Green Tea in Human Cancer

By Farabegoli, F

Green tea contains active polyphenols called catechins, thought to have beneficial effects on human health including a chemo- preventive action. The present review will summarize epidemiologic in vivo and in vitro studies that support this hypothesis. Epidemiologic studies, mostly conducted in Asia, demonstrated green tea had a chemo-preventive effect on prostate, ovary and breast cancer. In contrast, conflicting data have been published about gastrointestinal, lung and bladder cancer. Before achieving conclusive evidences, similar studies need to be also undertaken in Western countries and evaluated taking into an account of differences in genetic background, exposure to environmental agents, alimentation and style of life. In animals, where these differences are limited, there is general agreement that green tea catechins were able to limit and delay tumor development in many organs and tissues (oral mucosa, esophagus, stomach, intestine, pancreas, liver, bladder, lung and breast) after exposure to numerous carcinogens. Green tea catechins were also demonstrated to reduce metastasis development after cancer cells inoculation animals (nude and nonobese diabetic/severe combined inimunodeficient NOD/SCID mice) or in animal models spontaneously developing metastasis. In vitro studies demonstrated that green tea catechins selectively killed cancer cells at doses comparable to those present in the human body of a usual drinker. Although the molecular targets of green tea catechins are far to be fully disclosed, NF-kB, p53, PI3K/ Akt, EGF/Her-2 pathways and downstream molecules involved in cell cycle, apoptosis, adhesion, invasion, angiogenesis and metastasis have been demonstrated to be affected by EGCG treatment. Green tea is cheap, widely diffuse and has a few toxic side-effects: these characteristics and the evidences so far achieved make it as an ideal candidate for studies aimed to better define its anti-cancer action, for a rational use in human health.

KEY WORDS: Green tea * Catechins * Chemo-prevention * In vivo animal studies * In vitro.

Green tea catechins

Tea, derived from Camellia sinensis, is one of the most widely consumed beverages in different parts of the world. Black tea is consumed primarily in Western countries and in some Asian countries: it accounts 78% of the total amount of tea produced and consumed in the world. Green tea (20%) is consumed prevalently in China, Japan, India, and in a few countries in North Africa and Middle East. Oolong tea production and consumption is confined to South-Eastern China and Taiwan. Tea is rich in polyphenols, molecules known for their antioxidant and anti-inflammatory properties, able to protect with respect to various human diseases.1 Among the polyphenols, catechins show the most relevant therapeutic properties and are maximally conserved in green tea with respect to black and oolong tea. Green tea leaves are not fermented but steamed: fermentation converts catechins to theaflavins and thearubigins, consequently decreasing the catechin content. Black tea leaves are fermented whereas oolong tea leaves are partially fermented. For this reason green tea contains polyphenolic compounds that account for up 30% of its dry weight. Several molecules have been identified as active principle in green tea: epigallocatechin-3-gallate (EGCG, the major polyphenol molecule with potent antioxidant and chemo-preventive effect), epicatechin (EC), epicatechin gallate (ECG) and catechin gallate (CG), which are also involved in protective effect on human health. Green tea is very popular in Japan and China, and the first studies about beneficial effects on human health come from Japan. Green tea consume has been found to be protective on cardiovascular diseases (particularly heart diseases and stroke), to have control of obesity, atherosclerosis and diabetesinduced vascular damage, to be effective against infections (bacterial and viral), and particularly, to be effective as a chemo-preventive agent in human cancer. The concept of use of tea for promotion of human health and prevention and cure of diseases2 has become a subject of intense research in the last decade, as demonstrated by the progressive increase in the number of studies dedicated to the properties of these molecules, investigated as green tea beverage, green tea extract (GTE) or as single molecules (mostly EGCG). The relative low toxicity and availability of green tea make it as an ideal compound to this aim. Limited side effects documented include irritability, insomnia and tachycardia, all of them due to the caffeine present in green tea. Numerous studies investigated the most relevant effects of green tea as chemo-preventive agent and the importance of green tea consume in human cancer.

