Handbook of Green Tea and Health Research (Food and Beverage Consumption and Health)

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In: Food and Beverage Consumption and Health Series

HANDBOOK OF GREEN TEA AND HEALTH RESEARCH No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

FOOD AND BEVERAGE CONSUMPTION AND HEALTH SERIES

Handbook of Green Tea and Health Research Helen McKinley and Mark Jamieson 2009. ISBN 978-1-60741-045-4

In: Food and Beverage Consumption and Health Series

HANDBOOK OF GREEN TEA AND HEALTH RESEARCH

HELEN MCKINLEY AND

MARK JAMIESON EDITORS

Nova Science Publishers, Inc. New York

Copyright © 2009 by Nova Science Publishers, Inc.

All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS.

LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Handbook of green tea and health research / [edited by] Helen McKinley and Mark Jamieson. p. ; cm. -- (Food and beverage consumption and health) Includes bibliographical references and index. ISBN 978-1-60876-202-6 (E-Book) 1. Green tea--Health aspects. I. McKinley, Helen. II. Jamieson, Mark. III. Series: Food and beverage consumption and health. [DNLM: 1. Tea. 2. Camellia sinensis. 3. Catechin--analogs & derivatives. 4. Phytotherapy--methods. WB 438 H236 2009] RM251.H36 2009 615'.321--dc22 2009000178

Published by Nova Science Publishers, Inc. Ô New York

CONTENTS Preface

ix

Chapter 1

Central Functions of Green Tea Components M. Furuse,, N. Adachi, S. Tomonaga, H. Yamane and D. M. Denbow

Chapter 2

Green Tea Catechins: A Class of Molecules with Antimicrobial Activity P. Buzzini, P. Vignolini, M. Goretti, B. Turchetti, E. Branda, E. Marchegiani, P. Pinelli and A. Romani

Chapter 3

Chapter 4

Chapter 5

Chapter 6

Chapter 7

1

23

Lipid-soluble Green Tea Polyphenols: Stabilized for Effective Formulation Ping Chen, Douglas Dickinson and Stephen Hsu

45

Assessment of the Antioxidant Capacity of Green Teas: A Critical Review Camilo López-Alarcón and Eduardo Lissi

63

Design and Assessment of the in Vitro Anti-oxidant Capacity of a Beverage Composed of Green Tea (Camellia Sinensis L.) and Lemongrass (Cymbopogon Citratus Stap) D. Fernando Ramos Escudero, Luis Alberto Condezo Hoyos, Mónica Ramos Escudero and Jaime A. Yáñez

81

Teas Are not All the Same: In Vitro and in Vivo Antioxidant Activity and Appetite Modulation in Rats of Green Teas with High and Low Levels of Organic Selenium Abdul L. Molan, Zhuojian Liu and Wenhua Wei Anti-obesity Effects of (-)-Epigallocathchin-3-gallate and its Molecular Mechanism Cheol-Heui Yun, Gi Rak Kim, Min Ji Seo, Hyun-Seuk Moon and Chong-Su Cho

103

125

vi Chapter 8

Chapter 9

Contents Green Tea: Protective Action against Pesticides and other Xenobiotics Present in Human Diet Geetanjali Kaushik, Poonam Kaushik and Shivani Chaturvedi

157

New Method to Improve the Function and Industrial Applicability of Green Tea and Its Byproducts Using Irradiation Technology Cheorun Jo and Myung Woo Byun

177

Chapter 10

Green Tea Catechin as Angiogenesis Inhibitor Kiminori Matsubara and Yoshiyuki Mizushina

197

Chapter 11

Neuroprotective Effect of Theanine on Cerebral Ischemia Nobuaki Egashira,, Kenichi Mishima, Katsunori Iwasaki, Ryozo Oishi and Michihiro Fujiwara

207

Chapter 12

Characterization of the Neuroprotective Activity of the Polyphenol (-)-Epigallocatechin-3-gallate in the Brain Orly Weinreb, Tamar Amit, Moussa B. H. Youdim and Silvia Mandel

219

Chapter 13

Cardiovascular and Metabolic Effects of Green Tea Kamilla Kelemen

Chapter 14

Molecular Basis for the Anti-cancer Activity of EGCG in Vivo: Molecular-Targeting Prevention of Cancer by Green Tea Catechin Yoshinori Fujimura and Hirofumi Tachibana

257

Utility of Epigallocatechin Gallate in the Treatment and Prevention of Breast Cancer: Molecular Mechanisms for Tumor Suppression R. J. Rosengren

301

Chapter 15

Chapter 16

Green Tea Catechins in Colorectal Cancer Seung Joon Baek and Mugdha Sukhthankar

Chapter 17

Inhibitory Effect of Catechin Derivatives from Green Tea on DNA Polymerase Activity, Human Cancer Cell Growth, and TPA (12-O-tetradecanoylphorbol-13-acetate) -induced Inflammation Yuko Kumamoto-Yonezawa, Hiromi Yoshida and Yoshiyuki Mizushina

Chapter 18

Telomerase Regulation in Response to Green Tea Huaping Chen and Trygve O. Tollefsbol

Chapter 19

Green Tea and Chronic Obstructive Pulmonary Disease: A Casecontrol Study in Japan Fumi Hirayama and Andy H. Lee

243

325

347

363

383

Chapter 20

Green Tea and Diabetes Dongfeng Wang, Linge Wang and Li Zhang

393

Chapter 21

Green Tea and Type 2 Diabetes Jae-Hyung Park, Hye-Young Sung and Dae-Kyu Song

411

Contents Chapter 22

Chapter 23

Biocatalytic Conversion of Green Tea Catechins to Epitheaflagallin, Epitheaflagallin, 3-O-gallate, and Theaflavins: Production of Promising Functional Foods Nobuya Itoh and Yuji Katsube Preventive Effects of Green Tea Catechins on Dementia Michio Hashimoto, Md Abdul Haque, Kohinoor Begum Himi and Yukihiko Hara

vii

419 429

Short Commentary Green Tea and Potential Human Health Effects James E. Trosko Index

451 463

PREFACE After water, tea signifies the second most frequently consumed beverage worldwide. Teas are not all the same; among the many areas of research that are included in this book are the effects of selenium-containing green tea on food consumption and body weight gain. Research shows that tea consumption may have its strongest effect among patients with cardiovascular disease. A specific chapter investigates whether green tea intake can reduce the risk of chronic obstructive pulmonary disease. Research is presented to show that green tea and its major constituent epigallocatechin gallate (EGCG) have a potential chemopreventative and/or treatment for a variety of diseases including breast cancer. Other research sheds new light on the molecular basis for the cancerpreventive activity of EGCG in vivo and helps in the design of new strategies to prevent cancer. A further study presents an analysis assessing the progress of research on the mechanisms pertaining to how telomerase activity is regulated by green tea in cancer cells. Further chapters look at the relationship of tea to diabetes and a description of the beneficial effects of green tea catechins on neuronal functions and neuronal diseases such as dementia. To improve biological functions and industrial applicability of green tea and its byproducts, research is presented showing irradiation as a useful method. Chapter 1 - Tea (Camellia sinensis) is widely consumed throughout the world and has a number of biologically active substances such as caffeine, catechins, and L-theanine (γglutamylethylamide). Tea consumption is generally known to induce a feeling of relaxation which may be mediated by either catechins, L-theanine, or both, since caffeine stimulates locomotor activities. The catechin (-)-epigallocatechin gallate (EGCG) occurs abundantly in tea. Moreover, frequent consumption of green tea results in high levels of EGCG in the blood and brain. Catechins, which are flavonoids, affect the central nervous system (CNS). The therapeutic effects of flavonoids may involve their binding to γ-aminobutyric acid (GABA)A receptors, which is a major inhibitory neurotransmission system. Recently, EGCG was shown to bind to GABAA receptors in vitro and to induce a sedative effect through GABAA, but not GABAB, receptors in the brain. L-Theanine, a derivative of glutamate, is a unique amino acid occurring only in green tea and a few other plants. After administration L-theanine concentrations were increased in serum, liver and brain, suggesting that L-theanine can cross the blood-brain barrier. Intravenous administration of L-theanine was shown to affect the cortex, hippocampus and amygdala, and increase the alpha-band component of electroencephalograms in rats. More

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recently, it was shown that L-theanine could reduce stress via either inhibiting cortical neuron excitation in human subjects or influencing the secretion and function of neurotransmitters in the CNS. We discuss the central functions of green tea components such as EGCG and Ltheanine in the CNS. Chapter 2 - A significant part of scientific interest of academy or industry is focused on discovering novel natural antimicrobial drugs. This attention is essentially justified by the expectation that a few of them could play a role in supporting (or even in substituting) some antibiotics of current use. It has been estimated that, although about some tens of novel antimicrobial drugs (either of biological or synthetic origin) are currently launched each year, due to the increasing development of resistant microbial genotypes, their downturn is becoming very rapid. Taking into account these considerations, the enormous scientific and commercial interest in discovering and developing novel classes of molecules exhibiting more or less pronounced antimicrobial properties has oriented the work of a growing part of the scientific community toward large-scale screening programs aimed at discovering novel classes of bioactive molecules. The occurrence in some plants of secondary metabolites exhibiting a more or less pronounced antimicrobial activity is a well-known phenomenon. Among them, green tea polyphenols represent a reservoir of molecules characterized by antioxidant, antiradical and antimicrobial activity. In particular, catechins have proven to be effective towards both prokaryotic and eukaryotic microorganisms. Despite the large number of studies published so far, their actual potentialities and limitations as antimicrobial (mainly antibacterial and antimycotic) drugs have not been critically evaluated. The present chapter represents an overview of the recent literature on the antiviral and antimicrobial properties exhibited by polyphenols, particularly catechins, occurring in green tea composition. Chapter 3 - Green tea polyphenols (GTPs), also referred to as green tea catechins, possess properties that can provide unique health benefits to humans. As indicated in other chapters of this book, studies using molecular, cellular, and animal models, and in human subjects, have demonstrated that these phytochemicals from non-oxidized tea leaves have anti-cancer, antioxidant, anti-microbial, and anti-inflammatory properties. Recently, investigations in our and other laboratories indicated that topical application of GTPs could protect the epidermis against autoimmune disorders, such as psoriasis, prevent or repair UV-induced damage, and suppress scar tissue overgrowth. In addition, specific gene regulation by GTPs, especially epigallocatechin-3-gallate (EGCG), promotes skin cell differentiation, which could lead to improved homeostasis of the skin. Based on these facts, the topical use of products containing GTPs has become more popular, and manufacturers of cosmetic, health care, and household products are adding GTPs or EGCG to their formulations. However, it is important to note that studies described in this book always use “freshly prepared” GTPs or green tea, instead of “pre-prepared” materials. This is because GTPs are potent antioxidants that react rapidly with reactive oxygen species (ROS). As a result, GTPs in most commercially available products have been oxidized and/or epimerized; the biological effects of the resulting compounds are largely unknown. In addition, due to the highly water-soluble nature of these compounds, GTPs in their original form are not lipid-soluble, and therefore not permeable to the skin, a water-proof barrier. Another problem with formulation of GTPs for topical application is the coloration change and precipitation caused by oxidation. Thus, GTPs for topical application (e.g., on skin and

Preface

xi

mucous membranes) must be prepared and used immediately prior to oxidation, coloration and precipitation. These properties of GTPs make it difficult to formulate products containing them that have a reasonable shelf life and maintain their activity and effectiveness. In other words, most of the commercially available “green tea”–containing products are without the full benefits of green tea or GTPs. Therefore, strategies to stabilize and increase the bioavailability of GTPs are needed to provide the full benefits of GTPs to consumers or patients. Recently, it has been shown that lipid esters of GTPs can be formed either enzymatically or chemically. These green tea polyphenol-lipid esters, also referred to as lipid-soluble tea polyphenols (LTPs), could significantly improve formulations of consumer or health care products. We hypothesized that fatty acyl esterification of green tea polyphenol would protect the hydroxyl groups from oxidation and improve skin permeability. In the current study, we compared the activities of LTPs to GTPs for their anti-cancer and gene regulation properties. We examined whether LTPs can be converted into a free GTP (EGCG) in human skin keratinocyte cultures. In addition, the effects of LTPs in a mouse model for psoriasis were evaluated. The results indicate that LTPs effectively cause cancer cell death, induce caspase 14 gene expression both in vitro and in vivo, and improve the skin condition in an animal model for psoriasis. Consistent with these observations, HPLC analysis demonstrated that EGCG in its original form was released from LTPs in situ by human epidermal keratinocytes. These results suggest that LTPs, under appropriate conditions, function similarly to GTPs. More importantly, since the most reactive hydroxyl group(s) is/are protected, and the lipid solubility is dramatically increased by the fatty acyl groups, the biological activity of these compounds can be stabilized, and their bioavailability increased significantly. In conclusion, LTPs are a novel and more effective form of green tea polyphenols for topical applications and other purposes, especially in formulations that require a reasonable shelf life. In addition, LTPs can be a natural additive to consumable products such as salad oil, fish oil, and cooking oil as antioxidants. Chapter 4 - In the last decades, the beneficial influence of green tea on human health has been related to the antioxidant capacity (AC) of its phenolic constituents. The latter has originated systematic studies of the AC of green tea and/or its pure antioxidants. Different methodologies have been used with this purpose. The methods are based on: (1) Estimation of the consumption by additives of stable free radicals (DPPH, and ABTS radical cation); (2) Evaluation of the protection given by antioxidants to a target being oxidized by free radicals (ORAC, TOSC, LDL oxidation assay); (3) Estimation of the steady state of free radicals before and after addition of additives (TAR); (4)Estimation of the reducing power capacity of the additives (FRAP, CUPRAC). The assays differ in the experimental conditions and their chemistry. Therefore, different conclusions could be obtained depending on the methodology used. For example, green tea presents a lower AC than peumus boldus by ORAC (oxygen radical absorbance capacity) method when fluorescein is used as target molecule. However, if pyrogallol red is used as probe, green tea appears with an ORAC index six times higher than peumus boldus. In the present review, we discuss the advantages, and disadvantages of the different methodologies employed to evaluate the AC of green tea. Chapter 5 - Tea is one of the most popular and widely consumed beverages in the world and it is derived from the infusion of tea leaves (Camellia sinensis L.). Different commercial types of tea are available, including black tea, oolong tea (semi-fermented) and green tea,

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which differ on their processing and chemical composition. All these types of tea have been reported to prevent multiple diseases such as cancer, heart conditions, among others. On the other hand, lemongrass (Cymbopogon citratus Stap) is a rich source of essential oils, widely employed in infusions, soaps, and perfumes, and it has been reported to possess gastrointestinal and analgesic properties. In the present study, green tea (Camellia sinensis L.) and lemongrass (Cymbopogon citratus Stap) leaves were collected from Río Azul and Porvenir de Marona, Perú. The anti-oxidant capacity of green tea and lemongrass extracts was evaluated using the DPPH method and it was observed that the IC50 values for green tea was 32.4 ± 0.39 μg/mL and 1350 ± 47 μg/mL for lemongrass. These two plants (green tea and lemongrass) were employed to design multiple infused beverages and it was determined that an infused beverage containing 10 mg/mL total extract (50% green tea and 50% lemongrass) reported a total catechin content of 24.4 ± 0.65 mg/100mL, a DPPH inhibition percentage of 88.6%, and exhibited the greatest acceptance for sensory attributes such as flavor, color, and aroma (values of 6.8, 9.0, and 8.0 respectively) based on Friedman Multiple Comparisons test. The taste panel results also indicated that the optimized acidity and sweetness were to be set at pH 3.1 and 11°Brix, while the optimum infusion time based on the total catechin content was 7 minutes. The pasteurization profile at 90°C for 5 minutes achieved mesophilic microorganisms counts of 80%, catechins >80%, EGCG > 45%, caffeine < 1%). The authors observed that physiological performances and endurance ability in mice were greatly enhanced by GTE dietary administration. Independent of running, GTE apparently decreased serum creatine kinase, heart and gastrocnemius lipid peroxidation and increased gastrocnemius citrate

Green Tea Catechins: A Class of Molecules with Antimicrobial Activity

27

synthase activity of rats. Based on these data, the authors suggested that the antioxidant activity of GTE might be beneficial as therapeutic strategies to improve muscle function in mice. The antimicrobial activity of green tea polyphenols (particularly catechins) is well known. Both industry and academy have been increasingly concerned with the growing number of illness outbreaks caused by microbial pathogens. It has been estimated that, although about some tens of novel antimicrobial drugs (either of biological or synthetic origin) are currently launched each year, their downturn is becoming very rapid. Increasing antibiotic resistance of some pathogens associated with over-consumption of some existing antimicrobial drugs exacerbates this trend. Therefore, there has been an increasing interest in studying and developing novel types of effective plant-derived antimicrobials, including those present in green tea leaves (Friedman, 2007; Buzzini et al., 2007). The main objective of this chapter is to unify and interpret widely scattered information of literature on inhibitory activities of catechins occurring in green tea leaves against viruses, bacteria, yeasts and filamentous fungi.

FUNDAMENTALS OF BIOSYNTHESIS OF GREEN TEA CATECHINS Phenylpropanoid units derived from the shikimate pathway are common structural elements of all flavonoid compounds and of other classes of phenylpropanoids, such as lignin, stilbenes and cinnamate esters. The enzymes catalysing the individual steps of the “phenylpropanoid metabolism” are: phenylalanine ammonia-lyase, cinnamate 4-hydroxylase and 4-coumarate CoA ligase. In particular, flavan-3-ols originated from a branch pathway of anthocyanin biosynthesis. Early studies covering enzymatic aspects of proanthocyanidins biosynthesis have been carried out on Ginko biloba and Pseudotsuga menziesii. Such studies reported that dihydroflavonols [(+)-dihydromyricetin (DHM) or (+)-dihydroquercetin (DHQ)] are converted to the corresponding flavan-3,4-diols and catechin derivatives [(+)-GC, or (+)catechin, respectively] (Stafford and Lester, 1984, Stafford and Lester, 1985). The existence of a relationship between the formation of 2,3-trans, catechin-derived series of flavan-3-ols and the consecutive action of a dihydroflavonol reductase (DFR, which produces a leucoanthocyanidin) and a leucoanthocyanidin reductase (LAR) has been postulated. Leucocyanidin (produced by DFR via reaction with dihydroquercetin) was not observed with tea enzyme preparations. Instead, it is immediately further converted to catechin by the LAR present in the enzyme preparation (Punyasiria et al., 2004). The anthocyanidin reductase (ANR) enzyme, recently isolated from Arabidopsis spp. and Medicago spp., was shown to be present in tea leaves with very high activity. It produces EC and EGC from their respective anthocyanidins, thus explaining the very high contents of such compounds. The high ANR activity seems to be essential with respect to the dominance of EC and EGC (as well as their galloyl esters) as the major flavonoid components in tea leaves. EC and EGC, together with catechins, are also the building blocks of proanthocyanidins reported from tea (Kiehne et al., 1997). Thus, ANR together with LAR, may be of great importance for the biosynthesis of proanthocyanidins. The DFR/LAR two-step reaction converted the dihydroflavonols, dihydroquercetin and dihydromyricetin to catechin and GC, respectively, with a high activity. The pathways for the formation of many flavan 3-ol

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P. Buzzini, P. Vignolini, M. Goretti et al.

derivatives are not known. The occurrence of a galloyl transferase has been recently reported (Garcia et al., 2002). Catechins and EC, however, may also get hydroxylated by F3050H. In general, in green tea is demonstrated a high activity of flavonoid 3’5’-hydroxylase (F3’5’H), since there are more EGC and EGCG than EC and ECG in tea leaves. But also F3’H activity must be present as B-ring dihydroxylated flavonoids like catechin, epicatechin and derivatives are also quite prominent. Future studies will focus on the membrane-bound microsomal enzymes F3’H and F3’5’H that are important for B-ring hydroxylation of tea flavonoids.

ANTIVIRAL ACTIVITY OF GREEN TEA CATECHINS Among the possible modes of antiviral action of green tea polyphenols their ability to prevent viral binding and penetration into cells, and to trigger the host cell self-defence mechanisms are the mostly hypothesized (Friedman, 2007). Weber et al. (2003) studied the effect of four green tea catechins on adenovirus (also labelled as adenoidal-pharyngeal-conjuctival virus, APC) responsible for the infection of mucous membranes of the respiratory and urinary tracts. EGCG was the most effective (IC50 = 109 μM) when added to the cells during the transition from the early to the late phase of viral infection. The same compound exhibited a high selectivity index (SI). On the basis of these evidences, the authors postulated that EGCG could inhibit one or more later steps of viral infection and proposed its use for treating adenovirus infections in humans (Weber et al., 2003). A study by Savi et al. (2006) investigated the structure-activities relationships (SAR) between green tea catechins and Herpes simplex virus (HSV). HSV, also known as cold sore, causes blisters on the mouth and lips (oral and facial infections) and on genitals. The authors observed that catechins exhibit an in vitro anti-HSV activity. The number of hydroxyl groups on the B ring of the catechin molecules as well as the presence (or absence) of galloyl side chains affected the SI (ranging from 1.3 to 13). Prodelphinidin B-2 3’-O-gallate, isolated from green tea leaves, also exhibited an in vitro anti-HSV-2 activity (IC50 = 5.0 μM) (Cheng et al., 2002). In close analogy with studies on anti-APC activity, the proposed mechanism of antiHSV properties of green tea catechins involves the inhibition of viral attachment and penetration into cells. The disturbance of the late stage of viral infection has also been postulated as an auxiliary mechanism (Friedman, 2007). Likewise, EGCG (50 μM) inhibited the expression of lytic proteins of Epstein-Barr virus (EBV, also known as human HSV-4) causing infectious mononucleosis. The inhibition of transcription of early genes governing the initiation of the EBV lytic cascade has been proposed as the anti-EBV mechanisms of EGCG (Chang et al., 2003). The antiviral effects of green tea catechins can be targeted towards HIV (the retrovirus causing the widespread human acquired immunodeficiency syndrome, AIDS) infection. Early studies reported that ECG and EGCG, but not EC or EGC, are powerful antagonists of HIV reverse transcriptase (IC50 from 10 to 20 ng/mL) (Nakane and Ono, 1990). Further studies reported that EGCG is responsible for the observed anti-HIV-1 activity of green tea (Nakane and Ono, 1989; Fassina et al., 2002). Several mechanisms explaining the anti-HIV properties of EGCG have been proposed. Early hypothesis involves the inhibition of the biochemical activity of HIV-1 reverse transcriptase, which causes the blocking of HIV-1 replication in


29

human cells (Nakane and Ono, 1989). More recent studies suggested a possible interference of EGCG with HIV-1 viral infection by virion destruction and HIV-1 reverse transcriptase inhibition (Fassina et al., 2002), whereas Yamaguchi et al. (2002) hypothesized that EGCG could induce in vitro virion destruction by deforming phospholipids through the formation of specific bounds with the surface of the viral envelope. Likewise, Kawai et al. (2003) reported that EGCG apparently prevents the attachment of the HIV-1 virion (gp120), to CD4 molecules on T-helper cells. The authors found that EGCG (in concentrations from 25 to 250 μM/L) down-regulated the cell surface expression of CD4 by binding to the CD4 molecule, presumably at a binding site recognized by gp120. Other viruses causing different diseases have been studied as target for the antiviral activity of green tea catechins. Preventive and curative effects of green tea extracts on influenza virus have been claimed in a patent (Shimamura and Hara., 1991). ECGC apparently prevents infection caused by the influenza virus by binding to the viral hemagglutinin (HA) (Nakayama et al., 1993). So, the viral particles (bound by EGCG) cannot attach to the target receptor cells. In vitro studies postulated that changes of viral membrane properties could contribute to the antiviral effect of green tea catechins against the influenza virus. EGCG and ECG were found to be 10–15 times more active against the influenza virus than EGC. The role of 3-galloyl side chain as a factor enhancing antiviral activity of the parent catechin molecule has been hypothesized (Song et al., 2005). As a part of a SAR study, Song et al. (2007) screened the in vitro and in vivo antiviral activity of synthetic EGC and (+)-catechin (C) derivatives characterized by the presence of different alkyl chain length and aromatic ring substitutions at the 3-hydroxyl group. Pronounced activity was observed for derivatives carrying moderate chain length (7–9 carbons) as compared to those with aromatic rings. On the contrary, the 5’-hydroxyl group of the trihydroxy benzyl moiety did not significantly contribute to antiviral activity. The active derivatives exerted inhibitory effects for all influenza subtypes tested, including three major types of currently circulating human influenza viruses (A/H1N1, A/H3N2 and B type), H2N2 and H9N2 avian influenza virus. The observed antiviral activity appears to be mediated by the interaction of catechin derivatives with HA and viral membrane (Song et al., 2007). Hepatitis B virus (HBV) infection causes public health problems worldwide and is endemic in some geographical regions (e.g. Asia). Xu et al. (2008) studied the in vitro antiHBV efficacy of green tea catechins and EGCG: IC50 from 5.02 to 10.76 μg/mL were observed, whereas the 50% cytotoxic concentration (CC50) was as higher as 170 μg/mL. Additional studies involving plant and animal viruses have been carried out. EGCG and ECG bound to and inactivated tobacco and cucumber mosaic viruses that cause lesions in leaves (Okada et al., 1971; Okada et al., 1978). Other studies reported that green tea catechins prevent rotavirus and enterovirus from infecting monkey kidney cells in tissue cultures (Mukoyama et al., 1991). This evidence was attributed to the possible interference of green tea catechins with viral adsorption rather than a direct antiviral effect. Additionally, green tea catechins were also proven to be active against the bovine coronavirus and rotavirus (causing diarrhea and gastroenteritis in calves and cattle and resulting in significant losses to agriculture) (Clark et al., 1998).

30


ANTIBACTERIAL ACTIVITY OF GREEN TEA CATECHINS Green tea catechins have been tested as antimicrobial drugs against human pathogenic bacteria (e.g. against ocular and cariogenic bacterial microflora causing minor infections). ECGC inhibited gelatinase activity produced by a few ocular bacterial pathogens (IC50 = 200 μM) (Blanco et al., 2003). More recently, Friedman (2007) underlined that the inhibition can delay the invasive spread of the bacteria in the eyes thriving on a gelatin substrate. Moreover, some studies described anticariogenic effects of green tea compounds. Dental caries are caused by a group of acid-producing species of the genus Streptococcus, in particular Streptococcus mutans and Streptococcus sobrinus, which are reported to be the major infective agents of human dental plaque. A key role in such process is played by salivary amylase, which hydrolyses food starch to oligo- and monosaccharides (e.g. maltose, glucose). The fermentation of such carbohydrates by bacterial enzymes occurring in oral cavities provokes the formation of organic acids responsible to dental caries. Green tea components have been seen to inhibit salivary amylase and, consequently, intra-oral hydrolysis of starch (Zhang and Kashket, 1998). Another study showed that EGCG prevented lowering of pH induced by cariogenic bacteria (Hirasawa et al., 2006). Likewise, an extract of green tea was used for inhibiting growth of S. mutans. The analytical characterization of such extract revealed that the main antibacterial components were GC, EGC and EGCG: among them, GC was the most active component (MIC = 250 μg/mL) (Sakanaka et al., 1989). Among pathogenic bacteria, Mycobacterium tuberculosis is a species that causes tuberculosis in humans. Anand et al. (2006) observed that EGCG inhibited the expression of tryptophan-aspartate containing coat protein (TACO) gene in a dose-dependent manner. This effect was coupled with the inhibition of the survival of M. tuberculosis within host macrophages. The species Helicobacter pylori is a urease-producing gastric pathogen that may contribute to the formation of ulcers and to low-grade gastric lymphoma in humans. Mabe et al. (1999) found that EGCG displayed a strong activity against H. pylori (MIC50 = 8 μg/ml). This compound exhibited bactericidal activity at pH 7 but not at pH < 5.0. In vivo studies carried out on infected Mongolian gerbils, reported that H. pylori was eradicated in 10 to 36% of the catechin-treated animals, with significant decreases in mucosal hemorrhage and erosion (Mabe et al., 1999). Likewise, a screening carried out on green tea catechins revealed their anti-H. pylori activity (Shin et al., 2005). Further studies reported that ECGC apparently protects epithelial cells of gastric mucosa against H. pylori-induced apoptosis and DNA damage (Lee et al., 2004). The authors postulated the block of activation of cellular signaling pathways as the mechanism causing protecting activity of epithelial cells. Legionella pneumophila causes Legionnaire disease, an infection of the lungs and other organs. A few authors found that ECGC enhanced the in vitro resistance of alveolar macrophages to infection caused by L. pneumophila (Yamamoto et al., 2004). Similarly, Matsunaga et al. (2001) showed that concentrations as low as 0.5 μg/mL of EGCG inhibited the growth of L. pneumophila in macrophages without any direct antibacterial effect. EGCG selectively up-regulated the production of interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-α) and down-regulated the L. pneumophila-induced production of interleukin-10 (IL-10) by macrophages. The authors sauggested that EGCG selectively leads to an enhanced anti-L. pneumophila activity of macrophages.


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Green tea catechins have been also tested as antimicrobial drugs against food-borne pathogen and food-spoilage bacteria. In particular, food-spoilage bacteria can cause spoilage and undesirable changes in a wide range of foods, particularly in processed, preserved, and refrigerated food. Chou et al. (1999) used selected strains of food-borne pathogen and foodspoilage bacteria to test the antibacterial activity of extracts from various tea products. In general, antimicrobial activity decreased when the extents of tea fermentation increased: tea flush and green tea extracts exerted the strongest antibacterial activity. The activity of green tea catechins against the bacterial genera belonging to the Bacillaceae family (Bacillus and Clostridium) was extensively studied. Bacillus cereus is a widely distributed food-borne pathogen causing vomiting and diarrhea in mammals (including humans). Friedman et al. (2006) demonstrated that GCG, EGCG, CG and ECG exhibit antimicrobial activities at nano-Molar levels, whereas catechins without gallate side chains and gallic acid were inactive. Interestingly, most compounds were more active than commercially available antibiotics (e.g. tetracycline or vancomycin) at comparable concentrations. In the same way, studies carried out by Hara et al. (1989; 1995) and by Ahn et al. (1991) showed that green tea catechins strongly inhibited growth of Bacillus stearothermophilus and Clostridium thermoaceticum in vitro. These compounds also reduced the heat-resistance of both species growing in vending machines, which causes sour spoilage in milk and other drinks (Sakanaka et al., 2000). Likewise, Sakanaka et al. (2000) studied the inhibitory action of green tea catechins towards the germination of Bacillus spp. and Clostridium spp. spores. The heat resistance of B. stearothermophilus and C. thermoaceticum spores was reduced by the addition of EGCG. More recently, Juneja et al. (2007) found that green tea extracts characterized by different catechin concentrations (from 141 to 697 mg of total catechins/g of extract) determined the inhibition of Clostridium perfringens spore germination in thawed beef, chicken, and pork. The authors underlined the catechins from green tea can reduce the potential risk of C. perfringens spore germination and outgrowth during abusive cooling. Staphylococcus aureus is a highly pathogenic, toxin-producing, food-borne organism. An early study reported that ECGC inhibits the growth of methicillin-resistant S. aureus (MRSA) strains (Toda et al., 1991). Likewise, Ikigai et al. (1993) showed that EC was much less active than EGCG against S. aureus strains. The MICs of EGCG and EC were 73 and 573 μg/mL, respectively, and the bactericidal effect of EGCG was attributed to membrane perturbation (Ikigai et al., 1993; Hamilton-Miller, 1995). The role of catechins (in particular EGC, EGCG ECG) on the anti-MRSA strains was also confirmed by Yam et al. (1997). More recently, Yoda et al. (2004) observed MICs from 50 to 100 μg/mL of EGCG against some species belonging to the genus Staphylococcus (S. aureus, Staphylococcus epidermis, Staphylococcus hominis and Staphylococcus haemolyticus). The authors postulated that the different structure of the cell wall as well as the variable affinities of ECGC to some cell wall components (e.g. peptidoglycans) might determine a differential activity of EGCG against such bacterial species. Furthermore, Si et al. (2006) found that ECG and EGCG were the most active compounds against S. aureus: EGCG exhibited the highest activity (MIC90 = 58 and 37 μg/L for MSSA and MRSA, respectively). Scanning electron microscopy (SEM) studies showed that both ECG and EGCG altered bacterial cell morphology, which might have resulted from disturbed cell division. Analogously, Kim et al. (2004) found that green tea catechins

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exhibited an approximately 5.0 log CFU/mL suppression of S. aureus strains (compared with the control). Physical factors can enhance the anti-S. aureus activity of green tea catechins. An et al. (2004) found that irradiated (at 40 kGy) catechins extracted from green tea leaves increased their antibacterial activities against S. aureus and S. mutans, whereas the major antioxidative activities (e.g. electron donating, inhibition of xanthine-oxydase, metal ion chelating, and inhibition of lipid oxidation) were unchanged after irradiation. The strain O157:H7 of the species Escherichia coli is a food-borne, toxin-producing enteropathogen responsible for a hemorrhagic form of colitis, bloody diarrhea, and hemolytic uremic syndrome. Isogai et al. (1998) reported that green tea catechins protect mice against neurologic and systemic symptoms caused by infection with E. coli O157:H7. Similar studies found that green tea extracts exhibit a wide spectrum of activity against a series of pathogenic bacteria, including strains of E. coli (Yam et al., 1997; Yam et al., 1998). A more recent study (Si et al., 2006) found that green tea extracts strongly inhibit the growth of E. coli O157:H7, Salmonella typhimurium, Listeria monocytogenes, S. aureus, and B. cereus. On the contrary, Kim et al. (2004) found that green tea extracts suppressed the growth of only L. monocytogenes (by approximately 3.0 log CFU/mL), whereas no influence on E. coli O157:H7 and Salmonella enteritidis growth has been noted. Yam et al. (1997) reported that a green tea extract inhibited the growth of several species of food-spoilage bacteria (Proteus vulgaris, Pseudomonas aeruginosa and Serratia marcescens) known to adversely affect the quality of some foods. More recently, Yoda et al. (2004) found that higher levels of EGCG (MIC = 800 μg/mL) were needed to inhibit the growth of some Gram-negative food-borne pathogenic and food-spoilage bacteria (E. coli, Klebsiella pneumoniae, Salmonella typhi, Proteus mirabilis, P. aeruginosa, and S. marcescens). In close analogy with above reported data, ECG and ECGC were also found to inhibit the growth of some phytopathogenic bacteria belonging to the genera Agrobacterium, Clavibacter, Pseudomonas, Erwinia, and Xanthomonas (which contaminates eggplants, grapes, cabbage, lettuce, onions, potatoes, tomatoes, etc.). MIC of about 100 μg/mL were observed (Fukai et al., 1991). Table 1. Some additional examples of antibacterial activity of green tea catechins Species Bacillus anthracis

Active References compound(s) EGCG Dell'Aica et al., 2004

Bacillis cereus

catechins

Hamilton-Miller, 1995; Friedman et al., 2006

Escherichia coli

EGCG

Taguri et al., 2004

Helicobacter pylori

ECG, EGCG Setiawan et al., 2001; Yee et al., 2002; Yahiro et al., 2005

Legionella pneumophila Staphylococcus aureus Vibrio cholerae

EGCG

Matsunaga et al., 2002; Rogers et al., 2005

EGCG

Stapleton et al., 2004; Taguri et al., 2004

EGC, EGCG Ikigai et al., 1990; Toda et al., 1992; Taguri et al., 2004; Bandyopadhyay et al., 2005


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In relation to the activity of green tea catechins on bacteria of technological importance, Goto et al. (1998) reported that these compounds are able to positively affect intestinal dysbiosis in nursing home patients by raising levels of Lactobacillus spp. and Bifidobacterium spp. while lowering levels of Enterobacteriaceae and Bacteroidaceae. Some additional examples of antibacterial activity of green tea catechins are reported in Table 1. Some hypotheses have been recently proposed for explaining the mechanism of antibacterial action of green tea catechins. Accordingly, detailed physicochemical studies suggested that the bactericidal activities of galloylated green tea catechins at the cell membrane level might be due to their specific perturbations carried out over the ordered structure of phosphatidylcholine and phosphatidylethanolamine bilayers constituting bacterial cell wall membranes (Nakayama et al., 2000; Caturla et al., 2003). ECGC was found to be the most effective catechin in causing leakage on E. coli-isolated membranes. Moreover, another study suggested that EGCG could increase antibiotic susceptibility in S. aureus through the inhibition of penicillinase produced by such bacterium (Zhao et al., 2002). In more recent studies additional hypothesis supporting the antibacterial action of green tea catechins have been proposed. Arakawa et al. (2004) and Hayakawa et al. (2004) suggested that the bactericidal action of EGCG may also depend on hydrogen peroxide derived from the reaction of EGCG with oxygen (pro-oxidative activity). Based on the above mentioned hypotheses, Friedman (2007) concluded that the observed antimicrobial effects arise from the interactions of catechins with oxygen, genes, cell membranes, and enzymes.

ANTI-YEAST AND ANTIFUNGAL ACTIVITY OF GREEN TEA CATECHINS Only a few studies have been carried out so far on the antimycotic activity of green tea tannins. Okubo et al. (1991) early examined EGCG for their antifungal and fungicidal activity against pathogenic filamentous fungi belonging to the species Trichophyton mentagrophytes and Trichophyton rubrum. This compound showed fungistatic activity against both Trichophyton species. More recently, Hirasawa and Takada (2004) have studied the susceptibility of the opportunistic pathogenic yeast Candida albicans to various green tea catechin under varying pH conditions. Analogously, Park et al. (2006) reported that EGCG exhibits antimycotic activity against 21 Candida spp. isolates. Among them, the strains belonging to the species Candida glabrata were the most susceptible. Both studies reported that the anti-yeast activity of catechins was pH dependent. The two studies reported contradictory results. Hirasawa and Takada (2004) observed that the MIC90 of EGCG was 2000 μg/mL at pH 6.0, 500–1000 μg/mL at pH 6.5 and 15.6–250 μg/mL at pH 7.0, whereas Park et al. (2006) found that the MIC of EGCG increased several folds as the pH was reduced from 7.0 to 6.0. As a result of a large-scale screening survey on the antimycotic activity of some plant extracts, Turchetti et al. (2005) observed that green tea extracts inhibited growth of yeasts belonging to the species C. glabrata Cryptococcus laurentii, Clavispora lusitaniae, Issatchenkia orientalis, Filobasidiella neoformans and Saccharomyces cerevisiae, as well as

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that of yeast-like microorganisms Prototheca wickerhamii. The authors concluded that the compounds responsible of the observed anti-yeast activity are ECG and EGCG. The mode of action of catechins on eukaryotic microorganisms has been little studied. In early investigations, Toyoshima et al. (1993) suggested that catechins are able to attack the cell membrane of Trichosporon mentagrophytes causing lysis of the conidia and hyphae.

SYNERGISTIC INTERACTIONS BETWEEN CATECHINS AND ANTIBIOTICS Current literature reports studies describing that the combined use of antibiotics and green tea catechins can increase the antimicrobial activity of the formers through specific synergistic interactions. Combinations of EGCG + β-lactames antibiotics exhibited synergistic activities against MRSA strains (Hu et al., 2002). Zhao et al. (2001) suggested that EGCG synergistically increases the activity of β-lactam antibiotics against S. aureus by binding to the peptidoglycan component of the bacterial cell wall. Likewise, Sudano Roccaro et al. (2004), postulated that EGCG acts in synergy with β-lactam antibiotics by reversing tetracycline resistance in Staphylococcus spp. isolates and by inhibiting the specific efflux pump Tet(K). The authors observed an increased accumulation of tetracycline inside bacterial cells as a visible effect of synergy with EGCG. On the contrary, Yanagawa et al. (2003) noted only additive interactions between the β-lactam antibiotic amoxicillin and EGCG against nonresistant and antibiotic-resistant isolates of H. pylori. More recently, Friedman (2007) reported that non-galloylated catechins also potentiated the activity of oxacillin against S. aureus. The combined use of green tea extracts with butylated hydroxyanisole (BHA) was more effective against bacteria and fungi than green tea alone (Simonetti et al., 2004). Glycolic extract from green tea showed a certain activity against E. coli, but only limited activity against S. mutans and no activity against C. albicans. Sub-inhibitory concentrations of BHA increased the microbicidal activity of green tea extracts against S. mutans, non-susceptible E. coli and C. albicans. In addition, green tea extracts in combination with BHA reduced the hydrophobicity of S. mutans and greatly inhibited the formation of pseudomycelium in C. albicans. The authors postulated that the increased antimicrobial activity of green tea extracts (due to synergy with BHA) is related to an impairment of the barrier function in microorganisms and a depletion of thiol groups. Other groups of antibiotics have been studied for their synergistic interactions with green tea catechins. Lee et al. (2005) observed that a combination of catechins and the fluoroquinolone antibiotic ciprofloxacin acts synergistically to alleviate chronic bacterial prostatitis in rats. Likewise, a further study showed that a green tea extract exhibited in vivo synergy with the antibiotic levofloxacin against infection of mice caused by E. coli O157:H7 (Isogai et al., 2001). Hirasawa and Takada (2004) reported that the addition of EGCG to sub-inhibitory concentrations of amphotericin B (AMB, belonging to the group of polyenes) increased the anti-yeast activity of AMB against AMB-susceptible or -resistant C. albicans. Combined treatment with EGCG + AMB (3.12–12.5 and 0.5 μg/mL, respectively) markedly decreased the growth of AMB-resistant C. albicans strains. Since sub-inhibitory concentrations of AMB


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stimulate yeast membrane permeability, the authors suggested that the combined use of AMB and EGCG might stimulate catechin uptake into the cell, and, consequently, the increased intracellular catechin concentration could act as a anti-yeast agent. The same authors also observed that when azole-susceptible C. albicans strains were treated with EGCG + fluconazole (25–50 and 0.125–0.25 μg/mL, respectively), its growth was inhibited by 93.0– 99.4% compared with the growth observed in the presence of fluconazole alone. The combined use of EGCG + fluconazole (12.5 and 10–50 μg/mL, respectively) also inhibited the growth of fluconazole-resistant C. albicans by 98.5–99.7% (Hirasawa and Takada, 2004).

CONCLUDING REMARKS Current literature herein highlight the fact that green tea polyphenols include a series of compounds (e.g. catechins) extensively studied as potential antiviral and antimicrobial drugs. Yet, although some promising results have been reported, most studies remains so far confined to the laboratory scale. In other words, in relation to the commercial exploitation of green tea catechins as antimicrobial drugs a few questions remain still open. First of all, the lack of a rigorous analytical determination of the chemical nature of active components of some green tea extracts is a common feature in most of the literature cited in this chapter. Studies using purified (or partially purified) molecules (e.g. catechins) are still very few in number. The improvement of analytical techniques will indisputably lead to a better knowledge of the antiviral and antimicrobial properties of individual compounds occurring in green tea composition. Hence, the high cost of commercially available pure standards (e.g. EGCG) represents a supplementary problem, whereas synthetic strategies, so far used only for SAR studies carried out at the laboratory-scale, are too costly and timeconsuming procedures. Secondly, the assay protocols employed for the in vitro evaluation of antimicrobial activity of green tea components is an additional problem. Most of the studies reporting MICs did not apply standard CLSI guidelines (CLSI, 2002a; CLSI, 2002b) essential for monitoring the accuracy of the method. As a result, some contradictory results could be the consequence of their low reproducibility level. The well-documented strain-related susceptibility of microbial genotypes is an additional problem. In this sense, the use of undetermined (or even unidentified) strains should be discouraged. Finally, as recently underlined by Friedman (2007), another critical aspect is to determine whether the antimicrobial activities of green tea compounds observed in vitro can be duplicated in vivo. It is important to remember that most of the literature cited in this chapter reported results of in vitro investigations. Even though useful as a preliminary survey, in vivo activity is clearly more significant. As pointed out by some authors (Kawai et al., 2003; Nance and Shearer, 2003), the most crucial aspect of translating the observed in vitro effects of green tea compounds to pharmacologically relevant strategies, is the requirement to achieve physiologically relevant concentrations. Due to the well-known poor bioavailability of some green tea compounds, most of the ingested EGCG does not get into the blood, and a significant fraction is eliminated presystemically (Chow et al., 2001; Pisters et al., 2001; Lee et al., 2002). So, even though the use of capsular administration of green tea or EGCG has been proposed, some authors doubted that, in this form, the blood EGCG concentration could

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remain at a low level than that used in the current in vitro studies (Kawai et al., 2003; Nance and Shearer, 2003). Additional studies should be consequently carried out to clarify these crucial aspects for the possible exploitation of green tea preparations for therapeutic purposes.

