Figure 1). Chemotherapeutic agents in clinical evaluation inhibit either the activities providing mevalonatederived intermediates (statins, phenylacetate, and phenylbutyrate) or the activity transferring the farnesyl moiety to small G proteins (farnesyl protein transferase inhibitors). The inhibitory actions attributed to the cyclic isoprenoids, perillyl alcohol and d-limonene, include inhibition of farnesyl protein transferase and activation of allyl diphosphate diphosphatase with a concomitant increase in the signaling molecule farnesol, triggering 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase degradation and inhibiting translation of HMG-CoA reductase mRNA. An overexpressed and sterol-feedback resistant HMG-CoA reductase activity in tumors, coupled with post-transcriptional downregulation of reductase activity triggered by isoprenoids, may afford isoprenoids tumor-targeted growth-suppressive potential.

ISOPRENOIDS: PURE AND MIXED Members of a broad class of plant products collectively termed isoprenoids, differing in size, complexity, and function derived from diverse mevalonate pathway activities operative in plants, suppress the growth of cultured cells, with selectivity for tumor cells and in preclinical studies suppress tumor growth with no evidence of host toxicity (Adany et al., 1994; Yazlovitskaya and Melnykovych, 1995;


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ACETATE Acyclic isoprenoids Farnesol Farnesol mimetics?

Cholesterol

3-HYDROXY-3-METHYLGLUTARYL CoA Transcriptional HMG CoA Reductase: Degradation Translation

HMG CoA Reductase

Cholesterol Control

Statins (-)

MEVALONIC ACID Cyclic isoprenoids d-Limonene? Perillyl alcohol? β-Ionone Thymol Carvacrol

Mevalonate PP decarboxylase Na phenylacetate (-) Na phenylbutyrate

Nascent Lamin B Farnesylated Lamin B

Allyl PP diphosphatase FARNESOL

FARNESYL PP

Farnesyl Protein Transferase

Peptidomimetics Non-peptidomim etics Perillyl alcohol? (-) d-Limonene?

Nascent Small G-proteins Farnesylated Small G-proteins Nascent Proteins Geranylgeranylated Proteins

Geranylgeranyl PP

N-Linked Glycosylation of Growth Factor Receptors

Dolichylation of Proteins

FIGURE 1 The mevalonate pathway supports pools of farnesyl diphosphate, geranylgeranyl diphosphate, and dolichol phosphate, products essential for cell survival and proliferation. Chemotherapeutic agents in clinical evaluation inhibit either the activities providing mevalonate-derived intermediates (statins, phenylacetate, and phenylbutyrate) or the activity transferring the farnesyl moiety to small G proteins (farnesyl protein transferase inhibitors). The inhibitory actions attributed to the cyclic isoprenoids, perillyl alcohol and d-limonene, include inhibition of farnesyl protein transferase, activation of allyl diphosphate diphosphatase with a concomitant increase in the signaling molecule, farnesol, triggering HMG-CoA reductase degradation and inhibiting translation of HMG-CoA reductase mRNA. An overexpressed and sterol-feedback–resistant HMG-CoA reductase activity in tumors, coupled with post-transcriptional downregulation of reductase activity triggered by isoprenoids, may afford isoprenoids tumor-targeted growth suppressive potential.

1998; Miyazawa et al., 2001) are reported to have very significant tumor-suppressive activity. Pure and mixed isoprenoids play regulatory roles in germination, growth, differentiation, flowering, senescence, abscission, and dormancy; provide antioxidant activity; attract pollinating insects; and provide a defense against predatory insects and fungi (Hemming, 1983; Dahiya et al., 1984; Janssen et al., 1987; De Rosa et al., 1994; Tahara et al., 1994; Gagnon et al., 1995; Rahalison et al., 1995; Klepzig et al., 1996; Saito et al., 1996; Pare and Tumlinson, 1997; Sadof and Grant, 1997). Consumers continue to enjoy the nutritional, antimicrobial, and sensory properties a number of diverse isoprenoids contribute to the food supply.

Sadly missing are the databanks required for estimating the quantities of pure and mixed isoprenoids presented in a typical diet (Tatman and Mo, 2002).

Isoprenoids Suppress Growth of Tumor Cells As a consequence of the structural diversity encompassing differences in number of isoprene residues, double bonds, bond configuration, cyclization, and head group polarity, isoprenoids differ substantially in tumorsuppressive potency (Elson, 1995, 1996; Elson et al., 1999; Mo et al., 1999, 2000; Tatman and Mo, 2002) and diverse tumor cell lines differ substantially in sensitivity to an indi-

Isoprenoids: Pure and Mixed

vidual isoprenoid (Mo and Elson, 1999; Mo et al., 1999). Although the differential impact of isoprenoids on the growth of tumor and normal cells has been broadly demonstrated, there remains uncertainty regarding the initiating event triggered by isoprenoids (Crowell and Elson, 2001). The growth of tumor cells reflects a “positive” balance between two factors, cell division and cell death. Definitive studies employing cell cycle analysis demonstrate that isoprenoids have an impact on both sides of the “balance” equation (Crowell and Elson, 2001).

Isoprenoids Arrest Tumor Cells at the G1/S Interface of the Cell Cycle Farnesol (Chakrabarti et al., 1991; Voziyan et al., 1993; Burke et al., 1997), farnesol derivatives (Mo et al., 2000; McAnally et al., 2003), geraniol (Shoff et al., 1991; Carnesecchi et al., 2004), geraniol derivatives (McAnally et al., 2004), β-ionone (Mo and Elson, 1999; Liu et al., 2004a, 2004b), perillyl alcohol (Bardon et al., 1998; Ferri et al., 2001; Shi and Gould, 2002; Elegbede et al., 2003), perillaldehyde (Elegbede et al., 2003), perillic acid (Beaupre et al., 2003), γ-tocotrienol (Mo and Elson, 1999), and menaquinone-3 (Miyazawa et al., 2001) slow the progress of diverse lines of tumor cells through the cell cycle with a resultant buildup of cells in the G1 phase. The G1 phase of the cell cycle represents the interval (gap) between mitosis (M phase) and DNA replication (S phase) in proliferating cells. Passage from G1 to S is regulated by a family of cyclins that act as regulatory subunits for cyclin-dependent kinases (cdks). Myc, a mitogenactivated transcription factor with downstream target genes encoding cyclins D2, D1, and E, modulates the cyclin/cdk complex that regulates the G1-to-S progression of cells. The cyclin/cdk complex is activated by the sequential phosphorylation and dephosphorylation of key residues located primarily in the cdk subunits. The cyclin/cdk complex of early G1 consists either of cdk2, cdk4, or cdk6 bound to a cyclin D isoform. When stabilized as a consequence of DNA damage, p53 upregulates expression of the cdk inhibitor, p21Waf1/Cip1, thereby holding cells in G1. Activation of transforming growth factor-β (TGF-β) receptors also leads to the inhibition of cyclin D/cdk activity preventing the phosphorylation of the retinoblastoma protein (Rb). Transition to the S phase is triggered by the phosphorylation of Rb following activation of the cyclin D/cdk complex. Phosphorylated Rb dissociates from transcription factor E2F, which is then free to initiate myc expression and DNA replication. Cyclin E/cdk2 accumulates during late G1 phase and triggers passage into S phase. An indirect action of myc is the sequestration of the cyclin E/cdk2 inhibitor, p27kip1, into cyclin D/cdk4 complexes (Harbour and Dean, 2000; Bartek and Lukas, 2001).

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Cells exposed to the cyclic monoterpenes, d-limonene or perillyl alcohol, geraniol, and β-ionone are arrested in G1 with concomitant decreased expression of c-myc (Giri et al., 1999), cyclin D1 (Bardon et al., 1998, 2002; Park et al., 2001; Shi and Gould, 2002), cyclin E (Ariazi et al., 1999), cdk2 (Bardon et al., 1998, 2002; Ariazi et al., 1999; Shi and Gould, 2002), and cdk4 (Bardon et al., 2002). Also reported as a consequence of exposure to cyclic monoterpenes (Ariazi et al., 1999; Bardon et al., 2002; Shi and Gould, 2002) and tocotrienols (Agarwal et al., 2004) is the increased expression of a cdk inhibitor, p21Waf1/Cip1; the resulting inhibition of cyclin D/cdk–mediated phosphorylation of Rb (Bardon et al., 2002; Shi and Gould, 2002) blocks the dissociation of the inactive Rb/E2F complex. Geraniol and β-ionone downregulate the expression of cyclins D1, E, and A1 and cdk2 (Duncan et al., 2004). Tocotrienols also downregulate the expression of c-myc (Nesaretnam et al., 2000). Acyclic isoprenoid derivatives suppress E2F activity (Ura et al., 1998) and induce p27kip1 expression (Miyazawa et al., 2001; Reuveni et al., 2003) with a concomitant suppression of cyclin E/cdk2 activity (Reuveni et al., 2003). The tocotrienol (Shun et al., 2004) and perillyl alcohol– stimulated upregulation of TGF-β2 and insulin-like growth factor-2 (IGF-2) receptors (Jirtle et al., 1993; Mills et al., 1995; Ariazi and Gould, 1996; Ariazi et al., 1999) may facilitate the trafficking of mitogens into lysosomes for degradation (Ariazi and Gould, 1996). All of the aforementioned reported responses to cyclic monoterpenes presage the arrest of cells in G1.

Isoprenoids Initiate Apoptotic Cell Death On the other side of the equation, farnesol (Adany et al., 1994; Haug et al., 1994; Voziyan et al., 1995; Miquel et al., 1996, 1998; Stayrook et al., 1997; Rioja et al., 2000), farnesyl derivatives (Perez-Sala et al., 1998; Ura et al., 1998; Mo et al., 2000; Smalley and Eisen, 2002; McAnally et al., 2004), geraniol (Shoff et al., 1991; Duncan et al., 2004), carotenoids (Yaguchi et al., 1998; Zhang et al., 1999; Briviba et al., 2001; Kotake-Nara et al., 2002; McAnally et al., 2004; Li et al., 2002; Palozza et al., 2002a, 2002b), βionone (Mo and Elson, 1999; Duncan et al., 2004; Liu et al., 2004a), limonene (Jirtle et al., 1993), perillyl alcohol (Mills et al., 1995; Reddy et al., 1997; Ariazi et al., 1999; Elegbede et al., 2003), perillic acid (Beaupre et al., 2003), and γtocotrienol (Mo and Elson, 1999; Sylvester and Shah, 2005; Birringer et al., 2003; Shah et al., 2003; Agarwal et al., 2004; Sakai et al., 2004; Takahashi and Loo, 2004) initiate apoptotic cell death. The relative abundance of proapoptotic (Bad, Bak, Bax, Bid, Bim) and antiapoptotic (Bcl-2, Bcl-XL) proteins determines the susceptibility of cells to programmed death. The proapoptotic proteins trigger caspase activation. Cyclic and acyclic carotenoids (Li et al., 2002; Palozza et al., 2002a,

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2002b), cyclic monoterpene, perillyl alcohol (Broitman et al., 1996; Burke et al., 2002), the acyclic sesquiterpene, farnesol (Broitman et al., 1996; Burke et al., 2002), and tocotrienols (Agarwal et al., 2004) suppress the expression of antiapoptotic Bcl-2; β-carotene (Palozza et al., 2002b), perillyl alcohol (Broitman et al., 1996; Burke et al., 2002), and farnesol (Broitman et al., 1996; Burke et al., 2002) suppress Bcl-XL expression. Tocotrienols (Agarwal et al., 2004), a finding requiring additional work (Takahashi and Loo, 2004), and perillyl alcohol induce expression of proapoptotic Bad, Bak, and Bax (Stayrook et al., 1997; Ariazi et al., 1999; Burke et al., 2002). Caspase activity is activated in cells incubated with perillyl alcohol (Beaupre et al., 2003), farnesol (Flach et al., 2000), tocotrienols (Sylvester and Shah, 2005; Shah et al., 2003; Sakai et al., 2004), and a farnesol mimetic vitamin K2 (Miyazawa et al., 2001). Expression of c-jun and c-fos, signal-transducing transcription factors of the AP-1 family, which appear to play roles in both cell proliferation and cell death (Ameyar et al., 2003; Eferl and Wagner 2003), are induced transiently in cells following exposure to perillyl alcohol (Ariazi et al., 1999; Satomi et al., 1999). Tocotrienols (Shun et al., 2004) and farnesylamine, an acyclic sesquiterpene derivative (Ura et al., 1998), similarly induce c-jun. The dynamic balance between fos- and jun-related proteins might play a decisive role in whether a cell survives or undergoes apoptosis (Bossy-Wetzel et al., 1997). The mechanism of the Rbregulated p53-dependent apoptotic pathway requires study (Harbour and Dean, 2000); as delineated earlier, the cyclin D/cdk–mediated phosphorylation of Rb and its subsequent dissociation from E2F is suppressed in cells incubated with perillyl alcohol. The isoprenoid-mediated initiation of apoptosis may be independent of a p53 function. β-Ionone and γ-tocotrienol initiate apoptosis in B16 and MCF-7 cells (Mo and Elson, 1999) that express wild-type p53 (Parker et al., 1994; David-Pfeuty et al., 1996; Gudas et al., 1996), in Caco-2 cells that express mutated p53 (Gartel et al., 1996), and in p53-null HL-60 cells (Koeffler et al., 1986; Parker et al., 1994). The suppressed post-translational processing of lamin B in nuclei of promyelocytic leukemia cells exposed to β-ionone interferes with the assembly of daughter nuclei and renders DNA available to p53-independent apoptotic endonuclease activities (Mo and Elson, 1999). Annexin I is upregulated in cells exposed to perillyl alcohol (Ariazi et al., 1999); upregulation of annexin I and facilitation of the recognition phosphatidylserine by the phosphatidylserine receptor (Fadok and Henson, 2003) might explain the redistribution of phosphatidylserine in cellular membranes (Clark et al., 2002) and changes in MAPK (Clark et al., 2003; Shun et al., 2004), as well as caspase-3 (Flach et al., 2000; Miyazawa et al., 2001; Shah et al., 2003; Agarwal et al., 2004; Sakai et al., 2004) activities in cells following exposure to isoprenoids. All of these reported responses to isoprenoids are proapoptotic.

PART A: POSTULATED ISOPRENOID-MEDIATED ACTION: SUPPRESSION OF FARNESYL PROTEIN TRANSFERASE ACTIVITY The diverse responses described earlier tend to be those that occur at midpoint or late in signal transduction pathways; this suggests that isoprenoids modulate an initiating event that has downstream impact on signal transduction. Interest has focused on the inhibition of farnesyl protein transferase (Figure 1), the activity catalyzing the transfer of a farnesyl moiety to the cysteine residue in the conserved carboxyl-terminus sequence (generally CAAX) of the small G proteins that are early components of interlocking signaling cascades (Coleman et al., 1997). Reviews of the effects of various farnesyl protein transferase inhibitors cited changes in the cell cycle with cells accumulating in G1 in some cell lines and G2 in others and the induction of apoptosis (Vitale et al., 1999; Tamanoi et al., 2001). The early investigations of the mechanism underlying the tumorsuppressive action of perillyl alcohol recorded a lower incorporation of C14- mevalonate into p21ras and other 21–26 kDA proteins in cells incubated with lovastatin and perillyl alcohol when compared with that in cells incubated with lovastatin (Crowell et al., 1991). These findings reflected either the inhibition of farnesyl protein transferase or the inhibition of the synthesis of farnesyl diphosphate and geranylgeranyl diphosphate. These mevalonate-derived substrates and a third, dolichyl phosphate, are required for the post-translational modification, membrane anchoring, and biological activity of small G proteins (Zhang and Casey, 1996), nuclear lamins (Hutchison et al., 1994), and growth factor receptors (Girnita et al., 2000), activities prominent in rapidly proliferating cells (Figure 1). Subsequent findings that perillyl alcohol suppressed C14-mevalonate incorporation into Rap1 and Rab6 but not into Ras suggest that perillyl alcohol inhibits type I and type II geranylgeranyl protein transferases but not farnesyl protein transferase (Ren et al., 1997). At physiologically attainable levels perillic acid methyl ester, a minor metabolite of perillyl alcohol, proved to be a potent inhibitor of the incorporation of farnesyl diphosphate and geranylgeranyl diphosphate into Ras and G25K proteins by mammalian farnesyl protein transferase and geranylgeranyl protein transferase (Gelb et al., 1995). The potency of diverse monoterpenes in suppressing protein isoprenylation parallels their potency in suppressing tumor cell proliferation (Crowell et al., 1994), albeit the latter action requires substantially lower concentrations of the metabolites. Monoterpenes suppress the prenylation of other proteins that may be oncogenic, for example, rasrelated TC21/R-Ras2 (Graham et al., 1994) and the PRL1/PTPCAAX tyrosine phosphatases (Cates et al., 1996). In one study, perillyl alcohol arrested the growth and initiated apoptosis in Bcr/Abl-transformed hematopoietic cells in the

Part A: Postulated Isoprenoid-Mediated Action

absence of an inhibitory impact on ras prenylation and ras activity (Clark et al., 2003). Acyclic isoprenoid derivatives suppress farnesyl protein transferase activity. Farnesylamine (Ura et al., 1998), but not the sesquiterpene, trans, trans-farnesol (Miquel et al., 1998), has an impact on the prenylation of small G proteins. Cellular kinases may provide substrate for the farnesylation of the small G proteins by activating farnesol (Westfall et al., 1997; Bentinger et al., 1998). Another derivatized form of farnesol farnesylthiosalicyclic acid is variously reported to inhibit in vitro methyltransferase activity, the final step in the post-translational processing of ras (Marciano et al., 1995), to prevent the membrane attachment of mature ras (Haklai et al., 1998) or dislodge membrane-bound ras (Egozi et al., 1999; Gana-Weisz et al., 2002), therein facilitating its degradation. Another farnesol derivative, farnesylpyridinium, appears to have detergent-like properties (Hamada et al., 2002).

Clinical Evaluations of Farnesyl Protein Transferase Inhibitors Phase I studies of d-limonene (Vigushin et al., 1998; Chow et al., 2002) and phase I (Ripple et al., 1998, 2000; Hudes et al., 2000; Murren et al., 2002; Azzoli et al., 2003) and II (Bailey et al., 2002; Liu et al., 2003; Meadows et al., 2002) studies of perillyl alcohol revealed dose-limiting toxicities: nausea, vomiting, anorexia, unpleasant taste, and eructation. Hypokalemia due to decreased absorption of dietary potassium was reversed with modest oral potassium supplementation (Hudes et al., 2000). Stable disease and/or modest clinical responses were attained in trials with doses ranging from 1.2 to 8.4 g/m2/day administered in three (Ripple et al., 1998; Hudes et al., 2000; Murren et al., 2002) or four (Ripple et al., 2000; Azzoli et al., 2002; Bailey et al., 2002; Morgan-Meadows et al., 2003) doses in some, but not all, trials (Liu et al., 2003). The maximum tolerated dose determined for perillyl alcohol was determined to be ~15–16 g (~8.4–9.0 g/m2) per day delivered orally in four doses (Azzoli et al., 2003; Morgan-Meadows et al., 2003). Although the gastrointestinal toxicity associated with the maximum dose, 16.2 g/day, was not dose limiting, a dose of 8.1 g/day (4 × 2.025 g) was recommended for Phase II trials, as no pharmacological advantage was achieved with higher doses (Morgan-Meadows et al., 2003). Four non-peptidomimetic farnesyl protein transferase inhibitors, R115777 (Zujewski et al., 2000; Karp et al., 2001; Punt et al., 2001; Crul et al., 2002; Johnston et al., 2003; Adjei et al., 2003; Cohen et al., 2003), SCH66336 (Adjei et al., 2000; Eskens et al., 2001; Awada et al., 2002; Sharma et al., 2002), BMS-214662 (Haluska et al., 2002; Caponigro et al., 2003), and tipifarnib (Patnaik et al., 2003), are undergoing clinical trials with each achieving significant clinically relevant responses. Shared dose-limiting toxicities

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include nausea, vomiting, and diarrhea. Agent-specific toxicities include myelosuppression, neurological complications, and skin sensitivity (R115777); myelosuppression and renal and neurological complications (SCH66336); abdominal cramping, anorexia, fatigue, fever, and gastrointestinal and liver toxicity (BMS-214662); and myelosuppression (tipifarnib). Clinical investigations of a peptidomimetic FT inhibitor (L-778,123) were discontinued because of evidence of drug-related cardiac conduction abnormalities (Britten et al., 2001; Hahn et al., 2002; Haluska et al., 2002). Alternative to the Suppression of Farnesyl Protein Transferase: Suppression of Mevalonic Acid–Pyrophosphate Decarboxylase Activity An alternative to the inhibition of farnesyl protein transferase is to suppress the synthesis of isopentenyl diphosphate (Figure 1), the precursor of farnesyl diphosphate and geranylgeranyl diphosphate; the substrates required for the post-translational modification of small G proteins and nuclear lamins; and dolichyl phosphate, the substrate required for the dolichylation of proteins and glycosylation of growth factor receptors. Cells incubated with an inhibitor of mevalonic acid–pyrophosphate decarboxylase, sodium phenylacetate, or sodium phenylbutyrate (Samid et al., 1994) accumulate in the G1 phase of the cell cycle or undergo apoptotic cell death (Harrison et al., 1998; DiGiuseppe et al., 1999; Finzer et al., 2003).

Clinical Evaluations of Mevalonic Acid–Pyrophosphate Decarboxylase Inhibitors Preclinical evaluations demonstrated the significant chemotherapeutic potential of high-dose sodium phenylacetate (550 mg/kg of body weight) (Samid et al., 1994, Harrison et al., 1998); the results of clinical trials have shown less promise. These inhibitors of mevalonic acid– pyrophosphate decarboxylase activity have been evaluated in Phase I (Thibault et al., 1994, 1995; Piscitelli et al., 1995; Carducci et al., 1996) and Phase II (Buckner et al., 1999; Chang et al., 1999) studies. Although modest responses were recorded with circa 300–400 mg/kg/day infusions, toxicities including emesis, central nervous system depression, transient somnolence, confusion, and fatigue were dose limiting.

Alternative to the Suppression of Farnesyl Protein Transferase: Suppression of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity A second approach employs the inhibition of HMG-CoA reductase activity (Figure 1), the rate-limiting activity in

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the pathway (Brown and Goldstein, 1980; Goldstein and Brown, 1990). As a consequence of mevalonate starvation, cells incubated with diverse inhibitors of HMG-CoA reductase, the statins, accumulate in the G1 phase of the cell cycle or undergo apoptotic death (reviewed by Elson et al., 1999; Mo and Elson, 1999; Mo et al., 1999). Findings that supplemental mevalonate reverses the geraniol-mediated arrest of cells grown in suspension culture support the concept offered herein (Shoff et al., 1991), whereas findings that cells grown on monolayers (Shoff et al., 1991; Duncan et al., 2004) may point to an isoprenoid-mediated impact at a later site in the mevalonate pathway, allyl diphosphate diphosphatase (Case et al., 1995) (Figure 1).

Statins Arrest Cells at the G1/S Interface of the Cell Cycle Cells exposed to statins are arrested in G1 with concomitant decreased expression of c-myc (Dricu et al., 1997; Park et al., 2001; Denoyelle et al., 2003), cyclin D1 (Oda et al., 1999; Park et al., 2001; Wachtershauser et al., 2001; Denoyelle et al., 2003), cyclin E (Oda et al., 1999; Park et al., 1999; Garcia-Roman et al., 2001; Ukomadu et al., 2003a), cdk2 (Gray-Bablin et al., 1997; Rao et al., 1998; Park et al., 1999), and cdk4 (Rao et al., 1998; Park et al., 1999; Wachtershauser et al., 2001), and increased expression of a cdk inhibitor, p21Waf1/Cip1 (Gray-Bablin et al., 1997; Lee et al., 1998; Ghosh et al., 1999; Fenig et al., 2002; Denoyelle et al., 2003; Ukomadu and Dutta, 2003b; Wang and Macaulay, 2003). Inhibition of the cyclin/cdk–mediated phosphorylation of Rb (Lee et al., 1998; Lukas et al., 1996; Ghosh et al., 1999; Ukomadu and Dutta, 2003a; Ukomadu and Dutta, 2003b) blocks the dissociation of the inactive Rb/E2F complex. Statins suppress E2F activity (Park et al., 2001) and induce p27kip1 expression (Gray-Bablin et al., 1997; Ghosh et al., 1999; Oda et al., 1999; Park et al., 1999, 2001; Garcia-Roman et al., 2001; Wang and Macaulay, 2003) with a concomitant suppression of cyclin E/cdk2 activity (Ghosh et al., 1999). Statins Initiate Apoptotic Cell Death Statins suppress the expression of antiapoptotic Bcl-2 (Agarwal et al., 1999; Park et al., 1999; Dimitroulakos et al., 2000; Garcia-Roman et al., 2001; Blanco-Colio et al., 2002) and Bcl-XL (Blanco-Colio et al., 2003) and induce proapoptotic Bax expression (Agarwal et al., 1999) and caspase-7 activity (Marcelli et al., 1998). On one hand, statins induce the expression of c-fos (Martinez-Gonzalez et al., 1997) and, on the other, suppress AP-1 binding activity (Vrtovsnik et al., 1997; Wang et al., 2000).