Green tea as chemo-preventive agent: epidemiologic evidences

The first study on chemo-preventive action of green tea came from Japan, where green tea assumption is a daily habit.3 On 1986, Nakachi and Imai conducted a prospective study with 8 552 individuals that were interviewed about their living habits, including daily consumption of green tea. Death for cancer during the following 9 years were registered and compared on the basis of green tea consumption, evaluated as number of cups a day. The authors found a negative association between green tea consumption and cancer incidence, especially among females drinking more than 10 cups a day. Ten cups a day correspond to 0.8-1.3 g GTE a day, and this dose was considered to be effective. The effect was evident for colon, liver and lung cancer, but not for stomach cancer.4 Following studies reported conflicting results on stomach cancer: a protective effect on stomach and colorectal carcinoma was found by Inoue 5 while a decreased risk of gastric cancer was observed only among women affected by tumor in the distal portion.6 No association was indeed found by two studies in Japan.7,8 The question is still open, although it is known that antioxidants have a protective effect on cancer developing in gastrointestinal tract and green tea has been demonstrated to scavenge reactive nitrogen species derived from acidic nitrite, known to be cancerogenetic in the stomach.9 A chemo- preventive effect on oesophageal10 and both pancreatic and colorectal cancer11 was found in Shangai population. Catechins intake was inversely associated with rectal cancer incidence in a cohort of 34 651 postmenopausal cancer-free women aged 55-69 years followed from 1986 to 1998.12 In contrast, other studies did not confirm these findings: critical analysis of 21 epidemiological investigations about green tea as a chemo-preventive agent in gastrointestinal cancer only found a protective effect on adenomatous polyps and chronic atrophie gastritis formations, but not on stomach and intestinal cancer.13 It has been suggested that many other factors (diets, alcohol consumption, smoking, type of tea, total quantity of tea consumption, interactions of tea with other dietary factors) may have influenced the results obtained in these studies and may explain the discrepancies found.

Even more conflicting data concern lung cancer: epidemiologic evidences regarding the association between the consumption of green tea and lung cancer are limited, although experimental studies have shown that tea preparations and tea polyphenols may inhibit lung cancer development in animal models. Consumption of green tea was associated with a reduced risk of lung cancer among non smoking women in a study conducted using the population-based Shanghai Cancer Registry.14 Nagano et al. failed to find any inverse correlation between green tea intake and lung and other human cancer.15 Indeed, high consumption of green tea was found to be a risk factor for developing lung cancer.16 Studies including larger number of individuals and a detailed and critical inclusion of parameters concerning style of life, diet and environmental factors are mandatory for deciphering the real impact of high consumption of green tea and lung cancer.

A few studies deals with bladder cancer and green tea: tea consumption was found to increase bladder cancer risk in Southern Taiwan 17 and Japan,18 no prognostic significance was reached in another study, whereas a reduced risk was suggested for the intake of black tea and matcha (powdered green tea) in females, and of fruit juice in males, in the population of Metropolitan Nagoya.1?

Green tea polyphenols are included in the list of promising bioactive food components being investigated at the National Cancer Institute in prevention clinical trials to reduce breast cancer risk, together with indole-3-carbinol, sulforaphanes, phytoestrogen isofiavones, perillyl alcohol.20 Epidemiologic evidences showed a significantly reduced risk of breast cancer in Chinese, Japanese and Filipino women in Los Angeles County: the protective effect was maintained after adjusting for age, specific Asian ethnicity, birthplace, age at menarche, parity, menopausal status, use of menopausal hormones, body size and intake of total calories and black tea and remained after further adjustment for other potential confounders, including smoking, alcohol, coffee and black tea intake, family history of breast cancer, physical activity and intake of soy and dark green vegetables.21 As catechol-containing tea polyphenols are very rapidly O-methylated by human catechol-O- methyltransferase (COMT), the effect of green tea was strongest among persons wh\o had the lowest activity COMT alleles, suggesting these individuals were less efficient in eliminating tea catechins and may derive the most benefit from these compounds.22 Breast cancer patients at stage I and II, consuming over 5 cups of green tea a day, showed a lower recurrence rate and a lower number of axillary lymph nodes metastasis. The difference was not statistically significant for patients at stage III. This suggested that green tea was more effective in early stages of the diseases and able to reduce recurrence.2325 A potential explanation of this effect on breast cancer might be due to the relation between green tea intake and estrogen circulating levels: in a study of healthy postmenopausal Chinese women in Singapore plasma estrone levels were 13% lower in regular green-tea drinkers and 19% higher in regular black tea drinkers with respect to non-or irregular tea drinkers. One study only, a pooled analysis of two prospective studies with 35 004 Japanese women, did not find green tea intake associated with a lower risk of breast cancer.26