REFERENCES Ahn, YJ; Kawamura, T; Kim M; Yamamoto T; Mitsuoka T. Tea polyphenols: selective growth inhibitors of Clostridium spp. Agric. Biol. Chem. 1991, 55, 1425–1426. Alschuler, L. Green Tea: Healing tonic. Am. J. Natur. Med. 1998, 5, 28-31. An BJ; Kwak JH; Son JH; Park JM; Lee JY; Jo C; Byun MW. Biological and anti-microbial activity of irradiated green tea polyphenols. Food Chem. 2004, 88, 549–555. Anand, PK; Kaul, D; Sharma, M. Green tea polyphenol inhibits Mycobacterium tuberculosis survival within human macrophages. Int. J. Biochem. Cell Biol. 2006, 38, 600–609. Bandyopadhyay, D; Chatterjee, TK; Dasgupta, A; Lourduraja, J; Dastidar, SG. In vitro and in vivo antimicrobial action of tea: The commonest beverage of Asia, Biol. Pharm. Bull. 2005, 28, 2125–2127. Beers, MH. Merck Manual of Diagnosis and Therapy, 18th Edition, Whitehouse Station, NJ, USA, Merck Research Laboratories, 2006. Blanco, AR; La Terra Mule, S; Babini, G; Garbisa, S; Enea, V.; Rusciano, D. (–)Epigallocatechin-3-gallate inhibits gelatinase activity of some bacterial isolates from ocular infection, and limits their invasion through gelatine, Biochim. Biophys. Acta 2003, 1620, 273–281. Buzzini, P; Turchetti, B; Ieri, F; Goretti, M; Branda, E; Mulinacci, N; Romani, A. Proantocyanidins: naturally occurring O-heterocycles with antimicrobial activity. In: Khan D. editor. Topics in Heterocyclic Chemistry: Bioactive Heterocycles. Berlin, Germany, Springer-Verlag, 2007, 239-263. Caturla, N; Vera-Samper, E; Villalain, J; Mateo, CR; Micol, V. The relationship between the antioxidant and the antibacterial properties of galloylated catechins and the structure of phospholipid model membranes. Free Radic. Biol. Med. 2003, 34, 648–662. Chang, LK; Wei, TT; Chiu, YF; Tung, CP. Chuang, JY; Hung, SK; Li, C; Liu, ST. Inhibition of Epstein-Barr virus lytic cycle by (–)-epigallocatechin gallate, Biochem. Biophys. Res. Commun. 2003, 301, 1062–1068. Cheng, HY; Lin, CC; Lin, TC. Antiviral properties of prodelphinidin B-2 3’-O-gallate from green tea leaf. Antivir. Chem. Chemother. 2002, 13, 223–229. Chou, CC; Lin, LL ; Chung, KT. Antimicrobial activity of tea as affected by the degree of fermentation and manufacturing season. Int. J. Food Microbiol. 1999, 48, 125 –130. Chow, H; Cai, Y; Alberts, DS; Hakim, I; Dorr, R; Shahi, F. Phase I pharmacokinetic study of tea polyphenols following single-dose administration of epigallocatehin gallate and polyphenon E. Cancer Epi. Biol. Prev. 2001, 10, 53-58. Clark, KJ; Grant, PG; Sarr, AB; Belakere, JR; Swaggerty, CL; Phillips TD; Woode, GN. An in vitro study of theaflavins extracted from black tea to neutralize bovine rotavirus and bovine coronavirus infections, Vet. Microbiol. 1998, 63, 147–157. CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically: Approved standard. Document M7-A7: Wayne, PA, USA, 2002.


37

CLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Approved standard. Document M27-A2: Wayne, PA, USA, 2002. Cooper, R; Morre, DJ; Morre, DM. Medicinal benefits of green tea. I: review of non-cancer health benefits. J. Altern. Complement. Med. 2005, 11, 521-528. Cooper, R; Morre, DJ; Morre, DM. Medicinal benefits of green tea. II: review of anticancer properties. J. Altern. Complement. Med. 2005, 11, 639-652. Dell'Aica, I; Donà, M; Tonello, F; Piris, A; Mock, M; Montecucco, C; Garbisa, S. Potent inhibitors of anthrax lethal factor from green tea. EMBO Rep. 2004, 5, 418–422. Dulloo, AG; Seydoux, J; Girardier, L; Chantre, P; Vendermander, J. Green tea and thermogenesis: interactions between catechin-polyphenols, caffeine, and sympathetic activity. Int. J. Obes. Relat. Metab. Disord. 2000, 24, 252-258. Fassina, G ; Buffa, A; Benelli, R; Varnier, OE; Noonan, DM; Albinai, A. Polyphenolic antioxidant (-)-epigallocatechin-3-gallate from green tea as a candidate anti-HIV agent. AIDS 2002, 16, 939-941. Frei, B; Higdon, JV. Antioxidant activity of tea polyphenols in vivo: evidence from animal studies. J. Nutr. 2003, 133, 3275S-3284S. Friedman, M. 2007. Overview of antibacterial, antitoxin, antiviral, and antifungal activities of tea flavonoids and teas. Mol. Nutr. Food Res. 2007, 51, 116–134. Friedman, M; Levin, CE; Choi, SH; Kozukue, E; Kozukue, N. HPLC analysis of catechins, theaflavins, and alkaloids in commercial teas and green tea dietary supplements: Comparison of water and 80% ethanol/water extracts. J. Food Sci. 2006, 71, C328–C337. Fukai, K; Ishigami, T; Hara Y. Antibacterial activity of tea polyphenols against phytopathogenic bacteria. Agric. Biol. Chem. 1991, 55, 1895–1897. Garcia, R; Brooks, S. In: El Hadrami I. editor. Polyphenol Communications 2002, XXI Int. Conf. on Polyphenols, Marrakech-Morocco, September 9–12, 2002, 33–34. Goto. K; Kanaya, S; Nishikawa, T. Green tea catechins improve gut flora. Ann Long-Term Care 1998, 6, 1-7. Graham; HN. Green tea composition, consumption, and polyphenol chemistry. Prev. Med. 1992, 21, 334-350. Hamilton-Miller, JMT. Antimicrobial properties of tea (Camellia sinensis L.). Antimicrob. Agents Chemother. 1995, 39, 2375–2377. Hara, Y; Luo, SJ ; Wickremasinghe, RL; Yamanishi, T. Use and benefits of tea. Food Rev. Int. 1995, 11, 527–542. Hara, Y; Watanabe, M; Sakaguchi, G. The fate of Clostridium botulinum spores inoculated into tea drinks. Shokuhin Kogyo Gakkaishi 1989, 36, 375–379. Hayatsu, H; Inada, N; Kakutani, T; Arimoto, S; Negishi, T; Mori, K; Okuda, T; Sakata, I. Suppression of genotoxicity of carcinogens by epigallocatechin gallate. Prev. Med. 1992, 21, 370–376. Haslam, E. Thoughts on thearubigins. Phytochem. 2003, 64, 61-73. Hirasawa, M; Takada, K. Multiple effects of green tea catechin on the antifungal activity of antimycotics against Candida albicans. J. Antimicrob. Chemother. 2004, 53, 225-229. Hirasawa, M; Takada, K; Otake, S; Inhibition of acid production in dental plaque bacteria by green tea catechins. Caries Res. 2006, 40, 265–270. Hirose, M; Hoshiya, T; Akagi, K; Takahashi, S; Hara. Y; Ito, N. Effects of green tea catechins in a rat multi-organ carcinogenesis model. Carcinogenesis.1993, 14, 15491553.

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Hu, ZQ; Zhao, WH; Yoda, Y; Asano, N; Hara, Y; Shimamura, T. Additive, indifferent and antagonistic effects in combinations of epigallocatechin gallate with 12 non-β-lactam antibiotics against methicillin-resistant Staphylococcus aureus. J. Antimicrob. Chemother. 2002, 50, 1051-1054. Ichihashi, M; Ahmed, NU; Budiyanto, A; Wu, A; Bito, T; Ueda, M; Osawa, T. Preventive effect of antioxidant on ultraviolet-induced skin cancer in mice. J. Dermatol. Sci. 2000, 23, 45-50. Ikeda, I; Imasato, Y; Sasaki, E; Nakayama, M; Nagao, H; Takeo, T; Yayabe, F; Sugano, M. Tea catechins decrease micellar solubility and intestinal adbsorption of cholesterol in rats. Biochim. Biophys. Acta 1992, 1127, 141–146. Ikigai, H; Nakae, T; Hara, Y; Shimamura, T. Bactericidal catechins damage the lipid bilayers. Biochim. Biophys. Acta 1993, 1147, 132–136. Ikigai, H; Toda, M; Okubo, S; Hara, Y; Shimamura, T. Relationship between the antihemolysin activity and the structures of catechins and theaflavins. Nippon Saikingaku Zasshi 1990, 45, 913 –919. Isogai, E; Isogai, H; Hirose, K; Hayashi, S; Oguma, K; In vivo synergy between green tea extract and levofloxacin against enterohemorrhagic Escherichia coli O157 infection. Curr. Microbiol. 2001, 42, 248 –251. Isogai, E; Isogai, H; Takeshi, K; Nishikawa, T; Protective effect of Japanese green tea extract on gnotobiotic mice infected with an Escherichia coli O157:H7 strain. Microbiol. Immunol. 1998, 42, 125–128. Jarrod AC; Voelker, KA; Wolff, AV; McMillan, RP; Evans, NP; Hulver, MW; Talmadge, RJ; Grange, RW. Endurance capacity in maturing mdx mice is markedly enhanced by combined voluntary wheel running and green tea extract. J. Appl. Physiol. 2008 (in press). Juhel, C; Armand, M; Pafumi, Y; Rosier, C; Vandermander, J; Lairon, D. Green tea extract (AR25) inhibits lipolysis of triglycerides in gastric and duodenal medium in vitro. J. Nutr. Biochem. 2000, 11, 45-51. Juneja, VK; Bari, M; Inatsu, Y; Kawamoto, S; Friedman, M. Control of Clostridium perfringens spores by green tea leaf extracts during cooling of cooked ground beef, chicken, and pork. J. Food Prot. 2007, 70, 1429-1433. Katiyar, SK; Mukhtar, H; Tea antioxidants in cancer chemoprevention. J. Cell Biochem. 1997, 27, S59-S67. Kawai, K; Tsuno, NH; Kitayama, J; Okaji, Y; Yazawa, K; Asakage, M; Hori, N; Watanabe, T; Takahashi, K; Nagawa, H. Epigallocatechin gallate, the main component of tea polyphenol, binds to CD4 and interferes with gp120 binding, J. Allergy Clin. Immunol. 2003, 112, 951–957. Khafif, A; Schantz, SP; al-Rawi, M; Edelstein, D; Sacks, PG. Green tea regulates cell cycle progression in oral leukoplakia. Head Neck 1998, 20, 528-534. Kiehne, A; Lakenbrink, C; Engelhardt, H. Analysis of proanthocyanidins in tea samples. Lebensm. Unters. Forsch. 1997, 205, 153–157. Kim, S; Ruengwilysup, C; Fung, DY. Antibacterial effect of water-soluble tea extracts on foodborne pathogens in laboratory medium and in a food model. J. Food Prot. 2004, 67, 2608–2612. Kris-Etherton, PM; Keen, CL. Evidence that the antioxidant flavonoids in tea and cocoa are beneficial for cardiovascular health. Curr. Opin. Lipidol. 2002, 13, 41-49.


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Lambert, JD; Sang, S; Lu, AY; Yang, CS. Metabolism of dietary polyphenols and possible interactions with drugs. Curr. Drug Metab. 2007, 8, 499–507. Lee, IP; Kim, YH; Kang, MH. Chemo-preventative effect of green tea (Camellia sinensis) against cigarette smoke-induced mutations (SCE) in humans. J. Cell Biochem. 1997, 27, S68-S75. Lee, KW; Lee, HJ; Lee, CY. Antioxidant activity of black tea vs. green tea. J. Nutr. 2002, 132, 785-786. Lee, KM; Yeo, M; Choue, JS; Jin, JH; Park, SJ; Cheong, JY; Lee, KJ; Kim, JH; Hahm, KB. Protective mechanism of epigallocatechin-3-gallate against Helicobacter pylori-induced gastric epithelial cytotoxicity via the blockage of TLR-4 signaling, Helicobacter 2004, 9, 632–642. Lee, MJ; Maliakal, P; Chen, L; Meng, X; Bondoc, FY; Prabhu S, Lambert, G; Mohr, S; Yang, CS. Pharmacokinetics of tea catechins after ingestion of green tea and (-)epigallocatechin-3-gallate by humans: formation of different metabolites and individual variability. Cancer Epi. Biol. Prev. 2002, 11, 1025-1032. Lee, YS; Han, CH; Kang, SH; Lee, SJ; Kim, SW; Shin,OR; Sim, YC; Lee, SJ; cho, YH. Synergistic effect between catechin and ciprofloxacin on chronic bacterial prostatitis rat model, Int. J. Urol. 2005, 12, 383–389. Leung, LK; Su, Y; Chen, R; Zhang, Z; Huang, Y; Chen, ZY. Theaflavins in black tea and catechins in green tea are equally effective antioxidants. J. Nutr. 2001, 131, 2248-2251. Mabe, K; Yamada, M; Oguni, I; Takahashi, T. In vitro and in vivo activities of tea catechins against Helicobacter pylori. Antimicrob. Agents Chemother. 1999, 43, 1788–1791. Matsunaga, K; Klein, TW; Friedman, H; Yamamoto, Y. Epigallocatechin gallate, a potential immunomodulatory agent of tea components, diminishes cigarette smoke condensateinduced suppression of anti-Legionella pneumophila activity and cytokine responses of alveolar macrophages. Clin. Diagn. Lab. Immunol. 2002, 9, 864–871. Matsunaga, K; Klein, TW; Friedman, H; Yamamoto, Y. Legionella pneumophila replication in macrophages inhibited by selective immunomodulatory effects on cytokine formation by epigallocatechin gallate, a major form of tea catechins. Infect. Immun. 2001, 69, 3947– 3953. Min, Z; Peigen, X. Quantitative analysis of the active constituents in green tea. Phytother. Res. 1991, 5, 239–240. Mukoyama, A; Ushijima, H; Nishimura, S; Koike, H; Toda, M; Hara, Y; Shimamura, T. Inhibition of rotavirus and enterovirus infections by tea extracts. Jpn. J. Med. Sci. Biol. 1991, 44, 181–186. Nakane, H; Ono, K. Differential inhibitory effects of some catechin derivatives on the activities of human immunodeficiency virus reverse transcriptase and cellular deoxyribonucleic and ribonucleic acid polymerases. Biochemistry 1990, 29, 2841–2845. Nakayama, M; Suzuki, K; Toda, M; Okubo, S; Hara, Y; Shimamura, T. Inhibition of infectivity of influenza virus by tea polyphenols, Antiviral Res. 1993, 21, 289–299. Nakayama, T; Hashimoto, T; Kajiya, K; Kumazawa, S. Affinity of polyphenols for lipid bilayers. Biofactors 2000, 13, 147–151. Nance, CL; Shearer, WT. Is green tea good for HIV-1 infection? J. Allergy Clin. Immunol. 2003, November, 851-853. Okada, F. Antiviral effects of tea catechins and black tea theaflavins on plant viruses. Jpn. Agric. Res. Q. 1978, 12, 27–32.

40


Okada, F; Furuya, K. Inhibitory effect of tea catechins on some plant virus diseases. Jap. Tea Res. Sta. Stud. Tea 1971, 42, 39–46. Okubo, S; Toda, M; Hara, Y; Shimamura, T. Antifungal and fungicidal activities of tea extract and catechin. Jpn. J. Bacteriol. 1991, 46, 509–514. Otsuka, T; Ogo, T; Eto, T; Asano, Y; Suganuma, M; Niho, Y. Growth inhibition of leukemic cells by (-)-epigallocatechin gallate, the main constituent of green tea. Life Sci. 1998, 63, 1397-1403. Park, BJ; Park, JC; Taguchi, H; Fukushima, K; Hyon, SH; Takatori, K. Antifungal susceptibility of epigallocatechin 3-O-gallate (EGCG) on clinical isolates of pathogenic yeasts. Biochem. Biophys. Res. Comm. 2006, 347, 401-405. Picard, D. The biochemistry of green tea polyphenols and their potential application in human skin cancer. Altern. Med. Rev. 1996, 1, 31-42. Pisters, KM; Newman, RA; Coldman, B; Shin, DM; Khuri, FR; Hong, WK; Glisson, BS; Lee, JS. Phase 1 trial of oral green tea extract in adult patients with solid tumors. J. Clin. Oncol. 2001, 19, 1830-1838. Punyasiria, PAN; Abeysinghea, ISB; Kumarb, V; Treutterc, D; Duyd, D; Goschd, C; Martense, S; Forkmannd, G; Fischerd, TC. Flavonoid biosynthesis in the tea plant Camellia sinensis: properties of enzymes of the prominent epicatechin and catechin pathway. Arch. Biochem. Biophys. 2004, 431, 22–30. Ramassamy, C. Emerging role of polyphenolic compounds in the treatment of neurodegenerative diseases: a review of their intracellular targets. Eur. J. Pharmacol. 2006, 545, 51–64. Rimm, EB; Stampfer, MJ. Diet, lifestyle, and longevity - the next steps? JAMA 2004, 292, 1490-1492. Rogers, J; Perkins, I; van Olphen, A; Burdash, N; Klein, TW; Friedman, H. Epigallocatechin gallate modulates cytokine production by bone marrow-derived dendritic cells stimulated with lipopolysaccharide or muramyldipeptide, or infected with Legionella pneumophila. Exp. Biol. Med. 2005, 230, 645–651. Sakagami, H; Asano, K; Hara, Y; Shinamura, T. Stimulation of human monocyte and polymorphonuclear cell iodination and interleukin-1 production by epigallocatechin gallate. J. Leukocyte Biol. 1992, 51, 478–483. Sakanaka, S; Juneja, LR; Taniguchi, M. Antimicrobial effects of green tea polyphenols on thermophilic spore-forming bacteria. J. Biosci. Bioeng. 2000, 90, 81–85. Sakanaka, S; Kim, M; Taniguchi, M; Yamamoto, T. Antibacterial substances in Japanese green tea extract against Streptococcus mutans, a cariogenic bacterium. Agric. Biol. Chem. 1989, 53, 2307–2311. Sato, D. Inhibition of urinary bladder tumors induced by N-butyl-N-(4-hydroxybutyl)nitrosamine in rats by green tea. Int. J. Urol. 1999, 6, 93-99. Savi, LA; Barardi, CR; Simoes, CM. Evaluation of antiherpetic activity and genotoxic effects of tea catechin derivatives. J. Agric. Food Chem. 2006, 54, 2552–2557. Schroeter, H; Spencer, JP; Rice-Evans, C; Williams, RJ. Flavonoids protect neurons from oxidized low-density-lipoprotein-induced apoptosis involving c-Jun N-terminal kinase (JNK), c-Jun and caspase-3. Biochem. J. 2001, 358: 547-557.


41

Setiawan, VW; Zhang, ZF; Yu, GP; Lu, QY; Wang, MR; Guo, CH; Yu, SZ;

Kurtz, RC; Hsieh, CC. Protective effect of green tea on the risks of chronic gastritis and stomach cancer. Int. J. Cancer 2001, 92, 600–604. Shimamura, T; Hara, Y. March Preventive and curative medicament against infection with influenza virus, containing tea or tea polyphenols. 1991, European patent EP 417385 A2. Shin, JE; Kim, JM; Bae, EA; Hyun, YJ; Kim, DH. In vitro inhibitory effect of flavonoids on growth, infection and vacuolation of Helicobacter pylori. Planta Med. 2005, 71, 197– 201. Si, W; Gong, J; Tsao, R; Kalab, M; Yang, R; Yin, Y. Bioassay-guided purification and identification of antimicrobial components in Chinese green tea extract. J. Chromatogr. A 2006, 1125, 204–210. Simonetti, G; Simonetti, N; Villa, A. Increased microbicidal activity of green tea (Camellia sinensis) in combination with butylated hydroxyanisole, J. Chemother. 2004, 16, 122 – 127. Song, JM; Lee, KH; Seong, BL. Antiviral effect of catechins in green tea on influenza virus, Antiviral Res. 2005, 68, 66–74. Stagg, GV. Tea. The elements of a cuppa. Nutr. Bull. 1980, 29, 233–245. Stagg, GV; Millin, DJ. The nutritional and therapeutic value of tea. A review. J. Sci. Food Agric. 1975, 26, 1439–1459. Stafford, HA; Lester, HH. Flavan-3-ol biosynthesis. The conversion of (+)-dihydroquercetin and flavan-3,4-cis-diol (leucocyanidin) to (+)-catechin by reductases extracted from cell suspension cultures of Douglas fir. Plant Physiol. 1984, 76, 184-186. Stafford, HA; Lester, HH. Flavan-3-ol biosynthesis. The conversion of (+)-dihydromyrecetin to its flavan-3,4-diol (leucodelphinidin) and to (+)-gallocatechin by reductases extracted from tissue cultures of Ginko biloba and Pseudotsuga menziesii. Plant Physiol. 1985, 78, 791-794. Stapleton, P. D; Shah, S; Anderson, JC; Hara, Y; Hamilton-Miller, JMT; Taylor, PW. Modulation of β-lactam resistance in Staphylococcus aureus by catechins and gallates. Int. J. Antimicrob. Agents 2004, 23, 462–467. Sudano Roccaro, A; Blanco, AR; Giuliano, F; Rusciano, D; Enea, V. Epigallocatechin-gallate enhances the activity of tetracycline in staphylococci by inhibiting its efflux from bacterial cells. Antimicrob. Agents Chemother. 2004, 48, 1968–1973. Sugiyama, T; Sadzuka, Y. Enhancing effects of green tea components on the antitumor activity of adriamycin against M5076 ovarian sarcoma. Cancer Lett. 1998, 133, 19-26. Taguri, T; Tanaka, T; Kouno, I. Antimicrobial activity of 10 different plant polyphenols against bacteria causing food-borne disease. Biol. Pharm. Bull. 2004, 27, 1965–1969. Tanaka, T; Mine, C; Kouno, I. Structures of two new oxidation products of green tea polyphenols generated by model tea fermentation. Tetrahedron 2002, 58, 8851–8856. Tanaka, T; Kouno, I. Oxidation of tea catechins: chemical structures and reaction mechanism. Food Sci. Tech. Res. 2003, 9, 128–133. Toda, M; Okubo, S; Hara, Y; Shinamura, T. Antibacterial and bactericidal activities of tea extracts and catechins against methicillin-resistant Staphylococcus aureus. Jpn. J. Bacteriol. 1991, 46, 839–845.

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Toda, M; Okubo, S; Ikigai, H; Suzuki, T; Suzuki, Y; Hara, Y; Shimamura, T. The protective activity of tea catechins against experimental infection by Vibrio cholerae O1. Microbiol. Immunol. 1992, 36, 999 – 1001. Toyoshima, Y; Okubo, S; Toda, M. Effect of catechin on the ultrastructure of Trichophyton mentagrophytes. Kansenshogaku Zasshi. 1993, 68, 295-303. Turchetti, B; Pinelli, P; Buzzini, P; Romani, A; Heimler, D; Franconi, F. In vitro antimycotic activity of some plant extracts towards yeast and yeast-like strains. Phytother. Res. 2005, 19, 44-49. Uchida, S; Ozaki, M; Suzuki, K; Shikita, M. Radioprotective effects of (2) epigallocatechin 3O-gallate (green tea tannin) in mice. Life Sci. 1992, 50, 147–152. Weber, JM; Ruzindana-Umunyana, A; Imbeault, L; Sircar, S. Inhibition of adenovirus infection and adenain by green tea catechins. Antiviral Res. 2003, 58, 167–173. Xu, J; Wang, J; Deng, F; Hu, Z; Wang, H. Green tea extract and its major component epigallocatechin gallate inhibits hepatitis B virus in vitro. Antiviral Res. 2008, 78, 242– 249. Xu, Y; Ho, CT; Amin, SG; Han, C; Chung, FL. Inhibition of tobacco-specific nitrosamineinduced lung tumorigenesis in A/J mice by green tea and its major polyphenol as antioxidants. Cancer Res. 1992, 52, 3875–3879. Yahiro, K; Shirasaka, D; Tagashira, M; Wada, A; Morinaga, N; Kuroda, F; Choi, O; Inoue, M; Aoyama, N; Ikeda, M; Hirayama, T; Moss, J; Noda, M. Inhibitory effects of polyphenols on gastric injury by Helicobacter pylori VacA toxin. Helicobacter 2005, 10, 231–239. Yam, TS; Hamilton-Miller, JM; Shah, S. The effect of a component of tea (Camellia sinensis) on methicillin resistance, PBP2’synthesis, and β-lactamase production in Staphylococcus aureus. J. Antimicrob. Chemother. 1998, 42, 211–216. Yam, TS; Shah, S; Hamilton-Miller, JM. Microbiological activity of whole and fractionated crude extracts of tea (Camellia sinensis), and of tea components, FEMS Microbiol. Lett. 1997, 152, 169 –174. Yamaguchi, K; Honda, M; Ikigai, H; Hara, Y; Shimamura, T. Inhibitory effects of (-)epigallocatechin gallate on the life cycle of human immunodeficiency virus type 1 (HIV1). Antiviral Res. 2002, 53, 19-34. Yamamoto, Y; Matsunaga, K; Friedman, H. Protective effects of green tea catechins on alveolar macrophages against bacterial infections. Biofactors 2004, 21, 119 –121. Yanagawa, Y; Yamamoto, Y; Hara, Y; Shimamura, T. A combination effect of epigallocatechin gallate, a major compound of green tea catechins, with antibiotics on Helicobacter pylori growth in vitro. Curr. Microbiol. 2003, 47, 244 – 249. Yee, YK; Koo, MW; Szeto, ML. Chinese tea consumption and lower risk of Helicobacter infection. J. Gastroenterol. Hepatol. 2002, 17, 552–555. Yoda, Y; Hu, ZQ; Zhao, WH; Shimamura, T. Different susceptibilities of Staphylococcus and Gram-negative rods to epigallocatechin gallate, J. Infect. Chemother. 2004, 10, 55–58. Zaveri, NT. Green tea and its polyphenolic catechins: medicinal uses in cancer and noncancer applications. Life Sci. 2006, 78, 2073-2080. Zhang, J; Kashket, S. Inhibition of salivary amylase by black and green teas and their effects on the intraoral hydrolysis of starch. Caries Res. 1998, 32, 233 –238.


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Zhao, WH; Hu, ZQ; Okubo, S; Hara, Y; Shimamura, T. Mechanism of synergy between epigallocatechin gallate and β-lactams against methicillin-resistant Staphylococcus aureus, Antimicrob. Agents Chemother. 2001, 45, 1737 – 1742. Zhao,WH; Hu, ZQ; Hara, Y; Shimamura, T. Inhibition of penicillinase by epigallocatechin gallate resulting in restoration of antibacterial activity of penicillin against penicillinaseproducing Staphylococcus aureus, Antimicrob. Agents Chemother. 2002, 46, 2266–2268. Zhao, BL; Li, XJ; He, RG; Cheng, SJ; Xin, WJ. Scavenging effect of extracts of green tea and natural antioxidants on active oxygen radicals. Cell. Biophys. 1989, 14: 175-185.

In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson

ISBN 978-1-60741-045-4 © 2009 Nova Science Publishers, Inc.

Chapter 3

LIPID-SOLUBLE GREEN TEA POLYPHENOLS: STABILIZED FOR EFFECTIVE FORMULATION Ping Chen2, Douglas Dickinson1 and Stephen Hsu1 1

Medical College of Georgia, USA 2 Zhejiang University, China

ABSTRACT Green tea polyphenols (GTPs), also referred to as green tea catechins, possess properties that can provide unique health benefits to humans. As indicated in other chapters of this book, studies using molecular, cellular, and animal models, and in human subjects, have demonstrated that these phytochemicals from non-oxidized tea leaves have anti-cancer, anti-oxidant, anti-microbial, and anti-inflammatory properties. Recently, investigations in our and other laboratories indicated that topical application of GTPs could protect the epidermis against autoimmune disorders, such as psoriasis, prevent or repair UV-induced damage, and suppress scar tissue overgrowth. In addition, specific gene regulation by GTPs, especially epigallocatechin-3-gallate (EGCG), promotes skin cell differentiation, which could lead to improved homeostasis of the skin. Based on these facts, the topical use of products containing GTPs has become more popular, and manufacturers of cosmetic, health care, and household products are adding GTPs or EGCG to their formulations. However, it is important to note that studies described in this book always use “freshly prepared” GTPs or green tea, instead of “preprepared” materials. This is because GTPs are potent antioxidants that react rapidly with reactive oxygen species (ROS). As a result, GTPs in most commercially available products have been oxidized and/or epimerized; the biological effects of the resulting compounds are largely unknown. In addition, due to the highly water-soluble nature of these compounds, GTPs in their original form are not lipid-soluble, and therefore not permeable to the skin, a water-proof barrier. Another problem with formulation of GTPs for topical application is the coloration change and precipitation caused by oxidation. Thus, GTPs for topical application (e.g., on skin and mucous membranes) must be prepared and used immediately prior to oxidation, coloration and precipitation. These properties of GTPs make it difficult to formulate products containing them that have a reasonable shelf life and maintain their activity and effectiveness. In other words, most of the commercially available “green tea”–containing products are without the full benefits

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Ping Chen, Douglas Dickinson and Stephen Hsu of green tea or GTPs. Therefore, strategies to stabilize and increase the bioavailability of GTPs are needed to provide the full benefits of GTPs to consumers or patients. Recently, it has been shown that lipid esters of GTPs can be formed either enzymatically or chemically. These green tea polyphenol-lipid esters, also referred to as lipid-soluble tea polyphenols (LTPs), could significantly improve formulations of consumer or health care products. We hypothesized that fatty acyl esterification of green tea polyphenol would protect the hydroxyl groups from oxidation and improve skin permeability. In the current study, we compared the activities of LTPs to GTPs for their anti-cancer and gene regulation properties. We examined whether LTPs can be converted into a free GTP (EGCG) in human skin keratinocyte cultures. In addition, the effects of LTPs in a mouse model for psoriasis were evaluated. The results indicate that LTPs effectively cause cancer cell death, induce caspase 14 gene expression both in vitro and in vivo, and improve the skin condition in an animal model for psoriasis. Consistent with these observations, HPLC analysis demonstrated that EGCG in its original form was released from LTPs in situ by human epidermal keratinocytes. These results suggest that LTPs, under appropriate conditions, function similarly to GTPs. More importantly, since the most reactive hydroxyl group(s) is/are protected, and the lipid solubility is dramatically increased by the fatty acyl groups, the biological activity of these compounds can be stabilized, and their bioavailability increased significantly. In conclusion, LTPs are a novel and more effective form of green tea polyphenols for topical applications and other purposes, especially in formulations that require a reasonable shelf life. In addition, LTPs can be a natural additive to consumable products such as salad oil, fish oil, and cooking oil as antioxidants.

INTRODUCTION Green tea polyphenols (GTPs) are under extensive study for their unique properties that can provide protection against a number of human diseases, including neoplastic, cardiovascular, autoimmune, and infectious diseases. During the past decade, anti-cancer, anti-inflammatory, anti-aging and anti-photo-damaging properties of GTPs, especially epigallocatechin-3-gallate (EGCG), have been identified [1-11]. These findings have prompted the addition of GTPs to cosmetic and health care products for topical application. The ingredient labels of many cosmetic and household products list various descriptions of GTPs, such as “green tea extract”, “extract of camellia sinensis”, “green tea polyphenols”, “green tea catechins”, “green tea leaf extracts”, and “EGCG”. In addition, due to their strong anti-oxidant activity, this type of naturally-occurring compound can potentially be used as natural antioxidant food additives in various products, including dietary oils. Unfortunately, certain properties of GTPs create problems for their commercial application. In traditional green tea-consuming countries such as China and Japan, green tea is consumed immediately after it is brewed, i.e., “freshly prepared”. In an effort to bring green tea’s benefits to modern populations, bottled green tea was invented, and it has become popular even in traditionally “green tea brewing” countries. However, an aqueous environment favors oxidation of GTPs, leading to epimerization and polymerization of the catechin monomers. When the monomers are oxidized, their anti-oxidant and biological activities are reduced significantly. A study of bottled tea beverages confirmed that the majority of these drinks no longer contain significant amount of GTPs, but rather the epimer gallocatechin gallate (GCG) and oxidized compounds [12]. In this study, dry leaves of green

Lipid-soluble Green Tea Polyphenols

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tea had an average EGCG content of 6.87% (see Table 1 in reference 12). In most brandnamed tea beverages tested by this study, the EGCG content was at “trace” levels (< 0.00001%) to 0.00082%. One exception had a higher level (0.058%) [12]. In other words, due to the required step of sterilization by autoclaving, the anti-oxidant and other beneficial effects of most tea beverages have been destroyed prior to their consumption. A second problem with GTPs is that they are highly hydrophilic, resulting in very low solubility in dietary oils, and they form dark precipitates in these oils [13]. Formulation experts have tried to emulsify GTPs with lipid material in order to increase their solubility in dietary oils, but the results have not been satisfactory due to precipitation and coloration [13]. A third problem for the use of GTPs is their relatively very low bioavailability in humans after oral administration as measured by serum concentration analysis [14]. In fact, the majority of GTPs is excreted from the digestive and urinary system, and less than 10 µM of GTPs concentration can be achieved in the circulation [14, 15]. This poor serum bioavailability decreases the potential anti-cancer and anti-inflammatory effectiveness of GTPs, which have been described in many studies that have used much higher concentrations of GTPs. In addition, since the epidermis is not vascularized, the benefit of orally consumed GTPs to the human epidermis is highly questionable [16]. Therefore, for the skin, topical application with high concentrations of GTPs/EGCG has been considered as an optional route of delivery (11, 10% EGCG). However, topical application of a high concentration of GTPs could cause significant skin coloration, although this effect could be used for skin tanning (US Patent 6399046). Other methods, such as “electrical assisted” methods, have been developed to increase the skin permeability to GTPs, but they may not be a suitable strategy to improve skin condition [17]. Taken together, the hydrophilic nature of the water-soluble and unstable polyphenolic GTP molecules prevents them from penetrating lipid barriers such as the stratum corneum. This compounds the fact that GTPs in these products are undergoing oxidation, epimerization, and/or polymerization prior to reaching the customers. Thus, the instability of GTPs in their original monomer form in an aqueous environment at physiological pH, and their impermeability to lipid barriers and insolubility in a hydrophobic environment, prevent their delivery at levels that provide effective benefits to human health. Stability and bioavailability are therefore two major challenges in the development of any effective product with a reasonable shelf life. Green tea researchers and formulation specialists identified these limitations, and attempts to preserve the chemical integrity and activity of GTPs began in the 1990s [13]. Esterification has been proven effective for other bio-molecules such as vitamins C (ascorbyl palmitate) and A (retinyl palmitate), and bioactive peptides, which are widely used in today’s consumer and cosmetic products [18, 19, and international patent WO 1992014830]. Researchers proposed that modification of GTPs with fatty acyl esterification would increase their stability and skin permeability without significant reduction of the antioxidant property. One group of Chinese researchers led by Professor Shuxiong Wang invented a method using an acylation reaction (patent CN1197786A) that chemically modifies GTPs to make GTPesters. This method renders the final product (lipid-soluble tea polyphenols [LTPs] in a dark brown gel-like form) soluble in oil and many hydrophobic solvents. Another method was later developed in China by Jian Hua Zhong and Ping Chen which produces GTP-esters in a solid powder form (CN1231277A). In Japan, similar GTP derivatives were made using the reverse reaction of lipases from Streptomyces rochei (Japanese patent JP6279430). Recently, Kunihiro Kaihatsu’s group in Osaka University synthesized a series of EGCG-esters by an

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enzymatic method using lipase PL from Alcaligenes sp [20]. In 2002, one of us (P Chen) first synthesized and identified the chemical structure of EGCG-4’-hexadecanate [21]. Subsequently, in 2003, the first systematic synthesis, purification, and analysis of EGCGacyl-derivatives in the laboratory was reported, and the purified LTP compound epigallocatechin-3-O-4’-O-hexadecanate was obtained (referred to as EGCG-palmitate hereafter) [22]. The antioxidant activity and stability of EGCG-palmitate was tested in dietary oils. It was found that the antioxidant activity of EGCG-palmitate in soybean salad oil and canola oil is greater than that of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), and similar to that of tertiary butylhydroquinone (TBHQ) [23]. In addition, the potential toxicity of EGCG-palmitate was tested by the Zhejiang Center for Disease Control & Prevention of China. The acute toxicology test result in mice was LD50>5.0 g/kg, and the Ames Test result for mutagenicity was negative [23]. The anti-cancer property of EGCG-palmitate was compared with GTPs and LTPs by determining the inhibition of growth of the human ovarian cancer cell line HO-8910 [24]. The 50% inhibitory concentration (IC50) for EGCG-palmitate was 78.2 µg/ml, while for GTPs it was 72.2 µg/ml, and 88.9 µg/ml for LTP [24]. In a separate study, EGCG-myristate (epigallocatechin-3-O-4’O-myristate) was synthesized [25]. This is an EGCG ester with a 14 carbon fatty acyl chain. The physical characteristics of EGCG-myristate are similar to EGCG-palmitate. It has a whitish powder appearance and is readily soluble in lipid, but not in water. The antioxidant activity of EGCG-myristate is almost identical to EGCG-palmitate when compared with TBHQ, BHA and BHT (in soy bean salad oil) [25]. That is, the food industry, especially dietary oil manufacturers, could benefit from the use of LTPs as an antioxidant. However, it is not known if LTP enters human cells, or if it can be converted to GTPs to induce similar biological effects. It is also not clear whether a stable LTP formulated topical application can improve the skin condition in a mouse model for psoriasiform lesions (the flaky skin mouse), as shown for EGCG. The current study used a powdered form of 18 carbon fatty acyl (stearoyl) and a gel-like 18 carbon monounsaturated fatty acyl (oleic acyl)ester of green tea polyphenols (referred to as LTPStearate and LTPOleate hereafter) to tests their biological effects in vitro and in vivo.

MATERIAL AND METHODS Chemicals and Antibodies EGCG was purchased from Sigma-Aldrich (St. Louis, MO). The solid-form LTPStearate (>90% stearoyl tea polyphenol) was purchased from Yuyao Huidelong Biological BioProducts, Co., Ltd, Zhejiang, China. The gel-form LTPOleate (>90% oleic acyl tea polyphenols) was purchased from Zhejiang Cereals Oils & Foodstuffs Import & Export Company, Ltd, Zhejiang, China. A topical formulation of LTPStearate was made of 0.1% LTPStearate in glycerin and stored at 4o C for daily topical use (the preparation of this formulation is not disclosed due to pending patent). The anti-caspase 14 and anti-human actin (I-19) antibodies were obtained from Santa Cruz Biotechnology, Santa Cruz, CA.


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Cell Lines and Cell Culture Pooled normal human primary epidermal keratinocyte (NHEK) cells were purchased from Cambrex (East Rutherford, NJ) and sub-cultured in the specific growth media (KGM-2) provided by the manufacturer. The OSC2 cell line was isolated from a metastatic lymph node of a patient with oral squamous cell carcinoma [26]. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/Ham’s F12 50/50 MIX medium (Cellgro, Kansas City, MO) supplemented with 10 % (v/v) fetal bovine serum, 100 I.U/ml penicillin, 100 µg/ml streptomycin and 5 µg/ml hydrocortisone.

Morphological Analysis of Cells Treated with LTPStearate OSC2 or NHEK cells were seeded in 24-well tissue culture plates at 2X105 cells/well and incubated overnight. LTPStearate dissolved in vehicle (100% ethanol) at different concentrations was added to each well. Control wells were incubated with vehicle only. At the end of incubation, wells were washed with PBS and photographed at 100X magnification.

Western Blotting To determine changes in the protein level of caspase 14 upon activation by EGCG or LTPStearate, NHEK were exposed to 50 µM EGCG or LTPStearate for various times. Cell lysates were prepared, and samples containing 30 µg protein were separated on 10 % SDS polyacrylamide gels. Western blot analysis was performed using anti-human caspase 14 and actin antibodies (for normalization). The method for Western analysis was previously described [27]. The resulting bands were visualized by enhanced chemiluminescent staining using ECL Western blotting detection reagents (Amersham Pharmacia Biotech Inc., Piscataway, NJ).

Animal Treatment and Immunohistochemistry Flaky skin mice were treated daily for consecutive ten days with an LTPStearate topical preparation (0.1% w/v in glycerin) in an area on the back of the animal. On the opposite side of the animal, glycerin was applied topically as control. The skin samples were collected by a biopsy puncture under anesthesia, and the animals were euthanized thereafter. Processing and immunostaining of skin samples have been described previously [28].

HPLC Analysis of EGCG from Cell Lysates Cultures of NHEK cells were divided into two and treated with 0.1% w/v of either LTPOleate or LTPStearate. At 0.5 h, 1.0 h, 2.0 h, and 6.0 h, cells were washed with PBS and lysed

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with 0.5 ml RIPA buffer (pH adjusted to 3.35). An equal amount of cell lysate was also collected from NHEK treated with 100 μM EGCG for 0.5 h as a control for HPLC detection. HPLC detection of EGCG in cell lysates was performed according to a method previously described [29]. The Discovery C-18 reverse-phase column was purchased from Supelco, Sigma-Aldrich (St. Louis, MO). Briefly, the column was eluted with Solution A (30 mM NaH2PO4 in 1.75% CH3CN and 0.125% tetrahydrofuran (pH 3.35) and Solution B (15 mM NaH2PO4 in 58.5% CH3CN and 6.25% tetrahydrofuran (pH 3.45), as described previously [29].

RESULTS LTPStearate Induces Tumor Cell Death as Efficiently as GTPs Our previous study demonstrated that GTPs at 1 mg/ml are a powerful inducer of cell death in OSC2 oral carcinoma cells [30, 31]. To test the effectiveness of LTPStearate, OSC2 cells were incubated with 1 mg/ml LTPStearate (0.1% w/v) for 24 hr. As shown in Figure 1, almost all tumor cells were eliminated by LTPStearate treatment, while control OSC2 cells treated with vehicle only remained viable. This result indicates that LTPStearate possesses the ability of a cell death-inducer for OSC2 cells. The anti-tumor cell effect of LTPStearate was also significant at a lower concentration (0.01% w/v), although less pronounced that at 0.1% w/v (Figure 2), consistent with dose-dependency.

Figure 1. 0.1% LTPStearate induced massive cell death in OSC2 cells. OSC2 oral carcinoma cells were incubated with vehicle or 0.1% LTPStearate (pre-dissolved in 100 ethanol) in cell culture medium for 24 h. The culture dishes were washed by PBS and photographed under light microscope (100 X).


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Figure 2. 0.01% LTPStearate induced significant cell death in OSC2 cells. OSC2 oral carcinoma cells were cultured until reaching confluency, and incubated with vehicle or 0.01% LTPStearate (pre-dissolved in 100 ethanol) in cell culture medium for 24 h. The culture dishes were washed by PBS and photographed under light microscope (100 X).

Figure 3. 0.1% LTPStearate failed to induce cell death in normal human epidermal keratinocytes (NHEK). NHEK cells were incubated with either vehicle or 0.1% LTPStearate for 24 h. The culture dishes were washed with PBS and photographed under light microscope (100 X).

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LTPStearate does not Induce Cell Death in Normal Epidermal Keratinocytes LTPStearate at 1 mg/ml (0.1% w/v) was incubated with primary NHEK (Figure 3). In contrast to the results with the tumor cell line OSC2, no sign of cell death or detachment was observed, indicating that LTPStearate did not induce cytotoxicity in NHEK.

Dose-dependent Induction of Caspase 14, a Marker for Epidermal Terminal Differentiation and Barrier Formation, by LTPStearate The expression of caspase 14 was compared between 50 µM EGCG-treated and 0.01% w/v LTPStearate-treated NHEK. Since 0.01% w/v of LTPStearate contains approximately 0.0025% w/v green tea polyphenols, the polyphenol content was comparable between the two treatments. Figure 4 demonstrated that both materials induced the expression of caspase 14 at the 20 h time point, and continued expression of caspase 14 was observed at 24 h. Western blotting also showed that LTPStearate at 0.001% w/v to 0.01% w/v (10-100 ppm) induced caspase 14 expression in NHEK cells in a dose-dependent manner (Figure 5).

LTPStearate Induces Caspase 14 Expression in Vivo in a Mouse Model for Psoriasiform Lesions Immunostaining of mouse skin treated with LTPStearate in glycerin demonstrated increased caspase 14 expression in the supra-basal layers of the epidermis (Figure 6). This result is consistent with our previous observation that GTPs induce and activate caspase 14 in vivo [28].

Figure 4. Western blot results demonstrate both EGCG and LTPStearate induced caspase 14 expression in NHEK. NHEK cells were treated with either EGCG (50 µM) or LTPStearate for 0, 2, 6, and 24 h. The cell lysates were collected for Western blot using rabbit anti-human caspase 14 and goat anti-human actin antibodies.


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Figure 5. LTPStearate dose-dependently induced caspase 14 protein expression in NHEK cells. Various concentrations of were prepared with DMSO, diluted with cell culture medium, and incubated with NHEK for 24 h. Cell lysates were collected for Western blot with antibodies against human caspase 14, and human actin as an internal control.

