甲磺酸阿帕替尼对结肠癌HCT-116细胞增殖的抑制作用及其机制

摘要

目的 检测阿帕替尼对结肠癌HCT-116细胞的抑制作用,探讨其可能的作用机制及其影响的信号通路.方法 MTT方法检测不同浓度(0、0.5、1、1.5、2 μmol/L)的阿帕替尼对结肠癌HCT-116细胞的毒性作用,并设置卡培他滨阳性对照组;Annexin V-FITC/PI双染法检测上述不同浓度的阿帕替尼处理后结肠癌HCT-116细胞的凋亡情况,实时荧光定量PCR及Western Blotting技术检测其对凋亡相关基因及蛋白Bcl-2,Bax,Caspase-3的影响,Western blotting技术检测其对Akt、p-Akt、Erk 1/2和p-Erk1/2蛋白表达的影响.结果 MTT细胞毒性检测结果表明,阿帕替尼在体外能有效抑制HCT-116细胞的增殖,IC50为1.335 μmol/L.Annexin-V/PI双染法细胞凋亡检测结果表明,阿帕替尼能显著的诱导HCT-116细胞凋亡,并且呈浓度依赖性实时荧光定量PCR和Western blotting结果表明,阿帕替尼可以诱导促凋亡基因 Bax和Caspase-3的表达,并抑制抗凋亡基因Bcl-2的表达.Western blotting检测信号通路蛋白的结果表明,在阿帕替尼处理后p-Akt 和p-Erkl/2的表达显著降低,而Akt和Erk总蛋白水平没有变化.结论 阿帕替尼可通过抑制MAPK/Erk、PI3K/Akt信号转导通路来实现诱导细胞凋亡,从而达到抑制HCT-116细胞增殖的目的.%Objective To investigate the inhibitory effects of apatinib on colorectal carcinoma HCT-116 cells in vitro and the signaling pathways involved.Methods The cytotoxicity of different concentrations (0,0.5,1,1.5,and 2 μmol/L) of apatinib in HCT-116 cells was assessed by MTT assay,using capecitabine as the positive control.The apoptosis rate of apatinib-treated HCT-116 cells was detected using flow cytometry,and the expressions of Bcl-2,Bax,and caspase-3 were determined with quantitative real-time PCR and Western blotting.The effect of apatinib on the expressions of Akt,pAkt,Erkl/2 and pErk1/2 in HCT-116 cells was evaluated using Western blotting.Results Apatinib significantly inhibited the proliferation of HCT-116 cells in a concentration-dependent manner with an IC50 value of 1.335 μmol/L.Flow cytometric analysis showed that apatinib significantly increased the apoptotic rate of HCT-116 cells dose-dependently.Apatinib induced the expression of the pro-apoptotic genes Bax and caspase-3 at both the mRNA and protein levels while inhibited the expression of the antiapoptotic gene Bcl-2.The expressions of p-Akt and p-Erkl/2 were decreased in HCT-116 cells after apatinib treatment,but the total protein levels did not undergo obvious changes.Conclusion Apatinib inhibits the proliferation and induces apoptosis of HCT-116 cells by suppressing the phosphorylation of Erkl/2 and Akt in the MAPK/Erk and PI3K/Akt signaling pathways.