CANCER RESEARCH 57. 4414-4419. October 1. 19971

ABSTRACT

(—)..Epigallocatechin gallate (EGCG) and theaflavins are believed to bekey active components in tea for the chemoprevention against cancer.However, the molecular mechanisms by which EGCG and theaflavinsblock carcinogenesis are not clear. We have used the JB6 mouse epidermal

cell line, a system that has been used extensivelyas an in vitromodel fortumor promotion studies, to examine the anti-tumor promotion effects of

EGCG and theaflavins at the molecular level. EGCG and theaflavins

inhibited epidermal growth factor- or 12-O-tetradecanoylphorbol-13-acetate-induced cell transformation in a dose-dependent manner. At the doserange (5—20,zM)that inhibitedcelltransformation, EGCG and theaflavinsalso inhibited AP-1-dependent transcriptional activity and DNA bindingactivity. The inhibition of AP-I activation occurs through the inhibition ofa c-Jun NH2-terminal Idnase-dependent, but not an extracellular signalregulated protein kinase (Erk) 1-dependent or Erk2-dependent, pathway.Because the transcription factor AP-1 is important for tumor promoterinduced neoplastic transformation, the inhibitory effects on AP.1 activation by EGCG and theaflavins may further explain the anti-tumor promotion action of these tea constituents.

INTRODUCTION

Prevention of carcinogenesis is one of the major strategies forcancer control. Many studies have shown the inhibitory actions ofgreen tea, black tea, and tea polyphenol preparations against carcinogenesis in rodent models (1—3).These include cancers of the skin,lung, esophagus, stomach, liver, duodenum and small intestine, pancreas, and mammary gland. The most extensively studied system isthe skin carcinogenesis model caused by chemicals, UV light, andTPA3 (4—11). In some of the studies, TPA or UV light was used as atumor promoter. The antipromoting effect of a major green tea constituent, EGCG, has been demonstrated (4, 7, 9, 11). However, theunderlying mechanisms responsible for these cancer-preventive activities have not been clearly elucidated.

Different tea preparations may contain various amounts of teapolyphenols, also known as catechins, among which EGCG is themost well studied. A cup of green tea (2.5 g of dried green tea leavesbrewed in 200 ml of water) may contain 90 mg of EGCG. In addition,it contains a similar or slightly smaller amount of (—)-epigallocat

echin, about 20 mg each of ( —)-epicatechin 3-gallate and (—)-epicatechin, and about 50 mg of caffeine (11). In black tea, the above teacatechins are reduced to about one-tenth to one-third of those in greentea, and theaflavins account for 1 to 2% of the total dry mauer (1).

Received 4/30/97: accepted 7/30/97.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I This research was supported by the Hormel Foundation and NIH Grant CA56673.

2 To whom requests for reprints should be addressed, at The Hormel Institute. Uni

versity of Minnesota, 801 16th Ave NE, Austin, MN 55912. Phone: (507) 437-9640; Fax:(507) 437-9606: E-mail: zgdong@wolf.co.net.

3 The abbreviations used are: TPA, l2-O-tetradecanylphorbol-13-acetate; AP-1, acti

vator protein I ; EGCG. (—)-epigallocatechin 3-gallate; EGF, epidermal growth factor;Erk, extracellular signal-regulated protein kinase; MAPK, mitogen-activated protein Idnase; JNK, c-Jun NH2-terminal kinase; P@, promotion sensitive; FBS, fetal bovine serum;BME, Basal Medium Eagle.

About 10—20%of the dry weight of black tea is composed of thearubigins, which are not well characterized chemically (1). One cup ofblack tea (2.5 g of dried black tea leaves brewed in 200 ml of water)may contain 12—15mg of theaflavins (11). Under conditions in whichthe antitumorigenesis of tea has been demonstrated, the plasma EGCGlevels in rats and mice are 37 and 124 ng/ml, respectively (2, 12).More recently, Huang et a!. (13) reported that caffeine is an importantcomponent in green and black tea on the inhibition of UVB-inducedcarcinogenesis. These catechins and theaflavins are generally considered to be the effective components for the inhibition of carcinogenesis, but the mechanisms are not well characterized (1, 2). A commonly discussed mechanism is the antioxidative activities of thesepolyphenolic compounds (1, 4). The inhibitory activities of tea catechins on the growth of tumor cell lines have been shown (14, 15).The antioxidative and antimutagenic effects of theaflavins have beenreported (16). The antipromotion activity of EGCG has been demonstrated, and its possible effects on the signal transduction pathwayhave been suggested (2, 17, 18).

