Akt磷酸化在A549细胞抵抗rsTRAIL蛋白诱导细胞凋亡的研究

摘要

目的：研究Akt磷酸化抵抗rsTRAIL蛋白诱导肺癌A549细胞株凋亡的作用机制。方法：rsTRAIL蛋白和哌立福新单独和联合作用于细胞株A549，Western blot检测A549细胞中Akt磷酸化水平变化、c-FLIPLL表达水平变化、caspase-8蛋白活化变化情况。流式细胞术进行细胞凋亡检测。 MTT法检测A549细胞增殖率。结果：Western blot结果显示，rsTRAIL蛋白可以导致A549细胞中Akt磷酸化水平的增加，哌立福新能够抑制A549细胞中Akt的磷酸化水平及c-FLIPLL表达水平，增强A549细胞中caspase-8的活化。哌立福新能提升rsTRAIL蛋白诱导A549细胞的细胞凋亡率至（76．5±3．02）％和杀伤活性到（83．2±2．54）％。结论：Akt磷酸化是导致A549细胞对抵抗rsTRAIL 诱导细胞凋亡的重要原因，抑制其活性可以促进rsTRAIL蛋白发挥抗NSCLC的活性。%Objective:To investigate mechanism of TRAIL-resistance in A549 cells ( a cell line of non-small cell lung carcino-ma cells) due to Akt phosphorylation .Methods:A549 cells were treated with Akt inhibitor Perifosine and rsTRAIL protein individual-ly and in combination.The expressions of Akt phosphorylation(p-Akt),c-FLIPLL and caspase-8 were detected by Western blot.The apoptotic rate of the A549 cells treated was detected by flow cytometry and the cell proliferation was evaluated by MTT assay .Results:A549 cells showed the increased level of Akt phosphorylation mediated by rsTRAIL protein .Treatment with the Akt inhibitor Perifosine induced a suppression of Akt activation in A 549 cells and a concomitant decrease in the expression of c-FLIPLL .As a result, Perifosine significantly enhanced TRAIL-induced apoptosis rate of (76.5 ±3.02)%and cytotoxic rate of (83.2 ±2.54)%by promoting the activ-ity of caspase-8.Conclusion:Akt activity promotes A549cells survival against TRAIL-induced apoptosis and that the cytotoxic effect of rsTRAIL protein can be enhanced by modulating the Akt phosphorylation in human non -small cell lung carcinoma cells .