Inhibition of Tumor Promoter-Induced Activator Protein 1 Activation and Cell Transformation by Tea Polyphenols, (Â€”)-Epigallocatechingallate, and Theaflavins'

CANCER RESEARCH 57. 4414-4419. October 1. 19971 Inhibition of Tumor Promoter-induced Activator Protein 1 Activation and Cell Transformation by Tea Polyphenols, (â€”)-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

ABSTRACT About 10â€”20%of the dry weight of black tea is composed of thea rubigins, which are not well characterized chemically (1). One cup of (â€”)..Epigallocatechin gallate (EGCG) and theaflavins are believed to be black tea (2.5 g of dried black tea leaves brewed in 200 ml of water) key active components in tea for the chemoprevention against cancer. may contain 12â€”15mgof theaflavins (11). Under conditions in which However, the molecular mechanisms by which EGCG and theaflavins the antitumorigenesis of tea has been demonstrated, the plasma EGCG block carcinogenesis are not clear. We have used the JB6 mouse epidermal cell line, a system that has been used extensivelyas an in vitromodel for levels in rats and mice are 37 and 124 ng/ml, respectively (2, 12). tumor promotion studies, to examine the anti-tumor promotion effects of More recently, Huang et a!. (13) reported that caffeine is an important EGCG and theaflavins at the molecular level. EGCG and theaflavins component in green and black tea on the inhibition of UVB-induced inhibited epidermal growth factor- or 12-O-tetradecanoylphorbol-13-ace carcinogenesis. These catechins and theaflavins are generally consid tate-induced cell transformation in a dose-dependent manner. At the dose ered to be the effective components for the inhibition of carcinogen range (5â€”20,zM)that inhibitedcelltransformation, EGCGand theaflavins esis, but the mechanisms are not well characterized (1, 2). A com also inhibited AP-1-dependent transcriptional activity and DNA binding monly discussed mechanism is the antioxidative activities of these activity. The inhibition of AP-I activation occurs through the inhibition of polyphenolic compounds (1, 4). The inhibitory activities of tea cat a c-Jun NH2-terminal Idnase-dependent, but not an extracellular signal echins on the growth of tumor cell lines have been shown (14, 15). regulated protein kinase (Erk) 1-dependent or Erk2-dependent, pathway. Because the transcription factor AP-1 is important for tumor promoter The antioxidative and antimutagenic effects of theaflavins have been induced neoplastic transformation, the inhibitory effects on AP.1 activa reported (16). The antipromotion activity of EGCG has been demon tion by EGCG and theaflavins may further explain the anti-tumor pro strated, and its possible effects on the signal transduction pathway motion action of these tea constituents. have been suggested (2, 17, 18). Previously, we and others have obtained evidence that activation of AP-l by phorbol ester-type tumor promoters plays a key role in tumor INTRODUCTION promotion (19â€”25).On the basis of the importance of AP-l activity in Prevention of carcinogenesis is one of the major strategies for tumor promoter-induced JB6 cell transformation and the antitransfor cancer control. Many studies have shown the inhibitory actions of mation effect of tea polyphenols, we have hypothesized that the green tea, black tea, and tea polyphenol preparations against carcino anti-tumor promotion activity of EGCG or theaflavins may be through genesis in rodent models (1â€”3).These include cancers of the skin, the inhibition of AP-1 activity. To test this hypothesis, we investigated lung, esophagus, stomach, liver, duodenum