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目的探讨自噬对吉非替尼(Gefitinib)诱导EGFR突变型NSCLC PC-9细胞凋亡的影响及机制。方法MTT法检测Gefitinib对PC-9细胞的生长抑制作用;AO染色观察经Gefitinib处理后PC-9细胞嗜酸性自噬泡(acidic vesicular organelles,AVOs)的变化情况;Western blot检测自噬标记物LC3、凋亡相关蛋白PARP、Caspase-3以及Akt/mTOR信号通路的表达;流式细胞术检测Gefitinib及Gefitinib联合自噬诱导剂Rapamycin作用下细胞凋亡情况。结果 MTT及流式细胞术显示Gefitinib呈剂量依赖性抑制PC-9细胞生长并促进其凋亡,AO染色后,经Gefitinib处理的PC-9细胞内可观察到红染的AVOs,Western blot显示Gefitinib能够诱导PC-9细胞自噬标记物LC3表达。Gefitinib联合Rapamycin显著增强Gefitinib对于PC-9细胞的杀伤作用,并且降低PC-9细胞中Akt/mTOR的磷酸化水平。结论 Gefitinib能够诱导PC-9细胞发生自噬,增强细胞自噬能够促进Gefitinib杀伤PC-9细胞的作用。
Objective To investigate the effect and mechanism of autophagy on apoptosis of EGFR mutant NSCLC PC-9 cells induced by gefitinib. Methods The growth inhibition of Gefitinib on PC-9 cells was detected by MTT assay. The changes of acidic vesicular organelles (AVOs) in PC-9 cells treated with Gefitinib were observed by AO staining. The expression of autophagy marker LC3 , Apoptosis related protein PARP, Caspase-3 and Akt / mTOR signaling pathway. Flow cytometry was used to detect the apoptosis of cells treated with Gefitinib and Gefitinib combined with Rapamycin. Results MTT and flow cytometry showed that Gefitinib could inhibit PC-9 cell growth and induce apoptosis in a dose-dependent manner. After AO staining, red stained AVOs were observed in Gefitinib-treated PC-9 cells. Western blot showed that Gefitinib Can induce PC-9 cell autophagy marker LC3 expression. Gefitinib in combination with Rapamycin significantly enhanced the killing effect of Gefitinib on PC-9 cells and decreased the phosphorylation of Akt / mTOR in PC-9 cells. Conclusion Gefitinib can induce autophagy in PC-9 cells and enhance the autophagy of cells to promote the killing effect of Gefitinib on PC-9 cells.