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Green tea and cancer chemoprevention
Mutation Research 428 Ž1999. 339–344 www.elsevier.comrlocatermolmut Community address: www.elsevier.comrlocatermutres
Green tea and cancer chemoprevention Masami Suganuma, Sachiko Okabe, Naoko Sueoka, Eisaburo Sueoka, Satoru Matsuyama, Kazue Imai, Kei Nakachi, Hirota Fujiki ) Saitama Cancer Center Research Institute, Ina, Kitaadachi-gun, Saitama 362-0806, Japan Received 29 November 1998; accepted 1 March 1999
Abstract Worldwide interest in green tea as a cancer preventive agent for humans has increased, because it is non-toxic and it is effective in a wide range of organs. Žy.-Epigallocatechin gallate ŽEGCG. is the main constituent of green tea; the others are Žy.-epicatechin gallate, Žy.-epigallocatechin and Žy.-epicatechin ŽEC.. This paper reports the results of our latest pharmacological and biochemical studies with 3 H-EGCG, along with studies on human subjects. The study on bioavailability of 3 H-EGCG in mice revealed the wide distribution of radioactivity in multiple organs. Specifically, radioactivity was found in all reported target organs of EGCG and green tea extract Ždigestive tract, liver, lung, pancreas, mammary gland and skin. as well as other organs Žbrain, kidney, uterus and ovary or testes. in mice. Recently, we demonstrated that EC enhanced incorporation of 3 H-EGCG into human lung cancer cell line PC-9 cells. EC along with another cancer preventive agent sulindac also synergistically enhanced apoptosis in PC-9 cells induced by EGCG. Moreover, a case-control study on breast cancer patients revealed that high daily consumption of green tea was associated with a lower recurrence rate among Stages I and II patients. All the results suggest that consumption of green tea is a practical and effective cancer preventive both before cancer onset and after cancer treatment. q 1999 Elsevier Science B.V. All rights reserved. Keywords: EGCG; Tea polyphenols; Apoptosis; Synergistic effect; Sulindac
1. Introduction Since 1983, when research in cancer prevention began in Japan, we have sought to establish original Japanese cancer preventive agents. To find non-toxic preventive agents, we used a two-stage carcinogenesis experiment consisting of initiation and tumor AbbreÕiations: EGCG, Žy.-epigallocatechin gallate; ECG, Žy.-epicatechin gallate; EGC, Žy.-epigallocatechin; EC, Žy.-epicatechin; TNF-a, tumor necrosis factor-a ) Corresponding author
promotion on mouse skin. We found several inhibitors of tumor promotion on mouse skin, such as penta-O-galloyl-b-D-glucose and Žy .-epigallocatechin gallate ŽEGCG. w1–3x. EGCG inhibited tumor promotion of 12-O-tetradecanoylphorbol-13acetate ŽTPA.-type, teleocidin and also that of okadaic acid w3x. Since then, various studies of the effects of EGCG and green tea extract on rodent carcinogenesis and in cell culture systems and human epidemiology have been conducted by numerous researchers in Japan and the USA w4–6x. Overall, the results indicate the following important features
0027-5107r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 4 2 Ž 9 9 . 0 0 0 5 9 - 9
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M. Suganuma et al.r Mutation Research 428 (1999) 339–344
of EGCG and green tea as cancer preventives: Ž1. their non-toxicity for rodents and humans; Ž2. a wide range of target organs, such as digestive tract including esophagus, stomach, duodenum and colon, plus liver, lung, pancreas, skin, breast, bladder and prostate w4,5x; Ž3. growth inhibition of various human cancer cell lines w7,8x; Ž4. inhibition of lung metastasis in mice w9x. Furthermore, a prospective cohort study in Saitama Prefecture, Japan, produced cancer-preventive results w6x, and another test with human volunteers in Japan demonstrated that green tea taken in tablet form had no serious adverse effects w10x. To study the bioavailability of tea polyphenols, we used 3 H-EGCG, which was administered into mouse stomach w11x. Subsequently, radioactivity was found in various mouse organs. This correlated well with the wide range of target organs for green tea as a cancer preventive agent. Radioactivity was also found in membrane, cytosol and nuclei of PC-9 cells after incubation with 3 H-EGCG w7x. Incorporation of 3 H-EGCG in the cells was inhibited by non-radioactive EGCG, Žy.-epicatechin gallate ŽECG. and Žy.-epigallocatechin ŽEGC., whereas Žy.