Proposed Mechanism Underlying Statin-Mediated Suppression of Cell Growth As a consequence of the reduced pools of farnesyl diphosphate, geranylgeranyl diphosphate, and dolichyl phosphate imposed by the inhibition of HMG-CoA reductase activity by statins, activities early in the signal transducing pathways requiring the prenylation of small G proteins (Hohl and Lewis, 1995; Coleman et al., 1997; Holstein et al., 2002a, 2002b; Wang et al., 2000; Blanco-Colio et al., 2002) and N-linked glycosylation of growth factor receptors (Engstrom and Larsson, 1988; Larsson, 1993, 1994; Dricu et al., 1997; McCarty, 2001) are attenuated as are mitotic activities requiring the prenylation of the nuclear lamins (Beck et al., 1988; Hancock et al., 1989; Schafer et al., 1989). Clinical Evaluations of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors Although preclinical evaluations demonstrated the significant chemotherapeutic potential of high-dose statins (15–70 mg/kg of body weight), the results of clinical trials have shown only modest promise and significant doselimiting toxicity (Thibault et al., 1996; Larner et al., 1998; Kawata et al., 2001; Kim et al., 2001; Minden et al., 2001). Toxicities associated with high-dose lovastatin include gastrointestinal dysfunction, myalgia, muscle weakness, elevated creatine phosphokinase, anorexia, and ulcerative lesions. Doses approaching levels normally prescribed for cholesterol control (20–80 mg/day) may have limited efficacy (Anonymous, 1993, Blais et al., 2000).

PART B: POSTULATED ISOPRENOID-MEDIATED ACTION: SUPPRESSION OF HMG CoA REDUCTASE ACTIVITY The isoprenoid-mediated impact on signal transduction pathway, cell division, and apoptosis might be explained, like that of the statins, by mevalonate starvation. Farnesol (Correll and Edwards, 1994; Meigs et al., 1996; Meigs and Simoni, 1997), farnesol derivatives (farnesyl acetate (Bradfute and Simoni, 1994), ethyl farnesyl ether (Bradfute and Simoni, 1994), farnesol homologs (tocotrienols) (Parker et al., 1993), and geranylated tocol analogs (Pearce et al., 1994) accelerate the degradation of HMG-CoA reductase by a nonlysosomal cysteine protease. Tocotrienols (Parker et al., 1993) and farnesol (Peffley and Gayen, 1997) decrease the efficiency of HMG-CoA reductase mRNA translation. Geraniol suppresses HMG-CoA reductase activity by suppressing both HMG-CoA reductase transcription and mRNA translation (Peffley and Gayen, 2003). Limonene (Clegg

Part B: Postulated Isoprenoid-Mediated Action

et al., 1982; Peffley and Gayen, 2003) and perillyl alcohol (Peffley and Gayen, 2003) decrease reductase mass by decreasing the efficiency of HMG-CoA reductase mRNA translation (Peffley and Gayen, 2003). Geranyl geraniol stimulates the Insig-dependent ubiquitination and degradation of HMG-CoA reductase (Sever et al., 2003); β-carotene also decreases HMG-CoA reductase mass via a posttranslational action (Moreno et al., 1995).

Parallel Responses to Statins and Isoprenoids The statins and isoprenoids suppress HMG-CoA reductase activity and concomitantly the post-translational processing of small G proteins, nuclear lamins, and growth factor receptors required for their biological activities. A brief recap of responses triggered by statins and by pure and mixed isoprenoids underlying the arrest of cells in G1 includes the suppression of various activities including the N-linked glycosylation of growth factor receptors, cyclin D1 and E expression, cdk expression, cyclin D1/cdk and cyclin E/cdk activity, c-myc expression, and Rb phosphorylation, and the induction of p21CiP1/WAF1, p27Kip1, and c-fos expression. A recap of responses triggered by statins and by pure and mixed isoprenoids underlying the initiation of apoptosis includes the suppression of the expression of antiapoptotic Bcl-2 and Bcl-xL expression and induction of proapoptotic bax expression and caspase activity.

Opposing Responses to Statins and Isoprenoids; Attenuation of Lovastatin-Mediated Responses by Isoprenoids The statins and isoprenoids suppress HMG-CoA reductase activity, the former by inhibition (Goldstein and Brown, 1990), the latter by post-transcriptional downregulation (Clegg et al., 1982; Parker et al., 1993; Bradfute and Simoni, 1994; Correll and Edwards, 1994; Pearce et al., 1994; Meigs and Simoni, 1997; Chao et al., 2002; Peffley and Gayen, 2003) of the enzyme. As a consequence of the depletion of cellular cholesterol by lovastatin, HMG-CoA reductase synthesis is upregulated and reductase stability increased (Brown and Goldstein, 1980). The post-transcriptional actions triggered by isoprenoids attenuate the statinmediated upregulation of HMG-CoA reductase (Parker et al., 1993; Bradfute and Simoni, 1994; Correll and Edwards, 1994; Yu et al., 1994; Meigs et al., 1996; Meigs and Simoni, 1997; Peffley and Gayen, 2003). Depletion of the farnesyl diphosphate pool secondary to statin-mediated mevalonate deprivation attenuates ras farnesylation (Wang et al., 2000; Blanco-Colia et al., 2002). In response to the indiscriminant inhibition of HMG-CoA reductase imposed by lovastatin, the depleted pools of cel-

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lular prenyl diphosphate upregulate the synthesis of small G proteins with a resultant accumulation of nascent small G proteins (Holstein et al., 2002b). Co-incubation with an isoprenoid (perillyl alcohol) suppresses this upregulation (Holstein et al., 2002a; Holstein and Hohl, 2003). As previously delineated, findings that the incorporation of labeled farnesyl diphosphate into small G proteins in cells incubated with perillyl alcohol and lovastatin is less than that recorded in cells incubated with lovastatin have been widely interpreted as being a consequence of perillyl alcohol–mediated inhibition of farnesyl protein transferase activity; findings now demonstrate that it is the limiting pool of small G proteins rather than the inhibition of farnesyl protein transferase activity that is responsible (Holstein et al., 2002a; Holstein and Hohl, 2003).

Tumor-Specific Action of the Isoprenoids The differential impact on the growth of malignant cells compared with normal cells, noted with isoprenoids (Adany et al., 1994; Yazlovitskaya and Melnykovych, 1995; Stayrook et al., 1997; Ura et al., 1998; Yaguchi et al., 1998; Ariazi et al., 1999; Elson et al., 1999; Mo and Elson, 1999; Mo et al., 1999; Rioja et al., 2000; Sahin et al., 1999; Crowell and Elson, 2001; McIntyre et al., 2000; Smalley and Eisen, 2002; Clark et al., 2003; Beaupre et al., 2003; Duncan et al., 2004; Sakai et al., 2004) but not with the indiscriminant inhibitors of HMG-CoA reductase activity, the statins (Brown and Goldstein, 1980) traces, we propose, to a fundamental lesion of malignant cells, the uncoupling of HMGCoA reductase activity from sterol-mediated feedback regulation, thereby permitting the synthesis of the nonsterol products of the mevalonate pathway essential for cell survival in a sterol-rich environment (reviewed by Elson and Yu, 1994; Elson, 1995; Elson, 1996; Elson et al., 1999; Mo et al., 1999). The dysregulation of reductase activity in tumors (Siperstein and Fagan, 1964; Chen et al., 1978; George and Goldfarb, 1980; Gregg et al., 1982; Yachnin et al., 1984; Bruscalalupi et al., 1985; Engstrom and Schofield, 1987; Erickson et al., 1988; Azrolan and Coleman, 1989; Kawata et al., 1990; Bennis et al., 1993) is confirmed by our findings of several-fold higher copies of HMG-CoA reductase mRNA in human colon tumor (Caco2) and leukemic (CEM) cells compared with levels present in normal colon cells (CCD18) and normal human lymphocytes (Hentosh et al., 2001). Findings that HMG-CoA reductase promoter activity was threefold higher in Caco2 tumor cells than in CCD18 normal cells suggest that the differential binding of transcription factor(s) on the reductase promoter is responsible for the attenuation of the normal sterol-mediated regulation of reductase activity (Hentosh et al., 2001). Observations of the inverse correlation between DNA methylation and gene expression levels (Laird and Jaenisch, 1994), the

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hypomethylation of protooncogenes, Ha-ras, Ki-ras, c-fos, c-myc, Erb-A1, and bcl-2 in human leukemias and liver tumors (Laird and Jaenisch, 1994) and of the hypomethylation of Ha-ras, c-fos, c-myc, and HMG-CoA reductase in nodules in livers of rats exposed to diverse carcinogens (Coni et al., 1992; Rossiello et al., 1994) led us to examine the methylation status of the promoter regions of HMG-CoA reductase genes from solid tumor, leukemic, and normal cells. On finding that the reductase promoter sequences in both normal and malignant cells were hypomethylated, we concluded that an aberrant methylation pattern does not alter the binding of transcription factors to the promoter region of the HMG-CoA reductase gene (Hentosh et al., 2001). We, therefore, propose alternate hypotheses to explain the overexpression of HMG-CoA reductase gene in tumor cells, cells with membranes enriched in cholesterol (Coleman et al., 1997). A perturbation, either in the precursors of the sterol regulatory element-binding proteins (SREBPs) or of the SREBP cleavage activation protein (SCAP), might support the overexpression of HMG-CoA reductase (Brown and Goldstein, 1997; Osborne, 2000; Horton et al., 2002). Cholesterol depletion in cultured cells initiates the proteolytic cascade leading to the elevation of transcription factors SREBP-1a and SREBP-2, increased HMG-CoA reductase mRNA transcription and translation with the resultant increase in HMG-CoA reductase mass (Shimano et al., 1996). The addition of sterols suppresses the processing of SREBP-2 but not of SREBP-1a (Hannah et al., 2001). Contrary to the relative abundance of SREBP-1c, a transcription factor targeting lipogenic genes, to SREBP-1a in liver tissue (Shimano et al., 1997; Shimomura et al., 1997), in embryonic and hepatoma cells, the expression of SREBP-1a is reported to exceed that of SREBP-1c (Brown and Goldstein, 1997; Shimomura et al., 1997; Osborne, 2000; Hannah et al., 2001). SREBP-1a, expressed in tumors (Hannah et al., 2001), does not respond to sterol-mediated downregulation. Alternatively, a mutation in SCAP, a G → A transition at codon 443 of SCAP, changing aspartic acid to asparagine, enhances its cleavage-stimulating activity, thereby rendering HMG-CoA reductase resistant to sterol regulation (Hua et al., 1996). The overexpression of members of the type 1 receptor tyrosine kinase family, especially ErbB-2 (HER-2, Neu) has been implicated in multiple forms of cancer (Hainsworth et al., 2000; Hellstrom et al., 2001; Yarden, 2001; Hempstock et al., 1998). Incubation of ErbB-2–expressing cells with tyrosine kinase inhibitors induces concentration- and time-dependent reductions in HMG-CoA reductase activity (Asslan et al., 1998; Sindermann et al., 2001). Both HMGCoA reductase mRNA levels and the rate of HMG-CoA reductase synthesis are significantly lower (Asslan et al., 1998). In normal cells treated with lovastatin, there is a compensatory increase in HMG-CoA reductase mRNA and reductase mass (Brown and Goldstein, 1980); the over-

expression is tentatively associated with an increase in phosphotyrosine levels (Sindermann et al., 2001) and tyrosine kinase activity (Mutoh et al., 1999). The metabolic error underlying the widely reported upregulation of HMG-CoA reductase activity in tumors (reviewed by Elson and Yu, 1994; Elson, 1995, 1996; Elson et al., 1999; Mo et al., 1999) remains to be delineated. Although the basis of the dysregulated activity remains to be resolved, the activity retains high sensitivity to isoprenoid-mediated post-transcriptional downregulation (Parker et al., 1993, 1994; Peffley and Gayen, 2003).

Preclinical and Clinical Evaluation of Isoprenoids Preclinical evaluations of farnesyl protein transferase, mevalonic acid–pyrophosphate decarboxylase, and HMGCoA reductase inhibitors yield dramatic responses. When administered at maximum tolerated levels in clinical trials, the responses are not encouraging. The dose-limiting toxicity noted with perillyl alcohol, gastrointestinal discomfort, differs from those toxicities recorded for the other mevalonate pathway–targeted agents. With revised formulations and dosing schedules, the severity of the dose-limiting toxicities elicited by perillyl alcohol might be eased, thereby permitting the administration of chemotherapeutically effective doses. The application of more potent isoprenoids might prove clinically effective absent the gastrointestinal discomfort accompanying high-dose perillyl alcohol. Another approach envisions the potentiation of the statin action with the co-administration of a tumor-targeted isoprenoid.

CANCER PREVENTION Efficacy and follow-up studies provide evidence that cancer incidence is modestly lower in hypercholesterolemic populations undergoing lovastatin therapy (Anonymous, 1993). A search for volatile isoprenoid constituents of fruits, vegetables, and herbs spanning seven plant families identified 179 monoterpenes and sesquiterpenes. Of these, 41 were screened for tumor-suppressive potency. Volatile isoprenoids differed substantially in potency as determined with a melanoma B16 screen; IC50 values fell generally within the range 200–400 µmol/liter. Sixteen of the isoprenoids evaluated proved to have greater potency than perillyl alcohol and 33 had greater potency than d-limonene, the two isoprenoids advanced to clinical evaluation. The finding that blends of isoprenoids suppressed tumor cell proliferation with efficacy greater than that of the sum of individual agents demonstrates the cumulative impact of diverse volatile isoprenoid constituents of the diet (Tatman and Mo, 2002).

Summary

The modest protection from breast cancer associated with dietary vitamin E may be due to the effects of the associated tocotrienols. Laboratory studies show that α-tocopherol alone has little effect on mammary tumors, whereas the tocotrienols have potent antiproliferative and proapoptotic effects (Guthrie et al., 1997; Netsaretnam et al., 1998; Mo and Elson, 1999; McIntyre et al., 2000), which would be expected to reduce risk of breast cancer (Schwenke, 2002). Isoprenoids complement the tumor-suppressive activity of other dietary agents. Incubation of tumor cells with tyrosine kinase inhibitors induces concentration- and timedependent reductions in HMG-CoA reductase activity (Asslan et al., 1998; Sindermann et al., 2001). Incubation of prostate tumor cells with isoprenoids or genistein, a tyrosine kinase inhibitor in soy products, yielded a concentrationdependent suppression of growth. When applied in combination, the resulting suppression of growth was significantly greater than that predicted by the sum of the activities of the two classes of agents (Mo, unreported observations).

SUMMARY Contrary to the promise shown in preclinical studies, dose-limiting toxicities diminish the prospects for the development of chemotherapeutic agents targeting the mevalonate pathway. An aberrant pathway activity, an overexpressed sterol feedback-resistant HMG-CoA reductase activity broadly present in tumors, offers a novel target for chemopreventive and nutritional intervention. Isoprenoids impacting only on the post transcriptional regulatory actions controlling reductase activity effectively starve tumor cells of the mevalonate-derived products required for the maturation and biological activity of proteins having essential roles in maintaining cell proliferation and blocking apoptosis.

Acknowledgments This work was supported by the Texas Woman’s University (TWU) Research Enhancement Program, TWU Chancellor’s Research Fellow Program, Human Nutrition Research Fund, Texas Food and Fiber Commission, American Cancer Society–University of North Texas Institutional Grant, and the Wisconsin Alumni Research Foundation.

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39 Cancer Anorexia and Cachexia DAVID HEBER AND N. SIMON TCHEKMEDYIAN

INTRODUCTION

The metabolic, hormonal, and inflammatory abnormalities associated with cancer anorexia and cachexia can overlap with the effects of chronic infections and surgery and result in marked effects on protein, carbohydrate, and lipid metabolism. Cytokines released by tumor cells, immune cells, and stromal cells mediate many of the observed changes in metabolism leading to anorexia and cachexia. Pharmacological agents that improve appetite, interfere with cytokine production, and inhibit inflammation have been combined with advanced nutritional technologies in attempts to produce weight gain, increase lean tissue, and improve the quality of life. Although pharmacological and adjunctive nutritional therapy of the malnourished cancer patient can ameliorate the impact of malnutrition on the quality of life, the search continues for additional therapeutic targets for reversing, delaying, or preventing cancer anorexia and cachexia.

As a result of advances in our understanding of the metabolic and nutritional effects of many common forms of cancer, therapeutic options have been developed to reverse or reduce the effects of cancer anorexia and cachexia. Such options are becoming recognized as an integral part of the care of cancer patients to improve both their quality of life and the outcome of cancer therapies. The development of early and appropriate nutritional interventions holds the promise of improving the cancer patient’s ability to undergo and tolerate definitive oncological therapies, including surgery, irradiation, chemotherapy, and the increasing variety of newer treatments, including biological response modifiers, angiogenesis inhibitors, monoclonal antibodies, and other targeted therapies. Implementation of nutritional management can provide a constructive and empowering experience for the patient and family during cancer therapy. In contrast to uncomplicated starvation, cachexia is an advanced state of wasting marked by excess loss of skeletal muscle mass relative to total body weight loss. Because humans are well adapted to starvation, weight loss occurring gradually because of decreased calorie intake occurs with relative maintenance of lean body mass at the expense of body fat as an energy source. Because of metabolic and nutritional effects accompanying advanced cancer, these adaptations are inhibited or impaired, ultimately leading to advanced malnutrition and life-threatening cachexia. Certain common forms of cancer are more frequently associated with marked weight loss at the time of diagnosis than others, but weight loss is typically present in up to half of all untreated cancer patients.


MALNUTRITION AND CACHEXIA IN CANCER Malnutrition and cachexia are frequently associated with cancers of the pancreas, lung, head and neck, stomach, and prostate. The overall incidence of malnutrition in cancer patients varies widely, occurring in between 30 and 87% of different populations studied (Shils, 1977; Nixon et al., 1980). For example, in patients with pancreatic and stomach cancers, up to 85% are cachectic, whereas among breast cancer and sarcoma patients, only 40% are cachectic (Monitto et al., 2001). In our experience with 644 mostly ambulatory cancer patients, we observed weight loss of >5% of baseline body

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weight in 59% of the cases (Tchekmedyian, 1995). Fiftynine percent of patients had decreased appetite, 67% had decreased food intake, and 54% were underweight when their weight was compared with the calculated ideal body weight. The advanced starvation state resulting from decreased food intake and hormonal/metabolic abnormalities characteristic of the interaction between tumor and host has been called cancer cachexia (Brennan, 1977). A retrospective analysis of patient body weight in early cooperative group chemotherapy trials determined that the presence of >6% weight loss from usual body weight was a significant prognostic factor predictive of a poorer survival (De Wys et al., 1980). In this study, the apparent effect of weight loss at the time of diagnosis in this study on median survival for certain cancers was greater than the impact of chemotherapy. Cachexia is directly associated with poorer survival in patients with many types of cancer, including advanced pancreatic and lung cancer (Persson et al., 2002). Poor quality of life, fatigue, weakness, and poorer responses to cancer therapies are all associated with cachexia.

RELATIVE ROLES OF ANOREXIA AND METABOLIC ABNORMALITIES Anorexia clearly plays a major role in weight loss in the cancer patient but cannot explain all of the weight loss noted. Anorexia complicates cancer in 15–40% of patients at the time of diagnosis (De Wys, 1972; Evans et al., 1985). However, malnourished patients with localized cancers under metabolic ward conditions fail to gain weight even when given apparently adequate calories for anabolism, suggesting that these patients are hypermetabolic. Moreover, treatment with megestrol acetate, while improving appetite and leading to weight gain, fails to increase lean body mass (LoPrinzi et al., 1993). Decreased caloric intake is a marker of poorer survival in patients with pancreatic cancer (Okusaka et al., 1998), but it is not clear whether this is simply secondary to advanced disease status and an inability to eat. By contrast, the metabolic and body composition changes seen with cachexia are profound with marked reductions in skeletal muscle and adipose tissue resembling those found with surgical trauma, burns, infections, acquired immunodeficiency syndrome (AIDS), and injury rather than those seen with uncomplicated starvation. Despite the development of advanced technology and delivery systems for total parenteral nutrition and continuous enteral nutrition, nutrition therapy alone does not reverse the weight loss and metabolic abnormalities of cancer malnutrition. Although nutritional rehabilitation can be demonstrated in selected patients who respond to antineoplastic therapy, the application of parenteral or enteral nutrition as adjuncts to chemotherapy in cancer patients has not resulted in increased survival or predictable weight gain

(Brennan, 1981; Shike et al., 1984). Although a decrease in caloric intake is characteristically observed in malnourished cancer patients, clinical evidence suggests that decreased food intake alone cannot account for all the weight loss noted in cancer patients. Means of reversing the relentless catabolic progression due to metabolic abnormalities reviewed later in this chapter could potentially have meaningful effects on nutrition and survival time. Increased whole body protein breakdown, increased lipolysis, and increased gluconeogenesis have been repeatedly demonstrated in malnourished cancer patients. A number of observations made with regard to fat metabolism in cachectic cancer patients may help explain the disproportionate decrease in fat tissue mass including increased lipolysis, decreased lipogenesis, hypertriglyceridemia, increased hepatic secretion of very low density lipoprotein (VLDL), increased de novo fatty acid synthesis, and a futile cycle of fatty acids between liver and adipose tissue. High turnover rates of both glycerol and free fatty acids (FFAs) have been observed combined with elevated fatty acid release into the circulation (Shaw and Wolfe, 1987). Fasting plasma FFAs are elevated in weight-losing cancer patients by comparison with weight-stable individuals, and there is increased sensitivity to the lipolytic effects of adrenaline (Yam et al., 1994). In malnourished cancer patients, basal fatty acid turnover was found to be 25% higher than in noncancer controls and was found to be similar to the rate observed in patients with severe burns (Legaspi et al., 1987). Overall, lipolysis was increased by 40%, and there was a 20% increase in fatty acid oxidation in cancer patients with fat loss. A disproportionate loss of muscle mass, as is the case with fat cell mass, is also seen in malnourished cancer patients. A 75% decrease in skeletal muscle mass has been observed with a 30% loss of weight from preillness weight.

Attempts to Reverse Metabolic Abnormalities Because predictable renutrition of the cancer patient has not been possible, a great deal of research has been conducted concerning specific hormonal and metabolic abnormalities that could interfere with renutrition. Over the past 15 years, research on the basic pathophysiology of cancer cachexia has resulted in the definition of several metabolic and hormonal abnormalities in malnourished cancer patients. These abnormalities include hypogonadism in male cancer patients (Chlebowski and Heber, 1982), increased glucose production (Holroyde et al., 1975; Chlebowski and Heber, 1986), increased protein catabolism (Heber et al., 1982; Burt et al., 1984), increased lipolysis and fatty acid oxidation (Jeevanandam et al., 1986; Shaw and Wolfe, 1987), and insulin resistance (Bennegard et al., 1986; Byerley et al., 1991).

Relative Roles of Anorexia and Metabolic Abnormalities

Based on the metabolic abnormalities observed, a number of strategies using hormonal and metabolic agents were tested to reverse these abnormalities. Hydrazine sulfate has been tested based on its ability to inhibit gluconeogenesis (Chlebowski et al., 1984), and insulin infusion has been attempted to counteract apparent insulin resistance (Moley et al., 1985). Neither of these treatments resulted in weight gain.

Host–Tumor Interactions and the Development of Cachexia Based on autopsy studies performed in the 1920s (Warren, 1932; Terepka and Waterhouse, 1956) and animal studies done in the 1950s (Fenninger and Mider, 1954), it was postulated that tumors acted to siphon off needed energy and protein from the host. In the 1970s and 1980s, specific abnormalities of intermediary metabolism were identified in cancer patients that could account for the common observation that such patients lost weight even in the face of apparently adequate nutrition. Studies conducted in a number of laboratories, including our own, have demonstrated that maladaptive metabolic abnormalities occur frequently in patients with cancer. In 1983, we demonstrated that adequate calories and protein administered to six patients with active localized head and neck cancer via forced continuous enteral alimentation under metabolic ward conditions for 29 days failed to lead to significant weight gain (Heber et al., 1986). The observed failure of these patients to gain weight despite adequate caloric intake under metabolic ward conditions supports the concept that malnourished cancer patients are hypermetabolic, which may be due in turn to futile cycling with resulting energy inefficiency.

