苦马豆素对胶质瘤细胞U87凋亡及HSP90/AKT通路的影响

摘要

Objective To explore the influence of swainsonine (SW) on apoptosis of glioma cell U87 and the related mechanism. Methods U87 cell line was cultured with different concentrations of swainsonine (0 μg/mL, 4 μg/mL, 8 μg/mL, 12 μg/mL), named as the control group, SWlow-dose group, SWmid-dose group and SWhighdose group. After 48 h incubation, morphological changes were observed by cell staining, and U87 cells apoptosis rate was detected by flow cytometry, and caspase 3, HSP90 and AKTexpression were tested by real-time quantitative PCR (qRT-PCR) and Western blot (WB). Results The morphology of U87 cells in control group varied and was in different sizes, and there was protrusions extending around the cells, and the chromatin distributed evenly. Cell number of swainsonine in SWmid-dose group and SWhigh-dose group decreased, cell and nuclear volume was significantly reduced, cytoplasmic concentrated, and there was some apoptotic bodies. Apoptosis rates in SWmid-dose group and SWhigh-dose group were significantly higher than that of control group, showing a dose-dependent manner (P＜0. 05). Caspase 3 expression level in SWmid-dose group and SWhigh-dose group was significantly higher than that of control group, showing a dose-dependent manner (P＜0. 05). HSP90 and AKTexpression levels in SWmid-dose group and SWhigh-dose group were significantly lower than those of control group, in a dose-dependent manner (P＜0. 05). Conclusion SWcan inhibit HSP90 and AKTproteins expression in HSP90/AKTsignaling pathway, and promote the apoptosis of glioma cell line U87. It is predicted that SWpromotes the apoptosis of U87 glioma cells by inhibiting the activity of HSP90/AKTsignaling pathway.%目的 探究苦马豆素 (Swainsonine, SW) 对胶质瘤细胞U87凋亡的影响及相关机制.方法 用不同浓度苦马豆素 (0、4、8、12 μg/mL) 培养U87细胞系, 依次命名为对照组、苦马豆素低剂量组、苦马豆素中剂量组、苦马豆素高剂量组, 培养48h后, 采用细胞染色观察其形态的变化, 应用流式细胞术检测U87细胞的凋亡率, 使用实时荧光定量PCR (qRT-PCR) 和蛋白免疫印迹 (WB) 检测caspase 3、HSP90和AKT的表达情况.结果 对照组U87细胞形态多样, 大小不一, 胞体上有突起向周围延伸, 染色质均匀分布; 苦马豆素中剂量组和苦马豆素高剂量组细胞数目明显减少, 细胞和细胞核体积明显缩小, 细胞质浓缩, 细胞内染色质固缩, 有的细胞出现凋亡小体.苦马豆素中剂量组和苦马豆素高剂量组细胞凋亡率显著高于对照组, 呈现剂量依赖性 (P＜0. 05); 苦马豆素中剂量组和苦马豆素高剂量组caspase 3的表达水平显著高于对照组, 呈现剂量依赖性 (P＜0. 05);苦马豆素中剂量组和苦马豆素高剂量组HSP90、AKT的表达水平显著低于对照组, 呈现剂量依赖性 (P＜0. 05).结论 苦马豆素能够抑制HSP90/AKT信号通路中HSP90和AKT蛋白的表达、促进胶质瘤细胞U87凋亡, 推测苦马豆素是通过抑制HSP90/AKT信号通路的活性促进胶质瘤细胞U87凋亡.