Previously, we and others have obtained evidence that activation ofAP-l by phorbol ester-type tumor promoters plays a key role in tumorpromotion (19—25).On the basis of the importance of AP-l activity intumor promoter-induced JB6 cell transformation and the antitransformation effect of tea polyphenols, we have hypothesized that theanti-tumor promotion activity of EGCG or theaflavins may be throughthe inhibition of AP-1 activity. To test this hypothesis, we investigatedthe effect of EGCG, theaflavins, and caffeine on EGF- or TPAinduced AP- 1 activity. The JB6 cell system, a well-developed cellculture model for the study of tumor promotion, was used to study theinhibition of AP-l activation as a molecular mechanism for theanti-tumor promotion activity of EGCG and theaflavins.

MATERIALS AND METHODS

Materials. Eagle's MEM and FBS were from Whittaker Biosciences; Lglutamine was from Life Technologies, Inc.; gentamicin was from QualityBiological, mc; formamide was from Fluka; and luciferase assay substrate wasfrom Promega. EGCG (purity >98%) was a gift from Dr. Yukihiko Ham ofMitsui Norm Co. (Fujied, Japan). Theaflavins (a mixture of theaflavin, theafla

yin 3-gallate, theaflavin 3'-gallate, and theaflavin 3,3'-digallate, accounting for

21, 30, 15, and 28%, respectively) were gifts from Thomas J. Lipton Co.(Englewood Cliffs, NJ). Caffeine and TPA were obtained from Sigma Chemical Co. (St. Louis, MO).

Cell Culture. Tumor promoters, such as TPA and EGF, induce the transformation of JB6 P@ cells in soft agar at a high frequency. JB6 P@ mouse

epidermal cell line, Cl 41, and its AP-l luciferase reporter transfectant P@ 1—1were cultured in monolayers at 37°C and 5% CO2 using Eagle's MEM

containing 5% FCS, 2 mist L-glutamlne, and 25 @g/mlgentamicin (18—22).Tomaintain the luciferase reporter construct, P@ ll cells were cultured inmedium containing the neomycin analogue Geneticin (G418, Life Technologies, Inc.) at 300 @Lg/mi.

Assay for AP-1 Activity. JB6 AP-1-Iuciferase stable transfectant cells(P'@1-1) were used to assay the AP-1 activity (18—22,25). Viable cells(8 X lOs) suspended in 100 pi of 5% FBS MEM were added to each well of

a 96-well plate. Plates were incubated at 37°C in a humidified atmosphere of

5% CO2and95% air.Twelve to 24 h later,cells were starvedby being cultured4414

Inhibition of Tumor Promoter-induced Activator Protein 1 Activation and

Cell Transformation by Tea Polyphenols, (—)-EpigallocatechinGallate,and Theaflavins'

Zigang Dong,2 Wei-ya Ma, Chuanshu Huang, and Chung S. Yang

The Hormel Institute. Unis'ersitv of Minnesota, Austin, Minnesota 55912 fZ D., W-y. M.. C. 11.1, and Laboratory for Cancer Research, College of Pharmacy, Rutgers University,Piscatawav, New Jersey 08855-0789 (C. S. Y.J

on March 26, 2020. © 1997 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

A j DEGF B DEGF C DEGFDTPA @TPA @TPA

IC

I

I I

@ II

,/@

@@ -‘,. ,

_0_@ 1 5 10 1 5 10 20 0 10 20

EGCG(/2M) Theaflavins(,uM) Caffeine(,uM)4415

20 0

TEA POLYPHENOLS INHIBIT AP-I AND CELL TRANSFORMATION

in 0.1% FBS MEM for 12 h. The cells were treated with EGCG, theaflavins,or caffeine for 30 mm. Then, the cells were exposed to 20 ng/ml TPA or 10ng/ml EGF in the presence of EGCG, theaflavins, or caffeine for 24 h. Thecells were extracted with lysis buffer, and the luciferase activity was measuredas described previously by using a luminometer (Monolight 2010). AP-l

activity is presented relative to medium control cells (25—29).

Assay for Cell Proliferation. The cell proliferation was determined by[3H]thymidine incorporation assay (26, 27). JB6 Cl 41 cells (5 X l0@)wereseeded in 96-well plates in the presence or absence of different concentrations

of inhibitors (EGCG, theaflavins, or caffeine). After the cells were cultured for36 h, [3H]thymidine (0.5 @Ci/we1l)was added to each well. The cells wereharvested 12 h later, and incorporation of [3H]thymidine was quantified usinga liquid scintillation counter.

Anchorage-Independent Transformation Assay. The effects of EGCG,theaflavins, or caffeine on TPA- or EGF-induced cell transformation wereinvestigated in JB6 Cl 41 cells. Cells (1 X 10@)were exposed to 20 ng/ml TPAor 10ng/mlEGFwith or withoutdifferentconcentrationsof inhibitors(EGCG,theaflavins, or caffeine) in 1 ml ofO.33% BME agar containing 10% FBS over3.5 ml of 0.5% BME agar medium containing 10% FBS. The cultures were

maintained in an incubator at 37°Cand 5% CO2 for 14—21days, and the cellcolonies were scored by the methods described previously (19—22).

