曲古霉素A体外诱导膀胱癌细胞凋亡及细胞周期阻滞的机制探讨

Trichostatin A inducing apoptosis and cell cycle arrest of bladder cancer cells:the in vitro mechanism

目的:观察组蛋白去乙酰化酶(HDAC)抑制剂曲古霉素A(TSA)对体外培养膀胱癌细胞生长情况及相关基因表达的影响,并探讨其可能的作用机制。方法:MTT法检测不同浓度(0.05、0.1、0.2、0.4、0.8μmol/L)的TSA对人膀胱癌T24细胞生长的影响。透射电镜观察TSA(0.4μmol/L)诱导后膀胱癌细胞的形态学变化;流式细胞术检测处理后膀胱癌细胞周期分布及凋亡率的变化;Western印迹法检测处理后膀胱癌细胞组蛋白乙酰化水平的变化;FQ-PCR检测处理后膀胱癌细胞p21CIP1/WAF1c、yclin A和cyclin E mRNA的表达。结果:TSA体外能明显抑制T24细胞生长,且抑制作用呈明显的剂量、时间依赖性。TSA(0.4μmol/L)诱导后,透射电镜下可见大量具有凋亡形态特征的T24细胞;流式细胞术检测示细胞阻滞于G0/G1期,并且出现典型的亚二倍体(Sub-G1)峰;TSA可明显提高组蛋白乙酰化水平,并诱导p21CIP1/WAF1的mRNA表达和抑制cy-clin A的mRNA表达,而对cyclin E无明显作用。结论:TSA可通过诱导细胞凋亡及细胞周期阻滞而发挥体外抗膀胱癌作用,其作用机制可能涉及组蛋白乙酰化水平以及相关基因(p21CIP1/WAF1、cyclin A)表达的调控。

Objective： To investigate the influence of trichostatin A （TSA）, a histone deacetylase （HDAC） inhibitor, on the growth of human bladder cancer cells and on the expression of related genes, and to explore the mechanism involved. Methods： MTT assay was employed to evaluate the inhibitory effect of TSA（0. 05, 0. 1, 0.2, 0. 4, 0. 8 μmol/L）on growth of human T24 bladder cancer cells. The morphological changes of T24 cells were observed by transmission electron microscope after treated with 0. 4 μmol/L TSA ; the cell cycle distribution and apoptotic ratio were determined by flow cytometry. The acetyl level of histone after TSA treatment was detected by Western blot; the rnRNA expression of p21^CIP1/WAF1 , cyclin A, and cyclin E was measured by FQ-PCR. Results： MTT assay revealed TSA inhibited the growth of T24 cells in a concentration- and time-dependent manner. Typical morphological changes of apoptotic cells were observed by electron microscope after treatment with 0.4 μmol/L TSA. Flow cytometry showed that the cells were blocked at Go/G1 phase and typical Sub-G1 peak appeared. TSA obviously promoted the acetyl level of histone, induced expression of p21^CIP1/WAF1 mRNA, and inhibited expression of cyclin A, but had no obvious influence on expression of cyclin E. Conclusion： TSA can inhibit bladder cancer cells through inducing cell apoptosis and cell cycle arrest in vitro, which might be related to the acetyl level of histone and the expression of p21^CIP1/WAF1 and cyclin A.