土槿乙酸诱导人胃癌AGS细胞凋亡机制的研究

Study on action mechanisms for the effect of pseudolaric acid B in inducing apoptosis of human gastric carcinoma cell line AGS

目的:研究土槿乙酸诱导人胃癌AGS细胞凋亡的作用机制。方法:采用Hoechst33342/PI核荧光双染色法,DNA片段化分析技术检测细胞凋亡的变化;FCM分析细胞周期的变化;Western印迹法和RT-PCR法检测与细胞凋亡和细胞周期相关的蛋白及mRNA的表达。结果:土槿乙酸明显诱导人胃癌AGS细胞的凋亡,细胞周期被阻滞在G2/M期;10μmol/L土槿乙酸作用AGS细胞12h后p53表达增加,在36h时达到峰值,24h后可见p21WAF1/CIP1表达明显增加。土槿乙酸作用于AGS细胞后,增加了Fas/APO-1蛋白表达和促凋亡蛋白Bax mRNA的表达,降低了抑凋亡蛋白Bcl-2 mRNA表达,提高了caspase-3活性。结论:土槿乙酸可通过p53激活Bcl-2介导的线粒体途径和Fas/APO-1介导的死亡受体途径诱导人胃癌AGS细胞凋亡。

Objective：The aim of this study was to investigate the mechanisms for pseudolaric acid B （PLAB）-induced apoptosis in human gastric carcinoma AGS cells.Methods：Apoptotic cells were detected using Hoechst33342/PI double staining, and confirmed by DNA fragmentation assay. Cell cycle was analyzed by flow cytometry. The expression of genes involved in apoptosis and cell cycle alteration was analyzed by using RT-PCR. The level of the protein was detected with Western blotting analysis. Results：PLAB significantly induced apoptosis and G2/M phase arrest of AGS cells. PLAB 10 μmol/L increased expression of p53 protein after 12 h and the expression of p53 protein reached the peak at 36 h. The expression of protein levels of p21^WAF1/CIP1 markedly increased at 24 h. PLAB increased the protein expression of Fas/APO-1 and the mRNA expression of pro-apoptosis proteins Bax, decreased the mRNA expression of anti-apoptosis protein Bcl-2, and elevated the activity of caspase-3. Conclusion： PLAB induced apoptosis of human gastric carcinoma AGS cells via two pathways： p53-activated mitochondrial pathway mediated by Bcl-2 and Fas/APO-1-mediated death receptor pathway.