LTPOleate and LTPStearate are Converted to Intracellular GTPs in NHEK HPLC analysis of cell lysates of NHEK demonstrated that intracellular EGCG appeared in both LTPOleate and LTPStearate - treated NHEK (Table 1). However, the release time was different between the two (Figure 7). The majority of EGCG released from LTPOleate appeared in the cell lysate collected at 1 h after the addition of LTPOleate, while the majority of EGCG released from LTPStearate appeared at 6 h after treatment (Figure 7). Table 1. Retention time comparison (min) between the two types of LTP and EGCG

*Retention time of EGCG: 28.92 min. Time of EGCG peak was recorded by HPLC using samples from cell lysates collected at different time points after the LTPs was introduced into the cell culture medium.. ND: not determined.

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Figure 6. Immunostaining of skin samples from vehicle-treated and LTPStearate -treated (flaky skin mice) using caspase 14 antibody. Nuclear caspase 14 staining appeared in the junction of the granular layer and the cornified layer in the LTPStearate-treated sample, while the vehicle-treated sample did not exhibit nuclear staining of caspase 14. The supra-basal layers of the LTPStearate-treated sample showed consistent immunostaining of caspase 14, but those of the vehicle-treated sample only showed sporadic caspase 14 expression. The epidermal portion of the samples was photographed at a 400X magnification.

Figure 7. EGCG release from NHEK demonstrates time differences between LTPStearate and LTPOleate. Cell lysates were collected at indicated time points after LTPs was introduced into the cell culture medium. HPLC analysis of the samples showed different release patterns between the two types of LTPs. LTPS: LTPStearate. LTPO: LTPOleate.


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DISCUSSION The earliest Chinese literature indicates that the Chinese grew and used tea plants 3,000 years ago. According to legend, in 2737 B.C., the Chinese Emperor Shen-Nung placed camellia blossom tips into a cup of boiled water and pronounced the beverage healing and refreshing. Tea consumption became popular in China after it was said to cure the ailing troops of General Zhu Ge Liang (181-234 A.D.). Tea was described as able to prevent pandemics and treat patients with eye diseases. During the Tang Dynasty (618-907 A.D.), tea became an essential component of Chinese culture. Chinese author Lu Yu (733-804 A.D.) published the book, Cha Jing (literally, The Book of Tea), systematically chronicling the planting, processing, preparation and consumption of tea. From then, green tea (as well as many other types of teas derived from green tea), became the most popular beverage (second to water) in the world. Today, the medicinal use of green tea and its compounds is being re-visited by scientists and clinicians, and the benefits are being unveiled almost daily. In 2006, the FDA approved an ointment, VeregenTM (MediGene AG, Polyphenon® E Ointment), for treatment of genital warts, an epidermal sexually transmitted disease cause by human papilloma virus (HPV). The active ingredient(s) in this topical ointment is 10-15% Polyphenon E, a highly purified and defined GTPs mixture developed by Dr. Yukihiko Hara and colleagues at Mistui Norin Co. Ltd of Japan [32]. It is important to note that this ointment contains 10-15% of GTPs, far higher than any GTP-containing beverages or topical products. Therefore, this topical ointment treatment for this skin lesion, with an interrupted stratum corneum, still requires a physician’s prescription due to potential side effects. However, it represents the beginning of an era in which green tea components can be used for therapeutic purposes. It also suggests that the anti-microbial, especially the anti-viral, effects of GTPs need to be explored further. Recently, results from various laboratories confirmed that GTPs or EGCG, the most abundant green tea polyphenol, possesses potent inhibitory effects on influenza A and B viruses [33, 34], hepatitis B virus [35], herpes simplex virus (HSV) [36], Epstein-Barr virus [37], adenovirus [38], and human immunodeficiency virus (HIV) [39, 40]. In 2008, Dr. Kaihatsu at Osaka University and colleagues reported that EGCG-palmitate is 24 times more effective than EGCG when compared for their anti-influenza activities [20]. Their findings suggest that EGCG-palmitate (or other types of esters) are suitable candidates for anti-viral formulations. Due to the stability and skin permeability of LTPs in topical formulations, it would only require less than 1% w/v to deliver similar effects in comparison to 10-15% w/v of Polyphenon E. We reported previously that, as measured by the MTT cell viability assay, EGCGpalmitate possesses anti-cancer activity similar to GTPs in the human ovarian cancer cell line HO-8910 [24]. Here we show that LTPStearate at 0.1% w/v induced massive cell death in an oral cancer cell line OSC2 (Figure 1). When the concentration of LTPStearate was decreased to 0.01% w/v, cell death was still clearly evident (Figure 2). In contrast, no cell death was observed in NHEK under identical conditions (Figure 3). This result is consistent with our previous reports that GTPs or EGCG selectively induce apoptosis in cancer cells but not in normal cells [41-47]. The significance of this finding is that the absorption of LTPs, a lipophilic complex, is different than that of GTPs, and it could therefore target internal cancers, such as liver cancer, with higher bioavailability by oral administration. Although the

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delivery route of LTPs into the human body is still not clear, it is postulated to be via the chylomicron pathway. If so, LTPs would be associated with lipoprotein particles only, which significantly reduces potential binding with serum proteins, but increases the level in lipoproteins such as LDL prior to internalization by hepatocytes. That is, in comparison to GTPs the anti-cancer and cardiovascular benefits of LTPs could be better. LTPStearate, even at just 0.01% w/v, significantly induced the expression of caspase 14, a gene product specifically associated with cell differentiation and barrier formation and that causes cell death in cancer cells [48]. The effect of LTPStearate on caspase 14 expression is comparable to that of EGCG (Figure 4), and the induction of caspase 14 by LTPStearate is dosedependent (Figure 5). These results indicate that the effects of LTPStearate on the expression of caspase 14 in epidermal keratinocytes are consistent with the results generated from EGCG [49-51]. However, due to its water-soluble nature EGCG is not bioavailable to human skin by oral consumption [14, 15] or topical application at 50 µM (0.0023% w/v). The concentrations to be delivered in formulations for effectiveness are documented at 10% w/v for EGCG [11] and 10-15% w/v for GTPs [32], which could be very costly (EGCG costs $4000 to $9000 per Kg) and very difficult to formulate. In contrast, bioavailability can be better achieved by LTPs through topical application due to their lipid-soluble nature ( 14 cups per day and coffee drinkers. However, this study didn’t differentiate between the different kinds of teas (black tea, green tea, white tea…). The first phase of the study found that tea consumption was associated with lower mortality among patients with acute myocardial infarction whereas coffee intake showed no effect on mortality outcome.

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Figure 2. EGCG blocks HERG potassium channels, that is the molecular correlate of the cardiac rapid delayed rectifier potassium current IKr. By blocking IKr ,which results in smaller currents, repolarization will be prolonged.

Unexpectedly the investigators also found that tea consuming patients had a lower prevalence of ventricular arrhythmia after myocardial infarction. However, this question was not the focus of the study, so that no adjusted analysis was conducted and a second phase of the Onset Study was started, where the occurrence of ventricular arrhythmia was assessed in detail and correlated with the different tea drinker groups and the coffee drinker group with adjustment for potentially confounding factors. The investigators found that of the 3882 patients 445 patients developed ventricular arrhythmia during hospitalization, in detail 370 patients developed ventricular tachycardia and 87 ventricular fibrillation. The results of this second phase of the study were that moderate tea intake was associated with a lower prevalence of ventricular arrhythmia and coffee drinkers had a higher incidence of ventricular arrhythmia. Heavy tea drinkers had an intermediate prevalence of ventricular arrhythmia. Possible explanations for a lower incidence of ventricular arrhythmia among tea drinkers might be an improved gap junctional intercellular communication due to the flavonoid content in tea. An experimental study with rat liver epithelial cells [60] showed that with increasing levels of epicatechin, which is found in green tea, in cell culture the number of communicating gap junctional cells increased so that gap junctional communication was improved. Since intercellular communication in the heart is also similar to the liver, with connexins playing the most important role, it might be assumed that cardiac intercellular gap junctional communication might also be positively affected by flavonoids. Another

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experimental study in rat hearts [61] describes the positive minimizing effect of catechins on ischemia-reperfusion injury and thus leading to less ischemia-triggered arrhythmia. In our own in vitro study in xenopus laevis oocytes [7] we demonstrated for the first time, that green tea flavonoid EGCG blocks the human ether a-go-go related gene (HERG), which is the molecular correlate of the cardiac rapid delayed rectifier potassium current IKr. Prolonging repolarization of the cardiac action potential is the mechanism of many antiarrhythmic agents, e.g. dofetilide [62], a class III-antiarrhythmic agent. Characteristically, class III-antiarrhythmic drugs prolong the cardiac refractory period and make the heart less susceptible for cardiac arrhythmias by blocking IKr. According to a study by Prineas et al. [63] coffee intake however is associated with a greater prevalence of premature ventricular contractions.

CONCLUSION Tea consumption had its origin in China almost 5,000 years ago. Green tea, a worldwide consumed beverage, has been used as traditional medicine in areas such as China, Japan, India and Thailand. Green tea has gained scientific attention due to its antioxidant, antinflammatory, antihypertensive, antidiabetic and antimutagenic properties. Due to its high content of polyphenolic flavonoids, mainly EGCG, green tea has especially shown exciting cardiovascular health benefits. A number of animal as well as randomized human studies have proven the benefits of green tea for cardiovascular and metabolic diseases concluding that 200-300 mg of EGCG or 5-6 cups of green tea per day protects cardiovascular and metabolic health. In times of growing cardiovascular and metabolic disease, a beverage that has scientific evidence for its health protective properties and that is easily available for everybody might have a tremendous impact on the health of the world population and an increased impact on the economic budget. A balanced diet combined with regular green tea consumption and physical activity as well as life style changes may offer primary prevention against cardiovascular disease.

REFERENCES [1]

[2] [3]

[4]

Geleijnse JM, Launer LJ, Hofman A, Pols HA, Witteman JC. Tea flavonoids may protect against atherosclerosis: the Rotterdam Study. Arch Intern Med 1999 Oct 11;159(18):2170-2174. Sesso HD, Gaziano JM, Buring JE, Hennekens CH. Coffee and tea intake and the risk of myocardial infarction. Am J Epidemiol 1999 Jan 15;149(2):162-167. Duffy SJ, Keaney JF, Jr., Holbrook M, Gokce N, Swerdloff PL, Frei B, et al. Short- and long-term black tea consumption reverses endothelial dysfunction in patients with coronary artery disease. Circulation 2001 Jul 10;104(2):151-156. Hertog MG, Feskens EJ, Hollman PC, Katan MB, Kromhout D. Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study. Lancet 1993 Oct 23;342(8878):1007-1011.

Cardiovascular and Metabolic Effects of Green Tea [5]

[6] [7]

[8]

[9]

[10]

[11]

[12] [13]

[14] [15]

[16]

[17]

[18] [19]

[20]

253

de Whalley CV, Rankin SM, Hoult JR, Jessup W, Leake DS. Flavonoids inhibit the oxidative modification of low density lipoproteins by macrophages. Biochem Pharmacol 1990 Jun 1;39(11):1743-1750. Tijburg LB, Mattern T, Folts JD, Weisgerber UM, Katan MB. Tea flavonoids and cardiovascular disease: a review. Crit Rev Food Sci Nutr 1997 Dec;37(8):771-785. Kelemen K, Kiesecker C, Zitron E, Bauer A, Scholz E, Bloehs R, et al. Green tea flavonoid epigallocatechin-3-gallate (EGCG) inhibits cardiac hERG potassium channels. Biochem Biophys Res Commun 2007 Dec 21;364(3):429-435. Iso H, Date C, Wakai K, Fukui M, Tamakoshi A. The relationship between green tea and total caffeine intake and risk for self-reported type 2 diabetes among Japanese adults. Ann Intern Med 2006 Apr 18;144(8):554-562. Polychronopoulos E, Zeimbekis A, Kastorini CM, Papairakleous N, Vlachou I, Bountziouka V, et al. Effects of black and green tea consumption on blood glucose levels in non-obese elderly men and women from Mediterranean Islands (MEDIS epidemiological study). Eur J Nutr 2008 Feb;47(1):10-16. Alvarez E, Campos-Toimil M, Justiniano-Basaran H, Lugnier C, Orallo F. Study of the mechanisms involved in the vasorelaxation induced by (-)-epigallocatechin-3-gallate in rat aorta. Br J Pharmacol 2006 Feb;147(3):269-280. Diepvens K, Kovacs EM, Nijs IM, Vogels N, Westerterp-Plantenga MS. Effect of green tea on resting energy expenditure and substrate oxidation during weight loss in overweight females. Br J Nutr 2005 Dec;94(6):1026-1034. Rumpler W, Seale J, Clevidence B, Judd J, Wiley E, Yamamoto S, et al. Oolong tea increases metabolic rate and fat oxidation in men. J Nutr 2001 Nov;131(11):2848-2852. Dulloo AG, Duret C, Rohrer D, Girardier L, Mensi N, Fathi M, et al. Efficacy of a green tea extract rich in catechin polyphenols and caffeine in increasing 24-h energy expenditure and fat oxidation in humans. Am J Clin Nutr 1999 Dec;70(6):1040-1045. Balentine DA, Wiseman SA, Bouwens LC. The chemistry of tea flavonoids. Crit Rev Food Sci Nutr 1997 Dec;37(8):693-704. Lee MJ, Wang ZY, Li H, Chen L, Sun Y, Gobbo S, et al. Analysis of plasma and urinary tea polyphenols in human subjects. Cancer Epidemiol Biomarkers Prev 1995 Jun;4(4):393-399. Lee MJ, Maliakal P, Chen L, Meng X, Bondoc FY, Prabhu S, et al. Pharmacokinetics of tea catechins after ingestion of green tea and (-)-epigallocatechin-3-gallate by humans: formation of different metabolites and individual variability. Cancer Epidemiol Biomarkers Prev 2002 Oct;11(10 Pt 1):1025-1032. Yang CS, Chen L, Lee MJ, Balentine D, Kuo MC, Schantz SP. Blood and urine levels of tea catechins after ingestion of different amounts of green tea by human volunteers. Cancer Epidemiol Biomarkers Prev 1998 Apr;7(4):351-354. Miyazawa T. Absorption, metabolism and antioxidative effects of tea catechin in humans. Biofactors 2000;13(1-4):55-59. Nakagawa K, Okuda S, Miyazawa T. Dose-dependent incorporation of tea catechins, ()-epigallocatechin-3-gallate and (-)-epigallocatechin, into human plasma. Biosci Biotechnol Biochem 1997 Dec;61(12):1981-1985. Ruckdaeschel S, Berger K, Szucs T. Koronare Herzkrankheit - Sozioökonomisches Problem? Müncher Medizinische Wochenzeitschrift 1996;138:404-408.

254

Kamilla Kelemen

[21] American Heart Association. Heart Disease and Stroke Statistics - 2005 Update. Dallas, Texas; 2004. [22] Kuriyama S, Shimazu T, Ohmori K, Kikuchi N, Nakaya N, Nishino Y, et al. Green tea consumption and mortality due to cardiovascular disease, cancer, and all causes in Japan: the Ohsaki study. JAMA 2006 Sep 13;296(10):1255-1265. [23] Kono S, Shinchi K, Ikeda N, Yanai F, Imanishi K. Green tea consumption and serum lipid profiles: a cross-sectional study in northern Kyushu, Japan. Prev Med 1992 Jul;21(4):526-531. [24] Imai K, Nakachi K. Cross sectional study of effects of drinking green tea on cardiovascular and liver diseases. BMJ 1995 Mar 18;310(6981):693-696. [25] Yang M, Wang C, Chen H. Green, oolong and black tea extracts modulate lipid metabolism in hyperlipidemia rats fed high-sucrose diet. J Nutr Biochem 2001 Jan;12(1):14-20. [26] Hertog MG, Kromhout D, Aravanis C, Blackburn H, Buzina R, Fidanza F, et al. Flavonoid intake and long-term risk of coronary heart disease and cancer in the seven countries study. Arch Intern Med 1995 Feb 27;155(4):381-386. [27] Ohmori R, Momiyama Y, Takahashi R. Inverse association of green tea intake with myocardial infarction (MI) and its genetic variation. Atherosclerosis Supplement 2003;4:21. [28] Hirano R, Momiyama Y, Takahashi R, Taniguchi H, Kondo K, Nakamura H, et al. Comparison of green tea intake in Japanese patients with and without angiographic coronary artery disease. Am J Cardiol 2002 Nov 15;90(10):1150-1153. [29] Ojo OO, Ladeji O, Nadro MS. Studies of the antioxidative effects of green and black tea (Camellia sinensis) extracts in rats. J Med Food 2007 Jun;10(2):345-349. [30] Antonello M, Montemurro D, Bolognesi M, Di PM, Piva A, Grego F, et al. Prevention of hypertension, cardiovascular damage and endothelial dysfunction with green tea extracts. Am J Hypertens 2007 Dec;20(12):1321-1328. [31] Erba D, Riso P, Bordoni A, Foti P, Biagi PL, Testolin G. Effectiveness of moderate green tea consumption on antioxidative status and plasma lipid profile in humans. J Nutr Biochem 2005 Mar;16(3):144-149. [32] Yang YC, Lu FH, Wu JS, Wu CH, Chang CJ. The protective effect of habitual tea consumption on hypertension. Arch Intern Med 2004 Jul 26;164(14):1534-1540. [33] Stensvold I, Tverdal A, Solvoll K, Foss OP. Tea consumption. relationship to cholesterol, blood pressure, and coronary and total mortality. Prev Med 1992 Jul;21(4):546-553. [34] Brunner H, Cockcroft JR, Deanfield J, Donald A, Ferrannini E, Halcox J, et al. Endothelial function and dysfunction. Part II: Association with cardiovascular risk factors and diseases. A statement by the Working Group on Endothelins and Endothelial Factors of the European Society of Hypertension. J Hypertens 2005 Feb;23(2):233-246. [35] Targonski PV, Bonetti PO, Pumper GM, Higano ST, Holmes DR, Jr., Lerman A. Coronary endothelial dysfunction is associated with an increased risk of cerebrovascular events. Circulation 2003 Jun 10;107(22):2805-2809. [36] Rice-Evans CA, Miller NJ, Bolwell PG, Bramley PM, Pridham JB. The relative antioxidant activities of plant-derived polyphenolic flavonoids. Free Radic Res 1995 Apr;22(4):375-383.

Cardiovascular and Metabolic Effects of Green Tea

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[37] Fitzpatrick DF, Hirschfield SL, Ricci T, Jantzen P, Coffey RG. Endothelium-dependent vasorelaxation caused by various plant extracts. J Cardiovasc Pharmacol 1995 Jul;26(1):90-95. [38] Lorenz M, Wessler S, Follmann E, Michaelis W, Dusterhoft T, Baumann G, et al. A constituent of green tea, epigallocatechin-3-gallate, activates endothelial nitric oxide synthase by a phosphatidylinositol-3-OH-kinase-, cAMP-dependent protein kinase-, and Akt-dependent pathway and leads to endothelial-dependent vasorelaxation. J Biol Chem 2004 Feb 13;279(7):6190-6195. [39] Hakim IA, Harris RB, Brown S, Chow HH, Wiseman S, Agarwal S, et al. Effect of increased tea consumption on oxidative DNA damage among smokers: a randomized controlled study. J Nutr 2003 Oct;133(10):3303S-3309S. [40] Yokogoshi H, Kato Y, Sagesaka YM, Takihara-Matsuura T, Kakuda T, Takeuchi N. Reduction effect of theanine on blood pressure and brain 5-hydroxyindoles in spontaneously hypertensive rats. Biosci Biotechnol Biochem 1995 Apr;59(4):615-618. [41] Yokogoshi H, Kobayashi M. Hypotensive effect of gamma-glutamylmethylamide in spontaneously hypertensive rats. Life Sci 1998;62(12):1065-1068. [42] Gu K, Cowie CC, Harris MI. Mortality in adults with and without diabetes in a national cohort of the U.S. population, 1971-1993. Diabetes Care 1998 Jul;21(7):1138-1145. [43] Tsuneki H, Ishizuka M, Terasawa M, Wu JB, Sasaoka T, Kimura I. Effect of green tea on blood glucose levels and serum proteomic patterns in diabetic (db/db) mice and on glucose metabolism in healthy humans. BMC Pharmacol 2004 Aug 26;4:18. [44] Hosoda K, Wang MF, Liao ML, Chuang CK, Iha M, Clevidence B, et al. Antihyperglycemic effect of oolong tea in type 2 diabetes. Diabetes Care 2003 Jun;26(6):1714-1718. [45] Shimada K, Kawarabayashi T, Tanaka A, Fukuda D, Nakamura Y, Yoshiyama M, et al. Oolong tea increases plasma adiponectin levels and low-density lipoprotein particle size in patients with coronary artery disease. Diabetes Res Clin Pract 2004 Sep;65(3):227234. [46] Serisier S, Leray V, Poudroux W, Magot T, Ouguerram K, Nguyen P. Effects of green tea on insulin sensitivity, lipid profile and expression of PPARalpha and PPARgamma and their target genes in obese dogs. Br J Nutr 2008 Jun;99(6):1208-1216. [47] Babu PV, Sabitha KE, Shyamaladevi CS. Therapeutic effect of green tea extract on oxidative stress in aorta and heart of streptozotocin diabetic rats. Chem Biol Interact 2006 Aug 25;162(2):114-120. [48] Babu PV, Sabitha KE, Shyamaladevi CS. Green tea impedes dyslipidemia, lipid peroxidation, protein glycation and ameliorates Ca2+ -ATPase and Na+/K+ -ATPase activity in the heart of streptozotocin-diabetic rats. Chem Biol Interact 2006 Aug 25;162(2):157-164. [49] Yamabe N, Yokozawa T, Oya T, Kim M. Therapeutic potential of (-)-epigallocatechin 3-O-gallate on renal damage in diabetic nephropathy model rats. J Pharmacol Exp Ther 2006 Oct;319(1):228-236. [50] Sabu MC, Smitha K, Kuttan R. Anti-diabetic activity of green tea polyphenols and their role in reducing oxidative stress in experimental diabetes. J Ethnopharmacol 2002 Nov;83(1-2):109-116.

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Kamilla Kelemen

[51] Wu LY, Juan CC, Ho LT, Hsu YP, Hwang LS. Effect of green tea supplementation on insulin sensitivity in Sprague-Dawley rats. J Agric Food Chem 2004 Feb 11;52(3):643648. [52] Chantre P, Lairon D. Recent findings of green tea extract AR25 (Exolise) and its activity for the treatment of obesity. Phytomedicine 2002 Jan;9(1):3-8. [53] Kovacs EM, Lejeune MP, Nijs I, Westerterp-Plantenga MS. Effects of green tea on weight maintenance after body-weight loss. Br J Nutr 2004 Mar;91(3):431-437. [54] Choo JJ. Green tea reduces body fat accretion caused by high-fat diet in rats through beta-adrenoceptor activation of thermogenesis in brown adipose tissue. J Nutr Biochem 2003 Nov;14(11):671-676. [55] Hasegawa N, Yamda N, Mori M. Powdered green tea has antilipogenic effect on Zucker rats fed a high-fat diet. Phytother Res 2003 May;17(5):477-480. [56] Kristen AV, Dengler TJ, Hegenbart U, Schonland SO, Goldschmidt H, Sack FU, et al. Prophylactic implantation of cardioverter-defibrillator in patients with severe cardiac amyloidosis and high risk for sudden cardiac death. Heart Rhythm 2008 Feb;5(2):235240. [57] Hunstein W. Epigallocathechin-3-gallate in AL amyloidosis: a new therapeutic option? Blood 2007 Sep 15;110(6):2216. [58] Heiser V, Scherzinger E, Boeddrich A, Nordhoff E, Lurz R, Schugardt N, et al. Inhibition of huntingtin fibrillogenesis by specific antibodies and small molecules: implications for Huntington's disease therapy. Proc Natl Acad Sci U S A 2000 Jun 6;97(12):6739-6744. [59] Mukamal KJ, Alert M, Maclure M, Muller JE, Mittleman MA. Tea consumption and infarct-related ventricular arrhythmias: the determinants of myocardial infarction onset study. J Am Coll Nutr 2006 Dec;25(6):472-479. [60] Ale-Agha N, Stahl W, Sies H. (-)-Epicatechin effects in rat liver epithelial cells: stimulation of gap junctional communication and counteraction of its loss due to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Biochem Pharmacol 2002 Jun 15;63(12):2145-2149. [61] Modun D, Music I, Katalinic V, Salamunic I, Boban M. Comparison of protective effects of catechin applied in vitro and in vivo on ischemia-reperfusion injury in the isolated rat hearts. Croat Med J 2003 Dec;44(6):690-696. [62] Kiehn J, Karle C, Thomas D, Yao X, Brachmann J, Kubler W. HERG potassium channel activation is shifted by phorbol esters via protein kinase A-dependent pathways. J Biol Chem 1998 Sep 25;273(39):25285-25291. [63] Prineas RJ, Jacobs DR, Jr., Crow RS, Blackburn H. Coffee, tea and VPB. J Chronic Dis 1980;33(2):67-72.



Chapter 14

MOLECULAR BASIS FOR THE ANTI-CANCER ACTIVITY OF EGCG IN VIVO: MOLECULAR-TARGETING PREVENTION OF CANCER BY GREEN TEA CATECHIN Yoshinori Fujimura1 and Hirofumi Tachibana1,2,3,* 1

Innovation Center for Medical Redox Navigation, Kyushu University, 3-3-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan 2 Bio-Architecture Center, 3Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

ABSTRACT For the past two decades, many researchers have been investigated the potential cancer-preventive and therapeutic effects of green tea. (–)-Epigallocatechin-3-O-gallate (EGCG) has been shown to be the most active and major polyphenolic compound from green tea. The mechanisms of action of EGCG have been extensively investigated, but the mechanisms for the cancer-preventive activity of EGCG are not completely characterized and many features remain to be elucidated. Recently we have identified 67kDa laminin receptor (67LR) as a cell-surface EGCG receptor that confers EGCG responsiveness to many cancer cells at physiological concentrations. This article reviews some of the reported mechanisms and possible targets for the action of EGCG. Especially, we focus the current understanding of signaling pathway for physiologically relevant EGCG through the 67LR for cancer prevention. This information shed new light on the molecular basis for the cancer-preventive activity of EGCG in vivo and helps in the design of new strategies to prevent cancer.

*

Corresponding author: Hirofumi Tachibana, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. E-mail address: [email protected]; Tel/Fax: +81-92-642-3008

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1. INTRODUCTION Tea is one of the most widely consumed beverages in the world. Green tea, black tea, and oolong tea are all derived from the leaves of Camellia sinensis plant and contain an assortment of compounds, the most significant components of which are polyphenols. Among all teas consumed in the world, green tea is best studied for its health benefits. It has been demonstrated that tea constituents exhibit various biological and pharmacological properties such anti-carcinogenic, anti-oxidative, anti-allergic, anti-virus, anti-hypertensive, antiatherosclerosis, and anti-hypercholesterolemic activities [1-8]. Major principles for these activities were shown to be a group of polyphenols, catechin. A typical green tea beverage, prepared in a proportion of 1 g leaf to 100 ml water in a 3-min brew, usually contains 250350 mg tea solids, and catechins account for 30-42% of the dry weight of the solids [9]. Catechins contain a benzopyran skeleton with a phenyl group substituted at the 2-position and a hydroxyl (or ester) function at the 3-position. Variations to catechin structure include the stereochemistry of the 2,3-substituents and the number of hydroxyl groups in the B- and Dring. Belonging to the flavan-3-ol class of flavonoids, major catechins found in tea leaves are (–)-epigallocatechin-3-O-gallate (EGCG), (–)-epigallocatechin (EGC), (–)-epicatechin-3-Ogallate (ECG), (–)-epicatechin (EC) and their structures are shown in Figure 1. Among the green tea catechins, EGCG is the most abundant, representing ~16.5 wt% of the water extractable fraction of green tea leaves, and most active catechin in various kinds of physiological activities. Because EGCG is not found to a plant except tea, EGCG is regarded as a constituent characterizing green tea. Recently, double-blind, placebo-controlled study on oral administration of green tea catechins (EGCG, 50%) in volunteers with high-grade prostate intraepithelial neoplasia demonstrated that green tea catechins have potent in vivo chemoprevention activity for human prostate cancer [10,11]. This impressive evidence has fueled interest in the role of EGCG as chemoprevention of cancer. This chapter will discuss the effects of green tea polyphenol EGCG on signal transduction pathways that are related to cancer chemoprevention based on the biological importance of the target, and especially we focused the current understanding of EGCG signaling pathway through the 67-kDa laminin receptor (67LR) as the target molecule mediating anti-cancer effect of the physiologically relevant EGCG

2. ANTI-OXIDANT AND PRO-OXIDANT Tea polyphenols such as EGCG are well known for their anti-oxidant activities. They have been reported to inhibit carcinogen-induced DNA damage and tumor promoter-induced oxidative stress [12,13]. These results are consistent with the commonly mentioned idea that tea prevents cancer because tea polyphenols are anti-oxidants. It is unclear, however, whether this is a general mechanism for cancer prevention, especially in human carcinogenesis when strong carcinogenesis and tumor promoters are not known to be involved. When carcinogenactivation and tumor promotion were active areas of research, these events had been proposed as the targets of tea polyphenol action. With the advancement of research on signal transduction pathways targeted by tea polyphenols, many studies have been carried out in cell

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lines. However, whether some of the phenomena observed in cell lines occur in vivo still remains unclear.

Figure 1. Chemical structures of green tea catechins and their analogues.

There two major problems in extrapolating results observed in cell lines to animal models. (i) The concentration used in cell line systems, for example, EGCG at 10-100 μM, or

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higher concentrations, are much higher than those observed in the plasma or tissues in experimental animals or humans after ingestion of tea or related tea preparations [12]. (ii) The oxygen partial pressure in a cell culture system (160 mmHg) is much higher than that in the blood or tissues ( GCG > ECG > CG. The reduction rates of inhibitory activities of pyrogallol-type catechins (GCG and EGCG) were higher than those of catechol-type catechins (CG and ECG). This 67LR-dependency was similar to a result of the dependency on their cell-surface bindings. The influence of different configuration of the B-ring on the 67LR-dependency was much smaller than those of different hydroxylation of the B-ring. These results suggest that the 67LR is involved in the inhibitory effect of four galloylated catechins on histamine release, and that the pattern of B-ring hydroxylation is a major structural determinant for the 67LRdependency of galloylated catechins. Taken together, our findings suggest that the combination of galloyl group with basic flavan-3-ol structure may be necessary for the binding of tea polyphenols to the cell-surface 67LR

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7. GREEN TEA CATECHIN RECEPTOR SIGNALING MOLECULES 7.1. Genetic Suppressor Element (GSE) Methodology In an attempt to elucidate the pathways involved in the anticancer action of EGCG, we applied genetic suppressor element (GSE) methodology. GSEs are short cDNA fragments encoding peptides acting as dominant inhibitors of protein function or antisense RNAs inhibiting gene expression [186]. GSEs behave as dominant selectable markers for the phenotype associated with the repression of the gene from which they derived, thus allowing identification of this gene. For example, this strategy previously allowed the demonstration that kinesin heavy chain is involved in the control of cell response to various DNA-damaging agents [187]. For identifying genes mediating cell sensitivity to EGCG, we selected GSEs conferring resistance to EGCG. To search for the mediators of EGCG-induced cell growth inhibition in B16 mouse melanoma cells, we utilized a targeted genetic screen with a GSE complementary DNA library, which was prepared from a mouse embryo. Among genetic elements protecting cells from EGCG-induced cell growth inhibition, we isolated a GSE that encoded the N terminus of eukaryotic translation elongation factor 1A (eEF1A) [17]. eEF1A is an important component of the eukaryotic translation apparatus and is also known as a multifunctional protein that is involved in a large number of cellular processes [188].

7.2. Eukaryotic Translation Elongation Factor 1A (eEF1A) To investigate the role of eEF1A in EGCG-induced cell growth inhibition, we used stable RNAi [189] to silence eEF1A expression in B16 cells. Remarkably, silencing of eEF1A attenuated the inhibitory effect of 1 μM EGCG on cell growth [17]. In contrast, overexpression of eEF1A enhanced the inhibitory effects of 1 μM EGCG on cell growth. This concentration is similar to the amount of EGCG found in human plasma after drinking more than two or three cups of green tea [150]. EGCG is the only known polyphenol present in plasma in large proportion (77-90%) in a free form, although the other catechins are highly conjugated with glucuronic acid and/or sulfate group [190]. Based on these considerations, the activities observed at 1 μM EGCG are relevant to the in vivo situations. Given this, we investigated the effect of oral administration of EGCG on subcutaneous tumor growth in C57BL/6N mice challenged with eEF1A-ablated B16 cells as shown in Figure 4B [17]. Tumor growth was significantly retarded in EGCG-administered mice implanted with the B16 cells harboring a control shRNA, whereas tumor growth was not affected by EGCG in the mice implanted with eEF1A-ablated B16 cells, indicating that eEF1A is involved in EGCG-induced cancer prevention. EGCG induces growth inhibition in many cell lines; however, the efficacy of inhibition varied, depending on the cell lines used [191]. We hypothesized that the expression level of eEF1A in a cell line correlates to the efficacy of EGCG-induced cell growth inhibition in that cell line. We investigated the expression levels of eEF1A in B16 cells and the following human cancer cell lines: hepatoma HepG2, breast carcinoma MCF-7, cervical carcinoma HeLa, and squamous cell carcinoma A431 [17]. The levels of eEF1A expression in B16 cells, HepG2 cells, and MCF-7 cells were relatively higher than those in HeLa cells and A431 cells.


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EGCG appeared to display different efficacies of growth inhibition in these cell lines, with estimated IC50 values of 9.7 μM for MCF-7 cells, 22.7 μM for HepG2 cells, 51.6 μM for HeLa cells, and 52.8 μM for A431 cells, respectively. The expression level of eEF1A is elevated in cell lines that are more sensitive to the effect of EGCG. These results support our conclusion that eEF1A serves as a mediator for EGCG-induced cancer prevention.

7.3. Myosin Phosphatase-targeting Subunit (MYPT1) and Remodeling of Actin Cytoskeleton MRLC phosphorylation controls the activity of myosin II, a major motor protein in animal cells, which is involved in a wide range of processes, including muscle contraction, cell locomotion, cell division, and receptor capping [192]. The phosphorylation of MRLC is regulated by two classes of enzymes: MLC kinases and myosin phosphatase [193]. Myosin light chain kinase and Rho-kinase seem to be the two major kinases that phosphorylate MRLC in vitro as well as in vivo [193]. Myosin phosphatase is composed with three subunits: a 37-kDa catalytic subunit, a 20-kDa subunit of unknown function, and a 110-130-kDa myosin phosphatase-targeting subunit (MYPT1) [194]. The activity of myosin phosphatase is known to be regulated by phosphorylation of MYPT1, and two major sites, Thr-696 and Thr853, have been extensively investigated and identified as an inhibitory site [194]. Previously, we reported that EGCG-induced cell growth inhibition may result from the reduction of the phosphorylation of myosin regulatory light chain (MRLC) at Thr-18/Ser-19 through 67LR in HeLa cells [19]. The activity of myosin phosphatase is known to be inhibited by phosphorylation of its targeting subunit MYPT1 at Thr-696 and Thr-853 [194]. We tested the effect of EGCG on the phosphorylation of MYPT1 at Thr-696 and Thr-853 [17]. Intriguingly, although the phosphorylation level at Thr-853 was unaffected by EGCG, EGCG induced the dephosphorylation of MYPT1 at Thr-696. Further, this effect correlated with EGCG-induced reduction of the MRLC phosphorylation, suggesting that EGCG activates myosin phosphatase by reducing the MYPT1 phosphorylation level at Thr-696. Next, we investigated whether MYPT1 is involved in anticancer action of EGCG in vivo. In B16 cells, physiological concentrations of EGCG reduced the MYPT1 phosphorylation at Thr-696 and the MRLC phosphorylation. We confirmed both silencing of MYPT1 by stable RNAi in B16 cells and attenuation of the inhibitory effect of 1 μM EGCG on cell growth in MYPT1-ablated B16 cells in vitro. We tested the effect of oral administration of EGCG on subcutaneous tumor growth in C57BL/6N mice challenged with MYPT1-ablated B16 cells (Figure 4C). Tumor growth was significantly retarded in EGCG-administered mice implanted with the B16 cells harboring a control shRNA, whereas tumor growth was not affected by EGCG in the mice implanted with MYPT-1-ablated B16 cells, suggesting that MYPT1 is indispensable for EGCG-induced cancer prevention. In addition to this role of MYPT1 in EGCG-induced inhibition of cancer cell growth, a study on RNAi-mediated knockdown of MYPT1 expression demonstrated the importance of MYPT1 as the signaling component mediating the inhibitory effect of EGCG on histamine release and MRLC phosphorylation on the basis of the defect of both activities (unpublished data).

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Figure 7. Model of possible EGCG signaling pathway through 67LR in vivo.

It has been reported that MYPT1 directs the action of myosin phosphatase to not only MRLC but also other F-actin-binding proteins that influence cell contractility, morphology, and proliferation [195-198]. We found that EGCG induces a dynamic remodeling of actin cytoskeleton. The cells treated with EGCG exhibited a filopodial-like structure, and then after 3 h, the cell body retracted and left intracellular gaps, suggesting that the EGCG-induced filopodial-like projections are simple residual contact sites that have not yet been released from the substrate [17]. Together, it is suggested that EGCG-induced actin cytoskeleton remodeling results from not only the reduction of the MRLC phosphorylation but also the activation of myosin phosphatase.

7.4. A Hierarchy of EGCG Signaling Molecules Further, to establish whether MYPT1 is indeed involved in the suppressive effect of EGCG on MRLC phosphorylation and cell growth, we used stable RNAi to silence MYPT1 expression in HeLa cells [17]. Western blot analysis indicated that stable RNAi for MYPT1 specifically silenced MYPT1 protein expression in HeLa cells with no effect on the expression of 67LR and eEF1A. Silencing of MYPT1 prevented both EGCG-induced reduction of the MRLC phosphorylation and cell growth inhibition, suggesting that EGCGinduced dephosphorylation of MYPT1 at Thr-696 results in the activation of myosin phosphatase and inhibition of cell growth.


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It has been reported that eEF1A binds to the ankyrin repeat of MYPT1 [198]. It is tempting to speculate that both 67LR and eEF1A are upstream signaling components responsible for EGCG-induced dephosphorylation of MYPT1 at Thr-696. To test this hypothesis, we used stable RNAi to silence the expression of 67LR or eEF1A in HeLa cells. We confirmed specific silencing of each target protein by stable RNAi in HeLa cells and attenuation of the inhibitory effect of EGCG on cell growth in these cells [17]. In both 67LRablated HeLa cells and eEF1A-ablated HeLa cells, the inhibitory effect of EGCG on both the phosphorylation of MYPT1 at Thr-696 and the phosphorylation of MRLC was attenuated. In addition, EGCG-induced actin cytoskeleton rearrangement was no longer observed in MYPT1-, eEF1A-, or 67LR-ablated HeLa cells. The involvement of MYPT1 in downstream EGCG-triggered signaling from both 67LR and eEF1A was further documented by confirming abrogation of 1 μM EGCG-induced reduction of the MYPT1 phosphorylation level at Thr-696 and the MRLC phosphorylation in 67LR- or eEF1A-ablated B16 cells. These results suggest that MYPT1 is involved in downstream EGCG signaling from both 67LR and eEF1A (Figure 7). It has been reported that MYPT1 binds to eEF1A [198], and more than half of the total eEF1A (>60%) binds to the actin cytoskeleton [199]. Because other findings indicate that eEF1A is also implicated in microtubule binding, bundling, or severing [200,201], a potential role for the protein in regulating cytoskeleton organization has also been proposed. Characterizing the mechanisms by which EGCG induces reduction of the MYPT1 phosphorylation at Thr-696 and reorganization of actin cytoskeleton through eEF1A should help in more precise understanding of cytoskeleton organization, although further experiments are necessary.

8. CONCLUSION Recent studies have highlighted the importance of genetically determined factors in evaluating the role of green tea intake in the development of breast cancer [202-204]. In a case-control study conducted among Asian-American women, green tea intake appeared to reduce breast cancer risk [203]. Reduction in risk was strongest among persons who had the low-activity catechol-O-methyltransferase (COMT) alleles, but not high-activity COMT alleles, suggesting that these individuals were less efficient in eliminating green tea catechins and may derive the most benefit from these compounds. Yuan et al. reported a low risk of breast cancer among women with higher green tea intake and the low-activity genotype of angiotensin-converting enzyme gene among Singapore Chinese women [202]. A nested casecontrol study suggested protective effect of green tea against breast cancer among women with high-activity genotypes of the methylenetetrahydrofolate reductase and thymidylate synthase genes [204]. This effect was even stronger among those who were low consumers of dietary folate. These observations indicate that further study to elucidate the key molecules determining EGCG responsiveness is indispensable for a better understanding of EGCG activity in vivo. Chemoprevention by edible phytochemicals is now considered to be an inexpensive, readily applicable, acceptable, and accessible approach to cancer control and management [205]; however, little is known about the mechanism of the chemopreventive action of most phytochemicals, including EGCG. Although previous studies have proposed various different

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mechanisms for cancer-preventive action of EGCG [205-207], it remains unclear which EGCG-induced molecular events are relevant in vivo. The activities affected by lower concentrations of EGCG are likely to be more relevant in vivo because of the limited bioavailability of EGCG. The essence is the identification of the primary target and the demonstration of specific mechanisms of action in animal models and human tissues. Here we described that each of 67LR, eEF1A, and MYPT1 is indispensable for EGCG-induced cancer prevention in vivo, and these proteins mediate physiological concentrations of EGCGtriggered unique signaling for cancer prevention. Our findings suggest that these proteins are "master proteins," which determine the efficacy of cancer-preventive activity of EGCG and have important implications for development and use of EGCG as a cancer-chemopreventive agent. Probably, only a tumor with a high expression level of these "master proteins" has sensitivity to physiological concentrations of EGCG, while lower expression of those molecules causes “EGCG-resistance”. Our results not only illuminate the mechanisms for the cancer-preventive activity of EGCG but should help in the design of new strategies to prevent cancer and underscore the importance of tailoring cancer therapy on the basis of tumor genotype. The integrity of lipid rafts has been shown to be important for the pathogenesis of cancer [115,208]. Epigenetic regulation of genes encoding raft components and its roles in cell transformation, angiogenesis, immune escape, and metastasis has been reviewed earlier [115,138,140,208-212]. The role of rafts in prostate cancer is intriguing and discussed recently [213,214]. These contributions would help to best understanding current concerns in raft-associated signaling in regulation of cancer, and will help to select strategies for better management of neoplasm. Therefore, both orienting research toward newly proposed interactions (EGCG-67LR-eEF1A-MYPT1-Cytoskeleton axis), as shown in Figure 7, and investigation focused on lipid rafts as a signaling platform regulating the 67LR-mediated action of EGCG may unravel some of the complex aspect of EGCG-induced anti-cancer signaling. Finally, in addition to cancer-chemopreventive and anti-allergic properties, EGCG has been shown to possess diverse physiological activities, and we are curious to know whether EGCG signaling through the 67LR relates to other beneficial effects of EGCG.

