BioFactors 13 (2000) 67–72
`IOS Press
`
`Original report
`
`67
`
`Mechanisms of cancer prevention by tea
`polyphenols based on inhibition of TNF-α
`expression
`
`Masami Suganuma, Eisaburo Sueoka1, Naoko Sueoka, Sachiko Okabe and Hirota Fujiki∗
`Saitama Cancer Center Research Institute, Ina, Kitaadachi-gun, Saitama 362-0806, Japan
`
`Abstract. Among various biochemical and biological activities of tea polyphenols, we believe inhibition of the expression and
`release of tumor necrosis factor-α (TNF-α) is crucial, since our study with TNF-α-deficient mice has revealed that TNF-α is an
`essential factor in tumor promotion. We found that EGCG dose-dependently inhibited AP-1 and NF-κB activation in BALB/3T3
`cells treated with okadaic acid, resulting in inhibition of TNF-α gene expression. Furthermore, treatment with 0.1% green tea
`extract in drinking water reduced TNF-α gene expression as well as TNF-α protein level in the lung of TNF-α transgenic mice;
`and IL-1β and IL-10 gene expression in the lung was also inhibited by treatment with green tea extract, indicating that green
`tea inhibits both TNF-α and the cytokines induced by TNF-α in organs. We recently found synergistic effects of EGCG and
`cancer preventive agents such as tamoxifen and sulindac, on cancer preventive activity. Taken together, the results show that
`green tea is efficacious as a non-toxic cancer preventive for humans.
`
`Keywords: EGCG, sulindac, tamoxifen, tumor promotion
`
`1. Introduction
`
`Green tea is rapidly being acknowledged as one of the most practical cancer preventives, based on
`results of various rodent carcinogenesis experiments on the inhibitory effects of (−)-epigallocatechin
`gallate (EGCG) and green tea extract, along with results of a prospective cohort study of humans [1–
`3].
`In 1987, we first reported the anti-tumor promoting activity of EGCG, the main constituent of
`green tea, in a two-stage carcinogenesis experiment on mouse skin [4]. Topical applications of EGCG
`before treatment with various tumor promoters – teleocidin, one of the 12-O-tetradecanoylphorbol-
`13-acetate – (TPA)-types, or okadaic acid – inhibited tumor promotion on mouse skin initiated with
`7,12-dimethylbenz(a)anthracene [1]. Based on these results, we next looked at how EGCG inhibits the
`process of tumor promotion. Our study of tumor promotion with okadaic acid had provided strong
`indications that tumor necrosis factor-α (TNF-α) is an endogenous tumor promoter and the essential
`
`1Present address: Saga Medical School, 5-1-1 Nabeshima, Saga 849-0937, Japan.
`∗Correspondence to: Hirota Fujiki, Saitama Cancer Center Research Institute, Ina, Kitaadachi-gun, Saitama 362-0806, Japan.
`Fax: +81 48 722 1739.
`
`0951-6433/00/$8.00  2000 – IOS Press. All rights reserved
`
`FUSTIBAL Ex. 1011
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`cytokine for tumor promotion [5]. Recently we were able to prove our hypothesis using TNF-α-deficient
`(TNF-α−/−) mice, which were established by Lloyd J. Old’s group in 1997 [6,7]: Repeated applications
`of okadaic acid did not induce any tumors in TNF-α −/− mice by 19 weeks of tumor promotion, whereas
`100% of TNF-α+/+ mice developed tumors by week 17. These results clearly demonstrated that TNF-α
`is the key cytokine for tumor promotion in mouse skin. We have also found that EGCG as well as cancer
`preventive agents inhibited TNF-α release from BALB/3T3 cells treated with okadaic acid [8]. Based on
`all of our results, we think that, among various biochemical and biological activities of tea polyphenols,
`inhibition of TNF-α production is crucial. So, we examined inhibitory mechanisms of EGCG on TNF-α
`production.
`Next, inhibitory effects of EGCG on cytokine production in vivo were studied using TNF-α transgenic
`mice. The TNF-α transgenic mice overexpressed TNF-α only in the lung and developed interstitial
`pneumonitis resembling idiopathic pulmonary fibrosis in humans [9]. Treatment with green tea extract
`in drinking water reduced TNF-α mRNA expression and its protein level in the lung of these mice, along
`with reduction of IL-1β and IL-10 gene expression. This is the first evidence that green tea inhibits
`TNF-α production as well as other cytokine production in mouse organs.
`More recently, we found synergistic effects of EGCG and (−)-epicatechin (EC) on cancer preventive
`activity [10]. We extended our investigation of synergistic effects by examining EGCG with other cancer
`preventive agents, such as tamoxifen and sulindac. That is, cotreatment with EGCG and tamoxifen
`or EGCG and sulindac enhanced cancer preventive activity. Tamoxifen is the first cancer preventive
`drug for breast cancer, approved by FDA in the United States in 1998 [11]; and sulindac is a cancer
`preventive agent for colon cancer and a nonsteroidal anti-inflammatory agent [12]. This paper presents
`our recent findings with tea polyphenols and green tea extract looking mainly at inhibition of TNF-α
`gene expression in the cells and in TNF-α transgenic mice. The synergistic effects of EGCG and sulindac
`are also discussed.