and small intestine, pan the effect of EGCG, theaflavins, and caffeine on EGF- or TPA creas, and mammary gland. The most extensively studied system is induced AP- 1 activity. The JB6 cell system, a well-developed cell the skin carcinogenesis model caused by chemicals, UV light, and culture model for the study of tumor promotion, was used to study the TPA3 (4â€”11).In some of the studies, TPA or UV light was used as a inhibition of AP-l activation as a molecular mechanism for the tumor promoter. The antipromoting effect of a major green tea con anti-tumor promotion activity of EGCG and theaflavins. stituent, EGCG, has been demonstrated (4, 7, 9, 11). However, the underlying mechanisms responsible for these cancer-preventive activ MATERIALS AND METHODS ities have not been clearly elucidated. Different tea preparations may contain various amounts of tea Materials. Eagle's MEM and FBS were from Whittaker Biosciences; L polyphenols, also known as catechins, among which EGCG is the glutamine was from Life Technologies, Inc.; gentamicin was from Quality Biological, mc; formamide was from Fluka; and luciferase assay substrate was most well studied. A cup of green tea (2.5 g of dried green tea leaves from Promega. EGCG (purity >98%) was a gift from Dr. Yukihiko Ham of brewed in 200 ml of water) may contain 90 mg of EGCG. In addition, Mitsui Norm Co. (Fujied, Japan). Theaflavins (a mixture of theaflavin, theafla it contains a similar or slightly smaller amount of (â€”)-epigallocat yin 3-gallate, theaflavin 3'-gallate, and theaflavin 3,3'-digallate, accounting for echin, about 20 mg each of (â€”)-epicatechin 3-gallate and (â€”)-epicat 21, 30, 15, and 28%, respectively) were gifts from Thomas J. Lipton Co. echin, and about 50 mg of caffeine (11). In black tea, the above tea (Englewood Cliffs, NJ). Caffeine and TPA were obtained from Sigma Chem catechins are reduced to about one-tenth to one-third of those in green ical Co. (St. Louis, MO). tea, and theaflavins account for 1 to 2% of the total dry mauer (1). Cell Culture. Tumor promoters, such as TPA and EGF, induce the trans formation 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@ 1â€”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 were cultured in monolayers at 37Â°C and 5% CO2 using Eagle's MEM charges. This article must therefore be hereby marked advertisement in accordance with containing 5% FCS, 2 mist L-glutamlne, and 25 @g/mlgentamicin (18â€”22).To 18 U.S.C. Section 1734 solely to indicate this fact. maintain the luciferase reporter construct, P@ ll cells were cultured in I This research was supported by the Hormel Foundation and NIH Grant CA56673. medium containing the neomycin analogue Geneticin (G418, Life Technolo 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: gies, Inc.) at 300 @Lg/mi. (507) 437-9606: E-mail: [email protected]. Assay for AP-1 Activity. JB6 AP-1-Iuciferase stable transfectant cells 3 The abbreviations used are: TPA, l2-O-tetradecanylphorbol-13-acetate; AP-1, acti (P'@1-1)were used to assay the AP-1 activity (18â€”22,25). Viable cells vator protein I; EGCG. (â€”)-epigallocatechin 3-gallate; EGF, epidermal growth factor; (8 X lOs) suspended in 100 pi of 5% FBS MEM were added to each well of Erk, extracellular signal-regulated protein kinase; MAPK, mitogen-activated protein Id nase; JNK, c-Jun NH2-terminal kinase; P@, promotion sensitive; FBS, fetal bovine serum; a 96-well plate. Plates were incubated at 37Â°C in a humidified atmosphere of BME, Basal Medium Eagle. 5% CO2and95% air.Twelve to 24 h later,cells were starvedby being cultured 4414