Nuclear Protein Analysis. Gel shift assays were used to detect AP-lbinding activity after exposure to the tumor promoter TPA with or withoutEGCG, theaflavin, or caffeine (22). Nuclear extracts were prepared as de

scribed previously (22). In brief, the cells were lysed with 500 @.dof lysis

buffer (50 mMKC1,0.5% NP4O,25 mt@iHEPES, 1 msi phenylmethylsulfonylfluoride, 10 i@gof leupeptinper ml, 20 @gof aprotininper ml, and 100 @tmDL-D1T). After centrifugation at 14,000 rpm for 1 mm, the nuclei were washedwith the 500 @tlof the same buffer without NP4O. Then placed into 300 pA of

extraction buffer (500 mMKC1and 10%glycerol with the same concentrationofHEPES, phenylmethylsulfonyl fluoride, leupeptin, aprotinin, and DL-DTI'asthe lysis buffer). After centrifugation at 14,000 rpm for 5 mm, the supernatantwas harvested as nuclear protein extract and stored at —70°C.An AP-lbinding sequence from the human collagenase promoter region, 5 ‘-AGCATGAGTCAGACACCTCTGGC-3', was synthesized and labeled with[32P]dCTP using the Klenow fragment (Life Science Co., Gaithersburg, MD).Three ,.@gof nuclear protein extracted from cells exposed to TPA for theindicated time intervals were added to the DNA-binding buffer, which conmined S X iO@cpm 32P-labeledoligonucleotide probe, I.5 @.tgof poly(dIdC),and 3 i@gof BSA. The reaction mixture was incubated on ice for 10 mm

2600

2400

U) 2200

a)0 2000

Va).@ 1800

a)C,) 1600

“J'01@ 1400

I 1200!

@8O0

: 600CI) 400

followed by incubation at room temperature for 20 mm. The DNA-proteincomplexes were resolved in a 6% nondenaturing acrylamide gel. The gel was

dried and exposed to X-ray film at —70°Covernight.Erki and Erk2 Analysis. Immunoblotfor Erkl, Erk2,and theirphospho

rylated proteins was carried out as described previously (25). In brief, JB6 Cl41 cells were cultured in monolayers in six-well plates. The cells were starvedin 0. 1% FBS MEM for 48 h at 37°C and then changed to fresh 0. 1% FBS

MEM for another 3—4h at 37°C.Before the cells were exposed to TPA orEGF, they were not treated or were treated with EGCG, theaflavins. or caffeinefor 30 mm. Then, 20 ng/ml TPA or 10 ng/ml EGF was added and incubated for

different time periods at 37°C. The cells were then lysed, and immunoblot

analysis was performed by using antibodies against Erkl and Erk2 (P44 andP42) or phospho-specific MAPK antibodies against phosphorylated tyrosine

204 of Erkl and Erk2 (New England Biolabs).

iNK Assay. JB6 Cl 41 cells were cultured in 100-mm dishes. The cells

were treated with TPA or EGF with or without EGCG, theaflavins, or caffeineas described above for the Erk assay. Activation of JNK was assayed asdescribed in the manufacturer's protocols of SAPK/JNK assay kit (NewEngland Biolabs). In brief, JNK proteins were precipitated by using the c-Jun

fusion protein beads. After removal of the nonspecific bound proteins, the

kinase reaction was carried out in the presence of ATP in the kinase buffer

(New England Biolabs). The JNK-catalyzed phosphorylation of c-Jun was

measured by antiphospho-c-Jun (Ser63) antibodies (New England Biolabs).

RESULTS

EGCG and Theaflavins but not Caffeine Inhibited TPA- or

EGF.induced Cell Transformation. As reportedpreviously,EGForTPA induces 1000—2000transformed colonies in soft agar, whereas inthe solvent control group (<0.1% DMSO) there is no soft agar colonyformation. EGF- or TPA-induced JB6 cell transformation was significantly blocked by EGCG or theaflavins at the concentration rangefrom 5—20@LM(P < 0.05; Fig. 1, A and B), whereas only the higherconcentration of caffeine (20 pM) showed a slight inhibition of celltransformation (P < 0.05; Fig. 10. The inhibition of cell transformation by EGCG or theaflavins was not caused by growth inhibition,because the concentration range that inhibited cell transformation didnot inhibit cell proliferation as measured by [3H]TdR incorporation(data not shown).