Energy Balance in the Cancer Patient If metabolic abnormalities promote the development of malnutrition or interfere with renutrition, then there should be some evidence of abnormally increased energy expenditure. A number of investigators have used indirect calorimetry and the abbreviated Weir formula to calculate energy expenditure at rest and then compared this with the basal energy expenditure (BEE) determined using the Harris–Benedict formulas. Long et al. (1981) demonstrated a mean difference of 2% when this comparison was performed in 20 normal controls. In 1980, Bozetti et al. (1980) found that 60% of a group of patients with advanced cancer had basal metabolic rates increased 20% above predicted. Dempsey et al. (1984) studied energy expenditure in a group of 173 malnourished gastrointestinal cancer patients. Fiftyeight percent had abnormal resting energy expenditure (REE) by indirect calorimetry compared with BEE, but a greater percentage were hypometabolic rather than hypermetabolic (36 vs 22%). Knox et al. (1983) studied 200

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patients with a variety of cancers and found abnormal energy metabolism in 59% but found more hypometabolic than hypermetabolic individuals (33% vs 26%). Lean body mass rather than fat mass correlates with the individual variations observed in measured REE. The hypothesis that the malnourished cancer patient may be hypermetabolic relative to the amount of lean body mass remaining has been examined. Peacock et al. (1987) studied REE in noncachectic patients with sarcomas. These patients had no prior treatment, had large localized sarcomas, and no weight loss or history of decreased food intake. REE corrected for body cell mass (BCM) determined by total body potassium counting or body surface area was significantly greater in male sarcoma patients compared with controls. This difference was due to both a decrease in BCM and an increase in REE in these patients before the onset of weight loss.

Glucose and Protein Metabolism in Cancer Patients Tumors have been demonstrated to increase the rate of glucose utilization in a number of tissues (Heber, 1989). Because there are only ~1200 kcal stored in the body as liver and muscle glycogen, blood glucose levels would be expected to fall. This does not occur, because there is also an increase in hepatic glucose production in cachectic and anorectic tumor-bearing animals and humans. The regulation of protein metabolism is tightly linked to carbohydrate metabolism because these processes are critical to the normal adaptation to starvation or underfeeding. During starvation, there is a decrease in glucose production, protein synthesis, and protein catabolism. The decrease in glucose production occurs because fat-derived fuels, primarily ketones, are used for energy production. Although there are, on average, ~54,000 kcal of protein stored in the BCM, only about half of this is available for energy production. In fact, depletion below 50% of body protein stores is incompatible with life. Whole body protein breakdown is increased in lung cancer patients and has been shown to correlate with the degree of malnutrition such that more malnourished patients have greater elevations of their whole body protein breakdown rates expressed per kilogram of body weight (Heber et al., 1982). Hydrazine sulfate is a noncompetitive inhibitor of gluconeogenesis. When this drug was administered to lung cancer patients in one study, not only did whole body glucose production decrease as expected, but there was also a decrease in the whole body protein breakdown rate (Tayek et al., 1987). Hydrazine sulfate has not been found to improve nutritional parameters, quality of life parameters, or survival in randomized trials in cancer patients, despite these seemingly promising effects (Kosty et al., 1994; Loprinzi et al., 1994).

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Glucoregulatory Hormones The normal adaptation to malnutrition involves changes in the secretion of a number of glucoregulatory hormones, including insulin, growth hormone, cortisol, and thyroid hormones. Because the changes observed in malnourished cancer patients are in the opposite direction of the adaptations observed in uncomplicated starvation, we have studied the secretion of these hormones under standardized conditions in a metabolic ward. In all cases, cancer patients received enteral nutrition calculated to maintain body weight for at least 3 days before study. Growth hormone levels were measured in the fasting state in 27 patients with lung cancer or colorectal cancer. The mean fasting level was 55.7 ± 10.6 (SEM) pg/ml with a wide range from 30 to 232 pg/ml. In other studies, Tayek and Brasel (1995) have reported that more severely malnourished colorectal cancer patients have more elevated levels of growth hormone. Plasma insulin and growth hormone were also measured following an infusion of 0.5 g/kg of arginine for 30 minutes. There was a rise in growth hormone to normal levels observed 60 minutes after the end of the infusion. Therefore, growth hormone appears to respond normally to fasting and arginine infusion. A standard oral glucose load of 40 g/m2 was also administered to a group of colon and lung cancer patients. Plasma glucose levels were elevated above the normal range into a range consistent with impaired glucose tolerance, but not diabetes. Plasma growth hormone levels were suppressed in the early part of the test, when insulin and glucose levels were elevated, but rose at 5 hours after the oral glucose load. Plasma cortisol levels were in the normal range. Thus, there was a generally normal response to an oral glucose load except for an elevation of insulin and glucose levels in the first 2 hours of the test. These data are consistent with an insulin-resistant state. Insulin resistance has been observed in malnourished cancer patients using the euglycemic insulin clamp technique (Bennegard et al., 1986). The infusion of tumor necrosis factor-α (TNF-α) for 12 hours in rats results in both increased uptake of glucose and insulin resistance (Lang et al., 1992). These observations suggest that cytokines, including tumor TNF-α, may play a role in the pathogenesis of the metabolic abnormalities noted in the malnourished cancer patient.

Thyroid Hormones The production of triiodothyronine (T3) from thyroxine (T4) is reduced significantly in uncomplicated starvation, short-term fasting, or malnutrition (Danforth et al., 1978). It has been proposed that the failure to adequately reduce T3 production during starvation in cancer patients could lead to a hypermetabolic state (Danforth et al., 1978). We examined

thyroid function tests in 27 cancer patients and 18 controls after at least 3 days of adequate nutrition under metabolic ward conditions. There were no differences in any of the measured thyroid hormone levels including T4, T3, or reverse T3 between cancer patients and control subjects observed under conditions of adequate nutrition. Therefore, there was no evidence in these studies of increased thyroid hormone levels during refeeding that could impair renutrition. Other studies conducted in outpatient settings have found decreased levels of thyroid hormone as expected in undernourished patients (Persson et al., 1985). However, these changes secondary to malnutrition are apparently reversible in cancer patients with renutrition.

Lipid Metabolism in Cancer Cachexia Net synthesis of adipose tissue triglyceride normally occurs when there is a net surplus of energy intake over expenditure. This can occur, however, also in the absence of increased food intake, as demonstrated in animals given progesterone under conditions of constant food intake (Hervey and Hervey, 1967). Under these circumstances, increased body fat deposition may result from a decrease in energy expenditure via modulation of the metabolism of fat away from fatty acid oxidation toward more energy efficient storage. In obese subjects, it has been proposed that increased fat mass results from the failure of increased fatty acid oxidation to keep pace with increased fat intake (Schutz et al., 1992). In the cancer patient who cannot maintain or gain weight, the opposite situation of excess fatty acid oxidation, which cannot be eclipsed by fat and calorie intake, may impair renutrition. Under normal conditions of feeding and fasting, the uptake and breakdown of fat from adipose tissue is controlled by two adipose tissue enzymes: hormone-sensitive lipase (HSL) (Holm et al., 1988) and lipoprotein lipase (LPL) (Eckel, 1987). During early fasting, decreased insulin levels and increased glucagon and epinephrine result in cyclic adenosine monophosphate (cAMP) activation of a protein kinase, which phosphorylates and activates HSL. When activated, HSL hydrolyzes the triglyceride in the lipid droplet of the adipocyte into FFAs to be released into the circulation. Conversely, activated LPL hydrolyzes the core of circulating triglyceride-rich lipoproteins into FFAs and monoacylglycerol (Tayek et al., 1987). These fatty acids are the major source of substrate for adipocyte triglyceride synthesis, because adipocytes synthesize very small amounts of fatty acids de novo (Knittle et al., 1977). Studies using stable isotopes have found that weightlosing cancer patients have increased rates of glycerol and FFA turnover when compared with normal patients and cancer patients without weight loss (Shaw and Wolfe, 1987). A glucose infusion failed to suppress lipolysis in the weight-losing cancer patients, as it did in normal subjects.

Relative Roles of Anorexia and Metabolic Abnormalities

Hypertriglyceridemia, depletion of carcass fat stores, and decreased LPL levels have been observed in tumor-bearing animals and malnourished cancer patients (Axelrod and Costa, 1980; Cohn et al., 1981; Devereaux et al., 1984; Thompson et al., 1984). In animals bearing a mammary adenocarcinoma (AC33) for 18 days, decreased adipose tissue LPL activity was observed, together with a decrease in fat cell size but not number, and a decrease in serum insulin levels compared wtih non–tumor-bearing control animals consuming similar amounts of food (Lanza-Jacoby et al., 1984). Increased serum-FFAs, cholesterol, and triglycerides were observed consistent with the decrease in LPL activity.

Tumor and Host Factors Mediating Malnutrition and Cachexia While studying cachexia in chronic infection, Beutler and Cerami (1986, 1988) and Oliff (1988) found that rabbits infected with trypanosomes lost weight and developed hypertriglyceridemia. They traced this to an inhibition of LPL and found that the serum of animals treated with lipopolysaccharide (LPS) contained the same inhibitor of LPL, suggesting it resulted from immune activation. In view of its potential role in wasting, it was called “cachectin.” In earlier studies, it had been reported that the serum of mice treated with BCG and endotoxin had antitumor activity. The factor responsible for this effect was isolated from macrophages and called “tumor necrosis factor” (Norton et al., 1985). The amino acid and genetic sequences of TNF and cachectin were found to be identical, and the protein was called TNF/cachectin. Using recombinant DNA methodology, it was possible to produce large amounts of TNF for evaluation of its biological properties. Intravenous administration of TNF caused all the manifestations of toxic shock (Socher et al., 1988). Sublethal doses produced many of the metabolic abnormalities seen with cancer cachexia in a dose-related fashion (Stovroff et al., 1988; Tracey et al., 1988). Oliff et al. (1987) transfected a tumor with the gene for human TNF and implanted the tumor with or without the transfection leading to continuous production of TNF. The mice bearing the TNF-α–secreting tumor developed progressive anorexia, weight loss, fat depletion, and earlier death compared with the animals bearing the nonsecreting tumor. In human studies where TNF was given to cancer patients intermittently, it did not cause weight loss (Blick et al., 1987). This failure may have been due to the failure to consistently elevate TNF levels. However, anorexia was noted consistent with the idea that anorexia secondary to cytokines precedes malnutrition. As the immune system attempts to clear the body of tumor cells, inflammation is seen in many forms of cancer in various tissues. Both tumor and host factors are released locally and into the circulation during this process. These factors can promote profound metabolic abnormalities that

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lead to anorexia, malnutrition, and cachexia. These factors are cytokines and include TNF-α interleukin (IL)-1, IL-2, and IL-6, interferon-γ (IFN-γ), and proteolysis-inducing factor (PIF). These cytokines are produced by tumor, immune, and stromal cells and in many cases provide one means of intercellular communication between tumor cells and the cells of the microenvironment in which they grow. A number of hormones regulate lipolysis, including insulin, adrenocorticotrophic hormone (ACTH), epinephrine, growth hormone, insulin-like growth factors (IGFs), and others (Nilsson-Ehle et al., 1980). Cytokines are produced, though not exclusively, by host macrophages and lymphocytes in response to the tumor (Moertel, 1986). These substances usually act in a paracrine fashion in the local environment of the tumor. With disease progression, it is possible that cytokines may circulate to other tissues and act as endocrine factors. Several cytokines have also been shown to increase lipolysis, including TNF-α, IL-1β, and IL-2, IL-6, IFN-γ, lipid-mobilizing factor (LMF), and PIF. For example, LMF and PIF were originally thought to be produced only by tumor cells, but LMF has been shown to be produced by both white and brown adipose tissue in addition to tumor cells (Bing et al., 2004). Other cytokines find their way into the circulation where they can ultimately cross the blood–brain barrier, including TNF-α, IL-1α, IL-2β, and IL-6. IL-1 is an inflammatory cytokine produced by macrophages in response to endotoxin, but it has also been implicated in the pathogenesis of cancer cachexia. Hellerstein et al. (1989) demonstrated that IL-1 can reduce food intake in meal-fed rats. Moldawer et al. (1988a) showed that doses of IL-1 one-tenth of those that cause fever can reduce food intake in mice. IL-1 can also cause a change in hepatic protein synthesis similar to that seen in tumor-bearing animals. Moldawer et al. (1988b) found IL-1 activity in the plasma of only 1 of 23 cancer patients, but in 5 of 6 patients with septic shock. Jensen et al. (1990) demonstrated increased IL-1 gene expression in the livers of cachectic tumor-bearing rats despite undetectable circulating levels of IL-1. Evidence for IL-6 playing a role in cancer cachexia comes from the observation that sarcoma-bearing mice have elevated levels of IL-6 (Jablons et al., 1989). In addition, under certain circumstances IL-6 levels are elevated following TNF infusion, so IL-6 may act to mediate in part the effects of TNF (Broukaert et al., 1989). IFN-γ is produced by activated T cells and is a potent stimulator of macrophages. IFN enhances the effects of TNF and increases the mRNA expression triggered by TNF in macrophages exposed to endotoxin (Koerner et al., 1987). Sarcoma-bearing rats treated with antibody against IFN have reduced degree of weight loss and improved survival compared with control tumor-bearing rats (Langstein et al., 1989).

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LMF (Hirai et al., 1998) and PIF (Todorov et al., 1996) appear to have direct effects on fat cells in the periphery and can produce weight loss via lipolysis in the absence of anorexia. Increased production of cytokines may also account for the increased production of acute-phase proteins and increased oxidation products in the circulation of cancer patients. In general, increases in serum levels of cytokines including TNF-α, IL-1, IL-6, and IFN-γ, though detected, are not correlated with the degree of weight loss in patients with advanced cancer (Maltoni et al., 1997). TNF has been detected in the circulation in some, but not all, cancer patients (Flick and Gifford, 1984). However, in a study of pancreatic cancer patients, PIF was detected in the urine of 80% of patients, and those patients with detectable PIF in their urine were more malnourished by comparison with those with no PIF detected (Wigmore et al., 2000). PIF has also been found in urine from patients with breast, ovarian, lung, colon, and rectal cancer who have progressive weight loss of ≥1 kg/mo (Caruik et al., 1997). In one study of breast cancer patients, serum levels of TNF-α were found to correlate with stage of disease rather than weight loss (Karayainnakis et al., 2001). In another study (Sheen-Chen et al., 1997), TNF-α serum levels were measurable in 36.5% of a group of patients with pancreatic cancer and higher levels were found in patients with metastatic disease by comparison with localized disease. In a study of gastric and colorectal cancer patients, the serum levels of soluble IL-2 receptor were most markedly elevated in patients with cachexia (Shibata and Takekawa, 1997).

Effects of Cytokines on Protein Metabolism We have previously demonstrated increased whole body protein turnover in lung cancer patients (Heber et al., 1982), and it is likely that protein wasting is mediated by cytokines (Melville et al., 1990). Ubiquitin is a 76 amino acid protein with multiple functions, among which is the attachment to proteins that are to be degraded in proteasomes. The ubiquitin–proteasome proteolytic pathway is considered to play the major role in intracellular protein degradation in muscles. In this process, ubiquitin becomes activated and attached to the protein substrate and the polyubiquitinated protein is recognized for degradation by the 26S proteasome complex. TNF-α (Garcia-Martinez et al., 1993) and PIF (Lorite et al., 1997) inhibit protein synthesis and increase protein degradation in skeletal muscle. PIF (Lorite et al., 2001), TNF-α, or IFN-γ (Llovera et al., 1998), when infused intravenously, cause an increased expression of ubiquitin, while leukemia-inhibitory factor (LIF) and IL-6 do not change ubiquitin expression. IL-6 has also been implicated in the loss of lean body mass seen in cachectic mice bearing tumors such as the colon-26 adenocarcinoma. Administration of an anti-IL-6 receptor antibody reduced loss of muscle weight and suppressed enzymatic activity of

cathepsins B and L (Fujita et al., 1996). On the other hand, some studies suggest that IL-6 does not cause skeletal muscle protein degradation when studied in vitro (GarciaMartinez et al., 1994). Goodman (1994) has shown that rats given IL-6 acutely activated both total and myofibrillar protein degradation in muscle, while mice receiving murine IL-6 over a 7-day period at a dose of 250 µg/kg of body weight per day showed no depression of body weight or food intake (Espat et al., 1996). IL-1 isolated from adherent human monocytes was shown to stimulate muscle protein degradation in intact muscles by a mechanism sensitive to inhibition of lysosomal thiol proteases (Baracos et al., 1983). However, recombinant human IL-1β was not able to reproduce this effect, which suggests that it was due to another cytokine that was not TNF-α or IFN-α, IFN-β, or IFN-γ (Goldberg et al., 1988). Infusion of IL-1β, together with TNF-α, to rats with Yoshida sarcoma reduced the synthesis rate of tumor fractional protein but had no effect on muscle protein metabolism (Ling et al., 1991). Pale muscle fibers are affected more than red fibers, and fiber loss is predominantly from myofibrillar protein, determined by the release of 3-methylhistidine (Mitch and Goldberg, 1996). Studies demonstrate specific targeting of myosin heavy chain in muscle (Acharyya et al., 2004). In addition, cancer patients have reduced serum albumin levels and increased levels of acute-phase proteins, such as Creactive protein (CRP) (Fearon et al., 1998). Elevated levels of CRP are associated with a shorter survival time (Falconer et al., 1995). Both depression of protein synthesis and increased protein degradation contribute to the muscle atrophy. Protein synthesis may be impaired due to an imbalance of amino acids caused by increased utilization of specific amino acids, as well as depression of branched-chain amino acids such as leucine (Yoshizawa, 2004), which not only are substrates for protein synthesis but have the unique ability to initiate signal transduction pathways that modulate the initiation of translation. The ubiquitin–proteasome proteolytic pathway has been shown to play a significant role in muscle protein degradation in cachexia (Bossola et al., 2003). Ubiquitin also has a role in DNA repair (Jentsch et al., 1987) and mitosis (Glotzer et al., 1991). In addition, the lysosomal pathway has been shown to play a role in lung cancer patients referred for curative resection with average weight losses of 29%. In these patients, an increased expression of the lysosomal protease cathepsin B was observed in skeletal muscle biopsies (Jagoe et al., 2002). In the ubiquitin–proteasome pathway, proteins are marked for degradation by the addition of a polyubiquitin tag and are cleaved within a 26S proteasome structure, which is a cylinder with proteolytic enzymes on the inner surface to prevent their mixing with the cellular cytoplasm. The protein is transferred to the inside of the cylinder by a specific 19S particle. Specific ubiquitin ligases (E3) are also

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Cancer Anorexia

important in the breakdown of myofibrillar proteins in cachexia (Bodine et al., 2001). Tumor or host factors that mediate malnutrition and cachexia are believed to upregulate the ubiquitin–proteasome pathway in skeletal muscle. In vitro studies with TNF-α showed that it stimulated ubiquitin conjugation of muscle proteins (Li et al., 1998) and directly induced protein degradation, as determined by the release of tyrosine or 3-methylhistidine (Llovera et al., 1997) and a decrease in myosin, but there was no change in expression of proteasome subunits. Both PIF (Whitehouse and Tisdale, 2003) and TNF-α (Li and Reid, 2000) are thought to stimulate protein degradation through increased nuclear binding of the transcription factor nuclear factor-κB (NFκB), which has a central role in inflammation as well.

Effects of Cytokines on Lipid Metabolism Cytokines can induce loss of adipose tissue and increases in serum triglycerides and fatty acid oxidation. The enzyme LPL extracts fatty acids from plasma lipoproteins for storage in fat cells so that its inhibition would lead to elevated levels of triglycerides in the circulation. On the other hand, HSL releases FFAs back into the circulation by hydrolyzing stored triglycerides and these fatty acids can be oxidized or taken up by the liver for synthesis into triglycerides. The activities of both LPL and HSL are affected by numerous hormones and cytokines. Hypertriglyceridemia is commonly observed in cancer patients, and stimulation of hepatic lipogenesis has been clearly demonstrated (Grunfeld and Feingold, 1991). Studies have examined the adipose cells of cancer patients to investigate the relative regulation of LPL and HSL. The mRNA for LPL and the total LPL enzyme activity have not been found to be significantly different between cancer patients and healthy controls, and serum TNF-α levels were also normal (Thompson et al., 1993). On the other hand, there was a doubling of mRNA levels for HSL in adipose tissue of cancer patients. The cancer patients also exhibited a twofold elevation of serum triglyceride and fatty acid levels. There was a significant correlation of the serum fatty acid level with the expression of HSL mRNA in adipose tissue, consistent with increased mobilization of stored triacylglycerides and an increased release of FFAs into the circulation. Nonetheless, all of the cytokines demonstrate the ability to inhibit LPL (Strassman and Kambayashi, 1995). Short-term administration of TNF in animals and in some human experiments causes hypertriglyceridemia in addition to increased oxygen consumption, temperature, and whole body protein breakdown (Michie et al., 1988). Thus, there is no single mechanism accounting for fat mobilization. Because fat mobilization is so essential to surviving starvation, it is not surprising that many mechanisms regulating this process are dysregulated in cancer malnutrition and cachexia.

Fatty acid mobilization may be partly due to increased activation of β-adrenergic receptors in the fat cells of cancer patients (Drott et al., 1989). LMF causes lipolysis in adipocytes independent of a direct effect on LPL or HSL (Hirai et al., 1998). Instead, LMF stimulates a β-adrenergic receptor, which increases lipolysis in white adipose tissue and thermogenesis in brown adipose tissue (Russell et al., 2002). In fact, LMF has been shown to be identical to a plasma protein identified as zinc α2-glycoprotein (ZAG) by Todorov et al. (1996). The levels of ZAG mRNA are elevated 10-fold in the adipose tissue of cachectic mice implanted with MAC16 tumor cells (Bing et al., 2004). In studies of fat cells obtained from cachectic cancer patients, ZAG has not been detected. However, both ZAG mRNA and immunoreactivity have been detected in human omental and subcutaneous white adipose tissue in cancer-free healthy individuals. TNF-α acts through a membrane-bound receptor, which activates mitogen-activated protein kinase (MEK) and extracellular signal-related kinase (ERK) to stimulate lipolysis rather than acting through the adrenergic receptor (Zhang et al., 2002).

CANCER ANOREXIA Anorexia is simply defined as reduced appetite for food resulting in reduced food intake. It is a common condition among cancer patients with malnutrition and in many cases precedes the development of malnutrition (Moley et al., 1988). Parabiosis experiments, in which a nonmetastasizing tumor is implanted in one animal and its circulation is connected surgically to another non–tumor-bearing animal, demonstrate that the same anorexia, metabolic changes, weight loss, and cachexia occur in the non–tumor-bearing animal, despite no evidence of metastatic tumor at necropsy (Norton et al., 1985). The tumor-bearing and non–tumorbearing animal only share 1.5% of their total circulation, strongly suggesting a humoral factor such as a cytokine. In humans, anorexia is often multifactorial and may result primarily from effects of cytokines; tumor-related mechanical gastric and bowel compression, such as is seen with hepatomegaly and peritoneal metastases; disorders in hunger satiety regulatory systems; taste disorders often caused by drugs (such as metallic taste or oral mucosal disruption also known as mucositis); nausea and vomiting; or psychological problems, including depression (Padilla, 1986). Although there are no established approaches to reversing cytokine actions on anorexia, there are a number of approaches to treating anorexia, and its early identification in the cancer patient is central to the prevention, or at least amelioration, of the malnutrition associated with cancer. Even in cancer patients where food intake appears to be normal, there is a failure to adapt to metabolic needs if there is coexistent weight loss. Metabolic abnormalities in cancer

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often lead to an increase in REE, and this occurs in the absence of any increase in food intake. For example, in the latter half of the menstrual cycle (called the luteal phase), there is an increase in progesterone production, which leads to an increase in body temperature, appetite, and calorie intake, often associated with increased sweet and fat cravings, resulting in an increase in food intake. This increased food intake can be viewed as adaptive in response to the increased metabolic rate resulting from the increase in body temperature. The failure to adapt to increased metabolic demands in the cancer patient has been identified as relative hypophagia even when food intake measured using current instruments, including food records and food frequency questionnaires, is normal. These instruments are relatively inaccurate and may lead to underestimates of total energy intake as great as 25% when compared with total energy expenditure measured directly with doubly labeled water (Thomson et al., 1990). An additional problem in assessing anorexia is that food intake is rarely measured before the development of cancer (Peacock et al., 1987).

Central Nervous System Mechanisms Food intake in humans is carefully regulated in the ventromedial nucleus of the hypothalamus. Several neuropeptides including leptin, neuropeptide Y, insulin, galanin, endorphins, and cholecystokinin have been shown to affect both food intake and energy expenditure (Rosenbaum et al., 1997). It is clear from findings on the actions of leptin that there is careful regulation of body fat because circulating blood levels of leptin are proportional to body fat percentage over a significant physiological range (Considine et al., 1996). Leptin is produced by adipocytes in response to the net energy balance at the fat cell (Klein et al., 1996). In fact, glucose or citrate incubated with fat cells has been shown to be the direct signal for leptin secretion rather than insulin, which had been proposed to be the signal. In a number of studies of short-term calorie restriction and refeeding, leptin levels and insulin levels both decrease together with restriction and then increase again with refeeding. The balance of neurotransmitters in the hypothalamus can also affect food intake. Agents that affect serotonin and norepinephrine levels in the neuronal synapse affect appetite. Amphetamines, amphetamine analogs including phentermine, phenylpropanolamine, and norepinephrine reuptake inhibitors reduce appetite and have modest effects on energy expenditure. A number of common selective serotonin reuptake inhibitors (SSRIs) including fluoxetine (Prozac) and sertraline (Zoloft) reduce food intake and body weight at least transiently in obese subjects, especially when weight gain is a symptom of depression. Sibutramine (Meridia) is a combined norepinephrine and serotonin reuptake inhibitor and reduces food intake. On the other hand, other agents used to treat depression including monoamine oxidase

(MAO) inhibitors such as amitriptyline (Elavil) and lithium result in an increase in body weight and food intake in some individuals. As discussed earlier, progesterone increases food intake in women in the latter part of the menstrual cycle and has been associated with mild depression, and the glucocorticoids affect food intake by a separate mechanism in the hypothalamus where the type II glucocorticoid receptor has been identified. Appetite, physical activity, and food intake are regulated as carefully as body temperature by a combination of neurotransmitters, neuropeptides, and steroid hormones in the central nervous system. It may be possible to alter these mechanisms in a desired fashion as specific agents are explored in cachexia and obesity research to modulate food intake.