Ovarian cancer risk declined with increasing frequency and duration of overall tea consumption. Tea consumption, evaluated post- diagnosis, enhanced survival in a cohort of 254 patients with epithelial ovarian cancer followed up for a minimum of 3 years, in Hangzhou, China. At least 2 g of dried green tea a day had to be consumed to have better outcome.27

Prostate Cancer Prevention Trial (PCPT) took into consideration green tea as a potential chemo-preventive agent: other substances included selenium, vitamin E, vitamin D, 5-oc-reductase inhibitors, cyclooxygenase-2 inhibitors and lycopene. Some of these agents are being tested in new large-scale phase III clinical trials.28 Geographical observations suggest that the incidence of prostate carcinoma is lower in Japanese and Chinese populations that consume green tea regularly. The prostate cancer risk declined with increasing frequency, duration and quantity of green tea consumption in South-west China.29 A decrease in risk was observed with tea intake of more than 500 mg per day in a study conducted in 3 geographical areas of Canada, but with no distinction between black and green tea.30 Finally, limited antineoplastic activity, as defined by a decline in prostate specific antigen (PSA) levels, was found in patients with androgen independent prostate carcinoma in a phase II study.3i Pooling together these different studies, it seems that endocrine-related cancer (breast, ovary and prostate) might benefit from green tea effects, whereas lung and gastrointestinal carcinoma require additional studies in order to define whether green tea intake may have a real protective effect. Due to the influence of dietary factors on the development of gastrointestinal and lung cancer, the epidemiologic analysis can be very complex and require a very high number of individuals. The limited number of green tea drinkers in Western countries also restrains the application of epidemiologic studies. Convincing evidences come from studies conducted on Asian individuals settled in Western countries and therefore exposed to the same environmental factors of non- Asian people. This could be helpful to define the relative contribute of environmental and genetic component to the final effect.

Green tea chemo-preventive effect on animal models

Experimental evidences of a chemo-preventive effect of green tea on animal models are numerous. Chung et al.32 and Crespy et al.33 published two very detailed reviews about the protective effect of catechins, investigated as green tea, GTE and EGCG in animal models (hamster, mouse and rat). Both the reviews concluded that green tea catechins tea have been shown to block tumorigenesis in at least one of the multiple stages, although some bioassays did not distinguish the stages (initiation, promotion and progression) in which tumorigenesis was inhibited by tea.

Green tea polyphenol supplementation was found to enhance the cellular thiol status thereby mitigate oral cancer in 4- Nitroquinolinel-oxide-(4-NQO) induced rats.34 In azoxymethane (AOM)- induced colon carcinogenesis in mice on a high corn oil diet, green tea inhibited colon tumors, suggesting its possible inhibitory effect on colon carcinogenesis in populations consuming high amounts of fat.35 In two different mouse models of intestinal tumorigenesis, the data support a chemo-preventive role for tea and sulindac against intermediate and late stages of colon cancer, via effects on the (catenin/Tcf signaling pathway.36 The protective effect of green tea on lung cancer development was demonstrated after administration of 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone) (NKK) by two different studies: treatment with 0.6% tea preparation significantly reduced lung tumor multiplicity induced in female A/J mice.37 In F344 rat, a relevant model for lung cancer in smokers, 2% green tea inhibited NNK-induced lung tumors per mouse by (45% as compared with the NNK group given only water. EGCG appeared to be the major active ingredient in green tea because it reduced lung tumor multiplicity by 30%.32 Co-administration of 2% GT was able to prevent increase of incidences and multiplicities of pentachlorophenol and diethylnitrosamine-induced hepatocellular tumors and also to arrest the progression of cholangiocellular tumors.38 Crude extracts of GT or components of GT, such as polyphenols, are known to have des- mutagenic and anti-carcinogenic effects against benzo-a-pyrene, aflatoxin Bl, 3-methylcholanthrene (MC). GT may work to bio- transform CYPlA-inducing carcinogens (i.e. polycyclic aromatic hydrocarbons PAHs, and aryl or heterocyclic amines) into non- carcinogenic metabolites by modulation of other microsomal enzymes than CYPlAs whereas the selective inhibition of CYPlAs may reduce the initiation. 39 in a N-Nitrosobis(2-oxopropyl)amine (BOP) model, 0.1% aqueous solution green tea catechins was found to reduce the formation of 8-oxo-2′-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, in nuclear DNA of hamster liver, but not in pancreas.40

