Review Effects of tea polyphenols on signal transduction ...

Mutation Research 555 (2004) 3–19

Review

Effects of tea polyphenols on signal transduction pathwaysrelated to cancer chemoprevention

Zhe Hou, Joshua D. Lambert, Khew-Voon Chin, Chung S. Yang∗

Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, RutgersUniversity, 164 Frelinghuysen Road, Piscataway, NJ 08854 8020, USA

Received 28 April 2004; received in revised form 9 June 2004; accepted 10 June 2004

Abstract

The inhibition of carcinogenesis by tea and tea polyphenols has been demonstrated in different animal models by manyinvestigators. The mechanisms of this inhibitory activity have also been investigated extensively, mostly in cell culture systems,but no clear conclusion can be reached concerning the cancer preventive mechanisms in vivo. In this article, we reviewed thepossible mechanisms, which include the inhibition of specific protein kinase activities, blocking receptor-mediated functions,and inhibition of proteases. These events may lead to cell cycle regulation, growth inhibition, enhanced apoptosis, inhibitionof angiogenesis, and inhibition of invasion and metastases. The possible complications of translating results obtained in cellculture studies to animals and humans are discussed. It is likely that multiple signal transduction pathways are involved in

rmined in

; COX,F,

; TFdiG,

the inhibition of carcinogenesis by tea constituents. The relative importance of these pathways needs to be detevivo.© 2004 Elsevier B.V. All rights reserved.

Keywords:Tea polyphenol; EGCG; Theaflavins; Signal transduction; Cancer prevention

Abbreviations: AA, arachidonic acid; AP-1, activator protein-1; BMP, bone morphogenetic protein; CDK, cyclin dependent kinasecyclooxygenase; EC, (−)-picatechin; ECG, (−)-epicatechin-3-gallate; EGC, (−)-epigallocatechin; EGCG, (−)-epigallocatechin-3-gallate; EGepidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular regulated kinase; JNK, c-JUN NH2-terminal kinase; LOX,lipoxygenase; LT, leukotriene; MMP, matrix metalloproteinase; NF-�B, nuclear factor�B; PDGF-�, platelet-derived growth factor�; PDGF-R�, PDGF-�, receptor; PGE2, prostaglandin E2; SOD, superoxide dismutase; STAT3, signal transducer and activator of transcription 3theaflavin-3-3′-gallate; TGF-�, transforming growth factor�

∗ Corresponding author. Tel.: +1 732 445 3400x244; fax: +1 732 445 0687.E-mail address:[email protected] (C.S. Yang).

0027-5107/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.mrfmmm.2004.06.040

4 Z. Hou et al. / Mutation Research 555 (2004) 3–19

Contents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

2. Inhibition of mitogen activated protein (MAP) kinases and activator protein-1 (AP-1). . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3. Blocking growth factor receptor-mediated pathways. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

4. Cell cycle arrest. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

5. Inhibition of nuclear factor-�B (NF-�B) signaling pathway. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

6. Induction of apoptosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

7. Novel signaling mechanisms of EGCG identified by expression genomics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

8. Inhibition of aberrant arachidonic acid metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

9. Inhibition of protease activities. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

10. Other possible targets. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

11. Concluding remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

1. Introduction

Tea (Camellia senesis)is a popular beverage world-wide. Historically, tea has also been used for medicinalpurposes. In recent years, extensive studies have been

lung, oral cavity, esophagus, stomach, small intestine,colon, liver, pancreas, bladder, and prostate (reviewedin [1–3]). The mechanisms of the chemopreventiveactivity, however, are not clearly understood. Althoughthe cancer preventive activity of tea polyphenols has

t inof

ts

e-tedns

theri-areycediventntsIt is

conducted on tea and tea constituents because oftheir potential beneficial health effects. In addition tocaffeine, green tea contains characteristic polyphenolconstituents, generally known as catechins, whichinclude (−)-epigallocatechin-3-gallate (EGCG); (−)-epicatechin-3-gallate (ECG); (−)-epigallocatechin(EGC) and (−)-epicatechin (EC). Of these catechins,EGCG is the most abundant and the most biologicallyactive compound. In making black tea, the “fermenta-tion” process causes the oxidation and polymerizationof most of the catechins to form theaflavins, includingtheaflavin, theaflavin-3-gallate, theaflavin-3′-gallate,and theaflavin-3,3′-digallate (TFdiG), as well asthe poorly characterized larger polymers known asthearubigins as the major products. The structuresof some of these compounds are shown inFig. 1.Tea has been shown to inhibit tumorigenesis in manyanimal models, including those for cancer of the skin,

been demonstrated in many experimental systems[2],caffeine has been shown to be the active ingrediensome other systems; for example, in the inhibitionUV-light induced skin tumorigenesis in mice[4] andchemically induced lung tumorigenesis in F-344 ra[5].

This chapter will discuss the effects of tea polyphnols on signal transduction pathways that are relato cancer chemoprevention. Mechanistic investigatioof the cancer chemopreventive activity depend onmethodologies that are available at different time peods. For example, EGCG and other tea polyphenolswell known for their antioxidant activities. Indeed, thehave been demonstrated to inhibit carcinogen-induDNA damage and tumor promoter-induced oxidatstress (reviewed in[1,2,6]). These results are consistewith the commonly mentioned idea that tea prevecancer because tea polyphenols are antioxidants.

