| 2-Methoxyestradiol (2-ME) is a normal physiological metabolite of 17β-estradiol, and was reported to have anti-proliferative and anti-angiogenic activities in a variety of cells and an ex vivo model. However, the cellular mechanisms underlying the antiproliferative activity of 2-ME are unclear. The purpose of this study is to examine the antitumor activities of 2-ME on the well-differentiated EC9706 esophageal carcinoma (EC) cells, then approach its mechanism.1. The in vitro effects of proliferation and apoptosis of 2-ME in EC9706 cellsEC9706 cells were incubated with 0.1,0.5,1,2,5 and 10μmol/L 2-ME for 24h,48h and 72h, respectively. MTT assay was used to detect cell growth inhibition. The results are expressed as percentage of cell growth with reference to the untreated. Cell proliferation was most significantly inhibited when treated with 1-10μmol/L 2-ME, 2-ME inhibited EC9706 cell proliferation in a time-and concentration-dependent manner. The survival rate of group 5μmol/L and 10μmol/L are 50.03% and 40.33% after 72 h. The IC50 values of EC9706 cells were 5.37μmol/L and 4.14μmol/L after treated with 2-ME for 48 and 72 hours. The morphological changes of induced cells were observed with an inverted microscope. Morphological alterations such as rounding, cell shrinkage and retraction from neighboring cells consistent with apoptotic cell death were observed. Cell cycle distribution was observed by flow cytometry, which showed that 2-ME causes cells to accumulate in G2/M phase, moreover the S and G1 phase decreased distinguished. Cell apoptosis was assayed by flow cytometry with Annexin V-FITC/PI, after treatment with 5μmol/L 2-ME for 24 hours, the percentage of Annexin-V+/PI+and Annexin-V+/PI cells are 2.26% and 6.41%, and treatment with 10μmol/L 2-ME for 24 h, the percentage of Annexin-V+ /PI+and Annexin-V+/PI- cells are 9.41% and 19.74%. So we concluded that 2-ME could inhibit cell proliferation, arrest cell cycle and induce apoptosis of EC9706 cell line.2. The effects of 2-ME on the expression of cyclin B1 and c-Myc mRNA In this section, total RNAs were isolated from EC9706 cells treated with 2-ME by the Trizol reagent. The mRNA expression of Bcl-2, Bax, cyclin B1, Tp53 and c-Myc were measured by RT-PCR analysis. Data showed the expression of cyclin B1 mRNA was significantly increased in cells treated with 10μmol/L 2-ME for 24h, and the expression of c-Myc mRNA was higher after treated with 2-ME for longer times. The mRNA expression of Bcl-2, Bax and TP53 were measured at the same time, but 2-ME didn't affect the expression of Bcl-2, Bax and TP53 mRNA. These results implicate that 2-ME induced apoptosis and G2/M cell cycle arrest is associated with up-regulation of cyclin B1 mRNA in EC9706 cells. Further,2-ME induced apoptosis of EC9706 cells is associated with up-regulation of c-Myc mRNA.3. Effects of 2-ME on the expression of c-Myc and cyclin B1 proteinTo determine whether c-Myc and cyclin B1 protein were also up-regulated in EC9706 cells, the expressions of the two proteins in EC9706 cells were investigated by western blot analysis. We found that cyclin B1 protein is significantly increased in EC9706 cells 24 hours after treatment with 1-10μmol/L 2-ME, and c-Myc protein expression was up-regulated after 24,48 and 72 hours of treatment with 1μm 2-ME. These results suggest that up-regulation of cyclin B1 might be involved in 2-ME-induced apoptosis and G2/M cell-cycle arrest of the EC9706 cells, and it was precedes the onset of apoptosis. Moreover, the up-regulation of c-Myc protein would be involved in 2-ME-induced apoptosis in EC9706 cells.
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