Beside carcinogenesis, animal models have also been used to investigate the anti-metastatic potential of green tea in vivo. In NOD/SCID mice transplanted intraperitoneally with three human non- Hodgkin’s lymphoma cell lines (Namalwa, RAPl-EIO and HS-Sultan) green tea prevented 50% of Namalwa tumors and significantly inhibited RAPl-EIO and HS-Sultan tumor growth.41 The autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP) model, which spontaneously develops metastatic prostate carcinoma, is a model that mimics progressive forms of human disease. Using male TRAMP mice, oral infusion of a polyphenolic fraction isolated from green tea (at a dose equivalent to 6 cups of green tea per day) significantly inhibited prostate carcinoma development and increases survival in these mice.42 The oral administration of theanine or green tea enhanced the antitumor activity of doxorubicin (DOX) in M5076 ovarian sarcoma-bearing mice and increased the DOX concentration in the tumour.43 p53 heterozygous knockout mice and p53 transgenic mice carrying a dominant negative mutant were crossed with the A/J mouse, which is highly susceptible to lung tumor induction. Mice treated with dexamethasone/myoinositol and green tea displayed an average of 70% and 50% inhibition of lung tumors, respectively, regardless of p53 status.44 The per-oral administration of green tea infusion reduced the number of lung colonies of mouse Lewis lung carcinoma cells in a spontaneous metastasis system.45 In animal experiments, EGCG alone reduced lung metastases in mice bearing Bl6-F3m melanomas. However, a combination of EGCG and dacarbazine was more effective than EGCG alone in reducing the number of pulmonary metastases and primary tumor growths, and increased the survival rate of melanoma-bearing mice.46 The immortalized KS cell line KS-IMM forms highly angiogenic tumors when injected in male nude mice: treatment with EGCG or green tea extract reduced the tumor growth as compared with controls receiving water alone. All treated mice developed slowly growing tumors, limited in size, whereas 90% control animals had larger tumors. Mice treated with EGCG showed a 50% reduction in tumor size; interestingly, the reduction was even more evident in animals treated with green tea extract.47 Investigation on the molecular mechanisms triggered by EGCG showed that TRAMP-Cl cell proliferation was restrained in a dose-dependent manner and the inflammatory involvement of prostate carcinoma was lowered, then reducing the invasive potential.48

Molecular targets of green tea catechins: in vitro studies

In vitro studies provided evidences on the most important biologic effects occurring in cancer cells after green tea catechins treatment: cell growth arrest and cell death by apoptosis, inhibition of adhesion, invasion, migration and metastasis. Numerous molecules, known to be responsible of cancer cell altered functions, have been demonstrated to be target of green tea catechins. Although the exact mechanism of action is far to be disclosed, these findings also support the hypothesis green tea catechins have chemopreventive activity.

Cell cycle arrest and apoptosis

Apoptosis was demonstrated to occur in numerous human and mouse cancer cells after in vitro treatment of EGCG.49’57 The effect was cancer-specific, as shown in the human colorectal cancer cell line Caco2 and in the breast cancer cell line Hs578T: 40 M EGCG completely inhibited cancer cell growth without affecting the normal counterpart.58 Selective effect was also demonstrated on normal mammary cells with respect to cancer cells.59 Apoptosis followed cell cycle arrest6o and was associated with dose-dependent increase of p53, induction of cyclin kinase inhibitor WAFl/p21, p27 and other Cdk inhibitors.52,61-6? Growth arrest followed by apoptosis appears as cons\equence of a complex interplay of factors, which can be elicited in different cell lines, having different genetic alterations and biologic features. Several molecules have been focused as important actors of this process. EGCG caused decrease in the cyclin Dl protein, increase in the p21(Cipl) and p27(Kipl) proteins, and a reduction in the hyperphosphorylated form of pRB, changes that may account for the arrest in G(I). EGCG also caused a decrease in Bcl-2 and BcI-X(L) proteins, an increase in Bax protein, and activation of caspase 9, suggesting that EGCG induces apoptosis via a mitochondrial pathway.68, 69 EGCG was found to inhibit NF-kB, with significant activation of caspases and consequent apoptosis.3,62,70,71 Depolarization of mitochondrial membranes and mitochondria damage were found in pancreatic cancer cells 72 and HT- 29 human colon cancer cells.73 p53 does not seem to be the primary molecular target, although it can be actively involved.74 In HepG2 cells the antiproliferative activity of EGCG was related to the induction of p53 and the activity of the Fas/FasL apoptotic system.75 In human prostate carcinoma LNCaP cells, EGCG-induced apoptosis was mediated by modulation of two related pathways: a) stabilization of p53 by phosphorylation on critical serine residues and pl4ARF-mediated downregulation of murine double minute 2 (MDM2) protein, and b) negative regulation of NF-kB activity, thereby decreasing the expression of the proapoptotic protein Bcl-2.76 Using isogenic cell lines and siRNA, EGCG activated growth arrest and apoptosis primarily via a p53-dependent pathway that involved the function of both p21 and Bax.77