REFERENCES [1] [2]

[3] [4]

Lambert, J.D. and Yang, C.S. (2003). Cancer chemopreventive activity and bioavailability of tea and tea polyphenols. Mutat Res 523-524, 201-8. Chisaka, T., Matsuda, H., Kubomura, Y., Mochizuki, M., Yamahara, J. and Fujimura, H. (1988). The effect of crude drugs on experimental hypercholesteremia: mode of action of (-)-epigallocatechin gallate in tea leaves. Chem Pharm Bull (Tokyo) 36, 22733. Cao, Y. and Cao, R. (1999). Angiogenesis inhibited by drinking tea. Nature 398, 381. Maeda-Yamamoto, M., Suzuki, N., Sawai, Y., Miyase, T., Sano, M., Hashimoto-Ohta, A. and Isemura, M. (2003). Association of suppression of extracellular signal-regulated kinase phosphorylation by epigallocatechin gallate with the reduction of matrix


[5]

[6] [7]

[8] [9] [10]

[11]

[12] [13] [14]

[15]

[16]

[17]

[18]

[19]

287

metalloproteinase activities in human fibrosarcoma HT1080 cells. J Agric Food Chem 51, 1858-63. Sazuka, M., Murakami, S., Isemura, M., Satoh, K. and Nukiwa, T. (1995). Inhibitory effects of green tea infusion on in vitro invasion and in vivo metastasis of mouse lung carcinoma cells. Cancer Lett 98, 27-31. Bors, W. and Saran, M. (1987). Radical scavenging by flavonoid antioxidants. Free Radic Res Commun 2, 289-94. Sano, M., Takahashi, Y., Yoshino, K., Shimoi, K., Nakamura, Y., Tomita, I., Oguni, I. and Konomoto, H. (1995). Effect of tea (Camellia sinensis L.) on lipid peroxidation in rat liver and kidney: a comparison of green and black tea feeding. Biol Pharm Bull 18, 1006-8. Hodgson, J.M., Puddey, I.B., Burke, V., Beilin, L.J. and Jordan, N. (1999). Effects on blood pressure of drinking green and black tea. J Hypertens 17, 457-63. Khan, N. and Mukhtar, H. (2007). Tea polyphenols for health promotion. Life Sci 81, 519-33. Bettuzzi, S., Brausi, M., Rizzi, F., Castagnetti, G., Peracchia, G. and Corti, A. (2006). Chemoprevention of human prostate cancer by oral administration of green tea catechins in volunteers with high-grade prostate intraepithelial neoplasia: a preliminary report from a one-year proof-of-principle study. Cancer Res 66, 1234-40. Brausi, M., Rizzi, F. and Bettuzzi, S. (2008). Chemoprevention of Human Prostate Cancer by Green Tea Catechins: Two Years Later. A Follow-up Update. Eur Urol 54, 472-3. Yang, C.S., Maliakal, P. and Meng, X. (2002). Inhibition of carcinogenesis by tea. Annu Rev Pharmacol Toxicol 42, 25-54. Higdon, J.V. and Frei, B. (2003). Tea catechins and polyphenols: health effects, metabolism, and antioxidant functions. Crit Rev Food Sci Nutr 43, 89-143. Hong, J., Lu, H., Meng, X., Ryu, J.H., Hara, Y. and Yang, C.S. (2002). Stability, cellular uptake, biotransformation, and efflux of tea polyphenol (-)-epigallocatechin-3gallate in HT-29 human colon adenocarcinoma cells. Cancer Res 62, 7241-6. Sang, S., Lee, M.J., Hou, Z., Ho, C.T. and Yang, C.S. (2005). Stability of tea polyphenol (-)-epigallocatechin-3-gallate and formation of dimers and epimers under common experimental conditions. J Agric Food Chem 53, 9478-84. Hou, Z., Sang, S., You, H., Lee, M.J., Hong, J., Chin, K.V. and Yang, C.S. (2005). Mechanism of action of (-)-epigallocatechin-3-gallate: auto-oxidation-dependent inactivation of epidermal growth factor receptor and direct effects on growth inhibition in human esophageal cancer KYSE 150 cells. Cancer Res 65, 8049-56. Umeda, D., Yano, S., Yamada, K. and Tachibana, H. (2008). Green tea polyphenol epigallocatechin-3-gallate signaling pathway through 67-kDa laminin receptor. J Biol Chem 283, 3050-8. Umeda, D., Yano, S., Yamada, K. and Tachibana, H. (2008). Involvement of 67-kDa laminin receptor-mediated myosin phosphatase activation in antiproliferative effect of epigallocatechin-3-O-gallate at a physiological concentration on Caco-2 colon cancer cells. Biochem Biophys Res Commun 371, 172-6. Umeda, D., Tachibana, H. and Yamada, K. (2005). Epigallocatechin-3-O-gallate disrupts stress fibers and the contractile ring by reducing myosin regulatory light chain

288

[20]

[21]

[22]

[23]

[24] [25] [26]

[27] [28]

[29]

[30]

[31]

[32]

[33] [34]

Yoshinori Fujimura and Hirofumi Tachibana phosphorylation mediated through the target molecule 67 kDa laminin receptor. Biochem Biophys Res Commun 333, 628-35. Fujimura, Y., Umeda, D., Yano, S., Maeda-Yamamoto, M., Yamada, K. and Tachibana, H. (2007). The 67kDa laminin receptor as a primary determinant of antiallergic effects of O-methylated EGCG. Biochem Biophys Res Commun 364, 79-85. Fujimura, Y., Umeda, D., Yamada, K. and Tachibana, H. (2008). The impact of the 67kDa laminin receptor on both cell-surface binding and anti-allergic action of tea catechins. Arch Biochem Biophys 476, 133-8. Sang, S., Yang, I., Buckley, B., Ho, C.T. and Yang, C.S. (2007). Autoxidative quinone formation in vitro and metabolite formation in vivo from tea polyphenol (-)epigallocatechin-3-gallate: studied by real-time mass spectrometry combined with tandem mass ion mapping. Free Radic Biol Med 43, 362-71. Yang, C.S., Chen, L., Lee, M.J., Balentine, D., Kuo, M.C. and Schantz, S.P. (1998). Blood and urine levels of tea catechins after ingestion of different amounts of green tea by human volunteers. Cancer Epidemiol Biomarkers Prev 7, 351-4. Chen, L., Lee, M.J., Li, H. and Yang, C.S. (1997). Absorption, distribution, elimination of tea polyphenols in rats. Drug Metab Dispos 25, 1045-50. Feng, W.Y. (2006). Metabolism of green tea catechins: an overview. Curr Drug Metab 7, 755-809. Lee, M.J. et al. (2002). Pharmacokinetics of tea catechins after ingestion of green tea and (-)-epigallocatechin-3-gallate by humans: formation of different metabolites and individual variability. Cancer Epidemiol Biomarkers Prev 11, 1025-32. Lambert, J.D. and Yang, C.S. (2003). Mechanisms of cancer prevention by tea constituents. J Nutr 133, 3262S-3267S. Kim, S., Lee, M.J., Hong, J., Li, C., Smith, T.J., Yang, G.Y., Seril, D.N. and Yang, C.S. (2000). Plasma and tissue levels of tea catechins in rats and mice during chronic consumption of green tea polyphenols. Nutr Cancer 37, 41-8. Lee, M.J., Wang, Z.Y., Li, H., Chen, L., Sun, Y., Gobbo, S., Balentine, D.A. and Yang, C.S. (1995). Analysis of plasma and urinary tea polyphenols in human subjects. Cancer Epidemiol Biomarkers Prev 4, 393-9. Chow, H.H. et al. (2005). Effects of dosing condition on the oral bioavailability of green tea catechins after single-dose administration of Polyphenon E in healthy individuals. Clin Cancer Res 11, 4627-33. Dong, Z., Ma, W., Huang, C. and Yang, C.S. (1997). Inhibition of tumor promoterinduced activator protein 1 activation and cell transformation by tea polyphenols, (-)epigallocatechin gallate, and theaflavins. Cancer Res 57, 4414-9. Chung, J.Y., Huang, C., Meng, X., Dong, Z. and Yang, C.S. (1999). Inhibition of activator protein 1 activity and cell growth by purified green tea and black tea polyphenols in H-ras-transformed cells: structure-activity relationship and mechanisms involved. Cancer Res 59, 4610-7. Nam, S., Smith, D.M. and Dou, Q.P. (2001). Ester bond-containing tea polyphenols potently inhibit proteasome activity in vitro and in vivo. J Biol Chem 276, 13322-30. Garbisa, S., Sartor, L., Biggin, S., Salvato, B., Benelli, R. and Albini, A. (2001). Tumor gelatinases and invasion inhibited by the green tea flavanol epigallocatechin-3-gallate. Cancer 91, 822-32.


289

[35] Annabi, B., Lachambre, M.P., Bousquet-Gagnon, N., Page, M., Gingras, D. and Beliveau, R. (2002). Green tea polyphenol (-)-epigallocatechin 3-gallate inhibits MMP2 secretion and MT1-MMP-driven migration in glioblastoma cells. Biochim Biophys Acta 1542, 209-20. [36] Leone, M., Zhai, D., Sareth, S., Kitada, S., Reed, J.C. and Pellecchia, M. (2003). Cancer prevention by tea polyphenols is linked to their direct inhibition of antiapoptotic Bcl-2-family proteins. Cancer Res 63, 8118-21. [37] Fang, M.Z., Wang, Y., Ai, N., Hou, Z., Sun, Y., Lu, H., Welsh, W. and Yang, C.S. (2003). Tea polyphenol (-)-epigallocatechin-3-gallate inhibits DNA methyltransferase and reactivates methylation-silenced genes in cancer cell lines. Cancer Res 63, 756370. [38] Tachibana, H., Koga, K., Fujimura, Y. and Yamada, K. (2004). A receptor for green tea polyphenol EGCG. Nat Struct Mol Biol 11, 380-1. [39] Navarro-Peran, E., Cabezas-Herrera, J., Garcia-Canovas, F., Durrant, M.C., Thorneley, R.N. and Rodriguez-Lopez, J.N. (2005). The antifolate activity of tea catechins. Cancer Res 65, 2059-64. [40] Ermakova, S., Choi, B.Y., Choi, H.S., Kang, B.S., Bode, A.M. and Dong, Z. (2005). The intermediate filament protein vimentin is a new target for epigallocatechin gallate. J Biol Chem 280, 16882-90. [41] Dashwood, W.M., Carter, O., Al-Fageeh, M., Li, Q. and Dashwood, R.H. (2005). Lysosomal trafficking of beta-catenin induced by the tea polyphenol epigallocatechin3-gallate. Mutat Res 591, 161-72. [42] Long, L.H., Clement, M.V. and Halliwell, B. (2000). Artifacts in cell culture: rapid generation of hydrogen peroxide on addition of (-)-epigallocatechin, (-)epigallocatechin gallate, (+)-catechin, and quercetin to commonly used cell culture media. Biochem Biophys Res Commun 273, 50-3. [43] Liang, Y.C., Lin-Shiau, S.Y., Chen, C.F. and Lin, J.K. (1999). Inhibition of cyclindependent kinases 2 and 4 activities as well as induction of Cdk inhibitors p21 and p27 during growth arrest of human breast carcinoma cells by (-)-epigallocatechin-3-gallate. J Cell Biochem 75, 1-12. [44] Suganuma, M., Okabe, S., Kai, Y., Sueoka, N., Sueoka, E. and Fujiki, H. (1999). Synergistic effects of (--)-epigallocatechin gallate with (--)-epicatechin, sulindac, or tamoxifen on cancer-preventive activity in the human lung cancer cell line PC-9. Cancer Res 59, 44-7. [45] Wiseman, S.A., Balentine, D.A. and Frei, B. (1997). Antioxidants in tea. Crit Rev Food Sci Nutr 37, 705-18. [46] Dashwood, W.M., Orner, G.A. and Dashwood, R.H. (2002). Inhibition of betacatenin/Tcf activity by white tea, green tea, and epigallocatechin-3-gallate (EGCG): minor contribution of H(2)O(2) at physiologically relevant EGCG concentrations. Biochem Biophys Res Commun 296, 584-8. [47] Kuruto-Niwa, R., Inoue, S., Ogawa, S., Muramatsu, M. and Nozawa, R. (2000). Effects of tea catechins on the ERE-regulated estrogenic activity. J Agric Food Chem 48, 6355-61. [48] Fukuda, I., Mukai, R., Kawase, M., Yoshida, K. and Ashida, H. (2007). Interaction between the aryl hydrocarbon receptor and its antagonists, flavonoids. Biochem Biophys Res Commun 359, 822-7.

290


[49] He, Z. et al. (2008). Fyn is a novel target of (-)-epigallocatechin gallate in the inhibition of JB6 Cl41 cell transformation. Mol Carcinog 47, 172-83. [50] Kuzuhara, T., Sei, Y., Yamaguchi, K., Suganuma, M. and Fujiki, H. (2006). DNA and RNA as new binding targets of green tea catechins. J Biol Chem 281, 17446-56. [51] Kawai, K. et al. (2003). Epigallocatechin gallate, the main component of tea polyphenol, binds to CD4 and interferes with gp120 binding. J Allergy Clin Immunol 112, 951-7. [52] Liang, Y.C., Lin-shiau, S.Y., Chen, C.F. and Lin, J.K. (1997). Suppression of extracellular signals and cell proliferation through EGF receptor binding by (-)epigallocatechin gallate in human A431 epidermoid carcinoma cells. J Cell Biochem 67, 55-65. [53] Jankun, J., Selman, S.H., Swiercz, R. and Skrzypczak-Jankun, E. (1997). Why drinking green tea could prevent cancer. Nature 387, 561. [54] Shim, J.H., Choi, H.S., Pugliese, A., Lee, S.Y., Chae, J.I., Choi, B.Y., Bode, A.M. and Dong, Z. (2008). (-)-Epigallocatechin gallate regulates CD3-mediated T-cell receptor signaling in leukemia through the inhibition of ZAP-70 kinase. J Biol Chem [55] Khan, N. and Mukhtar, H. (2008). Multitargeted therapy of cancer by green tea polyphenols. Cancer Lett [56] Yang, C.S., Lambert, J.D., Hou, Z., Ju, J., Lu, G. and Hao, X. (2006). Molecular targets for the cancer preventive activity of tea polyphenols. Mol Carcinog 45, 431-5. [57] Garbisa, S., Biggin, S., Cavallarin, N., Sartor, L., Benelli, R. and Albini, A. (1999). Tumor invasion: molecular shears blunted by green tea. Nat Med 5, 1216. [58] Dell'Aica, I., Dona, M., Tonello, F., Piris, A., Mock, M., Montecucco, C. and Garbisa, S. (2004). Potent inhibitors of anthrax lethal factor from green tea. EMBO Rep 5, 41822. [59] Smith, D.M., Daniel, K.G., Wang, Z., Guida, W.C., Chan, T.H. and Dou, Q.P. (2004). Docking studies and model development of tea polyphenol proteasome inhibitors: applications to rational drug design. Proteins 54, 58-70. [60] Montagut, C., Rovira, A. and Albanell, J. (2006). The proteasome: a novel target for anticancer therapy. Clin Transl Oncol 8, 313-7. [61] Katiyar, S.K., Afaq, F., Azizuddin, K. and Mukhtar, H. (2001). Inhibition of UVBinduced oxidative stress-mediated phosphorylation of mitogen-activated protein kinase signaling pathways in cultured human epidermal keratinocytes by green tea polyphenol (-)-epigallocatechin-3-gallate. Toxicol Appl Pharmacol 176, 110-7. [62] Chung, J.Y., Park, J.O., Phyu, H., Dong, Z. and Yang, C.S. (2001). Mechanisms of inhibition of the Ras-MAP kinase signaling pathway in 30.7b Ras 12 cells by tea polyphenols (-)-epigallocatechin-3-gallate and theaflavin-3,3'-digallate. Faseb J 15, 2022-4. [63] Salomon, D.S., Brandt, R., Ciardiello, F. and Normanno, N. (1995). Epidermal growth factor-related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 19, 183-232. [64] Mendelsohn, J. and Baselga, J. (2000). The EGF receptor family as targets for cancer therapy. Oncogene 19, 6550-65. [65] Noble, M.E., Endicott, J.A. and Johnson, L.N. (2004). Protein kinase inhibitors: insights into drug design from structure. Science 303, 1800-5.


291

[66] Sah, J.F., Balasubramanian, S., Eckert, R.L. and Rorke, E.A. (2004). Epigallocatechin3-gallate inhibits epidermal growth factor receptor signaling pathway. Evidence for direct inhibition of ERK1/2 and AKT kinases. J Biol Chem 279, 12755-62. [67] Esteller, M. (2002). CpG island hypermethylation and tumor suppressor genes: a booming present, a brighter future. Oncogene 21, 5427-40. [68] Jones, P.A. and Baylin, S.B. (2002). The fundamental role of epigenetic events in cancer. Nat Rev Genet 3, 415-28. [69] Jones, P.A. and Takai, D. (2001). The role of DNA methylation in mammalian epigenetics. Science 293, 1068-70. [70] Lichtenstein, A.V. and Kisseljova, N.P. (2001). DNA methylation and carcinogenesis. Biochemistry (Mosc) 66, 235-55. [71] Nguyen, C.T., Gonzales, F.A. and Jones, P.A. (2001). Altered chromatin structure associated with methylation-induced gene silencing in cancer cells: correlation of accessibility, methylation, MeCP2 binding and acetylation. Nucleic Acids Res 29, 4598-606. [72] Rice, J.C., Massey-Brown, K.S. and Futscher, B.W. (1998). Aberrant methylation of the BRCA1 CpG island promoter is associated with decreased BRCA1 mRNA in sporadic breast cancer cells. Oncogene 17, 1807-12. [73] Robertson, K.D., Ait-Si-Ali, S., Yokochi, T., Wade, P.A., Jones, P.L. and Wolffe, A.P. (2000). DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters. Nat Genet 25, 338-42. [74] Rountree, M.R., Bachman, K.E. and Baylin, S.B. (2000). DNMT1 binds HDAC2 and a new co-repressor, DMAP1, to form a complex at replication foci. Nat Genet 25, 26977. [75] Clark, S.J. and Melki, J. (2002). DNA methylation and gene silencing in cancer: which is the guilty party? Oncogene 21, 5380-7. [76] Christman, J.K. (2002). 5-Azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene 21, 5483-95. [77] Zhou, L., Cheng, X., Connolly, B.A., Dickman, M.J., Hurd, P.J. and Hornby, D.P. (2002). Zebularine: a novel DNA methylation inhibitor that forms a covalent complex with DNA methyltransferases. J Mol Biol 321, 591-9. [78] Cheng, J.C., Matsen, C.B., Gonzales, F.A., Ye, W., Greer, S., Marquez, V.E., Jones, P.A. and Selker, E.U. (2003). Inhibition of DNA methylation and reactivation of silenced genes by zebularine. J Natl Cancer Inst 95, 399-409. [79] Bender, C.M., Pao, M.M. and Jones, P.A. (1998). Inhibition of DNA methylation by 5aza-2'-deoxycytidine suppresses the growth of human tumor cell lines. Cancer Res 58, 95-101. [80] Reed, J.C. (1995). Regulation of apoptosis by bcl-2 family proteins and its role in cancer and chemoresistance. Curr Opin Oncol 7, 541-6. [81] Wang, J.L. et al. (2000). Structure-based discovery of an organic compound that binds Bcl-2 protein and induces apoptosis of tumor cells. Proc Natl Acad Sci U S A 97, 7124-9. [82] Kitada, S., Leone, M., Sareth, S., Zhai, D., Reed, J.C. and Pellecchia, M. (2003). Discovery, characterization, and structure-activity relationships studies of proapoptotic

292

[83] [84] [85] [86] [87] [88]

[89] [90]

[91]

[92]

[93]

[94] [95] [96]

[97]

[98] [99] [100]

Yoshinori Fujimura and Hirofumi Tachibana polyphenols targeting B-cell lymphocyte/leukemia-2 proteins. J Med Chem 46, 425964. Matsuzawa, K. et al. (1997). Domain-specific phosphorylation of vimentin and glial fibrillary acidic protein by PKN. Biochem Biophys Res Commun 234, 621-5. Okayama, N. et al. (2000). Characterization of JOK-1, a human gastric epithelial cell line. In Vitro Cell Dev Biol Anim 36, 228-34. Chou, Y.H. and Goldman, R.D. (2000). Intermediate filaments on the move. J Cell Biol 150, F101-6. Helfand, B.T., Chang, L. and Goldman, R.D. (2003). The dynamic and motile properties of intermediate filaments. Annu Rev Cell Dev Biol 19, 445-67. Yoon, M., Moir, R.D., Prahlad, V. and Goldman, R.D. (1998). Motile properties of vimentin intermediate filament networks in living cells. J Cell Biol 143, 147-57. Ando, S., Tanabe, K., Gonda, Y., Sato, C. and Inagaki, M. (1989). Domain- and sequence-specific phosphorylation of vimentin induces disassembly of the filament structure. Biochemistry 28, 2974-9. Dass, K., Ahmad, A., Azmi, A.S., Sarkar, S.H. and Sarkar, F.H. (2008). Evolving role of uPA/uPAR system in human cancers. Cancer Treat Rev 34, 122-36. Harvey, S.R., Lawrence, D.D., Madeja, J.M., Abbey, S.J. and Markus, G. (1988). Secretion of plasminogen activators by human colorectal and gastric tumor explants. Clin Exp Metastasis 6, 431-50. Jankun, J., Keck, R.W., Skrzypczak-Jankun, E. and Swiercz, R. (1997). Inhibitors of urokinase reduce size of prostate cancer xenografts in severe combined immunodeficient mice. Cancer Res 57, 559-63. Narasimhan, S., Armstrong, M.Y., Rhee, K., Edman, J.C., Richards, F.F. and Spicer, E. (1994). Gene for an extracellular matrix receptor protein from Pneumocystis carinii. Proc Natl Acad Sci U S A 91, 7440-4. Iwabuchi, K., Nagaoka, I., Someya, A. and Yamashita, T. (1996). Type IV collagenbinding proteins of neutrophils: possible involvement of L-selectin in the neutrophil binding to type IV collagen. Blood 87, 365-72. Menard, S., Tagliabue, E. and Colnaghi, M.I. (1998). The 67 kDa laminin receptor as a prognostic factor in human cancer. Breast Cancer Res Treat 52, 137-45. Sanjuan, X. et al. (1996). Overexpression of the 67-kD laminin receptor correlates with tumour progression in human colorectal carcinoma. J Pathol 179, 376-80. Mafune, K. and Ravikumar, T.S. (1992). Anti-sense RNA of 32-kDa laminin-binding protein inhibits attachment and invasion of a human colon carcinoma cell line. J Surg Res 52, 340-6. Vande Broek, I., Vanderkerken, K., De Greef, C., Asosingh, K., Straetmans, N., Van Camp, B. and Van Riet, I. (2001). Laminin-1-induced migration of multiple myeloma cells involves the high-affinity 67 kD laminin receptor. Br J Cancer 85, 1387-95. Tanaka, M. et al. (2000). Expression of the 37-kDa laminin binding protein in murine lung tumor cell correlates with tumor angiogenesis. Cancer Lett 153, 161-8. Huard, T.K., Malinoff, H.L. and Wicha, M.S. (1986). Macrophages express a plasma membrane receptor for basement membrane laminin. Am J Pathol 123, 365-70. Yoon, P.S., Boxer, L.A., Mayo, L.A., Yang, A.Y. and Wicha, M.S. (1987). Human neutrophil laminin receptors: activation-dependent receptor expression. J Immunol 138, 259-65.


293

[101] Singer, II, Scott, S., Kawka, D.W. and Kazazis, D.M. (1989). Adhesomes: specific granules containing receptors for laminin, C3bi/fibrinogen, fibronectin, and vitronectin in human polymorphonuclear leukocytes and monocytes. J Cell Biol 109, 3169-82. [102] Thompson, H.L., Burbelo, P.D., Segui-Real, B., Yamada, Y. and Metcalfe, D.D. (1989). Laminin promotes mast cell attachment. J Immunol 143, 2323-7. [103] Chen, A., Ganor, Y., Rahimipour, S., Ben-Aroya, N., Koch, Y. and Levite, M. (2002). The neuropeptides GnRH-II and GnRH-I are produced by human T cells and trigger laminin receptor gene expression, adhesion, chemotaxis and homing to specific organs. Nat Med 8, 1421-6. [104] Thompson, H.L., Burbelo, P.D. and Metcalfe, D.D. (1990). Regulation of adhesion of mouse bone marrow-derived mast cells to laminin. J Immunol 145, 3425-31. [105] Chen, J., Carcamo, J.M., Borquez-Ojeda, O., Erdjument-Bromage, H., Tempst, P. and Golde, D.W. (2003). The laminin receptor modulates granulocyte-macrophage colonystimulating factor receptor complex formation and modulates its signaling. Proc Natl Acad Sci U S A 100, 14000-5. [106] Gauczynski, S. et al. (2001). The 37-kDa/67-kDa laminin receptor acts as the cellsurface receptor for the cellular prion protein. Embo J 20, 5863-75. [107] Kim, K.J., Chung, J.W. and Kim, K.S. (2005). 67-kDa laminin receptor promotes internalization of cytotoxic necrotizing factor 1-expressing Escherichia coli K1 into human brain microvascular endothelial cells. J Biol Chem 280, 1360-8. [108] Wang, K.S., Kuhn, R.J., Strauss, E.G., Ou, S. and Strauss, J.H. (1992). High-affinity laminin receptor is a receptor for Sindbis virus in mammalian cells. J Virol 66, 49925001. [109] Ludwig, G.V., Kondig, J.P. and Smith, J.F. (1996). A putative receptor for Venezuelan equine encephalitis virus from mosquito cells. J Virol 70, 5592-9. [110] Thepparit, C. and Smith, D.R. (2004). Serotype-specific entry of dengue virus into liver cells: identification of the 37-kilodalton/67-kilodalton high-affinity laminin receptor as a dengue virus serotype 1 receptor. J Virol 78, 12647-56. [111] Akache, B., Grimm, D., Pandey, K., Yant, S.R., Xu, H. and Kay, M.A. (2006). The 37/67-kilodalton laminin receptor is a receptor for adeno-associated virus serotypes 8, 2, 3, and 9. J Virol 80, 9831-6. [112] Tsuchiya, H. (1999). Effects of green tea catechins on membrane fluidity. Pharmacology 59, 34-44. [113] Nakayama, T., Hashimoto, T., Kajiya, K. and Kumazawa, S. (2000). Affinity of polyphenols for lipid bilayers. Biofactors 13, 147-51. [114] Simons, K. and Ikonen, E. (1997). Functional rafts in cell membranes. Nature 387, 569-72. [115] Simons, K. and Ehehalt, R. (2002). Cholesterol, lipid rafts, and disease. J Clin Invest 110, 597-603. [116] Smart, E.J., Ying, Y.S., Mineo, C. and Anderson, R.G. (1995). A detergent-free method for purifying caveolae membrane from tissue culture cells. Proc Natl Acad Sci U S A 92, 10104-8. [117] Liu, P., Ying, Y., Ko, Y.G. and Anderson, R.G. (1996). Localization of platelet-derived growth factor-stimulated phosphorylation cascade to caveolae. J Biol Chem 271, 10299-303.

294


[118] Parkin, E.T., Turner, A.J. and Hooper, N.M. (1999). Amyloid precursor protein, although partially detergent-insoluble in mouse cerebral cortex, behaves as an atypical lipid raft protein. Biochem J 344 Pt 1, 23-30. [119] Nguyen, D.H. and Hildreth, J.E. (2000). Evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts. J Virol 74, 3264-72. [120] Lisanti, M.P., Scherer, P.E., Vidugiriene, J., Tang, Z., Hermanowski-Vosatka, A., Tu, Y.H., Cook, R.F. and Sargiacomo, M. (1994). Characterization of caveolin-rich membrane domains isolated from an endothelial-rich source: implications for human disease. J Cell Biol 126, 111-26. [121] Wang, P.Y., Kitchens, R.L. and Munford, R.S. (1995). Bacterial lipopolysaccharide binds to CD14 in low-density domains of the monocyte-macrophage plasma membrane. J Inflamm 47, 126-37. [122] Sargiacomo, M., Sudol, M., Tang, Z. and Lisanti, M.P. (1993). Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells. J Cell Biol 122, 789-807. [123] Chun, M., Liyanage, U.K., Lisanti, M.P. and Lodish, H.F. (1994). Signal transduction of a G protein-coupled receptor in caveolae: colocalization of endothelin and its receptor with caveolin. Proc Natl Acad Sci U S A 91, 11728-32. [124] Vey, M. et al. (1996). Subcellular colocalization of the cellular and scrapie prion proteins in caveolae-like membranous domains. Proc Natl Acad Sci U S A 93, 14945-9. [125] Shaul, P.W., Smart, E.J., Robinson, L.J., German, Z., Yuhanna, I.S., Ying, Y., Anderson, R.G. and Michel, T. (1996). Acylation targets emdothelial nitric-oxide synthase to plasmalemmal caveolae. J Biol Chem 271, 6518-22. [126] Smart, E.J., Mineo, C. and Anderson, R.G. (1996). Clustered folate receptors deliver 5methyltetrahydrofolate to cytoplasm of MA104 cells. J Cell Biol 134, 1169-77. [127] Stahl, A. and Mueller, B.M. (1995). The urokinase-type plasminogen activator receptor, a GPI-linked protein, is localized in caveolae. J Cell Biol 129, 335-44. [128] Lee, S.J., Liyanage, U., Bickel, P.E., Xia, W., Lansbury, P.T., Jr. and Kosik, K.S. (1998). A detergent-insoluble membrane compartment contains A beta in vivo. Nat Med 4, 730-4. [129] Wu, C., Butz, S., Ying, Y. and Anderson, R.G. (1997). Tyrosine kinase receptors concentrated in caveolae-like domains from neuronal plasma membrane. J Biol Chem 272, 3554-9. [130] Kawabuchi, M., Satomi, Y., Takao, T., Shimonishi, Y., Nada, S., Nagai, K., Tarakhovsky, A. and Okada, M. (2000). Transmembrane phosphoprotein Cbp regulates the activities of Src-family tyrosine kinases. Nature 404, 999-1003. [131] Lang, D.M. et al. (1998). Identification of reggie-1 and reggie-2 as plasmamembraneassociated proteins which cocluster with activated GPI-anchored cell adhesion molecules in non-caveolar micropatches in neurons. J Neurobiol 37, 502-23. [132] Laux, T., Fukami, K., Thelen, M., Golub, T., Frey, D. and Caroni, P. (2000). GAP43, MARCKS, and CAP23 modulate PI(4,5)P(2) at plasmalemmal rafts, and regulate cell cortex actin dynamics through a common mechanism. J Cell Biol 149, 1455-72. [133] Prinetti, A., Iwabuchi, K. and Hakomori, S. (1999). Glycosphingolipid-enriched signaling domain in mouse neuroblastoma Neuro2a cells. Mechanism of gangliosidedependent neuritogenesis. J Biol Chem 274, 20916-24.


295

[134] Orito, A., Kumanogoh, H., Yasaka, K., Sokawa, J., Hidaka, H., Sokawa, Y. and Maekawa, S. (2001). Calcium-dependent association of annexin VI, protein kinase C alpha, and neurocalcin alpha on the raft fraction derived from the synaptic plasma membrane of rat brain. J Neurosci Res 64, 235-41. [135] Mineo, C., James, G.L., Smart, E.J. and Anderson, R.G. (1996). Localization of epidermal growth factor-stimulated Ras/Raf-1 interaction to caveolae membrane. J Biol Chem 271, 11930-5. [136] Babitt, J., Trigatti, B., Rigotti, A., Smart, E.J., Anderson, R.G., Xu, S. and Krieger, M. (1997). Murine SR-BI, a high density lipoprotein receptor that mediates selective lipid uptake, is N-glycosylated and fatty acylated and colocalizes with plasma membrane caveolae. J Biol Chem 272, 13242-9. [137] Schnitzer, J.E., Oh, P., Jacobson, B.S. and Dvorak, A.M. (1995). Caveolae from luminal plasmalemma of rat lung endothelium: microdomains enriched in caveolin, Ca(2+)-ATPase, and inositol trisphosphate receptor. Proc Natl Acad Sci U S A 92, 1759-63. [138] Patra, S.K. and Bettuzzi, S. (2007). Epigenetic DNA-methylation regulation of genes coding for lipid raft-associated components: a role for raft proteins in cell transformation and cancer progression (review). Oncol Rep 17, 1279-90. [139] Foster, L.J., De Hoog, C.L. and Mann, M. (2003). Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors. Proc Natl Acad Sci U S A 100, 5813-8. [140] Gulbins, E. and Kolesnick, R. (2003). Raft ceramide in molecular medicine. Oncogene 22, 7070-7. [141] Gajate, C. and Mollinedo, F. (2005). Cytoskeleton-mediated death receptor and ligand concentration in lipid rafts forms apoptosis-promoting clusters in cancer chemotherapy. J Biol Chem 280, 11641-7. [142] Ehehalt, R., Keller, P., Haass, C., Thiele, C. and Simons, K. (2003). Amyloidogenic processing of the Alzheimer beta-amyloid precursor protein depends on lipid rafts. J Cell Biol 160, 113-23. [143] Yankner, B.A., Dawes, L.R., Fisher, S., Villa-Komaroff, L., Oster-Granite, M.L. and Neve, R.L. (1989). Neurotoxicity of a fragment of the amyloid precursor associated with Alzheimer's disease. Science 245, 417-20. [144] Choi, Y.T. et al. (2001). The green tea polyphenol (-)-epigallocatechin gallate attenuates beta-amyloid-induced neurotoxicity in cultured hippocampal neurons. Life Sci 70, 603-14. [145] Kilsdonk, E.P., Yancey, P.G., Stoudt, G.W., Bangerter, F.W., Johnson, W.J., Phillips, M.C. and Rothblat, G.H. (1995). Cellular cholesterol efflux mediated by cyclodextrins. J Biol Chem 270, 17250-6. [146] Fujimura, Y., Tachibana, H. and Yamada, K. (2004). Lipid raft-associated catechin suppresses the FcepsilonRI expression by inhibiting phosphorylation of the extracellular signal-regulated kinase1/2. FEBS Lett 556, 204-10. [147] Fujimura, Y., Umeda, D., Kiyohara, Y., Sunada, Y., Yamada, K. and Tachibana, H. (2006). The involvement of the 67 kDa laminin receptor-mediated modulation of cytoskeleton in the degranulation inhibition induced by epigallocatechin-3-O-gallate. Biochem Biophys Res Commun 348, 524-31.

296


[148] Leulier, F. and Lemaitre, B. (2008). Toll-like receptors--taking an evolutionary approach. Nat Rev Genet 9, 165-78. [149] Fujimura, Y., Yamada, K. and Tachibana, H. (2005). A lipid raft-associated 67kDa laminin receptor mediates suppressive effect of epigallocatechin-3-O-gallate on FcepsilonRI expression. Biochem Biophys Res Commun 336, 674-81. [150] Yang, C.S. (1997). Inhibition of carcinogenesis by tea. Nature 389, 134-5. [151] Goeckeler, Z.M. and Wysolmerski, R.B. (1995). Myosin light chain kinase-regulated endothelial cell contraction: the relationship between isometric tension, actin polymerization, and myosin phosphorylation. J Cell Biol 130, 613-27. [152] Ikebe, M., Reardon, S., Scott-Woo, G.C., Zhou, Z. and Koda, Y. (1990). Purification and characterization of calmodulin-dependent multifunctional protein kinase from smooth muscle: isolation of caldesmon kinase. Biochemistry 29, 11242-8. [153] Iwasaki, T., Murata-Hori, M., Ishitobi, S. and Hosoya, H. (2001). Diphosphorylated MRLC is required for organization of stress fibers in interphase cells and the contractile ring in dividing cells. Cell Struct Funct 26, 677-83. [154] Glotzer, M. (2001). Animal cell cytokinesis. Annu Rev Cell Dev Biol 17, 351-86. [155] Masuda, M., Suzui, M. and Weinstein, I.B. (2001). Effects of epigallocatechin-3gallate on growth, epidermal growth factor receptor signaling pathways, gene expression, and chemosensitivity in human head and neck squamous cell carcinoma cell lines. Clin Cancer Res 7, 4220-9. [156] Yang, G.Y., Liao, J., Kim, K., Yurkow, E.J. and Yang, C.S. (1998). Inhibition of growth and induction of apoptosis in human cancer cell lines by tea polyphenols. Carcinogenesis 19, 611-6. [157] Calle, E.E., Rodriguez, C., Walker-Thurmond, K. and Thun, M.J. (2003). Overweight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. adults. N Engl J Med 348, 1625-38. [158] Yuan, J.M., Gao, Y.T., Yang, C.S. and Yu, M.C. (2007). Urinary biomarkers of tea polyphenols and risk of colorectal cancer in the Shanghai Cohort Study. Int J Cancer 120, 1344-50. [159] Radtke, F. and Clevers, H. (2005). Self-renewal and cancer of the gut: two sides of a coin. Science 307, 1904-9. [160] Orsulic, S., Huber, O., Aberle, H., Arnold, S. and Kemler, R. (1999). E-cadherin binding prevents beta-catenin nuclear localization and beta-catenin/LEF-1-mediated transactivation. J Cell Sci 112 ( Pt 8), 1237-45. [161] Ju, J. et al. (2005). Inhibition of intestinal tumorigenesis in Apcmin/+ mice by (-)epigallocatechin-3-gallate, the major catechin in green tea. Cancer Res 65, 10623-31. [162] Shammas, M.A. et al. (2006). Specific killing of multiple myeloma cells by (-)epigallocatechin-3-gallate extracted from green tea: biologic activity and therapeutic implications. Blood 108, 2804-10. [163] Metzger, H. (1992). The receptor with high affinity for IgE. Immunol Rev 125, 37-48. [164] Ravetch, J.V. and Kinet, J.P. (1991). Fc receptors. Annu Rev Immunol 9, 457-92. [165] Turner, H. and Kinet, J.P. (1999). Signalling through the high-affinity IgE receptor Fc epsilonRI. Nature 402, B24-30. [166] Rivera, J. (2002). Molecular adapters in Fc(epsilon)RI signaling and the allergic response. Curr Opin Immunol 14, 688-93.


297

[167] Choi, O.H., Adelstein, R.S. and Beaven, M.A. (1994). Secretion from rat basophilic RBL-2H3 cells is associated with diphosphorylation of myosin light chains by myosin light chain kinase as well as phosphorylation by protein kinase C. J Biol Chem 269, 536-41. [168] Pfeiffer, J.R., Seagrave, J.C., Davis, B.H., Deanin, G.G. and Oliver, J.M. (1985). Membrane and cytoskeletal changes associated with IgE-mediated serotonin release from rat basophilic leukemia cells. J Cell Biol 101, 2145-55. [169] Edgar, A.J. and Bennett, J.P. (1997). Circular ruffle formation in rat basophilic leukemia cells in response to antigen stimulation. Eur J Cell Biol 73, 132-40. [170] Ludowyke, R.I., Peleg, I., Beaven, M.A. and Adelstein, R.S. (1989). Antigen-induced secretion of histamine and the phosphorylation of myosin by protein kinase C in rat basophilic leukemia cells. J Biol Chem 264, 12492-501. [171] Oliver, J.M., Seagrave, J., Stump, R.F., Pfeiffer, J.R. and Deanin, G.G. (1988). Signal transduction and cellular response in RBL-2H3 mast cells. Prog Allergy 42, 185-245. [172] Apgar, J.R. (1991). Association of the crosslinked IgE receptor with the membrane skeleton is independent of the known signaling mechanisms in rat basophilic leukemia cells. Cell Regul 2, 181-91. [173] Apgar, J.R. (1991). Regulation of the antigen-induced F-actin response in rat basophilic leukemia cells by protein kinase C. J Cell Biol 112, 1157-63. [174] Spudich, A. (1994). Myosin reorganization in activated RBL cells correlates temporally with stimulated secretion. Cell Motil Cytoskeleton 29, 345-53. [175] Sahara, N., Siraganian, R.P. and Oliver, C. (1990). Morphological changes induced by the calcium ionophore A23187 in rat basophilic leukemia (2H3) cells. J Histochem Cytochem 38, 975-83. [176] Dombrowicz, D., Flamand, V., Brigman, K.K., Koller, B.H. and Kinet, J.P. (1993). Abolition of anaphylaxis by targeted disruption of the high affinity immunoglobulin E receptor alpha chain gene. Cell 75, 969-76. [177] Fujimura, Y., Tachibana, H. and Yamada, K. (2001). A tea catechin suppresses the expression of the high-affinity IgE receptor Fc epsilon RI in human basophilic KU812 cells. J Agric Food Chem 49, 2527-31. [178] Sano, M., Suzuki, M., Miyase, T., Yoshino, K. and Maeda-Yamamoto, M. (1999). Novel antiallergic catechin derivatives isolated from oolong tea. J Agric Food Chem 47, 1906-10. [179] Tachibana, H., Sunada, Y., Miyase, T., Sano, M., Maeda-Yamamoto, M. and Yamada, K. (2000). Identification of a methylated tea catechin as an inhibitor of degranulation in human basophilic KU812 cells. Biosci Biotechnol Biochem 64, 452-4. [180] Suzuki, M., Yoshino, K., Maeda-Yamamoto, M., Miyase, T. and Sano, M. (2000). Inhibitory effects of tea catechins and O-methylated derivatives of (-)-epigallocatechin3-O-gallate on mouse type IV allergy. J Agric Food Chem 48, 5649-53. [181] Maeda-Yamamoto, M., Ema, K. and Shibuichi, I. (2007). In vitro and in vivo antiallergic effects of 'benifuuki' green tea containing O-methylated catechin and ginger extract enhancement. Cytotechnology 55, 135-142. [182] Maeda-Yamamoto, M. et al. (2004). O-methylated catechins from tea leaves inhibit multiple protein kinases in mast cells. J Immunol 172, 4486-92. [183] Fujimura, Y., Tachibana, H., Maeda-Yamamoto, M., Miyase, T., Sano, M. and Yamada, K. (2002). Antiallergic tea catechin, (-)-epigallocatechin-3-O-(3-O-methyl)-

298

[184]

[185] [186]

[187]

[188]

[189] [190]

[191] [192] [193]

[194] [195]

[196]

[197]

[198]

Yoshinori Fujimura and Hirofumi Tachibana gallate, suppresses FcepsilonRI expression in human basophilic KU812 cells. J Agric Food Chem 50, 5729-34. Yano, S., Fujimura, Y., Umeda, D., Miyase, T., Yamada, K. and Tachibana, H. (2007). Relationship between the biological activities of methylated derivatives of (-)epigallocatechin-3-O-gallate (EGCG) and their cell surface binding activities. J Agric Food Chem 55, 7144-8. Conlay, L.A., Conant, J.A., deBros, F. and Wurtman, R. (1997). Caffeine alters plasma adenosine levels. Nature 389, 136. Roninson, I.B. et al. (1995). Genetic suppressor elements: new tools for molecular oncology--thirteenth Cornelius P. Rhoads Memorial Award Lecture. Cancer Res 55, 4023-8. Gudkov, A.V., Kazarov, A.R., Thimmapaya, R., Axenovich, S.A., Mazo, I.A. and Roninson, I.B. (1994). Cloning mammalian genes by expression selection of genetic suppressor elements: association of kinesin with drug resistance and cell immortalization. Proc Natl Acad Sci U S A 91, 3744-8. Negrutskii, B.S. and El'skaya, A.V. (1998). Eukaryotic translation elongation factor 1 alpha: structure, expression, functions, and possible role in aminoacyl-tRNA channeling. Prog Nucleic Acid Res Mol Biol 60, 47-78. Brummelkamp, T.R., Bernards, R. and Agami, R. (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550-3. Manach, C., Williamson, G., Morand, C., Scalbert, A. and Remesy, C. (2005). Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. Am J Clin Nutr 81, 230S-242S. Yang, C.S., Chung, J.Y., Yang, G., Chhabra, S.K. and Lee, M.J. (2000). Tea and tea polyphenols in cancer prevention. J Nutr 130, 472S-478S. Maciver, S.K. (1996). Myosin II function in non-muscle cells. Bioessays 18, 179-82. Somlyo, A.P. and Somlyo, A.V. (2003). Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev 83, 1325-58. Hartshorne, D.J., Ito, M. and Erdodi, F. (2004). Role of protein phosphatase type 1 in contractile functions: myosin phosphatase. J Biol Chem 279, 37211-4. Fukata, Y., Kimura, K., Oshiro, N., Saya, H., Matsuura, Y. and Kaibuchi, K. (1998). Association of the myosin-binding subunit of myosin phosphatase and moesin: dual regulation of moesin phosphorylation by Rho-associated kinase and myosin phosphatase. J Cell Biol 141, 409-18. Kimura, K., Fukata, Y., Matsuoka, Y., Bennett, V., Matsuura, Y., Okawa, K., Iwamatsu, A. and Kaibuchi, K. (1998). Regulation of the association of adducin with actin filaments by Rho-associated kinase (Rho-kinase) and myosin phosphatase. J Biol Chem 273, 5542-8. Eto, M., Wong, L., Yazawa, M. and Brautigan, D.L. (2000). Inhibition of myosin/moesin phosphatase by expression of the phosphoinhibitor protein CPI-17 alters microfilament organization and retards cell spreading. Cell Motil Cytoskeleton 46, 222-34. Izawa, T., Fukata, Y., Kimura, T., Iwamatsu, A., Dohi, K. and Kaibuchi, K. (2000). Elongation factor-1 alpha is a novel substrate of rho-associated kinase. Biochem Biophys Res Commun 278, 72-8.