-epicatechin ŽEC. enhanced incorporation w12x. This suggested to us the existence of a possible synergistic effect with EGCG and EC, as well as with EGCG and other cancer preventive agents, and this paper includes a summary of these synergistic effects on induction of apoptosis in PC-9 cells. The multiple inhibitory effects of green tea on carcinogenesis, cancer cell growth and metastasis led us to study whether malignant phenotypes of cancer are influenced by patients’ consumption of green tea. In one study, we found that a high consumption of green tea is associated with a lower recurrence rate among Stages I and II breast cancer patients w13x. Such results strongly indicate that green tea is a suitable preventive agent for high-risk groups and the general population as well. 2. Materials and methods 2.1. Distribution of 3H-EGCG in mouse tissue 3
H-EGCG Ž48.1 GBqrmmol. was labeled with tritium gas ŽAmersham Aylesbury, UK., as described previously w11x. CD-1 mouse ŽCharles River Japan,
Kanagawa, Japan. was given 200 ml of 0.05% EGCG solution containing 3.7 MBq 3 H-EGCG by gastric tube and kept in a metabolic cage with controlled temperature, humidity and light. After administration, radioactivity of samples of digestive tract, blood, various organs, feces and urine were measured by oxidizer ŽPackard Japan, Tokyo, Japan. as described previously w11x. 2.2. Induction of apoptosis in PC-9 cells PC-9 cells Ž2 = 10 5 cellsrml. in 24-well plates were incubated with various tea polyphenols for 2 days. DNA fragmentation was measured by quantitation of cytosolic oligonucleosome-bound DNA by using ELISA kit ŽBoehringer Mannheim, Mannheim, Germany., as described previously w12x. 2.3. Inhibition of tumor necrosis factor-a (TNF-a ) release from BALBr 3T3 cells BALBr3T3 cells Ž2 = 10 5 cellsr0.5 ml. were preincubated with various tea polyphenols for 1 h, and then treated with 0.2 mM okadaic acid for another 24 h. Concentration of TNF-a in medium was determined by ELISA ŽGenzyme, MA, USA., as described previously w14x.
3. Results and discussion 3.1. Wide distribution of 3H-EGCG in mouse organs To determine the bioavailability of EGCG, we used 3 H-EGCG with specific activity of 48.1 GBqrmmol. After direct administration of 3 H-EGCG into mouse stomach, radioactivity in blood, feces, urine and various organs was measured by oxidizer. Results in both sexes were similar, and this paper presents results with female mice. Radioactivity in blood was measurable 1 h after administration and reached about 2% of total administered radioactivity at 6 h, sustained 24 h after administration. The measured radioactivity indicated 3 H-EGCG itself, its metabolites and their protein-bound forms. In digestive tract, about 75.2% of total administered radioactivity was found 1 h after intubation. This subsequently decreased, but 14.5% of radioactivity re-
M. Suganuma et al.r Mutation Research 428 (1999) 339–344 Table 1 Distribution of radioactivity in organs of female mice after an oral administration of 3 H-EGCG
Stomach Small intestine Colon Blood Žrml. Brain Lung Heart Liver Kidney Spleen Pancreas Uterus and ovary Mammary gland Bladder Bone Skin
Radioactivity=10y3 dpmr100 mg tissuea
Percentage of total administered radioactivity Ž%.
328.0"168.3 213.7"54.4 269.0"183.5 288.0"2.0 22.5"10.4 22.0"5.8 24.4"8.1 27.3"3.7 24.7"6.5 20.0"7.1 21.4"12.3 21.8"8.0 18.4"4.3 6.6"3.6 18.5"6.5 19.3"0.4
3.9 6.0 4.5 – 0.32 0.28 0.10 0.89 0.28 0.03 0.07 0.12 – 0.01 – –
Each mouse was given 200 ml of 0.05% EGCG solution containing 3.7 MBq 3 H-EGCG. At 24 h after administration, samples of each organ were taken for measurements of radioactivity. a Each value represents the mean of two mice"variation.
mained after 24 h ŽTable 1., indicating that some of the EGCG remained in the digestive tract after drinking green tea. Within 24 h, 6.6% of total administered radioactivity had been excreted in urine, 37.1% in feces. Significant amounts of radioactivity were found in various organs 1 h after administration, and the levels gradually increased up to 24 h in almost all these organs. Radioactivity was found in what are assumed to be the target organs of EGCG and green tea extract, including digestive tract, lung, liver, pancreas, mammary gland and skin, 24 h after oral administration of 3 H-EGCG ŽTable 1.. Furthermore, similar amounts of radioactivity were found in other organs, such as kidney, brain, uterus and ovary and testes, all of which are probable additional target organs for cancer prevention with green tea. This wide distribution of 3 H-EGCG in mouse organs suggests that EGCG and green tea extract have a broad range of target organs for cancer prevention in humans w11x.