Z. Hou et al. / Mutation Research 555 (2004) 3–19 5

Fig. 1. Structures of tea polyphenols.

unclear, however, whether this is a general mechanismfor cancer prevention, especially in human carcinogen-esis when strong carcinogens and tumor promoters arenot known to be involved. In the 1980s when carcino-gen activation and tumor promotion were active areasof research, these events had been proposed as the tar-gets of tea polyphenol action. With the advancementof research on signal transduction, many current ef-forts have been focused on the effect of tea polyphenolson signal transduction pathways. Most of these studieshave been carried out in cell lines. It is not clear whethersome of the phenomena observed in cell lines occur invivo.

There are two major problems in extrapolating re-sults observed in cell lines to animal models. (1) Theconcentration of the test compound used in cell linesystems, for example, EGCG at 20, 50, 100�M, orhigher concentrations, are much higher than those ob-served in the plasma or tissues in experimental ani-mals or humans after ingestion of tea or related teapreparations[2]. (2) The oxygen partial pressure in acell culture system (160 mmHg) is much higher than

that in the blood or tissues (<40 mmHg)[7]. Under cellculture conditions, EGCG is not stable, with a half-life less than 2 h[8]. The half-life can be extendedseveral fold by the addition of superoxide dismutase(SOD), suggesting a role for superoxide radical in theoxidation and polymerization of EGCG. A proposedmechanism of EGCG auto-oxidation is shown inFig. 2.Similar to many other antioxidants, EGCG and othertea polyphenols may also act as pro-oxidants. Theycan be oxidized to form phenolic radicals, superox-ide radical and hydrogen peroxide. These species maytrigger a variety of biochemical reactions and biologi-cal responses. As will be discussed in subsequent sec-tions, hydrogen peroxide may contribute to cell apop-tosis and the radical species may contribute to the in-activation of epidermal growth factor receptor (EGFR)and telomerase as reported in the literature. It is notclear whether these pro-oxidation of EGCG generatedreactions occur in low oxygen partial pressure con-ditions in vivo in cells, which generally have stronganti-oxidative capacity and low oxygen partial pres-sure.


Fig. 2. Auto-oxidation of EGCG and generation of reactive species.Under neutral or slightly alkaline pH in the cell culture medium,EGCG is oxidized by molecular oxygen to form superoxide radical(O2

•−) and EGCG radical (EGCG•) in a reaction probably catalyzedby trace metal ions such as Cu2+. The O2

•− can then react withanother EGCG molecule to form EGCG•. EGCG• may collide withanother EGCG• to form an EGCG dimmer. More likely, EGCG•may react with EGCG to form EGCG dimer radical, which has thepotential to react with molecular oxygen to generate EGCG dimerand O2

•−. The O2•− can then oxidize another molecule of EGCG

to propagate the chain reaction. The O2•− can also be converted to

H2O2, especially in the presence of superoxide dismutase.

2. Inhibition of mitogen activated protein(MAP) kinases and activator protein-1 (AP-1)

Previously, we found that 5–20�M of EGCGor theaflavin inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)- or epidermal growth factor (EGF)-induced transformation of mouse epidermal cell lineJB6, and the activity was closely related to the in-hibition of the activation of the transcription factorAP-1 [9]. This activity was associated with the inhi-bition of c-Jun NH2-terminal kinase (JNK) phospho-rylation, but not ERK1/2 phosphorylation. Since MAPkinases play key roles in growth and neoplasia, theeffects of tea polyphenols on this cascade were in-vestigated subsequently. In 30.7b Ras12 cells (H-rastransformed JB6 cells), AP-1 was highly activated, andmost tea polyphenols inhibited the transcription activ-ity of AP-1 [10]. The presence of a galloyl group inthe catechin structure was associated with a high in-hibitory ability. The presence of a trihydroxy structureon the B-ring, such as in EGCG, conferred a higherinhibitory activity than those with a dihydroxy struc-ture, such as in ECG. EGCG and TFdiG inhibited the

phosphorylation of MEK1/2, ERK1/2, and ELK-1, aswell as c-Jun (Fig. 3), but not that of JNK[10,11].Further studies suggested that EGCG decreased theassociation between RAF-1 and MEK1, and EGCGcompetitively inhibited the phosphorylation of ELK-1by ERK1/2 possibly by competing for the binding siteon ERK1/2.

These results led us to hypothesize that EGCGinhibits selected protein kinase activities by compet-itively binding to the protein substrate binding site,possibly involving proline-rich sequences[11]. Thishypothesis is supported by the recent finding that,in the prevention of UV-induced damage in the skinof SKH-1 hairless mice by topically applied greentea polyphenols (5 mg in 200�l acetone/mouse),decreased phosphorylation of ERK1/2 and JNK andreduced protein level of p38 were observed[12]. Inthe same mouse strain, green tea polyphenols, appliedin topical hydrophilic ointment or through drinkingwater, prevented UVB-induced depletion of antioxi-dant enzymes in skin. UVB-induced phosphorylationof ERK1/2, JNK and p38 was also blocked by thetreatment[13]. EGCG and theaflavin were also shownto inhibit PI3K pathway by decreasing the levelsof PI3K and phosphorylation of Akt in two humanprostate cancer cell lines, DU145 and LNCaP cells[14].

