哇巴因通过阻遏激光激酶信号途径抑制肝癌细胞生长机制探索

The mechanism study of ouabain in inhibiting the growth of hepatocellular carcinoma cells by inhibiting the laser kinase signaling pathway

目的观察钠钾ATP酶抑制剂哇巴因抑制肝癌HepG2细胞的增殖和分裂效果,并探讨其作用机制。方法不同浓度(0.1、1.0、10.0μmol/L)哇巴因作用HepG2细胞24 h,以HepG2细胞为对照组,通过细胞活力检测试剂盒(CCK-8法)检测哇巴因对HepG2细胞增殖的抑制作用,细胞免疫荧光共定位(ICC法)检测细胞染色体分离状态,Western印迹检测极光激酶A(AURKA)、雷帕霉素靶蛋白(mTOR)、p-mTOR、促分裂原活化蛋白激酶(ERK)、p-ERK蛋白表达。结果0.1、1、10μmol/L哇巴因作用细胞24 h后,细胞生长抑制率分别为(23.50±4.57)%、(49.80±5.32)%和(72.10±5.62)%,与对照组相比差异均有统计学意义,抑制效果呈现浓度依赖性(F=32.8,均P<0.05);细胞免疫荧光共定位显示,1μmol/L哇巴因作用细胞24 h后,染色体分离发生障碍。与对照组相比,加药组细胞纺锤体直径显著较低,差异有统计学意义(t=9.58,P<0.05)。Western印迹结果显示,1μmol/L哇巴因作用HepG2细胞24 h后,与对照组相比,AURKA、p-mTOR、p-ERK表达较低(F=16.26,8.32,33.59;P<0.05),哇巴因可阻抑肝癌细胞在裸鼠体内生长(F=370.20,P<0.05)。结论哇巴因通过阻遏激光激酶信号途径,诱发肝癌HepG2细胞染色体分裂障碍、抑制肝癌细胞生长。

Objective To investigate the effect of Na+/K+-ATPase inhibitor ouabain on the proliferation and division of liver cancer HepG2 cells,and to explore the anticancer mechanism.Methods HepG2 cells were exposed with different concentrations of ouabain(0.1,1,10μmol/L)for 24 h,the proliferation ability was appraised using CCK-8,and the HepG2 cells was as a control group.The status of chromosome separation was detected with cell immunofluorescence(ICC)coupled to confocal microscope.The expression levels of AURKA,mTOR,p-mTOR,ERK and p-ERK protein were analyzed using western blot.Results After treating with 0.1,1 and 10μmol/L of ouabain for 24 h,the inhibitory rate of cells were(23.5±4.57)%,(49.80±5.32)%,and(72.10±5.62)%,respectively.Ouabain could significantly inhibit the proliferation of HepG2,and presented in a dose-dependent manner(F=32.8,P<0.05).The ICC results showed that the chromosome separation disorders occurred in HepG2 cells treated with 1μmol/L for 24 h,and the spindle diameter of HepG2 cells with ouabain treatment was decreased significantly compared with the control group(t=9.58,P<0.05).The results of western blot showed that the expression levels of AURKA,p-mTOR and p-ERK expressions in HepG2 cells treated with 1μmol/L of ouabain were significantly decreased compared with the control group(F=16.26,8.32,33.59,P<0.05).Ouabain inhibited the growth of hepatocellular carcinoma cells in nude mice(F=370.20,P<0.05).Conclusion Ouabain can induce chromosome division disorder and inhibit the proliferation in liver cancer HepG2 cells by inhibiting AURKA signaling pathway.