shBAG3对紫草素杀伤人U87胶质瘤细胞作用的影响及机制

The effect and mechanism of sh BAG3 on the cytotoxicity of shikonin in human U87 glioma cells

目的:研究shB AG3对紫草素杀伤人U87胶质瘤细胞作用的影响及相关分子机制。方法:紫草素(2μmol/L)对人U87细胞分别处理0h、4h、8h、12h和24h后,应用Real-time PCR和Western blot方法检测紫草素作用下U87细胞中BAG3的表达变化;免疫荧光显微镜下观察紫草素作用下BAG3在细胞内的表达分布;CCK-8法检测shB AG3对紫草素降低细胞活力的影响;应用Western blot检测shB AG3联合紫草素作用下U87细胞中Cytosolic cyto C、caspase-9和Cleaved caspase-9的表达变化。结果:紫草素作用24h内BAG3在mRNA和蛋白表达上均显著升高。与单独应用紫草素组相比,联合shB AG3可显著增强紫草素对U87细胞活力的抑制作用。紫草素作用24h内BAG3的表达分布逐渐从U87细胞的胞浆中转移至线粒体。此外,联合shB AG3可导致U87细胞浆中Cytosolic cyto C和Cleaved caspase-9的表达显著增加。结论:紫草素杀伤U87细胞过程中产生的BAG3高表达可能是紫草素抗胶质瘤过程中的促存活机制之一;联合shB AG3能够显著增强紫草素对胶质瘤细胞的肿瘤杀伤作用,其机制可能与BAG3参与调节线粒体凋亡途径有关。

Objective： To explore the effect and mechanism of sh BAG3 on the cytotoxicity of shikonin in human U87 glioma cells. Methods： The alteration of BAG3 expression by shikonin treatment（ 2μmol / L） for 0h,4h,8h,12 h and 24 h was evaluated by Real- time PCR and Western blot assay. The expression and distribution of BAG3 were examined by immunofiuorescent staining. The alteration of cell viability by combination shikonin with sh BAG3 was tested by CCK- 8 assay. We used Western blot assay to detect the alteration of Cytosolic cyto C,caspase- 9 and Cleaved caspase- 9 expression by combination with sh BAG3 in shikonin treated U87 cells. Results： The expression of BAG3 was increased both at mRNA and protein level in shikonin treated U87 cells. Combination with sh BAG3 significantly enhanced the inhibitory effect of shikonin on cell viability. The distribution of BAG3 was gradually co- localized with mitochondria,most obvious at 12 h. Combination of sh BAG3 markedly increased the expression of Cytosolic cyto C and Cleaved caspase- 9. Conclusion： The high expression of BAG3 in antitumor process of shikonin toward glioma cells is one of the pro- survival mechanisms. Combination with sh BAG3 significantly enhances the inhibitory effect of shikonin toward glioma cells,in which the regulation of mitochondrial apoptotic pathway by BAG3 is involved.