There have been several reports of EGCG activat-ing ERK1/2 in different cancer cell lines[14,15]. An-tioxidants, such as glutathione andN-acetyl-l-cysteine,w RKa d byE s inv

ane s ofE b-i tingE axb hu-m andt thatE udys r-m r, thecd nes,d

ere able to block this activation, suggesting that Ectivation was caused by oxidative stress induceGCG. It is unclear whether such an effect occurivo.

A very interesting result, observed in normal humpidermal keratinocytes, is that low concentrationGCG (<1�M) increased cell proliferation and inhi

ted UV-induced apoptosis, possibly through activaRK and Akt pathways and changing the Bcl-2/Balance[16]. When EGCG was applied to agedan skin, it resulted in increased cell proliferation

hickening of the skin. This leads to the proposalGCG may prevent the aging of the skin. Another sthowed that adding 100�M EGCG to normal epideal keratinocytes did not induce caspase 3; rathe

ells showed clear differentiated features[17]. Theseata, contrasting the observation in cancer cell lieserve further mechanistic investigation.


Fig. 3. EGFR and MAP kinase cascades as targets for tea polyphenols. Activation of EGFR or HER-2/neu elicits the autophosphorylation ofthe receptors, and the phosphotyrosine residues are recognized and bound by other protein kinases in the cytosol. These kinases transduce thesignal by phosphorylating downstream protein kinases. Some of the phosphorylated signal transducers translocate into the nucleus to activatetranscription factors (such as AP-1), which regulate the expression of selected sets of genes. EGCG and TFdiG have been shown to block theautophosphorylation of EGFR and HER-2/neu as well as the MAP kinase cascade, such as RAS/RAF/MEK/ERK, JNK pathways, and the P13Kpathway.

3. Blocking growth factor receptor-mediatedpathways

The inhibition of EGF-induced transformation ofJB6 cells and associated MAP kinase signaling couldalso be explained if we assume that EGCG blocksthe function of EGFR. Some of the EGFR signalingpathways are shown inFig. 3. Indeed, inhibition ofEGF binding and EGF-induced autophosphorylationof EGFR have been observed in A431 epidermoid car-cinoma cells[18,19]. EGCG appeared to block thebinding of EGF to EGFR. Nevertheless, a 30 min pre-incubation of EGCG with the cells was needed to pro-

duce these inhibitory effects. In vitro kinase assaysshowed that receptor tyrosine kinase activity was in-hibited by EGCG (IC50 = 1–2�M) [19].

EGFR is often overexpressed in neoplastic cells, ac-tivating signal transduction pathways that promote cellproliferation and tumor progression[20,21]. Antago-nists to EGFR are currently under intensive investiga-tion for cancer therapy[22]. In recent studies, EGCGwas also found to inhibit EGFR autophosphorylationin YCU-N861 and YCU-H891 head and neck carci-noma cells and MDA-MB-231 breast carcinoma cells[23,24]. Downstream events, the phosphorylation ofERK, STAT3, and Akt were subsequently blocked by


the treatment with EGCG. In these experiments, how-ever, the cells were pre-incubated with EGCG for 6 or18 h before the addition of TGF-�. The inhibition ofEGFR-dependent STAT3 activation subsequently re-tarded VEGF synthesis in the cancer cells. The in-hibitory effect of NF-�B by EGCG also contributedto the overall VEGF down-regulation[24]. The resultssuggested that blocking the EGFR signaling by EGCGwould potentially inhibit both cancer cell proliferationand angiogenesis. Overexpression of HER-2/neu, anoncogene in the EGFR tyrosine kinase superfamily,is observed in many human cancers, and it is recog-nized as a target for cancer therapy[25]. In cell linesYCU-H891 and BT-474 (a breast carcinoma cell line)EGCG (20 and 60�M) blocked the HER-2/neu onco-genic pathway, including the activation of STAT3 andc-fos,andcyclin D1promoter activity[26].

Similarly green tea and black tea polyphenols in-hibited the activation of platelet-derived growth factor� (PDGF-�) receptor in A431 cells, mouse NIH3T3fibroblast cells, and A172 human glioblastoma[18,27].In rat hepatic stellate cells, PDGF-� receptor (PDGF-R�) activation was also blocked by EGCG[28]. Theinhibitory effect was correlated with a reduction in pro-liferation of hepatic stellate cells and an anti-fibrotic ac-tivity. Mechanistic evidence suggested that the inhibi-tion was through the blockade to tyrosine phosphoryla-tion. It was suggested that cell membrane-incorporatedEGCG preferably binds to PDGF-BB, one of the PDGFisoforms, lowering the ligand binding and leading to thei

re-i n-s ust ilart ane celll Gw n ofE pre-i re-v res-e thatt tedtT fore tentw as

decreased by 60% after pre-incubation of 240 min. Akey question is whether the inhibition or inactivationof growth factor receptor by EGCG occurs in animaltissues, or it is just a phenomenon in cell culture stud-ies due to auto-oxidation of EGCG in the presence ofhigh oxygen partial pressure and absence of an effec-tive anti-oxidative system.