Increased p53, p21(WAF-l), and p27(KIP-l) levels, reduced cyclin E level and CDK2 kinase activity also followed epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) pathway inactivation. EGCG selectively inhibited multiple EGFdependent kinases and EGFR-dependent activation of the mitogen-activated protein kinases ERK1/2. EGCG also inhibited EGFR-dependent Akt activity. EGCG directly inhibited ERK1/2 and Akt, and the inhibition was selective, since EGCG did not affect the EGFR-dependent activation of Jun N-terminal kinase (JNK).78 EGCG inhibited mouse mammary tumor virus-Her-2/neu NF639 cell growth in culture and soft agar. EGCG reduced signaling via the phosphatidylinositol 3-kinase (PIK3), Akt kinase and NF-kB pathway because of inhibition of basal Her-2/neu receptor tyrosine phosphorylation.7? In human head and neck squamous cell cancer (HNSCC) and breast carcinoma cell lines, which display constitutive activation of HER-2, 10-30 g of EGCG caused 50% inhibition of growth and marked inhibition of HER-2 phosphorylation in both cell lines, inhibition of Stat3 activation, inhibition of c-fos and cyclin Dl promoter activity, and decreased cellular levels of the cyclin Dl and Bel-XL proteins.80

This body of evidences strongly suggests that several pathways, known to be altered in cancer cells, are target of EGCG action: NF- kB, p53, PI3K/Akt, EGF/Her-2 and downstream molecules involved in cell cycle and apoptosis regulation. Other molecules were found to be affected by green tea catechins and supposed to have a role in EGCG mechanism of action, but validation of these data is necessary to achieve conclusive evidence. Green tea components induced apoptosis via a TGF-β superfamily protein, non-steroidal anti- inflammatory drug activated gene (NAG-1), in a p53-independent manner.81 EGCG inhibited telomerase activity (40-55%) leading to the suppression of cell viability,82 and topoisomerase I, but not topoisomerase II.8^ Production of H^sub 2^O^sub 2^ was thought to mediate apoptosis and contribute to the growth inhibitory activities of tea polyphenols, since apoptosis was completely inhibited by exogenousIy added catalase.84 EGCG, ECG and EGC were found to compete with ^sup 3^H-β-Estradiol for binding to estrogen receptor (ER) α and β and to elicit reporter gene activity in MCF-7 cells. Green tea catechins show a molecular structure potentially able to interact with ER, but only EGCG was found to elicit ER-mediated gene expression in vitro.85 This effect was dose- related: EGCG was anti-estrogenic at high doses but co-estrogenic at low dosed for ERa and β.86 As estrogen exposition is considered a risk factor in breast cancer development, and ERα is a target of currently used breast cancer therapy (tamoxifen), the relation between EGCG and ER ought to be better investigated.

Adhesion

EGCG and ECG in the culture medium were found to inhibit cell adhesion: affinity chromatography revealed a binding between laminin and EGCG. By using surface plasmon resonance, EGCG was demonstrated to bind to the 67-kDa laminin receptor. Expression of the metastasis- associated 67-kDa lamina receptor confers EGCG responsiveness to cancer cells at physiologically relevant concentrations.87 EGCG inhibited phosphorylation of vimentin at serines 50 and 55, and phosphorylation of vimentin by Cdc2 and PKA. EGCG specifically inhibited cell proliferation by binding to vimentin. Because vimentin concurs to maintain cellular functions important in the structure and mechanical integration of the cellular space, the inhibitory effect of EGCG on vimentin may further explain the anti- tumor-promoting effect of EGCG.88 GTE exerts an effect on cytoskeletal actin remodeling. Alteration of actin polymerization and loss of actin filaments is a marker of cellular dedifferentiation and early malignant transformation. GTE antagonized carcinogen 4-ABP induced actin depolymerization and stress fiber disruption in human uroepithelial cells. In MC-TIl cells, GTE inhibited 4-ABP induced motility by increasing cell adhesion and focal adhesion complex formation. The effect of GTE on actin remodeling seems to be mediated by the stimulation of small guanosine triphosphatebinding protein Rho activity.89 In the medulloblastoma cell line DAOY, EGCG could antagonize cell migration on collagen by increasing cell adhesive ability through specific gene and protein upregulation of the β-l integrin subunit.?0

