| 【Objective】Radiation therapy is one of the most effective therapeutic strategies for esophageal squamous cell carcinoma(ESCC). How to promote radiation sensitivity in ESCC deserves consideration. Numerous reports have shown that EGCG could enhance radiosensitivity on nasopharyngeal carcinoma. However, whether EGCG could sensitize ESCC to radiation has not been elucidated. In this study, we aimed to investigate the role of autophagy and apoptosis,which plays a key role in EGCG-induced radiation sensitivity of human esophageal squamous carcinoma TE-1 cells.【Methods】(1) EGCG with different concentrations(20、40、60、80μg/ml) and times(24、48、72h)were applied to human esophageal squamous cancer cell lines TE-1 and determined EGCG best work concentration. TE-1 cell were treated with 80μg/ml EGCG and X-rays with different doses(2、4、8Gy) in combination and determined radiation best work dose. Cell viability was detected by MTT assay. Pretreatment with autophagy inhibitor3-methyladenine(3-MA),cell proliferation was examined by MTT after EGCG combining with X-rays irradiation.(2) TE-1 cells were divided into four groups of control, 80μg/ml EGCG treatment, 4Gy X-rays irradiation, and 80μg/ml EGCG combining with 4Gy X-rays irradiation. Number of cell colonies above 50 cells was tested by clonogenic survival assay, cell apoptosis was detected by flow cytometry. By X-rays irradiation alone or in combination on ESCC TE-1cells, radiosensitization was evaluated by clone forming assay.(3) The protein expression levels of Cleaved caspase-3, Bcl-2, LC3-II and Beclin1 were evaluated by western blot analysis, and formation of autophagosome was determined by fluorescence staining after TE-1 cells were treated.(4) After treatment with small interfering RNA(si RNA-Beclin1), the cell proliferation was tested by MTT assay, and the protein expression of LC3-II by western blotting.【Results】(1) EGCG inhibited the growth of TE-1 esophageal squamous cancer cell in a time-dependent and dose-dependent manner, and 80μg/ml EGCG for 48 hours was a best work concentration. EGCG dramatically increased the cytoxicity of X-rays irradiation in a synergistic manner, and 4Gy X-rays radiation was a best work dose. 3-MA as a kind of autophagy inhibitors could attenuate the effect of EGCG on cell growth.(2) Compared with the control group, 80μg/ml EGCG treatment group, 4Gy X-rays irradiation group, the rate of number of cell colonies and cell apoptosis ratio in the group of80μg/ml EGCG combining with 4Gy X-rays irradiation were remarkly decreased. The cell survival curve demonstrated that D0 and Dq in the group of 80μg/ml EGCG combining with irradiation were lower than that in other treatment groups, sensitization enhancement ratio was 1.19.(3) The expression level of LC3-II, Beclin1 were augmented in a dose-dependent manner after EGCG-treated ESCC TE-1 cells for 48 hours. Compared with the control group, 80μg/ml EGCG treatment group and 4Gy X-rays irradiation group, the level of Bcl-2 was decreased, while the level of Cleaved caspase-3, LC3-II, Beclin1 and the number of autophagosome fluorescent foci were significantly increased in the group of 80μg/ml EGCG combining with 4Gy X-rays irradiation.(4) After treatment with si RNA-Beclin1, EGCG combining with irradiation inducedLC3-II upregulation and growth inhibition were attenuated.【Conclusions】(1) Both EGCG and X-rays are cytotoxic to human esophageal squamous cancer cell lines TE-1.(2) EGCG could increase the radiosensitization of human esophageal squamous cancer lines TE-1.(3) The mechanism of EGCG enhance radiosensitization on TE-1 cells may be to induce apoptosis and autophagy through down regulation of the expression of Bcl-2 and up regulation the expression of Beclin1. |
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