黄芪甲苷对非小细胞肺癌细胞A549、SPC-A1增殖凋亡的调控作用及其机制

Regulatory effects of astragaloside IV on proliferation and apoptosis of non-small-cell lung cancer cells A549 and SPC-A1

目的探讨黄芪甲苷对非小细胞肺癌细胞A549、SPC-A1增殖凋亡的调控作用及潜在的作用机制。方法分别用0、10、20、40、60、80、100μmoL/L的黄芪甲苷处理非小细胞肺癌细胞A549和SPC-A124 h后,用四甲基偶氮唑蓝染色法测算A549和SPC-A1细胞存活率,计算出黄芪甲苷的半数抑制浓度(IC_(50))。将IC_(50)的黄芪甲苷加入非小细胞肺癌细胞A549和SPC-A124 h,同时设立空白对照,采用原位末端转移酶标记法测算A549和SPC-A1细胞的凋亡率。实时荧光定量PCR检测Ki67、PCNA、Caspase-3、Caspase-9基因,免疫印迹法检测Cleaved Caspase3、Cleaved Caspase9、total-Akt、p-Akt蛋白。结果不同浓度黄芪甲苷处理后24 h,A549和SPC-A1的细胞活性均明显受到了抑制(P均<0.05),A549和SPC-A1细胞增殖的IC50分别为32.6μmol/L和29.4μmol/L。与空白对照比较,IC50的黄芪甲苷加入后,A549和SPC-A-1细胞凋亡率明显升高(P均<0.05)。与空白对照比较,黄芪甲苷加入后,A549、SPC-A1细胞增殖相关基因Ki67、PCNA下降(P均<0.05),凋亡相关基因Caspase-3和Caspase-9的mRNA和蛋白的表达均升高(P均<0.01),p-Akt表达下降(P<0.01)。结论黄芪甲苷可抑制非小细胞肺癌A549、SPC-A1的增殖,促进其凋亡,呈浓度依赖性;抑制Akt信号通路中p-Akt的表达可能是黄芪甲苷的作用机制之一。

Objective To explore the regulatory effects of astragaloside Ⅳ(As-Ⅳ) on proliferation and apoptosis of non-small-cell lung cancer(NSCLC) cells A549 and SPC-A1 and its potential mechanism.Methods NSCLC cells(A549 and SPC-A1) were treated with 0,10,20,40,60,80 and 100 μmol/L astragaloside Ⅳ for 24 h,respectively.The survival rates of A549 and SPC-A1 cells were measured by MTT staining,and the IC50 of astragaloside Ⅳ was calculated.Astragaloside Ⅳ with IC50 was added to A549 and SPC-A1 cells for 24 h,and meanwhile,the blank control group was set up.The apoptosis rates of A549 and SPC-A1 cells were tested by TUNEL.Quantitative real-time PCR(qRT-PCR) was used to test Ki67,PCNA,Caspase-3 and Caspase-9 genes,and Western blotting was used to test Cleaved-Caspase3,Cleaved-Caspase9,total Akt,and p-Akt proteins.Results After treatment with different concentrations of astragaloside Ⅳ for 24 h,the cell activities of A549 and SPC-A1 cells were significantly inhibited,and the IC50 of A549 and SPC-A1 cells were 32.6 μmol/L and 29.4 μmol/L,respectively;meanwhile,compared with the control group,the apoptosis rates of A549 and SPC-A-1 cells in the astragaloside Ⅳ group increased significantly(all P<0.05).Compared with the control group,after astragaloside Ⅳ was added,the proliferation-related genes Ki67 and PCNA of A549 and SPC-A1 cells decreased(all P<0.05),the expression of apoptosis-related genes Caspase-3 and Caspase-9 mRNA and protein increased(all P<0.01),and the expression of p-Akt decreased(P<0.01).Conclusion Astragaloside Ⅳ can regulate the proliferation and apoptosis of A549 and SPC-A1 cells in a dose-dependent manner probably by inhibiting the expression of Akt in Akt signaling pathway.