341
3.2. Synergistic effects of EGCG with EC or sulindac As reported previously, green tea polyphenols, such as EGCG, ECG, and EGC, inhibit cell growth of PC-9 cells w7x. EGCG, ECG and EGC also induced apoptosis of PC-9 cells, but EC did not ŽFig. 1.. Their potencies for apoptosis correlated well with their inhibition of cell growth. Since EC did not induce apoptosis, we inferred that EC is an inert polyphenol. When PC-9 cells were incubated with 3 H-EGCG, radioactivity was incorporated time- and dose-dependently. Incubation of 3 H-EGCG with nonradioactive EGCG, ECG and EGC decreased the incorporated radioactivity dose-dependently ŽFig. 2.. The concentrations for 50% inhibition ŽIC 50 . were 2.5 mM for EGCG, 40 mM for ECG and 100 mM for EGC, indicating that the other tea polyphenols with galloyl-moiety are incorporated into the cells in a manner similar to EGCG. However, EC at concentrations over 100 mM increased 3 H-EGCG incorporation. The increase with 1 mM EC was 1.5-fold, which led us to believe that EC might enhance the cancer preventive activity of EGCG. To determine the synergistic effects of EGCG with EC, induction of apoptosis was quantitatively measured by ELISA for cytosolic oligonucleosomebound DNA. The results clearly showed that EC Ž50
Fig. 1. Induction of apoptosis with various green tea polyphenols. PC-9 cells were treated with ECG Žv ., EGCG Ž`., EGC Ž^. or EC Ž'. for 2 days. DNA fragmentation was measured by ELISA kit for cytosolic oligonucleosome-bound DNA, as described in Section 2.
342
M. Suganuma et al.r Mutation Research 428 (1999) 339–344
Fig. 2. Enhancement of 3 H-EGCG incorporation into PC-9 cells by EC w12x. PC-9 cells were incubated with 100 mM 3 H-EGCG in the presence of EGCG Ž`., ECG Žv ., EGC Ž^., EC Ž'. and caffeine Ž=..
to 200 mM. dose-dependently enhanced apoptosis of PC-9 cells induced by 75 mM EGCG: 200 mM EC enhanced it 7.2-fold ŽTable 2.. EC also stimulated apoptosis induced by ECG and EGC about twofold and threefold, respectively ŽTable 2.. On the basis of our previous finding that TNF-a is an endogenous tumor promoter and cancer mediator w14x, we next examined whether EC enhances inhibition of TNF-a release from BALBr3T3 cells treated with okadaic acid, a known tumor promoter. EC alone did not inhibit TNF-a release at concentrations up to 500 mM, but cotreatment with EGCG and 100 mM EC reduced the IC 50 of EGCG from 60 to 15 mM, indicating a fourfold enhancement ŽTable 2.; EC also enhanced the inhibitory effects of ECG
Fig. 3. Synergistic effect with EGCG and sulindac or EGCG and sulindac sulfide. PC-9 cells were incubated with sulindac or sulindac sulfide in the presence or absence of 75 mM EGCG. ., sulindacq EGCG alone Žopen rectangle., sulindac alone Ž ., EGCGq EGCG Žfilled rectangle., sulindac sulfide alone Ž .. sulindac sulfide Ž
ŽIC 50 from 30 to 7 mM.. The results clearly indicated that EC stimulated the cancer preventive activity of EGCG and other polyphenols mediated through enhancement of their incorporation into the cells, demonstrating that green tea itself is a more effective and practical cancer preventive than EGCG alone. Next, we studied whether established cancer preventive agents could enhance anti-cancer activity by EGCG in a manner similar to that of EC. Fig. 3 shows the results of cotreatment with EGCG and sulindac or its active metabolite, sulindac sulfide. Sulindac at concentrations up to 200 mM induced almost no apoptosis of PC-9 cells; 10 or 100 mM sulindac with 75 mM EGCG induced apoptosis over
Table 2 Synergistic effects of cotreatment with EC and other tea polyphenols on apoptosis and TNF-a release Induction of apoptosisa Ž A 415 .
EGCG Ž75 mM c . ECG Ž50 mM c . EGC Ž200 mM c . a
Inhibition of TNF-a release b wIC 50 ŽmM.x
Without EC
With EC Ž200 mM.
Without EC
With EC Ž100 mM.
– 0.07 " 0.04 0.27 " 0.13 0.14 " 0.03
0.10 " 0.03 0.56 " 0.17 0.53 " 0.18 0.43 " 0.08 e
– 60 30 n.d.
) 500 d 15e 7 n.d.