4. Cell cycle arrest

Dysregulation of the cell cycle check points andoverexpression of growth promoting cell cycle factorssuch as cyclin D1 and cyclin dependent kinases (CDK)are associated with tumorigenesis[31,32]. Some keyproteins involved in cell cycle control are shown inFig. 4. Studies have shown that EGCG can induceG0/G1 phase cell cycle arrest in several human tu-mor cell lines, including breast, epidermoid, prostate,and head and neck squamous cell cancers[23,33–37].Liang et al. reported that treatment of MCF7 breast can-cer cells with 30�M EGCG resulted in G0/G1 phasecell cycle arrest[38]. EGCG also induced expression ofp21 and p27, inhibited the activity of CDK2 and CDK4,and caused Rb hypophosphorylation. In prostate can-cer cells, EGCG (10–80�M) increased the expressionof p16, p18, p21, and p53, which are associated withnegative regulation of cell cycle progression[35,39].EGCG also reduced the protein expression of cyclinD1, cyclin E, CDK2, CDK4, and CDK6, but not cy-c cellsw CG;E n-c ee pres-s

um-b n, iti hicha ctlyi bi-t iono bet ionsu ved inb re-m rrestm

nhibition of the PDGF-R signaling pathways[29,30].The fact that a 30 min or longer period of p

ncubation of EGCG with cells is required for demotrating the inhibition of EGFR signaling ledo study the effect of this pre-incubation. Simo previous results, we observed that, with humsophageal squamous cell carcinoma KYSE 150

ine, a pre-incubation of 30–240 min with EGCas needed to demonstrate a clear-cut inhibitioGF-activated signal transduction pathway. This

ncubation-induced inhibiting effect, however, was pented if the pre-incubation was conducted in the pnce of superoxide dismutase (SOD), suggesting

he inhibition of the EGFR signaling pathway is relao the auto-oxidation of EGCG as depicted inFig. 2.he possible inactivation of EGFR in this situation,xample by superoxide or EGCG radicals, is consisith our observation that the EGFR protein level w

lin D2. Head and neck squamous cell carcinomaere found to be more sensitive to the effects of EGGCG induced G0/G1 phase cell cycle arrest at coentrations lower than 20�M [23]. EGCG induced thxpression of p21 and p27 while decreasing the exion of cyclin D1 and the phosphorylation of Rb.

Although EGCG has been shown to affect a ner of factors associated with cell cycle progressio

s unclear which of these are direct effects and wre indirect effects. EGCG has been shown to dire

nhibit CDKs [38]. It may be proposed that the inhiion of the CDKs is the primary event. The inductf various negative regulators of the cell cycle may

he consequence of this inhibition. The concentratsed in these studies were higher than those obserlood and tissues following consumption of tea. Itains to be demonstrated whether this cell cycle aechanism by EGCG occurs in vivo.


Fig. 4. Cell cycle and possible modulation of tea polyphenols. Stimulation of cells with growth factors or stress signals results in the movementfrom the quiescent G0 phase of the cell cycle into the G1 phase, where sequential phosphorylation of Rb first by the cyclin D1/CDK4 complex andthen the cyclin E/CDK2 complex occurs. This allows release of the transcription factor E2F, which is responsible for transcription of early genesnecessary for cell division. The cyclin A/CDK2 complex then promotes progression of the cell cycle into the S-phase with replication of cellularDNA. Cyclin A/CDC2 and cyclin B/CDC2 are responsible for promoting movement through G2 and into mitosis (M) where cellular divisionoccurs. P16INK4, p27KIP1, and p21WAF1 negatively regulate movement of cells through G1 and into S-phase. Tea catechins, especially EGCG,have been shown to inhibit the activity of CDK2 and 4 and inhibit the phosphorylation of Rb. Further EGCG has been shown to up-regulate theexpression of p16, p21, p27, and p53. These activities contribute to the ability of EGCG to inhibit cell cycle progression at G1.

5. Inhibition of nuclear factor- �B (NF-�B)signaling pathway

The transcription factor NF-�B-induced signalingis well known for its importance in inflammation[40].Recent results also point to its pivotal roles in sup-pressing apoptosis in cancer cells[41]. NF-�B is inac-tive when bound to I-�B in the cytosol (Fig. 5). Thephosphorylation of I-�B by IKKs leads to proteasome-dependent degradation of I-�B, setting NF-�B free.NF-�B can then translocate into the nucleus to ac-tivate the expression of a set of NF-�B responsivegenes. EGCG has been shown to inhibit the constitu-tive activation of NF-�B in H891 head and neck carci-noma cells and MDA-MB-231 breast carcinoma cells[24]. In A431 epidermoid carcinoma cells, treatmentof EGCG dose- and time-dependently increased I-�Blevel, and inhibited NF-�B nuclear translocation[42].UVB irradiation-induced NF-�B activation in normalhuman epidermal keratinocytes was associated withincreased I-�B phosphorylation and degradation and

EGCG was shown to block NF-�B activation and nu-clear translocation[43].