Nausea and Acquired Taste Aversions Nausea and acquired taste aversions are common problems in the malnourished cancer patient. De Wys and Walters (1975) specifically identified an inability to taste sugar and a heightened sensitivity to bitterness in cancer patients and associated these with observed red-meat aversion and elevated threshold to sweet taste that had been observed clinically. Such taste abnormalities have been associated with reductions in the levels of circulating trace metals, including zinc and nickel. In our own studies, we examined >100 cancer patients and were able to identify increased sweet threshold and red-meat aversion with heightened sensitivity to bitter tastes. However, by using age-matched controls without cancer, all differences in taste observed in our studies could be accounted for by age, sex, and smoking status (Larsen et al., 1983). Nonetheless, patients continue to report taste abnormalities, including metallic tastes secondary to chemotherapy and general losses of taste following head and neck irradiation. Some of these changes are clearly due to direct damage to the papilla in the tongue where the taste receptors are located (Mattes et al., 1992). In addition to these global taste changes, it is possible for patients to develop specific taste aversions. In experiments by Bernstein (1978), children were exposed to nut-flavored ice cream during chemotherapy, which is a type of ice cream not usually eaten by children. Following the appropriately controlled study designs, Bernstein (1978) was able to demonstrate that children developed a specific acquired taste aversion to nut-flavored ice creams, but not to strawberry, chocolate, or vanilla. As a result of these studies, it is recommended that patients avoid their favorite foods on the days they receive chemotherapy, particularly if they have nausea. This food intake then has to be made up through supplementation as they recover from the effects of chemotherapy. Fortunately, there are newer agents to deal with nausea specifically, including serotonin antagonists such as ondansetron and centrally acting drugs (metoclo-

Approaches to the Treatment of Anorexia and Cachexia

pramide, dexamethasone), which minimize nausea, acquired taste aversion, and the malnutrition associated with chemotherapy (Rolla et al., 1992). Physicians should consider the effects of frequent tests, procedures, and treatments on a patient’s ability to maintain adequate intake over time.

Psychological Causes Including Depression Stress and depression can lead to reduced food intake in some patients. One physiological basis for this is the increased levels of corticotropin-releasing factor (CRF) found in the cerebrospinal fluid of depressed patients who decrease their food intake by comparison with depressed patients who increase their food intake (Nemeroff et al., 1984). Infusion of CRF into the third ventricle of rats or mice leads to reduced food intake. Similar studies of cerebrospinal fluid have not been performed in cancer patients, but there are a number of surveys documenting the importance of depression in the cancer patient. This depression leads to a reduced quality of life and can lead to anorexia and reduced food intake. Careful attention to the psychological and social aspects of the patient and the family can often be helpful in ameliorating the effects of depression on food intake. However, in some cases pharmacological treatment of depression is required (Haig, 1992). In these cases, careful attention to the effects of the antidepressant therapy on food intake should be maintained.

APPROACHES TO THE TREATMENT OF ANOREXIA AND CACHEXIA In the 1920s, prior to the era of modern cancer treatment, it was believed that malnutrition was the means by which tumors killed patients (Warren, 1932). Therefore, great effort was expended in force-feeding patients with terminal cancer (Terepka and Waterhouse, 1956). In the 1970s following the introduction of total parenteral nutrition, force-feeding by the parenteral route was utilized in cancer patients with malnutrition. As reviewed by Klein et al. (1986), the net effect of total parenteral nutrition was negative with an increased incidence of hyperglycemia and infection. These two complications are likely related because it is known that hyperglycemia impairs white cell function. In the 1980s, the mechanisms by which cytokines could alter metabolism were demonstrated, as outlined in previous sections. However, no major breakthrough has been made that can reverse the effects of the cytokines released by the immune system in response to cancer. The successful treatment of cancer leads to a regain of lost weight and a reversal of the metabolic abnormalities associated with cancer cachexia. Therefore, the emphasis has now shifted to early diagnosis and recognition of malnutrition, adjunctive treatment of the malnourished patient with

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antiemetics, appetite enhancement, and enteral nutrition when possible. Pharmacological agents developed to combat anorexia were selected for study on the basis that they result in weight gain as a side effect in other conditions. Testosterone can be given in injectable form, particularly in patients with hypogonadism associated with cachexia. Anabolic androgens were tested based on their ability to build muscle and to counteract the metabolic effects of hypogonadism observed in male cancer patients (Chlebowski et al., 1986). Oxandrolone, an oral anabolic steroid, has been used to promote weight gain in patients who have had extensive surgery, chronic infection, or severe trauma (Berger et al., 1996). Appetite improvement and a trend toward weight gain were seen in a trial of oxandrolone in patients with AIDS (Berger et al., 1996). Danazol is a synthetic steroid known to cause weight gain. It was assessed in combination with tamoxifen in a randomized double-blind, placebo-controlled trial in patients with advanced cancer (Bishop et al., 1993). It was found that the combination stabilized body weight in patients that did not demonstrate weight gain in response to tamoxifen alone. In the 1980s, it was serendipitously noted (Tchekmedyian et al., 1986, 1987) that a high dose of megestrol acetate, a progestational steroid, increased appetite and led to weight gain in 30 of 33 patients with advanced breast cancer. Preliminary observations also demonstrated weight gain in 13 of 15 AIDS patients (von Roenn et al., 1988). Two subsequent randomized placebo-controlled trials demonstrated significant improvements in weight, appetite, well-being, and quality of life parameters in patients with AIDS (Oster et al., 1994; von Roenn et al., 1994). Based on these studies, the Food and Drug Administration (FDA) approved the use of megestrol acetate oral suspension in patients with AIDS anorexia. Additional randomized placebo-controlled trials showed appetite improvement in cancer patients given megestrol acetate (Berenstein et al., 2005). Although it appears that the primary effect of megestrol acetate is to enhance food intake, there may be important effects of megestrol acetate specific to adipocyte lipid metabolism. In animals, an increase in fat mass has been observed in the absence of a change in food intake following progesterone administration (Hervey and Hervey, 1967), and in cell culture studies megestrol acetate induces adipocyte development of lipogenic enzymes (Hamburger et al., 1988). A traditional medical maneuver of family physicians to increase appetite has been the administration of prednisone, a glucocorticoid. However, randomized trials of prednisone have revealed short-term (4 weeks) improvements in appetite, without weight gain (Moertel et al., 1974). Dronabinol, a marijuana derivative, has been found to improve appetite and mood in patients with AIDS and anorexia, without significant weight gain. FDA approval of this compound for use in AIDS anorexia was based on a placebo-

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controlled trial (Beal et al., 1995). Treatment with the serotonin antagonist cyproheptadine has been reported to have a weight-enhancing effect, but in a randomized, placebocontrolled, double-blind trial, it had no effect on progressive weight loss in cachectic cancer patients (Kardinal et al., 1990). Another line of investigation has examined the effects of drugs with anti-inflammatory effects. The nonsteroidal antiinflammatory drug (NSAID) indomethacin acts by inhibiting cyclooxygenase activity and has been shown to prolong survival of patients with solid tumors and improve some parameters of malnutrition such as hand-grip strength without an increase in body weight (Lundholm et al., 1994). Another NSAID, ibuprofen, has been shown to reduce REE and CRP levels in patients with pancreatic cancer (Wigmore et al., 1995). When combined with megestrol acetate and administered to patients with advanced gastrointestinal cancer, ibuprofen produced an increase in body weight (2.3 kg), compared with megestrol acetate alone, which in this study demonstrated a decrease in body weight (2.8 kg) (McMillan et al., 1999). Agents that interfere with TNF-α production such as pentoxifylline have been reported to decrease TNF-α mRNA levels in cancer patients. However, pentoxifylline did not affect either anorexia or cachexia in a study of 35 cancer patients (Goldberg et al., 1995). Thalidomide can also reduce production of TNF-α by increasing its degradation rate and has been shown to promote weight gain in human immunodeficiency virus (HIV)–infected patients who are receiving treatment for tuberculosis. No results for its use in cancer cachexia have been reported. A single study reported that the pineal hormone melatonin reduced the mean serum levels of TNF-α in patients with solid tumors and significantly reduced the incidence of high weight loss (>10%) compared with supportive care alone (Lissoni et al., 1996). However, there have been no randomized trials of melatonin. Eicosapentaenoic acid (EPA), an omega-3 polyunsaturated fatty acid, has been shown to antagonize the action of PIF. In cachectic mice bearing the MAC16 tumor, EPA attenuated protein degradation by downregulation of the increased expression of the proteasome subunits and ubiquitin-conjugating enzyme without any effect on protein synthesis (Whitehouse et al., 2001). EPA acts by interfering with intracellular signalling events, eventually preventing nuclear accumulation of NFκB (Whitehouse et al., 2003). EPA has been investigated as an anticachectic agent, both as a triglyceride (Wigmore et al., 1996) and as the free acid (Wigmore et al., 2000), in patients with pancreatic cancer and was found to produce weight stabilization in patients losing weight at the rate of 2.0–2.9 kg/mo. Combined with nutritional supplementation, EPA produced significant weight gain at 3 weeks (1 kg) and 7 weeks (2 kg) (Barber et al., 1999). Both performance status and appetite were sig-

nificantly improved, and there was a fall in REE normalized to lean body mass with a weight gain primarily as lean body mass. After 3 weeks of consuming a fish-oil–enriched nutritional supplement formula, a significant fall in IL-6 production, a rise in serum insulin with a fall in the cortisol-to-insulin ratio, and a fall in the proportion of patients that excreted PIF in the urine were observed (Barber et al., 2001). A randomized trial confirmed that there was a net gain of weight and lean tissue and improved quality of life (Fearon et al., 2003).

CLINICAL RECOMMENDATIONS FOR ANOREXIA AND CACHEXIA PREVENTION AND TREATMENT Early recognition of malnutrition is essential. Improved biomarkers are needed for the detection of early anorexia and metabolic changes associated with the host response to the tumor. Many of the available tools are so insensitive that global assessment of malnutrition clinically is often as discerning as obtaining biochemical measures of malnutrition. For clinical purposes, one should bear in mind that a 5% unintended weight loss is very significant and indicates a late manifestation of this process. Up to two-thirds of patients with advanced cancer have some degree of weight loss, and more than half are underweight, have loss of appetite, or complain of a decrease in food intake. More than 1.3 million new invasive cancer cases are diagnosed yearly in the United States alone, with an estimated 560,000 yearly cancer deaths. These statistics emphasize the enormous frequency of anorexia and cachexia (Cancer Statistics, 2004). The need for nutritional support depends on the clinical context, patient preferences, and economic considerations. The first step in management is nutritional evaluation, dietary counseling, and follow-up. Severe, persistent anorexia that does not respond to dietary counseling can be reversed with pharmacological treatment such as megestrol acetate. Nutritional support is available in the form of homemade or commercial food supplements, enteral nutrition, and parenteral nutrition. Conservative cost estimates for these interventions range from $8000/mo for home parenteral nutrition. Clinicians must be familiar with the benefits, risks, and costs of these therapies to suggest appropriate options. Table 1 summarizes the most common interventions utilized and presents benefits, risks, included services and products, and costs. One should note that with regard to androgen replacement, injectable testosterone is rather inexpensive ($2 for a 200-mg dose) compared with oral anabolic steroids. Transdermal testosterone patches have an intermediate cost (Tchekmedyian, 1998). As research progresses in the field of obesity for the development of various agonists and antagonists that affect

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Clinical Recommendations for Anorexia and Cachexia Prevention and Treatment

TABLE 1 Intervention Counseling

Food supplements: Homemade

Commercial Appetite stimulants: Megestrol acetate oral suspension Dronabinol Prednisone

Oxandrolone

Enteral nutrition

Home parenteral

Benefits, Risks, and Costs of Nutrition Interventions

Benefits

Included services and products

Risks

Average monthly charges or costs ($) (range) 190 (140–210)a

Patient satisfaction Patient education Nutrition maintenance Adherence to protocols

None

One initial and one follow-up visit by registered dietitian

Nutrition maintenance Avoid or delay need for more expensive therapy

Limited risks: diarrhea (lactose intolerance), nausea

Same

Patients may not like taste

Three 8-oz servings = 750 kcal/day Example: Carnation Instant Breakfast + milk Three 8-oz servings = 750–1080 kcal/day

Improved appetite, weight, well being

Male impotence, vaginal bleeding, deep-vein thrombosis Euphoria, somnolence, dizziness, confusion Hypokalemia, muscle weakness, cushingoid features, hyperglycemia, immune suppression, others Liver toxicity; contraindicated in prostate cancer

200 mg/day, 1-mo supply 400 mg/day, 1-mo supply 800 mg/day, 1-mo supply 2.5 mg/day, 1-mo supply 5 mg/day, 1-mo supply 40 mg/day, 1-mo supply

92 (88–109)c 184 (177–219) 368 (354–438) 161 (130–180)c 305 (220–354) 15 (4–21)c

5 mg/day, 1-mo supply 15 mg/day, 1-mo supply

344 (327–370)c 912 (817–1134)

Requires nasogastric, gastrostomy, or jejunostomy tube placement; aspiration, diarrhea, nausea, bloating, infection, bleeding Catheter-related pneumothorax, sepsis, thrombosis, bleeding; hepatic dysfunction, fluid and electrolyte imbalance

Feeding supply kit

469e, f

Improved appetite, no significant weight change Short-term (4-wk) appetite stimulation

Increased appetite at 15-mg/day dose; no significant weight change Maintenance of nutrition via enteral route when oral route is not available

Maintenance of nutrition when no other alternative is appropriate; no evidence of improved survival in end-stage cancer

Costs of indwelling venous devices and their placement, as well as care of complications not included

64 (49–80)b

168 (120–240)b

6517g

a

Charges calculated based on prevailing hospital- and office-based dietitian compensation; overhead not included. Average retail prices in five grocery stores in Long Beach, California (2005). c Average retail prices in five pharmacies in Long Beach, California (2005). d Medicare-allowable charges (Durable Medical Equipment Regional Center, Region D. Supplier Manual. Nashville, TN, Cigna, February 2005). e Based on intake of 2000 kcal/day. (Patient receiving 500-ml bolus four times a day using a 1 kcal/ml feeding.) Basic formula; specialized formulas for specific comorbidities can increase costs substantially. f Feedings are administered through a syringe as bolus feedings. g Estimated needs based on a 70-kg person receiving 25 kcal/kg and 0.8–1.0 g of protein/kg/day. Source: Modified and updated with permission from Tchekmedyian (2005). b

appetite and energy expenditure, the field of cancer cachexia research may benefit by the development of newer pharmacological agents based on this knowledge. In addition to the compounds presented in Table 1, research on the inflammatory response has progressed considerably as well, and it is now clear that the central nervous system senses inflammation via the direct actions of cytokines on brain centers. It has not been possible to separate the peripheral and central

aspects of the inflammatory response, but this area deserves further research. Further research with anticytokine drugs such as thalidomide and anabolic compounds such as growth hormone are ongoing. Research efforts are also focused on the nutritional modulation of the process of tumor progression following initial treatment. This work includes the use of low-fat, highfiber diets, micronutrients, and some phytochemicals found

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to have antitumor effects in experimental model systems. Ongoing clinical trials should provide new information on the use of nutritional interventions for the prevention of cancer recurrence and/or progression. In addition, it may be possible to separate the beneficial aspects of the immune response directed against the tumor and the undesirable spillover effects on host metabolism.

Acknowledgment This research was supported by the UCLA Clinical Nutrition Research Unit, National Institutes of Health grant no. CA 47210.

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40 Weight Management in the Breast Cancer Survivor ANNE MCTIERNAN

INTRODUCTION

mates percent body fat), fat calipers (can be used to estimate overall and regional body fat) (Roche et al., 1996). Other body composition measures give more reliable and valid measures of body composition but are more invasive and expensive. These latter include underwater weighing (considered the “gold standard” for body composition), DEXA scan (measures lean mass, bone mass, and fat mass), computed tomography (CT) and magnetic resonance imaging (MRI) scans (used to estimate intra-abdominal and subcutaneous abdominal fat, typically measured with one crosssectional slice to reduce radiation exposure and costs). BMI is the most common measure of adiposity used in studies of obesity and breast cancer and, for comparison purposes, is the one used throughout this chapter. Obesity experts have developed the following categories of adiposity based on BMI (Flegal et al., 1998): underweight (140 lbs (baseline ≤140 lbs); no difference was seen among postmenopausal women. RR of death for all obese women = 1.4* (baseline BMI ≤30.4) Survival was 71%** for women 66 kg; type of treatment was not shown to influence survival For all stages, 5-year survival rates were associated with BMI (χ2 = 15.02, p < .001); blacks had a lower probability of survival for 5 years (57%) than whites (76%), and a higher average BMI

RR for recurrence in women >130 lbs = 1.44**; RR for recurrence in women >130 lbs with negative axillary nodes = 2.65** Rate of recurrence = 41%** for obese and 8%** for nonobese; 40%** for women ≥160 lbs and 11%** for women 45 years, recurrence-free survival was significantly longer for those who received adjuvant ovarian ablation (χ2 = 5.75*) Cumulative 5-year disease-free survival = 49%* for women ≥150 lbs and 67%* for 154 lbs; 80% for women 154 lbs (baseline 29.7 in QI

10-year survival rates for premenopausal women = 80.8% (NS) (lean), 70.4%* (ordinary), and 75.1%* (obese); for postmenopausal women = 87.5%* (lean), 80.2%* (ordinary), and 68.8%* (obese) RR for death = 3.20* for body weight >60 kg (baseline 1.5 (baseline 1.2 (baseline 8.0 (baseline 25 (baseline 27 in QI; adjusted for age, stage, tumor size, nodal status, hormone receptor status, and level of treatment Restricted study to women with natural menopause; measured triceps and subscapular skinfolds to determine fat distribution

Quetelet score = (weight/height); obese defined as having Quetelet score >2.45 All patients had advanced breast cancer and were treated with tamoxifen (88%) or by ovarian ablation (12%) subsequent to their first relapse; menopausal status did not affect the results Calculations of BSA and QI were based on 536 patients for whom both weight and height were available

—, Not reported. *p < .05. **p < .01. NS, Not significant at α = 0.05 level. RR, relative risk; OR, odds ratio; HR, hazard ratio; QI, Quetelet index; BSA, body surface area; BMI, body mass index; FFQ, food frequency questionnaire; RW, relative weight; OI, obesity index.

Pittsburgh, PA

Katoh et al. (1994)

Population based

Hospital based

Manchester, UK

Denmark

Hospital based

Type of study

Breast Cancer Studies Showing Nonsignificant Results for the Associations among Survival or Recurrence and Obesity

Louisville, KY

Geographic location

Kamby et al. (1989)

Sohrabi et al. (1981) Williams et al. (1988)

Study

TABLE 3

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Potential Mechanisms for an Adverse Prognostic Effect of Obesity

include increased levels of circulating hormones such as estrogen and androgens, reduced levels of sex hormone–binding globulin (SHBG), which thereby increases the levels of free estradiol and free testosterone, increased levels of insulin and insulin-like growth factors (IGFs), reduced levels of IGF-binding globulin, increased levels of cortisol and leptin, increased levels of cytokines, effects of diet, reduced immune functioning, and chemotherapy underdosing in obese patients. Although some biological evidence exists to support many of these potential mechanisms, there is little direct evidence of their role.

669

FIGURE 5 Estrone and estradiol concentrations according to body mass index: the HEAL population-based cohort of stage 0–3a breast cancer patients (N = 505 postmenopausal cases).

Estrogens Postmenopausal women produce estrogens in fat and other tissue through the aromatization of androgens to estrogens (Siiteri, 1987). The enzyme aromatase is abundantly present in adipose tissue, especially subcutaneous fat. Estrogens are tumor promoters in vitro and in vivo, and women with high circulating levels of estrogens are at increased risk of developing breast cancer (Endogenous Hormones and Breast Cancer Collaborative Group, 2002; Key et al., 2003). Postmenopausal women who are overweight or obese have elevated levels of estrogens compared with lighter weight women (Cauley et al., 1989; Verkasalo et al., 2001). In a population-based cohort of 505 postmenopausal women with stage 0–3a breast cancer (the HEAL Study), adiposity was positively and statistically significantly associated with circulating levels of estrone, estradiol, and free estradiol (McTiernan et al., 2003) (Figures 5 and 6). Women were identified to this study through the SEER cancer registries of western Washington and New Mexico and were primarily non-Hispanic and Hispanic Whites. Between 4 and 12 months after diagnosis, anthropometric measures and blood draws were obtained on all women and DEXA scans were obtained on 415 women. Obese women (BMI ≥ 30 kg/m2) had 35% higher concentrations of estrone and 130% higher concentrations of estradiol, compared with lighter women (BMI < 22.0 kg/m2) (p trend, .005 and .002, respectively). Similar associations were observed for DEXA-derived body fat mass and percent body fat and waist circumference. Concentrations of free estradiol were doubled to tripled in overweight and obese women compared with lighter weight women (p trend = .0001).

Androgens Overweight, obese, and sedentary postmenopausal women have elevated concentrations of circulating total and free androgens (Cauley et al., 1989; Newcomb et al., 1995), and one report suggests that this association may be due to increased amounts of 17β-hydroxysteroid dehydrogenase in subcutaneous and intra-abdominal fat (Corbould et al., 1998). A combined analysis of nested case–control studies

FIGURE 6 Free estradiol concentrations according to body mass index: the HEAL population-based cohort of stage 0–3a breast cancer patients (N = 505 postmenopausal cases).

within nine cohort studies, which included data from 663 breast cancer cases and 1765 women without breast cancer, found that postmenopausal women with serum hormone concentrations in the top quintile for testosterone, androstenedione, dehydroepiandrosterone (DHEA), and DHEA-sulfate (DHEA-S) were approximately twice as likely to develop breast cancer compared with women with serum hormones in the bottom quintile (Endogenous Hormones and Breast Cancer Collaborative Group, 2002). In the same analysis, a doubling of androgen concentration resulted in a 20–40% increase in risk for breast cancer. When estradiol and testosterone were included in the same model, the effect of doubling of testosterone on breast cancer risk was greater than that of estradiol (RR 1.32 and 1.18, respectively), and similar results were observed for androstenedione when combined in a model with estradiol. These androgens may increase cell proliferation by being converted to estradiol and estrone in the circulation or target tissue (Siiteri, 1987). In addition, androgens may affect breast cancer risk by directly stimulating the growth and division of breast cells (Endogenous Hormones and Breast Cancer Collaborative Group, 2002).

670

Chapter 40/Weight Management in the Breast Cancer Survivor

FIGURE 7 Testosterone concentrations according to body mass index: the HEAL population-based cohort of stage 0–3a breast cancer patients (N = 505 postmenopausal cases).

stage, tumor stage, tumor grade, hormone receptor status, adjuvant chemotherapy, adjuvant tamoxifen). The effect of insulin on survival was independent of BMI. Circulating IGF-1, IGF-2, IGF-binding protein (IGFBP)-1 and IGFBP3, and estradiol did not exert independent effects on distant recurrence or death. Hyperinsulinemia could affect breast cancer prognosis in several ways. Insulin has been found to stimulate ER-α in breast cancer cells in vitro (Yee and Lee, 2000). Insulin also stimulates the production of estrogens and androgens, increasing production of these by adipose tissue, and downregulates sex hormone–binding protein, the major carrier molecule for estradiol and testosterone (Plymate et al., 1990).

Abdominal Fat

FIGURE 8 Free testosterone concentrations according to body mass index: the HEAL population-based cohort of stage 0–3a breast cancer patients (N = 505 postmenopausal cases).

In the HEAL cohort of breast cancer patients, overweight and obese women had statistically significantly elevated levels of testosterone, free testosterone, and DHEA-S (McTiernan et al., 2003) (Figures 7 and 8). Levels of DHEAS and free testosterone were higher in women in the top quartiles for body fat mass compared with the leanest women, although the trend was only statistically significant for free testosterone.

