重楼皂苷Ⅰ诱导G_2/M期阻滞及干扰微管结构抗结肠癌HCT116细胞作用机制

Mechanism of Polyphyllin I in Inhibiting HCT116 Cells Based on Inducing G_2/M Arrest and Interfering Microtubule Structure

目的:探讨重楼皂苷Ⅰ(PPI)对人结肠癌HCT116细胞周期的影响,并研究其作用机制。方法:采用四甲基偶氮唑盐比色法(MTT)检测PPI对HCT116细胞的体外抑制活性;采用Hoechst33258染色法观察PPI对HCT116细胞核数量的影响;采用流式细胞仪检测PPI对HCT116细胞周期的影响;采用蛋白免疫印迹法(Western blot)检测细胞中细胞周期蛋白依赖性激酶(CDC2)和细胞分裂周期蛋白25同源蛋白C(CDC25C)蛋白的表达和活性,利用细胞免疫荧光和激光扫描共聚焦显微镜检测PPI对HCT116细胞微管的影响。结果:PPI以剂量-时间依赖的方式抑制HCT116的体外增殖,Hoechst 33258染色发现PPI处理组双核细胞数量明显增加(P<0.05,P<0.01);将其细胞周期阻滞于G2/M期;PPI未能抑制G2/M期相关蛋白CDC2,CDC25C的表达和活性,却有促进作用;PPI可导致细胞微管结构紊乱。结论:PPI会导致HCT116细胞阻滞在G2/M期,其作用机制与干扰细胞内微管结构有关。

Objective： To study the effect of polyphyllin I（ PPI） on the cell cycle of human colon cancer cell line HCT 116,and investigate the mechanism. Method： 3-（ 4,5-dimethyl-2 thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide（ MTT） assay was used to detect the inhibitory activity of PPI on HCT116 cells in vitro,then effect of PPI on the number of HCT116 cellular nuclear was observed by Hoechst 33258 staining; flow cytometry was used to detect the cell cycle changes of HCT116 cells. Western blot assay was used to detect the expression and activity of cell division cyclin2（ CDC2）,cell division cyclin25 homolog C（ cdc25c） protein in cells; and immunofluorescence and confocal laser scanning microscope were used to detect the effect of PPI on cell microtubules of HCT116 cells. Result： MTT assay showed that PPI inhibited the proliferation of HCT116 in a dosetime dependent manner. Hoechst 33258 nuclear staining showed that there the number of dual-core cells in PPI group was significantly increased（ P〈0. 05,P〈0. 01）; flow cytometry showed that the cell cycle was arrested in G2/ M phase. Western blot assay indicated that PPI could not inhibit but could promote the expression and activity of the G2/ M phase related proteins CDC2 and CDC25 C. In addition,in confocal laser scanning microscope detection,we found that PPI could lead to cell microtubules disorder. Conclusion： PPI could make the HCT116 cells arrested in G2/ M phase,and the action mechanism is related to interference with microtubule structure.