Mechanistic Studies on the Activities of EGCG in Cell Lines

Mechanistic Studies on the Activities of EGCG in Cell Lines

In general, if an effect can be observed in vitro at concentrations lower or similar to those observed in vivo, then the event may occur in vivo. Inhibition of telomerase and matrix metalloproteinases has been demonstrated with rather low concentrations of EGCG (IC₅₀ in the range of 0.5–1 μM). EGCG has also been found to bind to 67-kDa laminin receptor, Bcl-2, vimentin, and glucose-regulated protein 78 with high affinity [50-53]. However, there are big differences between the effective concentrations determined with pure enzymes and those in cell lines or tissues, possibly due to the nonspecific binding of EGCG to many proteins and the limited amount of EGCG that can enter the cells. When a small amount of pure enzyme is used in an enzymatic assay, inhibition may be observed with nanomolar concentrations of EGCG, but it may take much higher concentrations of EGCG to inhibit the activity in cell lines or tissues. This point is illustrated in the inhibition of 20 s proteasome chymotryptic activities by EGCG; i.e. the IC₅₀ observed in a cell-free system was 0.1–0.2 μM, but it was 1–40 μM in tumor cell lines [54]. EGCG was reported to bind to the 67-kDa laminin receptor with a K_d of 0.04 μM, to vimentin with a K_d of 3.3 nM, and interact with Bcl-2 with a K_i of 0.33 μM [50-52]. In all these studies, there were experiments demonstrating the biological relevance of the effects in their specific experimental systems, but it required much higher concentrations of EGCG to cause growth inhibition and induce apoptosis. The general applicability of these mechanisms for cancer prevention is still not known.

Another concern in the use of redox-sensitive compounds in a cell culture system is the oxidation and stability of the compound. When added to the cell culture medium, EGCG is oxidized to produce superoxide radical and H₂O₂ [55]. We have demonstrated that, depending on the cell lines and culture conditions, the EGCG-induced apoptosis can be completely or partially blocked by the addition of catalase in the culture medium, suggesting that the apoptosis is mediated by H₂O₂ [49]. Autooxidation of EGCG generated reactive species may inactivate epidermal growth factor receptor in cells in culture [55]. In a recent study on EGCG-induced gene expression changes using DNA microarrays [56], we found that the suppression of gene expression of the bone morphogenic protein-signaling pathway by EGCG was not affected by catalase, and was therefore considered to be hydrogen peroxide independent. On the other hand, many gene and cellular pathways, including genes of the transforming growth factor β signaling pathway were hydrogen peroxide dependent [56]. It is not clear whether the EGCG autooxidation-induced effects occur inside animal tissues, because these tissues are endowed with anti-oxidative enzymes and are usually under lower oxygen partial pressure (< 40 mm Hg) than the cell culture medium (152 mm Hg). This point was discussed in our previous publications [49, 55] and reviewed by Khan et al. [57].

Based on the above discussions, we summarize our understanding of the mechanisms of cancer prevention by EGCG as follows: 1) multiple mechanisms are likely to be involved in different experimental systems; 2) some of the proposed mechanisms based on studies in cancer cell lines may not be relevant to cancer prevention; 3) mechanisms of cancer prevention need to be demonstrated in relevant models or human tissues; and 4) many of the observed effects are probably secondary events or downstream events and it is important to identify the direct targets of EGCG action.

Studies on Tea and Human Cancer

The relationship between tea consumption and human cancer risk has been reviewed in many articles. In 1991, the Working Group of the International Agency for Research on Cancer (IARC) reviewed the effects of tea on cancers of different sites and concluded that “there is inadequate evidence for the carcinogenicity in human and experimental animals of tea drinking” [58]. In 1993, Yang and Wang reviewed more than 100 published papers and paid more attention to the possible cancer preventive effects of tea consumption [1]. This review summarized that, while some studies showed a negative association between tea consumption and cancer risk, others showed no association or positive association. It was suggested that the protective effect of tea may depend on the different etiological factors involved in different cancer types and even for the same cancer types in different geographical areas. Similar conclusions have been reached in subsequent reviews [59-62].

In the present article, we reviewed approximately 150 epidemiological studies regarding the association between tea consumption and human cancer risks of the colorectum, lung, stomach, esophagus, breast, kidney, bladder, prostate, ovary, pancreas and other sites. The results are summarized in Table 2.

Number of studies on tea consumption and the risk of human cancers

Colorectal cancer

The relationship between tea consumption and colorectal cancer risk has been the topic of several reviews [63-66]. Most of the reviews concluded that the studies did not provide consistent evidence to support the hypothesis that tea is a chemopreventive agent and that a negative association may be stronger in observational epidemiological studies of rectal cancer than colon cancer [65, 66]. Sun et al. [67] recently performed a meta-analysis on eight published studies with usable data. Reduced risk of colorectal cancer with intake of green tea was found (OR= 0.82, 95% CI=0.69–0.98); however, no association was found with black tea (OR = 0.99, 95% CI = 0.87–1.13).

