二烯丙基二硫通过磷酸化Chk1负调控Cdc25C/CyclinB1/CDK1通路阻滞白血病K562细胞G_(2)/M期

Phosphorylated Chk1 by DADS blocks the G_(2)/M phase of K562 cells via Cdc25C/CyclinB1/CDK1 pathway

目的:研究二烯丙基二硫(diallyl disulfide,DADS)诱导人白血病K562细胞周期阻滞及其分子机制。方法:采用CCK-8、细胞计数及流式细胞术观察DADS对K562细胞增殖与周期阻滞效应。Western Blot检测DADS对K562细胞PCNA、Chk1/2以及下游分子Cdc25C、CyclinB1与CDK1表达的影响。结果:CCK-8检测显示,15μmol/L、30μmol/L、60μmol/L、120μmol/L、240μmol/L DADS处理后,呈浓度依赖性抑制K562细胞增殖,抑制率分别为32.48%、59.34%、66.42%、77.06%、81.05%(P<0.05)。对照组与Tween80组无抑制作用(P>0.05)。细胞计数结果显示,30μmol/L、60μmol/L与120μmol/L DADS处理K562细胞后,群体倍增时间分别为22.71±0.29、36.69±0.93与73.02±0.87呈浓度依赖性增加(P<0.05),而Tween80组与对照组无明显差异(P>0.05)。流式细胞术检测显示,60μmol/L与120μmol/L DADS作用K562细胞24 h与48 h后,G_(2)/M期百分率分别增加到17.6%与28.5%和18.6%与34.4%,较对照组有显著性差异(P<0.05)。60μmol/L DADS作用K562细胞1 h、2 h、4 h、8 h和24 h后,PCNA表达呈时间依赖性表达下调(P<0.05)。p-Chk1表达呈时间依赖性上调(P<0.05),而Chk1、Chk2与p-Chk2表达无明显差异(P>0.05)。并且,Cdc25C、CyclinB1和CDK1分别呈时间依赖性下调(P<0.05),但是,14-3-3蛋白无明显改变(P>0.05)。结论:DADS可磷酸化Chk1通过Cdc25C/CyclinB1/CDK1通路抑制K562细胞增殖与阻滞G_(2)/M期。

Objective:To study the cell cycle arrest induced by diallyl disulfide(DADS)in human leukemia K562 and its molecular mechanism.Methods:CCK-8,cell counting and flow cytometry were used to observe the effect of DADS on K562 cell proliferation and cycle arrest.Western Blot analysis of the effects of DADS on the expression of PCNA,Chk1/2 and downstream molecules Cdc25C,CyclinB1 and CDK1 in K562 cells.Results:CCK-8 assay showed that 15μmol/L,30μmol/L,60μmol/L,120μmol/L,240μmol/L DADS treatment inhibited K562 cell proliferation in a concentration-dependent manner with inhibition rates of 32.48%,59.34%,66.42%,77.06%,81.05%,respectively(P<0.05).Control group and Tween80 group had no inhibitory effect(P>0.05).Cell counting results showed that the population doubling time of K562 cells treated with 30μmol/L,60μmol/L and 120μmol/L DADS was increased by 22.71±0.29,36.69±0.93 and 73.02±0.87 in a concentration-dependent way,respectively(P<0.05).There was no significant difference between Tween80 group and control group(P>0.05).Flow cytometry showed that the percentage of G_(2)/M phase increased to 17.6%and 28.5%and 18.6%and 34.4%after 60μmol/L and 120μmol/L DADS treatment of K562 cells for 24 h and 48 h,respectively,and there was a significant difference compared with the control group(P<0.05).PCNA expression in K562 cells was down-regulated in a time-dependent manner after 60μmol/L DADS treatment for 1 h,2 h,4 h,8 h and 24 h(P<0.05).The expression of p-Chk1 was up-regulated in a time-dependent manner(P<0.05),while the expression of Chk1,Chk2 and p-Chk2 had no significant differences(P>0.05).In addition,Cdc25C,CyclinB1 and CDK1 were down-regulated in a time-dependent manner(P<0.05),but 14-3-3 protein had no significant changes(P>0.05).Conclusion:DADS can phosphorylate Chk1 to inhibit K562 cell proliferation and block G_(2)/M phase through Cdc25C/CyclinB1/CDK1 pathway.