Although reactive oxygen species have been sug-gested to be involved in the activation of the NF-�Bsignaling system, and that its inhibition by EGCG isdue to the antioxidant activity, direct evidence for thismechanism is lacking. We propose that the inhibition ofNF-�B signaling by EGCG can be simply interpretedby the inhibition of IKK-catalyzed phosphorylation ofI-�B. Consistent with this proposal is the observationthat topical application of green tea polyphenols toUVB-irradiated SKH-1 hairless skin decreased phos-phorylation and degradation of I-�B and the subsequentactivation of NF-�B [12].

6. Induction of apoptosis

The balance between survival and apoptosis oftentips towards the former in cancer cells. Tea polyphenoltreatment has been shown to induce apoptosis in many


Fig. 5. NF-�B pathway and possible modulation by EGCG. NF-�B is inactive in the cytosol as a result of the binding of p50 and p65 to I-�B.When I-�B is phosphorylated by IKKs and degraded in a proteasome-dependent pathway, p50 and p65 are set free and are translocated into thenucleus to activate a specific set of genes. This pathway has been shown to be inhibited by EGCG both in vitro and in vivo, possibly by inhibitingIKK-catalyzed phosphorylation of I-�B.

cell lines, including leukemia, skin, lung, stomach, andprostate cancer cells[44–46]. The requirement of cas-pase 3 as an executor in green tea polyphenol-inducedapoptosis was demonstrated both directly and indi-rectly. Caspase 3 activity was augmented when cellsare treated with green tea polyphenols, and caspase 3-deficient tumor cells did not undergo apoptosis underthe same treatment[47].

Some recent studies have focused on primary eventsin tea polyphenol-induced apoptosis. The observationsthat inclusion of catalase in the cell culture systemprevented EGCG-induced apoptosis of human lungcancer cell line H661 and H-ras-transformed humanbronchial epithelial cell line 21 BES, suggest thatH2O2 generated from EGCG plays a role in apoptosisinduction [48,49]. Oxidative stress caused by highconcentrations (100�M) of tea polyphenols was foundto induce apoptosis, which could be blocked by freeradical scavengers (such asN-acetyl-l-cysteine andglutathione)[15,50]. In EGCG-treated LNCaP cells

and smooth muscle cells, p53 protein was stabilized,and NF-�B transcription activity was inhibited byEGCG [51,52]. As a consequence, LNCaP cellswere arrested in the G0/G1 phase, and the balancebetween pro- and anti-apoptotic Bcl-2 family pro-teins favored apoptosis[51]. A recent study withNMR spectroscopy showed the direct binding of teapolyphenols to the BH3 pocket of anti-apoptotic Bcl-2family proteins, suggesting a mechanism for EGCGto inhibit the anti-apoptotic function of Bcl-2 proteins[53]. The BH3 domain was recognized as one of thebinding sites of tea polyphenols; however, the func-tional importance of this binding still requires moreinvestigation.

An outstanding question is why EGCG inducedapoptosis more effectively in cancer cells than in nor-mal cells. A possible answer was suggested by Hsuet al.[17,54]who showed that p57/KIP2 was inducedby green tea polyphenols only in normal human ep-ithelial cells, which were less responsive to apopto-


sis induction by EGCG. In carcinoma cells that weretransfected and overexpressing p57/KIP2, resistance toEGCG-induced apoptosis was conferred.

In vivo studies showed that 0.6% green tea in drink-ing fluid (6 mg of tea solids/ml) increased the apoptosisindex in lung adenoma in chemically induced lung tu-mors in the A/J mouse model[55]. An increase in thenumber of apoptotic epidermal cells was also observedin an experiment, in which SKH-1 mice were admin-istered 0.6% green tea in drinking fluid prior to UVBexposure[56]. In this experiment, caffeine may be thekey active ingredient. Nevertheless, topical applicationof EGCG (6.5�mol) to the skin of SKH-1 mice in-creased the number of caspase-3 positive apoptotic tu-mor cells which were induced by prior irradiation withUVB [57].

7. Novel signaling mechanisms of EGCGidentified by expression genomics

Recent results using expression genomics ap-proaches facilitated the identification of biochemicalnetworks and pathways that mediate pharmacologicalresponse of cells to various small molecules. Genome-wide scan of the expression profiles of the H-ras trans-formed human bronchial epithelial 21BES cells in re-sponse to EGCG (25�M) showed distinct temporalchanges in gene expression, which can be classified intoearly, intermediate, and late-response genes (unpub-l lateda thatH thec thea com-p ionct nyg f thet -w erea of theb wayw wn-r up-r 06-b and8

We showed further that H2O2 transactivated TGF-�-response element promoter activity and this trans-activation was blocked by catalase; whereas EGCGtransactivated BMP-response element promoter, andthis effect was not influenced by catalase. These re-sults suggest that in addition to the network of path-ways described in the previous sections, the BMP-signaling pathway may be a novel target for EGCGto influence cell growth and differentiation. Recent ad-vances in the molecular biology of TGF-�/BMP super-family of cytokines reveal their roles in tumorigenicprogression[58,59]. BMP, its receptors, and SMADs,are targeted for genetic alteration in various cancers.Expression profile of genes of the BMP pathwaysin response to EGCG suggests possible mechanismsfor the cancer preventive effect of EGCG for furtherinvestigation.

