白藜芦醇联合顺铂对肺癌A549/DDP细胞的作用机制研究

Effects and Mechanism of Resveratrol Combined with Cisplatin on Lung Cancer A549/DDP Cells

目的基于PI3K/AKT/mTOR信号通路,以人非小细胞肺癌耐顺铂细胞株A549/DDP为研究对象,探讨白藜芦醇联合顺铂对A549/DDP细胞增殖、凋亡、自噬的影响。方法以CCK-8法检测白藜芦醇、顺铂以及两药联用对A549/DDP细胞增殖的影响,并通过Isobologram分析两药联用的协同作用;将A549/DDP细胞分为空白对照组、白藜芦醇组、顺铂组、白藜芦醇+顺铂组;以Hoechst 33342染色和Annexin V/PI双染实验检测细胞凋亡率;免疫荧光实验检测自噬标志物LC3的表达;Western Blot实验检测各组细胞中凋亡相关蛋白、自噬标志物蛋白、PI3K/AKT/mTOR通路蛋白表达。结果与顺铂组比较,白藜芦醇与顺铂两药联用可以明显降低顺铂对A549/DDP细胞的IC50,Isobologram分析结果也表明二者联用对A549/DDP细胞具有协同抑制作用;与空白对照组、白藜芦醇组、顺铂组3组比较,白藜芦醇和顺铂联用可以明显增加A549/DDP细胞凋亡率(P<0.01),降低抗凋亡蛋白Bcl-2表达水平(P<0.01),上调凋亡蛋白Bax、Caspase 3表达水平(P<0.01),及增加自噬标志物蛋白LC3Ⅱ/Ⅰ表达水平(P<0.01),降低p62表达水平(P<0.01);此外Western Bolt实验结果表明两药联用可以明显下调p-PI3K、p-AKT,p-mTOR蛋白表达水平(P<0.01)。结论白藜芦醇与顺铂联用可通过PI3K/AKT/mTOR通路,促进A549/DDP细胞自噬诱导细胞凋亡。

Objective From perspective of PI3 K/AKT/mTOR signaling pathway,the effects of resveratrol combined with cisplatin on proliferation,apoptosis and autophagy of A549/DDP cells were studied using cisplatin-resistant human non-small cell lung cancer cell line A549/DDP as a cell model.Methods CCK-8 assay was used to detect the effects of resveratrol,cisplatin and combination of the two drugs on the proliferation of A549/DDP cells,and the synergistic effect of the two drugs was analyzed by Isobologram.The A549/DDP cells were divided into blank control group,resveratrol group,cisplatin group and resveratrol+cisplatin group.The apoptosis rate of each group was detected by Hoechst 33342 staining and Annexin V/PI double staining.The expression of autophagy marker LC3 was detected by fluorescence assay.The expression of proteins related to apoptosis,autophagy marker and PI3 K/AKT/mTOR pathway of each group was detected by Western Blot assay.Results Compared with the cisplatin group,the combination of resveratrol and cisplatin could significantly reduce the IC50 of cisplatin on A549/DDP cells.Isobologram analysis also showed that the combination of the two drugs had synergistic inhibitory effect on A549/DDP cells.Compared with the blank control group,the resveratrol group and the cisplatin group,combination of resveratrol and cisplatin could significantly increase the apoptosis rate of A549/DDP cells(P<0.01),decrease the expression level of anti-apoptotic protein Bcl-2(P<0.01),up-regulate the expression levels of apoptotic protein Bax and Caspase 3(P<0.01),and increase the expression level of autophagy marker protein LC3Ⅱ/Ⅰ(P<0.01),decreased the expression level of p62(P<0.01).The results of Western Bolt assay showed that the combination of the two drugs could significantly down-regulate the protein expression levels of p-PI3 K,p-AKT and p-mTOR(P<0.01).Conclusion The combination of resveratrol and cisplatin can promote autophagy and induce apoptosis in A549/DDP cells through PI3 K/AKT/mTOR pathway.