A New Function of Green Tea: Prevention of Lifestyle-related Diseases

NAOKO SUEOKA, MASAMI SUGANUMA, EISABURO SUEOKA, SACHIKO OKABE, SATORU MATSUYAMA, KAZUE IMAI, KEI NAKACHI, AND HIROTA FUJIKI Saitama Cancer Center Research Institute, Ina, Kitaadachi-gun, Saitama 362-0806, Japan

ABSTRACT: In the normal human life span, there occur lifestyle-related diseases that may be preventable with nontoxic agents. This paper deals with the preventive activity of green tea in some lifestyle-related diseases. Green tea is one of the most practical cancer preventives, as we have shown in various in vitro and in vivo experiments, along with epidemiological studies. Among various biological effects of green tea, we have focused on its inhibitory effect on TNFgene expression mediated through inhibition of NF- B and AP-1 activation. Based on our recent results with TNF- -deficient mice, TNF- is an endogenous tumor promoter. TNF- is also known to be a central mediator in chronic inflammatory diseases such as rheumatoid arthritis and multiple sclerosis. We therefore hypothesized that green tea might be a preventive agent for chronic inflammatory diseases. To test this hypothesis, TNF- transgenic mice, which overexpress TNF- only in the lungs, were examined. The TNF- transgenic mouse is an animal model of human idiopathic pulmonary fibrosis which also frequently develops lung cancer. Expressions of TNF- and IL-6 were inhibited in the lungs of these mice after treatment with green tea in drinking water for 4 months. In addition, judging from the results of a prospective cohort study in Saitama Prefecture, Japan, green tea helps to prevent cardiovascular disease. In this study, a decreased relative risk of death from cardiovascular disease was found for people consuming over 10 cups of green tea a day, and green tea also had life-prolonging effects on cumulative survival. These data suggest that green tea has preventive effects on both chronic inflammatory diseases and lifestyle-related diseases (including cardiovascular disease and cancer), resulting in prolongation of life span. KEYWORDS: Cancer prevention; TNF- ; NF ; Life-prolonging effects

INTRODUCTION In 1987, we reported the first evidence that ()-epigallocatechin gallate (EGCG) inhibited tumor promotion in a two-stage carcinogenesis experiment on mouse skin.1 Later, the cancer preventive effect was accepted by the scientists throughout the world.2,3 The rodent carcinogenesis experiments showed that EGCG and green tea extract have preventive activities on carcinogenesis of various organs, such as diAddress for correspondence: Hirota Fujiki, M.D., Saitama Cancer Center Research Institute, Ina, Kitaadachi-gun, Saitama 362-0806, Japan. Voice: +81 48-722-1111; fax: +81 48-722-1739. hfujiki@cancer-c.pref.saitama.jp

