Chk1与Chk2高表达对DADS阻滞人胃癌BGC823细胞G2/M期的影响

Effect of Chk1/2 Overexpression on the G2/M Arrest in Gastric Cancer BGC823 Cells Induced by Diallyl Disulfide

目的:在建立Chk1/2基因高表达人胃癌BGC823细胞的基础上,探讨Chk1/2基因在二烯丙基二硫(DADS)诱导人胃癌BGC823细胞G2/M期阻滞中的作用。方法:采用流式细胞术检测DADS作用于Chk1/2高表达BGC823细胞周期分布情况。Western blot检测Chk1、p-Chk1、Chk2、p-Chk2、Cdc25C与Cyclin B1表达变化。结果:流式细胞术显示,Chk1高表达组G2/M期细胞较对照组与空载体组增加(P<0.05)。而15 mg·L^(-1) DADS处理后,各组G2/M期细胞较处理前增加,Chk1高表达组较空载体组差异有统计学意义(P<0.05)。Chk2高表达组G2/M期细胞较对照组与空载体组差异无统计学意义(P>0.05)。而DADS处理Chk2高表达组后,G2/M期细胞较对照组与空载体组差异有统计学意义(P<0.05)。Western blot显示,Chk1/2蛋白及p-Chk2表达水平不受DADS的影响,但p-Chk1呈时间依赖性上调(P<0.05)。DADS处理Chk1高表达BGC823细胞12、24、36、48 h后,Cdc25C磷酸酶与Cyclin B1表达较对照组呈时间依赖性下降(P<0.05)。结论:DADS阻滞人胃癌BGC823细胞G2/M期与激活Chk1/Cdc25C/Cyclin B1通路有关。Chk1高表达可增强DADS阻滞G2/M期细胞的作用,而Chk2高表达对DADS无影响。

Objective : To investigate the role of Chk1/2 on the G2/M phase arrest in gastric cancer BGC823 cells induced by diallyl disulfide (DADS) on the basis of the establishment of Chk1/2 gene overexpression BGC823 cells. Methods : Flow cytometry was used to detect the cell cycle distribution in Chk1/2 overexpression BGC823 cells treated by DADS.The expressions of Chk1, p-Chk1,Chk2, p-Chk2, Cdc25C and Cyclin B1 were detected by Western blot. Results :Flow cytometry showed that the G2/M phase cells in Chk1 overexpression group were increased compared with the control group and the vector group ( P <0.05).After 15 mg·L^-1 DADS treatment,G2/M phase cells in each group were increased compared with those before treatment,and there was statistically significant of the difference between the Chk1 overexpression group and the vector group ( P <0.05). The G2/M phase cells in the Chk2 overexpression group exhibited no statistically significant of the difference with the control group and the vector group ( P >0.05). The G2/M phase cells were different from those in the control group and the vector group after DADS treated Chk2 overexpression group ( P <0.05). Western blot showed that the expression level of Chk1/2 protein and p-Chk2 was not affected by DADS, but the p-Chk1 was time-dependent increased( P <0.05). The expression of Cdc25C and Cyclin B1 showed time-dependent decrease after DADS treated Chk1 overexpression BGC823 cells for 12,24,36 and 48 h ( P <0.05). Conclusion :DADS arrest the G2/M phase in BGC823 cells is related to the activation of Chk1/Cdc25C/Cyclin B1 pathway.Chk1 overexpression can enhance the role of DADS arrest G2/M phase, while Chk2 overexpression has no effect on DADS.