拓扑替康诱导卵巢癌细胞自噬的实验研究

Autophagy induced by topotecan in ovarian cancer cells

目的:检测拓扑替康(TPT)对卵巢癌OVCAR3细胞的增殖抑制作用,观察TPT诱导OVCAR3细胞自噬现象,初步探讨其发生的可能机制。方法:采用不同浓度的TPT处理卵巢癌OVCAR3细胞,MTT法测定TPT对OVCAR3细胞增殖的抑制作用,吖啶橙(AO)染色观察酸性小体(AVO)形成,MDC荧光染色检测自噬囊泡。免疫印迹法检测TPT对OVCAR3细胞LC3-Ⅱ和Beclin1蛋白的影响。结果:TPT对OVCAR3细胞生长有显著的抑制作用,且此作用呈明显的剂量依赖性,IC50为0.057μg/mL。TPT可诱导OVCAR3细胞产生AVO。MDC荧光染色显示,TPT可诱导OVCAR3细胞产生自噬囊泡。采用0.05μg/mL TPT处理OVCAR3细胞24h后,免疫印迹法显示,随着时间的推移LC3-Ⅱ和Beclin1蛋白水平表达个逐渐增加,提示TPT诱导的OVCAR3细胞自噬可能与Bec-lin1蛋白有关。结论:TPT对OVCAR3细胞有显著的抑制作用,TPT可以诱导OVCAR3细胞发生自噬,TPT诱导OVCAR3细胞自噬可能与Beclin1蛋白上调有关。

OBJECTIVE： To evaluate the growth inhibition of topotecan（TPT） on OVCAR3 cells and investigate autophagy induced by TPT.METHODS： The growth inhibition of TPT on OVCAR3 cells was detected by MTT assay.Formation of acidic vesicle organelles（AVO） was examined in fluorescence microscopy by staining with acridine orange（AO）.MDC staining was used to examine autophagosome.Western blot analysis was used to detect the expression of LC3-Ⅱ and Beclin1 induced by TPT in OVCAR3 cells.RESULTS： MTT assay showed that TPT could evidently inhibit the proliferation of OVCAR3 cells in a dose-dependent manner.The half maximal inhibitory concentration（IC50） value was 0.057 μg/mL.After TPT treatment for 24 h,AO staining was performed,and AVOs were increased compared to the control group.Then we performed MDC staining after TPT treatment.As the result showed,after TPT treatment for 24 h,autophagosomes were increased compared to the negative group.After TPT treatment for the indicated time,LC3-Ⅱ and Beclin1 were detected by Western blot assay.The results showed that the expression of LC3-Ⅱ and Beclin1 were gradually upregulated.CONCLUSIONS： TPT can strongly inhibit proliferation of OVCAR3.It can also induce an autophagic cell death in OVCAR3 cells.The autophagic cell death induced by TPT in OVCAR3 cells may be related to Beclin1 upregulation.