in 0.1% FBS MEM for 12 h. The cells were treated with EGCG, theaflavins, followed by incubation at room temperature for 20 mm. The DNA-protein or caffeine for 30 mm. Then, the cells were exposed to 20 ng/ml TPA or 10 complexes were resolved in a 6% nondenaturing acrylamide gel. The gel was ng/ml EGF in the presence of EGCG, theaflavins, or caffeine for 24 h. The dried and exposed to X-ray film at â€”70Â°Covernight. cells were extracted with lysis buffer, and the luciferase activity was measured Erki and Erk2 Analysis. ImmunoblotforErkl, Erk2,andtheirphospho as described previously by using a luminometer (Monolight 2010). AP-l rylated proteins was carried out as described previously (25). In brief, JB6 Cl activity is presented relative to medium control cells (25â€”29). 41 cells were cultured in monolayers in six-well plates. The cells were starved Assay for Cell Proliferation. The cell proliferation was determined by in 0. 1% FBS MEM for 48 h at 37Â°C and then changed to fresh 0. 1% FBS [3H]thymidine incorporation assay (26, 27). JB6 Cl 41 cells (5 X l0@)were MEM for another 3â€”4hat 37Â°C.Beforethe cells were exposed to TPA or seeded in 96-well plates in the presence or absence of different concentrations EGF, they were not treated or were treated with EGCG, theaflavins. or caffeine of inhibitors (EGCG, theaflavins, or caffeine). After the cells were cultured for for 30 mm. Then, 20 ng/ml TPA or 10 ng/ml EGF was added and incubated for 36 h, [3H]thymidine (0.5 @Ci/we1l)wasadded to each well. The cells were different time periods at 37Â°C. The cells were then lysed, and immunoblot harvested 12 h later, and incorporation of [3H]thymidine was quantified using analysis was performed by using antibodies against Erkl and Erk2 (P44 and a liquid scintillation counter. P42) or phospho-specific MAPK antibodies against phosphorylated tyrosine Anchorage-Independent Transformation Assay. The effects of EGCG, 204 of Erkl and Erk2 (New England Biolabs). theaflavins, or caffeine on TPA- or EGF-induced cell transformation were iNK Assay. JB6 Cl 41 cells were cultured in 100-mm dishes. The cells investigated in JB6 Cl 41 cells. Cells (1 X 10@)wereexposed to 20 ng/ml TPA were treated with TPA or EGF with or without EGCG, theaflavins, or caffeine or 10ng/mlEGFwith or withoutdifferentconcentrationsofinhibitors(EGCG, as described above for the Erk assay. Activation of JNK was assayed as theaflavins, or caffeine) in 1 ml ofO.33% BME agar containing 10% FBS over described in the manufacturer's protocols of SAPK/JNK assay kit (New 3.5 ml of 0.5% BME agar medium containing 10% FBS. The cultures were England Biolabs). In brief, JNK proteins were precipitated by using the c-Jun maintained in an incubator at 37Â°Cand 5% CO2 for 14â€”21days, and the cell fusion protein beads. After removal of the nonspecific bound proteins, the colonies were scored by the methods described previously (19â€”22). kinase reaction was carried out in the presence of ATP in the kinase buffer Nuclear Protein Analysis. Gel shift assays were used to detect AP-l (New England Biolabs). The JNK-catalyzed phosphorylation of c-Jun was binding activity after exposure to the tumor promoter TPA with or without measured by antiphospho-c-Jun (Ser63) antibodies (New England Biolabs). EGCG, theaflavin, or caffeine (22). Nuclear extracts were prepared as de scribed previously (22). In brief, the cells were lysed with 500 @.dof lysis RESULTS buffer (50 mMKC1,0.5% NP4O,25 mt@iHEPES,1 msi phenylmethylsulfonyl fluoride, 10 i@gofleupeptinper ml, 20 @gofaprotininper ml, and 100 @tm EGCG and Theaflavins but not Caffeine Inhibited TPA- or DL-D1T). After centrifugation at 14,000 rpm for 1 mm, the nuclei were washed EGF.induced Cell Transformation. As reportedpreviously,EGFor with the 500 @tlof the same buffer without NP4O. Then placed into 300 pA of TPA induces 1000â€”2000transformedcolonies in soft agar, whereas in extraction buffer (500 mMKC1and 10%glycerol with the same concentration the solvent control group (<0.1% DMSO) there is no soft agar colony ofHEPES, phenylmethylsulfonyl fluoride, leupeptin, aprotinin, and DL-DTI'as formation. EGF- or TPA-induced JB6 cell transformation was signif the lysis buffer). After centrifugation at 14,000rpm for 5 mm, the supernatant icantly blocked by EGCG or theaflavins at the concentration range was harvested as nuclear protein extract and stored at â€”70Â°C.An AP-l from 5â€”20@LM(P< 0.05; Fig. 1, A and B), whereas only the higher binding sequence from the human collagenase promoter region, 5 â€˜-AGCAT concentration of caffeine (20 pM) showed a slight inhibition of cell GAGTCAGACACCTCTGGC-3', was synthesized and labeled with transformation (P < 0.05; Fig. 10. The inhibition of cell transfor [32P]dCTP using the Klenow fragment (Life Science Co., Gaithersburg, MD). Three ,.@gof nuclear protein extracted from cells exposed to TPA for the mation by EGCG or theaflavins was not caused by growth inhibition, indicated time intervals were added to the DNA-binding buffer, which con because the concentration range that inhibited cell transformation did mined S X iO@cpm32P-labeledoligonucleotide probe, I.5 @.tgofpoly(dIdC), not inhibit cell proliferation as measured by [3H]TdR incorporation and 3 i@gof BSA. The reaction mixture was incubated on ice for 10 mm (data not shown).

