Cancer of the cervix is the third most common malignancy worldwide in women, and the most common gynecologic cancer in the developing world. In developed countries, prevention of cervical cancer achieved by the widespread and systematic use of cervical cytologic screening, has contributed to the successful decrease in the incidence of invasive cervical carcinomas. In the developing world, cervical cancer remains a common malignancy impacting the lives of women during their period of highest productivity. Especially in low-resource settings, an inexpensive dietary chemo-preventive intervention would be an attractive adjunct to existing cervical cancer prevention programs.
It is well-known that the regular consumption of fruits and vegetables is highly associated with the reduced epidemiologic risk of different types of cancer (18
) and green tea consumption is associated with lowering certain cancer incidences. This has not been demonstrated for cervical cancer specifically.
Polyphenol E (poly E) and (−)-Epigallocatechin gallate (EGCG) are one of the various polyphenols found in green tea. In this study, we investigated the chemopreventive effect of EGCG, the major component of green tea polyphenols, and poly E on the growth of HPV-positive (HeLa, adenocarcinoma and Me180, squamous carcinoma) and HPV-immortalized cervical epithelial (TCL-1) cells. Results showed that EGCG and poly E inhibit the growth of both immortalized TCL-1 cells and cancer cell lines HeLa and Me180. In cancer cell lines, cell growth rate was inhibited by the high concentration of poly E and EGCG (50 µg/ml: 76.9 and 100%) in squamous cell carcinoma cell line, Me180, whereas growth inhibition was less in adenocarcinoma cell lines, HeLa (67.5 and 85.6%). Suggesting that growth inhibition by treatment with green tea polyphenol may be modulated by cellular type.
The EGCG-induced growth inhibition was stronger than that of poly E. Nevertheless, the effect of poly E on cell growth was observed at highest concentrations (25 and 50 µg/ml) than that of EGCG (from 10 µg/ml). Most studies focused only on EGCG which is by far, the most pharmacologically active green tea polyphenol, and our findings are consistent with previous studies reporting the beneficial effects of EGCG on different types of human cancer and cancer cell lines (lung, colon, breast, cervix, etc.) (8
). Our result suggests that green tea poly E at high concentrations may be a potential chemo preventive agent.
In this study, the concentrations of EGCG and poly E: 0; 1; 5; 10; 25 and 50 µg/ml, was consistent with the previous studies showing an inhibition of growth of HPV-18 immortalized cervical cells and cervical cancer cell lines (14
). The dose-dependent effects of EGCG and poly E on growth inhibition were also consistent with previous reports (14
). At present, the physiological extra- and intra-cellular levels of green tea polyphenols are unknown. Even the amount of tea ingestion necessary to obtain noticeable benefits is still under debate. It has been reported that a cup of tea contains approximately 120–150 mg EGCG, which is equivalent to 300 µmoles (9
). For a single cup of tea, Lin et al. (26
) estimated a maximum of 60 µM EGCG in the human blood while Lee et al. (7
) measured a range of 0.2 to 0.65 µM in human blood plasma after consumption of 2–3 cups of green tea. The difference between these data may be attributed to the biodegradability of the ingested tea and polyphenols and/or the delay between ingestion and EGCG measurement. Other studies have suggested amounts of about 10 cups (27
). Our data suggest that a regular and high consumption of green tea may contribute to the prevention of cervical cancer.
Several mechanisms of cancer inhibition by green tea polyphenols have been proposed. We investigated the regulation of apoptosis and cell cycle progression, which could be important targets for cancer chemoprevention (9
). For this purpose, experiments were run one day before the onset of the significant inhibition of cell growth, allowing a better evaluation of events occurring downstream.
