二烯丙基二硫下调MCL-1诱导K562细胞G2/M阻滞及其机制

Downregulation of MCL-1 by Diallyl Disulfide Induces G_2/M Arrest in Human Leukemia K562 Cells and Its Mechanism

目的:研究二烯丙基二硫(diallyl disulfide,DADS)下调MCL-1诱导人白血病K562细胞周期阻滞及其机制。方法:采用CCK-8检测DADS对K562细胞增殖的作用;流式细胞术观察DADS与沉默MCL-1对K562细胞周期的影响;Western blot检测MCL-1、PCNA和CDK1表达;免疫共沉淀方法分析MCL-1与PCNA及CDK1结合作用。结果:15、30、60、120、240μmol/L DADS对K562细胞增殖抑制率分别为32.48%、59.34%、66.42%、77.06%和81.05%(P<0.05)。60和120μmol/L DADS分别作用K562细胞24和48 h后,G_2/M期细胞分别增加到18.6%与34.4%和17.5%与28.5%(P<0.05)。沉默MCL-1可阻滞K562细胞于G_2/M,沉默MCL-1加DADS(60μmol/L)较单独DADS和沉默MCL-1明显增强(P<0.05)。DADS可明显下调MCL-1、PCNA与CDK1表达(P<0.05)。DADS处理K562细胞8 h,MCL-1与结合的PCNA和CDK1较对照组显著下调(P<0.05)。结论:DADS可通过下调MCL-1继而降低PCNA与CDK1表达,抑制K562细胞增殖,细胞阻滞于G_2/M期,沉默MCL-1可增强DADS的作用。

Objective： To investigate the inducing effect of down-regulation of MCL-1 by diallyl disulfide（ DADS）on the G2/M arrest of human leukemia K562 cells and its mechanisms.Methods： CCK-8 was used to detect the effect of DADS on proliferation of K562 cells,flow cytometry was employed to observe the effect of cycle arrest by DADS and RNAi silencing MCL-1 gene in K562 cells.The expressions of MCL-1,PCNA and CDK1 in K562 cells treated with DADS were detected by Western blot.The amphigamy of MCL-1 with PCNA and CDK1 was detected by Coimmunoprecipitation.Results： CCK-8 detection showed that the inhibition rates of K562 cells treated with 15,30,60,120,240 μmol/L DADS were 32.48%,59.34%,66.42%,77.06%,81.05% respectively（ P〈0.05）.Flow cytometry analysis revealed that the perecentages of G2/M cells were increased to 18.6% and 34.4%,17.5% and 28.5%,respectively at 24 and 48 h after treating K562 cells with 60 and 120 μmol/L DADS（ P〈0.05）.And the perecentage of G2/M cells of silencing MCL-1 was significantly increased（ P〈0.05）.Silencing effects of MCL-1 ＋DADS on the cells were enhanced more significantly as compared with DADS or MCL-1 alone（ P〈0.05）.Western blot exhibited that DADS could markedly downregulate the expression of MCL-1, PCNA and CDK1（ P〈0.05）.Coimmunoprecipitation revealed that MCL-1 bound with PCNA and CDK1,then forming heterodimers,which were downregulated respectively more significantly than that in the control group after treating K562 cells with DADS for 8 h（P〈0.05）.Conclusion： DADS can inhibit the K562 cell proliferation and induce them arrest G2/M through downregulation of MCL-1, then decreasing the expression of PCNA and CDK1 in hunan leukemia K562 cells.Moreover,silencing MCL-1 can enhance the effect of DADS.