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gestive tract, skin, liver, pancreas, lung, prostate, and bladder, as well as pulmonary metastasis of melanoma cells.46 These data have been further supported by the results of human epidemiological studies. And recently our colleagues (K.I. and K.N.) reported that their prospective cohort study found cancer preventive effects of drinking green tea on the basis of an eleven-year follow-up study among 8,552 respondents. 7 From our intensive studies on tumor promotion with okadaic acid, we have demonstrated that TNF- is an endogenous tumor promoter.8 A two-stage carcinogenesis experiment on the skin of TNF--deficient mice clearly showed that TNF- is an essential cytokine in tumor promotion,9 and in a subsequent experiment, EGCG inhibited TNF- gene expression mediated through inhibition of NF-B and AP-1 activation.10 TNF- is a key cytokine not only in cancer but also in other diseases such as rheumatoid arthritis, Crohns disease, multiple sclerosis, and idiopathic pulmonary fibrosis,11 and TNF- is reported to contribute to diabetes mellitus, a major lifestyle-related disease.12 Based on accumlating evidence that TNF- is central mediator in various diseases which frequently occur among elderly people, we hypothesized that green tea, an inhibitor of TNF- production, might prevent the development of some of these diseases. To examine our hypothesis, we used TNF- transgenic mice, which overexpressed TNF- only in the lungs due to constant activation of surfactant protein-C (SP-C) promoter.13 In this paper, we review the cancer preventive effects of green tea and discuss its mechanisms of action from the point of view of inhibition of TNF- as an endogenous tumor promoter. We also present the preventive effects of green tea on other TNF--related diseases, using results of experiments with TNF- transgenic mice. CANCER PREVENTIVE EFFECTS OF GREEN TEA The key element in a cancer preventive agent is that it is nontoxic, since the target for cancer prevention is not only the high-risk group but also the general population.14 EGCG, the main constituent of green tea, was screened from among various natural products in two-stage carcinogenesis experiments on mouse skin. Repeated topical applications of EGCG completely inhibited tumor promotion of okadaic acid, a potent tumor promoter, on mouse skin initiated with 7,12-dimethylbenz(a)anthracene (DMBA).4 Since the first result published in 1987, EGCG and green tea extract have been shown to inhibit carcinogenesis of various organs of rodents, such as digestive tract including esophagus, stomach, duodenum, and colon, plus liver, lung, pancreas, and skin (TABLE 1).46 From our investigation of okadaic acid we concluded that TNF- is an endogenous tumor promoter.8,15 The mechanism of the action of okadaic acid is different from that of 12-O-tetradecanoylphorbol-13-acetate. The okadaic acid class compounds, which are potent inhibitors of protein phosphatases 1 and 2A, induced tumor promotion in three different organsmouse skin, rat glandular stomach, and rat liverinitiated with three different initiators.1618 TNF- was also induced in the target organs by the okadaic acid class compounds. And in a series of in vitro two-stage carcinogenesis experiments with BALB/3T3 cells initiated with 3-methylcholantrene, TNF- induced potent tumor promoting activity.8 To prove that TNF- is the essential tumor promoter, we conducted a two-stage carcinogenesis experiment on skin of TNF--deficient mice, which were established by Lloyd J.

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TABLE 1. Inhibition of carcinogenesis with EGCG and green tea extract Reduction of Tumor Incidence (%) 62.0 31.0 63.0 20.0 77.3 38.1 67.0 33.0 83.8 52.2 96.3 65.5 54.0 33.0 67.0 7.0

Organs Glandular stomach Duodenum Colon rat

Species

Carcinogens MNNG ENNG AOM MNU spontaneous NNK BOP UVB

Inhibitors EGCG EGCG EGCG GTE EGCG GTE GTE GTE

mouse C57BL/6 rat rat

Liver Lung Pancreas Skin

mouse C3H/HeN mouse A/J hamster house SKG-1

MNNG: N-methyl-N-nitro-N-nitrosoguanidine; ENNG:N-ethyl-N-nitro-N-nitrosoguanidine; AOM: azoxymethane; MNU: methylnitrosourea; NNK:4-(methylnitrosoamine)-1-(3-pyridyl)-1butanone; and BOP: N-nitrosobis-(2-oxopropyl)amine; GTE: green tea extract.

Olds research group in 1997.19 A single application of okadaic acid to mouse skin did not induce TNF- gene expression in skin of TNF--deficient mice, but it did induce TNF- in wild mice. Repeated applications of okadaic acid did not induce any tumors in TNF--deficient mice initiated with DMBA by 19 weeks of tumor promotion, while 100% of the wild mice developed tumors by week 17.9 These results clearly demonstrated that tumor promotion by okadaic acid is mediated through TNF-, and that TNF- is the key cytokine for tumor promotion on mouse skin. Since EGCG inhibited TNF- gene expression in BALB/3T3 cells and KATO III cells treated with okadaic acid, EGCGs action as a cancer preventive is mediated through its inhibition of TNF- production.10,15

EFFECTS OF GREEN TEA EXTRACT ON CYTOKINE PRODUCTION IN TNF- TRANSGENIC MICE Besides being a potent tumor promoter, TNF- also plays an important role in the development of other diseases, including rheumatoid arthritis, multiple sclerosis, and idiopathic pulmonary fibrosis.11,13 To examine whether green tea extract can inhibit TNF- production in an animal model, we used TNF- transgenic mice. These mice, which carry a chimeric gene consisting of human SP-C promoter and mouse TNF- gene, progressively develop interstitial pneumonitis that resembles idiopathic pulmonary fibrosis in humans.13,20 Because SP-C promoter is activated only in alveolar type II epithelial cells, TNF- is overexpressed only in the lungs. We divided the process of interstitial pneumonitis into three stages according to histological change: early stage (1-month-old), middle stage (7-month-old), and late stage (13month-old). In the early stage, lymphocytic dominant infiltration was observed in alveolar septa; in the middle stage this was followed by macrophage accumulation in both interstitial regions and alveolar spaces; and in the late stage, hyperplastic