The dose-dependent increase of apoptosis in immortalized and cancer cells in the presence of EGCG are in agreement with other reports on human carcinoma cells (11
). The oxidative properties of EGCG in cancer cells have been suggested to explain the increase of apoptosis (34
). Indeed, higher concentrations (100–200 µM) of EGCG appear to be associated with an intracellular production of free radicals whereas low concentrations (10 µM) have anti-oxidative properties (14
). In the present study, 5 µg/ml (equivalent of 10 µM) EGCG induced a significant increase of apoptosis in both immortalized TCL-1 and squamous carcinoma Me180 cell lines, while only 25 and 50 µg/ml (50 or 100 µM) EGCG induced the same significant effect with adenocarcinoma HeLa cells. These results also suggest a differential sensitivity of cervical cancer cell types to EGCG exposure. Some studies have reported an association between the induction of apoptosis and disruption or loss of telomere structure (36
). A loss of telomerase activity has been observed in cervical adenocarcinoma cancer cells (HeLa: 13 and OMC-4/TMCC1) (39
) exposed to the same range of EGCG concentrations (50 µM and/or 100 µM). A similar pattern in apoptosis induction was not observed with poly E treatment, where only a slight increase was found at 50 µg/ml. However in a parallel experiment, we found that the concentration of 100 µ
/ml poly E induced a high proportion of apoptotic cells (data not shown). These data suggest that poly E is less effective than EGCG to induce apoptosis in these cells.
The ability of EGCG to modify cell cycle progression has been reported by many groups (40
), however, the effect of poly E on the cell cycle is still not clear. In the present study, only the effect of poly E (50 µg/ml) on the cell cycle was evaluated. All cell lines showed a modified cell cycle profile, which was characterized by an accumulation of cells in G1
-phase and a decrease of those in S-phase. The same pattern was observed both in immortalized (TCL-1) and cancer (Me180 and HeLa) cells. These results are comparable with the EGCG effect on cell cycle, as reported in the literature. EGCG is known to induce cell cycle arrest at G0
phase, with a perturbation of expression of cell cycle regulatory genes.
Several cell cycle check-point genes are involved in the regulation of cell growth and apoptosis (42
). Here, we evaluated the expression of p53, p27KIP1
proteins on the squamous cervical carcinoma Me180 cells, which were the most sensitive to the lowest concentrations of EGCG. The p53 and p21WAF1/Cip1
protein expression increased in a dose dependent fashion in the presence of EGCG (0; 5; 25 and 50 µg/ml). Similar dose- and time-dependent increases have been observed by Gupta et al. (42
) The up regulation of p53 by EGCG at low concentrations (1 to 5 µg/ml) has been suggested to induce an arrest of mitosis and DNA damage repair, whereas at higher concentrations (25–50 µg/ml), it may act for induction of apoptosis. Many studies have shown that certain exogenous stimuli may result in a p53-dependent or p53-independent induction of p21WAF1/Cip1
, an inhibitor of cyclin dependent kinases (cdks) (45
). This in turn may trigger a series of events such as cell cycle arrest and apoptosis (44
). Therefore, our data showing an increase of p21WAF1/Cip1
by EGCG appears p53-dependent in Me180 cancer cell line with wild type of p53.
The effect of the two green tea compounds on the high-risk HPV E7 oncoprotein expression in cervical immortalized and adenocarcinoma cell lines was also evaluated. The E7 protein is one of the early proteins expressed by the viral genome to ensure its reproduction in a differentiated cell. The binding of E7 to proteins involved in the cell cycle (i.e., pRB, p107RB) lead to the degradation of pRb. The release of its transcription factor E2F allows the cell to enter into the S phase, resulting in uncontrolled cell proliferation (48
). Our results show a down-regulation of E7 expression by both EGCG and poly E, irrespective of the cancer cell lines. However, the mechanism by which these two green tea polyphenol compounds exert their effects is still unknown. The hypothesis of a down-regulation of E7 by p27 protein as observed by Lee et al. (52
) in HeLa cells is unlikely since the p27 protein level was not affected by EGCG treatment. Therefore, the repression of E7 protein level by p27 might not be a general mechanism.