PI3K特异性抑制剂LY294002增强rsTRAIL蛋白对A549细胞的杀伤作用

Enhanced effect of rsTRAIL-based therapy for A549 cells by phosphatidylinositol 3′-kinase inhibitor LY294002

目的:探讨磷脂酰肌醇3激酶(PI3K)参与非小细胞肺癌A549细胞株抵抗重组可溶性肿瘤坏死因子相关凋亡诱导配体(rsTRAIL)蛋白诱导凋亡的机制,寻找增强rsTRAIL蛋白杀伤非小细胞肺癌细胞的新方法。方法:选取对数生长期A549细胞,随机分为rsTRAIL蛋白组和PI3K特异性抑制剂LY294002联合rsTRAIL蛋白(LY294002+rsTRAIL蛋白)组。MTT法检测2组A549细胞生长抑制率,流式细胞技术检测2组A549细胞周期和细胞凋亡的变化,蛋白质印迹法检测2组A549细胞中p-Akt(Ser473)、c-FLIPL、Bcl-2和Bax蛋白表达水平。结果:与rsTRAIL蛋白组(5.16%±0.32%)比较,LY294002+rsTRAIL蛋白组A549细胞生长抑制率(74.6%±2.63%)明显升高(P<0.05)。与rsTRAIL蛋白组比较,LY294002+rsTRAIL蛋白组处理2h后G0/G1期A549细胞百分比增加(P<0.05),S期细胞百分比降低(P<0.05)。LY294002+rsTRAIL蛋白组A549细胞凋亡率为(61.50±3.02)%,显著高于rsTRAIL蛋白组(3.21%±0.96%)(P<0.05)。蛋白质印迹检测,与rsTRAIL蛋白组比较,LY294002+rsTRAIL蛋白组A549细胞中p-Akt、c-FLIPL和Bcl-2蛋白表达水平降低(P<0.05),Bax/Bcl-2比值升高(P<0.05)。结论:LY294002能够抑制PI3K活性,增强rsTRAIL蛋白对A549细胞的杀伤作用。

Objective To explore the potential mechanisms of non-small cell lung carcinoma cells to rsTRAIL protein-induced apoptosis by phosphatidylinositol 3′-kinase （PI3K/Akt）inhibitor LY294002,and to provide new ways to increase killing activities of rsTRAIL protein for non-small cell lung cancer.Methods The A549 cells at logarithmic growth phase were selected and randomly divided into rsTRAIL group and LY294002＋rsTRAIL group. The inhibitory rate of growth of the A549 cells was tested by MTT assay.The cell cycle and apoptotic rate were detected by flow cytometry analysis. The expression levels of Ser473 phosphorylated form of Akt （p-Akt）,c-FLIPL protein and Bcl-2 protein in the A549 cells in two groups were analyzed by Western blotting method. Results The inhibitory rate of growth of the A549 cells in LY294002＋rsTRAIL group （74.6 %± 2.63%）was higher than that in rsTRAIL group （5.61% ± 0.32%） （P〈 0.05 ）. Compared with rsTRAIL group, the percentage of the cells at G0/G1 phase in LY294002＋rsTRAIL group was increased（P〈0.05）and the percentage of the cells at S phase was decreased（P〈0.05）.The apoptotic rate of the A549 cells in LY294002＋rsTRAIL group （61.5%±3.02%）was higher than that in rsTRAIL group （3.21%±0.96%）（P〈0.05）. The Western blotting results showed that the expression levels of p-Akt, c-FLIPL and Bcl-2 proteins in the A549 cells in LY294002＋rsTRAIL group were decreased （P〈0.05 ）and the ratio of Bax/Bcl-2 was increased （P〈0.05 ） compared with rsTRAIL group.Conclusion LY294002 can increase the killing activity of rsTRAIL protein in A549 cells by inhibiting the activity of PI3K.