8. Inhibition of aberrant arachidonic acidmetabolism

Cytosolic phospholipase A2 (cPLA2) is phosphory-lated in response to growth stimuli or pro-inflammatorymediators (Fig. 7). The phosphorylated enzymetranslocates to the membranes of the endoplasmicreticulum (ER) and nucleus where it catalyzes therelease of arachidonic acid (AA) from membranephospholipids. This free AA then undergoes furthermetabolism by cyclooxygenase (COX, constitutiveC ),om ofpc ex-p esh

ffects bero in-d dep ty ofC -d e( hes int

ished results). Close to 300 genes were up-regund 16 genes were down-regulated. It was shown2O2 was produced when EGCG was added toell culture system. By treating cells with EGCG inbsence or presence of added catalase, which deoses H2O2, we further distinguished gene expresshanges into those that are mediated by H2O2 fromhose that are H2O2-independent. We found that maenes and cellular pathways, including genes o

ransforming growth factor� (TGF-�)-signaling pathay, were H2O2-dependent, because the effects wbolished by catalase. Gene expression changesone morphogenetic protein (BMP)-signaling pathere not affected by catalase, which include do

egulation of the type II receptor of BMP, as well asegulation of its negative modulators including FK5inding protein 5, dual specificity phosphatase 5, and SMAD 7 (Fig. 6).

OX-1 or inducible COX-2), lipoxygenase (LOXr cytochrome P450 monooxygenase (Fig. 7). COX-2ediated metabolism resulting in the formationrostaglandin E2 (PGE2) and 5-, 12-, or 15-LOXatalyzing the formation of leukotrienes (LT). Overression of COX-2, 5-LOX, and other LOX enzymave been observed in several types of cancer[60].

Green tea and EGCG have been shown to aome aspects of aberrant AA metabolism in a numf models. EGCG has been shown to inhibit theuction of COX-2 both in vitro and in vivo. Ahmet al. showed that EGCG (100–200�M) reduced therotein expression (24–48% decrease) and activiOX-2 following IL-1� stimulation of human chonrocytes[61]. Oral administration of EGCG to mic20–50 mg/kg) prior to the application of TPA to tkin significantly inhibited the expression of COX-2he skin[62].


Fig. 6. Effects of EGCG on the TGF/BMP signaling mechanisms. The effects on TGF-� pathway are H2O2-mediated; whereas the effects on theBMP-signaling mechanism are H2O2-independent. EGCG appeared to attenuate the BMP signaling pathway by down-regulating BMP receptortype II and up-regulating its negative modulators, including FK506-protein 5, dual specificity phosphatase 5 and 8, and SMAD7.

Lu et al. showed that treatment of SV-40 trans-formed WI38 human cells with 50�M theaflavinmonogallates results in blockade of serum-induced ex-pression of COX-2 mRNA and protein, but this ef-fect was not observed in non-transformed cells[63].These effects correlated with the increased sensitivityof the transformed cells to theaflavin monogallated-induced growth inhibition and apoptosis as comparedto the parental non-transformed cell line. Given thepoor bioavailability of the theaflavins, the presentlyused concentrations may only be relevant in the oraland gastrointestinal tract where direct contact betweentheaflavins in the lumen and the intestinal mucosa ispossible[64].

In contrast to the observed inhibitory effects of greentea components on COX-2 expression, 25�M EGCGhas been reported to significantly increase COX-2and PGE2 expression by non-stimulated Raw 264.7murine macrophages[65]. This effect was precededby an increase in the levels of phosphorylated ERK

and could be attenuated by co-treatment with the MEKinhibitor, PD098059. Further, addition of the proteintyrosine phosphatase inhibitor, sodium orthovanadate,enhanced the effect of EGCG. These data suggest thatactivation of ERK is critical for up-regulation of COX-2 in Raw 264.7 cells.

Our laboratory reported that catechins or theaflavins(30�g/mL) inhibited the formation of COX and LOX-dependent metabolites in human normal and tumormicrosomes[66]. ECG and EGCG were the most po-tent inhibitor (62–74% inhibition of LOX and COX).Interestingly, theaflavins inhibited the production ofthromboxanes and 12-hydroxyheptadecatrienoic acid,but significantly increased the production of PGE2.Further investigation showed that although theaflavinsinhibited the activity of purified ovine COX-2, itstimulated PGE2 production in a system consisting ofCOX-2 and microsomes, suggesting that theaflavinsenhance the interaction of COX-2 with PGE2synthetase.


