环孢素A联合Wortmannin对胃癌细胞SGC-7901增殖抑制作用及对蛋白激酶B信号通路的影响

Cyclosporin A plus Wortmannin inhibited proliferation of gastric cancer cell SGC-7901 and its influence on protein kinase B signaling pathway

目的:探讨环孢素A(cyclosporin A,CsA)联合Wortmannin对胃癌细胞SGC-7901增殖的抑制作用及对蛋白激酶B(protein kinase B,PKB/AKT1)活性的影响,并解释其可能的分子机制。方法:培养人胃癌SGC-7901细胞,采用CCK8(Cell Counting Kit-8)法检测不同浓度环孢素A(5,10,20,40,80μmol·L-1)和不同浓度Wortmannin(10,50,100,500,1000 nmol·L-1)作用于胃癌细胞SGC-7901不同时间(24,48,72 h)后的细胞增殖抑制情况,并计算半数抑制浓度(half-inhibitory concentration,IC_(50));根据2种药物的IC_(50)确定最佳药物作用浓度,并分为CsA组、Wortmannin组以及CsA+Wortmannin组,用CCK8法和流式细胞实验检测CsA联合Wortmannin对胃癌细胞的增殖和凋亡水平的影响;Western blot法检测各组细胞中cleaved caspase 3、p-AKT、PI3K以及PKB/AKT1蛋白表达量。结果:不同浓度CsA、Wortmannin对人胃癌SGC-7901细胞作用24,48,72 h后,细胞存活率相比0h显著下降(P<0.05),且随着作用时间延长,细胞存活率呈逐渐下降趋势(P<0.01)。与对照组相比,CsA组、Wortmannin组、CsA+Wortmannin组细胞存活率均明显降低、细胞凋亡率明显升高,且CsA+Wortmannin组细胞存活率明显低于CsA组和Wortmannin组,细胞凋亡率明显高于CsA组和Wortmannin组(P<0.05或P<0.01)。Western blot实验结果表明,CsA组、Wortmannin组、CsA+Wortmannin组的PKB/AKT1、PI3K、p-AKT蛋白表达均显著低于对照组,Cleaved caspase3蛋白表达率显著高于对照组(P<0.01),且CsA+Wortmannin组PI3K、p-AKT蛋白表达显著低于CsA组和Wortmannin组,cleaved caspase3蛋白表达率显著高于CsA组和Wortmannin组(P<0.01)。结论:CsA和Wortmannin均对胃癌细胞SGC-7901增殖具有明显抑制作用,且2者联用对胃癌细胞抑制作用显著强于单一药物,细胞增殖抑制作用的发挥可能是通过干扰PI3K/AKT1、PKB/AKT1信号通路和降低蛋白激酶B活性起效。

OBJECTIVE To explore the inhibitory effect of cyclosporin A(CsA)plus Wortmannin on the proliferation of human gastric cancer cell line SGC-7901 and the activity of protein kinase B(PKB/AKT1)and elucidate its possible molecular mechanism.METHODS SGC-7901 cells were cultured.CCK8(Cell Counting Kit-8)assay was performed for detect cellular proliferation inhibition of different concentrations of cyclosporin A(5/10/20/40/80μmol·L-1)and different concentrations of Wortmannin(10/50/100/500/1000 nmol·L-1)with different times(24/48/72 h)and half-inhibitory concentration(IC50)was calculated.According to the IC50of two drugs,optimal drug concentration was determined and divided into three groups of CsA,Wortmannin and CsA+Wortmannin.CCK8 assay and flow cytometry were employed for detecting the effect of CsA plus Wortmannin on cellular proliferation and apoptosis.Western blot was employed for detecting the expressions of cleaved caspase 3,p-AKT,PI3K and PKB/AKT1 protein in each group.RESULTS After 24/48/72 hours of different concentrations of CsA and Wortmannin,cellular survival rate declined markedly as compared with 0h(P<0.05)and downward trend continued gradually with elongating acting time(P<0.01).As compared with control group,cellular survival rate of CsA,Wortmannin and CsA+Wortmannin groups dipped markedly and apoptotic rate was obviously elevated.And cellular survival rate of CsA+Wortmannin group was significantly lower than that of CsA/Wortmannin group and apoptotic rate was significantly higher than that of CsA/Wortmannin group(P<0.05 or P<0.01).Western blot indicated that the expressions of PKB/AKT1,PI3K and p-AKT protein were significantly lower in CsA,Wortmannin and CsA+Wortmannin groups than those of control group.And the expression rate of cleaved caspase3 protein was significantly higher than that in control group(P<0.01).Moreover,the expressions of PI3K and p-AKT proteins were significantly lower in CsA+Wortmannin group than those in CsA/Wortmannin group.And the expression rate of cleaved caspase 3 protein was significantly higher than that of CsA/Wortmannin group(P<0.01).CONCLU-SION Both CsA and Wortmannin can significantly inhibit cellular proliferation and the combination of two can significantly inhibit gastric cancer cells than single drugs.The inhibition effect of cellular proliferation may be caused by interfering with PI3K/AKT1 and PKB/AKT1 signaling pathway and reducing the activity of protein kinase B.