Insulin and Insulin-Like Growth Factors High levels of insulin and C peptide have been associated with increased risk for some common cancers (Kaaks et al., 200), and insulin has mitogenic effects on breast cells (Belfiore et al., 1996; Papa and Belfiore, 1996). In a study of 535 women with early-stage breast cancer, Goodwin et al. (2002) investigated several of these potential mediators. Fasting insulin levels were significantly associated with both distant recurrence and death. Women in the highest quartile of insulin levels had a 2.1 times increased risk of distant recurrence compared with those in the lowest quartile (95% CI 1.2–3.6, p = .01) and a 3.3 times greater risk of death (95% CI 1.5–7.0, p = .002) (adjusted for age, nodal

The body fat that is stored in the intra-abdominal area is thought to have special physiological properties and is more associated than other body fat with risk factors for diabetes and cardiovascular disease. Increased intra-abdominal fat is associated with increased levels of insulin and total and lowdensity lipoprotein (LDL) cholesterol (Irwin et al., 2003b). The role of visceral fat on breast cancer prognosis has not been established. Increased intra-abdominal fat, however, is associated with increased circulating levels of serum insulin and glucose (Irwin et al., 2003b), and as described earlier, insulin may be a tumor growth promoter. Therefore, interventions that decrease intra-abdominal fat levels may be hypothesized to improve cancer prognosis. In a small study, a 2-month low-fat diet and structured exercise intervention in women with a history of breast cancer resulted in a significant decrease in waist circumference (McTiernan et al., 1998). In a clinical trial in 173 postmenopausal overweight/obese sedentary women without breast cancer, exercise preferentially and significantly decreased intraabdominal fat (Irwin et al., 2003b).

Cytokines, Inflammation, and Immune Function Obesity is also associated with inflammatory markers including C-reactive protein (CRP), serum amyloid A (SAA), interleukin (IL)-6, IL-1, and tumor necrosis factorα (TNF-α), some of which have been shown to be higher in women with metastatic breast cancer compared with normal controls and with women with early breast cancer (Blann et al., 2002; Mahmoud and Rivera, 2002; O’Hanlan et al., 2002). Despite a paucity of data, it seems plausible that women with depressed immune function might be at increased risk of breast tumor progression. Studies suggest decreased immune function in obese women (Nieman et al., 1999), in women who participate in “yo-yo” dieting (Shad et al., 2004), and increased immune function with exercise in breast cancer survivors (Nieman et al., 1995).

Western, US

Minnesota and Illinois

Birmingham, AL

Chlebowski et al. (1986)

Camoriano et al. (1990)

Levine et al. (1991)

Hospital based

Hospital based

Multicenter

Hospital based

Type of study

32

545

62

67

N

—

—

—

II

Stage

26–68

20–75

—

—

Ages

1.8 kg** (−3.6–10.35)

Median: 5.9 kg** (premenopausal; 3.6 kg** (treated postmenopausal); 1.8 kg** (observed postmenopausal)

3.7 kg for CMF group; 2.0 kg for 5-FU group

18 lbs

Mean weight gain observation (range)

In 1 year, 91% of women in CMF group gained weight, 3% lost weight; 74% of women in 5-FU group gained weight, 26% lost weight; none of the five women who gained >10 kg survived* For premenopausal women: RR of recurrence = 1.5 (NS) for women who gained more weight than the median after 60 weeks; RR of death = 1.6* for women who gained more weight than the median 69% of total sample gained weight. RR of recurrence within 2 years for women who gained weight (baseline = no weight gain) = 1.36 (NS)

62/67 women gained at least 5 lbs; weight gain analyzed as % of ideal weight was negatively associated to disease-free survival (χ2 = 12.34*)

Results

Breast Cancer Studies of Weight Gain after Diagnosis and Clinical Outcome

—, Not reported; *P < .05; **p < .01; NS, not significant. CMF, cyclophosphamide, methotrexate, and 5-fluorouracil; CMFVP, CMF, vincristine, prednisone; 5-FU, 5-fluorouracil.

Chicago, IL

Geographic location

Bonomi et al. (1984)

Study

TABLE 4

No differences were observed among type of chemotherapy, operation, or menopausal status

RR adjusted for nodal status, age, estrogen receptor status, tumor size, baseline Quetelet index, and nuclear grade

Out of 67 women receiving adjuvant chemotherapy, 47 of these women also received tamoxifen Overall survival rates were 31% in CMF group and 51% in 5-FU group

Notes

Potential Mechanisms for an Adverse Prognostic Effect of Obesity

671

672


METHODS FOR WEIGHT LOSS AND MAINTENANCE FOR BREAST CANCER PATIENTS Several methods for weight loss or control have been tested in the general population (Obesity Education Initiative, 1998) or in persons with other obesity-related conditions such as diabetes and cardiovascular disease (Tuomilehto et al., 2001; Knowler et al., 2002). Detailed guidelines for the identification, diagnosis, and treatment of overweight and obesity have been published by several institutes of the National Institutes of Health (Obesity Education Initiative, 1998) and from the U.S. Preventive Task Force (McTigue et al., 2003). There are no specific guidelines for weight loss or maintenance methods in breast cancer patients or survivors, however. The first step for clinicians should be the assessment of body composition including BMI (McTigue et al., 2003). For overweight (BMI 25.0–29.9 kg/m2) or obese (BMI ≥ 30 kg/m2) women, physicians can then apply a weight loss strategy as outlined by the National Heart, Lung, and Blood Institute (NHLBI) Obesity Education Initiative Expert Panel (1998). This panel recommends a weight loss treatment algorithm that combines dietary therapy, physical activity, and behavioral treatment, provided on an ongoing basis to promote weight loss and maintenance.

Behavioral Weight Loss Therapy Reviews of randomized trials in healthy obese individuals and in those at high risk for other diseases (prediabetics, hypertensives) (Perri and Fuller, 1995; Perri, 1998; Wadden and Sarwer, 2000) show that the combination of diet and behavioral treatment typically delivered in 15–24 weekly group sessions produces an average weight loss of ~8.5 kg (mean body weight reduction = 9%). This degree of weight loss is associated with significant improvements in blood pressure, blood glucose, and psychological well-being (Blackburn, 1992, 1999). In the year following behavioral treatment, participants regain typically 30–40% of their lost weight. However, relatively few studies have provided behavioral treatment lasting more than 6 months, and follow-up studies conducted 2–5 years after behavioral treatment have documented a gradual but reliable return to baseline weights (Clark et al., 1991, 1996; Perri and Fuller, 1995; Perri, 1998; Kumanyika et al., 2000). Long-term success is more likely when participants are provided with extended treatment programs. Support for the efficacy of extended lifestyle treatment has been well documented (Graham et al., 1983; Clark et al., 1991, 1996; Perri and Fuller, 1995; Perri, 1998; Wadden and Sarwer, 2000). Compared with behavior therapy without additional therapist contacts, extended treatment in the form of weekly or biweekly group therapy sessions improves the maintenance of treatment effects for as

long as 1 year following initial therapy. Similarly, multicomponent approaches that combine ongoing client– therapist contacts (whether in person or by telephone and mail) with relapse prevention training or social support programs have shown improved maintenance compared with behavioral treatment without such programs (Clark et al., 1991, 1996; Perri and Fuller, 1995; Perri, 1998). Continued adherence appears to be the mechanism responsible for the better outcomes observed in extended treatments.

Weight Loss Diets The success of most dietary weight loss therapies has rested on reducing caloric intake below that required for current weight maintenance (e.g., creating a negative energy balance). A low-fat, reduced-calorie diet has been shown to produce significant weight loss when combined with behavioral change counseling (NHLBI Obesity Education Initiative, 1998). Reduced calorie diets can be achieved through meal replacements, dietary pattern change, or a combination (NHLBI Obesity Education Initiative, 1998). The first step in most weight loss diets is self-monitoring, where the individual records all food eaten each day. Then, the daily intake of calories, fat, fiber, carbohydrates, or all of these can be tallied by the individual or weight loss counselor. Another key step is frequent and regular weighing by a health professional or weight loss counselor. A major key to sustained weight loss is to achieve lifelong dietary pattern changes, rather than short-term “crash” diets (NHLBI Obesity Education Initiative, 1998). There is preliminary evidence that very low carbohydrate diets may work through additional mechanisms beyond simple calorie restriction and produce greater weight loss compared with low-calorie diets (Foster et al., 2003; Samaha et al., 2003), although over a year, low-carbohydrate diets may not be more efficacious than reduced calorie diets (Foster et al., 2003). All diets have similar issues, in that most patients regain weight within a year after initial weight loss (McTigue et al., 2003).

Weight Loss Pharmacotherapy For obese patients, or for those with BMI ≥ 27 with serious co-morbidities, weight loss medications may be a useful adjunct to diet and exercise therapy (Arterburn and Noel, 2001; McTigue et al., 2003). Two medications are approved for weight loss, and both are efficacious in the short term, although long-term efficacy data are not available. The first is sibutramine, a dopamine, norepinephrine, and serotonin reuptake inhibitor that inhibits appetite through a central mechanism. In a review of seven randomized clinical trials, sibutramine combined with lifestyle change promoted weight loss of 2.8–4.2 kg over 8–52 weeks in healthy adults and those with controlled hypertension

Methods for Weight Loss and Maintenance for Breast Cancer Patients

(Arterburn and Noel, 2001). However, patients regained weight after cessation of treatment. The second, orlistat, a gastrointestinal lipase inhibitor, prevents fat absorption. In 10 randomized trials, it has produced an average 3.5 kg weight loss over 1–2 years, in excess of control (lifestyle alone) weight loss. Another review with updated trial data (McTigue et al., 2003) concluded that therapy with sibutramine or orlistat combined with lifestyle change produced weight loss of 3–5 kg over that of control (lifestyle alone), and that prolonged use continued this weight loss past 2 years. Two other medications, phentermine and mazindol, produced similar weight loss in the short term but are not Food and Drug Administration (FDA) approved for longterm use. This same review concluded that three additional medications showed mixed results: metformin, diethylpropion, and fluoxetine. None of these medications has been specifically tested in breast cancer patients or survivors, so their effects on prognosis or other aspects of the breast cancer experience are unknown.

Bariatric Surgery For severely obese persons (BMI ≥ 40.0) or for patients with serious co-morbidities for whom obesity poses an extreme risk, more invasive methods of weight loss can be considered such as bariatric surgery (Brolin, 2002). The effect of weight loss surgery, however, has not been tested in persons who have had cancer.

Exercise and Weight Loss and Maintenance Physical activity may provide a low-risk method of preventing weight gain and promoting maintenance of weight loss in overweight and obese men and women (Pronk and Wing, 1994). Several studies have shown losses in total body weight from exercise training without dieting (Keim et al., 1990; Katoh et al., 1994; Irwin et al., 2003a). In a review of several hundred studies of exercise training and weight loss, Wilmore (1996) concluded that the average weight loss over 12 months of exercise training would amount to only 3.2 kg. Unlike weight loss dietary interventions, physical activity also increases or maintains muscle mass (Andreoli et al., 2001) and increases cardiorespiratory fitness levels (Hardman et al., 1992).

Weight Loss Studies in Cancer Patients and Survivors Studies of weight loss reduction have been limited to breast cancer patients and survivors and have been few and with mixed results. The Mayo Clinic randomized 107 breast cancer patients to monthly nonintensive dietitian counseling. Median weight increase at 6 months was 2 kg in the counseling group versus 3.5 kg in the control group, a nonsignificant difference (Loprinzi et al., 1996). Goodwin et al.

673

(1995) evaluated a multidisciplinary approach combining group dietary sessions, psychological support groups, and exercise programs in 61 breast cancer patients. For women with BMI > 25 kg/m2, weight loss was 1.63 ± 4.22 kg, and aerobic exercise increase was a strong predictor of successful weight loss (Goodwin et al., 1995). In a study of 34 obese breast cancer survivors, a combination of individualized counseling and Weight Watchers program produced greater weight loss than either alone or control (Djuric et al., 2002). Weight change after 12 months of intervention was 0.85 kg in the control group, −2.6 kg in the Weight Watchers program group, −8.0 kg in the individualized counseling group, and −9.4 kg in the comprehensive group that used both individualized counseling and the Weight Watchers program. Weight loss relative to control was statistically significant in the comprehensive group 3, 6, and 12 months after randomization, whereas weight loss in the individualized group was significant only at 12 months. The study resulted in weight loss of 10% or more of initial body weight in 6 of 10 women in the comprehensive group after 12 months. These same researchers found that the comprehensive group experienced significant declines in leptin and improvements in lipids (Jen et al., 2004). Finally, de Waard et al. (1993) randomized 102 postmenopausal women (median BMI 27 kg/m2) with a recent breast cancer diagnosis to a weight loss program involving stepwise reduction in caloric intake versus a control group. After 1 year, median weight loss was 6.0 kg with the intervention (p < .05). Most studies targeting weight loss in breast cancer patients have not focused on metabolic and hormonal consequences of weight loss. Furthermore, most of the small number of weight loss studies in breast cancer patients have been in newly diagnosed patients undergoing treatment (i.e., during times when attention to diet and exercise are difficult). The emerging science of weight loss combining dietary change, exercise, behavioral intervention, and ongoing contact (Blackburn, 1999; Kiernan and Winkleby, 2000) suggests weight loss programs can now be successfully implemented in breast cancer patients.

Dietary Interventions in Breast Cancer Patients and Survivors Dietary fat intake represents one possible mediating mechanism for the poor outcome associated with obesity in breast cancer, assuming a greater dietary fat intake in obese than in nonobese persons (Zhang et al., 1995; Herbert et al., 1998; Saxe et al., 1999). Though not without controversy (Holmes et al., 1999), the current evidence (Schatzkin, 1997) on dietary fat intake and breast cancer recurrence supports two ongoing full-scale outcome studies in breast cancer survivors (Chlebowski et al., 1993; Pierce et al., 1997). Feasibility of maintaining dietary fat intake reduction in breast cancer patients and survivors has been established

674


(Chlebowski et al., 1993, 2000). In women without breast cancer, reduction in estrogen levels in postmenopausal women and decreased mammographic density (a marker of breast cancer risk) (Boyd et al., 1997; Knight et al., 1999) have been associated with fat reduction. The two ongoing clinical trials of dietary fat intake reduction in breast cancer survivors have not targeted weight loss, reduction in BMI, or prevention of weight gain as intervention targets, however. Indeed, these interventions are associated with modest or no weight loss (e.g., ~4-lb difference between intervention and control throughout 3 years in the Women’s Intervention Nutrition Study [Chlebowski et al., 2000] and no difference between intervention and control participants in the WHEL Study [Thomson et al., 2004]). Thus, existing studies of dietary fat intake reduction in breast cancer populations are not addressing the question of weight loss in breast cancer patients and survivors.

Exercise Interventions in Breast Cancer Patients and Survivors In one clinical trial, 121 stage I-II breast cancer patients were randomized to control or self-directed or supervised exercise (Segal et al., 2001). Physical functioning was favorably (p < .01) impacted by exercise. Weight loss was not an intervention target, however, and only in the subset “supervised exercise in women not receiving chemotherapy” was reduced body weight (−3.8 kg) seen. Several other randomized trials of exercise in breast cancer patients and survivors have been reported, and all involved much smaller study populations (Courneya, 2003). Typically the intervention lasted only ~12 weeks, with a goal of change in fitness. Thus, existing studies of exercise in cancer populations have not targeted or achieved substantial weight reduction. There may be several quality of life and other beneficial effects of exercise in breast cancer survivors beyond weight loss and maintenance, so it is important to include exercise as part of the weight loss intervention. Furthermore, in women without breast cancer, exercise has been shown to promote weight maintenance after a weight loss intervention (NHLBI Obesity Education Initiative, 1998).

SUMMARY Overweight and obesity are associated with poor prognosis in breast cancer patients. There have been no randomized clinical trials testing the effect of weight loss on recurrence or survival in overweight or obese breast cancer patients, however. In the absence of clinical trial data, most individual patients should be advised to avoid weight gain through the cancer treatment process. In addition, weight loss is probably safe, and perhaps helpful, for overweight and obese breast cancer survivors who are otherwise healthy.

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lesterol, body mass index, height and cigarette smoking: a populationbased study. Eur J Cancer Prev 27(5): 641–646. Verkasalo, P.K., Thomas, H.V., Appleby, P.N., Davey, G.K., and Key, T.J. 2001. Circulating levels of sex hormones and their relation to risk factors for breast cancer: a cross-sectional study in 1092 pre- and postmenopausal women (United Kingdom). Cancer Causes Control 12(1): 47–59. Wadden, T.A., and Sarwer, D.B. 2000. Behavioral treatment of obesity. New approaches to an old disorder. In “The Management of Eating Disorders” (D. Goldstein, ed.). Humana Press, Totowa, NJ. Wee, C., McCarthy, E., Davis, R., and Phillips, R. 2000. Screening for cervical and breast cancer: is obesity an unrecognized barrier to preventive care? Ann Intern Med 132(9): 697–704. Williams, G., Howell, A., and Jones, M. 1988. The relationship of body weight to response to endocrine therapy, steroid hormone receptors and survival of patients with advance cancer of the breast. Br J Cancer 58: 631–634. Wilmorem, J.H. 1996. Increasing physical activity: alterations in body mass and composition. Am J Clin Nutr 63(3 Suppl): 456S–460S. Yancik, R., Ganz, P.A., Varricchio, C.G., and Conley, B. 2001a. Perspectives on comorbidity and cancer in older patients: approaches to expand the knowledge base. J Clin Oncol 19(4): 1147–1151. Yancik, R., Wesley, M.N., Ries, L.A., Havlik, R.J., Edwards, B.K., and Yates, J.W. 2001b. Effect of age and comorbidity in postmenopausal breast cancer patients aged 55 years and older. JAMA 285(7): 885– 892. Yee, D., and Lee, A. 2000. Crosstalk between the insulin-like growth factors and estrogens in breast cancer. J Mammary Gland Biol Neoplasia 5(107–15). Zhang, S., Folsom, A.R., Sellers, T.A., Kushi, L.H., and Potter, J.D. 1995. Better breast cancer survival for postmenopausal women who are less overweight and eat less fat. The Iowa Women’s Health Study. Cancer 76(2): 275–283.

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41 Nutrition Support of the Adult Cancer Patient HEIDI J. SILVER, EVE CALLAHAN, AND GORDON L. JENSEN

INTRODUCTION

catheter into a peripheral or central vein (parenteral nutrition support [PN]). The primary goal of nutrition support in the cancer patient is to reverse undernutrition in a safe and cost-effective manner (American Society for Parenteral and Enteral Nutrition [ASPEN] Board of Directors and the Clinical Guidelines Task Force, 2002). Despite aggressive attempts to provide nutrition support in patients with cancer cachexia, most patients fail to gain weight consistently (Heber et al., 1992). Secondary goals of nutrition support focus on minimizing antineoplastic treatment side effects and enhancing quality of life.

Twenty-five years ago, Bistrian et al. (1976) recognized that among hospitalized patients, those with cancer had the highest prevalence of undernutrition. Presently, more than half of all cancer patients and ~80% of those with gastrointestinal (GI) malignancies lose weight during the course of their disease (National Cancer Institute [NCI], 2004). More than two-thirds of these patients are adults 65 years or older with coexisting diseases. Weight loss and undernutrition contribute to morbidity and mortality. For example, Conti et al. (1977) found that weight loss in esophageal cancer patients with resectable tumors increased surgical risk. Additionally, Gianotti et al. (1995) showed in multiple regression analysis that preadmission weight loss of ≥10% of usual body weight was a strong predictor of postoperative infection (p = .02). Further, DeWys et al. (1980) observed 31–87% of 3047 patients enrolled in 12 chemotherapy protocols had weight loss before treatment and weight loss was related to decreased median survival. More recent estimates suggest that up to 40% of cancerrelated deaths are secondary to impaired nutritional status and mortality is highest in older patients (Zeman, 1991). The syndrome of cancer cachexia is characterized not only by weight loss but also anorexia and resultant decreased food intake, early meal satiety, alterations in taste and smell, weakness and fatigue, anemia, and edema (Heber et al., 1992). Within the options for medical nutrition therapy (MNT) interventions, nutrition support allows provision of nutrients (protein, carbohydrates, fat, water, vitamins, and trace elements) either through a tube into the stomach or small bowel (enteral nutrition support [EN]) or through a


ENTERAL NUTRITION SUPPORT Indications EN is typically provided to head, neck, and upper GI tract cancer patients in acute, community, and long-term care settings when patients are unable to chew, swallow, digest, and/or absorb adequately to meet nutrient requirements. One key reason EN is preferred to PN is that EN is more physiological; EN maintains gut mucosal barrier function and prevents endogenous gut bacterial translocation. For example, Jiang et al. (2003) randomized 40 patients with stomach or colon cancer into an EN or a PN group after tumor resection. Patients received isocaloric and isonitrogenous formulas initiated 3 days postoperatively. Intestinal permeability was measured by urinary excretion of lactulose and mannitol after administration on postoperative days 1, 7, and 12. Lactulose/mannitol ratio was significantly lower in the EN group on days 7 and 12, indicating increased gut mucosal integrity.

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Chapter 41/Nutrition Support of the Adult Cancer Patient

In a study conducted by Bozzetti et al. (2001), 317 GI cancer patients from 10 institutions in Italy were randomly assigned to isocaloric and isonitrogenous EN or PN solutions for 1 week after surgery. EN patients had significantly fewer total and infectious complications. Additionally, the mean length of hospital stay for the EN group was almost half as long as that of the PN group (5.7 ± 2.9 vs 10.4 ± 4.5 hospital days, respectively). In contrast, Fish et al. (1997) compared EN to PN of the same energy, nitrogen, and glutamine composition by randomly assigning 17 patients with gastric or pancreatic malignancies who were matched by age and body mass index (BMI) to receive either solution for 5 days postoperatively. No significant differences were observed for plasma amino acid profiles or nitrogen balance. Moreover, Braga et al. (2001) randomized 257 patients with upper GI tract cancer after surgery to early EN or PN solutions that were isocaloric and isonitrogenous for an average of 13 days. These authors observed no significant differences in nutritional variables (albumin, prealbumin, and retinol-binding protein) or immune function variables (delayed hypersensitivity response, polymorphonuclear phagocytosis, T lymphocytes, interleukin [IL]-2 receptors, IL-6, and C-reactive protein [CRP]) in a subgroup of these patients. Additionally, overall complication rate was similar (36% for EN vs 40% for PN). As in most studies comparing EN with PN, the EN group did not achieve their caloric goal as quickly as the PN group (4 days vs 1 day). However, a significant recovery of intestinal oxygen tension was observed earlier in EN patients and occurred from postoperative day 4 to study completion (p < .01). The potential benefits of administering EN have also been investigated in comparison with oral intake and with intravenous fluid without PN or oral intake. Bozzetti et al. (1998) compared EN with a standard oral diet in 50 undernourished patients receiving two cycles (32 days) of concomitant chemotherapy and radiation therapy for esophageal cancer. During the course of treatment, the EN group maintained body weight and visceral proteins, whereas the oral diet group had a significant decrease in body weight and visceral proteins (p = .01). Notably, the findings from this study may underestimate the benefits of EN, as the EN group was more undernourished with a greater pretreatment weight loss than the oral diet group (15.5% vs 12.3% of usual body weight, p < .02). In evaluating the provision of early EN (i.e., EN initiated within 24 hours postoperatively), Hochwald et al. (1997) demonstrated that early EN resulted in improved protein metabolism in 29 patients undergoing curative resection for an upper GI tract malignancy. In this study, patients were randomized to standard intravenous fluids (n = 17) or early EN (n = 12). The early EN patients had significantly decreased protein catabolism, as measured by continuous

leucine infusion, and improved net whole body protein balance. Similar results in achieving positive nitrogen balance with provision of EN were observed in studies conducted in the late 1980s in nonsurgical cancer patients (Daly et al., 1987). It is also generally agreed that EN is more cost effective than PN (ASPEN, 2002). However, only a few studies have compared the costs of EN with PN and the only one conducted with cancer patients exclusively was the aforementioned study by Braga et al. (2001). Like other studies, the main focus of these investigators was to compare EN and PN solution costs. Braga et al. (2001) estimated that using EN instead of PN would save $65 daily. In a retrospective comparison of the costs associated with home EN (HEN) to home PN (HPN) based on Medicare charges in 1996, Reddy and Malone (1998) found that the average annual cost per patient for EN solutions ($9535 ± 13,980) was about onefifth the cost of PN solutions ($55,193 ± 30,596). Yet, the entire cost of both EN and PN may be greater than what has been reported, as it would encompass other costs such as placement of the feeding tube or catheter, infusion pump loan, administration set and dressing kit, and costs of followup care including emergency room, physician office or clinic visits, home nursing and other professional therapeutic care, medication therapy, laboratory charges, and hospital readmissions.