2600 A j DEGF B DEGF C DEGF 2400 DTPA @TPA @TPA

U) 2200 a) I 0 2000 V C a) .@ 1800 a) I Fig. 1. Inhibition of TPA- or EGF-induced JB6 C,) 1600 P+ cell transformation by EGCG or theaflavin. Cl â€œJ' 41 cells (1 x l0@) were exposed simultaneously to 0 I I 20 ng/miTPA or 10 ng/mlEGFwithor without 1@ 1400 different concentrations of EGCG (A), theaflavins (B), or caffeine (C) in 0.33% agar containing 10% I 1200 FBS over 0.5% agar containing 10% FBS. Cell @ colonieswerescoredaftera 14-dayincubationat II 37Â°Cin 5% CO2. The inhibition ofcell transforma ! tion induced by EGF or TPA is expressed as de @8O0 scribed in â€œMaterialsand Methods.â€• : 600

CI) 400 @ ,/ 200 ,. , @@ 0 -â€˜ @ _0_ 1 5 10 20 0 1 5 10 20 0 10 20 EGCG(/2M) Theaflavins(,uM) Caffeine(,uM)

4415

TEA POLYPHENOLS INHIBIT AP-I AND CELL TRANSFORMATION

> Fig. 2. Inhibition of AP-l transcriptional activity by EGCG and theaflavin. JB6 cell AP-I reporter stable P+ 1-1 cells (8 X l0@)were seeded into each well of 96-well plates. After culture at 37'C ovemight, the cells a- were starved for 12 h by replacing the medium with 0. 1% FBS MEM. Then, cells were treated with 20 ng/ml a) TPA or 10 ng/ml EGF with or without different con > centrations of EGCG (A), theaflavins (B), or caffeine (C). The luciferase activity was determined, and the a) results were presented as relative luciferase activity. a:

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 vms may be important in their inhibitory activities against tumor Activity. As shown in Fig. 2, A and B, in a similar dose range for promoter-induced cell transformation. inhibition of cell transformation, EGCG and theaflavins inhibited EGCG and Theaflavins Inhibited Sequence-specific AP-1 DNA EGF- or TPA-induced AP-1 activity in a concentration-dependent Binding Activity. To study the molecular basis of the inhibition on manner. In contrast, caffeine, which shows slight antitransformation AP-1 activity by EGCG and theaflavins, we considered the possibil activity, showed no inhibition of AP-1 activity (P > 0.05). These ities that AP-l transactivation activity might have been altered by results indicate that inhibition of AP-l activity by EGCG and theafla these compounds. The AP-1 DNA binding activity was analyzed by

A B :@ TPA @ TPA(2Ong/ml)-++++EGCG(p@M)--20105 + +Ã· stimulated -Theaflavins -- NuclearExtract C 5AP-1--@( p,M)201 0 @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 binding activity was determined by gel-shift analysis using a 32P-labeled oligonucleotide containing the AP-l-binding site as described in â€œMaterialsandMethods.â€•Arrow, position of specific AP-l DNA binding activity. By a competition experiment, this band was shown to be compatible with unlabeled AP-l binding oligonucleotides (B). 4416

TEA POLYPHENOLS INHIBIT AP-I AND CELL TRANSFORMATION

TPA (20 ng/mI) - + + + + + Fig. 4. Inhibition of TPA-induced serine 73 EGCG(@M) - - 2010 5 1 phosphorylation of c-Jun protein. JB6 P@ Cl 41 cells (8 X 1O@)were seeded into each well of 6-well plates. After culture at 37'C for 24 h, the @ cells were starved for 48 h by replacing medium A i::: :@jâ€” phosphorylated with 0.1% FBS MEM. Two to 4 h before cells were exposed to TPA, the medium was replaced with c-Jun serum-free MEM. Then, the cells were exposed to 20 ng/ml TPA for 30 mm in the presence of dif TPA(2Ong/mI) - + + + + + ferent concentrations of EGCG (A) or theaflavin (B). The cells were extracted and c-Jun (senne Theaflavins ( @M) - 20 10 5 1 73)-phosphoiylated proteins were detected with the PhosphoPlus c-Jun (Ser73) antibody kit from New England Biolabs. @ B â€”-- .@ .* :@j- phosphorylated c-Jun