Fig. 7. Arachidonic acid metabolism and possible effects of tea polyphenols. Phosphorylation via MAP kinases or an increase in cytosolic Ca2+levels leads to translocation of cPLA2 to the membrane of the endoplasmic reticulum (ER). Whereas phosphorylation of cPLA2 is not neededfor the catalytic activity of cPLA2, it greatly enhances said activity. At the ER membrane, cPLA2 catalyzes the release of arachidonic acid(AA) from the membrane phospholipids. This molecule is then subject to further catalysis by cyclooxygenase (COX), lipoxygenase (LOX) andcytochrome P450 monooxygenase (CYP450) to produce thromboxanes (TXA), prostaglandins (PG), leukotrienes (LT) and other products. BothPG and LT have been shown to have important roles in the development of some cancers. EGCG and green tea have been shown inhibit theexpression and activity of COX and LOX both in vitro and in vivo.

Inhibition of aberrant AA metabolism in the colondue to ingestion of green tea has been correlated withreduced aberrant crypt foci in azoxymethane (AOM)-treated mice fed a high-fat diet[67]. In AOM-treatedmice, those receiving a high-fat diet had significantlyhigher colonic levels of 5-LOX, leukotriene A4 hydro-lase, and LTB4 in comparison with those on a low fatdiet. Following treatment with 0.6% green tea as thedrinking fluid for 10 weeks, there were significantlydecreased colonic levels of cPLA2, 5-LOX, and LTB4.

9. Inhibition of protease activities

EGCG has been shown to inhibit at least two im-portant classes of cellular protease activities: the pro-teasome and matrix metalloproteinases[3]. The pro-teasome is essential for the degradation of a number

of important cellular regulators including I-�B, cyclinD1, and other proteins; inhibition of this enzyme hasrecently been acknowledged as a valid approach to can-cer chemotherapy[68]. Nam et al., reported that EGCGand ECG, but not EGC and EC, potently inhibited thechymotryptic activity of the 20s proteasome both incell-free systems (IC50 = 90–200 nM) and in tumorcell lines (IC50 = 1–10�M) [69]. Treatment of LNCaPprostate cancer cells with EGCG resulted in G0/G1 cellcycle arrest and accumulation of p27 and I�B, bothof which are targets of the proteasome[69]. The de-crease in potency from cell-free to whole cell systemsmay be due to non-specific binding of EGCG to cel-lular proteins, and thus lowering the effective concen-tration of EGCG to the target enzyme. This issue addscomplications for correlating effective concentrationsfor in vitro and in vivo studies. Molecular modelingstudies showed that EGCG binds to the chymotrypsin


Fig. 8. Activation of MMP2 by MT-MMP and effects of tea polyphenols. Pro-MMP2 is synthesized in the cytosol and secreted into theextracellular space where it interacts with a complex of TIMP-2 and MT1-MMP. Under low TIMP-2 conditions, the pro-MMP2 is activatedby a nearby MT1-MMP not complexed to TIMP-2. Under high TIMP-2 conditions, TIMP-2 prevents this cleavage by occupying the freeMT1-MMP2. EGCG has been shown to inhibit secretion of pro-MMP2, up-regulated expression of TIMP-2, and inhibit the proteolytic activityof both MMP-2 and MT1-MMP.

site of the proteasome[70]. An interaction was pro-posed between the carbonyl carbon of EGCG and theactive site amino acid Thr 1. This may explain why thegalloyl-containing catechins are active, but EGC andEC are not. Inhibition of the proteasome represents anattractive mechanism, not only because of the relativelylow concentrations needed, but also because it suggestsmechanistic rationale for using EGCG in combinationwith other agents. Several researchers have reportedthat inhibitors of the proteasome enhance the antitumoractivity of gemcytabine and irinotecan[71,72]. Inhibi-tion of the proteasome by EGCG, however, remains tobe demonstrated in vivo.

The matrix metalloproteinases (MMPs) (Fig. 8) areinvolved in tumor progression and metastasis[73].Overexpression of these zinc-dependent proteases hasbeen shown to increase the invasive and metastatic po-tential of tumor cells. Conversely, pharmacological orgenetic ablation of these enzymes has been shown toinhibit tumor growth and invasion[74,75]. EGCG andother tea polyphenols have been shown to affect MMPactivity both directly and indirectly[3]. Isemura et al.showed that EGCG, ECG, theaflavin, and TFdiG in-hibit collagenase activity in vitro at concentrations of5–50�M [76]. EGCG also inhibited the invasion ofLewis lung cancer cells through a Matrigel basementmembrane in vitro. EGCG has been shown to inhibitthe activity of secreted MMP2 and MMP9 with IC50

values of 8–13�M [77]. This inhibition was shown tobe independent of Zn2+ concentration. EGCG also in-creased expression of the tissue inhibitor of metallopro-teinases (TIMP) 1 and 2 at even lower concentrations(∼1�M). These proteins are endogenous inhibitors ofMMPs (Fig. 8) and the dual activities of EGCG maylead to a more dramatic overall effect on MMP activ-ity. Several researchers showed that EGCG can inhibitthe activation of MMPs by MT1-MMP[78–80]. TheIC50 for this inhibition was reportedly as low as 19 nM.EGCG has also been shown to inhibit MMP2-inducedmigration in transfected COS-7 cells[80].

