扶正抗癌方通过PTEN/PI3K/Bad通路调控肺癌A549细胞增殖与凋亡

Effect of FZKA Decoction in Regulating A549 Proliferation and Apoptosis Via PTEN/PI3K/Bad

目的:观察扶正抗癌(FZKA)方对肺癌细胞A549增殖的调控作用,探讨其作用的分子机制。方法:以非小细胞肺癌细胞A549为研究对象,以FAKA方0.8,1.2,1.6,2.0,2.4,2.8,3.2 g·L^(-1)干预,空白组,分别孵育24,48,72 h后,设立采用噻唑蓝(MTT)比色法检测FZKA方对A549细胞活力的影响;以FAKA方1.2,1.6,2.0 g·L^(-1)干预,设立空白组,培养24 h后,采用实时荧光定量聚合酶链式反应(Real-time PCR)检测FZKA方对A549细胞人第10号染色体同源丢失性磷酸酶-张力蛋白(phosphatase and tensin homolog deleted on chromosome ten,PTEN)mRNA水平的影响;孵育24 h后,采用蛋白质免疫印迹法(Western blot)分析FZKA方对PTEN,磷脂酰肌醇3-激酶(phosphatidylinositol-3-kinase,PI3K),磷酸化的促凋亡蛋白Bad(Phospho-Bad,p-Bad)表达的影响并探讨其相关性。结果:与空白组比较,FZKA方能明显抑制A549细胞增殖,处理24 h后,FZKA从0.8 g·L^(-1)开始,处理48,72 h后,FZKA方从0.4 g·L^(-1)开始,细胞存活率明显降低(P<0.05,P<0.01);与空白组比较,处理24 h后,FZKA方从0.8 g·L^(-1)开始以浓度依赖性上调PTEN mRNA和蛋白的表达(P<0.05,P<0.01),FZKA方从1.6 g·L^(-1)开始以浓度依赖性下调PI3K蛋白表达(P<0.05,P<0.01),从2 h开始以时间依赖性上调p-Bad蛋白的表达(P<0.05,P<0.01)。结论:FZKA方可通过上调PTEN来调控下游PI3K/Akt/Bad通路相关蛋白的表达进而抑制A549的增殖,促进肺癌细胞凋亡。

Objective： To observe the effect of Fuzheng Kangai （FZKA） decoction on the proliferation and apoptosis, in order to discuss the molecular mechanism of apoptosis induced by FZKA decoction in A549 cells. Method： Non-small lung cancer cells （A549 cells） were taken as the research objects and treated for 24, 48, 72 h with 0 （ blank group） , 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2 g·L-1FAKA decoction. Then, methyl-thiazolyl-tetrazolium （MTT） assay was used to detect the effect of FZKA decoction on the cell proliferation and apoptosis. A549 cells were treated for 24 h with 0 （blank group）, 1.2, 1.6, 2.0 g·L-1. Then, quantitative Real-time PCR was used to detect the effect of FZKA decoction on PTEN mRNA expression. A549 ceils were treated for 24 h with 0 （blank group） , 0. 8, 1.2, 1.6, 2.0 g·L-1. Then, Western blot assay was used to detect the expressions of phosphatase and tensin homolog deleted on chromosome ten （PTEN）, phosphatidylinositol-3-kinase （PI3K） and phospho-Bad （p-Bad）, and discuss the relationship between them. Result： Compared with blank group, the proliferation of A549 cells were inhibited by FZKA decoction in concentration-dependent and time-dependent manners （P 〈 0.01 ）. Compared with blank group, mRNA and protein expressions of PTEN were up-regulated in a concentration-dependent manner （P 〈 0.05, P 〈 0.01 ）. Compared with blank group, the protein expression of PI3K was down-regulated in a concentration-dependent manner （P 〈 0.05, P 〈 0.01 ） , and p-Bad was down- regulated in a time-dependent manner （P 〈 0.05, P 〈 0.01 ）. Conclusion： Apoptosis is induced by FZKA decoction by up-regulating PTEN and regulating PI3K/Akt/Bad pass way in A549 cells.