PREVENTIVE

MEDlCINE

Anticarcinogenic

21, 503-509 (1992)

Effects

of (-)-Epigallocatechin

Gallate’~*

HIROTA FUJIKI, M.D.,*,3 SEIJI YOSHIZAWA, M.D.,* TAKAHIKO HORIUCHI, M.D.,? MASAMI SUGANUMA, PH.D.,* JUN YATSUNAMI, M.D.,* SHINJI NISHIWAKI, M.D.,* SACHIKO OKABE,” RIE NISHIWAKI-MATSUSHIMA,* TAKUO OKUDA, PH.D.,§ AND TAKASHI SUGIMURA, M.D.li *Cancer Prevention Division, National Cancer Center Research fEhime University School of Medicine, Ehime 791-02; #Faculty Okayama University, Okayama 700; and IiNational Cancer

Institute, Chuo-ku, Tokyo 104; of Pharmaceutical Sciences, Center, Tokyo 104, Japan

Background. Our research objective is to develop nontoxic cancer chemopreventive agents and to apply these agents in treating humans. We are identifying agents that inhibit the process of tumor promotion in two-stage carcinogenesis experiments on mouse skin. Methods. We review (a) the inhibitory effect of penta-0-galloyl-B-D-glucose (5GG) on tumor promotion by teleocidin, one of the 12-0-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoters (5GG is structurally similar to (-)-epigallocatechin gallate (EGCG) and is isolated from hydrolyzed tannic acid); (b) the inhibitory effects of EGCG, the main constituent of Japanese green tea, on tumor promotion with two tumor promoters, teleocidin and okadaic acid, a non-TPA-type tumor promoter; (c) the mechanisms of action of EGCG, a single application of which reduced the specific binding of r3H]TPA and r3H]okadaic acid to a particulate fraction of mouse skin; and (d) the anticarcinogenic effects of EGCG on duodenal carcinogenesis induced by N-ethyl-N’nitro-N-nitrosoguanidine in male C57BL/6 mice. EGCG is a nontoxic compound. Conclusion. We believe that the main constituent of Japanese green tea, EGCG, is a practical cancer chemopreventive agent available in everyday life. Q 1992 Academic Press, IIIC.

INTRODUCTION

Our study of (-)-epigallocatechin gallate (EGCG), the main constituent of green tea, for the purpose of cancer prevention started with the anticarcinogenic study of polyphenolic compounds derived from medicinal plants and drugs. After obtaining a few milligrams of each of 30 polyphenolic compounds, we had to select a test suitable for investigating the inhibitory effects of these various polyphenolic compounds on tumor promotion of 12-O-tetradecanoylphorbol-13acetate (TPA). Our first test was whether a compound shares the receptor with TPA. Two compounds, penta-0galloyl-P-D-glucose (5GG) and EGCG (Fig. I), inhibited the specific binding of 13H]TPA to a particulate fraction as a function of dose (Fig. 2). 5GG, which was obtained in large amounts from hydrolyzed tannic ’ This work was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Education, Science, and Culture, by a grant for the Program for a Comprehensive IO-Year Strategy for Cancer Control from the Ministry of Health and Welfare of Japan, and by grants from the Foundation for Promotion of Cancer Research, the Uehara Memorial Life Science Foundation, the Princess Takamatsu Cancer Research Fund, and the Smoking Research Foundation. * Presented at an International Symposium “Physiological and Pharmacological Effects of Camellia sinensis (Tea),” March 4 and 5, 1991, American Health Foundation, New York City. 3 To whom reprint requests should be addressed. 503 0091-7435/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form rescrvcd.

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OH

OH OH

Penta-Q-galloyl-BD-glucose

(-)-Epiga;l~z~?$in

gallate

(5GG) FIG.