Enteral Access Short-term and long-term techniques of enteral access are available for cancer patients who need EN. Short-time techniques such as nasogastric and nasojejunal feeding tube placement are considered easier to insert and lower in cost. However, these techniques are usually not used longer than 4–6 weeks because of the potential for patient discomfort (e.g., nasal irritation), technical failure (e.g., tube displacement), and/or long-term complications. Nasoenteric tubes are inserted through the nose, into the pharynx, down the esophagus, and into the stomach (nasogastric); or through the stomach into the duodenum (nasoduodenal); or through the duodenum into the jejunum (nasojejunal). Nasogastric feeding may be contraindicated for patients at risk of aspiration or those with delayed gastric emptying. For long-term EN, access techniques are typically through the abdominal wall (enterostomy) into the stomach (gastrostomy) or the jejunum (jejunostomy). A gastrostomy or jejunostomy can be performed by endoscopy, interventional radiology, or surgically. The percutaneous endoscopic gastrostomy (PEG) technique is the most common method for placing a gastrostomy tube. PEG tubes are usually placed in an endoscopy suite using intravenous conscious sedation and local anesthesia. Three methods have been described for PEG placement commonly referred to as “pull,” “push,” and

Enteral Nutrition Support

“poke” (Sacks et al., 1983; Russell et al., 1984; Ponsky, 1989). Contraindications to PEG tubes for EN are nonfunctioning gut, high risk for aspiration pneumonia, gastroesophageal reflux, ascites, or obstructions of the pharynx or esophagus that prevent passage of the endoscope (Vanek, 2002a). Percutaneous tubes can also be placed by interventional radiology. Two procedures are most commonly used for surgical placement of gastrostomy tubes in the operating room. The Stamm gastrostomy (1894) is an open surgical procedure that requires an incision through the abdominal wall, suturing the stomach to the abdominal wall, and inserting a catheter through that is sutured in place. The Witzel technique (1891) is more frequently used for jejunal feeding. In comparing the “push” PEG technique with surgical gastrostomy using the Stamm procedure, Steigmann et al. (1990) observed no significant differences in tube function, complication rates, mortality, or costs. Still, the PEG is used most frequently in the adult oncology population, as it can be performed as an ambulatory procedure. Gastric buttons, which are skin-level devices, are often preferable for children and more active ambulatory adult patients because they offer a low-profile tube with a shorter catheter length (Gauderer et al., 1980). For patients who require small-bowel feeding, such as those at high risk for aspiration or those who have had esophagectomy, small-bore percutaneous endoscopic jejunostomy or surgical jejunostomy tubes have been used with similar outcomes as PEG tubes (Bergstrom et al., 1995). Jejunostomy tubes are often anchored in place with an external suture to prevent migration or removal. Combination gastrojejunal tubes are placed endoscopically to allow simultaneous gastric decompression with jejunal feeding. Both gastrostomy and jejunostomy tubes can be replaced in an established tract.

Enteral Formula Selection A wide variety of liquid formulas are commercially available for enteral support of cancer patients. Some are also designed to be used as oral supplements depending on the palatability of the formulation. The choice of an enteral formula is typically patient specific. In addition to meeting energy, protein, and volume requirements, formula selection is influenced by the type of feeding access and the potential benefit from formulas enhanced by specialty nutrients. All formulas are supplemented with vitamins, minerals, and trace elements to meet recommended daily allowances (RDA) (Food and Nutrition Board [FNB], 1989). Formulas are typically categorized as polymeric, chemically defined, disease-specific, or specialty formulas. Polymeric (standard) formulas are usually lactose free and provide 1 kcal/ml. They are composed of intact nutrients

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(whole proteins, disaccharides, and triglycerides) and formulated so that 12–20% of the total calories are protein, 45–60% are carbohydrate, and 30–40% are fat. Some intact formulas are concentrated to provide more calories in less volume such as 1.5 or 2.0 kcal/ml or a higher nitrogen load per nonprotein calorie for patients with increased protein requirements. Most standard formulas were designed to be low in fiber and residue. Fiber-supplemented formulas are usually enriched with insoluble (soy polysaccharide) fiber and are often used in long-term EN because diarrhea is the most frequently occurring GI complication (Ciocon et al., 1992). Although the etiology of diarrhea is multifactorial and medications are a major factor (Edes et al., 1990), fibersupplemented formulas may contribute to resolving diarrhea, as fiber fermentation by anaerobic bacteria in the colon produces short-chain fatty acids, which increase sodium and water absorption (Zarling et al., 1994). Chemically defined formulas are often referred to as predigested or elemental formulas because the protein source is partially hydrolyzed by digestive enzymes. These formulas are primarily used for patients with reduced absorptive capacity or those with a jejunal feeding access and intolerant of polymeric formulas. The protein content of these formulas is provided as elemental amino acids, dipeptides, and/or tripeptides that can be directly absorbed by enterocytes via active transport. Because the sites on the small-bowel brush–border membrane for absorption of amino acids and small peptides are separate and separate carrier systems exist for transport, it is thought that enteral formulas containing both amino acids and peptides appear in the portal blood faster and allow greater amounts of total nitrogen absorption than formulas composed of amino acids alone (Johnson, 1997). One study conducted by Borlase et al. (1992) showed no significant difference in tolerance to either type of chemically defined formula. In disease-specific formulas, the nutrient composition has been modified to meet macronutrient or micronutrient needs in a specific disease state. For example, formulas that have increased amounts of the branched-chain amino acids (leucine, isoleucine, and valine) and reduced amounts of the aromatic amino acids (tryptophan, tyrosine, and phenylalanine) have been used in patients with hepatic encephalopathy. Likewise, formulations containing lower amounts of phosphorus, potassium, and sodium have been used in patients with kidney failure and medium-chain triglycerides have been used in lieu of long-chain triglycerides in patients with pancreatitis. Specialty formulas are designed to enhance immune function with specific nutrient substrates such as conditionally essential amino acids like glutamine and arginine or omega-3 fatty acids or ribonucleic acids (RNA). In 1988, Daly et al. found that supplementing EN with arginine in postoperative cancer patients significantly increased mean

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Chapter 41/Nutrition Support of the Adult Cancer Patient

T-lymphocyte response and CD4 count within 1 week of surgery. Similar benefits have been observed for pancreatic, gastric, and colorectal cancer patients who have undergone surgery (Di Carlo et al., 1999; Gianotti et al., 1999; Wu et al., 2001). In 1999, Heys et al. reported findings from a meta-analysis of 11 prospective randomized controlled trials comparing EN using a formula supplemented with “immune-enhancing” nutrients versus a standard enteral formula. Six of these eleven trials involved patients with GI malignancies who were treated postoperatively with early EN. The formula provided to patients in the experimental group in these six studies was enhanced with l-arginine, omega-3 fatty acids, and RNA (Impact). These 243 experimental patients experienced a 47% reduced incidence of postoperative infectious complications (i.e., major wound infections, intra-abdominal abscesses, and/or septicemia). However, the enhanced formula did not have significant effects on the incidence of nosocomial pneumonia or mortality. Yet these patients had significantly shorter hospital stays—by 2.4 hospital days. It is possible the decreased hospital stays provided economic benefits. In another study by Daly et al. (1995), the effect of immune-enhanced EN was evaluated on prostaglandin E2 (PGE2) synthesis, infectious complications, and hospital stay in 60 adults with upper GI tract malignancies who had jejunal tubes placed intraoperatively. Patients were stratified by disease and usual body weight and randomized to either standard enteral feedings or enteral feedings supplemented with l-arginine, omega-3 fatty acids, and RNA beginning 1 day postoperatively. Mean PGE2 production decreased significantly at day 7 only in the enhanced group. Infectious complications occurred in significantly fewer patients of the enhanced group (10%) compared with the standard group (43%). The enhanced group also had an average hospital stay that was significantly shorter (16 vs 22 hospital days). Although many cancer patients may benefit from administration of immune-enhanced EN, a meta-analysis conducted by Heyland et al. (1994) raises concerns that the potential benefit of reduced infections and complications may not extend to those patients who are critically ill. A number of studies suggest that provision of arginine, through increasing cytokine release and/or nitric oxide production, increases mortality in critically ill patients who have sepsis (Heyland and Samis, 2003). De Luis et al. (2003) investigated the effect of argininesupplemented early EN on ILs in oral and laryngeal cancer patients. Thirty-six patients who had a 5–10% weight loss before surgery were randomized to either standard EN or an isocaloric isonitrogenous arginine-enhanced EN for an average of 9 days. These investigators observed no significant differences in IL-6 and tumor necrosis factor-α (TNFα) levels of patients in the standard group compared with those receiving arginine supplementation.

Enteral Infusion Regimen Selecting a method for the EN infusion is based on the type of feeding tube, the enteral formula, the feeding route, and volume of the infusion. Continuous infusions are administered at a steady rate usually over a period of 16–24 hours in the acute-care setting and 8–16 hours in the home environment. Continuous feedings are beneficial for patients with delayed gastric emptying, risk for aspiration pneumonia, reduced absorptive capacity, or dumping syndrome. They are most frequently used for feeding into the duodenum or jejunum to prevent malabsorptive or osmotic diarrhea. Bolus or intermittent feedings allow infusing enteral formula at specific intervals throughout the day. Typically the total 24-hour volume of formula is divided into three to six feedings daily. Bolus feedings, for gastric access, are usually delivered by syringe or feeding reservoir into the tube with a maximum of 500 ml/feeding. Intermittent feedings are infused by gravity drip over a slower time period such as 30–120 minutes. After verification of feeding tube placement, EN is usually initiated full strength at a rate of 25–50 ml/h for standard formulas and 15–25 ml/h for hypertonic formulations or small-bowel feeding. The rate of feeding is advanced to the desired volume on the basis of patient tolerance, typically increased by 25 ml in 4–12 hour increments. As most cancer patients receiving EN are undernourished before tube placement, caution should be used in advancing enteral formula rate to prevent refeeding syndrome (i.e., aggressive feeding resulting in increased extracellular fluid volume, hypophosphatemia, hypokalemia, and hypomagnesemia). Gonzalez et al. (1996) observed hypophosphatemia of 1 month led to suppression of the hypothalamic–pituitary–adrenal axis (Cook and Meikle, 1985). Such secondary hypoadrenalism can be detected by measuring a morning cortisol level. A level of 180 days • Segmental mastectomy patient with dominant mass within ipsilateral breast remnant • Segmental mastectomy patient with tumor size >4 cm • Malignant breast cancer other than carcinoma or distant metastatic disease • Patients in the category of more advanced disease • Patients with previous breast malignancy • Patients consuming 25% reduction in calories from fat after the biweekly sessions, the sessions were repeated as needed over the next 2 months. After the initial intensive meetings, subsequent nutrition contacts (visits, calls, or mailings) were monthly through 8 months, then bimonthly through 12 months, and then quarterly through 24 months. The nutrition education sessions in the treatment arm were from the WINS low-fat eating plan (LFEP) that had been developed and pilot tested with postmenopausal breast cancer patients and detailed elsewhere (Buzzard et al., 1990). Briefly, the LFEP is a step-by-step individualized counseling approach to reduce total fat intake based on behavioral and social teaming conceptualizations of dietary behavioral change (Bandura, 1969). The LFEP includes education, goal setting, evaluation, and feedback components. The controls received minimal nutritional counseling except guidance on nutrient adequacy as defined by a nutrient intake that was 2 years with only a slight increase. Although there is not a significant group effect, there is a significant group by time interaction. During the first 3 months of the trial, the treatment group reduced their daily percentage fat calories from >32 to 60 g/day to just under 30 g/day by the third month. Over the following 21 months, their fat grams per day increased slightly to ~32 g/day. During the first 3 months of the trial, the treatment group lost a small amount of weight; however, by the end of the second year, they had gained it back. Applying the methodology of Leung and Elashoff (1996), we found that we could subclassify the treatment into two clusters—adherers and nonadherers—with respect to per-

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Chapter 45/Modern Statistical Methods in Clinical Nutrition

FIGURE 3 Observed data and mean response profiles for the treatment and control arms. Response: Weight. (From first edition of Nutritional Oncology.)

centage calories from fat and daily fat grams. The mean response profiles of these two clusters are shown in Figures 4 and 5, respectively. It is interesting to note that after the first 3 months of the trial, the mean profile for the treatment arm was ~20% calories from fat (see Figure 1). This mean is made up of the two clusters: adherers and nonadherers. The adherers, after the third month, have a mean of ~16% fat calories, while the nonadherers are >25%. A total of 44.4% (63/142) of the participants in the treatment group were classified as adherers with respect to percentage of fat calories. Because the cluster classification is based on the

expected posterior probability of belonging to a particular cluster, it may turn out that a participant is placed in Cluster 1 with a probability of 0.5–1.0. In this case, we are not very certain whether this participant belongs in this cluster or the other. Sixty-seven out of seventy-nine participants (84.8%) classified as nonadherent had a posterior probability of ≥60% of belonging to the nonadherent cluster. Sixty-two out of sixty-three participants (98.4%) classified as adherent had a posterior probability of ≥60% of belonging to the adherent cluster. This is a very high percentage of participants that were classified into a cluster with >60% probability, so we

731

Results of the Statistical Analysis of the Trial Data

TABLE 1

Comparison of Parameter Estimates and Standard Errors for the Different Methodologies; Outcome: Log of % Daily Energy from Fat

Linear regression

LME

GEE

Covariate

Est.

S.E.

P

Est.

S.E.

p

Est.

S.E.

p

Intercept bfata grpb BS1c BS2c bfat × BS1 bfat × BS2 grp × BS1 grp × BS2

−0.002 1.001 0.000 1.340 1.124 −0.407 −0.370 −0.450 −0.323

0.314 0.089 0.030 0.404 0.593 0.115 0.168 0.039 0.058

0.994 0.000 0.992 0.001 0.058 0.000 0.028 0.000 0.000

−0.003 1.001 0.000 1.327 1.330 −0.403 −0.426 −0.447 −0.325

0.207 0.059 0.020 0.384 0.572 0.109 0.163 0.037 0.056

0.990 0.000 0.986 0.001 0.020 0.000 0.009 0.000 0.000

−0.005 1.001 −0.001 1.318 1.210 −0.402 −0.387 −0.440 −0.350

0.003 0.001 0.000 0.304 0.602 0.085 0.170 0.032 0.052

0.103 0.000 0.085 0.000 0.045 0.000 0.023 0.000 0.000

a

Log of baseline % daily energy from fat. Treatment group coded 1 for treatment; 0 for control. c BS1, first B-spline; BS2, second B-spline. b

TABLE 2

Comparison of Parameter Estimates and Standard Errors for the Different Methodologies; Outcome: Log of Daily Fat Grams Linear regression

LME

GEE

Covariate

Est.

S.E.

p

Est.

S.E.

p

Est.

S.E.

p

Intercept bfata grpb BS1c BS2c bfat × BS1 bfat × BS2 grp × BS1 grp × BS2

−0.003 1.001 0.000 2.118 1.156 −0.555 −0.433 −0.570 −0.379

0.268 0.064 0.041 0.346 0.515 0.083 0.123 0.053 0.078

0.991 0.000 0.991 0.000 0.003 0.000 0.000 0.000 0.000

−0.002 1.000 0.000 2.062 1.636 −0.541 −0.460 −0.566 −0.398

0.189 0.045 0.029 0.337 0.488 0.081 0.117 0.052 0.074

0.993 0.000 0.985 0.000 0.001 0.000 0.000 0.000 0.000

−0.003 1.001 0.001 2.095 1.553 −0.550 −0.434 −0.559 −0.435

0.005 0.001 0.001 0.270 0.527 0.065 0.127 0.043 0.069

0.564 0.000 0.089 0.000 0.003 0.000 0.000 0.000 0.000

a

Log of baseline daily fat grams. Treatment group coded 1 for treatment; 0 for control. c BS1, first B-spline; BS2, second B-spline. b

TABLE 3

Comparison of Parameter Estimates and Standard Errors for the Different Methodologies; Outcome: Log of Weight

Linear regression

LME

GEE

Covariate

Est.

S.E.

p

Est.

S.E.

p

Est.

S.E.

p

Intercept bwta grpb BS1c BS2c bwt × BS1 bwt × BS2 grp × BS1 grp × BS2

−0.000 1.000 0.000 0.092 0.367 −0.020 −0.081 −0.028 −0.038

0.066 0.015 0.006 0.090 0.125 0.021 0.029 0.008 0.012

0.999 0.000 0.995 0.310 0.003 0.353 0.006 0.000 0.000

0.000 1.000 7.956 0.133 0.177 −0.029 −0.037 −0.027 −0.038

0.028 0.007 0.003 0.068 0.183 0.016 0.043 0.006 0.016

0.999 0.000 0.997 0.054 0.330 0.070 0.396 0.000 0.017

0.000 1.000 0.000 0.117 0.203 −0.025 −0.041 −0.027 −0.042

0.000 0.000 0.000 0.072 0.145 0.017 0.035 0.005 0.015

0.112 0.000 0.121 0.106 0.161 0.125 0.232 0.000 0.006

a

Log of baseline weight. Treatment group coded 1 for treatment; 0 for control. c BS1, first B-spline; BS2, second B-spline. b

732


FIGURE 4 Observed data and mean response profiles for the treatment clusters around percentage of calories from fat. (From first edition of Nutritional Oncology.)

think that these clusters represent a real finding and not some weak artificial outcome. With regard to total daily fat grams, 29.6% (42/142) of the participants in the treatment arm were classified into Cluster 2 (Figure 5), which we consider adherers. Of these adherers, 97.6% (41/42) of them were classified into this cluster with posterior probability >60%. Ninety-eight out of one-hundred (98%) of those classified as nonadherers were classified into this cluster with posterior probability >60%. We also clustered the controls into two groups with respect to percent fat calories and total fat grams. Non-

adherence in the control group is a little more difficult to assess. Figure 6 shows the two clusters of participants, with regard to percentage fat calories, with their mean responses. The participants in the first cluster clearly adhere (i.e., do not change their dietary habits). However, the participants in the second cluster changed their dietary habits and consumed a lower percentage of their calories from fat. By 2 years, the mean percentage fat calories in this cluster is 30 g/day during the first 3 months of the trial (see Figure 7). Classification probabilities again were quite high. A total of 107 (97.3%) out of 110 of the control participants that

were classified into Cluster 1 were done so with posterior probability >60%. It is interesting to look at how many participants in each arm of the trial were classified as adherers with respect to both percent fat calories and daily fat grams. Table 4 displays this cross-tabulation. In the treatment arm, 72.3% of the participants were classified the same with respect to both percentage fat calories and daily fat grams. In the control arm, 87.8% were classified the same. A possible explanation of why only 72.3% of the treatment arm participants were

734


FIGURE 6 Observed data and mean response profiles for the control clusters around daily fat grams. (From first edition of Nutritional Oncology.)

classified the same is that a large number, 30, of the participants were adherers with respect to percentage fat calories and nonadherers with respect to total fat grams. This suggests that these women may have achieved their goal with respect to percentage fat calories by keeping their total fat grams relatively constant while eating more. The strengths, weaknesses, and potential for errors in dietary assessment methods are well known (Beaton, 1994; Buzzard, 1994; Wynder et al., 1997). The possibility for inaccurate measurements exists with most measures because

they rely on self-report, as in the case of WINS use of 4DFR. The major strengths in food-record methods include the precision in measuring food intake and the limited reliance on memory in reporting intakes. The major limitation in 4DFR is the tendency for people to eat differently when recording their intake (Witschi, 1990; Schakel et al., 1998). This limitation is of particular concern in dietary intervention studies because of the potential for participants in the low-fat group to better adhere when recording their intake compared with other times. This behavior, generally known

Summary

735

FIGURE 7 Observed data and mean response profiles for the control clusters around daily fat grams. (From first edition of Nutritional Oncology.)

TABLE 4

Cross-Tabulation of Adherers and Nonadherers in Both Arms of the Trial (Treatment/Control) Percentage fat grams

Total fat grams Adherers Nonadherers Total

Adherers

Nonadherers

Total

33/107 30/15 63/122

9/3 70/23 79/26

42/110 100/38 142/148

as “response-set bias” (Kristal et al., 1998) cannot be controlled for and must be considered in the interpretation of the results herein. The WINS Feasibility Study was designed to investigate the viability of permanently changing the dietary habits of a randomly selected group of women who met certain inclusion/exclusion criteria (see Chlebowski et al., 1993). For the two responses investigated, weight and percentage of fat calories, the intervention was successful in changing dietary habits as evidenced by the group by time interaction.

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However, the specific goal of the study was to effect change that would result in a diet that consisted of 20% (or less) of daily calories from fat. Our cluster analysis shows that 44.4% of the women randomly assigned to the treatment group were successful (16.4% daily calories from fat). The remaining 55.6% reduced their percentage of calories from fat and achieved a mean of ~25% fat calories. This suggests that a more intensive intervention would be useful, followed by an aggressive maintenance program. It could be speculated that those women whose percentage fat calories were higher at baseline were those who could not adhere to the study protocol. However, we adjusted for baseline percentage of calories from fat in our model. This, in effect, removes any bias due to the baseline value. The mean baseline percentage of calories from fat for the adherers in the treatment group was 33.6% and that for the nonadherers was 34.4%. These are not significantly different from one another (p = 0.45, using the Wilcoxon Rank Sum Test) (Lehmann, 1975).

SUMMARY The usefulness of approaches to longitudinal analyses has been clearly shown. The newer methodologies initiated by Laird and Ware (1982), Jennrich and Schluchter (1986), and Liang and Zeger (1986) and further developed by almost a generation of statisticians have produced methods to importantly enhance our ability to obtain much more information from the longitudinal design. This work does not require the analyst to “fudge factor” the data; it makes minimal use of normal distributions (including some methodologies in which normal distribution assumptions are nowhere required); it does not require us to assume correlation structures of questionable accuracy; it enables us to make inferences with missing data; and it provides us with valid significance tests.

about the undetermined lymph node strata patient was not available. The power calculations were carried out non-parametrically using a flexible program that obtains power estimates for survival analyses via simulation. Options in the program include: up to 10 strata; general subject accrual proportions; two treatment groups; a variety of disease-free survival or survival distributions; drop-in or drop-out rates; loss to follow-up and withdrawal distributions; years of accrual and follow-up; and three methods of interim testing to maintain the overall significance level of the test (including Pocock, Tsiatis, and Fleming). The survival, loss-to-follow-up, withdrawal, and accrual distributions may be either empirical, Rayleigh, exponential, gamma, Weibull, or uniform. The power calculations for the WINS are based on a comparison of disease-free survival time between the two treatment groups (IIG and NIG) and the following assumptions: A. Stratification factor (only one stratification factor is used because the surgical consultant of SCU indicates that the three groups of systemic therapy do not have different disease-free survival rates.) Nodal status: Several accrual proportions by nodal status were considered. Nodal status Proportion of patients accrued

Sample size computations for this study are based on relapse-free survival as the primary outcome. Additionally, all subjects who are randomized, except the strata of undetermined lymph node group, will be included in the primary analysis within their randomized treatment assignments (i.e., an intent-to-treat analysis). Sample size calculations are not exact computations but are approximations based on an assumed model. We utilized published information from NSABP trials involving tamoxifen treatment in postmenopausal breast cancer populations to estimate the required survival probabilities that we expect to observe in the current study. The undetermined lymph node strata patients were not considered in sample size calculation because the survival or relapse-free survival information

+

10% 25% 55% 60%

90% 75% 45% 40%

B. Accrual and follow-up time: 417 subjects accrued per year for 6 years, with 3 years of additional follow-up. C. Drop-in rate from NIG to IIG and drop-out rate from IIG to NIG: Drop-in rate

Sample Size Considerations

Option 1 Option 2 Option 3 Option 4

−

0.10

Drop-out rate 0.30

D. Uniform accrual distributions E. Random censoring model F. No qualitative interaction G. Five interim analyses using the Fleming–Harrington method H. Two-sided significance level of .05 I. Withdrawals folded into the drop-out rate J. Upward adjustment of sample size to handle noncancer death rate without recurrence K. All centers follow the same survival distribution (i.e., there is no interaction of treatment by center) L. The final power was computed by carrying out 1000 simulations of the study design

737

References

TABLE 5

Assumed Cumulative Disease-Free Survival Rates

Nodal status

ER level

Time (years)

Group I

Group II

−

+

−

−

+

+

0.0 1.0 2.0 3.0 4.0 10.0 100.0 0.0 1.0 2.0 3.0 4.0 10.0 100.0 0.0 1.0 2.0 3.0 4.0 5.0 10.0 100.0 0.0 1.0 2.0 3.0 4.0 5.0 10.0 100.0

1.000 0.990 0.920 0.870 0.800 0.700 0.000 1.000 0.920a 0.900 0.880 0.850 0.750 0.000 1.000 0.950 0.850 0.750 0.700 0.650 0.500 0.000 1.000 0.900 0.750 0.650 0.600 0.550 0.400 0.000

1.000 0.995 0.970 0.940 0.870 0.800 0.000 1.000 0.950 0.940 0.930 0.900 0.850 0.000 1.000 0.970 0.920 0.820 0.780 0.730 0.600 0.000 1.000 0.950 0.820 0.720 0.680 0.630 0.500 0.000

+

−

TABLE 6

Results of Power Calculations

Accrual time Follow-up time No. of accrual patients per year Nodal status − : + % ER status − : + Drop in : out rate Power

6 years 3 years 417 60% : 40% 20% : 80% 10% : 30% 84.4%

M. The disease-free survival probabilities assumed within each stratum are summarized in Table 5; these values were based on published data from NSABP breast cancer trials involving tamoxifen. Table 6 summarizes the result of final power calculations. In summary, if 2500 subjects are accrued over a 6-year period and subsequently followed for 3 additional years, there would be 84% power to detect the assumed differences in disease-free survival at 9 years, using the assumed models. Based on these computations, our goal is to accrue at least 2502 subjects over a 6-year period.