Fig. 1. Inhibition of TPA- or EGF-induced JB6P+ cell transformation by EGCG or theaflavin. Cl41 cells (1 x l0@) were exposed simultaneously to20 ng/miTPA or 10 ng/mlEGFwithor withoutdifferent concentrations of EGCG (A), theaflavins(B), or caffeine (C) in 0.33% agar containing 10%FBS over 0.5% agar containing 10% FBS. Cellcolonieswerescoredaftera 14-dayincubationat37°Cin 5% CO2. The inhibition ofcell transformation induced by EGF or TPA is expressed as described in “Materialsand Methods.―

200

0

on March 26, 2020. © 1997 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

TEA POLYPHENOLS INHIBIT AP-I AND CELL TRANSFORMATION

>

a-

a)>

a)a:

Fig. 2. Inhibition of AP-l transcriptional activity byEGCG and theaflavin. JB6 cell AP-I reporter stableP+ 1-1 cells (8 X l0@)were seeded into each well of96-well plates. After culture at 37'C ovemight, the cellswere starved for 12 h by replacing the medium with0. 1% FBS MEM. Then, cells were treated with 20 ng/mlTPA or 10 ng/ml EGF with or without different concentrations of EGCG (A), theaflavins (B), or caffeine(C). The luciferase activity was determined, and theresults were presented as relative luciferase activity.

0 1 5 10 20 0 1 5 10 20 0 1 5 10 20 40

EGCG(/j,M) Theaflavins(,@M) Caffeine (MM)

EGCG and Theaflavins Inhibited TPA- or EGF-induced AP-1

Activity. As shown in Fig. 2, A and B, in a similar dose range forinhibition of cell transformation, EGCG and theaflavins inhibitedEGF- or TPA-induced AP-1 activity in a concentration-dependentmanner. In contrast, caffeine, which shows slight antitransformationactivity, showed no inhibition of AP-1 activity (P > 0.05). Theseresults indicate that inhibition of AP-l activity by EGCG and theafla

vms may be important in their inhibitory activities against tumorpromoter-induced cell transformation.

EGCG and Theaflavins Inhibited Sequence-specific AP-1 DNABinding Activity. To study the molecular basis of the inhibition onAP-1 activity by EGCG and theaflavins, we considered the possibilities that AP-l transactivation activity might have been altered bythese compounds. The AP-1 DNA binding activity was analyzed by

:@ TPA@ stimulated

C

+ ++ + +

A B

TPA(2Ong/ml)-+++ + +÷+EGCG(p@M)--20105

---Theaflavins

( p,M)201 05AP-1--@ NuclearExtract@P-AP-1Probes

Competitors

AP-1

+

- - AP-1@ -

$@@

A AkA@

Fig. 3. Inhibition of AP-l DNA binding activity by EGCG and theaflavins. JB6 cells were treated with EGCG or theaflavins as indicated (A). Sequence-specific AP-1 DNA bindingactivity was determined by gel-shift analysis using a 32P-labeled oligonucleotide containing the AP-l-binding site as described in “Materialsand Methods.―Arrow, position of specificAP-l DNA binding activity. By a competition experiment, this band was shown to be compatible with unlabeled AP-l binding oligonucleotides (B).

4416

on March 26, 2020. © 1997 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

TEA POLYPHENOLS INHIBIT AP-I AND CELL TRANSFORMATION

- + + + + +

- - 2010 5 1Fig. 4. Inhibition of TPA-induced serine 73

phosphorylation of c-Jun protein. JB6 P@ Cl 41cells (8 X 1O@)were seeded into each well of6-well plates. After culture at 37'C for 24 h, thecells were starved for 48 h by replacing mediumwith 0.1% FBS MEM. Two to 4 h before cells wereexposed to TPA, the medium was replaced withserum-free MEM. Then, the cells were exposed to20 ng/ml TPA for 30 mm in the presence of different concentrations of EGCG (A) or theaflavin(B). The cells were extracted and c-Jun (senne73)-phosphoiylated proteins were detected with thePhosphoPlus c-Jun (Ser73) antibody kit from NewEngland Biolabs.

A

B

i:::@ :@j— phosphorylated

— .@ .*@ :@j- phosphorylated--

ated serine 73 of c-Jun. As shown in Fig. 4, EGCG and theaflavins, ata concentration range that blocked AP-1 activity, inhibited c-Junphosphorylation at serine 73.

EGCG and Theaflavins Inhibited the Activation of iNKS but

not Erks. MAPKS Erks and JNKs are the upstream activatorkinases responsible for the phosphorylation of c-Jun proteins (33,34). In an in vitro assay, JNKs were shown to be effective kinasesfor the phosphorylation of c-Jun protein at serine 63/73. To testwhich class of MAPK is involved in the inhibition of c-Junphosphorylation and AP-l activation by EGCG or theaflavins, weexamined the influences of these compounds on the phosphorylation of Erki, Erk2, and JNK activity (Figs. 5 and 6). As shown inFig. 5, EGCG and theaflavins inhibited TPA- or EGF-induced JNKactivity in a dose-dependent manner at the same dose range forinhibition of TPA- or EGF-induced AP-1 activity, c-Jun phosphorylation, or cell transformation. On the other hand, EGCG ortheaflavins showed no inhibition on TPA- or EGF-induced phos