1. Structures of 5GG and EGCG.

acid, had antitumor promoting activity in a two-stage carcinogenesis experiment on mouse skin. EGCG is structurally similar to 5GG; it is a nontoxic compound ingested daily through consumption of green tea by Japanese people. The low standardized mortality ratio for all sites of cancer and for stomach cancer among both males and females in the Shizuoka area, Japan, is associated with a high intake of green tea (8). We found that EGCG significantly inhibits tumor promotion as strongly as 5GG on mouse skin. This discovery led to new studies of EGCG in cancer prevention. We review polyphenolic compounds such as 5GG and EGCG, in relation to anticarcinogenic effects, and discuss the possibility of green tea as a practical cancer chemopreventive material. INHIBITORY

EFFECTS

OF 5GG ON TUMOR

PROMOTION

Inhibition of specific binding of [3H]TPA to a particulate fraction of mouse skin containing the phorbol ester receptor was a proper test for screening for inhibitors

0

-9 \\, 0 . \I\ 4, ‘\ ‘\ 0, --. r!. .-! 1 o-9

1 o-’

Concentration

10-s

1 o-3

( M 1

2. Effects of SGG (m) and EGCG (0) on specific binding of [3H]TPA to a particulate fraction of mouse skin, compared with that of unlabeled TPA (0). FIG.

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of tumor promotion. Various polyphenolic compounds were subjected to this test. 5GG was as effective as EGCG, whereas ( +)-catechin, ellagic acid, and chlorogenie acid did not show any strong inhibitory effects (Fig. 3). Also, 5GG weakly inhibited the activation of protein kinase C, which serves as the phorbol ester receptor, induced by teleocidin (Fig. 4). These results indicated that 5GG might interact with the phorbol ester receptor in cell membranes differently from TPA, suggesting that 5GG inhibits tumor promotion of teleocidin. 5GG could be easily obtained in gram amounts, sufficient to carry out the inhibition of tumor promotion in a two-stage carcinogenesis experiment on mouse skin. Initiation was achieved by a single application of 100 kg of 7,12dimethylbenz[a]anthracene (DMBA). One week later, tumor promotion was performed by applications of 2.5 pg teleocidin (l), twice a week. Five milligrams 5GG was applied 15 min before each treatment of teleocidin. The 5GG treatment reduced the percentage of tumor-bearing mice from 100 to 53% in Week 20, and the average numbers of tumors per mouse from 3.3 to 0.9 (Fig. 5). INHIBITORY

EFFECTS

OF EGCG ON TUMOR

PROMOTION

The preparation of EGCG used contained from 80 to 85% EGCG, and the rest was (-)-epicatechin and (-)-epicatechin gallate (IO). It is generally accepted that a key property of polyphenolic compounds is their ability to precipitate watersoluble proteins such as a hemoglobin solution. A practical method for quantitative determination of a polyphenolic compound is the astringency of a compound to a hemoglobin solution, shown in Fig. 3 as its relative astringency to geraniin (9), used as the standard polyphenolic compound. EGCG showed values similar to those for 5GG in the two parameters, inhibition of specific binding of [3H]TPA and the relative astringency to geraniin, indicating that EGCG might act similarly to 5GG on the cells. Initiation was carried out by a single application of 50 p,g DMBA in this experiment, and tumor promotion was achieved by applications of 2.5 kg teleocidin, twice a week. Five milligrams EGCG, applied topically before each treatment

Inhibition binding

of specific of ‘H-TPA ( % )

Relative

astringency

FIG. 3. Inhibition by some polyphenolic compounds of specific binding of [3H]TPA to a particulate fraction and their relative astringency to geraniin.

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ET AL.

4. Inhibition by SGG of the activation of protein kinase C by 2.2 pM teleocidin.

with teleocidin, reduced the percentage of tumor-bearing mice from 53 to 13% in Week 25, and the average numbers of tumors per mouse from 2.1 to 0.1 (Fig. 6) (10). Recently, we have found that okadaic acid is as potent a tumor promoter as teleocidin and TPA on mouse skin, but acts by a different mechanism (2). Specifically, okadaic acid inhibits dephosphorylation of phosphoserine and phosphothreonine through protein phosphatases 1 and 2A, resulting in an increase of phosphoproteins, whereas teleocidin and TPA activate protein kinase C and thus induce phosphoproteins in larger amounts than in controls (3). EGCG treatment, using the same amount (5 mg) as that in the experiment with teleocidin, completely inhibited tumor promotion by okadaic acid on mouse skin (Yoshizawa et al., manuscript in preparation). These results indicated that repeated applications of EGCG inhibited tumor promotion of two tumor promoters operating through different mechanisms of action on mouse skin. B

10 Weeks

of promotion

5. Inhibition by SGG of tumor promotion of teleocidin in mouse skin. (A) Percentages of tumor-bearing mice. (B) Average numbers of tumors per mouse in the groups treated with DMBA plus teleocidin (0) and with DMBA plus teleocidin and 5GG (0). FIG.