Values represent the means" SD of two separate experiments performed in duplicate. Values are representative of two separate experiments performed in duplicate. c Concentration for experiment on apoptosis and growth inhibition. d IC 50 value of EC. e P - 0.01. b
M. Suganuma et al.r Mutation Research 428 (1999) 339–344 Table 3 Recurrence rate of breast cancer in relation to daily consumption of green tea Daily consumption of green tea Žcups. Stages I and II (390 patients) Recurrence rate Ž%. Disease-free years Stage III (82 patients) Recurrence rate Ž%. Disease-free years
F4
G5
24.3 2.8
16.7 3.6
48.8 1.9
58.5 1.9
10 times more strongly than sulindac alone; and sulindac sulfide at a concentration of 1 mM with EGCG resulted in marked enhancement. The implications are clear: a combination of sulindac and green tea can result in smaller doses of sulindac, and thus fewer adverse effects. This would make it feasible to administer green tea to a high-risk cancer population. 3.3. Cancer preÕentiÕe effects of green tea in a Japanese population Our prospective cohort study with 8552 individuals in Saitama, Japan, revealed that cancer onset for patients who had consumed over 10 Japanese cups of green tea per day was 7.3 years later among females, and 3.2 years later among males, than for patients who had consumed less than three cups per day w6x. We next examined the association between consumption of green tea prior to clinical cancer onset and recurrence rate of cancer w13x. A total of 472 breast cancer patients were histologically classified into Stages I, II and III, and consumption of green tea by the patients was roughly divided into two classes, F 4 cupsrday Žmean: 2 cupsrday. and G 5 cupsrday Žmean: 8 cupsrday.. Among Stages I and II patients, the group consuming G 5 cupsrday showed a lower recurrence rate, 16.7%, and a longer disease-free period, 3.6 years, than those consuming F 4 cupsrday ŽTable 3.. This was associated with decreased numbers of axillary lymph node metastases among premenopausal patients in Stages I and II, and with increased expression of progesterone
343
receptor and estrogen receptor. Stage III cancer patients did not show any significant associations. This suggests that green tea is more effective in the early stages of cancer development.
4. Conclusion Based on our current understanding of multi-stage carcinogenesis in humans, it is possible to introduce cancer prevention both before cancer onset and after cancer treatment w15,16x. Before cancer onset, cancer preventive agents can be given to prevent the growth of dormant and initiated cancer cells, resulting in delay of cancer onset. After treatment, cancer patients Žroughly 1% of the population of Japan. who are clinically healthy can drink green tea to reduce the risk of cancer recurrence, second primary tumors and metastasis. For both groups, we feel strongly that drinking green tea is the most practical method of cancer prevention.
Acknowledgements This work was supported by the following grantsin-aid: for Scientific Research on Priority Areas for Cancer Research from the Ministry of Education, Science, Sports and Culture, Japan; for a Second Term Comprehensive 10-Year Strategy for Cancer Control and for Comprehensive Research on Aging and Health from Ministry of Health and Welfare, Japan; for Selectively Applied and Developed Research from Saitama Prefecture, Japan; and by grants from the Uehara Memorial Life Science Foundation, the Smoking Research Fund, and the Plant Science Research Foundation of the Faculty of Agriculture, Kyoto University. We thank Ms. Aiko Inoue for her assistance with the experiments.
References w1x H. Fujiki, M. Suganuma, A. Komori, S. Okabe, E. Sueoka, N. Sueoka, T. Kozu, Y. Tada, Natural inhibitors of carcinogenesis, in: Y. Martinet, F.R. Hirsch, N. Martinet, J.-M. Vignand, J.L. Mulshine ŽEds.., Clinical and Biological Basis of Lung Cancer Prevention, 1998, pp. 285–290.
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w2x S. Yoshizawa, T. Horiuchi, H. Fujiki, T. Yoshida, T. Okuda, T. Sugimura, Antitumor promoting activity of Žy.-epigallocatechin gallate, the main constituent of ‘Tannin’ in green tea, Phytother. Res. 1 Ž1987. 44–47. w3x S. Yoshizawa, T. Horiuchi, M. Suganuma, S. Nishiwaki, J. Yatsunami, S. Okabe, T. Okuda, Y. Muto, K. Frenkel, W. Troll, H. Fujiki, Penta-O-galloyl-b-D-glucose and Žy.-epigallocatechin gallate, in: M.-T. Huang, C.-T. Ho, C.Y. Lee ŽEds.., Phenolic Compounds in Food and Their Effect on Health II, ACS Symposium Series 507, USA, 1992, pp. 316–325. w4x H. Fujiki, M. Suganuma, S. Okabe, A. Komori, E. Sueoka, N. Sueoka, T. Kozu, Y. Sakai, Japanese green tea as a cancer preventive in humans, Nutr. Rev. 54 Ž1996. S67–S70. w5x NCI, DCPC, Chemoprevention Branch, and Agent Development Committee, Clinical development plan: the extracts green tea polyphenols epigallocatechin gallate, J. Cell. Biochem. 26 Ž1996. 236–257. w6x K. Imai, K. Suga, K. Nakachi, Cancer-preventive effects of drinking green tea among a Japanese population, Prev. Med. 26 Ž1997. 769–775. w7x S. Okabe, M. Suganuma, M. Hayashi, E. Sueoka, A. Komori, H. Fujiki, Mechanisms of growth inhibition of human lung cancer cell line, PC-9 by tea polyphenols, Jpn. J. Cancer Res. 88 Ž1997. 639–643. w8x G.-Y. Yang, J. Liao, K. Kim, E.J. Yurkow, C.S. Yang, Inhibition of growth and induction of apoptosis in human cancer cell lines by tea polyphenols, Carcinogenesis 19 Ž1998. 611–616. w9x S.-I. Taniguchi, H. Fujiki, H. Kobayashi, H. Go, K. Miyado,