Angiogenesis

Many studies indicated that the consumption of green tea is associated with a reduced risk of developing certain forms of cancer and angiogenesis. The mechanism of inhibition of angiogenesis by green tea, however, has not been well-established. The cadherin- catenin adhesion system is implicated in cell recognition, differentiation, growth and migration of capillary endothelium. Vascular endothelial (VE)-cadherin and Akt, known downstream proteins in vascular endothelial growth factor (VEGF) receptor 2 (VEGFR-2)-mediated cascade, are proteins by which green tea catechins inhibit angiogenesis. Using tube formation of human microvascular endothelial cells in culture as an in vitro model of angiogenesis, VEGF-induced tube formation was inhibited by anti-VE- cadherin antibody and by green tea catechins dose-dependently. The effect was in part mediated through suppression of VE-cadherin tyrosine phosphorylation and inhibition of Akt activation during VEGF-induced tube formation.91 GTE or EGCG 40 g/ml could decrease the level of the angiogenic factor basic fibroblast growth factor in both human umbilical vein endothelial cells and in human breast cancer cells MDA-MB-231: this effect was dose dependent.92 Physiological concentrations (0.01-1 M) of EGCG, CG, and, to a lesser extent, ECG, induced a rapid and potent inhibition of VEGF- dependent tyrosine phosphorylation of VEGFR-2. The inhibition of VEGFR-2 by EGCG was similar to that induced by Semaxanib (SU5416), a specific VEGFR-2 inhibitor.?3 EGCG inhibited anchorage-independent Kaposi sarcoma cell growth and endothelial cell growth, chemotaxis, and invasion over a range of doses. EGCG inhibited the metalloprotease-mediated gelatinolytic activity produced by endothelial cell supernatants and the formation of new capillary- like structures in vitro.94 EGCG inhibited activation of EGFR and related signaling pathways, which is associated with angiogenesis. EGCG inhibited VEGF production by inhibiting both the constitutive activation of Stat3 and NF-kB in YCU-H891, HNSCC and MDA-MB-231 breast carcinoma cell lines. All of them display autocrine activation of transforming growth factor-α (TGF-α)XEGFR signaling and produce high levels of VEGF. EGCG also inhibited the constitutive activation of NF-kB in all cell lines.95

Invasion and metastasis

Human cancer needs proteolytic enzymes to invade cells and form metastases. One of these enzymes is urokinase CuPA). Inhibition of uPA can decrease tumor size or even cause complete remission of cancers in mice. The known uPA inhibitors are unlikely to be used in anticancer therapy because of their weak inhibitory activity or high toxicity. EGCG binds to uPA, blocking His 57 and Ser 195 of the uPA catalytic triad and extending towards Arg 35 from a positively charged loop of uPA. Such localization of EGCG would interfere with the ability of uPA to recognize its substrates and inhibit enzyme activity. 96 EGCG could regulate the uPA expression by at least two different mechanisms: EGCG may inhibit the ERK-1/2 and P38 MAPK, leading to suppression of the uPA promoter activity, and EGCG may destabilize the uPA mRNA in an ERK-1/2- and p38 MAPK-independent way.97 Overexpression of MMPs has been known to correlate closely with tumor cell invasion. Gelatin zymography showed that cancer cells secreted MMPs, probably including MMP-2 and MMP-9, which may be involved in tumor cell invasion and metastasis.?8 EGCG is a potent inhibitor of MMP-2 and MMP-9. EGCG concentration effective in inhibiting MMP-2 and MMP-9 was 1/500 lower than reported for uPA and could reduce tumor cell invasion by 50%. EGCG is a potent inhibitor of gelatinases: EGCG forms a reversible complex with MMP-2, resulting in the inhibition of gelatinolytic activity of MMP-2.99 Dose-dependent inhibition of \MTl-MMP (membrane-type 1 MMP, the receptor/activator of MMP-2) was demonstrated by a solution fluorimetric assay with activated MTl-MMP and gelatin-zymography at EGCG concentrations slightly lower than reported to inhibit MMP-2 and MMP-9.100 Suppression of ERK phosphorylation by EGCG was involved in the inhibition of expression for MMP-2 and MMP-9 mRNAs, leading to the reduction of their enzyme activities in cancer cells.101 EGCG was found to inhibit all the PSA activities in a dose- dependent manner: PSA is a serine-protease able to cleave extracellular matrix glycoproteins, thereby affecting cell migration and metastasis. In addition, PSA showed degradation of gelatin, degradation of type IV collagen in reconstituted basement membrane (Matrigel) and activation of progelatinase A (MMP-2), but not pro- MMP-9, at concentrations lower than the cytotoxic serine-protease inhibitor PMSF and close to levels measured in the serum following ingestion of green tea EGCG.102