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B

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0

20 Weeks

10

20

of promotion

FIG. 6. Inhibition by EGCG of tumor promotion of teleocidin in mouse skin. (A) Percentages of tumor-bearing mice. (B) Average numbers of tumors per mouse in the groups treated with DMBA plus teleocidin (0) and with DMBA plus teleocidin and EGCG (0).

MECHANISMS

OF ACTION

OF EGCG

With regard to mechanisms, we previously reported that EGCG application reduced the specific binding of [3H]TPA to a particulate fraction, compared with control mouse skin. This reduction of specific binding to the phorbol ester receptor was found immediately after application of EGCG (Fig. 7) (10). We measured the specific binding of [3H]okadaic acid to the same particulate fraction. A similar reduction of specific binding to the okadaic acid receptor was also found (Yoshizawa et al., manuscript in preparation). Therefore, inhibition of tumor promotion by EGCG was induced because of interference with receptor binding of either tumor promoter. A reduction of specific binding to the tumor promoter

0

5

40

120 Time

( min 1

FIG. 7. Time course of the specific binding of [3H]TPA to a particulate fraction of mouse skin treated with a single application of EGCG.

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receptor, that is, “sealing by EGCG of membranes” due to its astringency, seems to be the first biochemical event of inhibition of the tumor promotion process on mouse skin. Recently, Zhong et al. at New York University Medical Center reported that EGCG and 5GG inhibited H202 formation by TPA-activated human polymorphonuclear leukocytes, indicating that EGCG and 5GG partly act by suppressing oxyradical formation (11). Their results also showed that the presence of EGCG and 5GG in culture medium inhibits the interaction of TPA with the tumor promoter receptor. ANTICARCINOGENIC

EFFECTS

OF EGCG

Because EGCG is ingested orally as green tea we were able to determine whether EGCG actually inhibits carcinogenesis in the gastrointestinal tract. A model system of mouse duodenal carcinogenesis, which was developed by Matsuyama et al. (7), was used. One advantage of this experiment was that tumors are induced in the duodenum of male C57BL/6 mice in a shorter time than in other parts of the digestive tract. The neoplasms were histologically adenocarcinomas. A solution of 0.01% N-ethyl-N’-nitro-iV-nitrosoguanidine (ENNG) was given to mice for the first 4 weeks as drinking water. One week after ENNG treatment, the experimental group was given a solution of 0.005% EGCG orally, and the control group tap water. After 16 weeks, the EGCG treatment reduced the percentage of tumor-bearing mice from 63 to 20% and the average numbers of tumors per mouse as well (4). The 0.005% EGCG solution roughly corresponds to 1 g of EGCG/60 kg body weight, or about 10 cups of green tea per day. Kono et al. (6) reported that among patients with greater consumption of green tea, that is, 10 or more cups of green tea per day, a decreased risk of gastric cancer was found (Table 1). Recently, Muto et al. (manuscript in preparation) noted that oral intake of either 0.05% EGCG or 0.1% EGCG reduced the percentage of spontaneous hepatoma development in C3H/HeN mice. EGCG in drinking water was effective against some, but not all, carcinogenesis tests; EGCG inhibited carcinogenesis in the duodenum and liver, but not the development of spontaneous thymic lymphoma in AKR mice (Yoshizawa et al., manuscript in preparation) and of bladder cancer induced by N-butyl-N-(4-hydroxylbutyl)nitrosamine (Ohtani et al., manuscript in preparation). Therefore, it is now important to study the bioavailability of active compounds such as EGCG in green tea in the body. For such studies, the avail-

ADJUSTED

RELATIVE

TABLE 1 RISK OF GASTRIC CANCER

FROM

GREEN RR (95%

Factor

Comparison with hospital control

Green tea (210 cups/day)

0.5 (0.3-1.1)

TEA CONSUMPTION CI)

Comparison with general population control 0.3 (0.147)

Note. Adjusted for smoking and consumption of mandarin oranges and other fruit. RR, relative risks; CI, confidence intervals. Source. Ref. (6).

