去乙酰化酶抑制剂TSA介导Ku70乙酰化对结肠癌HT29细胞凋亡和自噬的影响

目的:探讨不同浓度组蛋白去乙酰化酶抑制剂TSA对结肠癌HT29细胞的增殖、凋亡和自噬影响及其机制研究。方法:取对数生长期人结肠癌HT29细胞,采用MTT法检测不同浓度TSA处理对其细胞活力影响,并根据IC50值确定适宜给药浓度;采用流式细胞术检测不同浓度TSA处理后结肠癌HT29细胞的凋亡情况;Western blot验证空白对照组与TSA给药处理组中凋亡标志蛋白Ku70、acetrl-Ku70、Caspase3、Bax、Bcl-2和自噬标志蛋白LC3和Beclin1的表达。结果:MTT法实验结果表明TSA对结肠癌HT29细胞具有时间和浓度依赖性抑制作用,根据IC50=1.12μM,本研究中TSA的给药浓度为0.5μM和1μM;流式细胞凋亡检测结果表明TSA能够显著促进结肠癌HT29细胞凋亡,且其促凋亡作用存在浓度依赖性;此外,Western blot检测结果证实,与空白对照组相比,TSA给药处理可显著上调上述细胞中acetrl-Ku70以及促凋亡蛋白Caspase3、Bax和自噬标志蛋白LC3和Beclin1的表达,下调抗凋亡蛋白Bcl-2的表达(P<0.05)。结论:组蛋白去乙酰化酶抑制剂(TSA)的体外抗结肠癌细胞的增殖、促进细胞凋亡和自噬作用与其上调Ku70蛋白乙酰化密切相关,有望成为临床潜在抗癌靶点。

Effect of TSA - mediated Ku70 Acetylation on Apoptosis and Autophagy of Colon Cancer HT29 Cells

Objective: To investigate the effect and mechanism of different concentrations of histone deacetylase inhibitor TSA on proliferation, apoptosis and autophagy in colon cancer HT29 cells. Methods: Human colon cancer HT29 cells in logarithmic growth phase were collected and the effect of TSA treatment at different concentrations on cell viability was determined by MTT assay, and the appropriate concentration was determined according to IC50 value. Flow cytometry was used to detect the apoptosis of colon cancer HT29 cells treated with different concentrations of TSA. Western blot was used to verify the expression of acetyl-Ku70 and apoptotic proteins Caspase3, Bax, Bcl-2 and autophagy proteins LC3 and Beclin1 in both control group and TSA treatment group. Results: The result of MTT assay showed that TSA could inhibit the viability of colon cancer HT29 cells in a time-and concentration-dependent manner. According to IC50=1.12 μM, the concentration of TSA used in this study were 0.5 μM and 1 μM respectively. Flow cytometry result showed that TSA could significantly promote the apoptosis of colon cancer HT29 cells in a concentration-dependent manner. In addition, Western blot analysis confirmed that compared with the control group, TSA treatment could significantly up-regulate the expression of acetyl-Ku70 and apoptotic proteins Caspase3 and Bax and autophagy proteins LC3 and Beclin1, and down-regulate the expression of anti-apoptotic protein Bcl-2(P<0.05). Conclusion: The effect of histone deacetylase inhibitor(TSA) on the proliferation, apoptosis and autophagy of colon cancer cells in vitro is closely related to the up-regulation of Ku70 acetylation, which is expected to be a potential clinical anticancer target.