gel-shift assay. As shown in Fig 3A, EGCG or theaflavins inhibited ated serine 73 of c-Jun. As shown in Fig. 4, EGCG and theaflavins, at TPA-induced AP-1 DNA binding activity in a concentration-depen a concentration range that blocked AP-1 activity, inhibited c-Jun dent manner. By a competition experiment with unlabeled AP-1 phosphorylation at serine 73. binding oligonucleotides, this band was shown to be compatible, EGCG and Theaflavins Inhibited the Activation of iNKS but whereas added unlabeled AP-2 binding oligonucleotides did not affect not Erks. MAPKS Erks and JNKs are the upstream activator the binding activity (Fig. 3B). The concentration range of EGCG or kinases responsible for the phosphorylation of c-Jun proteins (33, theaflavins required for inhibition of AP-1 DNA binding activity was 34). In an in vitro assay, JNKs were shown to be effective kinases similar to AP-1 transactivation activity (Fig. 3). It thus appears that for the phosphorylation of c-Jun protein at serine 63/73. To test the inhibition of AP-1 transactivation by EGCG and theaflavin is, at which class of MAPK is involved in the inhibition of c-Jun least in part, due to the inhibition of AP-1 DNA binding activity. phosphorylation and AP-l activation by EGCG or theaflavins, we Inhibition of the Phosphorylation of c-Jun by EGCG and examined the influences of these compounds on the phosphoryla Theaflavins. TPA, EGF, and other tumor promoters, such as UV tion of Erki, Erk2, and JNK activity (Figs. 5 and 6). As shown in light, induce signal transduction, which leads to phosphorylation of Fig. 5, EGCG and theaflavins inhibited TPA- or EGF-induced JNK the c-Jun protein at serine 63 and serine 73 on the NH2-terminal and activity in a dose-dependent manner at the same dose range for activated AP-1 complex (30â€”32).To test whether the phosphorylation inhibition of TPA- or EGF-induced AP-1 activity, c-Jun phospho of serine 73 is affected in the inhibition of AP-1 activation by EGCG rylation, or cell transformation. On the other hand, EGCG or and theaflavins, we used phospho-specific antibodies for phosphoryl theaflavins showed no inhibition on TPA- or EGF-induced phos

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

EGCG(@M) - - 20 10 5 1

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

. (ser 63) B Fig. 5. Inhibition ofTPA- or EGF-induced INK activation by EGCG and theaflavin. JB6 Cl 41 cells were treated with medium, 10 ng/mI EGF (A and B) EGF(2Ong/mI) - + + + + + or 20 ng/ml TPA (C) with or without different concentrations of EGCG or theaflavins at 37'C in Theaflavins( @M)- - 2010 5 5% CO2. The cells were harvested and JNK activity was assayed as described in â€œMaterialsand Meth odsâ€•by a INK assay kit from New England Bio ::D- phospho-c-Jun labs. (ser 63) C

TPA (20 ng/mI) - + + + + + + + + +

EGCG(@M) - - 20 10 5 1

Theaflavins( jIM) - - 20 10 5 1

.- â€” :@:l- phospho-c-Jun (ser 63)

4417

TEA POLYPHENOLS INHIBIT AP-I AND CELL TRANSFORMATION A

TPA (20 ng/mI) - + ++++

EGCG(@M) - - 20 1 0 5 1 phosphorylated P44MAPK

@ Fig. 6. No effect on TPA- or EGF-induced Erk -.-- j . . â€¢ :=:: phosphorylated phosphorylation by EGCG (A) and theaflavins (B). P42MAPK JB6 P@Cl 41 cells were treated as described in the legend to Fig. 4. The protein extracts used in the experiment shown in Fig. 4 were used for analysis of Erkl and Erk2 phosphorylated proteins (Phos B phoPlus MAPK antibody kit, New England Bio labs). EGF (20 ng/mI) - + + + + + + + + EGCG(@M) --201051--- Theaflavins( @M) 20 10 5 phosphorylated P44MAPK phosphorylated P42MAPK