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H. Sadano, R. Shimokawa, Effect of Žy.-epigallocatechin gallate, the main constituent of green tea, on lung metastasis with mouse B16 melanoma cell lines, Cancer Lett. 65 Ž1992. 51–54. K. Suga, K. Imai, N. Sueoka, K. Nakachi, Phase I clinical trial with green tea tablets in a Japanese healthy population, Cancer Prev. Intern. 3 Ž1998. 79–88. M. Suganuma, S. Okabe, M. Oniyama, Y. Tada, H. Ito, H. Fujiki, Wide distribution of 3 H-Žy.-epigallocatechin gallate, a cancer preventive tea polyphenol, in mouse tissue, Carcinogenesis 19 Ž1998. 1771–1776. M. Suganuma, S. Okabe, Y. Kai, N. Sueoka, E. Sueoka, H. Fujiki, Synergistic effects of Žy.-epigallocatechin gallate with Žy.-epicatechin, sulindac, or tamoxifen on cancer preventive activity in human lung cancer cell line, PC-9, Cancer Res. 59 Ž1999. 44–47. K. Nakachi, K. Suemasu, K. Suga, T. Takeo, K. Imai, Y. Higashi, Influence of drinking green tea on breast cancer malignancy among Japanese patients, Jpn. J. Cancer Res. 89 Ž1998. 254–261. M. Suganuma, S. Okabe, E. Sueoka, N. Iida, A. Komori, S.-J. Kim, H. Fujiki, A new process of cancer prevention mediated throgh inhibition of tumor necrosis factor a expression, Cancer Res. 56 Ž1996. 3711–3715. W.K. Hong, M.B. Sporn, Recent advances in chemoprevention of cancer, Science 278 Ž1997. 1073–1077. H. Fujiki, A. Komori, M. Suganuma, Chemoprevention of cancer, in: T. Bowden, S. Fischer ŽEds.., Comprehensive Toxicology, Vol. 12, Elsevier, UK, 1997, pp. 453–471.
Green tea and cancer chemoprevention Masami Suganuma, Sachiko Okabe, Naoko Sueoka, Eisaburo Sueoka, Satoru Matsuyama, Kazue Imai, Kei Nakachi, Hirota Fujiki ) Saitama Cancer Center Research Institute, Ina, Kitaadachi-gun, Saitama 362-0806, Japan Received 29 November 1998; accepted 1 March 1999
Abstract Worldwide interest in green tea as a cancer preventive agent for humans has increased, because it is non-toxic and it is effective in a wide range of organs. Žy.-Epigallocatechin gallate ŽEGCG. is the main constituent of green tea; the others are Žy.-epicatechin gallate, Žy.-epigallocatechin and Žy.-epicatechin ŽEC.. This paper reports the results of our latest pharmacological and biochemical studies with 3 H-EGCG, along with studies on human subjects. The study on bioavailability of 3 H-EGCG in mice revealed the wide distribution of radioactivity in multiple organs. Specifically, radioactivity was found in all reported target organs of EGCG and green tea extract Ždigestive tract, liver, lung, pancreas, mammary gland and skin. as well as other organs Žbrain, kidney, uterus and ovary or testes. in mice. Recently, we demonstrated that EC enhanced incorporation of 3 H-EGCG into human lung cancer cell line PC-9 cells. EC along with another cancer preventive agent sulindac also synergistically enhanced apoptosis in PC-9 cells induced by EGCG. Moreover, a case-control study on breast cancer patients revealed that high daily consumption of green tea was associated with a lower recurrence rate among Stages I and II patients. All the results suggest that consumption of green tea is a practical and effective cancer preventive both before cancer onset and after cancer treatment. q 1999 Elsevier Science B.V. All rights reserved. Keywords: EGCG; Tea polyphenols; Apoptosis; Synergistic effect; Sulindac
1. Introduction Since 1983, when research in cancer prevention began in Japan, we have sought to establish original Japanese cancer preventive agents. To find non-toxic preventive agents, we used a two-stage carcinogenesis experiment consisting of initiation and tumor AbbreÕiations: EGCG, Žy.-epigallocatechin gallate; ECG, Žy.-epicatechin gallate; EGC, Žy.-epigallocatechin; EC, Žy.-epicatechin; TNF-a, tumor necrosis factor-a ) Corresponding author
promotion on mouse skin. We found several inhibitors of tumor promotion on mouse skin, such as penta-O-galloyl-b-D-glucose and Žy .-epigallocatechin gallate ŽEGCG. w1–3x. EGCG inhibited tumor promotion of 12-O-tetradecanoylphorbol-13acetate ŽTPA.-type, teleocidin and also that of okadaic acid w3x. Since then, various studies of the effects of EGCG and green tea extract on rodent carcinogenesis and in cell culture systems and human epidemiology have been conducted by numerous researchers in Japan and the USA w4–6x. Overall, the results indicate the following important features
