马钱子碱对人结肠癌HT-29细胞增殖、细胞周期的影响及其作用机制

Effects of brucine on cell proliferation and cell cycle of human colon cancer HT-29 cells and its mechanism

目的:探讨马钱子碱对人结肠癌HT-29细胞增殖、细胞周期的影响及其作用机制。方法:体外培养HT-29细胞,加入不同浓度马钱子碱,用MTT法检测对HT-29细胞增殖的影响;应用流式细胞术检测对HT-29细胞周期的影响。采用Western blot法检测细胞中Bcl-2、caspase3、caspase9、P53、c-PARP的表达。结果:马钱子碱对人结肠癌HT-29细胞增殖有明显的抑制作用,且与浓度和作用时间呈正向关系,抑制作用在浓度为1 000μmol/L培养72h时最强,达到(98.54±0.68)%;马钱子碱(250μmol/L)干预48h后,可诱使HT-29细胞停滞在G1期(P<0.01)。250、500μmd/L马钱子碱可上调细胞中caspase3、caspase9、P53、c-PARP蛋白的表达,下调Bcl-2蛋白的表达,与空白对照组比较具有显著性差异(P<0.01)。结论:马钱子碱对人结肠癌细胞具有显著的抑制作用,且呈现剂量-效应和时间-效应关系,可诱导细胞在G1期发生阻滞,其抑制机制可能是阻滞细胞周期,影响促凋亡蛋白caspase3、caspase9、c-PARP、P53及抑癌蛋白Bcl-2的表达。

Objective:To determine cell proliferation,cell cycle and related mechanism of brucine on human colon cancer HT-29 cells.Methods:HT-29 cells were treated with brucine at various concentrations to determine cell proliferation by MTT assay in vitro;The effects on cell cycle of HT-29 cells were detected by flow cytometry.Western blot was employed to detect the expressions of Bcl-2,caspase3,caspase9,P53,c-PARP protein.Results:Brucine had inhibitory effect on the proliferation of HT-29 cells,and its inhibitory effect had a positive relationship with concentration and time,the most significant effect concentration was 1000μmol/L after cultured 72 h,the inhibitory rate was(98.54±0.68)%;HT-29 cells was arrested at the G1 phase with brucine’s(250μmol/L)intervention of 48 h(P<0.01).The expressions of caspase3,caspase9,P53,c-PARP protein were up-regulated by 250μmol/L and 500μmol/L brucine,the expressions of Bcl-2 protein were down-regulated,the difference was statistically significant compared with the blank control group(P<0.01).Conclusion:Brucine has significant inhibitory effect on human colon cancer cell,and has dose-dependent and time-dependent the effect,which could induce HT-29 cells in G1 phase,the mechanism of inhibitory effect may be related to the arrest of cell cycle,and effect on expression of apoptosis stimulating proteins caspase3,caspase9,c-PARP,P53 and suppressor protein Bcl-2.