CONCLUSIONS The statistical design is that of a randomized, two-arm study with two stratification factors: systemic therapy and nodal status. The primary endpoint of the study is diseasefree survival. A secondary endpoint includes overall survival defined as death from any cause. Both of these endpoints may be right-censored because of subjects who are lost to follow-up or who have not yet reached the study endpoint. Other secondary endpoints include the lipid profile measurements. These outcomes, as well as other measured covariates, may be binary, polychotomous, or continuous and may consist of repeated measurements over time.

References Bandura, A. 1969. “Principles of Behavior Modification.” Holt, Rinehart & Winston, New York. Beaton, G.H. 1994. Approaches to analysis of dietary data: relationship between planned analyses and choice of methodology. Am J Clin Nutr 59: 253s–261s. Buzzard, I.M., Asp, E.H., Chlebowski, R.T., Boyer, A.P., Jeffery, R.W., Nixon, D.W., Blackburn, G.L., Joehimsen, P.R., Scanlon, E.F., Insull, W.J., Elashoff, R.M., Butrum, R., and Wynder, E.L. 1990. Diet intervention methods to reduce fat intake: Nutrient and food group composition of self-selected low-fat diets. J Am Diet Assoc 90: 42– 50. Buzzard, I.M. 1994. Rationale for an international conference series on dietary assessment methods. Am J Clin Nutr 59(Suppl): 143s–145s. Cheney, W., and Kincaid, D. 1994. “Numerical Mathematics and Computing.” Brooks/Cole, Pacific Grove, CA. Chlebowski, R.T., Blackburn, G.L., Buzzard, I.M., Rose, D.P., Martino, S., Khandokar, J.D., York, R.M., Jeffery, R.W., Elashoff, M.E., and Wynder, E.L. 1993. Adherence to a dietary fat intake reduction program in postmenopausal women receiving therapy for early breast cancer. J Clin Oncol 11: 2072–2080. Cohen, L.A., Rose, D.P., and Wynder, E.L. 1993. A rationale for dietary intervention in postmenopausal breast cancer patients: an update. Nutr Cancer 90: 1–10. Davidson, M.H., Maki, K.C., Kong, J.C., Dugan, L.D., Torri, S.A., Hall, H.A., Drennan, K.B., Anderson, S.M., Fulgoni, V.Z., Saldanha, L.G., and Olson, B.H. 1998. Long term effects of consuming foods containing psyllium seed husks on serum lipids in patients with hypercholesterolemia. Am J Clin Nutr 67: 367–376. DeBoor, C. 1978. “A Practical Guide to Splines.” Springer-Verlag, New York. Diggle, P.J., Liang, K., and Zeger, S.L. 1994. “Analysis of Longitudinal Data.” Oxford University Press, New York. Jennrich, R.J., and Schluchter, M.D. 1986. Unbalanced repeated measures models with structured covariance matrices. Biometries 42: 805– 820. Kristal, A.F., Andrilla, H.A., Koepsell, T.D., Diehr, P.H., and Cheadle, A. 1998. Dietary assessment instruments are susceptible to interventionassociated response set bias. J Am Diet Assoc 98: 40–43. Laird, N.M., and Ware, J.H. 1982. Random-effects models for longitudinal data. Biometrics 38: 963–974. Lehmann, E.L. 1975. “Nonparametries: Statistical Methods Based on Ranks.” Holden-Day, San Francisco. Leung, M.K., and Elashoff, M.E. 1996. Generalized linear mixed-effects models with a finite-support random-effects distribution: a maximumpenalized likelihood approach. Biometr J 38: 135–151.

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Liang, K., and Zeger, S.L. 1986. Longitudinal data analysis using generalized linear models. Biometrika 73: 13–22. Lipkin, E.W. 1998. A longitudinal study of calcium regulation in a nonhuman primate model of parenteral nutrition. Am J Clin Nutr 67: 246–254. Little, R.J.A. 1995. Modeling the drop-out mechanism in repeatedmeasures studies. JASA J Am Stat Assoc 90: 1112–1121. Little, R., and Rubin, D. 2002. “Statistical Analysis with Missing Data,” 2nd edition. Wiley InterScience, Hoboken, NJ. Peto, R., Pike, M.C., Armitage, P., Breslow, N.E., Cox, D.R., Howard, S.V., Mantel, N., McPherson, K., Peto, J., and Smith, P.G. 1977. Design and analysis of randomized clinical trials requiring prolonged observation of each patient. II. analysis and examples. Br J Cancer 35: 1–39. Robins, J.M., Rotnitzky, A., and P, Z.L. 1995. Analysis of semiparametric regression models for repeated outcomes in the presence of missing data. JASA J Am Stat Assoc 90: 106–121. Rubin, D.B. 1976. Inference and missing data. Biometrika 63: 81–92. Savendahl, L., Mars, M.H., Underwood, L.E., and Zeisel, S.H. 1997. Prolonged fasting in humans results in diminished plasma choline concen-

trations but does not cause liver dysfunction. Am J Clin Nutr 66: 622–625. Schakel, S., Sievert, Y.A., and Buzzard, I.M. 1998. Sources of data for developing and maintaining a nutrient database. J Am Diet Assoc 88: 1268–1271. Shi, M., Weiss, R.E., and Taylor, J.M.G. 1996. An analysis of paediatric CD4 counts for acquired immune deficiency syndrome using flexible random curves. Appl Stat 45: 151–163. Wang, Y., and Taylor, J.M.G. 1995. Flexible methods for analysing longitudinal data using piecewise cubic polynomials. J Stat Comput Simul 52: 133–150. Witschi, J.C. 1990. Short-term dietary recall and recording methods. In “Nutrition Epidemiology” (W. Willett, ed.), pp. 42–50. Oxford University Press, New York. Wynder, E.L., Cohen, L.A., Muscat, J.E., Winters, B., Dwyer, J.T., and Blackburn, G. 1997. Breast cancer: weighing the evidence for a promoting role of dietary fat. J Natl Cancer Inst 89: 766–775. Zeger, S.L., and Liang, K. 1986. Longitudinal data analysis for discrete and continuous outcomes. Biometries 42: 121–130.

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46 Evidence-based Practice Management in Cancer Prevention and Treatment GEORGE L. BLACKBURN

INTRODUCTION

These new approaches include modification of traditional levels of evidence to accommodate outcomes from wellconducted observational studies (Blackburn et al., 2005) and evaluation of evidence beyond that of efficacy alone. The U.S. Preventive Services Task Force has acknowledged these changes by citing the need to consider evidence as a whole, including trade-offs among benefits, harms, costs, and net benefits relative to optimal allocation of resources (Harris et al., 2001; Blackburn et al., 2005).

Methodologies for summarizing, assessing, and judging the strength of scientific evidence on diet, nutrition, and the causation and prevention of cancer have evolved from Sir Austin Bradford Hill’s inference of etiology in a broad biological context (e.g., criteria for establishing scientific causation) (Bradford Hill, 1965) to evidence-based medicine’s (EBM) focus on efficacy of treatment. The evidence-based approach is considered state of the art in efforts to synthesize research findings as a basis for practice, guidelines, and recommendations (U.S. Preventive Services Task Force, 1996; Heggie et al., 2003; Morris and Carson, 2003; American Diabetes Association, 2005a) (Tables 1 and 2). The aim of EBM (Evidencebased Medicine Working Group, 1992) is to promote clinical decision-making based on the best available scientific evidence (Bero and Rennie, 1995; Blackburn et al., 2005). The standards against which evidence is judged are designed to be overt and predefined, with studies classified according to grades of evidence based on research design. The result is a hierarchy of evidence that puts the highest weighting on randomized controlled trials (RCTs) rather than on observational studies (Heggie et al., 2003) (Table 3). Considerable evolution has occurred in the evaluation of scientific evidence. Advances in study design methodology, along with data showing parity of outcomes between RCTs and observational interventions (Benson and Hartz, 2000; Concato et al., 2000), have challenged assumptions that underlie the hierarchy of evidence. In part, they have also served as an impetus for the development of new methods to assess the quality of research findings.


CANCER RATES AND CAUSATION Background Cancer is a major cause of mortality throughout the world. It accounts for 7.1 million deaths annually (12.5%) of the global total. Approximately 20 million people suffer from cancer—a figure projected to rise to 30 million within 20 years (World Health Organization, 2005a). The latest data from the American Cancer Society (ACS) (2006b) indicate that cancer is the leading cause of death among all Americans younger than 85 years. This year in the United States, the ACS estimates that 1,372,910 new cases of cancer will be diagnosed and that 570,260 people will die from the disease. Lung cancer, which is largely preventable, is the leading cause of cancer death, accounting for one in three deaths in men and one in four in women.

Trends In many countries, >25% of deaths are attributable to cancer. In 2000, 5.3 million men and 4.7 million women

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Chapter 46/Evidence-based Practice Management

TABLE 1 I II-1 II-2

II-3

III

U.S. Preventive Services Task Force Hierarchy of Research Design

Evidence obtained from at least one properly randomized controlled trial Evidence obtained from well-designed controlled trials without randomization Evidence obtained from well-designed cohort or case-control analytical studies, preferably from more than one center or research group Evidence from multiple time series with or without the intervention; dramatic results in uncontrolled experiments (such as results of the introduction of penicillin treatment in the 1940s) Opinions of respected authorities, based on clinical experience, descriptive studies and case reports, or reports of expert committees

Note: The Third U.S. Preventive Services Task Force added a threecategory rating (good, fair, poor) of internal validity to its standard hierarchy of research design. A well-performed randomized controlled trial, for example, would be rated 1-good; a fair cohort study would be rated II-2-fair (Agency for Healthcare Research and Quality, 2001; Harris et al., 2001).

developed a malignant tumor, and 6.2 million of them died from the disease. Cancer has emerged as a major public health problem in developing countries, with an impact equal to that in industrialized nations (International Agency for Research on Cancer[IARC], 2004); it is becoming an increasingly important factor in the global burden of disease. The number of new cancer cases annually is expected to increase an estimated 50%, rising from 10 million in 2000 to 15 million by 2020 (Stewart and Kleihues, 2003). This predicted rise will be due mainly to steadily aging populations in both developed and developing countries, as well as growing adoption of unhealthy lifestyles. Approximately 60% of new cancer cases will occur in the less developed parts of the world (WHO). Cancer is strongly associated with social and economic status, with risk factors highest in groups with the least education (WHO, 2005a). In most developed countries, cancer is the second leading cause of death after cardiovascular disease (CVD), and epidemiological evidence points to this trend emerging in the less developed world. This is particularly true of countries in “transition,” or middle income countries such as South America and Asia. Already more than half of all cancer cases occur in developing countries. The incidence of lung cancer and cancers of the colon, rectum, breast, and prostate generally increases in parallel with economic development, while stomach cancer declines.

Environmental Causes From a global perspective, the three main cancer-causing agents are tobacco, diet and exercise, and infections (IARC/WHO, 2006). Dietary modification and regular physical activity are significant elements in cancer prevention and control. Overweight and obesity are both serious risk

TABLE 2

American Diabetes Association Grading System for Clinical Practice Recommendations Level of Evidence Description

A Clear evidence from well-conducted, generalizable, randomized controlled trials that are adequately powered, including: • Evidence from a well-conducted multicenter trial • Evidence from a meta-analysis that incorporated quality ratings in the analysis • Compelling nonexperimental evidence, i.e., “all or none” rule developed by the Center for Evidence-Based Medicine at Oxforda Supportive evidence from well-conducted randomized controlled trials that are adequately powered, including: • Evidence from a well-conducted trial at one or more institutions • Evidence from a meta-analysis that incorporated quality ratings in the analysis B Supportive evidence from well-conducted cohort studies, including: • Evidence from a well-conducted prospective cohort study or registry • Evidence from a well-conducted meta-analysis of cohort studies Supportive evidence from a well-conducted case-control study C Supportive evidence from poorly controlled or uncontrolled studies, including: • Evidence from randomized clinical trials with one or more major or three or more minor methodological flaws that could invalidate the results • Evidence from observational studies with high potential for bias (such as case series with comparison with historical controls) • Evidence from case series or case reports Conflicting evidence with the weight of evidence supporting the recommendation E Expert consensus or clinical experience Note: The latest American Diabetes Association grading system assigns ratings of A, B, or C depending on the quality of evidence. Expert Opinion (E) is a separate category for recommendations that do not yet have clinical trial evidence, clinical trials might be impractical, or there is conflicting evidence. Source: From the American Diabetes Association, 2006. a Either all patients died before therapy and at least some survived with therapy, or some patients died without therapy and none died with therapy; for example, use of insulin in the treatment of diabetic ketoacidosis.

factors for cancer. After tobacco, overweight and obesity appear to be the most important avoidable causes of cancer (WHO, 2005a). Dietary factors account for up to 30% of cancers in Western countries (Doll and Peto, 1996). The proportion is thought to be ~20% in developing countries and is expected to grow (IARC, 2006). As developing nations become urbanized, patterns of cancer, particularly those most strongly associated with diet and physical activity, tend to shift toward those of economically developed countries. Cancer rates also change as populations move between countries and adopt different dietary patterns.

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Cancer Rates and Causation

TABLE 3

Evidence-based Medicine Conventional Hierarchy of Study Design

Experimental studies Meta-analysis of randomized controlled trials Randomized controlled trials Quasi-experimental Observational studies Cohort study Case-control study Cross-sectional study Before and after study Case series Note: Evidence-based medicine’s traditional hierarchy of evidence puts the highest weighting on randomized controlled trials rather than on observational studies. Source: From NHS Centre for Reviews and Dissemination, 2001.

Migrants from regions with low rates of colon cancer to those with high rates of colon carcinogenesis (or vice versa) show rapid changes in their risk of colon cancer. In the United States, colon and rectal cancers are increasing, with ~1 million new cases every year. These tumors are most prevalent in the economically developed world, but their incidence is rising in developing countries as well (Bingham et al., 2003; IARC, 2006). Dietary effects are presumed to underlie many of the large international differences in incidence seen for most cancers (McCullough and Giovannucci, 2004).

Dietary Factors and Cancer Alcohol consumption is an established cause of cancers of the oral cavity, pharynx, larynx, esophagus, liver, and breast. For each of these cancers, risk increases substantially with intake of more than two drinks per day (ACS, 2005a). Prolonged high consumption of red and processed meat is also thought to increase the risk of cancer in the distal portion of the large intestine (Chao et al., 2005). Data show an association between intake of dietary fiber and risk of colon and rectal cancers. Bingham et al. (2003) report that an approximate doubling of total fiber from foods could reduce the risk of colorectal cancer by 40%. Similarly, Peters et al. (2003) suggest that dietary fiber, particularly from grains, cereals, and fruits, is associated with decreased risk of distal colon adenoma. Findings from the Women’s Health Initiative (WHI) Dietary Modification Trial suggest that the risk of brest cancer may be modified by reducing the intake of total fat to 20% of total energy and increasing the daily consumption of vegetables and fruit and grain (Buzdar, 2006; Prentice et al., 2006). These data are complemented by initial results from another prospective dietary study, the Women’s Intervention Nutrition Study (WINS), which show that a lifestyle intervention resulting in dietary fat intake reduction can

improve the relapse-free survival of postmenopausal breast cancer patients (Chlebowski et al., 2005). Studies indicate that consumption of fruits and vegetables may protect against cancers of the mouth, pharynx, larynx, stomach, colorectum, and lung (IARC/WHO, 2005). However, van Giles et al. (2005) suggest that the intake of fruits and vegetables, once thought to protect against breast cancer, provides no significant risk reduction. These data run counter to those of the EPIC study (Miller et al., 2004). Obesity, insulin resistance, and low physical activity may mask or negate biologically consequential benefits from consumption of fruits and vegetables. Using the cumulative average of repeated dietary assessments could introduce systematic measurement error and further distort diet–cancer relations (Schatzkin and Kipnis, 2004). As originally reported in Food, Nutrition and the Prevention of Cancer: A Global Perspective (World Cancer Research Fund, 1997), confounding can interfere with the interaction of dietary factors and cancer in nutritional intervention studies, leading to misinterpretation of data, misleading results, and missed opportunities to prevent and/or control cancer. Further investigation is necessary to assess whether adjustment for these variables can lead to protection against cancer, earlier detection of existing cancers, or improved prognosis. A priority exists to prevent and treat obesity, insulin resistance, and physical inactivity so that patients can gain the health benefits contained within the 2005 Dietary Guidelines for Americans (Department of Health and Human Services [HHS] and the U.S. Department of Agriculture [USDA], 2005). Dietary patterns, physical activity, and weight control can substantially affect the risk of developing cancer and modify cancer risk at all stages of its development (ACS, 2006a).

The Obesity-Cancer Connection In the United States, current patterns of overweight and obesity are thought to account for 14% of all deaths from cancer in men and 20% of those in women. Calle et al. (2003) report a relation between increased body weight and higher death rates for all cancers combined and for cancers at multiple specific sites. Obesity has been linked to significantly higher death rates from cancers of the esophagus, colon, rectum, liver, gallbladder, pancreas, and kidney. It has also been implicated in higher death rates from non-Hodgkin’s lymphoma and multiple myeloma (Calle et al., 2003; ACS, 2006a). Significant trends of increasing risk with higher body mass index (BMI) have also been seen in death rates from stomach and prostate cancers in men, and cancers of the breast (in women), uterus, cervix, and ovaries (Calle et al., 2003; ACS, 2006a). A study indicates that obesity may be associated with more advanced-stage prostate cancer and lower overall survival rates (Baillargeon et al., 2005). A substantial body of evidence (Chlebowski et al., 2004) suggests that biological mechanisms for these associations

742


and trends include increased levels of endogenous hormones (sex steroids, insulin, and insulin-like growth factor-1 [IGF1]) associated with overweight and obesity. Longer exposure of breast tissue to circulating estrogen has been linked to increased risk of breast cancer (ACS, 2006a). Components of the metabolic syndrome—for example, hyperinsulinemia (Lawlor et al., 2004), insulin resistance (Muti et al., 2002), low high-density lipoprotein cholesterol (HDL-C) (Furberg et al., 2005)—are known to be associated with cancers of the breast, colon, and other sites (ACS, 2005a). Malin et al. (2004) report that insulin resistance and IGFs may synergistically increase breast cancer risk. These data are consistent with those from other studies (Chlebowski et al., 2004, 2005; Winters et al., 2004). As mechanisms underlying human carcinogenesis are better understood, dietary research will increasingly focus on intermediate markers, such as the IGFs and potential carcinogenic metabolites (McCullough and Giovannucci, 2004).

Prevalence of Obesity The prevalence of obesity worldwide has risen sharply during the past four decades. Substantial evidence shows a relation among overweight, obesity, and many types of cancer, such as those of the esophagus, colorectum, breast, endometrium, and kidney (WHO). In the United States, the number of obese adults doubled to ~63 million between 1976–1980 and 2001–2002. The ranks of those with severe obesity, with >100 lbs of excess weight, grew to nearly 11 million people in 2001/2002 (Lehman Center Weight Loss Surgery Expert Panel, 2005). The WHO estimates that of the more than one billion people who are overweight (BMI ≥ 30 kg/m2) worldwide, 300 million are obese (BMI ≥ 40 kg/m2) (SanchezCastillo et al., 2004). The 2005 Dietary Guidelines for Americans (Bero and Rennie, 1995) underscores the health risks associated with overweight, obesity, and a sedentary lifestyle by highlighting the need for physical activity and calorie control as well as a healthy balance of nutritious foods.

Physical Activity The nature of the associations among physical activity, obesity, nutrition, and cancer remains unclear. However, regular activity is thought to improve metabolic syndrome and reduce risk of colon, breast, endometrial, and prostate cancers (Calle et al., 2003; Chlebowski et al., 2004, 2005; Winters et al., 2004; WHO, 2004). Regular physical activity can contribute to the maintenance of a healthy body weight by balancing caloric intake and energy expenditure (ACS, 2006a). Data from the IARC and the WHO (IARC, 2006) indicate that limiting weight gain reduces the risk of cancer

at several sites, including breast (in postmenopausal women), endometrium, kidney (renal cell), and esophagus. Modern chronic diseases, including type 2 diabetes, coronary heart disease, and cancer are the leading killers in Westernized society and are increasing at a rampant pace in developing nations. Overwhelming evidence from a variety of sources—including epidemiological, prospective cohort, and intervention studies—links most chronic diseases seen in the world today to physical inactivity and inappropriate dietary consumption (Roberts and Barnard, 2005).

GRADING OF SCIENTIFIC EVIDENCE Overview of Causation Science Reliance on medical statistics and clinical applications of epidemiology are developments of the twentieth century, especially the latter half of the period. Through the 1940s, medical research was carried out by physician–investigators guided by observation of patients, pathophysiological reasoning, and small-scale experimentation at the bedside (Lewis, 1945; Bernard, 1966). The nature of clinical research changed in conjunction with, and in response to, advances in cellular analysis and molecular biology; development of quantification in therapeutic evaluation; the emergence of probabilistic thinking; and the application of statistical methods and theory (Chalmers, 2001). Bradford Hill’s influence on medical statistics and clinical research has been considerable. He authored an influential textbook on medical statistics, which was published in 1937 (Hill, 1937). He also played a pivotal role in the evaluation of streptomycin in the treatment of tuberculosis. The results of that trial, published in 1948 (Medical Research Council Streptomycin in Tuberculosis Trials Committee, 1948), marked the start of the modern era of clinical research. Bradford Hill and his colleague, Sir Richard Doll, also played key roles in the development of observational epidemiological research. Major case-control studies conducted in 1950 by Hill and Doll in the United Kingdom (Doll and Hill, 1950) and Wynder and Graham (1950) in the United States showed that smoking was a cause of most cancer deaths. In 1951, Hill and Doll (1954) also initiated the first major cohort study of British doctors, their smoking habits, and lung cancer. In 1965, Bradford Hill (1965) established criteria for scientific causation, broad guidelines that have served as a model for standards since developed to assess the quality of research evidence. Originally developed for use in occupational medicine, the criteria have been widely applied in a variety of fields (Rom, 1992). They include nine points of consideration, not all of which must be fulfilled to establish scientific causation. Key criteria, those with the highest priority in judging a causal relationship, are (1) consistency of

History of Evidence-Based Medicine

the association across studies, (2) strength of the association, (3) and temporal relationship. In the case of dietary fat intake and prostate cancer risk, for example, a series of in vitro laboratory, animal, and clinical studies were required to meet criteria for substantial scientific evidence. Though epidemiological investigations suggested an association of dietary fat intake with prostate cancer risk, there was limited supporting evidence from animal studies (Zhou and Blackburn, 1997). The failure of the Alpha-Tocopherol and Beta Carotene (ATBC) Cancer Prevention Study (Alpha-Tocopherol and Beta Carotene Cancer Prevention Study Group, 1994) underscores the need to develop strong supporting data in animal models (De Luca and Ross, 1996). Although conceived on the basis of epidemiological and mechanistic evidence, the trial went forward without published evidence that B-carotene prevented lung cancer in animal models (Wynder et al., 1997). The Causes of Cancer (Doll and Peto, 1981), a landmark review by British epidemiologists Richard Doll and Richard Peto, included estimates of the extent to which cancer in general, and specific cancers, could be avoided by changes in diet. Published 16 years after Hill defined causality, The Causes of Cancer concluded that environmental carcinogens, other than those in tobacco and diet, were relatively unimportant causes of cancer. Doll and Peto’s review, which is still frequently cited, set the agenda for current thinking on nutritional oncology.