0027-5107r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 4 2 Ž 9 9 . 0 0 0 5 9 - 9
340
M. Suganuma et al.r Mutation Research 428 (1999) 339–344
of EGCG and green tea as cancer preventives: Ž1. their non-toxicity for rodents and humans; Ž2. a wide range of target organs, such as digestive tract including esophagus, stomach, duodenum and colon, plus liver, lung, pancreas, skin, breast, bladder and prostate w4,5x; Ž3. growth inhibition of various human cancer cell lines w7,8x; Ž4. inhibition of lung metastasis in mice w9x. Furthermore, a prospective cohort study in Saitama Prefecture, Japan, produced cancer-preventive results w6x, and another test with human volunteers in Japan demonstrated that green tea taken in tablet form had no serious adverse effects w10x. To study the bioavailability of tea polyphenols, we used 3 H-EGCG, which was administered into mouse stomach w11x. Subsequently, radioactivity was found in various mouse organs. This correlated well with the wide range of target organs for green tea as a cancer preventive agent. Radioactivity was also found in membrane, cytosol and nuclei of PC-9 cells after incubation with 3 H-EGCG w7x. Incorporation of 3 H-EGCG in the cells was inhibited by non-radioactive EGCG, Žy.-epicatechin gallate ŽECG. and Žy.-epigallocatechin ŽEGC., whereas Žy.-epicatechin ŽEC. enhanced incorporation w12x. This suggested to us the existence of a possible synergistic effect with EGCG and EC, as well as with EGCG and other cancer preventive agents, and this paper includes a summary of these synergistic effects on induction of apoptosis in PC-9 cells. The multiple inhibitory effects of green tea on carcinogenesis, cancer cell growth and metastasis led us to study whether malignant phenotypes of cancer are influenced by patients’ consumption of green tea. In one study, we found that a high consumption of green tea is associated with a lower recurrence rate among Stages I and II breast cancer patients w13x. Such results strongly indicate that green tea is a suitable preventive agent for high-risk groups and the general population as well. 2. Materials and methods 2.1. Distribution of 3H-EGCG in mouse tissue 3
H-EGCG Ž48.1 GBqrmmol. was labeled with tritium gas ŽAmersham Aylesbury, UK., as described previously w11x. CD-1 mouse ŽCharles River Japan,
Kanagawa, Japan. was given 200 ml of 0.05% EGCG solution containing 3.7 MBq 3 H-EGCG by gastric tube and kept in a metabolic cage with controlled temperature, humidity and light. After administration, radioactivity of samples of digestive tract, blood, various organs, feces and urine were measured by oxidizer ŽPackard Japan, Tokyo, Japan. as described previously w11x. 2.2. Induction of apoptosis in PC-9 cells PC-9 cells Ž2 = 10 5 cellsrml. in 24-well plates were incubated with various tea polyphenols for 2 days. DNA fragmentation was measured by quantitation of cytosolic oligonucleosome-bound DNA by using ELISA kit ŽBoehringer Mannheim, Mannheim, Germany., as described previously w12x. 2.3. Inhibition of tumor necrosis factor-a (TNF-a ) release from BALBr 3T3 cells BALBr3T3 cells Ž2 = 10 5 cellsr0.5 ml. were preincubated with various tea polyphenols for 1 h, and then treated with 0.2 mM okadaic acid for another 24 h. Concentration of TNF-a in medium was determined by ELISA ŽGenzyme, MA, USA., as described previously w14x.
3. Results and discussion 3.1. Wide distribution of 3H-EGCG in mouse organs To determine the bioavailability of EGCG, we used 3 H-EGCG with specific activity of 48.1 GBqrmmol. After direct administration of 3 H-EGCG into mouse stomach, radioactivity in blood, feces, urine and various organs was measured by oxidizer. Results in both sexes were similar, and this paper presents results with female mice. Radioactivity in blood was measurable 1 h after administration and reached about 2% of total administered radioactivity at 6 h, sustained 24 h after administration. The measured radioactivity indicated 3 H-EGCG itself, its metabolites and their protein-bound forms. In digestive tract, about 75.2% of total administered radioactivity was found 1 h after intubation. This subsequently decreased, but 14.5% of radioactivity re-
M. Suganuma et al.r Mutation Research 428 (1999) 339–344 Table 1 Distribution of radioactivity in organs of female mice after an oral administration of 3 H-EGCG
Stomach Small intestine Colon Blood Žrml. Brain Lung Heart Liver Kidney Spleen Pancreas Uterus and ovary Mammary gland Bladder Bone Skin
Radioactivity=10y3 dpmr100 mg tissuea
Percentage of total administered radioactivity Ž%.