299

[199] Edmonds, B.T., Wyckoff, J., Yeung, Y.G., Wang, Y., Stanley, E.R., Jones, J., Segall, J. and Condeelis, J. (1996). Elongation factor-1 alpha is an overexpressed actin binding protein in metastatic rat mammary adenocarcinoma. J Cell Sci 109 ( Pt 11), 2705-14. [200] Durso, N.A. and Cyr, R.J. (1994). A calmodulin-sensitive interaction between microtubules and a higher plant homolog of elongation factor-1 alpha. Plant Cell 6, 893-905. [201] Murray, J.W., Edmonds, B.T., Liu, G. and Condeelis, J. (1996). Bundling of actin filaments by elongation factor 1 alpha inhibits polymerization at filament ends. J Cell Biol 135, 1309-21. [202] Yuan, J.M., Koh, W.P., Sun, C.L., Lee, H.P. and Yu, M.C. (2005). Green tea intake, ACE gene polymorphism and breast cancer risk among Chinese women in Singapore. Carcinogenesis 26, 1389-94. [203] Wu, A.H., Tseng, C.C., Van Den Berg, D. and Yu, M.C. (2003). Tea intake, COMT genotype, and breast cancer in Asian-American women. Cancer Res 63, 7526-9. [204] Inoue, M., Robien, K., Wang, R., van den Berg, D., Koh, W.P. and Yu, M.C. (2008). Green tea intake, MTHFR/TYMS genotype, and breast cancer risk: The Singapore Chinese Health Study. Carcinogenesis [205] Surh, Y.J. (2003). Cancer chemoprevention with dietary phytochemicals. Nat Rev Cancer 3, 768-80. [206] Yang, C.S., Sang, S., Lambert, J.D., Hou, Z., Ju, J. and Lu, G. (2006). Possible mechanisms of the cancer-preventive activities of green tea. Mol Nutr Food Res 50, 170-5. [207] Khan, N., Afaq, F., Saleem, M., Ahmad, N. and Mukhtar, H. (2006). Targeting multiple signaling pathways by green tea polyphenol (-)-epigallocatechin-3-gallate. Cancer Res 66, 2500-5. [208] Patra, S.K. (2008). Dissecting lipid raft facilitated cell signaling pathways in cancer. Biochim Biophys Acta 1785, 182-206. [209] Simons, K. and Toomre, D. (2000). Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 1, 31-9. [210] Galbiati, F., Razani, B. and Lisanti, M.P. (2001). Emerging themes in lipid rafts and caveolae. Cell 106, 403-11. [211] Maxfield, F.R. (2002). Plasma membrane microdomains. Curr Opin Cell Biol 14, 4837. [212] Brown, D.A. and London, E. (2000). Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J Biol Chem 275, 17221-4. [213] Freeman, M.R., Cinar, B. and Lu, M.L. (2005). Membrane rafts as potential sites of nongenomic hormonal signaling in prostate cancer. Trends Endocrinol Metab 16, 2739. [214] Freeman, M.R., Cinar, B., Kim, J., Mukhopadhyay, N.K., Di Vizio, D., Adam, R.M. and Solomon, K.R. (2007). Transit of hormonal and EGF receptor-dependent signals through cholesterol-rich membranes. Steroids 72, 210-7.



Chapter 15

UTILITY OF EPIGALLOCATECHIN GALLATE IN THE TREATMENT AND PREVENTION OF BREAST CANCER: MOLECULAR MECHANISMS FOR TUMOR SUPPRESSION R. J. Rosengren* Department of Pharmacology and Toxicology, University of Otago, Dunedin, New Zealand

ABSTRACT Green tea and its major constituent epigallocatechin gallate (EGCG) have been extensively studied as a potential chemopreventative and/or treatment for a variety of diseases including breast cancer. Experimental evidence is supported by epidemiological studies that have shown an inverse relationship between green tea consumption and the incidence of breast cancer. Numerous studies have demonstrated that EGCG is cytotoxic toward both estrogen receptor-positive and estrogen receptor-negative breast cancer cell lines. These studies have highlighted potential mechanisms for the actions of EGCG, such as the induction of apoptosis, the alteration of the expression of cell cycle regulatory proteins critical for cell proliferation, inhibition of the chymotrypsin-like proteasome, inhibition of angiogenesis, as well as inhibition of cell invasion and metastasis. Importantly, these effects occur independently of estrogen receptor expression. This chapter will provide evidence for these events and other molecular mechanisms that significantly contribute to the actions of EGCG in vitro. The utility of green tea extract or EGCG as a breast cancer treatment and chemopreventative has also been extensively investigated using various in vivo models of estrogen receptor-positive and estrogen receptor-negative breast cancer (ie., chemical carcinogenesis and xenograft models). Evidence for EGCG-mediated tumor suppression and the major molecular mechanisms for this effect, such as the induction of apoptosis, the inhibition of angiogenesis and *

Address for correspondence: Rhonda J. Rosengren. Department of Pharmacology & Toxicology. 18 Frederick Street, Adams Building, University of Otago, Dunedin, New Zealand, 9001. E-mail: [email protected]; Tel: +64 3 479 9141; Fax +64 3 479 9140.

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R. J. Rosengren modulation of the expression of cell signaling proteins are also fully examined. These in vivo studies have led to investigations which have focused on ways to improve the actions of EGCG, by either using it as part of a combination therapy or by synthesizing pro-drugs of EGCG. Both of these have been done in order to enhance the bioavailability, stability and efficacy of EGCG. These new compounds and drug combinations have significantly improved the tumor suppression potential of EGCG and provide an exciting future for this multi-faceted phytochemical in the prevention and treatment of breast cancer.

INTRODUCTION Flavanoids are plant-derived polyphenolic compounds found in fruits, vegetables, herbs, tea and wine [1] and are divided into several different classes based on small variations in their structure. One such class is the flavan-3-ols, also termed the catechins, which are differentiated by di- or tri-hydroxyl group substitutions on the B-ring and meta-5,7-dihydroxy substitution on the A ring [2]. Catechins are particularly abundant in green tea (Camellia sinensis), accounting for 30-40% of its dry weight [3-5]. The major catechins contained in green tea are (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), (-)epicatechin gallate (ECG), (-)-epicatechin (EC) and catechin, with EGCG comprising the greatest proportion of these catechins [4, 5]. Since tea is the second most widely consumed beverage next to water, both tea and EGCG are generally considered to be non-toxic. However, the literature on this topic is not consistent. For example, EGCG (500 mg/kg/d as part of the diet, for 13 weeks) did not produce weight loss in male or female Sprague-Dawley rats [6]. In contrast, green tea extract (GTE) (5% of the diet/d, for 90 d) caused a loss of body weight in male but not female rats [7] and EGCG (81 mg/kg/, 8d, ip) decreased body weight in both rat sexes [8]. Even though the LD50 of GTE in mice has been calculated to be 3.09 g/kg for females and 5 g/kg for males [7], weight loss, hepatotoxicity and mortality have been reported following the administration of much lower doses of EGCG. Specifically, a single dose of EGCG (150 mg/kg, ip) produced mortality in femake mice [9] and repeated administration of EGCG (50 mg/kg/d, 7d, ip) elicited hepatotoxicity, weight loss and mortality in male and female mice [10-12]. However, all three parameters were more severe in females compared to males [10]. While female mice were more susceptible to EGCG-mediated hepatotoxicity, this has not been demonstrated in rats. Specifically, female Wistar rats were administered GTE (2.5 g/kg/d, po, 6 weeks or 2 g/kg/d, po, 12 weeks) and no liver injury was seen in any treatment group [13]. The conflicting evidence in rodents makes extrapolation to humans difficult. However, there have been reports of liver injury in humans following consumption of GTE. For example, In Europe, there have been 14 reported cases of severe hepatitis following ingestion of an ethanolic extract of green tea, which caused the product to no longer be marketed [14]. A further 3 cases of liver injury have been reported following the consumption of either green tea (6 cups/day, 4 months) [15] or a micronized powder of Camellia sinensis) [16]. However, clinical trials with high doses of GTE (2.2 g/m2, tid, 6 months) have only shown minor sideeffects most likely related to caffeine in the extract (insomnia, restlessness, gastrointestinal complaints) [17]. Additionally, EGCG (1.6 g, as one bolus oral dose) was given to 8 volunteers without any clinical or biological adverse reactions [18]. Therefore, it is probably

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safe to conclude that green tea / EGCG can be consumed without unwanted side effects in most individuals. This is also supported by the numerous health benefits associated with green tea / EGCG consumption. These have been consistently demonstrated in numerous epidemiological studies which have examine the role of green tea consumption and breast cancer risk [19-21]. In this chapter, evidence will be given for the potential application of EGCG in the prevention and treatment of breast cancer as well as the molecular mechanisms that underpin these actions of EGCG.

EPIDEMIOLOGICAL EVIDENCE - BREAST CANCER RISK ASSOCIATION WITH GREEN TEA CONSUMPTION AND GENOTYPE Epidemiological studies that have analyzed green tea consumption and the risk of developing breast cancer have yielded conflicting results. Some studies have shown no association with green tea drinking [19-21], while others have demonstrated a chemopreventative effect [22] (Table 1). It is important to note that most of the published epidemiological studies were not specifically designed to examine the relationship between tea consumption and cancer risk and therefore the amount and frequency of tea consumption was not always accurately determined [23]. However, the results from a meta-analysis of 4 studies (three cohort studies from Japan and one case control study from the USA) showed that breast cancer risk was significantly reduced following green tea consumption (OR = 0.77, 95% CI = 0.61-0.97) [24]. Additionally, a decreased risk of recurrence of breast cancer has also been shown to correlate with green tea consumption [25, 26] (Table 1). Therefore, the overall evidence suggests a positive correlation between green tea consumption and the development and recurrence of breast cancer. In order to more fully understand the chemopreventative actions of green tea, studies have genotyped breast cancer patients to determine whether genetic polymorphisms could influence the actions of green tea. One such case-control study in Chinese-, Japanese-, and Filipino-American women demonstrated that green tea intake was associated with a reduction in breast cancer risk, but only in women possessing a low-activity allele of catechol-Omethyltransferase (COMT) [22]. COMT is responsible for the rapid methylation of the catechins in green tea and, therefore, differences in the methylation capacity between individuals may alter the chemopreventative activity of green tea. The findings of Wu et al. (2003) [22] suggest that chemoprevention by green tea in women possessing the low-activity COMT allele may result from decreased metabolism and thus an increased bioavailability of catechins. However, women with a low activity COMT would also be at a higher risk for developing breast cancer due to a decreased production of the pro-apoptotoic and antiangiogenic metabolite of estradiol, 2-methoxy-estradiol [28]. Therefore, further work needs to be conducted to determine if the reduction in risk is related to catechin metabolism or the overall higher risk of the patients. Other enzymes were also shown to be important as green tea consumption was also associated with a reduced risk of breast cancer in women who expressed the high-activity, but not the low-activity, angiotensin-converting enzyme [29]. Changes in the activity of this enzyme support the hypothesis that a possible mechanism of chemoprevention by EGCG and other green tea catechins involves their inhibition of reactive oxygen species via the

Table 1. Green Tea Intake and the Risk of the Development or Recurrence of Breast Cancer Type

Population Profile

Risk Ratio (95% CI)

Recurrence

472 Japanese women with Stage I, II or III breast cancer

Green tea consumption: Stage I and II Stage III Green tea consumption: Stage I ≤3 cups/day 3-5 cups/day ≥6 cups/day Stage II ≤3 cups/day 3-5 cups/day ≥6 cups/day Stage III ≤3 cups/day 3-5 cups/day ≥6 cups/day Green tea consumption: ≤ 1/day 2-4/day ≥ 5/day Green tea consumption: ≤ 1/day 2-4/day ≥ 5/day Green tea consumption: 1-2 cups/day 3-4 cups/day ≥ 5 cups/day Green tea consumption: 0 - 85.7 ml/day ≥ 85.7 ml/day

1,160 Japanese women

Development

34,759 women in Hiroshima & Nagasaki, Japan

23,667 women, members of the Life Span Study Cohort

Combined 2 cohort studies conducted in rural Japan -17,353 women cohort 1 -24,769 women cohort 2 Chinese, Japanese and Filipino women residing in the US

*Statistically significant. Table modified from Stuart et al., (2006) [27].

Outcome 0.56 (0.35 - 0.91)* 1.88 (0.79 - 4.54) 0.43 (0.22 - 0.84)* 0.37 (0.17 - 0.80)* 0.59 (0.23 - 1.52) 0.71 (0.35 - 1.44) 0.80 (0.38 - 1.69) 0.51 (0.18 - 1.46) 1.01 (0.50 - 2.05) 1.06 (0.51 - 2.17) 0.87 (0.33 - 2.27)

Reference

Green tea intake was associated with a reduced risk of recurrence of Stage I and II breast cancer Green tea intake was associated with a reduced risk of recurrence in Stage I breast cancer

[25]

No association

[19]

No association

[20]

No association

[21]

Green tea intake is associated with a reduced risk of the development of breast cancer

[22]

[26]


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angiotensin-converting enzyme. A further mechanism of chemoprevention by green tea catechins involves the alteration of circulating steroid hormone levels. Specifically, in a study of 130 postmenopausal Chinese women, it was shown that women who regularly consume green tea had lower plasma levels of estrone, estradiol and androstenedione compared with non- or irregular green tea-drinkers [30]. In this study, the changes in hormone levels were not dependent upon the genotype of COMT. Therefore, these findings suggest that alteration of steroid hormone levels may contribute to the chemopreventative activity of green tea. However, in order to obtain a more thorough picture of the effect of green tea / EGCG, more investigations must be conducted with both GTE and pure EGCG.

IN VITRO EFFECTS AND MOLECULAR MECHANISMS OF EGCG EGCG is cytotoxic toward breast cancer cells regardless of their ER status. Specifically, after EGCG treatment, cell number was significantly decreased from control in both ERpositive (MCF-7, T47-D and BT474) [31-34] and ER-negative (MDA-MB-231, Hs578t, MBA-MB-468 and BT-20) human breast cancer cells lines, [31-41]. Importantly, EGCG displayed a greater cytotoxic potency in ER-negative breast cancer cell lines compared to ERpositive cell lines [33, 34]. Results from in vitro ER binding and reporter gene assays as well as in vivo functional assays demonstrated that EGCG weakly bound both ERα and ERβ, but could not antagonize estradiol-mediated responses in vivo [11]. These results demonstrate that EGCG is not a strong antagonist of the ER and its cytotoxic action in breast cancer cells occurs independently of the ER. Furthermore, EGCG is cytotoxic toward breast cancer cells that grow in response to stimulation via HER2, an isoform of the epidermal growth factor receptor (EGFR), but are also resistant to trastuzumab immunotherapy. Specifically, EGCG (~87-349 µM) dose-dependently decreased the proliferation of both trastuzumab-resistant BT474 human breast cancer cells and JIMT-1 cells (derived from a patient who displayed clinical resistance to trastuzumab immunotherapy) [42]. Therefore, EGCG is cytotoxic toward a variety of human breast cancer cells and this effect is mediated via ER-independent mechanisms that are critical in the regulation of cell proliferation. The major mechanisms by which EGCG conveys its cytotoxicity toward breast cancer cells are outlined in the following sections.

EGCG-MEDIATED INDUCTION OF APOPTOSIS AND MODULATION OF CELL SIGNALING PATHWAYS There is an abundance of literature obtained from research using a variety of human cancer cell lines that has demonstrated that EGCG induces apoptosis [43-47]. It would then be expected that EGCG would induce apoptosis in most, if not all, breast cancer cell lines. However, the role of EGCG in breast cancer has only more recently become a focus of this potential drug and most of this work has been conducted in ER-negative breast cancer cells. Specifically, EGCG or green tea extract induced apoptosis in ER-negative MDA-MB-468 [38] and MDA-MB-231 cells [33, 40, 41, 48, 49], as well as trastuzumab–resistant BT474 and JINT-1 cells, but only following high concentrations in these cells (>170 µM) [42].

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Mechanisms through which EGCG-induced apoptosis was mediated includes; cell cycle arrest [31, 36, 39], changes in intracellular signaling cascades [50-53], inhibition of proteasomal chymotrypsin-like activity [49], upregulation of p53 and changes in the ratio of Bax/Bcl-2 [38]. For example EGCG has caused ER-negative breast cancer cells to arrest in the G1 phase of the cell cycle. Specifically, treatment of MDA-MB-231 cells with EGCG (87 µM) for 12 h produced a 34% increase in the number of cells in the G1 phase of the cell cycle compared to control [39], while a lower concentration of EGCG (25 µM) only increased the number of G1 phase cells by 4% [40, 41]. Therefore, the ability of EGCG to induce cell cycle arrest is clearly dose-dependent. Further studies have demonstrated that changes in the cell cycle were driven by EGCG-mediated alterations in the protein expression of the cyclins, cyclin dependent kinases (cdks) and their inhibitors (CDKIs). Specifically, studies in MDA-MB-231 cells have shown that EGCG (87 µM) decreased the protein expression of Cyclin D and Cylcin E as well as cdk4 and cdk1 by 50% [39], while the CDKIs, p21 and p27 were increased [36]. A similar result has also been reported in MCF-7 cells where 30 µM of EGCG-induced the expression of p21 and p27 and this correlated with an 1.5-fold increase in the number of cells arrested in the G1 phase of the cell cycle [31]. The induction of apoptosis and alterations in the cell cycle are likely to be the result of EGCG-mediated changes in intracellular pathways. It has been documented that EGCG alters the phosphorylative activity of the EGFR and its downstream targets in breast cancer cells (for an overview of EGFR and its downstream targets see Figure 1). Specifically, the treatment of MDA-MB-231 cells with EGCG inhibited both basal and TGF-α-induced autophosphorylation of the EGFR [36]. It was further established that EGCG inhibited constitutive and TGF-α-induced AKT and STAT3 activity in these cells, but not the expression of phosphorylated ERK. Furthermore, EGCG inhibited the activity of the HER2 and this correlated with a decrease in downstream effects of EGFR activation, such as c-fos promoter activity [37]. Another important protein that governs cell survival is the transfactor NF-κB. NF-κB has overlapping roles in many mitogenic signaling pathways as it is capable of promoting and repressing the expression of proteins involved in cell growth, apoptosis, inflammation, the stress response as well as other important physiological processes [54-56]. Therefore, it is vital for tumor growth, and is a key player in ER-negative breast cancer as it is overexpressed in both ER-negative breast cancer cell lines and in tumors from patients [57]. Therefore, it is an important target in ER-negative breast cancer treatment strategies. Studies in cancer cell lines have demonstrated that EGCG inhibits NF-κB [36, 50-53]. For example, MDA-MB-231 cells were used to demonstrate that EGCG inhibited both the basal and inducible activity of the NF-κB complex [36]. This supported previous work showing that EGCG elicited doseand time-dependent inhibition of both the activation and translocation of NF-κB via the suppression and cytoplasmic degradation of IkBa [52, 53]. Further studies demonstrated that EGCG exhibited a concurrent effect on p53 and NF-κB, which caused a change in the ratio of BAX/Bcl-2 and thus favored apoptosis [50]. This effect would significantly impact the growth of many breast cancer cell lines but would not be relevant to MDA-MB-231 cell growth, as these cells lack a functional form of p53 [58].


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Figure 1. Schematic diagram of intracellular cell signaling cascades that are activated following phosphorylation of the EGFR. Dimerization of the EGFR leads to the activation of various signaling pathways such as PI3K/Akt and Ras/Raf/ERK. These regulate key downstream regulators of cell growth such as NFκB and mTOR.

EGCG modulates other important cell survival pathways in MDA-MB-231 cells. One of these is the hepatocyte growth factor (HGF)/Met signaling pathway which is involved in proliferation, survival, motility and invasion [59]. Met is a tyrosine receptor kinase which autophosphorylates upon activation by HGF and its downstream targets include the PI3K/AKT and MAPK pathways [59-62]. Studies with EGCG have shown that concentrations as low as 0.6 µM inhibited HGF-induced Met phosphorylation, and subsequent AKT and ERK activation and this correlated with a 67% decrease in HGFinduced invasion in MDA-MB-231 cells [59]. Importantly, the authors also demonstrated that EGCG (5 µM) inhibited the basal invasion capabilities of MDA-MB-231 cells by 50%, which demonstrates that EGCG decreases both basal cancer cell invasion and that induced by HGF. Apoptosis can also be modulated in cancer cells by the chymotrypsin-like proteasome [63]. Inhibition of this specific proteasome rapidly and selectively induces apoptosis in cancer cells but not normal cells [64, 65]. Importantly, this effect has been shown in human cancer

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cells that are resistant to numerous other anticancer agents. In MDA-MB-231 breast cancer cells EGCG inhibited chymotrypsin-like activity by 24% and this correlated with an increase in ubiquitinated IkBa, p27 and Bax [49]. All three of these proteins work to decrease cell proliferation and increase apoptosis, as IkB prevents the activation of the transfactor NF-kB [66]. Overall these studies show that, EGCG induces apoptosis in breast cancer cells by modulating intracellular signaling pathways that control cell cycle progression, cell motility/invasion and proteasomal degradation. Since these effects are not dependent on the ER, EGCG is able to decrease cell growth and induce a strong apoptotic response in ERnegative breast cancer cells. Thus EGCG has the potential to be used as a drug that will positively impact on the lives of women with ER-negative breast cancer.

EGCG AS AN INHIBITOR OF ANGIOGENESIS Numerous studies have demonstrated that EGCG inhibits a variety of processes involved in angiogenesis. This action elicited by EGCG is extremely relevant to tumor suppression since tumor growth is an angiogenic-dependent process, with new blood vessels supplying nutrients, oxygen and growth factors that enhance tumor proliferation and expansion [67]. In breast cancer, neovascularization has been shown to contribute to the long-term aggressiveness of the tumor and correlates to increased metastasis [68, 69]. EGCG has been documented to inhibit endothelial cell growth in a dose-dependent manner in vitro, which correlated to a significant decrease in new blood vessel formation in an in vivo angiogenesis model [70]. Furthermore, in vitro studies have shown that EGCG inhibits the production of vascular endothelial growth factor (VEGF) and matrix metalloproteinases [71, 72]. All isoforms of VEGF have been established as potent angiogenic agents [73] and the levels of this growth factor are closely correlated to the induction and maintenance of the neovasculature in breast cancer [74, 75]. VEGF initiates angiogenesis via the binding to its receptors (VEGFR-1 and VEGFR-2) [76]. VEGFR-2 activation is responsible for most of the mitogenic and chemotactic effects of VEGF [77] and inhibition of angiogenesis occurs through the inhibition of both VEGF and VEGFR-2 [78, 79]. Importantly, EGCG has been shown to inhibit VEGFR-2 phosphorylation in vitro but this study was not conducted in human breast cancer cells [80] and thus, further work needs to be conducted to confirm this in breast cancer models. EGCG also decreases cancer cell invasion and metastasis [81, 82] by inhibiting cell adhesion function via an inhibition of E-cadherin. Macrophages and other inflammatory cells also promote angiogenesis and EGCG has decreased inflammation by suppressing the overexpression of both cyclooxgenase [83] and nitric oxide synthatase [84]. Another potential mechanism for EGCG is via binding to the metastatsis-associated 67-kDa laminin receptor. While this study was conducted in lung cancer cells, the results showed that growth inhibition occurred only in cells transfected with the 67-kDa laminin receptor and that EGCG bound to this receptor with a Kd of 39.9 nM [85]. Interestingly, none of the other green tea catechins were able to bind to this receptor and thus the effect was specific for EGCG. However, epicatechin gallate was not tested and thus the gallate moiety may prove to be critical for activity. Additionally, these experiments were not performed in breast cancer cells and thus it


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has yet to be determined if this response is relevant to the antiangiogenic effect of EGCG in breast cancer, as there are many additional facets to this process. In breast cancer, another factor involved in angiogenesis, invasiveness and metastasis is the oncogene Wnt. Constitutively active Wnt has been linked to increased proliferation and invasiveness in breast cancer through the stabilization of β-catenin. In breast cancer, high βcatenin levels correlates with poor prognosis [86-88] Additionally, the percentage of breast cancers with high β-catenin expression is estimated to be approximately 50% [89]. Therefore, modulation of Wnt could significantly impact on a large number of breast cancer patients. Importantly, in MDA-MB-231 cells EGCG (25-100 µM) blocked Wnt signaling in a dosedependent manner, via the induction of HBP1 [90]. Induction of HBP1 occurred via an increase in the stabilization of mRNA and not through transcriptional induction. HBP1 plays an important role in invasive breast cancer, as a loss HBP1 is associated with invasive breast cancer and 30% of patients with invasive disease express HBP1 mutants [89]. Importantly, downstream effects of Wnt signaling were also modulated by EGCG, as c-mcy (a relevant Wnt target gene) was also decreased [90]. These results correlated with a decrease in the migration of MDA-MB-231 cells. Specifically, EGCG (50 and 100 µM) decreased the migration of cells toward fibronectin treated medium by 50 and 75%, respectively and also decreased the invasion of cells through Matrigel [90]. This demonstrates that EGCG has the ability to modulate breast cancer invasiveness by increasing the stability of HBP1, which decrease Wnt signaling, c-mcy expression and ultimately cell migration. In breast cancer, invasion and metastasis is more extensive in breast cancer cells that lack the alpha isoform of the ER (ERα). Breast cancer cells that are positive for ERα have a more epithelial architecture while; ERα-negative cells have a more invasive phenotype. Importantly, this effect is not static and the transition from an epithelial to a more mesenchymal phenotype (EMT) has been shown to occur. During EMT, cancer cells lose the expression of E-cadherin and γ-catenin, proteins that promote cell to cell contact and gain markers of a mesenchymal, and thus more migratory and invasive phenotype, such as Snail, vimentin, N-cadherin and fibronetin [91, 92]. Recently it has been shown that EMT can be inhibited and the modulation of ERα signaling by FOXO3a (a forkhead family transcription factor) is a key mechanistic driver of this effect. Evidence for this includes the fact that ERα synthesis can be controled by FOXO3a, [93, 94] and FOXO3a is also greatly reduced in ERαnegative breast cancers that are driven by HER2 [72, 93]. Importantly, EGCG repressed the invasive phenotype of breast cancer cell growth that was driven by HER2 [95]. Specifically, treatment of NF639 breast cancer cells with EGCG (87 µM) increased the protein expression of E-cadherin, γ-catenin, FOXO3a and ERα and decreased the protein expression of Snail [95]. The increase in FOXO3a by EGCG promotes ERα signaling and a less malignant phenotype in breast cancer cells. The phenotype of these breast cancer cells changes to a more epithelial type partly due to the inhibition of Snail, which represses the expression of Ecadherin [92]. Further evidence for the role of FOXO3a in this phenotypic change is the fact that FOXO3a inhibited cell migration, invasiveness in Matrigel as well as TGF-β1 stimulated invasion [95]. Importantly, ERα signaling was required for this change toward a more epithelial phenotype. Therefore, EGCG plays an important role in decreasing the invasiveness of HER2 driven breast cancer cells by upregulating ERα through the stimulation of FOXO3a gene expression. These studies provide further evidence for the clinical usefulness of EGCG,

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as inactive FOXO3a expression was shown to correlate with the poor survival of breast cancer patients [96].

EGCG AS A NEW CLASS OF CHEMICAL CHAPERONES IN VITRO Since EGCG has a wide range of effects in a variety of cancer models, new modes of action have been proposed. One of these is a potential role for EGCG as a new class of chemical chaperones [97]. This theory was put forward due to the fact that both EGCG and protein chaperones interact transiently with numerous proteins. For example, both EGCG and chemical chaperones regulate endoplasmic reticulum stress, as the GADD153 gene is induced by both stress [98] and EGCG [99, 100]. More evidence for this theory is the structural similarity between the chemical chaperone trehalose and EGCG, as both have eight hydroxyl groups. To test this theory, in silico analysis was used to assess the mobility and flexibility of the galloyl group and the results showed that EGCG existed in variable conformations and thus could interact with numerous molecular targets [97]. The presence of the galloyl group also provided more possible conformations compared to the other green tea catechins. These results support previous surface plasmon resonance studies that demonstrated that the binding of EGCG to DNA and RNA was reversed [101, 102] in a similar manner to that of chemical chaperones. While this theory will be strengthened by further tertiary structural analysis of the EGCG-protein complex, the results to date provide evidence to suggest that EGCG acts as a new class of chemical chaperones.

IN VIVO EFFECTS AND MOLECULAR MECHANISMS OF EGCG The majority of studies investigating the effects of green tea constituents in in vivo breast cancer models have focused on green tea mixtures rather than purified individual catechins. The earliest studies focused on chemical-induced mammary carcinogenesis in rats and demonstrated a protective effect of green tea compounds on tumor burden and survival [35, 103-107]. However, it is still unclear whether this protection is greater at the pre- or postinitiation stage. Various studies using either GTE or purified EGCG have also been conducted using breast cancer cell xenografts in mice [39, 108, 109]. For example, MDA-MB-231 breast cancer cell xenografts were used to illustrate that tumor growth, tumor weight and endothelial vessel density decreased following GTE (1.25 – 2.5 g/l) consumption compared to control [108]. Delayed tumor growth onset, rate of tumor growth, tumor volume and metastasis was also shown following the administration of a green tea polyphenol mixture in the drinking water of BALB/c mice inoculated with 4T1 mouse mammary carcinoma cells [110]. These effects were associated with an increase in the Bax/Bcl2 ratio and caspase-3 activation, demonstrating that the induction of apoptosis is a major mechanism for the tumor suppression. There have also been studies that focused on the effects of purified EGCG. Liao et al. (1995) [109] was the first to demonstrate that EGCG (1mg/mouse/day, i.p., 14 days) reduced tumor size in female athymic nude mice inoculated with MCF-7 cells. More recently, Thangapazham et al. (2007) [39] conducted a study using female athymic nude mice


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inoculated with MDA-MB-231 human breast cancer cells. Mice received 3 mg of green tea polyphenols (GTP) in the drinking water or EGCG (1 mg) by oral gavage. The treatments began on the day of cell inoculation and continued for 10 weeks. This treatment protocol more closely represents chemoprevention because the drug treatment began before palpable tumors had formed. Additionally at the conclusion of the study the control tumors were onethird the size (~30 mm3) of tumors from typical xenograft studies that begin when the tumor volume reaches ~100 mm3. In this model, both EGCG and the green tea mixture suppressed tumor growth (45 and 61%, respectively) and decreased incidence (10 and 20%, respectively) and this suggests that EGCG is predominantly responsible for the chemopreventative actions of green tea. Further examination of tumor sections demonstrated that GTP increased the number of apoptotic cells by 3.5-fold and EGCG increased apoptotic cells by 2.6-fold [39]. PCNA staining also revealed that proliferation of tumor cells was decreased by both treatments. These findings have been supported by other more recent studies that followed a more standard treatment protocol, as treatment with EGCG (25-50 mg/kg/d, 5-10 weeks) began once palpable tumors (derived from MDA-MB-231 cell xenografts) had formed [34, 49, 111]. Results in all of these studies showed a modest but statistically significant suppression of ERnegative breast cancer xenografts following EGCG administration. Mechanisms for this suppression were determined and the results from both Western blotting and immunohistochemistry demonstrated that EGCG (25 mg/kg, ip) caused a significant decrease in Akt, bRaf, VEGF and VEGFR-1 [34, 111]. While no other markers for changes in apoptosis or angiogenesis were determined in these studies, Akt is critical for breast cancer cell proliferation [112] and numerous other studies have shown that EGCG inhibits angiogenesis [71, 72, 81, 82]. Apoptosis was also linked with tumor suppression following 50 mg/kg of EGCG for 31 days, as Bax, cleaved PARP and caspase-3 activity were increased in tumors from treated mice compared to tumors from control mice [49]. These authors also demonstrated that chymotrypsin-like activity was decreased and this correlated with a modest increase in IkBa protein expression. Importantly, this study also showed that apoptosis in cancer cells could be induced by in vivo inhibition of the chymotrypsin-like proteasome and these results were further supported by in vitro studies in MDA-MB-231 cells. Overall, recent studies in ER-breast cancer models have shown that EGCG elicits modest tumor suppression through an induction of apoptosis and modulation of cell signaling proteins.

IMPROVING THE EFFICACY AND BIOAVAILABILITY OF EGCG Many of the in vitro effects elicited by EGCG are produced following the use of high concentrations (~80-150 µM). Due to the instability of EGCG and its extensive first pass metabolism [113], these concentrations are not achievable in vivo where plasma concentrations are aproximately 1-10-fold lower [114, 115]. Therefore, clinical trials with EGCG and GTP have relied on extremely high doses. While patients in these clinical studies were relatively free from side effects, the sheer volume of the medication required in these trials was not well tolerated [17, 115] and the maximum tolerated dose of oral, once-daily GTE was determined to be 3 g/m2 per day [116]. Therefore, stability and bioavailability problems, which result in the need for very high doses, are significant hurdles that EGCG

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must overcome in order to make significant clinical progress. One way to circumvent this problem would be to provide EGCG as a pro-drug. The first attempts at this involved the introduction of peracetate groups, in order to protect the reactive hydroxyl groups of EGCG. In cell free conditions this peracetate-EGCG was efficiently converted back to the parent compound [117]. Further studies with this compound in MDA-MB-231 breast cancer cells demonstrated that it was converted to EGCG, which accumulated in 2.4-fold greater amounts compared to standard EGCG following treatment at 50 µM [49]. In all in vitro assays examined, peracetate-EGCG was more potent than standard EGCG (namely, chymotrysinlike activity, PARP cleavage, caspase-3 activity, and protein expression of Bax, p27 and IkBa) [49]. Importantly, these in vitro results correlated with tumor suppression in vivo. Specifically, peracetate-EGCG (50 mg/kg/d, 31d, sc) decreased tumor growth by 54% compared to control, while EGCG decreased tumor growth by 23% [49]. Mechanisms which were elicited in vitro were also shown to be relevant to tumor suppression as peractetateEGCG inhibited tumor proteasome activity in vivo, as IkBa, p27, and Bax proteins were all accumulated in tumors from peracetate treated mice [49]. This study showed that there are numerous advantages to administering EGCG in peracetate form. Specifically, peracetateEGCG was more stable in neutral and slightly alkaline pH than EGCG and more readily absorbed into the tumor cells where it was subsequently hydrolyzed back to EGCG. While these results are promising, peracetate-EGCG was parentally administered at a high dose, namely 50 mg/kg. Studies using a more realistic route of administration have also been performed. Specifically, orally administered O-acyl derivatives of EGCG have also shown anti-tumor activity in a skin cancer model. The results with these O-acyl derivatives are relevant because they have improved bioavailability, stability and also convert back to EGCG in vivo [118]. An important component of this work was that the O-acyl derivatives were synthesized using green tea leaves as the starting material. Four O-acyl derivatives were synthesized with increasing acyl side chains and one derivative contained a branched side chain. These derivatives were administered orally at 50-53 mg/kg for a total of 20 weeks. All of the derivatives reduced incidence, number of skin tumors and percent survival of the mice [119]. Furthermore, there was a significant decrease in the anti-tumor activity with an increase in the size and branching of the acyl side chain. One downfall of this study was that EGCG was not examined alongside the derivatives to demonstrate the increased potency of the derivatives. However, the study provides important evidence for anti-tumor activity of orally administered derivatives of EGCG. While these results are even more promising due to the fact that the derivative was given orally, the compounds need to be examined in breast cancer models in order to demonstrate efficacy toward this type of cancer. Other attempts to improve the effectiveness of EGCG have involved the use of combination therapy. These studies have used both a conventional breast cancer drug (tamoxifen) and a natural polyphenolic compound (curcumin) in combination with EGCG. Specifically, The combination of EGCG and curcumin was synergistically cytotoxic toward ER-negative, but not ER-positive breast cancer cells [34]. These results correlated with a 263±16% increase in the proportion of cells in G2/M phase and a 40±4% decrease in G0/G1 phase cells compared to control following EGCG (20 µM) + curcumin (3 µM) [34]. These in vitro results translated to in vivo efficacy as EGCG (25 mg/kg, ip) + curcumin (200 mg/kg, po) significantly decreased tumor volume compared to all other treatment groups and 49% compared to control in an MDA-MB-231 xenograft model of ER-negative tumorigenesis


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[34]. A decrease in angiogenesis was proposed as the major mechanism for this effect as the protein expression of VEGFR-1 was decreased 78% following treatment with EGCG + curcumin. Other combination studies with tamoxifen showed an even greater tumor suppression, as EGCG (25 mg/kg,ip) + tamoxifen (75 µg/kg, po) decreased tumor volume and tumor weight 71 and 78%, respectively compared to control [111]. The mechanism for this effect is likely to be initiated via modulation of EGFR activity, as protein levels of the EGFR and its active phosphorylated form were both significantly reduced (78% compared to control) by EGCG + tamoxifen treatment. The similar decrease in both the unphosphorylated and phosphorylated forms indicates that the signaling capacity of the pathway as a whole was reduced by approximately 78% by the combination treatment [111]. Combination treatment also caused a similar reduction in the protein expression of mammalian target of rapamycin (mTOR). The inhibition of mTOR is of particular significance, as this is a key regulator of protein synthesis within the cell (Figure 1). Furthermore, studies in other cancer models have shown that a dual suppression of EGFR and mTOR resulted in significant tumor suppression [120]. Importantly, the combination of EGCG and tamoxifen also decreased angiogenesis by decreasing the protein expression of both VEGF and VEGFR-1 in the tumor, as shown by both Western blotting and immunohistochemistry [111]. A final component to the mechanism involved a decrease in the protein expression of cytochrome P5401B1 (CYP1B1), a protein that plays a variety of roles in both the treatment and development of breast cancer. Specifically, inhibition of CYP1B1 is critical to breast tumor formation, as CYP1B1 null mice are protected against DMBA-induced mammary tumors compared to wild-type mice [121]. Additionally, CYP1B1 can inactivate the cancer dug flutamide [122] and is also induced in breast cancer cells following treatment with docetaxel [123]. Therefore, it plays an important role in the development of resistance to chemotherapy. Since there was a 78% decrease in the tumor protein expression of CYP1B1 following treatment with EGCG + tamoxifen, this provides another critical component to the mechanism of action of this drug combination. An important aspect of this combination therapy is the fact that the doses of both drugs were much lower than those typically used in similar studies. For example, combination therapy with a PDK-1/Akt inhibitor has been used to sensitize MDA-MB-231 cells to the effects of tamoxifen. In this study tamoxifen (60 mg/kg) was unable to suppress the growth of MDA-MB-231 xenografts, but the addition of OUS-03012 (100 mg/kg) showed significant tumor suppression of 50% compared to control [124]. This result further demonstrates that tamoxifen can be used in combination therapies for the treatment of ER-negative breast cancer. However, it was much more effective when combined with EGCG, as this combination produced a greater level of tumor suppression (71%) at a much lower dose of tamoxfien (75 µg/kg) [111]. Overall these combination studies have shown tumor suppression following the lowest dose of EGCG used to date and thus provide another role for the use of EGCG in ER-negative breast cancer. The only research using a drug combination that administered green tea orally was conducted with GTE and tamoxifen. In this study, GTE (2.5 g/l) was administered in the drinking water and tamoxifen (20 mg) was inoculated subcutaneously as a slow-release pellet. Treatment continued for 52 days after the establishment of MCF-7 cell xenografts in mice. The results showed that combination treatment elicited tumor suppression that was greater than each individual treatment and was also 81% smaller than tumors from control mice [48].

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These results correlated with a significant decrease (46%) in blood vessel density in the tumors from GTE + tamoxifen treated mice. Additionally, TUNEL analysis revealed a 2.5fold increase in the number of apoptotic tumor cells following combination treatment, compared to control [48]. Importantly, the author also reported a dose-dependent increase in the amount of three major green tea catechins (EC, ECG, EGCG) in the mammary fat pad of mice that drank GTE (0.625 – 2.5 g/l) for 4 days. Interestingly, EGCG (2.5 g/l) accumulated to the highest level of all the green tea catechins (45 ng/g) and this demonstrates that green tea catechins administered orally can reach the mammary tissue. Demonstrating that the drug reaches the target tissue is an important component of breast cancer drug design. However another important aspect will be providing the drug at a dose that is low enough to ensure that patient compliance remains high. This balance between high oral activity and a low oral dose is a significant challenge for future research investigating EGCG as a treatment for breast cancer.

CONCLUSION EGCG induces apoptosis in ER-positive and ER-negative breast cancer cells in vitro and the effect is not influenced by the hormone receptor status of the cell line. Many mechanisms contribute to this effect, which cumulate in the induction of apoptosis and the inhibition of angiogenesis. Importantly, many of the in vitro effects elicited by EGCG have also been documented in ER-negative xenograft models following treatment with either EGCG or GTE. This demonstrates that in vitro studies are useful for dictating / predicting in vivo results with this multifaceted phytochemical. The latest research with EGCG indicates that there are new roles for this compound in treatment-resistant breast cancer, including those cancers resistant to trastuzumab (Herceptin). This new in vitro role for EGCG suggests a promising future for this drug. Especially since in vivo studies have demonstrated that the efficacy of EGCG can be increased by synthesizing pro-drug analogs of EGCG as well as through the use of combination therapy with EGCG. These latest studies provide a basis for the development of highly active, orally available analogs of EGCG that could, one day, significantly impact on the lives of women with treatment-resistant breast cancer

REFERENCES [1]

[2]

[3]

Middleton, EJ; Kandaswami, C; Theoharides, TC. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacological Reviews 2000 52,673-751. Yang, CS; Chen, L; Lee, M-J; Landau, JM. Effects of tea on carcinogenesis in animal models and humans. In: American Institute for Cancer Research, editor. Dietary Phytochemicals in Cancer Prevention and Treatment. New York: Plenum Press; 1996; 51-61. Arts, ICW; van de Putte, B; Hollman, PC. Catechin contents of food commonly consumed in The Netherlands.1. Fruits, vegetables, staple foods and processed foods. Journal of Agricultural Food Chemistry, 2000 48,1746-1751.

Utility of Epigallocatechin Gallate … [4] [5] [6]

[7]

[8] [9]

[10]

[11]

[12] [13] [14]

[15] [16]

[17]

[18]

[19]

[20]

315

Graham, NH. Green tea composition, consumption, and polyphenol chemistry. Preventive Medicine, 1992 21,334-350. Muhktar, H; Wang, ZY; Katiyar, SK; Agarwal, R. Tea components: antimutagenic and anticarcinogenic effects. Preventive Medicine, 1992 21,351-360. Isbrucker, RA; Edwards, JA; Wolz, E; Davidovich, A; Bausch, J. Safety studies on epigallocatechin gallate (EGCG) preparations. Part 2: dermal, acute and short-term toxicity studies. Food and Chemical Toxicology, 2006 44,636-650. Yamane, T; Nakatani, H; Kikuoka, N; Matsumoto, H; Iwata, Y; Kitao, Y; Oya, K; Takahashi, T. Inhibitory effects and toxicity of green tea polyphenols for gastrointestinal carcinogenesis. Cancer, 1996 77,1662-1667. Kao, Y-H; Hiipakka, RA; Liao, S. Modulation of endocrine systems and food intake by green tea epigallocatechin gallate. Endocrinology, 2000 141,980-987. Galati, G; Lin, A; Sultan, AM; O'Brien, PJ. Cellular and in vivo hepatotoxicity caused by green tea phenolic acids and catechins. Free Radical Biology and Medicine, 2006 40,570-580. Goodin, MG; Bray, BJ; Rosengren, RJ. Sex- and strain-dependent effects of epigallocatechin gallate (EGCG) and epigallocatechin (ECG) in the mouse. Food and Chemical Toxicology, 2006 44,1496-1504. Goodin, MG; Fertuck, KC; Zacharewski, TR; Rosengren, RJ. Estrogen receptormediated actions of polyphenolic catechins in vivo and in vitro. Toxicological Sciences, 2002 69,354-361. Goodin, MG; Rosengren, RJ. Epigallocatechin gallate modulates CYP450 isoforms in the female Swiss Webster mouse. Toxicological Sciences, 2003 76,262-270. Bun, SS; Bun, H; Guédon, D; Rosier, C; Ollivier, E. Effect of green tea extracts on liver functions in Wistar rats. Food and Chemical Toxicology, 2006 44,1108-1113. Gloro, R; Hourmand-Ollivier, I; Mosquet, B; Mosquet, L; Rousselot, P; Salamé, E; Piquet, MA; Dao, T. Fulminant hepatitis during self-medication with hydroalcoholic extract of green tea. European Journal of Gastroenterrology and Hepatology, 2005 17,1135-1137. Jimenez-Saenz, M; Martines-Sanchez, MD. Acute hepatitis associated with the use of green tea infusions. Hepatology, 2006 44,616-617. Garcia-Moran, S; Saez-Royuela, F; Gento, E; Lopez-Morante, A; Arias, L. Acute hepatitis associated with Camellia thea and Orthosiphon stamineus ingestion. Gastroenterology and Hepatology, 2004 27,559-560. Pisters, KMW; Newman, RA; Coldman, B; Shin, DM; Khurik, FR; Hong, WK; Glisson, BS; Lee, JS. Phase I trial of oral green tea extract in adult patients with solid tumors. Journal of Clinical Oncology, 2001 19,1830-1838. Ullman, U; Haller, J; Decourt, JP; Girault, N; Girault, J; Richard-Caudron, AS. A sinlge ascending dose study of epigallocatechin gallate in healthy volunteers. Journal of International Medical Research, 2003 31,88-101. Key, TJ; Sharp, GB; Appleby, PN; Beral, V; Goodman, MT; Soda, M; Mabuchi, K. Soya foods and breast cancer risk: a prospective study in Hiroshima and Nagasaki, Japan. British Journal of Cancer, 1999 81,1248-1256. Nagano, J; Kono, S; Preston, DL; Mabuchi, K. A prospective study of green tea consumption and cancer incidence, Hiroshima and Nagasaki, Japan. Cancer Causes and Control, 2001 12,501-508.

