冬凌草甲素抑制人肝癌BEL-7402细胞生长及诱导细胞凋亡的机制研究

Mechanisms of growth inhibition and apoptosis inducement of oridonin on human hepatic carcinoma BEL-7402 cells

目的探讨冬凌草甲素体外对肝癌BEL-7402细胞生长抑制作用及诱导细胞凋亡作用机制。方法以8、16、24、32μmol/L冬凌草甲素作用于体外培养的BEL-7402细胞,MTT法检测细胞生长抑制率,应用流式细胞仪检测细胞凋亡,Hoechst33258荧光染色法和DNA凝胶电泳观察细胞凋亡时的形态学变化。应用Westernblotting观察caspase-3、Bcl-2、Bax蛋白表达变化。结果冬凌草甲素可显著地抑制BEL-7402细胞的生长,诱导细胞发生凋亡,并呈现出明显的量-效与时-效关系。冬凌草甲素作用60h后,在Hoechst33258荧光染色图片上可见核浓缩及核碎裂等典型的细胞凋亡特征,同时Westernblotting显示32000的caspase-3酶原蛋白的激活,出现20000的亚单位,凋亡过程中伴有Bcl-2蛋白表达的降低和Bax蛋白上调。结论冬凌草甲素能通过降低Bcl-2蛋白表达和上调Bax蛋白诱导细胞发生凋亡,抑制BEL-7402细胞的生长;冬凌草甲素可能成为一种潜在的抗肝癌药物。

Objective To investigate the mechanism of cell growth inhibition and apoptosis inducement of oridonin on human hepatocellular carcinoma BEL-7402 cells. Methods BEL-7402 cells in cultural medium in vitro were given 8, 16, 24, and 32 μmol/L oridonin. The inhibitory rate of cells was measured by MTT assay, cell apoptotic rate was detected by flow cytometry （FCM）, morphology of cell apoptosis was observed by Hoechst 33258 fluorescent staining and DNA gel electrophoresis, caspase-3, Bcl-2 and Bax protein expressions were detected by Western blotting. Results Oridonin could inhibit the grwoth of BEL-7402 cells and cause apoptosis significantly, the suppression was both in a time- and dosedependent manner. Marked morphological changes of cell apoptosis were observed very clearly by Hoechst 33258 fluorescent staining as well as DNA gel electrophoresis analysis after the cells exposed to oridonin for 60 h; Western blotting showed cleavage of the caspase-3 zymogen protein （32 000） with the appearance of its 20 000 subunit. Along with the apoptotic process Bcl-2 protein expression was down-regulated and Bax protein expression up-regulated concurrently observed by Western blotting. Conclusion Oridonin can inhibit cell growth by induction of apoptosis in BEL-7402 cells via activation of caspase-3 as well as downregulation of Bcl-2 and up-regulation of Bax protein expression, the results indicate that oridonin may be an important potential anti-hepatocellular carcinoma reagents.