c-Jun

TPA(2Ong/mI) - + + + + +

c-Jun

gel-shift assay. As shown in Fig 3A, EGCG or theaflavins inhibitedTPA-induced AP-1 DNA binding activity in a concentration-dependent manner. By a competition experiment with unlabeled AP-1binding oligonucleotides, this band was shown to be compatible,whereas added unlabeled AP-2 binding oligonucleotides did not affectthe binding activity (Fig. 3B). The concentration range of EGCG ortheaflavins required for inhibition of AP-1 DNA binding activity wassimilar to AP-1 transactivation activity (Fig. 3). It thus appears thatthe inhibition of AP-1 transactivation by EGCG and theaflavin is, atleast in part, due to the inhibition of AP-1 DNA binding activity.

Inhibition of the Phosphorylation of c-Jun by EGCG andTheaflavins. TPA, EGF, and other tumor promoters, such as UVlight, induce signal transduction, which leads to phosphorylation ofthe c-Jun protein at serine 63 and serine 73 on the NH2-terminal andactivated AP-1 complex (30—32).To test whether the phosphorylationof serine 73 is affected in the inhibition of AP-1 activation by EGCGand theaflavins, we used phospho-specific antibodies for phosphoryl

AEGF(2Ong/mI) -

EGCG(@M) -+ + + + +

- 20 10 5 1

. __ @... @D-phospho-c-Jun

. (ser 63)

B

C

Fig. 5. Inhibition ofTPA- or EGF-induced INKactivation by EGCG and theaflavin. JB6 Cl 41 cellswere treated with medium, 10 ng/mI EGF (A and B)or 20 ng/ml TPA (C) with or without differentconcentrations of EGCG or theaflavins at 37'C in5% CO2. The cells were harvested and JNK activitywas assayed as described in “Materialsand Methods―by a INK assay kit from New England Biolabs.

+ + + + +

- 2010 5

@ @-

::D- phospho-c-Jun(ser 63)

+ + + + + + +

- 20 10 5 1

+ +

- 20 10 5 1

@-@@@@ .@

.- — :@:l- phospho-c-Jun

(ser 63)

4417

TPA (20 ng/mI)EGCG(@M)

Theaflavins ( @M) - 20 10 5 1

EGF(2Ong/mI) -Theaflavins( @M)-

TPA (20 ng/mI) -EGCG(@M) -

Theaflavins( jIM) -

on March 26, 2020. © 1997 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

TEA POLYPHENOLS INHIBIT AP-I AND CELL TRANSFORMATION

Fig. 6. No effect on TPA- or EGF-induced Erkphosphorylation by EGCG (A) and theaflavins (B).JB6 P@Cl 41 cells were treated as described in thelegend to Fig. 4. The protein extracts used in theexperiment shown in Fig. 4 were used for analysisof Erkl and Erk2 phosphorylated proteins (PhosphoPlus MAPK antibody kit, New England Biolabs).

phorylation of Erkl and Erk2 (Fig. 6). In agreement with theresults of cell transformation, AP-1 activity, and c-Jun phosphorylation, caffeine did not inhibit JNK activity or Erk activity (datanot shown). These results suggest that inhibition of c-Jun phosphorylation (serine 63/73) and AP-l activation by EGCG andtheaflavin is through an inhibition of JNK-dependent, Erki- andErk2-independent pathway.

DISCUSSION

Green tea is widely used as a beverage in China, Japan, and otherAsian countries, whereas black tea is more popular in Western countries (1 , 13). In recent years, many animal studies and several epidemiological studies have suggested the anticarcinogenic effects of tea(1—3).Extractsofgreen,black,andotherteasinhibitedTPA-inducedJB6 cell transformation (35). However, the mechanisms of action bywhich these chemicals block carcinogenesis are not clear. The antioxidative activities of the tea polyphenols have been shown (1, 12,16). Inactivation of protein kinase C (4, 36) and prevention of tumorpromoter-induced inhibition of GAP junctional intercellular communication by the tea components have been reported as possible mechanisms by which the tea polyphenols might inhibit tumor promotion(37, 38). In this study, we investigated the anti-tumor promotion effectof EGCG and theaflavins in JB6 cells. EOCG and theaflavins inhibittumor promoter TPA- or EGF-induced cell transformation. In thesame dose range for inhibition of cell transformation, EGCG andtheaflavins also inhibit TPA- or EGF-induced AP-1 activity. Previously, we have reported that induced AP-l activity is required fortumor promoter-induced cell transformation in vitro (19—22,25, 26)and tumor promotion in an animal model (29). Therefore, the inhibition of AP-l activity may be functionally linked to the anti-tumorpromotion effects of EGCG and theaflavins.