316

R. J. Rosengren

[21] Suzuki, Y; Tsubono, Y; Nakaya, N; Suzuki, Y; Koizumi, Y; Tsuji, I. Green tea and the risk of breast cancer: pooled analysis of two prospective studies in Japan. British Journal of Cancer, 2004 90,1361-1363. [22] Wu, AH; Yu, MC; Tseng, C; Hankin, J; Pike, MC. Green tea and risk of breast cancer in Asian Americans. International Journal of Cancer, 2003 106,574-579. [23] Wu, Y; Hooper, AT; Zhong, Z; Witte, L; Bohlen, P; Rafii, S; Hicklin, DJ. The vascular endothelial growth factor receptor (VEGFR-1) supports growth and survival of human breast carcinoma. International Journal of Cancer, 2006 119,1519-1529. [24] Sun, CL; Yuan, JM; Koh, WP; Yu, MC. Green tea, black tea and breast cancer risk: a meta-analysis of epidemiological studies. Carcinogenesis, 2006 27,1310-1315. [25] Nakachi, K; Suemasu, K; Suga, K; Takeo, T; Imai, K; Higashi, Y. Influence of drinking tea on breast cancer malignancy among Japanese patients. Japanese Journal of Cancer Research, 1998 89,254-261. [26] Inoue, M; Tajima, K; Mizutani, M; Iwata, H; Iwase, T; Miura, S; Hirose, K; Hamajima, N; Tominaga, S. Regular consumption of green tea and the risk of breast cancer recurrence: follow-up study from the Hospital-based Epidemiologic Research Program at Aichi Cancer Center (HERPACC), Japan. Cancer Letters, 2001 167,175-182. [27] Stuart, EC; Scandlyn, MJ; Rosengren, RJ. Role of epigallocatechin gallate (EGCG) in the treatment of breast and prostate cancer. Life Sciences, 2006 79,2329-2336. [28] Liu, ZJ; Zhu, BT. Concentration-dependent mitogenic and antiproliferative actions of 2methoxyestradiol in estrogen receptor-positive human breast cancer cells. Journal of Steroid Biochemistry and Molecular Biology, 2004 88,265-275. [29] Yuan, J; Koh, W; Sun, C; Lee, H; Yu, MC. Green tea intake, ACE gene polymorphism and breast cancer risk among Chinese women in Singapore. Carcinogenesis, 2005 26,1389-1394. [30] Wu, AH; Arakawa, K; Stanczyk, FZ; Van Den Burg, D; Koh, W; Yu, MC. Tea and circulating estrogen levels in postmenopausal Chinese women in Singapore. Carcinogenesis, 2005 26,976-980. [31] Liang, Y-C; Lin-Shiau, S-Y; Chen, C-F; Lin, J-K. Inhibition of cyclin-dependent kinases 2 and 4 activities as well as induction of Cdk inhibitors p21 and p27 during growth arrest of human breast carcinoma cells by (-)-epigallocatechin-3-gallate. Journal of Cellular Biochemistry, 1999 75,1-12. [32] Morre', DJ; Bridge, A; Wu, L-Y; Morre', DM. Preferential inhibition by (-)epigallocatechin-3-gallate of the cell surface NADH oxidase and growth of transformed cells in culture. Biochemical Pharmacology, 2000 60,937-946. [33] Chisholm, K; Bray, BJ; Rosengren, RJ. Tamoxifen and epigallocatechin gallate (EGCG) are synergistically cytotoxic to MDA-MB-231 human breast cancer cells. Anticancer Drugs, 2004 15,889-897. [34] Somers-Edgar, TJ; Scandlyn, MJ; Stuart, EC; Le Nedelec, MJ; Valentine, SP; Rosengren, RJ. The combination of epigallocatechin gallate and curcumin suppresses ERα- breast cancer cell growth in vitro and in vivo. International Journal of Cancer, 2008 122,1966-1971. [35] Kavanagh, KT; Hafer, LJ; Kim, DW; Mann, KK; Sherr, DH; Rogers, AE; Sonenshein, GE. Green tea extracts decrease carcinogen-induced mammary tumor burden in rats and rate of breast cancer cell proliferation in culture. Journal of Cellular Biochemistry, 2001 82,387-398.


317

[36] Masuda, M; Suzui, M; Lim, JT; Deguchi, A; Soh, JW; Weinstein, IB. Epigallocatechin3-gallate decreases VEGF production in head and neck and breast carcinoma cells by inhibiting EGFR-related pathways of signal transduction. Jouranl of Experimental and Therapeutic Oncology, 2002 2,350-359. [37] Masuda, M; Suzui, M; Lim, JTE; Weinstein, IB. Epigallocatechin-3-gallate inhibits activation of HER-2/neu and downstream signaling pathways in human head and neck and breast carcinoma cells. Clinical Cancer Research, 2003 9,3486-3491. [38] Roy, AM; Baliga, MS; Katiyar, SK. Epigallocatechin-3-gallate induces apoptosis in estrogen receptor-negative human breast carcinoma cells via modulation in protein expression of p53 and Bax and caspase-3 activation. Molecular Cancer Therapeutics, 2005 4,81-90. [39] Thangapazham, RL; Singh, AK; Sharma, A; Warren, J; Gaddipati, JP; Maheshwari, RK. Green tea polyphenols and its constituent epigallocatechin gallate inhibits proliferation of human breast cancer cells in vitro and in vivo. Cancer Letters, 2007 245,232-241. [40] Stuart, EC; Larsen, L; Rosengren, RJ. Potential mechanisms for the synergistic cytotoxicity elicited by 4-hydroxytamoxifen and epigallocatechin gallate in MDA-MB231 cells. International Journal of Oncology, 2007 30,1407-1412. [41] Stuart, EC; Rosengren, RJ. The combination of raloxifene and epigallocatechin gallate suppresses growth and induces apoptosis in MDA-MB-231 cells. Life Sciences, 2008 82,943-948. [42] Eddy, S; Kane, S; Sonenshein, G. Trastuzumab-resistant HER2-driven breast cancer cells are sensitive to epigallocatechin-3 gallate. Cancer Research, 2007 67,9018-9023. [43] Ahmad, N; Feyes, DK; Nieminen, AL; Agarwal, R; Mukhtar, H. Green tea constituent epigallocatechin-3-gallate and induction of apoptosis and cell cycle arrest in human carcinoma cells. Journal of the National Cancer Institute, 1997 89,1881-1886. [44] Chen, C; Shen, G; Hebbar, V; Hu, R; Owuor, ED; Kong, A-NT. Epigallocatechin-3gallate-induced stress signals in HT-29 human colon adenocarcinoma cells. Carcinogenesis, 2003 24,1369-1378. [45] Chen, ZP; Schell, JB; Ho, CT; Chen, KY. Green tea epigallocatechin gallate shows a pronounced growth inhibitory effect on cancerous cells but not on their normal counterparts. Cancer Letters, 1998 129,173-179. [46] Gupta, S; Hussain, T; Mukhtar, H. Molecular pathway for (-)-epigallocatechin-3gallate-induced cell cycle arrest and apoptosis of human prostate carcinoma cells. Archives of Biochemistry and Biophysics, 2003 410,177-185. [47] Masuda, M; Suzui, M; Weinstein, IB. Effects of epigallocatechin-3-gallate on growth, epidermal growth factor receptor signaling pathways, gene expression, and chemosensitivity in human head and neck squamous cell carcinoma cell lines. Clinical Cancer Research, 2001 7,4220-4229. [48] Sartippour, MR; Pietras, R; Marquez-Garban, DC; Chen, H-W; Heber, D; Henning, SM; Sartippour, G; Zhange, L; Lu, M; Weinberg, O; Rao, JY; Brooks, MN. The combination of green tea and tamoxifen is effective against breast cancer. Carcinogenesis, 2006 27,2424-2433. [49] Landis-Piwowar, KR; Huo, C; Chen, D; Milacic, V; Shi, G; Chan, TH; Dou, QP. A novel prodrug of the green tea polyphenol (-)-epigallocatechin-3-gallate as a potential anticancer agent. Cancer Research, 2007 67,4303-4310.

318

R. J. Rosengren

[50] Hastak, K; Gupta, S; Ahmad, N; Agarwal, MK; M.L., A; Mukhtar, H. Role of p53 and NF-κB in epigallocatechin-3-gallate-induced apoptosis of LNCaP cells. Oncogene, 2003 22,4851-4859. [51] Yang, F; Oz, HS; Barve, S; de Villiers, WJS; McClain, CJ; Varilek, GW. The green tea polyphenol ()-epigallocatechin-3-gallate blocks Nuclear Factor-B activation by inhibiting IB kinase activity in the intestinal epithelial cell line IEC-6 Molecular Pharmacology, 2001 60,528-533. [52] Ahmad, N; Gupta, S; Mukhtar, H. Green tea polyphenol epigallocatechin-3-gallate differentially modulates nuclear factor κB in cancer cells versus normal cells. Archives of Biochemistry and Biophysics, 2000 376,338-346. [53] Afaq, F; Adhami, VM; Ahmad, N; Mukhtar, H. Inhibition of ultraviolet B-mediated activation of nuclear factor B in normal human epidermal keratinocytes by green tea constituent (-)-epigallocatechin-3-gallate. Oncogene, 2003 22,1035-1044. [54] Aggarwal, BB. Nuclear Factor-kappaB: the enemy within. Cancer Cell, 2004 6,203208. [55] Lin, A; Karin, M. NF-kappaB in cancer: A marked target. Seminars in Cancer Biology, 2003 13,107-114. [56] Monks, NR; Biswas, DK; Pardee, AB. 2004. Blocking anti-apoptosis as a strategy for cancer chemotherapy: NF-kappaB as a target. Journal of Cell Biochemistry, 2004 92,646-650. [57] Biswas, DK; Shi, Q; Baily, S; Strickland, I; Ghosh, S; Perdee, AB; Iglehart, JD. NF-kB activation in human breast cancer specimens and its role in cell proliferation and apoptosis. Proceedings of the National Academy of Sciences of the United States of America 2004 101,10137-10142. [58] Gartel, AL; Felicianok, C; Tyner, AL. A new method for determining the status of p53 in tumor cell lines of different origin. Oncology Research, 2003 13,405-408. [59] Bigelow, RL; Cardelli, JA. The green tea catechins, (-)-epigallocatechin-3-gallate (EGCG) and (-)-epicatechin (ECG), inhibit HGF/Met signaling in immortalized and tumorigenic breast epithelial cells. Oncogene 2006 25,1922-1930. [60] Casalini, P; Iorio, MV; Galmozzi, E; Menard, S. Role of HER family in development and differentiation. Journal of Cellular Physiology, 2004 200,343-350. [61] Osaki, M; Oshimura, M; Ito, H. PI3K-Akt pathway: its functions and alterations in human cancer. Apoptosis 2004 9,667-676. [62] Pearson, G; Robinson, F; Gibson, TB; Xu, B-E; Karandikar, M; Berman, K; Cobb, MH. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocrine Reviews, 2001 22,153-183. [63] Lopes, UG; Erhardt, P; Yao, R; Cooper, GM. p53-dependent Induction of apoptosis by proteasome inhibitors Journal of Biological Chemistry, 1997 272,12893-12896. [64] Adams, J. Potential for proteasome inhibition in the treatment of cancer. Drug Discovery Today, 2003 8,307-315. [65] Dou, QP; Smith, DM; Daniel, KG; Kazi, A. Interruption of tumor cell cycle progression through proteasome inhibtion: implications for cancer therapy. Progess in Cell Cycle Research, 2003 2003,572-579. [66] Biswas, DK; Inglehar, JD. Linkage between EGFR family receptors and nuclear factorkB (NF-kB) signaling in breast cancer. Journal of Cell Physiology, 2006 209,645-652.


319

[67] Folkman, J. Angiogenesis in cancer, vascular, theumatoid and other disease. Nature Medicine, 1995 1,27-31. [68] Guidi, AJ; Fischer, L; Harris, JR; Schnitt, SJ. Microvessel density and distribution in ductal carcinoma in situ of the breast. Jouranl of the National Cancer Institute, 1994 86,614-619. [69] Bosari, S; Lee, AKC; DeLellis, RA; Wiley, BD; Heatley, GJ; Silverman, MJ. Microvessel quantification and prognosis in invasive breast carcinoma. Human Pathology, 1992 23,755-761. [70] Cao, Y; Cao, R. Angiogenesis inhibited by drinking tea. Nature Medicine, 1999 398,381. [71] Jung, YD; Kim, MS; Shin, BA; Chay, KO; Ahn, BW; Liu, W; Bucana, CD; Gallick, GE; Ellis, LM. EGCG, a major component of green tea, inhibits tumour growth by inhibiting VEGF induction in human colon carcinoma cells. British Journal of Cancer, 2001 84,844-850. [72] Garbisa, S; Biggin, S; Cavallarin, N; Sartor, L; Benelli, R; Albini, A. Tumor invaion: molecular shears blunted by green tea. Nature Medicine, 1999 5,1216. [73] Connolly, DT; Heuvelman, DM; Nelson, R; Olander, JV; Eppley, BL; Delfino, JJ; Siegel, NR; Leimgruber, RM; Feder, J. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. Journal of Clinical Investigation, 1989 84,699-702. [74] Takahashi, Y; Kitadai, Y; Bucana, CD; Cleary, KR; Ellis, LM. Expression of vascular endothelial growth factor and its receptor, KDR, correclates with vascularity, metastasis, and proliferation of human colon cancer. Cancer Research, 1995 55,39643968. [75] Ellis, LM; Liu, W; Wilson, M. Down-regulation of vascular endothelial growth factor in human colon carcinoma cell lines by antisense transfection decreases endothelial cell proliferation. Surgery, 1996 120,871-878. [76] Qu, H; Nagy, JA; Senger, DR; Dvorak, HF; Dvorak, AM. Ultrastructural localization of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) to the abluminal plasma membrane and vesiculovacuolar organelles of tumor microvascular endothelium. Journal Histochemical Cytochemistry, 1995 43,381-389. [77] Ferra, N. Role of vaxcular endothelial growth factor in regulation of physiological angiogenesis. American Jouranl of Physiology and Cell Physiology, 2001 280,C1358C1366. [78] Appleton, IA; Brown, NJ; Willis, D; Colville-Nash, PR; Alam, C; Brown, JR; Willoughby, DA. The role of vascular endothelial growth factor in a murine chronic granulomatous tisue air pouch model of angiogenesis. Journal of Pathology, 1996 180,90-94. [79] Schaeppi, JM; Wood, JM. Targeting vascular endothelial growth factor (VEGF) for anti-tumor therapy, by anti-VEGF neutralizing monoclonal antibodies or by VEGF receptor tyrosine kinase inhibition. Cancer Metastasis Reviews, 1999 18,473-481. [80] Lamy, S; Gingras, D; Beliveau, R. Green tea catechins inhibit vascular endothelial growth factor receptor phosphorylation. Cancer Research, 2002 62,381-385. [81] Fassina, G; Venè, R; Morini, M; Minghelli, S; Benelli, R; Noonan, DM; Albini, A. Mechanisms of inhibition of tumor angiogenesis and vascular tumor growth by epigallocatechin-3-gallate. Clinical Cancer Research, 2004 10,4865-4873.

320

R. J. Rosengren

[82] Liu, JD; Chen, SH; Lin, CL; S.H., T; Liang, YC. Inhibition of melanoma growth and metastasis by combination with (-)-epigallocatechin-3-gallate and dacarbazine in mice. Journal of Cellular Biochemistry, 2001 83,631-642. [83] Hong, J; Smith, TJ; Ho, CT; August, DA; Yang, CS. Effects of purified green and black tea polyphenols on cyclooxygenase- and lipoxygenase-dependent metabolism of arachidonic acid in human colon mucosa and colon tumor tissues. Biochemical Pharmacology, 2001 62,1175-1183. [84] Lin, YL; Lin, JK. (-)-Epigallocatechin-3-gallate blocks the induction of nitric oxide synthase by down-regulating lipopolysaccharide-induced activity of transcription factor nuclear factor-B Molecular Pharmacology, 1997 52,465-472. [85] Tachibana, H; Koga, K; Fujimura, Y; Yamada, K. A receptor for green tea polyphenol EGCG. Nature Structural and Molecular Biology, 2004 11,380-381. [86] Wulf, G; Ryo, A; Liou, YC; Lu, KP. The prolyl isomerase Pin1 in breast development and cancer. Breast Cancer Research, 2003 5,76-82. [87] Lin, SY; Xia, W; Wang, JC; Kwong, KY; Spohn, B; Wen, Y; Pestell, RG; Hung, MC. β-Catenin, a novel prognostic marker for breast cancer: Its roles in cyclin D1 expression and cancer progression. Proceeding of the National Academy of Sciences of the United States of America, 2000 97,4262-4266. [88] Wulf, GM; Ryo, A; Wulf, GG; Lee, SW; Niu, T; Petkova, V; Lu, KP. Pin1 is overexpressed in breast cancer and cooperates with Ras signaling in increasing the transcriptional activity of c-Jun towards cyclin D1. The EMBO Journal, 2001 20,34593472. [89] Howe, LR; Brown, AMC. Wnt signaling and breast cancer. Cancer Biology and Therapy, 2004 3,36-41. [90] Kim, J; Zhang, X; Rieger-Christ, KM; Summerhayes, IC; Wazer, DE; Paulson, KE; Yee, AS. Suppression of Wnt signaling by the green tea compound (–)-epigallocatechin 3-gallate (EGCG) in invasive breast cancer cells: requirement of the transcriptional repressor HBP1. Journal of Biological Chemistry, 2006 281,10865-10876. [91] Garcia, M; Derocq, D; Freiss, G; Rochefort, H. Activation of estrogen receptor transfected into a receptor-negative breast cancer cell line decreases the metastatic and invasive potential of the cells. Proceeding of the National Academy of Sciences of the United States of America, 1992 89,11538-11542. [92] Fujita, N; Jaye, DL; Kajita, M; Geigerman, C; Moreno, CS; Wade, PA. MTA3, a Mi2/NuRD complex subunit, regulates an invasive growth pathway in breast cancer. Cell, 2003 113,207-219. [93] Guo, S; Sonenshein, GE. Forkhead box transcription factor FOXO3a regulates estrogen receptor alpha expression and is repressed by the Her-2/neu/Phosphatidylinositol 3Kinase/Akt signaling pathway. Molecular and Cellular Biology, 2004 24,8681-8690. [94] Madureira, PA; Varshochi, R; Constantinidou, D; Francis, RE; Coombes, RC; Yao, KM; Lam, EWF. The forkhead box M1 protein regulates the transcription of the estrogen receptor in breast cancer cells. Journal of Biological Chemistry, 2006 2814,25167-25176. [95] Belguise, K; Guo, S; Sonenshein, GE. Activation of FOXO3a by the green tea polyphenol epigallocatehin-3-gallate induces estrogen receptor α expression reversing invasive phenotype of breast cancer cells. Cancer Research, 2007 67,5763-5770.


321

[96] Hu, MCT; Lee, DF; Golfman, LS; Ou-Yang, F; Yang, JY; Zou, Y; Bao, S; Hanada, N; Saso, H; Kobayashi, R; Hung, MC. IκB kinase promotes tumorigenesis through Inhibition of forkhead FOXO3a. Cell, 2004 117,225-237. [97] Kuzuhara, T; Suganuma, M; Funiki, H. Green tea catechin as a chemical chaperone in cancer prevention. Cancer Letters, 2008 261,12-20. [98] Fornace Jr, AJ; Nebert, DW; Hollander, MC; Luethy, JD; Papathanasiou, M; Hargnoli, J; Holbrook, NJ. Mammalian genes coordinately regulated by growth arrest signals and DNA-damaging agents. Molecular and Cellular Biochemistry, 1989 9,4196-4203. [99] Fujiki, H. Green tea: health benefits as cancer preventative for humans. Chemical Record, 2005 5,119-132. [100] Suganuma, M; Kurusu, M; Suzuki, K; Tasaki, E; Fujiki, H. Green tea polyphenol stimulates cancer preventation effects of celecoxib in human lung cancer cells by upregulation of GADD153 gene. International Journal of Cancer, 2006 119,33-40. [101] Kuzuhara, T; Sei, Y; Yamaguchi, K; Suganuma, M; Fujiki, H. DNA and RNA as new binding targets of green tea catechins. Journal of Biological Chemistry, 2006 281,17446-17456. [102] Kuzuhara, T; Tanabe, A; Sei, Y; Yamaguchi, K; Suganuma, M; Hirota, F. Synergistic effects of multiple treatments, and both DNA and RNA direct bindings on, green tea catechins. Molecular Carcinogenesis, 2007 46,640-645. [103] Hirose, M; Hoshiya, T; Akagi, K; Futakuchi, M; Ito, N. Inhibition of mammary gland carcinogenesis by green tea catechins and other naturally occurring antioxidants in female Spraque-Dawley rats pretreated with 7,12-dimethylbenz[a]antracene. Cancer Letters, 1994 83,149-156. [104] Bhide, SV; Azuine, MA; Lahiri, M; Telang, NT. Chemoprevention of mammary tumor virus-induced and chemical carcinogen-induced rodent mammary tumors by natural plant products. Breast Cancer Research and Treatment, 1994 30,233-242. [105] Hirose, M; Hoshiya, T; Akagi, K; Takahashi, S; Hara, Y; Ito, N. Effects of green tea catechins in a rat multi-organ carcinogenesis model. Carcinogenesis, 1993 14,15491553. [106] Tanaka, H; Hirose, M; Kawabe, M; Sano, M; Takesada, Y; Hagiwara, A; Shirai, T. Post-initiation inhibitory effects of green tea catechins on 7,12dimethylbenz[a]anthracene-induced mammary gland carcinogenesis in female SpragueDawley rats. Cancer Letters, 1997 116,47-52. [107] Hirose, M; Nishikawa, A; Shibutani, M; Imai, T; Shirai, T. Chemoprevention of heterocyclic amine-induced mammary carcinogenesis in rats. Environmental and Molecular Mutagenesis, 2002 39,271-278. [108] Sartippour, MR; Heber, D; Ma, J; Lu, Q; Go, VL; Nguyen, M. Green tea and its catechins inhibit breast cancer xenografts. Nutrition and Cancer, 2001 40,149-156. [109] Liao, S; Umekita, Y; Guo, J; Kokontis, JM; Hiipakka, RA. Growth inhibition and regression of human prostate and breast tumors in athymic mice by tea epigallocatechin gallate. Cancer Letters, 1995 96,239-243. [110] Baliga, MS; Meleth, S; Katiyar, SK. Growth inhibitory and antimetastatic effect of green tea polyphenols on metastasis-specific mouse mammary 4T1 cells in vitro and in vivo. Clinical Cancer Research, 2005 11,1918-1927.

322

R. J. Rosengren

[111] Scandlyn, MJ; Stuart, EC; Somers-Edgar, TJ; Menzies, AR; Rosengren, RJ. A new role for tamoxifen in estrogen receptor negative breast cancer when it is combined with epigallocatechin gallate. British Journal of Cancer, 2008 99,1056-1063. [112] Ju, X; Katiyar, S; Wang, W; Liu, M; Jiao, X; Li, S; Zhou, J; Turner, J; Lisanti, MP; Russell, RG; Mueller, SC; Ojeifo, J; Chen, WS; Hay, N; Pestell, RG. Akt1 governs breast cancer progression in vivo. Proceedings of the National Academy of Sciences of the United States of America, 2007 104,7438-7443. [113] Lu, H; Meng, X; Yang, CS. Enzymology of methylation of tea catechins and inhibition of catechol-O-methyltransferase by (-)-epigallocatechin gallate. Drug Metabolism and Disposition, 2003 31,572-579. [114] Henning, SM; Niu, Y; Lee, NH; Thames, GD; Minutti, RR; Wang, H; Go, VL; Heber, D. Bioavailability and antioxidant activity of tea flavanols after consumption of green tea, black tea, or a green tea extract supplement. American Journal of Clinical Nutrition, 2004 80,1558-1564. [115] Chow, HH; Hakim, IA; Vining, DR; Crowell, JA; Ranger-Moore, J; Chew, WM; Celaya, CA; Rodney, SR; Hara, Y; Alberts, DS. Effects of dosing condition on the oral bioavailability of green tea catechins after single-dose administration of Polyphenon E in healthy individuals. Clinical Cancer Research, 2005 1511,4627-4633. [116] Laurie, SA; Miller, VA; Grant, SC; Kris, MG; Ng, KK. Phase I study of green tea extract in patients with advanced lung cancer. Cancer Chemotherapeutics and Pharmacology, 2005 55,33-38. [117] Lam, WH; Kazi, A; Kuhn, DJ; Chow, LMC; Chan, ASC; Dou, QP; Chan, TH. A potential prodrug for a green tea polyphenol proteasome inhibitor: evaluation of the peracetate ester of (−)-epigallocatechin gallate [(−)-EGCG]. Bioorganic and Medicinal Chemistry, 2004 12,5587-5593. [118] Landis-Piwowar, KR; Kuhn, DJ; Wan, SB; Chen, D; Chan, TK; Dou, QP. Evaluation of proteasome-inhibitor and apoptosis-inducing potencies of novel (-)-EGCG analogs and their prodrugs. Internation Journal of Molecular Medicine, 2005 15,735-742. [119] Vyas, S; Sharma, M; Sharma, PD; Singh, TV. Design, semisynthesis, and evaluation of O-acyl derivatives of (-)-epigallocatechin-3-gallate as antitumor agents. Journal of Agricultural Food Chemistry, 2007 55,6319-6324. [120] Buck, E; Eyzaguirre, A; Brown. E; Petti, F; McCormack, S; Haley, JD; Iwata, KK; Gibson, NW; Griffin, G. Rapamycin synergizes with the epderimal growth factor receptor inhibitor erlotinib in non-small-cell lung, pancreastic, colon, and breast tumors. Molecular Cancer Therapeutics, 2006 5,2676-2684. [121] Buters, JTM; Sakai, S; Richter, T; Pineau, T; Alexander, DL; Savas, U; Doehmer, J; Ward, JM; Jefcoate, CR; Gonzalez, FJ. Cytochrome P450 CYP1B1 determines susceptibility to 7,12-dimethylbenz[a]anthracene-induced lymphomas. Proceedings of the Nattional Academy of Sciences of the United States of America, 1999 96,1977-1982. [122] Rochat, BM; Murray, JM; Figg, GI; McLeod, WD. Human CYP1B1 and anticancer agent metabolism: mechanism for tumor-specific drug inactivation? Journal of Pharmacology and Experimental Therapeutics, 2001 296,537-541. [123] Martinez, VG; O'Connor, R; Clynes, M. CYP1B1 expression is induced by docetaxel: effect on cell viability and drug resistance. British Journal of Cancer, 2008 98,564-570.


323

[124] Weng, S-C; Kashida, U; Kulp, SK; Wang, D; Brueggemeier, RW; Shapiro, C; Chen, CS. Sensitizing estrogen receptor-negative breast cancer cells to tamoxifen with OSU03012, a novel celecoxib-derived phophoinositide-dependent protein kinase-1/Akt signaling inhibitor. Molecular Cancer Therapeutics, 2008 7,800-808.

In: Handbook of Green Tea and Health Research Editor: H. McKinley and M. Jamieson, pp.


Chapter 16

GREEN TEA CATECHINS IN COLORECTAL CANCER Seung Joon Baek and Mugdha Sukhthankar Laboratory of Environmental Carcinogenesis, Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA

ABSTRACT Colorectal cancer is a global problem that accounts for over 50,000 cancer-related deaths each year in the United States. Americans have about a one in 20 lifetime risk of developing colorectal cancer. It affects primarily those over 65, but risk starts increasing at age 40. Colorectal cancer develops following disruptions in key cancer-causing genes (oncogenes) like K-ras and β-catenin and tumor suppressor genes like gate keeper APC and p53, and early detection greatly increases the chances of survival. Most cancers are related to a combination of hereditary and environmental factors, and such factors can either contribute to the initiation of cancer or the prevention of tumor development. There is persuasive epidemiological and experimental evidence that a phytochemical-enriched diet may be involved in the prevention of colon cancer. Therefore, the use of dietary compounds for prevention and therapy of colorectal cancer would be of major importance with potentially fewer side effects than therapeutic drugs. Green tea has received much attention as a suitable dietary agent because of its anti-tumorigenic activity. The most active constituents of green tea are catechins, including epigallocatechin 3-gallate (EGCG), epigallocatechin (EGC), epicatechin-3-gallate (ECG) and epicatechin (EC). Many laboratories, including ours, have reported preventive effects with green tea components in cancers of the gastrointestinal tract, lung, skin, prostate, and breast. A mechanistic study indicated that green tea decreased the total levels of early carcinogenesis biomarkers and increased tumor suppressor proteins; in addition, reports related to new molecular targets affected by green tea in chemoprevention study have been increased. Since the preponderance of the data strongly indicates significant antitumorigenic benefits from green tea polyphenols, this chapter will summarize the current knowledge of molecular targets of green tea research in human colorectal cancer prevention.

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Seung Joon Baek and Mugdha Sukhthankar

1. INTRODUCTION Cancer, or malignant neoplasia, is an array of diseases wherein a group of cells display uncontrolled growth and metastasis. Cancer affects all age groups, even fetuses, but risk tends to increase with age. According to the American Cancer Society, cancer killed 7.6 million people in the world during 2007 [1; 2]. Cancer is caused by both external and internal factors that may work together or in a pattern to initiate or promote carcinogenesis. The lifetime risk (the risk of developing a disease during one’s lifetime or dying of the disease) for developing cancer in men is 1 in 2, and in women, it is 1 in 3. Cancer is second only to heart disease as the leading cause of death in the United States. Specifically, colorectal cancer is one of the most prevalent causes of cancer-related mortality in the western world [3]. The process of cancer development may be divided into at least three stages: initiation, promotion and progression [4]. Most advanced cancers are incurable, hence it is important to prolong or block the process of carcinogenesis through chemoprevention, which turns out to be a feasible strategy for cancer control and management [5]. Thus, further development of therapeutic and preventive means of controlling this disease are clearly needed, particularly as they pertain to gastrointestinal cancer [6; 7]. Epidemiological studies have suggested that nutrition plays an important role in carcinogenesis, and dietary factors have been estimated to account for up to 80% of cancers of the gastrointestinal tract. Approximately 30% of cancer morbidity and mortality might be prevented with proper adjustment of diets [8; 9; 10; 11; 12]. The basic theory of chemoprevention is to reduce the occurrence of cancer either by slowing, blocking, or reversing the development of the disease by the administration of natural or synthetic compounds [13]. In addition, the molecular mechanisms responsible for potential human health benefits derived from dietary components must be studied in vitro and validated in preclinical studies in animal models before strong support can be given for more extensive clinical trials.

2. COLORECTAL CANCER Colorectal cancer is the third most common cancer in the United States [14]. It develops slowly over many years and usually begins as a polyp: a benign growth of tissue that starts in the lining and grows towards the center of the colon or rectum. Early detection and removal of polyps may prevent formation of adenocarcinomas, which account for over 95% of colorectal cancers. Colorectal cancer is caused due to mutations in both tumor suppressors and oncogenes [15]. Broadly, there are two types of colorectal cancers: hereditary and sporadic.

2.1. Hereditary Cancers Familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPCC) are the two best-known hereditary colorectal cancers.

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Familial Adenomatous Polyposis Familial adenomatous polyposis is very rare. It causes less than 1% of all colorectal cancer cases. FAP is caused by mutations in the adenomatous polyposis coli (APC) gene, a tumor suppressor. One of the many roles of APC is regulating cell replication, and hence it earned the name “GATE KEEPER”. Around five hundred APC mutations have been identified so far [16]. The polyps themselves are benign but because of their usual high numbers, some of them might undergo a mutation in the normal copy of the APC gene and trigger the development of cancer. Hereditary Non-polyposis Colorectal Cancer Hereditary non-polyposis colorectal cancer (HNPCC, also known as Lynch syndrome) is caused by a mutation in one of the DNA mismatch repair (MMR) genes. The hMSH2 and hMLH1 are most commonly mutated MMR genes in HNPCC, and the other genes involved are hMSH6, PMS1 and PMS2 [17]. The aberrant operation of DNA mismatch repair systems lead to Microsatellite instability (MSI) and cause naturally occurring, highly repeated short DNA sequences, called microsatellites, to get shorter or longer than expected.

2.2. Sporadic Colorectal Cancer In most people without an inherited mutation predisposing them to colorectal cancer or a family history of it, a series of mutations is needed for colorectal cancer to develop (Fig. 1). It often takes many decades for these mutations to accumulate –hence the majority of colorectal cancer cases occur in the elderly. Mutations in APC are seen in 70 to 80% of sporadic tumors and often occur early in the development of colorectal cancer. During early stages of colorectal tumorigenesis, three ras genes are mutated (K-ras, N-ras, and H-ras) [18]. The p53

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gene is another tumor suppressor gene that is known to be mutated. The p53-encoding gene mutations in colorectal cancer occur in specific conserved regions of the gene and might be present in over 50% of the colorectal cancers [19]. In addition, the DCC (deleted in colorectal cancer) gene is mutated in 70% of colorectal cancers, and MMR genes are inactivated in around 15% of sporadic cases.

3. DIFFERENT SIGNALING PATHWAYS INVOLVED IN COLORECTAL CARCINOGENESIS Many signaling pathways play a pivotal role in colorectal tumorigenesis. Specifically, the cyclooxygenases (COX), p53, Wnt, and TGF-β pathways are important in the development of colorectal tumorigenesis (Fig. 2). COXs, key enzymes responsible for eicosanoid production, can profoundly influence cancer development, progression and therapeutic response. COX-2 is highly inducible by various cytokines, growth factors, and tumor promoters [20]. Elevated COX-2 levels are associated with inflammation, neoplastic transformation and metastasis [21; 22], and increased expression of mRNA and protein is found in the great majority of colorectal cancers [23; 24]. Mutations also commonly occur at the p53 tumor suppressor protein locus in many forms of cancer, including colorectal cancer. Thus, it is logical when studying colorectal cancer to consider the p53 mutation and the status of COX-2 expression.


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The over-activation of the Wingless/Wnt signaling pathway also leads to the expression of genes that favor cell growth and thus, contributes to many different cancers including colorectal cancer. APC and β-catenin are two major genes involved in this pathway [25]. In normal cells, APC interacts with β-catenin and forms a macromolecular complex with axin and glycogen synthase kinase-3 β (GSK-3β), in which β-catenin is subsequently directed towards ubiquitin-proteasomal degradation by phophorylation of GSK-3 β [26; 27; 28]. If this complex is not formed, then β-catenin increases in cytoplasm and translocates to the nucleus where it acts on transcription of many downstream genes. The β-catenin expression has been shown in 90% of the sporadic colorectal cancers and in 65% of HNPCC’s [29; 30]. Transforming growth factor-β (TGF- β) is part of a superfamily of proteins known as the transforming growth factor β superfamily. TGF- β initiates signaling by formation of heteromeric complexes of type I and type II serine/threonine kinase receptors [31; 32; 33]. The role of TGF-β in tumorigenesis is quite complex, but it appears that TGF-β functions both as a tumor suppressor in early tumor onset and as an oncogene during late tumor progression [34; 35]. TGF- β receptor II somatic mutations are found in patients with HNPCCs and in cancers that show microsatellite instability [34; 35]. De-regulation of one of the above pathways lies at the heart of the development and progression of human colorectal malignancies.

4. HEALTH BENEFITS OF GREEN TEA Green tea has been known to have many health benefits. It has been shown to decrease low-density lipoprotein and increase high-density lipoprotein [36; 37]. Subsequently, green tea has been shown to inhibit hypertension [38] and also to prevent cardiac hypertrophy [39]. Green tea has been used as a diet drink in women as it raises metabolic rates, fastens fat oxidation and ameliorates insulin sensitivity and glucose tolerance [40]. Green tea can even lead to the inhibition of HIV virus binding and hence be used as a supportive therapy for HIV patients [41] because oxalates present in tea help with HIV and general infections by cleaning up free iron, which leaves one less task for the immune system.

5. GREEN TEA AND CANCER Abundant experimental and epidemiological evidence accumulated mainly in the past decade from several centers worldwide provides a convincing argument that polyphenolic antioxidants present in green tea can reduce cancer risk in a variety of animal tumor bioassay systems. Tea consumption also assures protection against cancers induced by chemical carcinogens that involve the lung, forestomach, esophagus, duodenum, pancreas, liver, breast, colon, and skin in mice, rats, and hamsters [42]. In addition, many epidemiological studies suggest that green tea consumption shows beneficial effects on many cancers [43].

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6. GREEN TEA CATECHINS Four major polyphenol catechins in green tea include epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG) and, epicatechin (EC) (Fig. 3). In experimental models, catechins show a wide range of protective effects, including cardioprotective, chemoprotective, and antimicrobial properties. Catechin and epicatechin are epimers, with (-)-epicatechin and (+)-catechin being the most common optical isomers found in nature. Catechin derives its name from the plant catechu from where it was first isolated. Epigallocatechin and gallocatechin contain an additional phenolic hydroxyl group, which makes them stronger anti-oxidant agents compared to epicatechin and catechin, respectively, similar to the difference in pyrogallol compared to pyrocatechol. Catechin gallates are gallic acid esters of the catechins, such as EGCG (epigallocatechin gallate), which is the most abundant catechin in tea. EGCG is about 25-100 times more potent than Vitamin C in its antioxidant property [44].

7. GREEN TEA CATECHINS IN CANCER Green tea catechins have recently gained significant acceptance as cancer preventive agents. We and numerous other scientists have reported the cancer preventive activities of EGCG and green tea extract: inhibition of angiogenesis and prevention of cancer metastasis, induction of apoptosis, inhibition of inflammation, translocation of different genes, cell cycle regulation, growth prevention of tumors in vivo, inhibiting activation of IGF-1 and alteration of the expression of various oncogenes and tumor suppressor genes [45; 46; 47]. The role of green tea catechins in colorectal cancers is more significant as the catechins are absorbed into the gut [48]; however, their role in other cancers is also important. Recent studies point out that dietary cancer chemopreventive agents modulate multiple signaling pathways that interrupt the carcinogenic process and are also capable of extending one or more stages of carcinogenic process [13; 49].


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7.1. Effect on Angiogenesis and Cancer Metastasis Angiogenesis is a physiological process involving the growth of new blood vessels from pre-existing vessels in growth and development, as well as in wound healing. It is also an important step in the progression and conversion of tumors from a dormant state to a malignant state. Tumors induce blood vessel growth by secreting various growth factors which can induce capillary growth into the tumor and supply required nutrients allowing for tumor expansion [50]. There could be either mechanical or chemical stimulation of angiogenesis. Chemical stimulation, as the name suggests, is performed by various chemical substances or angiogenic proteins, including several growth factors like VEGF, bFGF, and TGF-β [51]. In addition, the role of various proteases such as urokinases, MMP, and tissue inhibitor of matrix metalloproteinases (TIMP) has also been extensively studied in tumor angiogenesis [52]. These proteins have been linked to be a molecular target of green tea catechins.

VEGF The vascular endothelial growth factor (VEGF) belongs to a sub-family of growth factors; the platelet-derived growth factor family, which are important signaling proteins involved in both vasculogenesis (the de novo formation of the embryonic circulatory system) and angiogenesis (the growth of blood vessels from pre-existing vasculature). VEGF has been known to be a key mediator of the abnormal angiogenesis in many hematologic malignancies and has been shown to be secreted by diverse types of malignant cells [53; 54; 55]. It is a small molecule (~45 kDa) with diverse biological activities that include the regulation of embryonic vascular development, extracellular matrix remodeling, generation of inflammatory cytokines, and enhancement of vascular permeability, through a VEGF receptor (VEGF-R) [56; 57]. EGCG inhibits transcriptional regulation of VEGF by decreasing the transcript levels of VEGF [58]. This effect is mediated by the VEGF promoter region, which contains several potential binding sites for the transcription factor activator protein-1 [59], cfos and c-jun [60]. ERK-1 and 2 have been reported to be important signaling cascades that lead to over-expression of VEGF mRNA [61]; however, EGCG inhibits kinases that cause activation of ERK-1 and 2 and hence decreases VEGF [62]. EGCG is also known to chelate strong metal ions [63] and some receptor kinases dependent on divalent cations for their activity, and EGCG can also inhibit the activity of receptor kinases by chelating the divalent ions [64]. Basic Fibroblast Growth Factor The bFGF protein (FGF-2) is a member of the fibroblast growth factor family [65]. In normal tissue, basic fibroblast growth factor is present in basement membranes and in the sub endothelial extracellular matrix of blood vessels. Our recent study in human colorectal cancer cells with EGCG treatment provided insight on a new mechanism by which EGCG downregulates bFGF both in vitro and in vivo [46]. The bFGF protein is degraded in the presence of EGCG through the ubiquitin/proteasome pathway. The proteasome complex includes three activities: chymotrypsin-like, trypsin-like and caspase-like [66] and chymotrypsin-like activity is involved in tumor survival [67]. Interestingly, our results showed that EGCG decreases the chymotrypsin-like activity and increases the trypsin-like activity of the 20S

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proteasome, subsequently degrading bFGF at the post-translational level. EGCG also increases ubiquitination in a dose-dependent manner [46]. In addition, bFGF suppression by EGCG is not only seen in culture but also seen in animal studies. We demonstrated the decrease in tumor volume and number in the APCMin/+ mice fed EGCG in their drinking water, as assessed by ELISA using the small intestinal tumor tissue lysates [46].

MMP and uPA MMP-9 is a collagenase involved in extracellular matrix degradation during tumor metastasis and inflammatory disorders [68]. It has been reported that EGCG inhibits MMP-9 secretion in cancer cells [69; 70]. Similary, uPA is a serine protease also called urokinase plasminogen activator. It is present in several physiological locations, such as the blood stream and the extracellular matrix. Plasminogen is the primary physiological substrate of plasmin. The activation of plasmin leads to activation of a proteolysis cascade leading to either thrombolysis or extracellular matrix degradation [71]. Swiercz et al. reported that EGCG inhibits uPA, thereby blocking metastasis and acting as a tumor inhibitor [72].

7.2. Effect on Apoptosis and Cancer Progression Programmed cell death in multicellular organisms is known as apoptosis. A series of biochemical events is involved in the process of apoptosis that leads to a variety of morphological changes, including blebbing, cell membrane asymmetry and attachment, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. Apoptosis helps to maintain tissue integrity and function and helps to eliminate damaged or unwanted cells [73]. EGCG induces apoptosis via different signaling pathways such as inhibiting NF-κB activation, binding to Fas, activating tumor necrosis factor-α, arresting cell cycle at G0/G1, and binding to and suppressing anti-apoptotic Bcl-2 family proteins [74; 75; 76]. EGCG inhibits growth of transformed cells, but not normal cells, primarily via induction of apoptosis [74; 76; 77] and by altering several protein expressions that are involved in apoptosis pathways.

NAG-1 NAG-1, or non-steroidal anti-inflammatory drug (NSAID)-activated gene is a proapoptotic and anti tumorigenic protein [78]. It is a secreted protein belonging to the TGF-β superfamily. NAG-1 expression causes the inhibition of cell growth of several epithelial cells as well as primitive hematopoietic progenitors [79]. NAG-1 is also known as macrophage inhibitory cytokine-1 (MIC-1) [80], placental transforming growth factor-β [81], prostatederived factor [82], growth differentiation factor 15 [83], and placental bone morphogenetic protein [84]. It has been shown to be highly expressed in mature intestinal epithelial cells; however, its expression is decreased in human colorectal carcinoma and neoplastic intestinal polyps of APCMin/+ mice [85]. It has been shown that EGCG or ECG can increase NAG-1 expression in human colorectal cancer cells and that NAG-1 induction is one of the mechanisms of catechin-induced apoptosis leading to blocking of colorectal tumorigenesis [45]. In addition, EGCG is known to produce oxidative DNA damage at high concentrations [86]. This DNA damage can cause induction of p53 and cause apoptosis.


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p53 The p53 gene encodes the transcription factor p53, which is a very important regulator of the cell cycle, plays a crucial role as a tumor suppressor, and hence is involved in preventing carcinogenesis [87]. The p53 protein can cause repair of damaged DNA by activating DNA repair proteins, and subsequently it can hold the cell cycle at the G1/S phase if DNA damage is detected, whereas it can initiate apoptosis if the DNA damage is irreversible. EGCG induces p53 tumor suppressor protein, followed by the induction of apoptotic related genes [88]. Amin et al. showed that EGCG can modulate the cancer cell growth pattern in a p53dependent manner [89]. In addition, EGCG induces p53 proteins not only in colorectal cancer cells, but also in other cancer cells [79].