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ability of radioactive EGCG would permit definition of the target organs of EGCG. Although the mechanisms of action of EGCG are not fully elucidated yet, the sealing of membranes by EGCG might interrupt the interaction of various growth factors and hormones with their receptors in membranes through autocrine and paracrine systems and result in a specific inhibition of tumor growth. For many centuries, Asian people have believed in the benefits of drinking tea. We suggest drinking green tea may be one of the most practical methods of cancer prevention available at the present (5). Cancer prevention includes two population targets, the general population and the high-risk population. Drinking green tea in larger amounts, such as 10 cups per day, would be possible for cancer prevention in the general population. For the high-risk groups of cancers of the liver and gastrointestinal tract, the use of EGCG should be further investigated. REFERENCES 1. Fujiki H, Suganuma M, Ninomiya M, Yoshizawa S, Yamashita K, Takayama S, Hitotsuyanagi Y, Sakai S, Shudo K, Sugimura T. Similar, potent tumor-promoting activity of all isomers of teleocidins A and B in a two-stage carcinogenesis experiment on the skin of CD-l mice. Cancer Res 1988; 48:4,211-4,214. 2. Fujiki H, Suganuma M, Sugimura T. Significance of new environmental tumor promoters. J Environ Sci Health Part C Environ Carcinog Rev, 1989; 7(l): l-51. 3. Fujiki H, Suganuma M, Nishiwaki S, Yoshizawa S, Winyar B, Sugimura T, Schmitz FJ. A new pathway of tumor promotion by the okadaic acid class compounds. In: Nishizuka Y, Endo M, Tanaka C, Eds. The Biology and Medicine of Signal Transduction. New York: Raven Press, 1990: 340-344. 4. Fujita Y, Yamane T, Tanaka M, Kuwata K, Okuzumi J, Takahashi T, Fujiki H, Okuda T. Inhibitory effect of (-)-epigallocatechin gallate on carcinogenesis with N-ethyl-N-nitro-Nnitrosoguanidine in mouse duodenum. Jpn J Cancer Res 1989; 80:503-505. 5. Fujiki H, Suganuma M, Yoshizawa S, Yatsunami J, Nishiwaki S, Furuya H, Okabe S, NishiwakiMatsushima R, Matsunaga S, Muto Y, Okuda T, Sugimura T. Sarcophytol A and (-)epigallocatechin gallate (ECCG), non-toxic inhibitors of cancer development. In: Boone C, Ed. Workshop of Cancer Chemoprevention, New York: CRC Press, 1991: in press. 6. Kono S, Ikeda M, Tokudome S, Kuratsune M. A case-control study of gastric cancer and diet in northern Kyushu, Japan. Jpn J Cancer Res (Gann) 1988; 79:1,067-1,074. 7. Matsuyama M, Nakamura T, Suzuki H, Nagayo T. Morphogenesis of duodenal adenocarcinomas induced by N-ethyl-N’-nitro-N-nitrosoguanidine in mice and rats. Gann Monogr Cancer Res 1975; 17:269-281. 8. Oguni I, Nasu K, Kanaya S, Ota Y, Yamamoto S, Nomura T. Epidemiological and experimental studies on the antitumor activity by green tea extracts. Jpn J Nutr 1989; 47:93-102. 9. Okuda T, Mori K, Hatano T. Relationship of the structure of tannins to the binding activities with hemoglobin and methylene blue. Chem Pharm Bull 1985; 33: 1,424-l ,433. 10. Yoshizawa S, Horiuchi T, Fujiki H, Yoshida T, Okuda T, Sugimura T. Antitumor promoting activity of (-)-epigallocatechin gallate, the main constituent of “tannin” in green tea. Phytother Res 1987; kU7. 11. Zhong Z, Tius M, Troll W, Fujiki H, Frenkel K. Inhibition of H,O, formation by human polymorphonuclear leukocytes (PMNs) as a measure of anti-carcinogenic activity. In: Abstract of the 82nd AACR Meeting, 1991; 32:127. Received August 21, 1991 Revised February 17, 1992 Accepted February 17, 1992