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FIGURE 1. Green tea extract inhibited cytokine production. After treatment with 0.1% green tea extract in drinking water for 4 months, protein levels of TNF- and IL-6 in the lungs were determined by enzyme-linked immunosorbent assay. The protein levels of these cytokines in the lungs of nontreated TNF- transgenic mice were shown as 100%.

changes of bronchiolar epithelial cells were found. The TNF- overexpression that started just after birth was continuous. IL-1 and IL-1 were overexpressed only in the early stage, while IL-6 production increased along with the progression of interstitial pneumonitis, suggesting that IL-6 contributed to the development of the disease.20 Based on our evidence that TNF- is an instigator of such diseases and that green tea has inhibitory effects on TNF- production, we hypothesized that green tea could prevent development of the diseases. To examine our hypothesis, TNF- transgenic mice were given 0.1% green tea extract in drinking water from 10 days before birth to 4 months old. After treatment with green tea extract, protein levels of TNF- and IL-6 were reduced by 70% and 80%, respectively (FIG. 1). The levels of mRNA expression of these cytokines were similar to those of protein levels, suggesting that green tea regulates the cytokine expression in transcriptional machinery. Since the level of SP-C mRNA did not change with treatment of green tea, the transcriptional regulation was performed in the endogenous promoter of either TNF- or IL-6 genes (data not shown). The animal model experiment indicates that green tea has preventive effects not only on development of cancer but also on that of other TNF--related diseases.

PREVENTIVE EFFECTS OF GREEN TEA ON CANCER AND LIFESTYLE-RELATED DISEASES ELUCIDATED BY EPIDEMIOLOGICAL STUDIES Along with the results of animal experiments, epidemiological studies have confirmed the preventive effects of green tea on cancer and other lifestyle-related dis-

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FIGURE 2. Effect of green tea on mean age of death from all causes. In the 11-year follow-up study, death from all causes was surveyed by death certificate; a total of 1,109 deaths from all causes was used in the analysis of the mean ages of death.

eases in humans. We conducted a prospective cohort study with 8,552 individuals in Yoshimi town in Saitama Prefecture beginning in 1986.21 During an 11-year followup, a total of 488 cancer patients (285 males and 203 females) were determined. Respondents were divided into three groups according to daily consumption of green tea: below 3 cups, from 4 to 9 cups, and over 10 cups. Individuals who consumed over 10 cups of green tea per day showed remarkable reduction of relative risk for lung, colon, and liver cancers;21 the relative risk of stomach cancer was also low, although not statistically significant. In addition to the cancer preventive effects, our data showed that increased green tea consumption was associated with decreased serum total cholesterol, decreased triglyceride levels, and decreased atherogenic index. Furthermore, both cardiovascular disease prevalence rate and diabetes mellitus prevalence rate were significantly lower among the population consuming over 10 cups of green tea per day.22 Since green tea apparently helped to prevent cancer, cardiovascular disease, and diabetes mellitus, all major lifestyle-related diseases, mean age at death from all causes was compared by consumption levels of green tea. We found a higher mean age at death among men with increased consumption of green tea. Among women, the mean age at death of the population consuming over 10 cups per day was 81, which was 6 years later than that of those consuming less than 3 cups. The same tendency among both sexes (FIG. 2),23 so these data clearly indicate that green tea reduces the risk of lifestyle-related diseases, resulting in a longer life span. In summary, green tea, an acknowledged cancer preventive, may also prevent other lifestyle-related diseases, and other TNF--related diseases. We know that green tea is nontoxic, and the inference now seems clear: for a longer, healthier life, drink at least 10 cups (about 2.0 g green tea extract) every day.24

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