phorylation of Erkl and Erk2 (Fig. 6). In agreement with the c-Jun nonresponsive to growth factor-, phorbol ester- and UV-induced results of cell transformation, AP-1 activity, and c-Jun phospho signaling pathways (34, 39, 40). Activation of MAPKS occurs through rylation, caffeine did not inhibit JNK activity or Erk activity (data phosphorylation of threonine and tyrosine (positions 202 and 204, not shown). These results suggest that inhibition of c-Jun phos respectively, in Erks; positions 183 and 185, respectively, in JNKs; phorylation (serine 63/73) and AP-l activation by EGCG and Refs. 34, 40, and 41). Our data indicate that there is no inhibitory theaflavin is through an inhibition of JNK-dependent, Erki- and effect of EGCG or theaflavins on Erki or Erk2. However, the phos Erk2-independent pathway. phorylation of c-Jun protein at Ser73 is inhibited by EGCG and theaflavins. Moreover, we found that the JNK activity was inhibited DISCUSSION by EGCG and theaflavins. These data suggest that the inhibition of tumor promoter-mediated AP-1 activity by EGCG or theaflavins may Green tea is widely used as a beverage in China, Japan, and other be through a JNK-dependent and Erk-independent pathway. The exact Asian countries, whereas black tea is more popular in Western coun reason for the inhibition of JNK activity is at present not known. tries (1, 13). In recent years, many animal studies and several epide In animal models, both green tea and black tea extracts showed miological studies have suggested the anticarcinogenic effects of tea anticarcinogenic effects (1â€”3).In the present work, we demonstrated (1â€”3).Extractsofgreen,black,andotherteasinhibitedTPA-inducedthat EGCG or theaflavins have strong inhibitory activity on tumor JB6 cell transformation (35). However, the mechanisms of action by promoter-induced cell transformation, whereas caffeine has slight which these chemicals block carcinogenesis are not clear. The anti inhibitory effect on the cell transformation. oxidative activities of the tea polyphenols have been shown (1, 12, In summary, we have provided evidence for a novel mechanism of 16). Inactivation of protein kinase C (4, 36) and prevention of tumor the anti-tumor promotion action by EGCG and theaflavins. Our cx promoter-induced inhibition of GAP junctional intercellular commu periments suggest that inhibition of tumor promoter-induced neoplas nication by the tea components have been reported as possible mech tic transformation in JB6 cells may be through the inhibition of AP-l anisms by which the tea polyphenols might inhibit tumor promotion transactivation. The inhibition of AP-1 activation of EGCG and (37, 38). In this study, we investigated the anti-tumor promotion effect theaflavins may be mediated through the inhibition of JNKs but not of EGCG and theaflavins in JB6 cells. EOCG and theaflavins inhibit Erks. These results provide insight into the biological actions of tea tumor promoter TPA- or EGF-induced cell transformation. In the and the molecular basis for the development of new chemoprotective same dose range for inhibition of cell transformation, EGCG and reagents for cancer. theaflavins also inhibit TPA- or EGF-induced AP-1 activity. Previ ously, we have reported that induced AP-l activity is required for tumor promoter-induced cell transformation in vitro (19â€”22,25, 26) ACKNOWLEDGMENTS and tumor promotion in an animal model (29). Therefore, the inhibi We thank Dr. Y. Hara for the generous gift of EGCG, T. J. Lipton Co. for tion of AP-l activity may be functionally linked to the anti-tumor theaflavins, and Jeanne A. Ruble for secretarial assistance. promotion effects of EGCG and theaflavins. AP-1 transcriptional activity is determined by the sequence specific AP-l DNA binding activity and the phosphorylation of REFERENCES AP-1 proteins (Jun/Fos; 36). Our results demonstrated that both of 1. Yang, C. S., and Wang, Z-Y. Tea and cancer: a review. J. Natl. Cancer Inst., 58: these determinants are involved for the inhibition activity of EGCG 1038â€”1049,1993. 2. Yang.C. S.,Yang,G-Y., Lee,M-L., andChen,L. Mechanisticconsiderationsofthe and theaflavins. inhibition of carcinogenesis by tea. In: H. Ohigashi (ed), Proceedings of the Inter MAPKs, including Erks and JNKs, are mediators in a protein national Conference on Food Factor in Cancer Prevention, pp. 113â€”117.Tokyo: Springer-Verlag, 1997. kinase cascade and in the regulation of transcription factor AP-1 3. Katiyar, S. K., and Mukhtar, H. Tea in chemoprevention of cancer: epidemiologic and proteins (Jun/Fos; 28â€”31). Mutation of Ser63/73 of c-Jun renders experimental studies. Int. J. Oncology, 8: 221â€”238,1996. 4418

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1997 American Association for Cancer Research. Inhibition of Tumor Promoter-induced Activator Protein 1 Activation and Cell Transformation by Tea Polyphenols, (-)-Epigallocatechin Gallate, and Theaflavins

Zigang Dong, Wei-ya Ma, Chuanshu Huang, et al.

Cancer Res 1997;57:4414-4419.

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