ATF3 Activating transcription factor 3 (ATF3) belongs to the mammalian activation transcription factor (ATF)/cAMP responsive element-binding (CREB) protein family of transcription factors. ATF3 is known to be activated by a variety of physiological and pathological stimuli to the cells [90]. Overexpression of ATF3 in human colorectal cancer cells lead to apoptosis and cell cycle arrest [91]. The human colorectal cancer cells, HCT-116 (p53 wild type) and SW480 (p53 mutant), treated with 50 µM of EGCG and ECG, respectively, caused a high induction of ATF3 [92]. This result implied that catechins can induce ATF3 in a p53-independent manner. Furthermore, it has been shown that catechins can act as both antioxidant and pro-oxidant agents [93], and that ECG generates oxidative stress in the media, followed by the induction of ATF3 [92]. EGR-1 Early growth response gene-1 is an inducible zinc finger transcription factor and an immediate-early gene induced by stress or injury, mitogens, growth factors, cytokines, hypoxia, and differentiation factors [94]. It is mainly involved in progress of vascular diseases. EGR-1 is also known to modulate the genes involved in growth control and survival. Expression of EGR-1 can have a dual effect; it can cause either promotion or inhibition of cell growth, which depends on cell type and environment. EGR-1 has been also shown to act as a tumor suppressor gene, and its loss can lead to progression of cancer. Many researchers have shown that EGR-1 induces apoptosis and is an important factor in neuronal apoptosis [95]. It exhibits its pro-apoptotic function by directly binding to p53 [96], NAG1[97] and PTEN promoters [98]. We have previously shown that EGR-1 phosphorylation enhances ATF3 expression in colorectal cancer cells [91], and then we demonstrated that EGR-1 played an important role in ECG-induced ATF3 expression in the human colorectal cancer cell line HCT-116 [92].

7.3. Effect on Cell Adhesion Cell or cellular adhesion is an interaction of a cell to another cell or to a surface. This interaction or adhesion is brought about by special molecules called cell adhesion molecules (CAM), which interact with other molecules on the other cells or on the other surface. The

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CAMs are transmembrane receptors and include molecules like integrins, cadherins and selectins.

Integrins Integrins are CAMs that interact with other cells and extracellular matrix and define cellular shape and mobility while playing an important role in regulating the cell cycle. They are usually attached to the cellular plasma membrane where they help to transfer a signal from one cell to another and influence cell growth and differentiation. All these properties make the integrins an important component in cell migration, invasion, and platelet interaction, hence its role in tumor growth and metastasis [99]. These integrin molecules regulate the interaction between tumor cells and other adhesion molecules such as fibronectin, laminin and collagens [100]. The adhesive interactions between tumor cells and these molecules lead to tumor growth, invasion and metastasis [101; 102]. EGCG has been shown to impair the adhesion between tumor cells and proteins such as fibronectin and laminin by binding to them [103; 104]. Suzuki et al. demonstrated the binding of EGCG to β1 integrin, and this binding led to disruption of β1 integrin and hence impairment of cell adhesion [105]. EGCG and ECG have also been shown to bind matrix proteins and inhibit smooth muscle cell adhesion with integrin receptor β1; EGCG can also inhibit laminin-induced smooth muscle cell migration [106]. Cadherins Cadherins play a crucial role in cell adhesion and do not allow cancer cells to migrate or metastasize. However, the perturbation of cadherin function can lead to temporary or permanent non-attachment of tumor cells and hence promote the invasion and metastasis of such loose cells. E-cadherin is also known to bind β-catenin, which is an intracellular anchoring protein. This adherence is important for epithelial cell homeostasis [107; 108]. In the development of intestinal tumors in APCMin/+ mice, aberrant β-catenin signaling is a key molecular event [109]. E-cadherin is a tumor and invasion suppressor protein that plays a crucial suppressive role in the progression from adenoma to carcinoma in colorectal cancers [110]. EGCG has been shown to increase E-cadherin protein levels in vitro in HT29 cells, and this is due to translocation of β-catenin from the cytoplasm to the nuclei and then to the plasma membrane. EGCG also increases E-cadherin protein levels by attenuating transcriptional repression, slug/snail zinc finger protein family, or by causing posttranscriptional modification of caveolin-1, a protein that plays an important role in the endocytosis of E-cadherin [111; 112; 113; 114].

7.4. Effect on Cell Cycle Regulators Cell cycle is a series of processes taking place in an eukaryotic cell that leads to its replication. There are two major events involved: interphase, divided into 4 phases, G0, G1, S, and G2, in which the cell grows and accumulates nutrients, and mitosis wherein the cells divide into two daughter cells. It is very important for the cell cycle to function properly, and certain check points are needed in order to detect and repair any kind of genetic damage so that the cell cycle does not go unchecked leading to uncontrolled growth. The regulatory


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molecules governing the ordered processes of cell cycle are cyclins and cyclin dependent kinases (CDKs). CDKs once activated by cyclins cause phosphorylation of target proteins, which either activates or inactivates the proteins or causes them to move into the next phase of the cell cycle.

Cyclin D1 Cyclin D1 is produced in response to growth factors. It is an important cell cycle regulator that is involved in G1-S transition and in regulation of proliferation and differentiation [115]. Dysregulated degradation of cyclin D1 is associated with its increased levels in various cancers [116]. Jeong et al. reported decreased expression of cyclin D1 in HT-29 human colorectal cancer cell lines by EGCG [117], and this result was confirmed by other groups, showing that EGCG and ECG decreased the expression of cyclin D1 [118]. In addition, we have reported that ECG can significantly suppress the expression of cyclin D1 in oral cancers [119]. GAK GAK (Cyclin G-associated kinase) is a serine/threonine kinase that is associated with cyclin G [120]. EGCG has been shown to down regulate the expression of GAK and hence arrest cell cycle progression from the G1 phase to the S phase by de-activating cyclin G [121]. p21 This regulator belongs to the CIP/KIP family which are inhibitors of cyclin-dependent kinases [122]. EGCG has been shown to increase the expression of p21 and hence lead to cell cycle arrest [123].

8. EGCG IN OTHER CANCERS Breast Cancer EGCG induces apoptosis and significantly decreases invasion of breast cancer cells, and induction of apoptosis is by increasing the pro-apoptotic agent BAX and decreasing the antiapoptotic agent BCL-2 [124]. EGCG also reduces invasion of cells by 24-28% on matrigel and decreases the expression of MMP-9 [125]. EGCG has also been shown to decrease the estrogen receptor α function, which is a pivotal pathway in breast tumorigenesis [126].

Lung Cancer EGCG has been shown to inhibit lung tumorigenesis in several different animal model systems. This includes lung tumorigenesis in A/J mice induced by 4-(methylnitrosamino)-1(3pyridyl)-1-butanone (NNK), N-nitrosodiethylamine, benzo[a]pyrene, N-nitrosomethylurea, or cisplatin [127]. EGCG mostly accounts for decreased tumor number, size and incidence. All these parameters are associated with the anti-proliferative, pro-apoptotic, and anti-

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angiogenic activities of EGCG and inhibition of protein kinases involved in signal transduction and cell cycle regulation.

Prostate Cancer Prostate cancer is the second leading cause of cancer-related death in men in the United States. Incorporating chemopreventive agents such as EGCG into the diet can delay the early onset of prostate cancer. EGCG exerts its chemopreventive effect in the prostate via regulation of the sex steroid receptor, growth factor-signaling, and inflammatory pathways. Harper et al. showed that EGCG inhibited early but not late stage prostate cancer. EGCG can also reduce cell proliferation and induce apoptosis by decreasing androgen receptor, IGF-1, COX-2 and MAPK signaling [128].

Pancreatic Cancer Pancreatic cancer can be a deadly disease and lead to higher mortality as it is very difficult to diagnose. By the time it is diagnosed, it has reached the late stages. EGCG has been shown to inhibit growth and induce apoptosis in human pancreatic cancer cells. EGCG can effectively inhibit viability, capillary tube formation and migration of HUVEC on its own and these effects are enhanced in presence of an ERK inhibitor in vitro. In the case of in vivo studies, AsPC-1 xenografted tumors treated with EGCG showed significant reduction in volume, proliferation, angiogenesis, VEGF-R2 and metastasis (MMP-2, MMP-7, MMP-9 and MMP-12) and induction of apoptosis, caspase-3 activity and growth arrest (p21/WAF1). In addition, there was a reduced ERK activity and enhanced p38 and JNK activities in tumor samples from EGCG-treated mice. Hence all these results show that EGCG inhibits pancreatic cancer growth, invasion, metastasis and angiogenesis and can be used in the management of pancreatic cancer prevention and treatment [129].

Skin Cancer Of all the possible causes of skin cancer, exposure to ultra violet radiation is the main one. Sevin et al. carried out a study in 12-week-old Wistar albino rats that were exposed to UV light and used ointment containing 2% EGCG. They found that topical application of EGCG was effective in preventing the skin cancer before UV exposure or had a protective effect; however, once damage was caused after exposure to UV, EGCG didn’t show any significant recovery [130]. Another study focused on EGCG-induced caspase-14 expression in an A431 human epidermoid cancer cell line. Caspase-14 is a member of the caspase family associated with epithelial cell differentiation, planned cell death, and barrier formation. Induction of caspase-14 by EGCG led to growth inhibition and reduction in tumorigenicity of A431 cells [131]. IL-12 is an interleukin known to induce immune responses and antiangiogenic activities. Based on these studies, EGCG can induce IL-12 and can prevent photocarcinogenesis through an EGCG-induced, IL-12-dependent, DNA repair mechanism [132].


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9. SUMMARY Most of the beneficial effects of green tea are due to the major catechin EGCG, which accounts for at least 50% of the total catechin content of green tea. EGCG has hydroxyl groups that make it a potent anti-oxidant agent, and its activity is 25-100% more potent than vitamin C and E. Other catechins also play a role in blocking colorectal carcinogenesis. ECG increases NAG-1 expression and induces apoptosis to prevent carcinogenesis. Colorectal cancer occurs due to mutations in both tumor suppressors as well as oncogenes. Different signaling pathways govern colorectal tumorigenesis. As shown in Fig. 4, many researchers have shown that EGCG acts on all these pathways in some way. EGCG also acts on other cancers such as lung, pancreas, breast, prostate, and skin and many other cancers. Green tea and its catechins have emerged up to be very effective dietary agents that are easily available, non- expensive, and most importantly have no side effects and can prevent cancer growth and metastasis.

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REFERENCES [1] [2]

[3] [4] [5]

[6] [7] [8]

[9] [10] [11] [12]

[13]

[14]

[15] [16]

[17]

Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun MJ. Cancer statistics, 2008. CA Cancer J Clin 2008;58:71-96. Woloshin S, Schwartz LM, Welch HG. The risk of death by age, sex, and smoking status in the united states: Putting health risks in context. J Natl Cancer Inst 2008;100:845-853. Bostick RM, Sprafka JM, Virnig BA, Potter JD. Knowledge, attitudes, and personal practices regarding prevention and early detection of cancer. Prev Med 1993;22:65-85. Pitot HC. The molecular biology of carcinogenesis. Cancer 1993;72:962-970. Chen D, Milacic V, Chen MS, Wan SB, Lam WH, Huo C, Landis-Piwowar KR, Cui QC, Wali A, Chan TH, Dou QP. Tea polyphenols, their biological effects and potential molecular targets. Histol Histopathol 2008;23:487-496. Burke CA, Bauer WM, Lashner B. Chemoprevention of colorectal cancer: Slow, steady progress. Cleve Clin J Med 2003;70:346-350. Lamprecht SA, Lipkin M. Mouse models of gastrointestinal tumorigenesis for dietary cancer prevention studies. Nutr Rev 2003;61:255-258. Garay CA, Engstrom PF. Chemoprevention of colorectal cancer: Dietary and pharmacologic approaches. Oncology (Williston Park) 1999;13:89-97; discussion 97100, 105. Holt PR. Studies of calcium in food supplements in humans. Ann N Y Acad Sci 1999;889:128-137. Mason JB. Diet, folate, and colon cancer. Curr Opin Gastroenterol 2002;18:229-234. Mason JB. Nutritional chemoprevention of colon cancer. Semin Gastrointest Dis 2002;13:143-153. Rahman KM, Sugie S, Tanaka T, Mori H, Reddy BS. Effect of types and amount of dietary fat during the initiation phase of hepatocarcinogenesis. Nutr Cancer 2001;39:220-225. Khan N, Afaq F, Saleem M, Ahmad N, Mukhtar H. Targeting multiple signaling pathways by green tea polyphenol (-)-epigallocatechin-3-gallate. Cancer Res 2006;66:2500-2505. Levin B, Lieberman DA, McFarland B, Smith RA, Brooks D, Andrews KS, Dash C, Giardiello FM, Glick S, Levin TR, Pickhardt P, Rex DK, Thorson A, Winawer SJ, for the American Cancer Society Colorectal Cancer Advisory Group tUSM-STF, the American College of Radiology Colon Cancer C. Screening and surveillance for the early detection of colorectal cancer and adenomatous polyps, 2008: A joint guideline from the american cancer society, the us multi-society task force on colorectal cancer, and the american college of radiology. CA Cancer J Clin 2008;58:130-160. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990;61:759-767. Aretz S, Stienen D, Uhlhaas S, Pagenstecher C, Mangold E, Caspari R, Propping P, Friedl W. Large submicroscopic genomic apc deletions are a common cause of typical familial adenomatous polyposis. J Med Genet 2005;42:185-192. Jiricny J, Nystrom-Lahti M. Mismatch repair defects in cancer. Curr Opin Genet Dev 2000;10:157-161.


339

[18] Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC, Leppert M, Nakamura Y, White R, Smits AM, Bos JL. Genetic alterations during colorectal-tumor development. N Engl J Med 1988;319:525-532. [19] Baker SJ, Fearon ER, Nigro JM, Hamilton SR, Preisinger AC, Jessup JM, vanTuinen P, Ledbetter DH, Barker DF, Nakamura Y, White R, Vogelstein B. Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas. Science 1989;244:217-221. [20] Taketo MM. Cox-2 and colon cancer. Inflamm Res 1998;47 Suppl 2:S112-116. [21] Tsujii M, DuBois RN. Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell 1995;83:493-501. [22] Tsujii M, Kawano S, Tsuji S, Sawaoka H, Hori M, DuBois RN. Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell 1998;93:705-716. [23] Marnett LJ. Recent developments in cyclooxygenase inhibition. Prostaglandins Other Lipid Mediat 2002;68-69:153-164. [24] Marnett LJ, DuBois RN. Cox-2: A target for colon cancer prevention. Annu Rev Pharmacol Toxicol 2002;42:55-80. [25] Kariola R, Abdel-Rahman WM, Ollikainen M, Butzow R, Peltomaki P, Nystrom M. Apc and beta-catenin protein expression patterns in hnpcc-related endometrial and colorectal cancers. Fam Cancer 2005;4:187-190. [26] Aberle H, Bauer A, Stappert J, Kispert A, Kemler R. Beta-catenin is a target for the ubiquitin-proteasome pathway. EMBO J 1997;16:3797-3804. [27] Kishida S, Yamamoto H, Ikeda S, Kishida M, Sakamoto I, Koyama S, Kikuchi A. Axin, a negative regulator of the wnt signaling pathway, directly interacts with adenomatous polyposis coli and regulates the stabilization of beta-catenin. J Biol Chem 1998;273:10823-10826. [28] Rubinfeld B, Albert I, Porfiri E, Fiol C, Munemitsu S, Polakis P. Binding of gsk3beta to the apc-beta-catenin complex and regulation of complex assembly. Science 1996;272:1023-1026. [29] Iwamoto M, Ahnen DJ, Franklin WA, Maltzman TH. Expression of beta-catenin and full-length apc protein in normal and neoplastic colonic tissues. Carcinogenesis 2000;21:1935-1940. [30] Miyaki M, Iijima T, Kimura J, Yasuno M, Mori T, Hayashi Y, Koike M, Shitara N, Iwama T, Kuroki T. Frequent mutation of beta-catenin and apc genes in primary colorectal tumors from patients with hereditary nonpolyposis colorectal cancer. Cancer Res 1999;59:4506-4509. [31] Ten Dijke P, Goumans MJ, Itoh F, Itoh S. Regulation of cell proliferation by smad proteins. J Cell Physiol 2002;191:1-16. [32] Attisano L, Wrana JL. Signal transduction by the tgf-beta superfamily. Science 2002;296:1646-1647. [33] Moustakas A, Souchelnytskyi S, Heldin CH. Smad regulation in tgf-beta signal transduction. J Cell Sci 2001;114:4359-4369. [34] Wakefield LM, Roberts AB. Tgf-beta signaling: Positive and negative effects on tumorigenesis. Curr Opin Genet Dev 2002;12:22-29. [35] Derynck R, Akhurst RJ, Balmain A. Tgf-beta signaling in tumor suppression and cancer progression. Nat Genet 2001;29:117-129.

340


[36] Bursill CA, Abbey M, Roach PD. A green tea extract lowers plasma cholesterol by inhibiting cholesterol synthesis and upregulating the ldl receptor in the cholesterol-fed rabbit. Atherosclerosis 2007;193:86-93. [37] Loest HB, Noh SK, Koo SI. Green tea extract inhibits the lymphatic absorption of cholesterol and alpha-tocopherol in ovariectomized rats. J Nutr 2002;132:1282-1288. [38] Antonello M, Montemurro D, Bolognesi M, Di Pascoli M, Piva A, Grego F, Sticchi D, Giuliani L, Garbisa S, Rossi GP. Prevention of hypertension, cardiovascular damage and endothelial dysfunction with green tea extracts. Am J Hypertens 2007;20:13211328. [39] Hao J, Kim CH, Ha TS, Ahn HY. Epigallocatechin-3 gallate prevents cardiac hypertrophy induced by pressure overload in rats. J Vet Sci 2007;8:121-129. [40] Venables MC, Hulston CJ, Cox HR, Jeukendrup AE. Green tea extract ingestion, fat oxidation, and glucose tolerance in healthy humans. Am J Clin Nutr 2008;87:778-784. [41] Williamson MP, McCormick TG, Nance CL, Shearer WT. Epigallocatechin gallate, the main polyphenol in green tea, binds to the t-cell receptor, cd4: Potential for hiv-1 therapy. J Allergy Clin Immunol 2006;118:1369-1374. [42] Mukhtar H, Ahmad N. Tea polyphenols: Prevention of cancer and optimizing health. Am J Clin Nutr 2000;71:1698S-1702S; discussion 1703S-1694S. [43] Ju J, Lu G, Lambert JD, Yang CS. Inhibition of carcinogenesis by tea constituents. Seminars in Cancer Biology 2007;17:395-402. [44] Rice-Evans CA, Miller NJ, Paganga G. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biology and Medicine 1996;20:933-956. [45] Baek SJ, Kim J-S, Jackson FR, Eling TE, McEntee MF, Lee S-H. Epicatechin gallateinduced expression of nag-1 is associated with growth inhibition and apoptosis in colon cancer cells. Carcinogenesis 2004;25:2425-2432. [46] Sukhthankar M, Yamaguchi K, Lee S-H, McEntee MF, Eling TE, Hara Y, Baek SJ. A green tea component suppresses posttranslational expression of basic fibroblast growth factor in colorectal cancer. Gastroenterology 2008;134:1972-1980. [47] Yang CS, Lambert JD, Hou Z, Ju J, Lu G, Hao X. Molecular targets for the cancer preventive activity of tea polyphenols. Mol Carcinog 2006;45:431-435. [48] Singh PN, Fraser GE. Dietary risk factors for colon cancer in a low-risk population. Am J Epidemiol 1998;148:761-774. [49] Surh YJ. Cancer chemoprevention with dietary phytochemicals. Nat Rev Cancer 2003;3:768-780. [50] Bergers G, Benjamin LE. Tumorigenesis and the angiogenic switch. Nat Rev Cancer 2003;3:401-410. [51] Smith SK. Angiogenesis, vascular endothelial growth factor and the endometrium. Hum Reprod Update 1998;4:509-519. [52] Singh AK, Seth P, Anthony P, Husain MM, Madhavan S, Mukhtar H, Maheshwari RK. Green tea constituent epigallocatechin-3-gallate inhibits angiogenic differentiation of human endothelial cells. Arch Biochem Biophys 2002;401:29-37. [53] Bellamy WT. Expression of vascular endothelial growth factor and its receptors in multiple myeloma and other hematopoietic malignancies. Semin Oncol 2001;28:551559. [54] Ferrara N. Role of vascular endothelial growth factor in physiologic and pathologic angiogenesis: Therapeutic implications. Semin Oncol 2002;29:10-14.


341

[55] Fiedler W, Graeven U, Ergun S, Verago S, Kilic N, Stockschlader M, Hossfeld DK. Vascular endothelial growth factor, a possible paracrine growth factor in human acute myeloid leukemia. Blood 1997;89:1870-1875. [56] Bikfalvi A, Bicknell R. Recent advances in angiogenesis, anti-angiogenesis and vascular targeting. Trends Pharmacol Sci 2002;23:576-582. [57] Gerber HP, Dixit V, Ferrara N. Vascular endothelial growth factor induces expression of the antiapoptotic proteins bcl-2 and a1 in vascular endothelial cells. J Biol Chem 1998;273:13313-13316. [58] Sartippour MR, Shao Z-M, Heber D, Beatty P, Zhang L, Liu C, Ellis L, Liu W, Go VL, Brooks MN. Green tea inhibits vascular endothelial growth factor (vegf) induction in human breast cancer cells. J Nutr 2002;132:2307-2311. [59] Tischer E, Mitchell R, Hartman T, Silva M, Gospodarowicz D, Fiddes JC, Abraham JA. The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Biol Chem 1991;266:11947-11954. [60] Hu YL, Tee MK, Goetzl EJ, Auersperg N, Mills GB, Ferrara N, Jaffe RB. Lysophosphatidic acid induction of vascular endothelial growth factor expression in human ovarian cancer cells. J Natl Cancer Inst 2001;93:762-768. [61] Jung YD, Nakano K, Liu W, Gallick GE, Ellis LM. Extracellular signal-regulated kinase activation is required for up-regulation of vascular endothelial growth factor by serum starvation in human colon carcinoma cells. Cancer Res 1999;59:4804-4807. [62] Kojima-Yuasa A, Hua JJ, Kennedy DO, Matsui-Yuasa I. Green tea extract inhibits angiogenesis of human umbilical vein endothelial cells through reduction of expression of vegf receptors. Life Sci 2003;73:1299-1313. [63] Yang CS, Wang ZY. Tea and cancer. J Natl Cancer Inst 1993;85:1038-1049. [64] Mahadevan D, Thanki N, Aroca P, McPhie P, Yu JC, Beeler J, Santos E, Wlodawer A, Heidaran MA. A divalent metal ion binding site in the kinase insert domain of the alpha-platelet-derived growth factor receptor regulates its association with sh2 domains. Biochemistry 1995;34:2095-2106. [65] Dow JK, deVere White RW. Fibroblast growth factor 2: Its structure and property, paracrine function, tumor angiogenesis, and prostate-related mitogenic and oncogenic functions. Urology 2000;55:800-806. [66] Naujokat C, Fuchs D, Berges C. Adaptive modification and flexibility of the proteasome system in response to proteasome inhibition. Biochim Biophys Acta 2007;1773:1389-1397. [67] Nam S, Smith DM, Dou QP. Ester bond-containing tea polyphenols potently inhibit proteasome activity in vitro and in vivo. J Biol Chem 2001;276:13322-13330. [68] McMillan JI, Weeks R, West JW, Bursten S, Rice GC, Lovett DH. Pharmacological inhibition of gelatinase b induction and tumor cell invasion. Int J Cancer 1996;67:523531. [69] Kim HS, Kim MH, Jeong M, Hwang YS, Lim SH, Shin BA, Ahn BW, Jung YD. Egcg blocks tumor promoter-induced mmp-9 expression via suppression of mapk and ap-1 activation in human gastric ags cells. Anticancer Res 2004;24:747-753. [70] Yang J, Wei D, Liu J. Repressions of mmp-9 expression and nf-kappa b localization are involved in inhibition of lung carcinoma 95-d cell invasion by (-)-epigallocatechin-3gallate. Biomed Pharmacother 2005;59:98-103.

342


[71] Rabbani SA, Xing RH. Role of urokinase (upa) and its receptor (upar) in invasion and metastasis of hormone-dependent malignancies. Int J Oncol 1998;12:911-920. [72] Swiercz R, Skrzypczak-Jankun E, Merrell MM, Selman SH, Jankun J. Angiostatic activity of synthetic inhibitors of urokinase type plasminogen activator. Oncol Rep 1999;6:523-526. [73] Ghobrial IM, Witzig TE, Adjei AA. Targeting apoptosis pathways in cancer therapy. CA Cancer J Clin 2005;55:178-194. [74] Ahmad N, Gupta S, Mukhtar H. Green tea polyphenol epigallocatechin-3-gallate differentially modulates nuclear factor kappab in cancer cells versus normal cells. Arch Biochem Biophys 2000;376:338-346. [75] Leone M, Zhai D, Sareth S, Kitada S, Reed JC, Pellecchia M. Cancer prevention by tea polyphenols is linked to their direct inhibition of antiapoptotic bcl-2-family proteins. Cancer Res 2003;63:8118-8121. [76] Yang GY, Liao J, Li C, Chung J, Yurkow EJ, Ho CT, Yang CS. Effect of black and green tea polyphenols on c-jun phosphorylation and h(2)o(2) production in transformed and non-transformed human bronchial cell lines: Possible mechanisms of cell growth inhibition and apoptosis induction. Carcinogenesis 2000;21:2035-2039. [77] Lu YP, Lou YR, Xie JG, Peng QY, Liao J, Yang CS, Huang MT, Conney AH. Topical applications of caffeine or (-)-epigallocatechin gallate (egcg) inhibit carcinogenesis and selectively increase apoptosis in uvb-induced skin tumors in mice. Proc Natl Acad Sci U S A 2002;99:12455-12460. [78] Baek SJ, Kim KS, Nixon JB, Wilson LC, Eling TE. Cyclooxygenase inhibitors regulate the expression of a tgf-beta superfamily member that has proapoptotic and antitumorigenic activities. Mol Pharmacol 2001;59:901-908. [79] Baek SJ, Eling TE. Changes in gene expression contribute to cancer prevention by cox inhibitors. Progress in Lipid Research 2006;45:1-16. [80] Bootcov MR, Bauskin AR, Valenzuela SM, Moore AG, Bansal M, He XY, Zhang HP, Donnellan M, Mahler S, Pryor K, Walsh BJ, Nicholson RC, Fairlie WD, Por SB, Robbins JM, Breit SN. Mic-1, a novel macrophage inhibitory cytokine, is a divergent member of the tgf-beta superfamily. Proc Natl Acad Sci U S A 1997;94:11514-11519. [81] Li PX, Wong J, Ayed A, Ngo D, Brade AM, Arrowsmith C, Austin RC, Klamut HJ. Placental transforming growth factor-beta is a downstream mediator of the growth arrest and apoptotic response of tumor cells to DNA damage and p53 overexpression. J Biol Chem 2000;275:20127-20135. [82] Paralkar VM, Vail AL, Grasser WA, Brown TA, Xu H, Vukicevic S, Ke HZ, Qi H, Owen TA, Thompson DD. Cloning and characterization of a novel member of the transforming growth factor-beta/bone morphogenetic protein family. J Biol Chem 1998;273:13760-13767. [83] Bottner M, Laaff M, Schechinger B, Rappold G, Unsicker K, Suter-Crazzolara C. Characterization of the rat, mouse, and human genes of growth/differentiation factor15/macrophage inhibiting cytokine-1 (gdf-15/mic-1). Gene 1999;237:105-111. [84] Hromas R, Hufford M, Sutton J, Xu D, Li Y, Lu L. Plab, a novel placental bone morphogenetic protein. Biochim Biophys Acta 1997;1354:40-44. [85] Kim KS, Baek SJ, Flake GP, Loftin CD, Calvo BF, Eling TE. Expression and regulation of nonsteroidal anti-inflammatory drug-activated gene (nag-1) in human and mouse tissue. Gastroenterology 2002;122:1388-1398.


343

[86] Johnson MK, Loo G. Effects of epigallocatechin gallate and quercetin on oxidative damage to cellular DNA. Mutat Res 2000;459:211-218. [87] Rallapalli R, Strachan G, Cho B, Mercer WE, Hall DJ. A novel mdmx transcript expressed in a variety of transformed cell lines encodes a truncated protein with potent p53 repressive activity. J Biol Chem 1999;274:8299-8308. [88] Baek SJ, Wilson LC, Eling TE. Resveratrol enhances the expression of non-steroidal anti-inflammatory drug-activated gene (nag-1) by increasing the expression of p53. Carcinogenesis 2002;23:425-434. [89] Amin AR, Thakur VS, Paul RK, Feng GS, Qu CK, Mukhtar H, Agarwal ML. Shp-2 tyrosine phosphatase inhibits p73-dependent apoptosis and expression of a subset of p53 target genes induced by egcg. Proc Natl Acad Sci U S A 2007;104:5419-5424. [90] Hai T, Wolfgang CD, Marsee DK, Allen AE, Sivaprasad U. Atf3 and stress responses. Gene Expr 1999;7:321-335. [91] Yamaguchi K, Lee S-H, Kim J-S, Wimalasena J, Kitajima S, Baek SJ. Activating transcription factor 3 and early growth response 1 are the novel targets of ly294002 in a phosphatidylinositol 3-kinase-independent pathway. Cancer Res 2006;66:2376-2384. [92] Cho KN, Sukhthankar M, Lee SH, Yoon JH, Baek SJ. Green tea catechin (-)epicatechin gallate induces tumour suppressor protein atf3 via egr-1 activation. Eur J Cancer 2007;43:2404-2412. [93] Yang CS, Hong J, Hou Z, Sang S. Green tea polyphenols: Antioxidative and prooxidative effects. J Nutr 2004;134:3181S. [94] Silverman ES, Collins T. Pathways of egr-1-mediated gene transcription in vascular biology. Am J Pathol 1999;154:665-670. [95] Pignatelli M, Luna-Medina R, Perez-Rendon A, Santos A, Perez-Castillo A. The transcription factor early growth response factor-1 (egr-1) promotes apoptosis of neuroblastoma cells. Biochem J 2003;373:739-746. [96] Nair P, Muthukkumar S, Sells SF, Han SS, Sukhatme VP, Rangnekar VM. Early growth response-1-dependent apoptosis is mediated by p53. J Biol Chem 1997;272:20131-20138. [97] Baek SJ, Kim JS, Moore SM, Lee SH, Martinez J, Eling TE. Cyclooxygenase inhibitors induce the expression of the tumor suppressor gene egr-1, which results in the upregulation of nag-1, an antitumorigenic protein. Mol Pharmacol 2005;67:356-364. [98] Virolle T, Adamson ED, Baron V, Birle D, Mercola D, Mustelin T, de Belle I. The egr1 transcription factor directly activates pten during irradiation-induced signalling. Nat Cell Biol 2001;3:1124-1128. [99] Mizejewski GJ. Role of integrins in cancer: Survey of expression patterns. Proc Soc Exp Biol Med 1999;222:124-138. [100] 1Hynes RO. Integrins: Versatility, modulation, and signaling in cell adhesion. Cell 1992;69:11-25. [101] 1Liotta LA, Rao CN, Wewer UM. Biochemical interactions of tumor cells with the basement membrane. Annu Rev Biochem 1986;55:1037-1057. [102] 1Turpeenniemi-Hujanen T, Thorgeirsson UP, Rao CN, Liotta LA. Laminin increases the release of type iv collagenase from malignant cells. J Biol Chem 1986;261:18831889.

344


[103] Ogata K, Mukae N, Suzuki Y, Satoh K, Narumi K, Nukiwa T, Isemura M. Effects of catechins on the mouse tumor cell adhesion to fibronectin. Planta Med 1995;61:472474. [104] Suzuki Y, Isemura M. Inhibitory effect of epigallocatechin gallate on adhesion of murine melanoma cells to laminin. Cancer Lett 2001;173:15-20. [105] Suzuki Y, Suzuki T, Minami T, Isemura M. Involvement of impaired interaction with β1 integrin in epigallocatechin gallate-mediated inhibition of fibrosarcoma ht1080 cell adhesion to fibronectin. Journal of Health Science 2006;52:103-109. [106] Lo HM, Hung CF, Huang YY, Wu WB. Tea polyphenols inhibit rat vascular smooth muscle cell adhesion and migration on collagen and laminin via interference with cellecm interaction. J Biomed Sci 2007;14:637-645. [107] Conacci-Sorrell M, Zhurinsky J, Ben-Ze'ev A. The cadherin-catenin adhesion system in signaling and cancer. J Clin Invest 2002;109:987-991. [108] Kemler R. From cadherins to catenins: Cytoplasmic protein interactions and regulation of cell adhesion. Trends Genet 1993;9:317-321. [109] Ju J, Hong J, Zhou JN, Pan Z, Bose M, Liao J, Yang GY, Liu YY, Hou Z, Lin Y, Ma J, Shih WJ, Carothers AM, Yang CS. Inhibition of intestinal tumorigenesis in apcmin/+ mice by (-)-epigallocatechin-3-gallate, the major catechin in green tea. Cancer Res 2005;65:10623-10631. [110] Perl AK, Wilgenbus P, Dahl U, Semb H, Christofori G. A causal role for e-cadherin in the transition from adenoma to carcinoma. Nature 1998;392:190-193. [111] Batlle E, Sancho E, Franci C, Dominguez D, Monfar M, Baulida J, Garcia De Herreros A. The transcription factor snail is a repressor of e-cadherin gene expression in epithelial tumour cells. Nat Cell Biol 2000;2:84-89. [112] Conacci-Sorrell M, Simcha I, Ben-Yedidia T, Blechman J, Savagner P, Ben-Ze'ev A. Autoregulation of e-cadherin expression by cadherin-cadherin interactions: The roles of beta-catenin signaling, slug, and mapk. J Cell Biol 2003;163:847-857. [113] Hennig G, Behrens J, Truss M, Frisch S, Reichmann E, Birchmeier W. Progression of carcinoma cells is associated with alterations in chromatin structure and factor binding at the e-cadherin promoter in vivo. Oncogene 1995;11:475-484. [114] Lu Z, Ghosh S, Wang Z, Hunter T. Downregulation of caveolin-1 function by egf leads to the loss of e-cadherin, increased transcriptional activity of beta-catenin, and enhanced tumor cell invasion. Cancer Cell 2003;4:499-515. [115] Izzo JG, Papadimitrakopoulou VA, Liu DD, den Hollander PL, Babenko IM, Keck J, El-Naggar AK, Shin DM, Lee JJ, Hong WK, Hittelman WN. Cyclin d1 genotype, response to biochemoprevention, and progression rate to upper aerodigestive tract cancer. J Natl Cancer Inst 2003;95:198-205. [116] Alao JP. The regulation of cyclin d1 degradation: Roles in cancer development and the potential for therapeutic invention. Mol Cancer 2007;6:24. [117] Jeong W-S, Kim I-W, Hu R, Kong A-NT. Modulation of ap-1 by natural chemopreventive compounds in human colon ht-29 cancer cell line. Pharmaceutical Research 2004;21:649-660. [118] Ju J, Hong J, Zhou J-n, Pan Z, Bose M, Liao J, Yang G-y, Liu YY, Hou Z, Lin Y, Ma J, Shih WJ, Carothers AM, Yang CS. Inhibition of intestinal tumorigenesis in apcmin/+ mice by (-)-epigallocatechin-3-gallate, the major catechin in green tea. Cancer Res 2005;65:10623-10631.


345

[119] Lim YC, Lee SH, Song MH, Yamaguchi K, Yoon JH, Choi EC, Baek SJ. Growth inhibition and apoptosis by (-)-epicatechin gallate are mediated by cyclin d1 suppression in head and neck squamous carcinoma cells. Eur J Cancer 2006;42:32603266. [120] Kanaoka Y, Kimura SH, Okazaki I, Ikeda M, Nojima H. Gak: A cyclin g associated kinase contains a tensin/auxilin-like domain. FEBS Letters 1997;402:73-80. [121] Ahn WS, Huh SW, Bae SM, Lee IP, Lee JM, Namkoong SE, Kim CK, Sin JI. A major constituent of green tea, egcg, inhibits the growth of a human cervical cancer cell line, caski cells, through apoptosis, g(1) arrest, and regulation of gene expression. DNA Cell Biol 2003;22:217-224. [122] Kei-ichi Nakayama KN. Cip/kip cyclin-dependent kinase inhibitors: Brakes of the cell cycle engine during development. BioEssays 1998;20:1020-1029. [123] Manson MM. Cancer prevention - the potential for diet to modulate molecular signalling. Trends in Molecular Medicine 2003;9:11-18. [124] Khan N, Afaq F, Mukhtar H. Apoptosis by dietary factors: The suicide solution for delaying cancer growth. Carcinogenesis 2007;28:233-239. [125] Thangapazham RL, Passi N, Maheshwari RK. Green tea polyphenol and epigallocatechin gallate induce apoptosis and inhibit invasion in human breast cancer cells. Cancer Biol Ther 2007;6:1938-1943. [126] Farabegoli F, Barbi C, Lambertini E, Piva R. (-)-epigallocatechin-3-gallate downregulates estrogen receptor alpha function in mcf-7 breast carcinoma cells. Cancer Detection and Prevention 2007;31:499-504. [127] Yang CS, Liao J, Yang GY, Lu G. Inhibition of lung tumorigenesis by tea. Exp Lung Res 2005;31:135-144. [128] Harper CE, Patel BB, Wang J, Eltoum IA, Lamartiniere CA. Epigallocatechin-3-gallate suppresses early stage, but not late stage prostate cancer in tramp mice: Mechanisms of action. Prostate 2007;67:1576-1589. [129] Shankar S, Ganapathy S, Hingorani SR, Srivastava RK. Egcg inhibits growth, invasion, angiogenesis and metastasis of pancreatic cancer. Front Biosci 2008;13:440-452. [130] Sevin A, Oztas P, Senen D, Han U, Karaman C, Tarimci N, Kartal M, Erdogan B. Effects of polyphenols on skin damage due to ultraviolet a rays: An experimental study on rats. J Eur Acad Dermatol Venereol 2007;21:650-656. [131] Hsu S, Qin H, Dickinson D, Xie D, Bollag WB, Stoppler H, Pearl H, Vu A, Watkins M, Koehler M, Schuster G. Expression of caspase-14 reduces tumorigenicity of skin cancer cells. In Vivo 2007;21:279-283. [132] Meeran SM, Mantena SK, Elmets CA, Katiyar SK. (-)-epigallocatechin-3-gallate prevents photocarcinogenesis in mice through interleukin-12-dependent DNA repair. Cancer Res 2006;66:5512-5520.



Chapter 17

INHIBITORY EFFECT OF CATECHIN DERIVATIVES FROM GREEN TEA ON DNA POLYMERASE ACTIVITY, HUMAN CANCER CELL GROWTH, AND TPA (12-OTETRADECANOYLPHORBOL-13-ACETATE) -INDUCED INFLAMMATION Yuko Kumamoto-Yonezawa1, Hiromi Yoshida1,2 and Yoshiyuki Mizushina1,2,* 1

Laboratory of Food & Nutritional Sciences, Department of Nutritional Science, KobeGakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan 2 Cooperative Research Center of Life Sciences, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan

ABSTRACT Green tea obtained from the leaves of the plant, Camellia sinensis, is one of the most popular beverages in the world. The major polyphenolic compounds in green tea are catechin derivatives (i.e., flavan-3-ols), and their composition varies depending on the season of harvest and the manufacturing process. The inhibitory activities against DNA polymerases (pols) of catechin derivatives such as (+)-catechin (C), (-)-epicatechin (EC), (-)-gallocatechin (GC), (-)-epigallocatechin (EGC), (+)-catechin gallate (Cg), (-)epicatechin gallate (ECg), (-)-gallocatechin gallate (GCg), and (-)-epigallocatechin gallate (EGCg) were investigated. Among these eight catechins, several inhibited mammalian pols with EGCg being the strongest inhibitor of pols α and λ with IC50 values of 5.1 and 3.8 μM, respectively. EGCg did not influence the activities of plant pols, such as pols α and λ, or prokaryotic pols, and had no effect on the activities of DNA metabolic enzymes such as calf primase of pol α, T7 RNA polymerase, T4 *

Correspondence to: Yoshiyuki Mizushina Ph.D., Laboratory of Food & Nutritional Sciences, Department of Nutritional Science, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan; Tel: +81-78-974-1551 (ext.3232); Fax: +81-78-974-5689; E-mail: [email protected]

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Yuko Kumamoto-Yonezawa, Hiromi Yoshida and Yoshiyuki Mizushina polynucleotide kinase, or bovine deoxyribonuclease I. Some tea catechins also suppressed human cancer cell growth and/or TPA (12-O-tetradecanoylphorbol-13acetate)-induced inflammation, and the tendency of the pol inhibitory activity for these compounds was the same as that of their anti-inflammatory activity rather than their anticancer activity. Based on these results, the relationship between the structure of tea catechins and their bioactivities is discussed.

Keywords: Green tea; Catechin derivatives; DNA polymerase; enzyme inhibitor; DNA replication; Cancer cell growth inhibition; Anti-cancer activity; Anti-inflammation

ABBREVIATIONS pol, DNA polymerase (E.C.2.7.7.7); C, (+)-catechin; EC, (-)-epicatechin; GC, (-)-gallocatechin; EGC, (-)-epigallocatechin; Cg, (+)-catechin gallate; ECg, (-)-epicatechin gallate; GCg, (-)-gallocatechin gallate; EGCg, (-)-epigallocatechin gallate; TPA, 12-O-tetradecanoylphorbol-13-acetate.

1. INTRODUCTION Green tea, a popular beverage, is consumed worldwide. It contains an infusion of the leaves from Camellia sinensis, which is rich in polyphenolic compounds known as catechins, especially, (-)-epigallocatechin gallate (EGCg), (-)-epicatechin gallate (ECg), (-)epigallocatechin (EGC), and (-)-epicatechin (EC) [1, 2]. The catechin composition of green tea varies depending on the season of harvest and the manufacturing process. Green tea leaves usually contain about 1 % EC, 2-3 % EGC, 1-2 % ECg, and 5-8 % EGCg [3]; therefore, the main constituent of catechin is EGCg. The human genome encodes 14 pols, which conduct cellular DNA synthesis [4]. Eukaryotic cells reportedly contain three replicative types (pols α, δ and ε, mitochondrial pol γ, and at least twelve repair types (pols β, δ, ε, ζ, η, θ, ι, κ, λ, μ, and σ, and REV1) [5]. We have been studying selective inhibitors of each pol from natural materials including foods such as vegetables for more than 10 years [6-13]. It is considered that food materials containing pol inhibitory compounds could be used as bioactive functional foods with anticancer, anti-virus, and immuno-suppression activities etc. Polyphenols, such as curcumin [7, 8] and anthocyanin compounds [12], were found to inhibit the activities of mammalian pols. This review reports the inhibitory effect of catechin derivatives [11], as contained in green tea, on bioactivities including DNA polymerase inhibition, human cancer cell growth inhibition, and anti-inflammation, and the relationship of these bioactivities of catechin derivatives is discussed.

EGCg Is a Potent Inhibitor of Mammalian DNA Polymerase

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2. EFFECT OF CATECHIN DERIVATIVES ON THE ACTIVITIES OF MAMMALIAN DNA POLYMERASES The assay method for pol activity was described previously [15, 16]. The substrates of the pols were poly(dA)/oligo(dT)12-18 and 2'-deoxythymidine 5'-triphosphate (dTTP) as the DNA template-primer and nucleotide substrate (i.e., 2'-deoxynucleotide 5'-triphosphate (dNTP)), respectively. As described above, during searches for natural inhibitors of mammalian pols it was found that catechin compounds from the leaves of dried green tea, Camellia sinensis, inhibited these activities. Eight analytical grade catechin derivatives (i.e., flavan-3-ols) were purchased commercially, including (+)-catechin (C, compound 1), (-)-epicatechin (EC, compound 2), (-)-gallocatechin (GC, compound 3), (-)-epigallocatechin (EGC, compound 4), (+)-catechin gallate (Cg, compound 5), (-)-epicatechin gallate (ECg, compound 6), (-)gallocatechin gallate (GCg, compound 7), and (-)-epigallocatechin gallate (EGCg, compound 8), and two part compounds of catechin derivatives as pyrogallol (compound 9) and gallic acid (compound 10). The chemical structures are shown in Figure 1. This study investigated whether compounds 1 - 10 inhibited the activities of mammalian pols α, β, and λ. The relative activity of each pol with a set concentration (100 μM) of the test compounds is shown in Figure 2. Immuno-affinity purified calf pol α was used as a replicative pol, and E. coli expressed recombinant mammalian pols β and λ were prepared and used as repair related pols. Compounds 1 (C) and 2 (EC) did not influence the activities of pols α, β or λ. Compounds 9 (pyrogallol) and 10 (gallic acid), which are the structural parts of flavan-3-ol, also did not inhibit these pol activities. Compounds 3 to 8 inhibited the pol activities, with the effects on pols α and λ being stronger than that on pol β. The compounds that inhibited the relative activity of pol α by more than 50 % were 3 (GC), 4 (EGC), 5 (Cg), 6 (ECg), 7 (GCg), and 8 (EGCg). Compound 8 (EGCg) had the strongest inhibitory effect on the three pols of all the catechin derivatives tested. Therefore, on the properties of EGCg were examined in the subsequent experiments. OH

OH

OH

OH HO

HO

O

O

OH O C

OH

OH

O

OH

OH Compound 1 (C)

Figure 1. (Continued)

OH

Compound 5 (Cg)

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Yuko Kumamoto-Yonezawa, Hiromi Yoshida and Yoshiyuki Mizushina

OH

OH

OH

OH HO

HO

O

OH

O OH

O C O

OH

OH

OH

OH Compound 2 (EC)

Compound 6 (ECg)

OH OH

OH OH HO

HO

O

O

OH OH

OH O C OH

OH

OH

O

OH

OH Compound 3 (GC)

Compound 7 (GCg)

OH OH

OH OH HO

HO

O

O

OH OH

OH OH

OH

O C O

OH

OH OH

Compound 4 (EGC)

Compound 8 (EGCg)

OH

OH HO

OH HOOC

OH OH

Compound 9 (Pyrogallol)

Compound 10 (Gallic acid)

Figure 1. Structure of catechin derivatives. Compound 1, (+)-catechin (C); compound 2, (-)-epicatechin (EC); compound 3, (-)-gallocatechin (GC); compound 4, (-)-epigallocatechin (EGC); compound 5, (+)catechin gallate (Cg); compound 6, (-)-epicatechin gallate (ECg); compound 7, (-)-gallocatechin gallate (GCg); compound 8, (-)-epigallocatechin gallate (EGCg); compound 9, pyrogallol; and compound 10, gallic acid.