Proteasome inhibition

A new green tea catechins function is emerging: ester bond- containing tea polyphenols, such as EGCG, potently and specifically inhibited the chymotrypsinlike activity of the proteasome in vitro and in vivo (1-10 M) at the concentrations found in the serum of green tea drinkers. Atomic orbital energy analyses and high performance liquid chromatography suggest that the carbon of the polyphenol ester bond is essential for targeting, thereby inhibiting the proteasome in cancer cells. This inhibition of the proteasome by EGCG in several tumor and transformed cell lines resulted in the accumulation of two natural proteasome substrates, p27(Kipl) and IkB- α, an inhibitor of transcription factor NF-kB, followed by growth arrest in the G(I) phase of the cell cycle. Proteasome is a cancer-related molecular target of tea polyphenols: inhibition of the proteasome activity by ester bondcontaining polyphenols may contribute to the cancer-preventive effect of tea.103 A new promising class of synthetic inhibitors of proteasome activity, analogous to green tea catechins, is now under investigation.104

Conclusions

Green tea, like other diet-derived compounds, represents a potentially interesting anticancer product, because it is widely available and cheap, non-toxic and it produces a few side-effects. The evidences obtained by epidemiologic, in vivo and in vitro studies, support the hypothesis that green tea exerts its action by mechanisms that link together inflammation, oxidative stress and cancer. Green tea only kills cancer cells, probably limiting those mechanisms that enable cancer to survive. In spite of these encouraging data, further evidences need to be achieved before considering green tea as a real chemo-preventive and anticancer agent. Epidemiologic studies ought to be extended in Western countries, encouraging people to assume controlled doses of green tea as a habit. This only could enable a serious comparison with epidemiologic studies undertaken in Eastern countries and to define whether green tea, assumed by a population with different genetic background, style of life, habits, alimentation, exposed to different environment agents, can retain the same chemo-preventive effect. The effect of green tea ought to be investigated in selected groups of individuals genetically predisposed to cancer, in cancer patients at an early stage of disease, in patients that, for any reason, could not have been treated after surgery and in patients treated for cancer. The most difficult part of the epidemiologic analysis on human individuals is the interaction with other substances, particularly assumed as aliments: the combination with other substances might be important to achieve the final result and ought to receive the adequate attention.

The main limitation on in vitro models is that cell cultures are only in part representative of the in vivo situation. The complexity of metabolism in human body and the interplay among green tea metabolites and many unknown factors cannot be simply summarized by cell lines and in vitro experiments. Furthermore, many studies on cell lines have been undertaken using the most active principle (EGCG) and not whole green tea infusion. This aspect could not be secondary, as green tea infusion contains other active principles, the combination of which could change or be important in achieving the final effect. In spite of these limitations, in vitro studies are the most powerful instrument we have presently to decipher the molecular mechanisms that makes green tea as an anti-inflammatory, anti-oxidant and anti-cancer agent. As demonstrated for many other anti-cancer agents, in vitro studies represent the base for any further development. As green tea is almost completely devoid of dangerous and toxic side-effects (a characteristic that, hopefully, any drug used for promoting human health ought to have) its use and study is encouraged.

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F. FARABEGOLI

Department of Experimental Pathology

University of Bologna, Bologna, Italy

This work was supported by RFO, University of Bologna funding for selected projects, Pallotti Legacy for Cancer Research.

Address reprint requests to: Dr. F. Farabegoli, Dipartimento di Patologia Sperimentale, Via S. Giacomo 14, 40126 Bologna, Italy. E- mail: frbfl@alma.unibo.it

Copyright Edizioni Minerva Medica Sep 2005