HISTORY OF EVIDENCE-BASED MEDICINE The traditional paradigm of medical education and practice considers understanding of basic pathophysiological mechanisms of disease, coupled with clinical experience (Haynes, 2002), sufficient grounds for clinical decisionmaking. In contrast, EBM promotes the centrality of scientific evidence as the basis for treatment and policy decisions. The underlying philosophy of EBM suggests that a formal set of rules must complement medical training and common sense for clinicians to effectively interpret the results of clinical research (Guyatt et al., 2000). However, challenges in applying new knowledge (e.g., abundance of literature, the tiny fraction of adequately tested and clinically valid reports, and limitations of time and resources) are considerable (Haynes, 2002). To help clinicians, health systems, and policy makers overcome these obstacles, advocates of EBM have created procedures to objectively summarize and assess the quality of scientific evidence, and make recommendations based on those assessments (Table 3). Charged with developing recommendations for clinical preventive services, the Canadian Task Force on the Periodic Health Examination published its first report in 1979, a seminal work using systematic rules of evidence to support

TABLE 4

743 Canadian Task Force on Preventive Health Care Levels of Evidence

Levels of evidence I Evidence from at least one well-designed randomized controlled trial II-1 Evidence from well-designed controlled trials without randomization II-2 Evidence from well-designed cohort or case-control analytical studies, preferably from more than one center or research group II-3 Evidence from comparisons between times or places with or without the intervention; dramatic results from uncontrolled studies could be included here III Opinions of respected authorities, based on clinical experience; descriptive studies or reports of expert committees Note: The 1979 report of the Canadian Task Force on the Periodic Health Examination was the first to use systematic rules of evidence to support the strength of recommendations for a wide variety of preventive services. The report applied a hierarchy of evidence to rank recommendations according to the available type and amount of evidence (Canadian Task Force on the Periodic Health Examination, 1979). Source: From Levine et al., 2001.

the strength of recommendations for a wide variety of preventive services (Table 4). The report applied a hierarchy of evidence to rank recommendations according to the available type and amount of evidence. The same approach, which was used to derive a grading of recommendations, appears to have been the first practical application of such levels of evidence (Centre for Evidence-based Medicine. Oxford-Centre for Evidence-based Medicine, 2001; Kroke et al., 2004) (Table 5). The U.S. Preventive Services Task Force (Task Force/USPSTF) (Table 1) was established in 1984 to extend the approach of the Canadian Task Force by systematically reviewing the scientific evidence for individual clinical preventive services, and making recommendations for practitioners about what services should be routinely offered. Its controversial Guide to Clinical Preventive Services, released in 1989, downplayed expert opinion as a basis for making recommendations and took a neutral position when evidence was lacking (USPSTF, 1989). It also accelerated a growing movement to replace traditional “expert consensus” methods for developing clinical recommendations with a systematic and explicit process for reviewing evidence and linking clinical practice guidelines directly to the quality of the science (Woolf, 1990). Within health care, the evidence-based paradigm has become all-pervasive. At both basic and continuing education levels, skills in finding and appraising evidence are now central to many curricula. Databases, journals, and Internet sites, often free at the point of delivery, provide an accessible infrastructure to support the adoption of evidence-based

744


TABLE 5

Level

Therapy/prevention: etiology/harm

Oxford Centre for Evidence-based Medicine Levels of Evidence (May 2001)

Prognosis

Diagnosis

Economic and decision analyses

SR (with homogeneitya) of prospective cohort studies

SR (with homogeneitya) of Level 1 economic studies

Prospective cohort study with good follow-upm

Analysis based on clinically sensible costs or alternatives; systematic review(s) of the evidence; and including multiway sensitivity analyses Absolute better-value or worse-value analysesj SR (with homogeneitya) of Level >2 economic studies

1a

SR (with homogeneitya) of RCTs

SR (with homogeneitya) of inception cohort studies; CDRb validated in different populations

1b

Individual RCT (with narrow confidence intervalc)

Individual inception cohort study with ≥80% follow-up; CDRb validated in a single population

1c

All or noned

All or none case series

Absolute SpPins and SnNoutsg

All or none case series

2a

SR (with homogeneitya) of cohort studies

SR (with homogeneitya) of Level >2 diagnostic studies

SR (with homogeneitya) of 2b and better studies

2b

Individual cohort study (including low-quality RCT; e.g., 300,000 reports of studies. Other evidence-based databases and resources include the Agency for Health Care Research and Quality: Evidence-based Practice, the National Guidelines Clearinghouse (AHRQ. National Guidelines Clearinghouse), and various practice guidelines developed by professional and academic medical societies (AHRQ. National Guidelines Clearinghouse).

746


HIERARCHIES OF EVIDENCE Current levels of evidence have been developed mainly to address clinical research questions and, therefore, place major emphasis on RCTs as the primary and most convincing evidence in the evaluation process (Kroke et al., 2004). However, problems (Claxton and Thompson, 2001) and limitations associated with this approach (Claxton and Thompson, 2001; Kroke et al., 2004) have prompted numerous initiatives to broaden concepts of evidence away from the rigor of research design alone. Many groups, including the USPSTF (Harris et al., 2001), have developed guidelines based on grading hierarchies and other approaches for classifying and using disparate sources of evidence (Agency for Healthcare Research and Quality, 2004; Lehman Center Weight Loss Surgery Expert Panel, 2005). The Executive Report from the Betsy Lehman Center for Patient Safety and Medical Error Reduction Expert Panel on

BOX 1

Types of Clinical Research

Overview Two types of studies—experimental and observational—are most relevant to hierarchies of evidence. The former includes controlled trials, either randomized or nonrandomized; and the latter, cohort, case-control, and cross-sectional studies. These are also known as analytical studies. Like controlled trials, they have comparison or control groups. The major difference between experimental and observational studies lies in whether investigators assign exposures (e.g., treatments) (Feinstein, 1985; Rothman, 1986; Hennekens and Buring, 1987; Hulley et al., 2001; Kelsey et al., 1996) or track them over time. Only comparative studies (both analytical and experimental) enable assessment of possible causal associations (Grimes and Schulz, 2002) (Figure 1). Of these study designs, clinical trials and cohort studies usually require large sample sizes and lengthy follow-up periods to provide adequate statistical power to measure differences in outcome (U.S. Preventive Services Task Force, 1996). As a result, they can be slow to yield results and very expensive. The following descriptions summarize the general strengths and weaknesses of the most frequently used experimental and observational study designs.

Randomized Controlled Trials

FIGURE 1 Algorithm for classifying types of clinical research. Each type of study design has particular strengths and weaknesses; all, however, are vital to a comprehensive understanding of the links between diet and cancer. The strongest evidence indicating that food and nutrition modify the risk of cancer comes from a combination of different types of epidemiological studies, supported by experimental findings and by identification of plausible biological pathways (World Cancer Research Fund and American Institute for Cancer Research, 1997). (Used with permission from Grimes and Schulz, 2002.)

Randomized controlled trials (RCTs) assign subjects by chance to either a study group (intervention) or a control (which receives standard treatment, e.g., no intervention or a placebo). Blinded trials conceal upcoming assignments from either investigators or subjects; double blind, from both. In nonrandomized controlled trials, assignments are made by some other allocation scheme, such as alternate assignment (Schulz et al., 1994). The design of RCTs eliminates confounding biases, both known and unknown, enhancing comparability between groups and providing a more valid basis for inferring that the intervention caused the observed outcome. Properly designed and implemented RCTs are likely to have internal validity (i.e., measure what they set out to measure and be free of all biases; e.g., selection, information, and outcome). External validity, the extent to which results can be generalized to the broader community, is a potential problem with RCTs; people who volunteer to participate in clinical trials may differ in important ways from those who do not (Lilienfeld and Lilienfeld, 1980). Ethical issues are another limiting factor: RCTs cannot be used if subjects would be harmed by withdrawal or denial of effective treatment or inten-

747

Hierarchies of Evidence

tionally exposed to noxious substances (e.g., toxins or bacteria). Nonrandomized Controlled Trials Nonrandomized controlled trials follow exposed and unexposed subjects forward in time to measure frequency of outcomes. Advantages of this study design include use of a concurrent control group and uniform measurement of outcomes for both groups. Selection bias, however, is a potential disadvantage. The U.S. Preventive Services Task Force (Agency for Healthcare Research and Quality, 2001) and the Canadian Task Force on Preventive Health Care (Canadian Task Force on the Periodic Health Examination, 1994) have designated the nonrandomized controlled trial design as class II-1, indicating less scientific rigor than RCTs, but more than analytical studies. Cohort, Case–Control, and Cross-Sectional Studies Cohort studies follow already-exposed individuals (e.g., smokers) forward over time (longitudinally) to measure outcomes (e.g., cancer). In contrast, case-control studies start with an outcome and then look back in time for exposures that might have caused the outcome. In case-control studies of cancer screening, for example, prior exposure to a cancer-screening test is compared between patients with cancer (cases) and those without (controls). Cross-sectional studies are sometimes called frequency surveys or prevalence studies (Last, 1988). They examine the presence or absence of disease and the presence or absence of exposure at one point in time (e.g., obesity in women with arthritis vs women without arthritis) (U.S. Preventive Services Task Force, 1996). All of these study designs are subject to potential systematic bias in that choice of treatments, risk factors, or other covariables might be made on the basis of important (or unrecognized) factors that could affect outcome. There is the potential for bias in the measurement and interpretation of data; the recall of medical histories or prior exposures; and the selection of patients who might differ from the general population in important ways. Confounding is a key concern with observational study designs. Unlike RCTs, which automatically adjust for known and unknown confounding variables, cohort and case-control study designs can only account for known confounders; the onus is, therefore, on investigators to make sure that such studies are free of bias.

BOX 2

Quantification of Risk

Cohort studies allow for calculation of true incident rates, relative risks, and attributable risks (i.e., the rate of a disease or other outcome in exposed individuals that can be attributed to the exposure). Relative risk compares the risk of disease among people with a particular exposure to that among people without that exposure. Relative risk above 1.0 indicates higher risk among exposed than unexposed persons; below 1.0 indicates an inverse association between a risk factor and the disease, or a protective effect. The higher the relative risk, the stronger the evidence of a causal relationship. A relative risk of 2.0 for high meat consumption implies a doubling of risk; a relative risk of 12.0 for certain levels of smoking and drinking implies a twelvefold increase in risk, as well as strong evidence of causation. Odds ratios, which provide a good proxy for relative risk, are used as the outcome measure in case-control studies. Absolute risk, the observed or calculated probability of an event in a population under study, is also important. A large relative risk of a rare cancer amounts to a small absolute risk. In contrast, a small relative risk may amount to a large absolute risk for common cancers. For example, a relative risk of 1.10 (or a 10% increased risk) translates into many extra cases of colon and breast cancer cases in Europe and North America, where those cancers are very common.

Weight Loss Surgery (WLS), for example, brought together multidisciplinary task groups to develop best practices in WLS. Each task group conducted a systematic review of the literature and prepared an evidence-based report. Most of evidence came from observational or experimental data. Traditional evidence-based analysis was used to critically assess all selected studies for internal validity; the studies were then ranked according to similar levels of evidence on the basis of study design. Expert opinion—including clinical experience, the opinions of respected authorities, reports from expert committees, and consensus of the Expert Panel—was used in conjunction with RCTs or observational studies to develop recommendations (Lehman Center Weight Loss Surgery Expert Panel, 2005). A 15-member Task Force (Guide to Community Preventive Services, 2005; Pappaioanou and Evans, 1998) charged with developing The Guide to Community Preventive Services: Systematic Reviews and Evidence-based Recommendations (the Guide) (Guide to Community Preventive Services, 2004) is using a similar approach to synthesize evidence derived primarily from observational or experimental data (Briss et al., 2000). The methodology allows for

748


a broad range of public health interventions to be evaluated in ways that incorporate scientific rigor, as well as the feasibility and appropriateness of the evaluation (Atkins et al., 2004b).

THE ROLES OF RCTs AND OBSERVATIONAL STUDIES For some years, the standard approach to evaluating the quality of individual studies has been based on the hierarchical grading system of research design in which RCTs receive the highest score (Table 3) (i.e., a stratification scheme that implies a trade-off between vulnerability to bias and external validity) (Hadorn et al., 1996). However, as critical appraisal techniques have matured, attention has been drawn to the limitations of this approach (Lohr and Carey, 1999), especially its focus on internal validity. Susceptibility to bias is an important indicator of the internal validity of a study, but lack of bias does not inform about the appropriateness of a study design, its external validity, or its relevance to the research question at hand (Kroke et al., 2004). In certain instances (e.g., studies of diseases that take many years to develop or lifestyle interventions), well-designed cohort studies may provide more compelling evidence than an inadequately powered or poorly conducted RCT. RCTs are also rarely available for lifestyle-related factors (Kroke et al., 2004) and might be inappropriate, unfeasible, or unethical to obtain. Observational studies dominate the literature (Funai et al., 2001) of preventive medicine and nutritional oncology and are primarily used to identify risk factors and prognostic indicators (Naylor and Guyatt, 1996). They have several advantages over RCTs—including lower cost, greater timeliness, and a broader range of patients (Feinstein, 1989)—but concern about inherent bias has limited their use in comparing treatments (Moses, 1995; Kunz and Oxman, 1998).

METHODOLOGY ADVANCES IN OBSERVATIONAL STUDIES The ascendancy of RCTs was hastened by a landmark article (Sacks et al., 1982) comparing published RCTs with those that used observational designs. That review of the literature concluded that biases in patient selection may irretrievably weight the outcome of historical controlled trials in favor of new therapies (Sacks et al., 1982). Other comparative studies (Chalmers et al., 1977, 1983; Sacks et al., 1982; Colditz et al., 1989; Miller et al., 1989) drew similar conclusions about cohort studies with concurrent selection of control subjects, as well as case-control designs. On the basis of these findings, many leading experts declared that

observational studies were inappropriate for use in defining evidence-based medical care. Historical milestones developed between 1662 and 1948 (Chalmers, 2001) challenge the fundamental criticism of observational studies (i.e., that unrecognized confounding factors may lead to distorted, and therefore, misleading results) (Benson and Hartz, 2000; Chalmers, 2001; Concato, 2004; Concato and Horwitz, 2004; Sacks et al., 1982). So do more recent data. In a comparison of observational studies and RCTs in certain clinical situations, Concato et al. (2000) concluded that data from well-conducted observational studies did not appear to produce results that were biased compared with randomized trials. Benson and Hartz (2000) reported similar findings from a comparison of observational studies and RCTs. Rigorous methodologies that mimic those used in the design of randomized clinical trials have much to do with those outcomes. A specific method to strengthen observational studies (the “restricted cohort” design) (Horwitz et al., 1990) adapts principles of RCT design for use in observational studies. It identifies a “zero time” for determining a patient’s eligibility and baseline features; uses inclusion and exclusion criteria similar to those of clinical trials; adjusts for differences in baseline susceptibility to the outcome; and uses statistical methods (e.g., intention-to-treat analysis) similar to those used in RCTs (Mannucci et al., 2004).

FACTORS PROMOTING EVOLUTION IN HIERARCHIES OF EVIDENCE The influence of dietary and nutritional exposures on the development of cancer has underscored the need to reassess criteria used to evaluate the quality of research. Cancer develops slowly, over years or decades. Consequently, single clinical trials, which rarely last >5 years, cannot address the whole span of cancer development. This does not mean that they are not an important component of the evidence base, only that they should not be seen as offering overriding evidence (Heggie et al., 2003). During the past decade, rapid expansion in a number of pertinent scientific fields and, in particular, the amount of population-based epidemiological evidence has helped clarify the role of diet in preventing and controlling morbidity and premature mortality resulting from cancer and other noncommunicable diseases (NCDs). Some of the specific dietary components that increase the probability of occurrence of these diseases in individuals, and interventions to modify their impact, have also been identified (Report of a Joint WHO/FAO Expert Consultation, 2003). Growing insight into the impact of diet and exercise on the risk of developing cancer has put preventive medicine at the center of efforts to reduce incidence rates. The most promising role for prevention in current medical practice

Evolutions in Evidence-Based Assessments of Research Quality

749

may lie in changing the personal health behaviors of patients long before clinical disease develops. The importance of this aspect of clinical practice is evident from a growing literature linking some of the leading causes of death to a handful of personal health behaviors (Manson et al., 2004). Following recommended diets, together with maintenance of physical activity and appropriate body mass, can reduce global incidence of cancer by 30–40% (World Cancer Research Fund, 1997). In the United States, excess weight and inactivity alone play such an important role in disease burden that Healthy People 2010, a nationwide health promotion and disease prevention agenda, lists physical inactivity and obesity as the top two health indicators, followed by tobacco use (U.S. Department of Health and Human Services, 2000). It is surprisingly rare for the evidence to show clearly and unambiguously what course of action should be recommended for any given question (Scottish Intercollegiate Guidelines Network, 2002). Nevertheless, organizations using evidence-based methodologies to develop guidelines on clinical preventive services are finding broad agreement on a core set of services of proven effectiveness that can be recommended to primary care providers and their patients (World Cancer Research Fund, 1997; National Institutes of Health, 1998; Report of a WHO Consultation, 2000; Report of a Joint WHO/FAO Expert Consultation, 2003). Basing preventive health care decisions on the evidence for their effectiveness is an important step in the progress of disease prevention and health promotion (U.S. Preventive Services Task Force, 1996).

The Canadian Task Force on Preventive Health Care has differentiated four research categories (i.e., therapy, prevention, etiology, and harm; prognosis; diagnosis; and economic analysis) with separate levels of evidence for each of these categories (Canadian Task Force on Periodic Health Examination, 1999). The Oxford Centre for Evidence-Based Medicine has adopted five categories related to diagnosis, prognosis, and harm, with 10 separate levels of evidence for each category (Atkins et al., 2004a; Oxford Centre for Evidence-based Medicine, 2001) (Table 5). Evolutions in the USPSTF hierarchy of evidence have led to revisions in some aspects of methodology (Atkins et al., 2001; Harris et al., 2001; Woolf and Atkins, 2001); consideration of elements of evidence beyond study design (Atkins et al., 2004a); and the use of outcomes tables to help categorize magnitude of benefits, harms, and net benefits from implementation of a preventive service (Harris et al., 2001). SIGN’s revised grading system (Table 6), introduced in 2000 (Scottish Intercollegiate Guidelines Network. Methodology Review Group, 1999; Harbour and Miller, 2001), combines study type and quality rating in the evidence level (Harbour and Miller, 2001) and allows more weight to be given to recommendations supported by good-quality observational studies where RCTs are not available for practical or ethical reasons. It also incorporates a considered judg-

EVOLUTIONS IN EVIDENCE-BASED ASSESSMENTS OF RESEARCH QUALITY

Levels of evidence 1++ High-quality meta-analyses, systematic reviews of RCTs, or RCTs with a very low risk of bias 1+ Well-conducted meta-analyses, systematic reviews of RCTs, or RCTs with a low risk of bias 1− Meta-analyses, systematic reviews of RCTs, or RCTs with a high risk of bias 2++ High-quality systematic reviews of case-control or cohort studies High-quality case-control or cohort studies with a very low risk of confounding, bias, or chance and a high probability that the relationship is causal 2+ Well-conducted case-control or cohort studies with a low risk of confounding, bias, or chance and a moderate probability that the relationship is causal 2− Case-control or cohort studies with a high risk of confounding, bias, or chance and a significant risk that the relationship is not causal 3 Nonanalytical studies, e.g., case reports, case series 4 Expert opinion

Shifting clinical priorities, methodology advances that reduce potential bias in observational studies, and rapid growth in the epidemiological database are validating the credibility of hierarchies that differ from conventional models and prompting reassessments of how scientific literature is categorized and judged. Trends include reordered hierarchies of evidence and growing awareness of the need to base appraisals of quality on a wider range of study designs. Levels of evidence have been updated to incorporate the understanding that different medical areas require different sets of levels of evidence. The developers of The Guide to Community Preventive Services: Systematic Reviews and Evidence-based Recommendations (the Guide) (Pappaioanou and Evans, 1998) are assessing the quality of study execution in detail and considering it along with study design. This approach allows a well-conducted case-control or prospective cohort study, for example, to receive greater weight than a poorly conducted RCT (Briss et al., 2000).

TABLE 6 Revised SIGN Grading System (Harbour and Miller, 2001)

Note: SIGN’s revised grading system, introduced in 2000 (Scottish Intercollegiate Guidelines Network. Methodology Review Group, 1999; Harbour and Miller, 2001), combines study type and quality rating in the evidence level and allows more weight to be given to recommendations supported by good-quality observational studies when RCTs are not available (Scottish Intercollegiate Guidelines Network, 2002).

750


ment process that allows guideline developers to downgrade a recommendation when they perceive that the evidence is weaker than a simple evaluation of the methodology would suggest (Scottish Intercollegiate Guidelines Network, 2002).

insufficient evidence is based on the findings of a few studies that are suggestive, but insufficient to establish an association between exposure and disease.

AN EMERGING STATE OF THE ART ADAPTING TO UNCERTAINTY Ideally, the definition of increased or decreased risk should be based on a relationship that has been established by multiple RCTs of interventions on populations that are representative of the target of a recommendation. In the absence of such evidence (Report of a Joint WHO/FAO Expert Consultation, 2003), the WHO/FAO has modified criteria used by the World Cancer Research Fund (World Cancer Research Fund, 1997) to include the results of controlled trials where relevant and available. In addition, consistent evidence on community and environmental variables that lead to behavior changes, and thereby modify risks, has been taken into account in categorizing risks. This applies in particular to the complex interaction among environmental factors that affect excess weight gain, a risk factor recognized as contributing to many chronic diseases and cancers. The WHO/FAO uses four categories to describe strength of evidence: convincing, probable, possible, and insufficient (Table 7). The first two categories rely on epidemiological data showing consistent associations between exposure and disease, with little or no evidence to the contrary. Available evidence is based on substantial numbers of studies, including prospective observational studies, and where relevant, RCTs of sufficient size, duration, and quality that show consistent effects. Possible evidence is based mainly on findings from case-control and cross-sectional studies, while

TABLE 7

It has been recognized for some time that inferring causation of chronic disease requires a comprehensive view of the evidence base in a broad biological context; indeed, Bradford Hill described the need to adapt existing models to the peculiarities of observational studies in 1965. In that evidence underpinning an inference of causation is inherently more diverse than that required to demonstrate clinical efficacy, an approach identical to EBM’s conventional hierarchy of study design (NHS Centre for Reviews and Dissemination, 2001) is inadequate to address the challenge of questions related to the influence of dietary and nutritional exposures on the development of cancer. Since the 1970s, a growing number of organizations have employed various systems to grade the quality (level) of evidence and the strength of recommendations. Methods for assessing quality differ in the characteristics considered (e.g., measures of internal, external, statistical, and construct validity) and in the way these characteristics are measured (Atkins et al., 2004a). The impact of various approaches on the quality of reported study evidence (Moher et al., 1998) and on the results of meta-analyses is an area of considerable research interest (Atkins et al., 2004b; Briss et al., 2000). In 2006, a rigorous assessment of thousands of published studies on the subject of diet and cancer is scheduled for publication by the American Institute for Cancer Research and the World Cancer Research Fund International (WCRF/AICR) (Heggie et al., 2003). That evidence-based

WHO/FAO Strength of Evidence

Convincing evidence: Evidence based on epidemiological studies showing consistent associations between exposure and disease, with little or no evidence to the contrary; a substantial number of studies, including prospective observational studies, and where relevant, randomized controlled trials of sufficient size, duration, and quality showing consistent effects; and biologically plausible associations. Probable evidence: Evidence based on epidemiological studies showing consistent associations between exposure and disease, with perceived shortcomings in the available evidence, or some evidence to the contrary that precludes a more definite judgment; usually supportive laboratory evidence; and biologically plausible associations. Shortcomings in the evidence may include insufficient duration of trials (or studies); insufficient trials (or studies) available; inadequate sample sizes; or incomplete follow-up. Possible evidence: Evidence based mainly on findings from case-control and cross-sectional studies, with insufficient randomized controlled trials, observational studies, or nonrandomized controlled trials available; supportive evidence from nonepidemiological studies, such as clinical and laboratory investigations; and biologically plausible associations. Additional trials are required to support tentative associations. Insufficient evidence: Evidence based on findings of a few studies that are suggestive but insufficient to establish an association between exposure and disease. Limited or no evidence is available from randomized controlled trials. More well-designed research is required to support tentative associations. Criteria used by the World Health Organization/UN Food and Agriculture Organization (WHO/FAO) include results from controlled trials, where relevant and available. They also take into account environmental factors that effect excess weight gain, a risk factor for many cancers (Report of a Joint WHO/FAO Expert Consultation, 2003).

751

References

report will admit data from all study designs (e.g., ecological, prospective cohort, case-control, and laboratory studies); human and animal mechanistic data (in vivo only) will be analyzed separately, giving more weight to the human mechanistic data (Wynder et al., 1997; Zhou and Blackburn, 1997; Heggie et al., 2003). Other leading-edge systems are also being developed for judging the quality of evidence. The latest American Diabetes Association (ADA) grading system assigns ratings of A, B, or C, depending on the quality of evidence (Table 2). Expert Opinion (E) is a separate category for recommendations when evidence from clinical trials is not yet available, clinical trials might be impractical, or there is conflicting evidence (ADA, 2006). The system used by the Grades of Recommendation Assessment, Development and Evaluation (GRADE) Working Group (Table 8) (Atkins et al., 2004b) takes into account study design, study quality, consistency, and directness of evidence for each important outcome. For randomized trials, for example, reviewers might use criteria such as the adequacy of allocation concealment, blinding, and follow-up. Overall quality of evidence is explicitly judged across outcomes on the basis of the lowest quality of evidence for any of the critical outcomes (Atkins et al., 2004a). Over the past 30 years, research on carcinogenesis has yielded a tremendous knowledge base (Go et al., 2004). The

TABLE 8

The GRADE Working Group System for Grading the Quality of Evidence

Criteria for assigning grade of evidence Type of evidence Randomized trial = high Observational study = low Any other evidence = very low Decrease grade if: • Serious (−1) or very serious (−2) limitation to study quality • Important inconsistency (−1) • Some (−1) or major (−2) uncertainty about directness • Imprecise or sparse data (−1) • High probability of reporting bias (−1) Increase grade if: • Strong evidence of association—significant relative risk of >2 (5 (

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