黄药子醇提物对人肝癌细胞凋亡的影响及其机制

Effect and mechanism of ethanol extract of xanthate on apoptosis of human hepatoma cells

目的探讨黄药子醇提物对人肝癌细胞SMCC-7721凋亡影响及其机制。方法将SMCC-7721细胞株(购自中国科学院)根据黄药子醇提物的不同浓度分为:空白对照组、低浓度组、高浓度组。药物处理48 h后,以反转录-聚合酶链反应(RT-PCR)、蛋白质印迹法(Western blot)检测各组细胞程序性细胞死亡因子4(PDCD4)、半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3表达;流式细胞仪检测细胞周期及凋亡变化;平板克隆实验检测细胞增殖;Transwell侵袭实验检测细胞侵袭力,组间比较采用t检验。结果 PDCD4、Caspase-3 mRNA表达空白对照组、低浓度组分别为0.47±0.06、0.45±0.07;1.15±0.12、1.02±0.13,高浓度组为2.38±0.31、2.26±0.28明显高于上述两组(t=6.830、5.140,P<0.05);PDCD4、Caspase-3蛋白表达空白对照组、低浓度组分别为0.51±0.09、0.48±0.08;1.23±0.13、1.18±0.10,高浓度组为2.51±0.28、2.48±0.22,明显高于上述两组(t=6.560、5.890,P<0.05);细胞周期结果显示G0/G1期与S期细胞空白对照组、低浓度组分别为[(53.46±4.32)%、(30.95±3.42)%],[(67.32±5.32)%、(21.49±3.46)%],高浓度组为[(77.12±6.34)%、(14.65±2.72)%],G0/G1期细胞明显高于上述两组(t=5.750、4.230,P<0.05),S期细胞数明显低于上述两组(t=5.280、4.460,P<0.05);高浓度组凋亡率[(22.62±1.24)%]明显高于空白对照组和低浓度组[(3.12±0.63)%、(11.25±0.85)%,t=5.280、4.720,P<0.05];平板克隆实验显示高浓度组细胞克隆形成数[(115.54±14.64)个]明显低于空白对照组和低浓度组[(214.35±18.52)、(171.95±16.83)个,t=5.280、4.760,P<0.05];穿膜细胞数高浓度组为(87.63±15.76)个,明显低于空白对照组和低浓度组[(208.36±23.72)、(122.46±21.42)个,t=6.870、6.150,P<0.05]。结论黄药子醇提物调高PDCD4、Caspase-3表达,抑制SMCC-7721细胞的增殖,促进细胞凋亡。

Objective:To investigate the effect and mechanism of ethanol extract of xanthate on apoptosis of SMCC-7721 cells.Methods:The purchased SMCC-7721 cell line was different concentrations of ethanol extract,it was divided into three groups:blank control group,low concentration group and high concentration group.After 48 hours of drug treatment,Reverse transcriptase-polymerase chain reaction(RT-PCR)and Western blotting were used to detect the expression of programmed cell death 4(PDCD4)and cysteinyl aspartate-specific protease(Caspase)-3;flow cytometry was used to detect cell cycle and apoptosis;plate cloning was used to detect cell proliferation;Transwell invasion was used to detect cell invasion.Results:The expression of PDCD4 and Caspase-3 mRNA in blank control group and low concentration group were 0.47±0.06,0.45±0.07;1.15±0.12,1.02±0.13,in high concentration group were 2.38±0.31,2.26±0.28 significantly higher than those in the above two groups(t=6.830,5.140,P<0.05);the expression of PDCD4 and Caspase-3 protein in blank control group and low concentration group were 0.51±0.09,respectively(0.48±0.08,1.23±0.13,1.18±0.10),high concentration group(2.51±0.28,2.48±0.22)were significantly higher than the above two groups(t=6.560、5.890,P<0.05);cell cycle results showed that cell cycle of G 0/G 1 phase and S phase blank control group,low concentration group[(53.46±4.32)%,(30.95±3.42)%;(67.32±5.32)%,(21.49±3.46)%],high concentration group[(77.12±6.34)%,respectively(14.65±2.72)%],the cell cycle was blocked in G 0/G 1 phase(t=5.750、4.230,P<0.05),and the number of cells in S phase was decreased(t=5.280,4.460,P<0.05);the apoptosis rate in high concentration group was(22.62±1.24)%,which was significantly higher than that in blank control group and low concentration group[(3.12±0.63)%,(11.25±0.85)%,t=5.280,4.720,P<0.05];The number of cell clone formation in high concentration group(115.54±14.64)was significantly lower than that in blank control group and low concentration group(214.35±18.52,171.95±16.83,t=5.280,4.760,P<0.05),and that in high concentration group(87.63±15.76)was significantly lower than that in blank control group and low concentration group(208.36±23.72,122.46±21.42,t=6.870,6.150,P<0.05).Conclusion:Ethanol extract of xanthate can increase the expression of PDCD4 and Caspase-3,inhibit the proliferation of SMCC-7721 cells and promote apoptosis.