328.0"168.3 213.7"54.4 269.0"183.5 288.0"2.0 22.5"10.4 22.0"5.8 24.4"8.1 27.3"3.7 24.7"6.5 20.0"7.1 21.4"12.3 21.8"8.0 18.4"4.3 6.6"3.6 18.5"6.5 19.3"0.4
3.9 6.0 4.5 – 0.32 0.28 0.10 0.89 0.28 0.03 0.07 0.12 – 0.01 – –
Each mouse was given 200 ml of 0.05% EGCG solution containing 3.7 MBq 3 H-EGCG. At 24 h after administration, samples of each organ were taken for measurements of radioactivity. a Each value represents the mean of two mice"variation.
mained after 24 h ŽTable 1., indicating that some of the EGCG remained in the digestive tract after drinking green tea. Within 24 h, 6.6% of total administered radioactivity had been excreted in urine, 37.1% in feces. Significant amounts of radioactivity were found in various organs 1 h after administration, and the levels gradually increased up to 24 h in almost all these organs. Radioactivity was found in what are assumed to be the target organs of EGCG and green tea extract, including digestive tract, lung, liver, pancreas, mammary gland and skin, 24 h after oral administration of 3 H-EGCG ŽTable 1.. Furthermore, similar amounts of radioactivity were found in other organs, such as kidney, brain, uterus and ovary and testes, all of which are probable additional target organs for cancer prevention with green tea. This wide distribution of 3 H-EGCG in mouse organs suggests that EGCG and green tea extract have a broad range of target organs for cancer prevention in humans w11x.
341
3.2. Synergistic effects of EGCG with EC or sulindac As reported previously, green tea polyphenols, such as EGCG, ECG, and EGC, inhibit cell growth of PC-9 cells w7x. EGCG, ECG and EGC also induced apoptosis of PC-9 cells, but EC did not ŽFig. 1.. Their potencies for apoptosis correlated well with their inhibition of cell growth. Since EC did not induce apoptosis, we inferred that EC is an inert polyphenol. When PC-9 cells were incubated with 3 H-EGCG, radioactivity was incorporated time- and dose-dependently. Incubation of 3 H-EGCG with nonradioactive EGCG, ECG and EGC decreased the incorporated radioactivity dose-dependently ŽFig. 2.. The concentrations for 50% inhibition ŽIC 50 . were 2.5 mM for EGCG, 40 mM for ECG and 100 mM for EGC, indicating that the other tea polyphenols with galloyl-moiety are incorporated into the cells in a manner similar to EGCG. However, EC at concentrations over 100 mM increased 3 H-EGCG incorporation. The increase with 1 mM EC was 1.5-fold, which led us to believe that EC might enhance the cancer preventive activity of EGCG. To determine the synergistic effects of EGCG with EC, induction of apoptosis was quantitatively measured by ELISA for cytosolic oligonucleosomebound DNA. The results clearly showed that EC Ž50
Fig. 1. Induction of apoptosis with various green tea polyphenols. PC-9 cells were treated with ECG Žv ., EGCG Ž`., EGC Ž^. or EC Ž'. for 2 days. DNA fragmentation was measured by ELISA kit for cytosolic oligonucleosome-bound DNA, as described in Section 2.
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Fig. 2. Enhancement of 3 H-EGCG incorporation into PC-9 cells by EC w12x. PC-9 cells were incubated with 100 mM 3 H-EGCG in the presence of EGCG Ž`., ECG Žv ., EGC Ž^., EC Ž'. and caffeine Ž=..