These data may provide a mechanistic basis forthe observed inhibition of metastasis and invasion ob-served following treatment of tumor-bearing mice withgreen tea or EGCG[81,82]. The concentrations usedin many of these studies are quite low. Further studiesin vivo analyzing expression of markers such as TIMPor activities of MMPs will provide better insight intothe relative importance of this mechanism.

10. Other possible targets

EGCG has been shown to have activity againstmany potentially important targets for cancer preven-tion and therapy. For example, in a recent communi-cation, it was reported that, in MCF-7 cells, expres-


sion of the metastasis-associated 67 kDa laminin re-ceptor conferred EGCG responsiveness at low micro-molar concentrations[83]. Binding of EGCG to the67 kDa laminin receptor with a nanomolar Kd valuewas observed with surface plasmon resonance exper-iment. We recently reported that EGCG (10–50�M)inhibited DNA methyltransferases and this inhibitionresulted in the reactivation of key tumor suppressiongene p16, retinoic acid receptor�, the DNA repairgene hMLH1, and methylguanine methyltransferase,which were inactivated by promoter hypermethylationin human esophageal squamous cell carcinoma cell lineKYSE 510[84]. Some of these genes were also reac-tivated in colon cancer cells HT29 and prostate cancercells PC3.

In other studies, Berger et al. reported that EGCG in-hibited topoisomerase I activity in various human coloncancer cells with IC50 values of 9–17�M [85]. Theseconcentrations were similar to those necessary to in-hibit cell growth. No inhibition of topoisomerase II wasobserved even at concentrations greater than 100�M.Suzuki et al. reported that EGCG inhibited not onlycalf thymus topoisomerase I (IC50 = 5�M), but alsohuman placental topoisomerase II (IC50 = 3�M) [86].The reason for this discrepancy and the significance ofthese observations remain to be determined.

Telomerase is important in maintaining the telomerenucleoprotein end caps of the chromosome structure[87]. This enzyme has been shown to be overexpressedin many human cancers and may be responsible fori -p d inc ata ina ro-m lyh -fi es-c itivec re-s m-p ear-i entalcM der-g canc sim-i s as

we discussed previously. It has not been demonstrated,however, if this phenomenon occurs in vivo.

11. Concluding remarks

As was discussed in preceding sections, many signaltransduction pathways have been proposed as targetsfor the cancer chemopreventive activity of tea polyphe-nols. Nevertheless, it is difficult to determine which arethe more relevant or important mechanisms in vivo.Some considerations are as follows:

1. It is possible that multiple mechanisms are involvedin the cancer preventive action and the relative im-portance of the different mechanisms may dependon the experimental systems studied. It is unlikelyfor EGCG to have a specific receptor that mediatesall the cancer preventive activity in all experimen-tal systems. Among the numerous mechanisms pro-posed in the literature, it is important to determinewhich are the primary events and which are the sub-sequent events. We have discussed the inhibition ofprotein kinases, including MAP kinases, IKKs, andCDKs, by EGCG as a possibility to illustrate thepoint that a biochemical mechanism is needed inorder for tea polyphenols to alter the cellular signaltransduction pathways. Nevertheless, this hypothe-sis needs to be carefully examined.

2. Some of the biological effects of the tea polyphe-InighCG,s ofon-xy-m isf thehe-

ve a

3 re-s ofseit is

ef-pe-in

ncreased replicative life-time[88]. It was recently reorted that EGCG inhibited telomerase in vitro anancer xenografts[89,90]. Naasani et al. reported tht neutral pH, EGCG inhibited telomerase activitycell-free system with high nanomolar to low micolar potency[89]. Long-term treatment with slightigher concentrations (5–10�M) of EGCG was sufcient to inhibit telomerase and induce cell senence. Treatment of mice bearing telomerase-posolon cancer xenografts with 1.2 mg/day EGCGulted in a 50% inhibition of tumor growth as coared to vehicle-treated control animals. Mice b

ng telomerase-negative tumors of the same parell line were unresponsive to EGCG treatment[89].echanistically, the authors suggest that EGCG unoes decomposition to form a galloyl radical whichovalently modify telomerase. This appears to belar to the EGCG auto-oxidation induced reaction

nols observed in vitro might not occur in vivo.many studies with cell culture systems, rather hconcentrations of tea polyphenols, such as EGare utilized. The blood and tissue concentrationEGCG may be much lower than the effective ccentrations observed in vitro. In addition, the ogen partial pressure in the tissue culture systehigher than those in the internal organs. Some oeffects caused by the auto-oxidation of tea polypnols may not occur in tissues, which usually hastrong anti-oxidative capacity.

. Because of the limited bioavailability of EGCG,actions that are affected by low concentrationEGCG are more likely to occur in vivo than thothat require high concentrations. Nevertheless,difficult to translate information concerning thefective concentration from in vitro studies, escially those with pure or isolated proteins, to


vivo situations, because of the nonspecific bindingof EGCG to cellular molecules.

4. Based on the above discussions, it is important todemonstrate the proposed mechanisms in vivo, es-pecially in short-term experiments. For example, ifwe propose that EGCG directly inhibit MAP ki-nases, then this effect should be seen in vivo in afew hours after the administration of EGCG.

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