`
`2. Materials and methods
`
`2.1. Inhibition of AP-1 and NF-κB activation by EGCG
`
`BALB/3T3 cells were pretreated with EGCG for one hr, and treated with 200 nM okadaic acid for
`another 8 hr. Binding activity of AP-1 and NF-κB in nuclear extract to each consensus sequence of DNA
`was examined by electrophoretic mobility gel-shift assay (EMSA) [13].
`
`2.2. Reduction of TNF-α in the lung of TNF-α transgenic mice by consumption of green tea extract
`
`Transgenic mice carry a chimeric gene consisting of the promoter region of the human surfactant
`protein-C (SP-C) gene and mouse TNF-α gene. Transgenic mice and transgenic negative littermates
`were given 0.1% green tea extract in drinking water from embryo to 4 month-old. Expression of TNF-α,
`IL-1β, and IL-10 genes was examined by reverse transcription (RT)-PCR in the presence of 32P-dCTP [9].
`TNF-α protein level in the lung was determined by enzyme-linked immunosorbent assay (ELISA) kit
`for mouse TNF-α (Genzyme Corporation, USA) [9].
`
`2.3. Induction of apoptosis of PC-9 cells by EGCG and sulindac
`
`PC-9 cells were incubated in sulindac with or without 75 µM EGCG for 2 days. DNA fragmentation
`was quantitatively measured using ELISA kit (Boehringer Mannheim, Mannheim, Germany) [10].
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`69
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`A
`
`B
`
`Fig. 1. Inhibition with EGCG of AP-1 and NF-κB activation induced by okadaic acid. Binding activity in nuclear extracts to
`AP-1 (A) and NF-κB (B) binding sites was examined by EMSA.
`
`5
`
`3
`
`1
`
`0
`
`Fig. 2. Reduction of TNF-α protein level in the lung of TNF-α transgenic mice. Transgenic mice were treated with 0.1% green
`tea extract in drinking water from embryo to 4 month-old. GTE: green tea extract.
`
`Control
`
`GTE
`
`3. Results
`
`3.1. Inhibition of TNF-α gene expression by EGCG
`
`Tea polyphenols, such as EGCG, (−)-epicatechin gallate (ECG) and (−)-epigallocatechin (EGC),
`inhibit TNF-α release from BALB/3T3 cells and KATO III cells treated with okadaic acid [8,13]. TNF-
`α gene expression was enhanced by treatment with okadaic acid in both BALB/3T3 cells and KATO
`III cells, and EGCG dose-dependently inhibited this TNF-α gene expression, just as it inhibited TNF-α
`release. Since TNF-α gene expression is regulated by several transcription factors, such as AP-1 and
`NF-κB, we examined inhibition of AP-1 and NF-κB activation with EGCG using EMSA: 200 nM
`okadaic acid significantly enhanced AP-1 and NF-κB binding to each consensus sequence of DNA
`in BALB/3T3 cells, and pretreatment with 500 µM EGCG clearly inhibited these binding activities, as
`shown in Fig. 1(A) and Fig. 1(B) [13]. These results demonstrated that EGCG inhibits TNF-α production
`by inhibiting AP-1 and NF-κB activation.
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`IL-1 β
`
`TNF- α
`
`I L - 1 0
`
`1500
`
`1000
`
`500
`
`0
`
`Relative expression
`
`Green tea extract
`
`+-
`
`+-
`
`+-
`
`Fig. 3. Effects of EGCG on expression of TNF-α, IL-1β, and IL-10 genes in the lung of TNF-α transgenic mice. Transgenic
`mice were treated with 0.1% green tea extract in drinking water from embryo to 4 month old.
`
`3.2. Reduction of TNF-α in the lung of TNF-α transgenic mice by consumption of green tea extract
`
`TNF-α transgenic mice had enhanced TNF-α gene expression just after birth and continuously over-
`expressed it in the lung. At 4 month-old, TNF-α protein level in the lung of TNF-α transgenic mice
`was significantly reduced, from 3.0 to 2.1 µg/mg protein, by treatment with 0.1% green tea extract in
`drinking water (Fig. 2). This reduction level was well correlated with reduction of TNF-α gene expres-
`sion (Fig. 3). Treatment with green tea extract also reduced expression of IL-1β and IL-10 genes about
`15–20% in the lung (Fig. 3). These results indicated that green tea inhibited TNF-α gene expression as
`well as expression of other cytokine genes induced by TNF-α.