to 200 mM. dose-dependently enhanced apoptosis of PC-9 cells induced by 75 mM EGCG: 200 mM EC enhanced it 7.2-fold ŽTable 2.. EC also stimulated apoptosis induced by ECG and EGC about twofold and threefold, respectively ŽTable 2.. On the basis of our previous finding that TNF-a is an endogenous tumor promoter and cancer mediator w14x, we next examined whether EC enhances inhibition of TNF-a release from BALBr3T3 cells treated with okadaic acid, a known tumor promoter. EC alone did not inhibit TNF-a release at concentrations up to 500 mM, but cotreatment with EGCG and 100 mM EC reduced the IC 50 of EGCG from 60 to 15 mM, indicating a fourfold enhancement ŽTable 2.; EC also enhanced the inhibitory effects of ECG
Fig. 3. Synergistic effect with EGCG and sulindac or EGCG and sulindac sulfide. PC-9 cells were incubated with sulindac or sulindac sulfide in the presence or absence of 75 mM EGCG. ., sulindacq EGCG alone Žopen rectangle., sulindac alone Ž ., EGCGq EGCG Žfilled rectangle., sulindac sulfide alone Ž .. sulindac sulfide Ž
ŽIC 50 from 30 to 7 mM.. The results clearly indicated that EC stimulated the cancer preventive activity of EGCG and other polyphenols mediated through enhancement of their incorporation into the cells, demonstrating that green tea itself is a more effective and practical cancer preventive than EGCG alone. Next, we studied whether established cancer preventive agents could enhance anti-cancer activity by EGCG in a manner similar to that of EC. Fig. 3 shows the results of cotreatment with EGCG and sulindac or its active metabolite, sulindac sulfide. Sulindac at concentrations up to 200 mM induced almost no apoptosis of PC-9 cells; 10 or 100 mM sulindac with 75 mM EGCG induced apoptosis over
Table 2 Synergistic effects of cotreatment with EC and other tea polyphenols on apoptosis and TNF-a release Induction of apoptosisa Ž A 415 .
EGCG Ž75 mM c . ECG Ž50 mM c . EGC Ž200 mM c . a
Inhibition of TNF-a release b wIC 50 ŽmM.x
Without EC
With EC Ž200 mM.
Without EC
With EC Ž100 mM.
– 0.07 " 0.04 0.27 " 0.13 0.14 " 0.03
0.10 " 0.03 0.56 " 0.17 0.53 " 0.18 0.43 " 0.08 e
– 60 30 n.d.
) 500 d 15e 7 n.d.
Values represent the means" SD of two separate experiments performed in duplicate. Values are representative of two separate experiments performed in duplicate. c Concentration for experiment on apoptosis and growth inhibition. d IC 50 value of EC. e P - 0.01. b
M. Suganuma et al.r Mutation Research 428 (1999) 339–344 Table 3 Recurrence rate of breast cancer in relation to daily consumption of green tea Daily consumption of green tea Žcups. Stages I and II (390 patients) Recurrence rate Ž%. Disease-free years Stage III (82 patients) Recurrence rate Ž%. Disease-free years
F4
G5
24.3 2.8
16.7 3.6
48.8 1.9
58.5 1.9
10 times more strongly than sulindac alone; and sulindac sulfide at a concentration of 1 mM with EGCG resulted in marked enhancement. The implications are clear: a combination of sulindac and green tea can result in smaller doses of sulindac, and thus fewer adverse effects. This would make it feasible to administer green tea to a high-risk cancer population. 3.3. Cancer preÕentiÕe effects of green tea in a Japanese population Our prospective cohort study with 8552 individuals in Saitama, Japan, revealed that cancer onset for patients who had consumed over 10 Japanese cups of green tea per day was 7.3 years later among females, and 3.2 years later among males, than for patients who had consumed less than three cups per day w6x. We next examined the association between consumption of green tea prior to clinical cancer onset and recurrence rate of cancer w13x. A total of 472 breast cancer patients were histologically classified into Stages I, II and III, and consumption of green tea by the patients was roughly divided into two classes, F 4 cupsrday Žmean: 2 cupsrday. and G 5 cupsrday Žmean: 8 cupsrday.. Among Stages I and II patients, the group consuming G 5 cupsrday showed a lower recurrence rate, 16.7%, and a longer disease-free period, 3.6 years, than those consuming F 4 cupsrday ŽTable 3.. This was associated with decreased numbers of axillary lymph node metastases among premenopausal patients in Stages I and II, and with increased expression of progesterone
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receptor and estrogen receptor. Stage III cancer patients did not show any significant associations. This suggests that green tea is more effective in the early stages of cancer development.
4. Conclusion Based on our current understanding of multi-stage carcinogenesis in humans, it is possible to introduce cancer prevention both before cancer onset and after cancer treatment w15,16x. Before cancer onset, cancer preventive agents can be given to prevent the growth of dormant and initiated cancer cells, resulting in delay of cancer onset. After treatment, cancer patients Žroughly 1% of the population of Japan. who are clinically healthy can drink green tea to reduce the risk of cancer recurrence, second primary tumors and metastasis. For both groups, we feel strongly that drinking green tea is the most practical method of cancer prevention.
Acknowledgements This work was supported by the following grantsin-aid: for Scientific Research on Priority Areas for Cancer Research from the Ministry of Education, Science, Sports and Culture, Japan; for a Second Term Comprehensive 10-Year Strategy for Cancer Control and for Comprehensive Research on Aging and Health from Ministry of Health and Welfare, Japan; for Selectively Applied and Developed Research from Saitama Prefecture, Japan; and by grants from the Uehara Memorial Life Science Foundation, the Smoking Research Fund, and the Plant Science Research Foundation of the Faculty of Agriculture, Kyoto University. We thank Ms. Aiko Inoue for her assistance with the experiments.
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