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Calf polƒ¿ Rat polƒÀ Human polƒÉ

Compound 1 (C) Compound 2 (EC) Compound 3 (GC) Compound 4 (EGC) Compound 5 (Cg) Compound 6 (ECg) Compound 7 (GCg) Compound 8 (EGCg) Compound 9 (Pyrogallol) Compound 10 (Gallic acid) 0

20

40

60

80

100

DNA polymerase activity (%) Figure 2. Inhibitory effect of catechin derivatives on the activities of mammalian DNA polymerases. Compounds 1 to 10 (100 μM each) were incubated with calf pol α, rat pol β and human pol λ (0.05 units each). Percent relative activity is shown. The enzymatic activities were measured as described previously [15, 16]. Pol activity in the absence of the test compounds was taken as 100%. Data are shown as the means ± SEM of three independent experiments.

3. EFFECTS OF EGCG ON THE ACTIVITIES OF DNA POLYMERASES AND OTHER DNA METABOLIC ENZYMES As shown in Table 1, EGCg inhibited the activities of all the mammalian pols tested, and calf pol α and human pol λ were strongly inhibited with IC50 values of 5.1 and 3.8 μM, respectively. The inhibitory effect on pols β and λ was the weakest and strongest, respectively, of the mammalian pols tested, and the inhibition of pol β was 12.3-fold weaker than that of pol λ, although the amino acid sequence of pol β is similar to pol λ, and these pols are both classified as family-X pols [1, 17]. On the other hand, the activity of plant pols

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such as cauliflower pol α and rice pol λ, prokaryotic pols such as the Klenow fragment of E. coli pol I, Taq pol, and T4 pol, and DNA metabolic enzymes such as calf primase of pol α, T7 RNA polymerase, T4 polynucleotide kinase, and bovine deoxyribonuclease I, were not influenced by EGCg. EGCg should be classified as an inhibitor of mammalian pols.

4. EFFECT OF CATECHIN DERIVATIVES ON CULTURED HUMAN CANCER CELL GROWTH Catechin derivatives are of interest in developing of functional foods for cancer chemotherapy. Replicative pols are regarded as targets of anti-cancer drugs because they play central roles in DNA replication, which is indispensable for the proliferation of cancer cells. Therefore, the growth effects of these compounds on a human cancer cell line (promyelocytic leukemia cell, HL-60) were investigated in culture using MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) assay [18]. Table 1. IC50 values of compound 8 (EGCg) on the activities of various DNA polymerases and other DNA metabolic enzymes Enzyme Mammalian DNA polymerases Calf DNA polymerase α Rat DNA polymerase β Human DNA polymerase γ Human DNA polymerase δ Human DNA polymerase ε Human DNA polymerase η Human DNA polymerase ι Human DNA polymerase κ Human DNA polymerase λ Plant DNA polymerases Cauliflower DNA polymerase α Rice DNA polymerase λ Prokaryotic DNA polymerases E. coli DNA polymerase I (Klenow fragment) Taq DNA polymerase T4 DNA polymerase Other DNA metabolic enzymes Calf primase of DNA polymerase α T7 RNA polymerase T4 polynucleotide kinase Bovine deoxyribonuclease I

IC50 value (μM) Compound 8 (EGCg) 5.1 ± 0.3 46.8 ± 1.8 11.0 ± 0.5 17.2 ± 0.9 17.6 ± 0.9 11.6 ± 0.6 12.7 ± 0.6 13.5 ± 0.7 3.8 ± 0.2 >100 >100 >100 >100 >100 >100 >100 >100 >100

The compound was incubated with each enzyme (0.05 units). Enzymatic activity in the absence of the compound was taken as 100 %.


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20

40

60

80

100

Rate of cancer cell growth (%)

Figure 3. Effects of catechin derivatives on HL-60 cancer cell growth. The compounds (100 μM each) were incubated with the human cancer cells (promyelocytic leukemia cell line, HL-60). Percent relative activity is shown. Cell viability was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay [18]. The growth rate of the cancer cells in the absence of the test compounds was taken as 100 %. Data are shown as the means ± SEM of five independent experiments.

As shown in Figure 3, 100 μM of compound 4 (EGC) had the strongest growth inhibitory effect on this cancer cell line of the compounds tested, and the compounds that prevented more than 50 % of cancer cell growth, were 3 (GC), 4 (EGC), 7 (GCg), and 9 (pyrogallol). Compounds 8 (EGCg) and 10 (gallic acid) moderately suppressed cell growth, although EGCg was the strongest inhibitor of mammalian pols (Figure 2). On the other hand, compounds 1 (C), 2 (EC), 5 (Cg), and 6 (ECg) had no effect on cancer cell growth.

5. EFFECT OF CATECHIN DERIVATIVES ON ANTI-INFLAMMATORY ACTIVITY Although TPA (12-O-tetradecanoylphorbol-13-acetate) promotes tumor formation [19], it is also known to cause inflammation and is generally used as an artificial inflammation inducer for the screening of anti-inflammatory agents [20]. Tumor promoter-induced inflammation can be distinguished from acute inflammation, which is exudative and accompanied by fibroblast proliferation and granulation. Using mouse inflammatory tests, the anti-inflammatory activities of catechin derivatives (compounds 1 – 10) were examined. The application of TPA (0.5 μg) to a mouse ear induced edema; the weight increase of the ear disk at 7 hr after application was 241 %. As expected, the inhibitory effect of compound 8 (EGCg) on inflammation was the strongest among the compounds tested at an applied dose of at least 250 μg, and the level of inhibition was 65.6 % (Figure 4). Compound 7 (GCg) was the second

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strongest inhibitor of inflammation with an inhibitory effect of 58.0 %. Compounds 3 (GC) and 4 (EGC) and compounds 5 (Cg) and 6 (ECg) moderately suppressed inflammation at levels of 30 to 32 % and 25 to 27 %, respectively. On the other hand, compounds 1 (C) and 2 (EC) and the part compounds of catechin derivatives, compounds 9 (pyrogallol) and 10 (gallic acid), had little effect on inflammation. These results suggested that some catechin derivatives caused a remarkable reduction in TPA-induced inflammation, indicating that EGCg possesses anti-inflammatory activity.


10

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30

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50

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Anti-inflammatory activity (%) Figure 4. Anti-inflammatory activity of catechin derivatives toward TPA-induced edema on mouse ear. Compounds 1 to 10 (250 μg each) were applied individually to one ear on each mouse, and after 30 min TPA (0.5 μg) was applied to both ears. Edema was evaluated after 7 hr. The inhibitory effect is expressed as the percentage of edema. Data are shown as the means ± SEM of five independent experiments.

6. RELATIONSHIP OF THE THREE BIOACTIVITIES OF CATECHIN DERIVATIVES To confirm if there is a relationship between mammalian pol inhibition, human cancer cell (promyelocytic leukemia cell line, HL-60) growth inhibition, and anti-chronic inflammation, catechin derivatives (compounds 1 - 10) were compared for their inhibitory effects on these three bioactivities (Figure 5). Pol λ inhibition had the largest correlation (correlation coefficient = 0.9979) with the anti-inflammatory activity of all combinations of bioactivities (Figure 5A). On the other hand, neither pol λ inhibitory activity nor antiinflammatory activity was related to the inhibition of human cancer cell growth because the correlation coefficient between these activities and the cell growth inhibitory activity was less


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than 0.2 (i.e., 0.1703 in Figure 5B and 0.1851 in Figure 5C, respectively). These results led to the speculation that TPA-induced inflammation may involve a process requiring pol λ.

R2 = 0.9979

Anti-inflammatory activity (%)

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8 7

60 50 40 3

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= 0.1703 7

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Pol λ inhibitory activity (%) Figure 5. (Continued)

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R2 = 0.1851

Anti-inflammatory activity (%)

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4

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10 1

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Cancer cell growth inhibitory activity (%)

100 (C)

Figure 5. The relationship between pol λ inhibition, cancer cell growth inhibition, and antiinflammation by catechin derivatives. (A) Pol λ inhibitory activity of 100 μM of each compound versus anti-inflammatory activity of 250 μg of the same compound. (B) Pol λ inhibitory activity of 100 μM of each compound versus cancer cell growth inhibitory activity of 100 μM of the same compound. (C) Cancer cell growth inhibitory activity of 100 μM of each compound versus anti-inflammatory activity of 250 μg of the same compound. Numbers 1 to 10 in the figures indicate compounds 1 to 10, respectively. The values of correlation coefficients are shown in each figure.

Pol λ is a pol X family polymerase [17]. Although the in vivo biochemical function of pol λ is unclear as yet, pol λ appears to work in a similar manner to pol β. Pol β, which is widely known to have roles in the short-patch base excision repair (BER) pathway [21-24], plays an essential role in neural development [25]. Recently, pol λ was found to contain 5'deoxyribose-5-phosphate (dRP) lyase activity, but no apurinic/apyrimidinic (AP) lyase activity [26] and to be able to substitute pol β in in vitro BER, suggesting that pol λ also participates in BER. Northern blot analysis indicated that transcripts of pol β were abundantly expressed in the testis, thymus, and brain in rats [27], but pol λ was efficiently transcribed mostly in the testis [17]. Bertocci et al. reported that mice in which pol λ was knocked down were not only viable and fertile but also displayed a normal hyper-mutation pattern [28]. TPA not only causes inflammation, but also influences cell proliferation and has physiological effects on cells because it is a tumor promoter [29]. Therefore, antiinflammatory agents are expected to suppress both mammalian cell proliferation and DNA replication / repair in nuclei related to the action of TPA. Since pol λ is a repair-related polymerase [30], the result that the molecular target of catechin derivatives was pol λ is in good agreement. If so, the pol λ inhibitor could be an inhibitor of chronic inflammation. A positive relationship between anti-inflammatory and pol λ-inhibitory activities was found,


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which may be useful as a new and convenient in vitro assay to screen for novel anti-chronic inflammatory compounds. On the other hand, the suppression of human cancer cell growth had almost the same tendency as the inhibition of mammalian pol α among the compounds (Figure 2 and Figure 3), suggesting that the cause of the cancer cell influence might be through the activity of replicative pols such as pols α, δ, and ε.

7. STRUCTURE AND ACTIVITY RELATIONSHIP OF CATECHIN DERIVATIVES Eight catechin derivatives (compounds 1 – 8) and two part compounds of them (compounds 9 and 10) were prepared, and compound 8 (EGCg) was the strongest inhibitor of pol λ and had the strongest anti-inflammation activity of the compounds tested. In the structure of EGCg (Figure 6), the essential moieties of the structure for these activities were considered as: the gallic acid moiety, and the hydroxyl group in the pyrogallol moiety. Compounds 7 (GCg) and 8 (EGCg) contain these moieties; therefore, these compounds should have the strongest activities of both pol λ inhibition and antiinflammation. On the other hand, catechin derivatives lacking moiety (i.e., compounds 1 (C), 2 (EC), 5 (Cg), and 6 (ECg)) had no effect on the growth inhibition of human cancer cells (promyelocytic leukemia cell line, HL-60), suggesting that this moiety must be essential for anti-cancer activity.

OH

OH HO

O

OH OH O C

OH

OH

O

OH

Figure 6. Chemical structure of compound 8 (EGCg). Essential groups (i.e., and ) for pol λ inhibitory activity and anti-inflammatory activity in catechin derivatives are shown.

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8. DISCUSSION Previous studies demonstrated that the existence of an effective daily amount of green tea polyphenols for cancer prevention in humans. Among green tea polyphenols comprising catechin derivatives, compound 8 (EGCg) has cancer preventive activities in vitro, in cell culture, and in vivo [31-36]. EGCg is able to inhibit the migration of bronchial tumor cells and could be an attractive candidate to treat tumor invasion and cell migration [37]. EGCg can also induce apoptosis and suppress the formation and growth of human cancers including colorectal cancers (CRC) [38]. Furthermore, green tea polyphenols were shown to affect several biological pathways. The anti-proliferative action of EGCg on pancreatic carcinoma is mediated through programmed cell death or apoptosis as evident from nuclear condensation, caspase-3 activation, and poly-ADP ribose polymerase (PARP) cleavage [39]. The biological effects of green tea and tea polyphenols, including anti-oxidative activity toward low-density lipoproteins [40], anti-carcinogenic [41], and anti-bacterial actions [4244], have been examined extensively both in vitro and in vivo. Catechin derivatives have also been receiving attention for their protective effects against cardiovascular disease and cancer [45-49]. The amounts required are assumed to be equivalent to 10 Japanese-size cups of green tea per day, and it contains 2.5 g green tea extract. Catechin derivatives such as EGCg have a wide-range of target organs, such as the digestive tract, lungs, liver, pancreas, breast, bladder, prostate, and skin [1, 2]. The human intake from the above sources would be approximately 0.2 g of EGCg per day, and this concentration in the human body (60 kg) is equivalent to 7.2 μM (3.3 μg/ml, molecular weight of EGCg is 458.37). This dose would be able to significantly inhibit the activities of mammalian pols such as pol λ in vitro, and might give the bioactive effects for human health in vivo. In conclusion, green tea, containing catechin derivative, is a potential functional food for preventing cancer and inflammation, and tea catechin derivatives, especially EGCg, could be considered as possible candidates for anti-cancer and anti-inflammation agents.

ACKNOWLEDGEMENTS We are grateful for the donations of calf pol α, rat pol β, human pol γ, human pols and ε, human pols η and ι, human pol κ, and human pol λ by Dr. M. Takemura of Tokyo University of Science (Tokyo, Japan), Dr. A. Matsukage of Japan Women's University (Tokyo, Japan), Dr. M. Suzuki of Nagoya University (Nagoya, Japan), Dr. K. Sakaguchi of Tokyo University of Science (Chiba, Japan), Dr. F. Hanaoka and Dr. C. Masutani of Osaka University (Osaka, Japan), Dr. H. Ohmori of Kyoto University (Kyoto, Japan), and Dr. O. Koiwai of Tokyo University of Science (Chiba, Japan), respectively. This work was supported in part by a Grant-in-Aid for Kobe-Gakuin University Joint Research (A), and the “Academic Frontier” Project for Private Universities: matching fund subsidy from the Ministry of Education, Science, Sports, and Culture of Japan (MEXT), 2006 - 2010, (H. Y. and Y. M.). Y. M. acknowledges a Grant-in-Aid for Young Scientists (A) (No. 19680031) from MEXT, Grants-in-Aid from the Nakashima Foundation (Japan), Foundation


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of Oil & Fat Industry Kaikan (Japan), and The Salt Science Research Foundation, No. 08S3 (Japan).

REFERENCES [1]

Fujiki, H. & Suganuma, M. (2002). Green tea and cancer prevention. Proc. Japan Acad., 78, 263-270. [2] Fujiki, H. (2005). Green tea: Health benefits as cancer preventive for humans. Chem. Rec., 5, 119-132. [3] Chen, Z., Zhu, Q. Y., Tsang, D. & Huang, Y. (2001). Degradation of green tea catechins in tea drinks. J. Agric. Food Chem., 49, 477-482. [4] Hubscher, U., Maga, G. & Spadari, S. (2002). Eukaryotic DNA polymerases. Annu. Rev. Biochem., 71, 133-163. [5] Kimura, S., Uchiyama, Y., Kasai, N., Namekawa, S., Saotome, A., Ueda, T., Ando, T., Ishibashi, T., Oshige, M., Furukawa, T., Yamamoto, T., Hashimoto, J. & Sakaguchi, K. (2002). A novel DNA polymerase homologous to Escherichia coli DNA polymerase I from a higher plant, rice (Oryza sativa L.). Nucleic Acids Res., 30, 1585-1592. [6] Nishida, M., Hada, T., Kuramochi, K., Yoshida, H., Yonezawa, Y., Kuriyama, I., Sugawara, F., Yoshida, H. & Mizushina, Y. (2008). Diallyl sulfides: Selective inhibitors of family X DNA polymerases from garlic (Allium sativum L.). Food Chem., 108, 551-560. [7] Mizushina, Y., Takeuchi, T., Kuramochi, K., Kobayashi, S., Sugawara, F., Sakaguchi, K. & Yoshida, H. (2007). Study on the molecular structure and bio-activity (DNA polymerase inhibitory activity, anti-inflammatory activity and anti-oxidant activity) relationship of curcumin derivatives. Curr. Bioactive Compounds, 3, 171-177. [8] Takeuchi, T., Ishidoh, T., Iijima, H., Kuriyama, I., Shimazaki, N., Koiwai, O., Kuramochi, K., Kobayashi, S., Sugawara, F., Sakaguchi, K., Yoshida, H. & Mizushina, Y. (2006). Structural relationship of curcumin derivatives binding to the BRCT domain of human DNA polymerase λ. Genes Cells, 11, 223-235. [9] Mizushina, Y., Nakanishi, R., Kuriyama, I., Kamiya, K., Satake, T., Shimazaki, N., Koiwai, O., Uchiyama, Y., Yonezawa, Y., Takemura, M., Sakaguchi, K. & Yoshida, H. (2006). β-sitosterol-3-O-β-D-glucopyranoside: a eukaryotic DNA polymerase λ inhibitor. J. Steroid Biochem. Mol. Biol., 99, 100-107. [10] Mizushina, Y., Yagita, E., Kuramochi, K., Kuriyama, I., Shimazaki, N., Koiwai, O., Uchiyama, Y., Yomezawa, Y., Sugawara, F., Kobayashi, S., Sakaguchi, K. & Yoshida, H. (2006). 5-(Hydroxymethyl)-2-furfural: a selective inhibitor of DNA polymerase λ and terminal deoxynucleotidyltransferase. Arch. Biochem. Biophys., 446, 69-76. [11] Mizushina, Y., Saito, A., Tanaka, A., Nakajima, N., Kuriyama, I., Takemura, M., Takeuchi, T., Sugawara, F. & Yoshida, H. (2005). Structural analysis of catechin derivatives as mammalian DNA polymerase inhibitors. Biochem. Biophys. Res. Commun., 333, 101-109. [12] Nakai, C. M., Kamiya, K., Satake, T., Kuriyama, I., Iijima, H., Ikawa, H., Yoshida, H. & Mizushina, Y. (2005). Novel topoisomerase I and II inhibitors, parameritannins A-2 and A-3, are selective human cancer cytotoxins. Int. J. Pharmacol., 1, 138-151.

360


[13] Murakami, C., Kumagai, T., Hada, T., Kanekazu, U., Nakazawa, S., Kamisuki, S., Maeda, N., Xu, X., Yoshida, H., Sugawara, F., Sakaguchi, K. & Mizushina, Y. (2003). Effects of glycolipids from spinach on mammalian DNA polymerases. Biochem. Pharmacol., 65, 259-267. [14] Mizushina Y, Kamisuki S, Kasai N, Ishidoh T, Shimazaki N, Takemura M, Asahara H, Linn S, Yoshida S, Koiwai O, Sugawara F, Yoshida H. & Sakaguchi K. (2002). Petasiphenol: a DNA polymerase λ inhibitor. Biochemistry, 41, 14463-14471. [15] Mizushina, Y., Tanaka, N., Yagi, H., Kurosawa, T., Onoue, M., Seto, H., Horie, T., Aoyagi, N., Yamaoka, M., Matsukage, A., Yoshida, S. & Sakaguchi, K. (1996). Fatty acids selectively inhibit eukaryotic DNA polymerase activities in vitro. Biochim. Biophys. Acta, 1308, 256-262. [16] Mizushina, Y., Yoshida, S., Matsukage, A. & Sakaguchi, K. (1997). The inhibitory action of fatty acids on DNA polymerase β. Biochim. Biophys. Acta, 1336, 509-521. [17] Garcia-Diaz, M., Dominguez, O., Lopez-Fernandez, L. A., de Lera, L. T., Saniger, M. L., Ruiz, J. F., Parraga, M., Garcia-Ortiz, M. J., Kirchhoff, T., del Mazo, J., Bernad, A. & Blanco, L. (2000). DNA polymerase λ (Pol λ), a novel eukaryotic DNA polymerase with a potential role in meiosis. J. Mol. Biol., 301, 851-867. [18] Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods, 65, 55-63. [19] Hecker, E. (1978). Carcinogenesis, Raben Press, NY, 2, Pp. 11-48. [20] Fujiki, H. & Sugimura, T. (1987) New classes of tumor promoters: teleocidin, aplysiatoxin, and palytoxin. Adv. Cancer Res., 49, 223-264. [21] Singhal, R. K. & Wilson, S. H. (1993). Short gap-filling synthesis by DNA polymerase β is processive. J. Biol. Chem., 268, 15906-15911. [22] Matsumoto, Y. & Kim, K. (1995). Excision of deoxyribose phosphate residues by DNA polymerase β during DNA repair. Science, 269, 699-702. [23] Sobol, R. W., Horton, J. K., Kuhn, R., Gu, H., Singhal, R. K., Prasad, R., Rajewsky, K. & Wilson, S. H. (1996). Requirement of mammalian DNA polymerase-β in baseexcision repair. Nature, 379, 183-186. [24] Ramadan, K., Shevelev, I. V., Maga, G. & Hubscher, U. (2002). DNA polymerase λ from calf thymus preferentially replicates damaged DNA. J. Biol. Chem., 277, 1845418458. [25] Sugo, N., Aratani, Y., Nagashima, Y., Kubota, Y. & Koyama, H. (2000). Neonatal lethality with abnormal neurogenesis in mice deficient in DNA polymerase β. EMBO J., 19, 1397-1404. [26] Garcia-Diaz, M., Bebenek, K., Kunkel, T. A. & Blanco, L. (2001). Identification of an intrinsic 5'-deoxyribose-5-phosphate lyase activity in human DNA polymerase λ: a possible role in base excision repair. J. Biol. Chem., 276, 34659-34663. [27] Hirose, F., Hotta, Y., Yamaguchi, M. & Matsukage, A. (1989). Difference in the expression level of DNA polymerase β among mouse tissues: high expression in the pachytene spermatocyte. Exp. Cell Res., 181, 169-180. [28] Bertocci, B., De Smet, A., Flatter, E., Dahan, A., Bories, J. C., Landreau, C., Weill, J. C. & Reynaud, C. A. (2002). Cutting edge: DNA polymerases μ and λ are dispensable for Ig gene hypermutation. J. Immunol., 168, 3702-3706.


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[29] Nakamura, Y., Murakami, A., Ohto, Y., Torikai, K., Tanaka, T. & Ohigashi, H. (1998). Suppression of tumor promoter-induced oxidative stress and inflammatory responses in mouse skin by a superoxide generation inhibitor 1'-acetoxychavicol acetate. Cancer Res., 58, 4832-4839. [30] Garcia-Diaz, M., Bebenek, K., Sabariegos, R., Dominguez, O., Rodriguez, J., Kirchhoff, T., Garcia-Palomero, E., Picher, A. J., Juarez, R., Ruiz, J. F., Kunkel, T. A. & Blanco, L. (2002). DNA polymerase λ, a novel DNA repair enzyme in human cells. J. Biol. Chem., 277, 13184-13191. [31] Nakachi, K., Matsuyama, S., Miyake, S., Suganuma, M. & Imai, K. (2000). Preventive effectts of drinking green tea on cancer and cardiovascular disease: epidemiological evidence for multiple targeting prevention. Bio-Factors, 13, 49-54. [32] Okuda, T. (2005). Systematics and health effects of chemically distinct tannins in medicinal plants. Phytochemistry, 66, 2012-2031. [33] Wang, Z. Y., Hong, J. Y., Huang, M. T., Reuhl, K. R., Conney, A. H. & Yang, C. S. (1992). Inhibition of N-nitrosodiethylamine- and 4-(methylnitrosamino)-1-(3-pyridyl)1-butanone-induced tumorigenesis in A/J mice by green tea and black tea. Cancer Res., 52, 1943-1947. [34] Yang, C. S. & Wang, Z. Y. (1993). Tea and cancer. J. Natl. Cancer Inst., 85, 10381049. [35] Lu, Y. P., Lou, Y. R., Lin, Y., Shih, W. J., Huang, M. T., Yang, C. S. & Conney, A. H. (2001). Inhibitory effects of orally administered green tea, black tea, and caffeine on skin carcinogenesis in mice previously treated with ultraviolet B light (high-risk mice): relationship to decreased tissue fat. Cancer Res., 61, 5002-5009. [36] Gupta, S., Hastak, K., Ahmad, N., Lewin, J. S. & Mukhtar, H. (2001). Inhibition of prostate carcinogenesis in TRAMP mice by oral infusion of green tea polyphenols. Proc. Natl. Acad. Sci. USA, 98, 10350-10355. [37] Hazgui, S., Bonnomet, A., Nawrocki, R. B., Milliot, M., Terryn, C., Cutrona, J., Polette, M., Birembaut, P. & Zahm, J. M., (2008). Epigallocatechin-3-gallate (EGCG) inhibits the migratory behavior of tumor bronchial epithelial cells. Respir Res ., 9, 33. [38] Kumar, N., Shibata, D., Helm, J., Coppola, D. & Malafa, M. (2007). Green tea polyphenols in the prevention of colon cancer. Front Biosci., 12, 2309-2315. [39] Qanungo, S., Das, M., Haldar, S. & Basu, A. (2005). Epigallocatechin-3-gallate induces mitochondrial membrane depolarization and caspase-dependent apoptosis in pancreatic cancer cells. Carcinogenesis, 26, 958-967. [40] Miura, S., Watanabe, J., Tomita, T., Sano, M. & Tomita, I. (1994). The inhibitory effects of tea polyphenols (flavan-3-ol derivatives) on Cu2+ mediated oxidative modification of low density lipoprotein. Biol. Pharm. Bull., 17, 1567-1572. [41] Dreosti, I. E., Wargovich, M. J. & Yang, C. S. (1997). Inhibition of carcinogenesis by tea: the evidence from experimental studies. Crit. Rev. Food Sci. Nutr., 37, 761-770. [42] Amarowicz, R., Pegg, R. B. & Bautista, D. A. (2000). Antibacterial activity of green tea polyphenols against Escherichia coli K 12. Nahrung, 44, 60-62. [43] Sakanaka, S., Kim, M., Taniguchi, M. & Yamamoto, T. (1989). Antibacterial substances in Japanese green tea extract against Streptococcus mutants, a cariogenic bacterium. Agric. Biol. Chem., 53, 2307-2311.

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[44] Mabe, K., Yamada, M., Oguni, I. & Takahashi, T. (1999). In vitro and in vivo activities of tea catechins against Helicobacter pylori. Antimicrob. Agents Chemother, 43, 17881791. [45] Ahmad, N. & Mukhtar, H. (1999). Green tea polyphenols and cancer: biologic mechanisms and practical implications. Nutr. Rev., 57, 78-83. [46] Buschman, J. L. (1998). Green tea and cancer in humans: a review of the literature. Nutr. Cancer, 31, 51-57. [47] Hertog, M. G., Feskens, E. J., Hollman, P. C., Katan, M. B. & Kromhout, D. (1993). Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study. Lancet , 342, 1007-1011. [48] Imai, K. & Nakachi, K. (1985). Cross sectional study of effects of drinking green tea on cardiovascular and liver disease. Biochem. Med. J., 310, 693-696. [49] Yang, C. S. (1999). Tea and health. Nutrition, 15, 946-949.



Chapter 18

TELOMERASE REGULATION IN RESPONSE TO GREEN TEA Huaping Chen1 and Trygve O. Tollefsbol1,2,3,4,* 1

Department of Biology, University of Alabama at Birmingham, AL 35294, USA 2 Center for Aging, University of Alabama at Birmingham, AL 35294, USA 3 Comprehensive Cancer Center, University of Alabama at Birmingham, AL 35294, USA 4 Clinical Nutrition Research Center, University of Alabama at Birmingham, AL 35294, USA

ABSTRACT The anti-tumor effect of green tea, especially its major constituent EGCG, has been demonstrated in several animal experiments and its ability to induce apoptosis of most of cancer cell lines has been further documented in cell culture models. A number of mechanisms for how green tea impacts cancer have been proposed. These mechanisms basically include intervention of cell signal transduction pathways or changes of cell epigenetic processes. Telomerase has been recognized as a novel target of green tea. This important enzyme is largely localized to cancer cells, is responsible for the maintenance of telomeres so that cancer cells can escape the replication problem due to their linear chromosomes, and it has been shown to be reactivated in almost all tumor tissues. Telomerase has been found to be inhibited by green tea in telomerase-positive cancer cell lines. This analysis assesses the progress on research of the mechanisms pertaining to how telomerase activity is regulated by green tea in cancer cells. A number of mechanisms for how green tea works through this pathway have been proposed. Since telomerase has been identified as a potential molecular target for cancer treatment, and green tea has been shown to inhibit telomerase, clarifying the specific mechanisms for how green tea functions in this pathway should shed new light on the potential to design effective and novel preventive or anti-cancer approaches using green tea.

*

Corresponding Author. Department of Biology, 175 Campbell Hall, 1300 University Boulevard, Birmingham, AL 35294-1170, USA. Tel: +1-205-934-4573; Fax: +1-205-975-6097; Email: [email protected]

364

Huaping Chen and Trygve O. Tollefsbol

Keywords: Green tea, EGCG, Telomerase, Cancer

INTRODUCTION Cancer still accounts for a high number of deaths worldwide. It is characterized by unlimited cell division, invasion into adjacent tissues, and may lead to metastasis which can spread to other tissues in the body through lymph or blood. The occurrence of cancer is a multistage process which includes initiation, promotion, and progression of carcinogenesis. Cancers can be caused by aberrant genetic material of the transformed cells. They are formed due in part to the interaction between genes of humans and environmental carcinogens, mutation in replication or inherited factors. These abnormalities include mutation of DNA sequences and epigenetic changes of the genome. The expression levels of two sets of genes are often changed in cancer. There are oncogenes which are responsible for the division of cells that have been activated, and tumor suppressor genes which are often inactivated as a result of disruption in the balance that controls proliferation of cells. Surgery, chemotherapy, radiation therapy, immunotherapy, and monoclonal antibody therapy have been used for treating cancer patients depending on the progress of the cancer and the health status of the patients as well as other factors.

EGCG

ECG

EC Figure 1. Molecular structures of the major polyphenol constituents of tea.

EGC

Telomerase Regulation in Response to Green Tea

frying or steaming

Fresh leaves

fermentation

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Prevent oxidation of polyphenols

Green tea

Partial oxidation of polyphenols

Oolong tea

Oxidation of polyphenols

Black tea

Figure 2. Processes by which various types of teas are prepared.

Tea is considered to be the most popular beverage worldwide next to water. Its use originates from China, and it is regularly consumed by a large number of individuals in the Asian countries. Tea is made from the leaves of Camellia sinensis. Several biologically active molecules are found in its leaves and the most important group is polyphenols, although other constituents include caffeine, flavonols such as quercetin and myricetin, and theobromine[1]. The primary polyphenols in tea are: epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC), epicatechin (EC), gallocatechin, and catechin (Figure 1). EGCG is the major and most active constituent of tea. There are currently three major types of tea: green tea, black tea, and oolong tea. They vary from each other in the procedure by which they are prepared and correspondingly the constituents they contain. Green tea is made by frying or steaming the fresh leaves of this plant. The oxidative enzymes in leaves can be heat-inactivated and thus the polyphenols in leaves can be protected in this way. Black tea is made by crushing the leaves and then letting the oxidation of polyphenols occur which is mediated by enzymes contained in the leaves, a process known as fermentation. Oolong tea is made by partial oxidation of the polyphenols in the leaves, and the ratio of the oxidized polyphenols in oolong tea lies between green tea and black tea (Figure 2). Thus, the distinguishing feature of green tea is that it is processed to prevent the oxidation of polyphenols, while the majority of the polyphenols in black tea have been oxidized into dimeric theaflavin molecules and polymeric thearubigins during its production, and theaflavins render black tea’s color and taste. Since traditional chemotherapy often produces unsatisfactory and toxic effects, non-toxic or less cytotoxic drugs for cancer prevention or treatment are therefore warranted. The effects of green tea have been investigated in a wide array of systems varying from animal models to cell lines, and inhibition of tumors and apoptosis of cancer cells have been observed. A variety of mechanisms have been proposed based on these studies. Here, we will briefly evaluate the biological effect of green tea first, and will then focus on the telomerase inhibition effect of green tea which has been regarded as a potential mechanism that imparts its chemopreventive and anticancer effects.

366


BENEFICIAL EFFECT OF GREEN TEA Epidemiological studies indicate that consuming green tea is associated with a decrease in the occurrence of cancer in Asian people. In addition, research from animal models shows that green tea can inhibit tumor formation in a number of tissues such as lung, oral cavity, esophagus, skin, stomach, liver, prostate, small intestine, colon, breast, and pancreas. Cell culture experiments also demonstrate that EGCG can inhibit the proliferation of cancer cells. Here we would like to briefly review those effects.

INHIBITION OF TUMOR GROWTH IN VIVO AND ANGIOGENESIS INHIBITION Tea and its constituents have been shown to inhibit tumor formation in different organs in animal models. The organs affected include but are not limited to lung, oral cavity, esophagus, skin, stomach, liver, prostate, small intestine, colon, breast, and pancreas [2]. EGCG has been shown to block angiogenesis which is the process involving the formation of blood vessels. This process therefore deprives the tumor cells of the nutrients that they need to grow [3, 4]. Oral administration of GTPs (green tea polyphenols) in the drinking water for mice can inhibit expression of angiogenic factors such as matrix metalloproteinases (MMPs), and their natural inhibitor, TIMP1, which is upregulated in response to GTPs [5].

PROLIFERATION INHIBITION AND INDUCTION OF APOPTOSIS OF CANCER CELLS IN VITRO Green tea and its major constituent, EGCG, have been shown to inhibit proliferation and induce apoptosis of a series of tumor cell lines. For instance, It has been shown that the exposure of human lymphoid leukemia Molt 4B cells to epigallocatechin gallate (EGCG) and epigallocatechin (EGC) led to both growth inhibition and the induction of programmed cell death (apoptosis) in a concentration- and time-dependent manner [6]. Green tea extract and EGCG have also been demonstrated to inhibit growth and induce apoptosis of human stomach cancer KATO III cells [7]. Another study has found that tea polyphenols (EGCG and EGC) can inhibit growth and induce apoptosis in human cancer cell lines such as lung tumor cell lines H661 and H1299, with estimated IC50 values of 22 µM, while the lung cancer cell line, H441, and colon cancer cell line, HT-29, have weak responses to these polyphenols, with IC50 values 2- to 3-fold higher [8]. Further work has indicated that tea polyphenol-induced apoptosis and the growth inhibitory activities may be mediated by hydrogen peroxide generated by polyphenols [8]. EGCG has also been shown to inhibit telomerase in MCF-7 breast cancer cells leading to suppression of cell viability and induction of apoptosis [9, 10].

Telomerase Regulation in Response to Green Tea

367

OTHER BENEFICIAL EFFECTS Besides the effects mentioned above, a series of other effects of green tea have also been reported, including antidiabetes properties, anti-inflammatory [11], anti-arthritic [12, 13], antibacteria, anti-viral [14, 15] and neuroprotective effects [16]. Due to the limitation of space, these effects will not be discussed in detail here.

POTENTIAL MECHANISMS PROPOSED To address those effects that have been observed in vivo and in vitro, a number of mechanisms pertaining to how EGCG mediates its cancer prevention properties have been proposed. These include the direct causes and the signal pathways that may be involved. The direct causes may include anti-oxidatant properties, telomerase inhibition, DNMT1 inhibition, and inhibition of the ubiquitin-proteasome and topoisomerase I [17]. Signal pathways involved may include mediation of growth factor-mediated pathways such as those initiated or carried out by epidermal growth factor receptor (EGFR) [18], the mitogen-activated protein (MAP) kinase-dependent proteins [19], activator protein 1 (AP-1) [20], nuclear factorB (NF-kappaB) [21], matrix metalloproteinases [5] and other potential targets.

ANTI-OXIDATION AND PRO-OXIDANT ACTIVITY Polyphenols have been shown to have anti-oxidation effects in several in vitro studies. For example, it has been demonstrated that 40 µmol/L of EGCG can inhibit the production of hydrogen peroxide in UVB-treated normal human keratinocytes (NHEK) by 66–80% [22]. This effect was correlated with inhibition of UVB-induced phosphorylation of ERK1/2, jun N-terminal kinase (JNK), and p38. Similar inhibitory activity of EGCG was observed when hydrogen peroxide was directly added to this cell culture system [22]. However, in vivo analyses of anti-oxidative effects of green tea on tumor inhibition are very limited in number. EGCG and theaflavin-3,3’-digallate (TFdiG) have also been shown to inhibit lipid peroxidation in vitro [23]. Meng Q et al [24] reported that treatment of middle-aged male Fischer 344 rats with high doses of EGCG resulted in significant decline in the concentration of hydroxy-2'-deoxyguanosine in the plasma while maintaining a better mitochondrial potential in the peripheral lymphocytes and preventing the deletion of the ND4 region from mitochondrial DNA in the liver compared with low dose treatment. EGCG has been recently shown to modulate the activity of nuclear factor erythroid 2 p45 (NF-E2)-related factor (Nrf2), which further induces expression of glutathione S-transferase, glutathione peroxidase, glutamate cysteine ligase, hemeoxygenase-1, and other antioxidant enzymes [25]. On the other hand, polyphenols have been demonstrated to have a pro-oxidant activity. For instance, the polyphenols may generate superoxide radicals and hydrogen peroxide in cell culture systems [26]. Interestingly, this is the cause for the instability of EGCG in cell culture conditions, where its halflife is less than 2 h [27], as the radicals generated due to this activity may cause an artificial impact on cell growth. Hong et al. [27] showed that administration of 50 µmol/L of EGCG to HT29 cells in McCoy’s 5A medium results in the production of up to

368


23 µmol/L of hydrogen peroxide. Moreover, adding catalase to the medium before EGCG treatment can partially or completely block apoptosis in H661 lung cancer cells and Rastransformed human bronchial cells [28, 29]. These data indicate that many of the effects observed in cell culture systems may originate from hydrogen peroxide production secondary to polyphenol administration. However, since oxygen partial pressure in vivo ( 2 cups/d

1.40

P for trend = 0.0006

1.20 1.00 0.80

*

0.60 0.40 0.20 0.00 n=170

n=108

n=725

Figure 2. Odds ratios (ORs) for the association between different frequencies of green tea consumption and cognitive impairment. The bars indicate adjusted ORs for the association between green tea consumption frequencies and cognitive impairment; error bar represent the corresponding 95% CIs. Multivariate logistic regression analysis was used to calculate ORs for cognitive impairment relative to the consumption frequencies of green tea, with the lowest frequency category (≤3 cups/wk) treated as the reference group. Cognitive impairment was defined as a Mini-Mental State Examination score < 26. *P< 0.001. 1 cup = 0.1L. Data from Kuriyama et al., (2006).

GREEN TEA CATECHINS AND BRAIN FUNCTION Influence of Green Tea Catechins on Cognitive Function Green tea catechins currently show a profound beneficial effect on cognitive function both in animal and humans. An epidemiological cross-sectional study involving 1003 Japanese subjects 70 years old or older, demonstrated the relationship between the consumption of green tea and cognitive function. The study clearly showed that a higher consumption of green tea is associated with lower prevalence of cognitive impairment in humans [Kuriyama et al., 2006]. Drinking more than 2 cups a day of green tea slashed odds of cognitive impairment in elderly Japanese men and women by 64% (Figure 2). The results might partly explain the relatively lower prevalence of dementia, especially AD, in Japan compared to Europe and North America [Ritchie and Lovestone, 2002].

Preventive Effects of Green Tea Catechins on Dementia A

B

4

Control (n=8)

4

3

2

a b

1

0

Working Memory Errors

Reference Memory Errors

435

Catechins (n=9) 3

2

1

a b

0 0

2

4

6

8

Blocks of Six Trials

10

0

2

4

6

8

10

Blocks of Six Trials

Figure 3. Reference (A) and working (B) memory-related learning ability in the radial maze task of rats administered water alone (control, n = 8), or green tea catechins (0.5% polyphenon E; n = 9) for 26 wk. Values are means ± SEM in each block of six trials. Groups without a common letter differ, P < 0.05. Data from Haque et al., (2006).

In animal experiments with rats, long-term administration of green tea catechins in the form of Polyphenon E (PE: EGCG 63%; EC 11%; EGC 6%; ECG 6%) mixed with water (0.5% w/v) improved spatial cognition learning ability when measuring eight-arm radial maze task (Figure 3). The radial maze estimates two types of memory function, reference and working memory without any harmful stress to the rats. Reference memory involves utilizing information that remains constant over time whereas working memory involves holding information that is pertinent only within a short period of time. The lower numbers of reference memory errors (RMEs; entry into unbaited arm within one trial) and working memory errors (WMEs; repeated entry into arms that had already been visited within same trial) implies a higher acquisition of spatial learning ability in rats. 0.5% PE-administered rats had significantly lower LPO levels both in plasma and hippocampus and higher ferric reducing antioxidant power (FRAP) levels (an indicator of plasma antioxidant status) (Table 2). A significant positive correlation between the hippocampal LPO levels and the number of RMEs, and a negative correlation between plasma FRAP levels and the number of RMEs were observed in block 10 of the radial maze task in controls and in 0.5% PE-administered rats (Figure 4). These results indicate that the lower LPO and higher FRAP levels, combined with higher acquisition of memory performance, are likely to be the effects of PE on scavenging and/or preventing radical formation at the neuronal level. Similarly, it is reported that dietary administration of green tea catechins prevents memory regression and DNA oxidative damage in aged mice [Unno et al., 2007]. Thus, these findings suggest that continued intake of green tea catechins might promote healthy ageing of the brain in older persons.

Michio Hashimoto, Md Abdul Haque, Kohinoor Begum Himi et al.

A r = - 0.570 p = 0.017

r = 0.520 p = 0.032

3.0 Reference M emory Errors

B 3.0.

2.0

2.0

1.0

1.0 Control (n=8) Catechins (n=9)

0.0 0

0.25

0.50

1

0.75

150

TBARS levels in hippocampus (nmol/mg protein)

Reference M emory Errors

436

0.0 200

250

300

350

FRAP levels in plasma ( mol/L)

Figure 4. Correlations between the numbers of reference memory errors and each of the hippocampal TBARS (Figure 4A) and the plasma FRAP (Figure 4B) levels in controls and green tea catechinsadministered rats. (o), control rats; (●), green tea catechins (0.5% polyphenon E)-administered rats. Data from Haque et al., (2006).

Table 2. Effects of green tea catechins (0.5% PE) administration on oxidative status of plasma, cerebral cortex and hippocampus in rats Plasma

Cerebral cortex

Hippocampus

TBARS

FRAP

TBARS

ROS

TBARS

ROS

Control (n=8)

4.0 ± 0.1

224 ± 10

1.45 ± 0.10

0.21 ± 0.03

0.63 ± 0.09

0.13 ± 0.03

Catechins (n=9)

3.0 ± 0.1*

271 ± 11*

1.27 ± 0.08

0.19 ± 0.04

0.33 ± 0.03*

0.05 ± 0.01*

Values are means ± SEM. Rats were orally administered either water (control rats) or green tea catechins (Polyphenon E, PE: EGCG 63%; EC 11%; EGC 6%; ECG 6%, catechins rats) for 26 weeks. The levels of lipid peroxide were measured as TBARS (thiobarbituric acid reactive substance) indicated in nmol malondialdehyde/mL for plasma and nmol malondialdehyde/mg protein for brain tissues. Reactive oxygen species (ROS) is indicated in pmol/min/mg protein. The antioxidant potential of plasma was mesured as ferric reducing antioxidation power of plasma (FRAP) is indicated in μmol/L. *P

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