冬凌草甲素对肝癌细胞增殖、凋亡及mTOR/P70S6K信号通路的影响

Effect of Oridonin on Proliferation and Apoptosis of Liver Cancer Cells as well as mTOR/P70S6K Signaling Pathway

目的:探讨不同浓度的冬凌草甲素对肝癌SMMC-7721细胞生物学行为及对哺乳动物雷帕霉素靶蛋白(mTOR)/P70S6激酶(P70S6K)信号通路的影响。方法:将人肝癌SMMC-7721细胞分为肝癌组及冬凌草甲素低、中、高剂量组(以下简称低、中、高剂量组)。肝癌组不作任何处理,低、中、高剂量组分别用浓度为5、20、40μmol/L的冬凌草甲素溶液处理。四甲基偶氮唑盐(MTT)法检测各组SMMC-7721细胞活性,流式细胞仪检测各组细胞凋亡率,蛋白免疫印迹法和PCR法分别检测各组SMMC-7721细胞中mTOR、P70S6K蛋白及mRNA表达。结果:细胞形态学观察显示,肝癌组细胞呈现单层生长,互相堆积,形态为梭形并呈现继续生长;低、中、高剂量组细胞形态逐渐发生改变,并随着药物浓度的提高,细胞数目减少,核缩严重并出现大面积凋亡。与肝癌组比较,中、高剂量组各时间点细胞抑制率均升高(P<0.05);与低剂量组比较,中、高剂量组各时间点细胞抑制率均升高(P<0.05);与中剂量组比较,高剂量组各时间点细胞抑制率升高(P<0.05);且中、高剂量组细胞抑制率均随着时间的增加而升高。与肝癌组比较,中、高剂量组细胞凋亡率升高(P<0.05),mTOR、P70S6K蛋白及mRNA表达均下降(P<0.05);与低剂量组比较,中、高剂量组细胞凋亡率升高(P<0.05),mTOR、P70S6K蛋白及mRNA表达均降低(P<0.05);与中剂量比较,高剂量组细胞凋亡率升高(P<0.05),mTOR、P70S6K蛋白及mRNA表达均降低(P<0.05)。结论:冬凌草甲素能够促进人肝癌SMMC-7721细胞凋亡、抑制其活性,其减少mTOR/P70S6K信号通路活性可能与细胞凋亡机制相关。

Objective:To discuss the effect of oridonin of different concentrations on biological behavior of liver cancer cells SMMC-7721 and mammalian target of rapamycin(mTOR)/P70 S6 kinase(P70 S6 K) signaling pathway. Methods:Human liver cancer cells SMMC-7721 were divided into the liver cancer group and the oridonin groups of low,medium,and high dose(low-dose group, medium-dose group, and high-dose group for short). The liver cancer group did not receive any treatment;the low-dose, medium-dose, and high-dose groups were given 5, 20, and 40 μmol/L oridonin solution respectively. Methyl thiazolyl tetrazolium(MTT) assay was used to detect cell activity in each group. Flow cytometry was used to detect rate of cell apoptosis in each group. The expression of protein and mRNA of mTOR/P70 S6 K in each group were detected by western blotting and PCR method respectively. Results:According to cytomorphology observation,in the liver cancer group,the cells displayed monolayer growth,accumulated on each other,and appeared morphologically spindleshaped,with a trend of continuous growth. According to cytomorphology observation,in the low-dose,medium-dose,and high-dose groups, cell morphology gradually changed;with the increase of concentration of the solution, the cell number was decreased,along with severe shrinkage of nucleus and extensive apoptosis. Compared with those in the liver cancer group,in the medium-dose and high-dose groups,the inhibition rates of cell at each time point were increased(P<0.05);compared with those in the low-dose group,in the medium-dose and high-dose groups,the inhibition rates of cell at each time point were increased(P<0.05);compared with those in the medium-dose group,in the high-dose group,the inhibition rates of cell at each time point were increased(P<0.05);the inhibition rates of cell were increased with time prolonging in the medium-dose and high-dose groups. Compared with those in the liver cancer group,in the medium-dose and high-dose groups, the rates of cell apoptosis were increased(P<0.05), and the expression of protein and mRNA of mTOR and P70 S6 K was decreased(P<0.05);compared with those in the low-dose group,in the medium-dose and highdose groups,the rates of cell apoptosis were increased(P<0.05),and the expression of protein and mRNA of mTOR and P70 S6 k was decreased(P<0.05);compared with those in the medium dose group,in the high-dose group,the rate of cell apoptosis was increased(P<0.05),and the expression of protein and mRNA of mTOR and P70 S6 k was decreased(P<0.05).Conclusion: Oridonin can promote apoptosis of human liver cancer cells SMMC-7721 and inhibit their activity. Lowering activity of mTOR/P70 S6 K signaling pathway by oridonin may be related to cell apoptosis mechanism.