We prospectively examined the associations between biomarkers of tea consumption and the risk of developing colorectal cancer among a cohort of 18,244 men in Shanghai, China, with 16 years of follow-up. EGC, 4′-O-methyl-EGC (4′-MeEGC) and EC, and their metabolites in baseline urine samples were measured on 162 incident colorectal cancer cases and 806 matched controls. Individuals with high prediagnostic urinary EGC and 4′-MeEGC levels had a lower risk of colon cancer. [68].

Lung cancer

Clark et al. [69] reviewed 15 epidemiological studies of tea consumption and lung cancer and discussed the related bias-producing factors. As with all epidemiological studies on lung cancer, the possible confounding effect of smoking or second hand smoking is a serious problem. In some recent studies, a protective effect of tea consumption against lung cancer was only observed in specific subpopulations. For example, green tea was protective in individuals with the OGG1 Cys (326) allele [70] and nonsmoking women [71], and black tea in nonsmoking women [72, 73]. These results point out the importance of considering genetic polymorphism and specific risk factors in future studies. A phase II chemoprevention trial is currently being conducted by a consortium of cancer centers and universities in Canada and the US in former heavy smokers using PPE [74].

Stomach and esophageal cancers

Fourteen cohort studies and 23 case-control studies have been performed on the relationship between tea drinking and stomach cancer since 1966. Results from early case-control studies on the possible stomach cancer preventive activities of tea encouraged many other studies in this topic. The results from cohort studies, however, have been disappointing. Of the eight cohort studies on green tea, two studies indicated a reduced risk in stomach cancer [75, 76]. Of six studies on black tea, one study showed increased risk of stomach cancer [77]. The other studies showed no association between tea consumption and stomach cancer risk. A nested case-control study in the Shanghai cohort showed that prediagnostic urinary EGC was inversely associated with gastric cancer (OR=0.52, 95% CI = 0.28-0.97) after adjustment for possible confounding factor [75]. In a review by Hoshiyama et al. [78], five out of eight case-control studies showed that tea consumption was associated with a significant risk reduction and two studies showed a non-significant risk reduction. Among six prospective studies, one showed a non-significant risk reduction, but the other five showed no association.

There have been three cohort studies and ten case-control studies on the relationship between tea consumption and esophageal cancer since 1974. The effects of tea consumption on esophageal cancer are inconsistent. The inconsistency can be mostly attributed to the temperature of the tea preparations [79], as consumption of hot food or beverage is known to be a risk factor in esophageal cancer [80]. It was further indicated that the higher the temperature of the tea or beverage, the greater the risk [81].

Breast Cancer

In a meta-analysis that included three cohort and one case-control studies on green tea, Sun et al. [82] found that breast cancer risk was significantly reduced (OR = 0.78, 95% CI = 0.61–0.98) with green tea intake but the risk reduction was weaker in the cohort studies (OR = 0.85, 95% CI =0.66–1.09). Black tea intake was positively associated with breast cancer risk in five cohort studies (OR=1.15, 95% CI=1.02–1.31), but inversely associated with the risk in eight case-control studies (OR = 0.91, 95% CI = 0.84–0.98). In another meta-analysis that included five cohort and two case-control by Seely et al. [83], drinking 5 or more cups of green tea a day showed a non-statistically significant trend towards the prevention of breast cancer.

Wu et al. [84] observed that the breast cancer risk reduction by green tea intake was only found in women with low activity allele of catechol-O-methyltransferase, which may result in increased tea catechin bioavailability. Another study [85] showed that green tea consumption was associated with a reduced risk of breast cancer in women possessing the high activity angiotensin-converting enzyme, but not the low activity enzyme. These studies suggest that the cancer preventive effect of green tea consumption is likely to be affected by genetic polymorphism.

Prostate cancer

The relationship between tea consumption and prostate cancer has been reviewed [86-89] and the possibility that green tea has greater chemopreventive potential than black tea is worth considering [89]. A recent double-blind study by Bettuzzi et al. [90] followed 200 individuals with high-grade prostate intraepithelial neoplasia (PIN) receiving either 600 mg of green tea catechins daily or placebo (100 individuals in each group) for 12 months. Only 3% of the patients in the catechin treatment group developed prostate cancer, whereas the rate of cancer development on the placebo group was 30%. No side or adverse effect was associated with the treatment. These results are very exciting, and the impact would be tremendous if the results could be reproduced in similar trials with larger numbers of subjects.

The above review of the literature indicates that a clear conclusion on the cancer preventive activity by tea consumption in humans cannot be reached. This is true even for specific types of cancer. Studies conducted in Asia, where green tea is consumed frequently, tend to show a beneficial effect on cancer prevention. Protective effects appear to be observed less frequently in European populations where intake of black tea predominates. Overall, more results on preventive effects were found in cancers of the gastrointestinal tract than other types of cancers, and in case-control studies than cohort studies.

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