AP-1 transcriptional activity is determined by the sequencespecific AP-l DNA binding activity and the phosphorylation ofAP-1 proteins (Jun/Fos; 36). Our results demonstrated that both ofthese determinants are involved for the inhibition activity of EGCGand theaflavins.

MAPKs, including Erks and JNKs, are mediators in a proteinkinase cascade and in the regulation of transcription factor AP-1proteins (Jun/Fos; 28—31). Mutation of Ser63/73 of c-Jun renders

- + + + + + + + +

--201051---20 10 5 phosphorylated

P44MAPK

@@@@@ — —@

c-Jun nonresponsive to growth factor-, phorbol ester- and UV-inducedsignaling pathways (34, 39, 40). Activation of MAPKS occurs throughphosphorylation of threonine and tyrosine (positions 202 and 204,respectively, in Erks; positions 183 and 185, respectively, in JNKs;Refs. 34, 40, and 41). Our data indicate that there is no inhibitoryeffect of EGCG or theaflavins on Erki or Erk2. However, the phosphorylation of c-Jun protein at Ser73 is inhibited by EGCG andtheaflavins. Moreover, we found that the JNK activity was inhibitedby EGCG and theaflavins. These data suggest that the inhibition oftumor promoter-mediated AP-1 activity by EGCG or theaflavins maybe through a JNK-dependent and Erk-independent pathway. The exactreason for the inhibition of JNK activity is at present not known.

In animal models, both green tea and black tea extracts showedanticarcinogenic effects (1—3).In the present work, we demonstratedthat EGCG or theaflavins have strong inhibitory activity on tumorpromoter-induced cell transformation, whereas caffeine has slightinhibitory effect on the cell transformation.

In summary, we have provided evidence for a novel mechanism ofthe anti-tumor promotion action by EGCG and theaflavins. Our cxperiments suggest that inhibition of tumor promoter-induced neoplastic transformation in JB6 cells may be through the inhibition of AP-ltransactivation. The inhibition of AP-1 activation of EGCG andtheaflavins may be mediated through the inhibition of JNKs but notErks. These results provide insight into the biological actions of teaand the molecular basis for the development of new chemoprotectivereagents for cancer.

ACKNOWLEDGMENTS

We thank Dr. Y. Hara for the generous gift of EGCG, T. J. Lipton Co. fortheaflavins, and Jeanne A. Ruble for secretarial assistance.

REFERENCES

1. Yang, C. S., and Wang, Z-Y. Tea and cancer: a review. J. Natl. Cancer Inst., 58:1038—1049,1993.

2. Yang.C. S.,Yang,G-Y., Lee,M-L., andChen,L. Mechanisticconsiderationsof theinhibition of carcinogenesis by tea. In: H. Ohigashi (ed), Proceedings of the International Conference on Food Factor in Cancer Prevention, pp. 113—117. Tokyo:Springer-Verlag, 1997.

3. Katiyar, S. K., and Mukhtar, H. Tea in chemoprevention of cancer: epidemiologic andexperimental studies. Int. J. Oncology, 8: 221—238,1996.

4418

- + ++++

- - 20 1 0 5 1 phosphorylated

P44MAPK

-.-- j@ . . • :=:: phosphorylated

P42MAPK

ATPA (20 ng/mI)

EGCG(@M)

BEGF (20 ng/mI)

EGCG(@M)Theaflavins( @M)

phosphorylatedP42MAPK

on March 26, 2020. © 1997 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

TEA POLYPHENOLS INHIBIT AP-1 AND CELL TRANSFORMATION

4. Yoshizawa, S., Horiuchi, T., Fujiki, H., Yoshida, T., Okuda, T., and Sugimura, T.Antitumor promoting activity of (—)-epigallocatechingallate, the main constituent of“tannin―in green tea. Photother. Res., I: 44—47,1987.

5. Wang, Z. Y., Khan, W. A., and Bickers, D. R. Protection against polycyclic aromatichydrocarbon-induced skin tumor initiation in mice by green tea polyphenols. Carcinogenesis (Lond.), 10: 411—415, 1989.

6. Gensler, H. L, Timmermann, B. N., Valcic, S., Wachter, G. A., Dorr, R., Dvorakova,K., and Alberta, D. S. Prevention of photocarcinogenesis by topical administration ofpure epigallocatechin gallate isolated from green tea. Nutr. Cancer. 26: 325—335,1996.

7. Huang, M-T., Ho, C-T., Wang, Z-Y., Ferraro, T., Finnegan-Olive, T., Lou, Y-R.,Mitchell, J. M., Laskin, J. D., Newmark, H., Yang, C. S., and Conney, A. H.Inhibitory effect of topical application of a green tea polyphenol fraction on tumorinitiation and promotion in mouse skin. Carcinogenesis (Lond.), /3: 947—954,1992.

