阿帕替尼对SMMC-7721肝癌细胞增殖的抑制作用及机制研究

Inhibiting effect of apatinib on the proliferation of SMMC-7721 hepatocellular carcinoma cell and its possible mechanism

目的观察阿帕替尼对SMMC-7721肝癌细胞株增殖的抑制作用,探讨其可能的作用机制及其影响的信号通路。方法MTT方法检测不同浓度的阿帕替尼对SMMC-7721肝癌细胞株增殖的抑制作用。Annexin V-FITC/PI双染色法检测不同浓度阿帕替尼处理后SMMC-7721肝癌细胞的凋亡情况。Real-time PCR检测不同浓度阿帕替尼处理组凋亡相关基因Bcl-2、Bax和Caspase-9的表达。Western blot检测不同浓度阿帕替尼处理组Bcl-2、Bax、Caspase-9、PI3K、p-PI3K、Akt、p-Akt、ERK、p-ERK蛋白的表达。结果MTT和Annexin V-FITC/PI双染色法检测结果表明,阿帕替尼有效抑制了SMMC-7721肝癌细胞的体外增殖并明显诱导其凋亡,且与阿帕替尼浓度呈正相关关系。Real-timePCR检测和Western blot检测结果表明,阿帕替尼诱导了促凋亡基因Bax和Caspase-9的表达,并抑制了抗凋亡基因Bcl-2的表达。Western blot检测相关信号通路蛋白结果表明,阿帕替尼处理后p-PI3K、p-ERK与p-Akt表达水平显著降低,PI3K、ERK与Akt的总蛋白表达水平无明显变化。结论阿帕替尼可以通过抑制MAPK/ERK、PI3K/Akt信号转导通路、上调Bax和Caspase-9的表达、下调Bcl-2的表达来抑制SMMC-7721肝癌细胞的增殖并诱导其凋亡。

Objective To explore the inhibiting effect of apatinib on the proliferation of the SMMC-7721 hepatocellular carcinoma cell,discuss its possible mechanism and the affected signal pathway.Methods The MTT assay was used to detect the inhibiting effect of different concentrations of apatinib on the proliferation of SMMC-7721 hepatocellular carcinoma cell.Annexin V-FITC/PI double staining method was performed to investigate the apoptosis of SMMC- 7721 cells after dealed with different concentrations of apatinib.Real-Time PCR was used to detect the expression of the apoptosis-related genes Bcl-2,Bax and caspase-9.The expression of Bcl-2,Bax,Caspase-9,PI3K,p-PI3K,Akt,p- Akt,ERK,p-ERK proteins was explored by Western blot.Results The results of MTT assay and Annexin V-FITC/PI double staining showed that apatinib inhibited the vitro proliferation of SMMC-7721 cells effectively and induced the obvious apoptosis of it,which was related to the concentration of apatinib.The results of Real-Time PCR and Western blot analysis showed that apatinib induced the expression of pro-apoptotic genes Bax and Caspase-9 and inhibited the expression of anti-apoptotic gene Bcl-2.The related signal pathway protein detected by western blot analysis showed that the expression of p- PI3K,p- ERK and p- Akt was significantly decreased after apatinib treatment,while the expression of PI3K,ERK and Akt had no significant difference.Conclusion By inhibiting the MAPK/ERK,PI3K/Akt signal pathway,up-regulating the expression of Bax and Caspase-9,down-regulating the expression of Bcl-2,apatinib is capable of suppressing the proliferation of SMMC-7721 hepatocellular carcinoma cells and promoting their apoptosis.