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

Inhibitory effect of apatinib on HCT-116 cells and its mechanism

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、2μmol/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 VFITC/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、Erk1/2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1.335μmol/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-Erk1/2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,Erk1/2 and p Erk1/2 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 proapoptotic genes Bax and caspase-3 at both the mRNA and protein levels while inhibited the expression of the anti-apoptotic gene Bcl-2. The expressions of p-Akt and p-Erk1/2 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 Erk1/2 and Akt in the MAPK/Erk and PI3K/Akt signaling pathways.