8. Wang, Z. Y., Huang, M-T., Ferraro, T., Wong, C. Q., Lou, Y-R., Reuhl, K.,Latropoulos, M., Yang, C. S., and Conney, A. H. Inhibitory effect of green tea in thedrinking water on tumorigenesis by ultraviolet light and 12-O-tetradecanoylphorbol13-acetate in the skin of SKH-l mice. Cancer Res., 52: 1162—1170, 1992.

9. Katiyar, S., Agarwal, R., and Wood, G. S. Inhibition of 12-O-tetradecanoyl-phorbol13-acetate-caused tumor promotion in 7,12-dimethylbenz(a)anthracene-initiatedSENCAR mouse skin by a polyphenolic fraction isolated from green tea. Cancer Res.,52: 6890—6897, 1992.

10. Katiyar, S. K., Agarwal, R., and Mukhtar, H. Protection against malignant conversionof chemically induced benign skin papillomas to squamous cell carcinomas inSENCAR mice by a polyphenolic fraction isolated from green tea. Cancer Res., 53:5409—5412,1993.

11. Wang, Z-Y., Huang, M-T., Lou, Y-R., Xie, J-G., Reuhl, K. R., Newmark, H. L., Ho,C-T., Yang, C. S., and Conney, A. H. Inhibitory effects of black tea, green tea,decaffeinated black tea, and decaffeinated green tea on ultraviolet B light-inducedskin carcinogenesis in 7,12-dimethylbenz[a]anthracene-initiated SKH-l mice. CancerRes., 54: 3428—3435. 1994.

12. Wang, Z. Y., Wang, L-D., Lee, M-J., Ho, C-T, Huang, M-T., H., C. A., and Yang,C. S. Inhibition of N-nitrosomethylbenzylamine-inducedesophageal tumorigenesis inrats by green and black tea. Carcinogenesis (Land.), 16: 2143—2148, 1995.

13. Huang, M-T., Xie, J-G., Wange, Z. Y., Ho, C-T., Lou, Y-R., Wang, C-X., Hard,G. C., and Conney, A. H. Effects of tea, decaffeinated tea, and caffeine on UVBlight-induced complete carcinogenesis in SKH-l mice: demonstration of caffeine asa biologically important constituent of tea. Cancer Res., 57: 2623—2629, 1997.

14. Graham, H. N. Green tea composition, consumption, and polyphenol chemistry. Prey.Med., 21: 334—350,1992.

15. Lea, M. A., Xiao, Q., Sadhukhan, A. K., ConIc, S., Wang, Z-Y., and Yang, C. S.Inhibitoryeffects of tea extracts and (—)-epigallocatechingallate on DNA synthesisandproliferationof hepatoma and eiythroleukemiacells. Cancer Len., 68: 231—236,1993.

16. Shiraki. M., Ham, Y., Osawa, T., Kumon, H., Nakayama, T., and Kawakishi, S.Antioxidative and antimutagemc effects of theaflavins from black tea. Mutat. Res.,323: 29—34,1994.

17. Lm, J-K., and Lee, S-F. Inhibition of tumor promotion through blocking signaltransduction (Review Article). Zool. Stud., 34: 67—81,1995.

18. Suganuma, M., Okabe, S., Sueoka, E., lids, N., Komori, A., Kim, S-J., and Fujiki, H.A new process of cancer prevention mediated through inhibition of tumor necrosisfactor a expression. Cancer Res., 56: 3711—3715, 1996.

19. Dong, Z., Birrer, M., Matrisian, L., and Colbum, N. H. Blocking tumor promoterinduced AP-l activity inhibits transformation of JB6 cells. Proc. NatI. Acad. Sci.USA, 91: 609—613,1994.

20. Dong, Z., Watts, R. G., Sun, Y., and Colburn, N. H. Progressive elevation of AP-1activity during preneoplastic-to-neoplastic progression as modeled in mouse JB6 cellvariants. tat. J. Oncol., 7: 359—364, 1995.

21. Huang, C., Ma, W., and Dong, Z. Inhibitory effects of ascorbic acid on AP-l activityand transformation of JB6 cells. Int. J. Oncol., 8: 389—393,1996.

22. Li, J-J., Dong, Z., Dawson, M. I., and Colbum, N. H. Inhibition of tumor promoterinduced transformation by retinoids that transrepress AP-l without transactivatingretinoic acid response element. Cancer Res., 56: 483—489,1996.

23. Alani, R., Brown, P., Binetniy, B., Dosaker,H., Rosenberg,R. K., AngeLP., Karin, M.,and Birrer, M. J. The transactivating domain of the c-Jun proto-oncoprotein is required forcotransformation of rat embryo cells. Mol. Cell. Biol., 11: 6286—6295, 1991.

