| Tea, made from the leaves of evergreen shrub tea plant, is a widely consumed beverage worldwide. Green tea, black tea, and oolong tea are the three major forms of tea. Due to the abundant resources of tea germplasm, Yunnan Province is the original center of tea plant and is also a principal area of production and consumption of green tea. The special production of green tea inactivates the enzymes and prevents the oxidation of tea polyphenols(TPs), which make TPs be maximally preserved. The natural product(-)-epigallocatechin-3-gallate(EGCG) is the major polyphenolic constituent found in green tea. More than 50% of TPs is composed of EGCG and it is responsible for the majority of the potential health benefits attributed to green tea consumption.Epidemiological, experimental animal models and in vitro studies have demonstrated that EGCG has potential anti-cancer properties, which can induce apoptosis in various cancers. EGCG can exert a dual pro- and anti-oxidant action in cell culture. Simultaneously, EGCG have shown to modulate various components of the epigenetic machinery, including inhibition of DNA methyl transferase(DNMTs)and alteration of histone modifications. These characteristics of EGCG imply it may effect on the cell growth and stability of genetic material in response to redox conditions change, and the change of epigenetic microenvironment induce the gene expression. But the relevant mechanism is not clear. This study, from genetic and epigenetic level, aimed at the different responses on EGCG treatment in different pathological state of the cells and discussed the mechanism of EGCG induction of apoptosis in cancer cells.First, Human colorectal normal cells(NCM460) and human colorectal adenocarcinoma cells(COLO205) were exposed to different doses of EGCG(5-40μg/ml) for 24-96 h, respectively. We studied the different effects of EGCG(5-40μg/ml) exposure(24-96h) on the chromosomal instability(micronuclei,nucleoplasmic bridges and nuclear buds), cell proliferation(nuclear division index)and cell apoptosis of NCM460 and COLO205.The results showed that:(1) EGCG(5-40 μg/ml) exposure(24-96h) had ability to significantly decrease the chromosomal instability of NCM460 cells(p<0.001), while induce the chromosomal instability of COLO205 cells, suggesting that EGCG has a protective and genetic damage effect in different pathological states cells;(2) 5 μg/ml of EGCG treatment could significantly increase the NDI of NCM460 cells, and subsequently decrease the NDI(p<0.05) in COLO205,suggesting that EGCG have different effects on cell growth pathways between the two kinds of tested cell;(3) ECGC treatment could significantly increase the frequency of apoptotic COLO205(p <0.001), while significantly decrease the frequency of apoptotic NCM460(p <0.001). Together, these data suggested EGCG exhibited opposed genetic and cytological effects in normal and cancer cells.Based on the above results, to further analysis the mechanism of these differences, studies further explored the differential effect of EGCG on the genetic damage repair and apoptosis pathway in the different pathological state cell, in the light of the epigenetic effect of EGCG. Under the same experimental conditions, we studied the effect of EGCG on the transcription of mismatch repair key genes h MLH1 and h MSH2 and apoptosis-related genes bcl-2, bax by q RT-PCR.The results showed that:(1) In NCM460, the 24 h exposure of EGCG could significantly increase the transcription of h MLH1 and h MSH2(p<0.05), and the transcription of h MLH1 and h MSH2 significantly increased after EGCG exposure48 h in COLO205(p<0.05), therefor, EGCG can up-regulate the mismatch repair key genes and that was more efficiently in NCM460 cells, suggesting EGCG may promote the DNA damage repair of normal cell, while the up-regulation mismatch repair genes may be a response to the genomic damage induced by EGCG in cancer cell;(2) EGCG has a significant effect on the transcription of apoptosis-related genes. After 40 μg/ml of EGCG exposure 48 h, the transcription level of bax was significantly decreased in NCM460, while that was increased in COLO205.Moreover, EGCG(5-40 μg/ml) exposure(24-96h) had ability to significantly increase the transcription of bcl-2 in NCM460. In COLO250, the transcription level of bcl-2 was significantly decreased after EGCG(5-40 μg/ml) exposure 72 h. EGCGpossessed different effects on the regulation of transcription of genes in bcl-2 family between the two test cell;(3) The average effect of EGCG dose that contributed to the changes of the bcl-2/bax ratio was discussed. In NCM460 cells, the ratio of bcl-2/bax was not significantly changed at the EGCG doses of 5 and 10 μg/ml but was significantly increased at the dose of 20 μg/ml and began to be decreased at the dose of 40 μg/ml. In COLO205, EGCG at 20 and 40 μg/ml significantly reduced the ratio of bcl-2/bax. These results suggesting middle/low concentration(5, 10, 20μg/ml) of EGCG had ability to inhibit apoptosis in NCM460, while middle/high concertration(20 and 40 μg/ml) of EGCG promoted apoptosis in COLO250.In summary, there was a dual effect of EGCG on the genome stability and the transcription of apoptosis related genes between the different pathological state cells.EGCG was beneficial to genetic damage repair and maintain normal cell genome stability, up-regulate bcl-2 and inhibit apoptosis; While the treatment of EGCG could significantly induce chromosomal stability, down-regulation bcl-2 and promote apoptosis in colorectal adenocarcinoma cells. Therefore, the different responses between these two cell types to genetic damage and repair system may be responsible for their different proliferation rate induced by EGCG, and we propose this may be the underlying mechanism of its anticancer action. Genomic instability is an important factor in causing cancer. EGCG could help to maintain normal cell genome stability, suggesting that EGCG may have the biological effects to prevention of tumorigenesis. This research lays the scientific foundation for developing health benefits of green tea purposefully and exploring its potential clinical effect. |
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