新型磺酰胺类肿瘤抑制剂DHW-51诱导宫颈癌细胞HeLa凋亡的作用机制

Mechanism of new sulfonamides tumor inhibitor DHW-51 in inducing apoptosis in HeLa cells

目的从新合成的甲磺酰胺类化合物库中筛选出一种新型肿瘤抑制剂DHW-51,初步探讨DHW-51对宫颈癌细胞HeLa凋亡的影响及其作用机制。方法采用细胞增殖抑制实验检测DHW-51作用HeLa细胞12、24和48h时的细胞存活率,分为对照组和10、15、20、25、30μmol/L实验组共6组;流式细胞术检测不同浓度DHW-51诱导HeLa细胞的凋亡率及细胞周期;检测细胞内Caspase 3、Caspase 8和Caspase 9活性及细胞凋亡相关蛋白pro-Caspase 3、pro-Caspase8和poly ADP-ribose polymerase-DNA修复酶(PARP)的表达,分析DHW-51的作用机制。结果DHW-51对HeLa细胞的增殖具有良好抑制作用。当DHW-51浓度为25μmol/L作用时间为24h时,HeLa细胞的存活率仅为(27.34±1.891)%,与对照组相比差异有统计学意义,F=1.369,P<0.001;随着浓度增加,作用时间延长细胞存活率更低。DHW-51能够有效诱导HeLa细胞凋亡,呈浓度和时间依赖关系;当DHW-51浓度为25μmol/L作用时间为24h时,细胞凋亡率为(92.10±1.683)%,与对照组相比差异有统计学意义,t=43.715,P<0.001;随着时间的延长,晚期凋亡的比例逐步增加,而对细胞周期则没有影响。当DHW-51浓度为20μmol/L时,Caspase 3[(1.87±0.063)%,t=12.43,P<0.001]和Caspase 9[(2.0±0.069)%,t=9.519,P<0.05]的活性增加,与对照组相比,差异有统计学意义;Caspase 8的活性无变化。蛋白质印迹结果显示,当DHW-51浓度为20μmol/L时,pro-Caspase 3[(0.33±0.005)%,t=-89.334,P<0.001]和pro-Caspase 9[(0.19±0.009)%,t=-48.308,P<0.001]蛋白表达量下调,pro-Caspase 8蛋白表达无变化,PARP[(0.35±0.012)%,t=14.961,P<0.001]出现断裂带。结论新型磺酰胺类化合物DHW-51可以诱导宫颈癌HeLa细胞凋亡。作用机制初步推测主要是通过由线粒体介导的Caspase依赖途径诱导HeLa细胞凋亡,进而实现抑制肿瘤细胞生长,这一过程与细胞周期阻滞无关。

Objective To screen a new tumor inhibitor,DHW-51,from the newly synthesized sulfonamide compounds,and explored its effect and mechanism on the apoptosis in HeLa cells.Methods First,this study detected the survival rate of HeLa cells treated with various concentrations of DHW-51 for 12,24 and 48 hby the cell proliferation inhibition experiments,there were 6 groups:control,10,15,20,25 and 30μmol/L.Flow cytometry was adopted to further analyze the apoptotic rate and cell cycle of Hela treated by DHW-51 with different concentrations.This study also examined the activity of intracellular Caspase 3,Caspase 8,Caspase 9 and the expression of apoptosis-related proteins including pro-Caspase 3,pro-Caspase 8,pro-Caspase 9 and PARP and analyzed the mechanism of DHW-51.Results DHW-51 showed a good inhibition on the proliferation of HeLa cells.When the cells were treated with DHW-51 at 25μmol/L for 24 hours,the survival rate of HeLa cells was only(27.34±1.891)%,compared to the control group(F=1.369,P<0.001),the difference was statistically significant.The cell survival rate of cells decreased with the increase of DHW-51 concentration and the extension of incubation time.DHW-51 can effectively induce the apoptosis of HeLa cells in a dose-and time-dependent manner.When 25μmol/L DHW-51 was added to the cells for 24 hours,the apoptosis rate of cells is(92.10±1.683)%,t=43.715,P<0.001,compared to the control group.The proportion of late apoptotic cells was gradually increased,without affecting the cell cycle.When the concentration of DHW-51 reached to 20μmol/L,the activities of Caspase 3[(1.87±0.063)%,t=12.43,P<0.001]and Caspase 9[(2.0±0.069)%,t=9.519,P<0.05]were increased,with statistically significant,and the activity of Caspase 8 remained unchanged,compared to the control group.Western blot results showed that DHW-51 is 20μmol/L,the expressions of pro-Caspase 3[(0.33±0.005)%,t=-89.334,P<0.001]and pro-Caspase 9[(0.19±0.009)%,t=-48.308,P<0.001]protein were down-regulated,the expression of pro-Caspase 8 protein remained unchanged,and PARP[(0.35±0.012)%,t=14.961,P<0.001]had a cleavage.Conclusion This new sulfonamide compound DHW-51 can effectively induce the apoptosis of HeLa cell through the mitochondria-mediated Caspase activation pathway,thereby inhibiting the growth of tumor cells,which has nothing to do with cell cycle.