24. Domann, F. E., Levy, J. P., Birrer, M. J., and Bowden, G. T. Stable expression of ac-Jun deletion mutant in two malignant mouse epidermal cell lines blocks cellularAP-l activity and tumor formation in nude mice. Cell Growth Differ., 5: 9—16,1994.

25. Dong, Z., Huang, C., Brown, R. E., and Ma, W-Y. Inhibition of AP-l activity andneoplastic transformation by aspirin. J. Biol. Chem., 272: 9962—9970, 1997.

26. Huang, C., Schmid, P. C., Ma, W-Y., Schmid, H. H. 0., and Dong, Z. Phosphatidylinositol-3 kinase is necessary for l2-O-tetradecanoylphorbol- 13-acetate-inducedtransformation and AP-l activation. J. Biol. Chem., 272: 4187—4194, 1997.

27. Huang, C., Ma, W-Y., Bowden, T., and Dong, Z. UVB-induced AP-l activation doesnot require EGF receptor, but is blocked by a dominative PKCA/L. J. Biol. Chem.,271: 31262—31268,1996.

28. Huang, C., Ma, W-Y., and Dong, Z. Requirement for phosphatidylinositol 3-kinase inepidermal growth factor induced AP-l transactivation and neoplastic transformationin JB6 cells. Mol. Cell. Biol., 16: 6427—6435,1996.

29. Huang, C., Ma. W-Y., Dawson, M. I., Rincon, M., Flavell, R. A., and Dong, Z.Blocking AP-l activity, but not activating RARE, is required for anti-tumor promotion effects by retinoic acid. Proc. Nail. Acad. Sci. USA, 94: 5826—5830, 1997.

30. Marshall, C. J. Specificity of receptor tyrosine kinase signaling: transient versussustained extracellular signal-regulated kinase activation. Cell, 80: 179—185,1995.

31. Hunter, T. Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell, 80: 225—236,1995.

32. Hill, C. S., and Triesman, R. Tranacriptional regulation by extracellular signals:mechanisms and specificity. Cell, 80: 199—211,1995.

33. Cowley, S., Paterson, H., Kemp P.. and Marshall, C. J. Activation of MAP kinasekinase is necessary and sufficient for PC12 differentiation and for transformation ofNIH 3T3 cells. Cell, 77: 841—852, 1994.

34. Sturgill, T. W., Ray, L. B., Erikson, E., and Maller, J. L. Insulin-stimulated MAP-2kinase phosphorylates and activates ribosomal protein 56 kinase II. Nature (Land.),334:715—718,1988.

35. Jam, A. K., Shimoi, K., Nakamura, Y., Sano, M., and Tomita, I. Effect of tea on12-O-tetradecanoyl-phorbol-13acetate (TPA) induced promotion of transformation inJB6 mouse epidermal cells. tad. J. Cancer, 26: 92—98,1989.

36. Hu, G., Han, C., and Chen, J. Inhibition of oncogene expression by green tea and(—)-epigallocatechingallate in mice. Nutr. Cancer, 24: 203—209,1995.

37. Sigler. K., and Ruch, R. J. Enhancement of gap junctional intercellular communication in tumor promoter-treated cells by components of green tea. Cancer Lett., 69:15—19,1993.

38. Ruch, R. J., Cheng, S. J., and Klaunig, J. E. Prevention of cytotoxicity and inhibitionof intercellular communication by antioxidant catechins isolated from Chinese greentea. Carcinogenesis (Land.), 10: 1003—1008,1989.

39. Angel, P. E., and Herrlich, P. A. (eds.), The FOS and JUN Families of Transcription.Boca Raton, FL: CRC Press, 1994.

40. Payne, D. M., Rossomando, A. J., Martino, P., Erickson, A. K., Her, J-H.,Shabano-witz, J., Hunt, D. F., Weber, M. J., and Sturgill, T. W. Identification of theregulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAPkinase). EMBO J., JO: 885—892,1991.

41. Derijard, B., Hibi, M., Wu, I-H., Barrett, T., Su, B., Deng. T.. Karin, M., and Davis,R. J. JNK1: a protein kinase stimulated by UVB light and Ha-Ras that binds andphosphorylates the c-Jun activation domain. Cell, 76: 1025—1037,1994.

4419

on March 26, 2020. © 1997 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

1997;57:4414-4419. Cancer Res   Zigang Dong, Wei-ya Ma, Chuanshu Huang, et al.   (-)-Epigallocatechin Gallate, and TheaflavinsActivation and Cell Transformation by Tea Polyphenols, Inhibition of Tumor Promoter-induced Activator Protein 1

  Updated version

  http://cancerres.aacrjournals.org/content/57/19/4414

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/57/19/4414To request permission to re-use all or part of this article, use this link

on March 26, 2020. © 1997 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from