`
`3.3. Enhanced effects of EGCG and tamoxifen or EGCG and sulindac on cancer preventive activity
`
`Based on our discovery that synergistic induction of apoptosis by EC and other tea polyphenols,
`EGCG, ECG and EGC, we examined synergistic effects of tea polyphenols with other cancer preventive
`agents, such as tamoxifen and sulindac. We found that cotreatment with EGCG and tamoxifen inhibited
`growth of MCF-7 cells and induction of apoptosis of PC-9 cells even more strongly than either did alone
`(data not shown) [10]. These results suggest that green tea enhances tamoxifen’s preventive activity
`against breast cancer in humans.
`We next looked at the synergistic effects of EGCG and sulindac or its metabolites, sulindac sulfide and
`sulindac sulfone, on induction of apoptosis of PC-9 cells. Sulindac at a concentration of 10 µM induced
`almost no apoptosis of PC-9 cells, while 10 µM sulindac with 75 µM EGCG induced apoptosis more than
`20 times as strongly as sulindac alone (Fig. 4). Sulindac sulfide, an inhibitor of cyclooxyganase (COX)-1
`
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`71
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`2
`
`1
`
`0
`
`EGCG
`
`Sulindac
`
`Sulindac
` sulfide
`
`Sulindac
` sulfone
`
`Fig. 4. Synergistic induction of apoptosis of PC-9 cells by cotreatment with EGCG and sulindac or its metabolites.
`
`and COX-2, and sulindac sulfone, an inactive metabolite, both synergistically induced apoptosis of the
`cells in the presence of 75 µM EGCG, whereas neither was effective alone (Fig. 4). We therefore believe
`that the synergistic effects on apoptosis by cotreatment with EGCG and sulindac are not directly related
`to inhibition of COX. Clearly, cotreatment with EGCG and sulindac will result in increased cancer
`preventive activity in humans.
`
`4. Discussion
`
`This paper reports that green tea extract in drinking water inhibits TNF-α gene expression and reduces
`TNF-α protein level in the lung of TNF-α transgenic mice. Since treatment with green tea extract did
`not inhibit expression of SP-C gene, we think that green tea inhibits expression of TNF-α gene and other
`cytokine genes subsequently induced by TNF-α through cytokine network. This important evidence
`strongly suggests that administration of green tea extract somehow inhibits TNF-α gene expression,
`resulting in reduction of an endogenous tumor promoter in the lung. We previously reported that 3H-
`EGCG orally administered was distributed into the lung as well [14], so it is reasonable to suppose
`that tea polyphenols reach the lung and inhibit TNF-α gene expression mediated through inhibition of
`AP-1 and NF-κB activation in the lung. It is now known that various cancer preventive agents, such
`as green tea polyphenols, sodium salicylate and curcumin, inhibit AP-1 and NF-κB activation [15–17]:
`Our results here indicate that inhibition of AP-1 and NF-κB activation is a further step toward inhibition
`of TNF-α gene expression.
`The synergistic effects of EGCG and sulindac were significant for cancer preventive activity in cells,
`and we recently demonstrated their synergistic effects on inhibition of tumor formation in C57BL/6J Min
`mice (manuscript in preparation). All our results show that green tea is a promising candidate for use in
`combination with cancer preventive agents such as sulindac and tamoxifen, for the purpose of reducing
`their adverse effects [10].
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`For cancer prevention in Japan, 10 Japanese-size cups of green tea per day, roughly 2.0 to 2.5 g
`green tea extract, is recommended based on the results of a prospective cohort study [3]. For those
`who cannot drink 10 cups per day, we have now prepared green tea tablets in collaboration with the Tea
`Experiment Station in Saitama Prefecture [18]. To determine whether the average person can consume
`tea polyphenols equal to ten cups of green tea per day without any changes in life-style, we conducted
`a trial using green tea tablets with 102 healthy volunteers in Saitama Prefecture. The number of green
`tea tablets was decided by each volunteer based on daily consumption of green tea, and most of the
`volunteers (91%) continued taking the tablets for 3 months, indicating that they can indeed drink the
`amounts of green tea for 10 cups per day.
`In summary, our concept for cancer prevention with green tea is as follows: For the purpose of cancer
`prevention, green tea consumption should be increased from the level for a daily beverage to the amount
`of a cancer preventive beverage: 10 Japanese-size cups of green tea daily is acceptable for most people,
`and will provide protection against cancer.
`
`Acknowledgments
`
`This work was supported by the following Grants-in-Aid: for Cancer Research from the Ministry
`of Health and Welfare, Scientific Research on Priority Areas for Cancer Research from the Ministry
`of Education, Science, Sports and Culture, Japan: for Comprehensive Research on Aging and Health
`from the Ministry of Health and Welfare, Japan: for Selectively Applied and Developed Research from
`Saitama Prefecture, Japan; and by the Smoking Research Fund.
`
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