Ji Xiao-Xia, Liu Fang, Xia Hong, He Jie, Tan Hui, Yi Lan, Su Qi
Department of Pathology,Yuncheng Muinicipal Central Hospital , Yuncheng 044000, Shanxi Province, China; Key Laboratory of Cancer And Moleculay Pathology, Institute of Oncology, University of South China, Hengyang 421001, Hunan Province, China.
Key Laboratory of Cancer And Moleculay Pathology, Institute of Oncology, University of South China, Hengyang 421001, Hunan Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2018 Jun;26(3):750-755. doi: 10.7534/j.issn.1009-2137.2018.03.020.
To investigate the inducing effect of down-regulation of MCL-1 by diallyl disulfide(DADS) on the G/M arrest of human leukemia K562 cells and its mechanisms.
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.
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 G/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 G/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).
DADS can inhibit the K562 cell proliferation and induce them arrest G/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.
探讨二烯丙基二硫化物（DADS）下调髓细胞白血病-1（MCL-1）对人白血病K562细胞G/M期阻滞的诱导作用及其机制。
采用CCK-8法检测DADS对K562细胞增殖的影响，运用流式细胞术观察DADS及RNA干扰沉默MCL-1基因对K562细胞周期阻滞的影响。采用蛋白质免疫印迹法检测DADS处理后K562细胞中MCL-1、增殖细胞核抗原（PCNA）和周期蛋白依赖性激酶1（CDK1）的表达。通过免疫共沉淀检测MCL-1与PCNA和CDK1的结合情况。
CCK-8检测显示，15、30、60、120、240 μmol/L DADS处理K562细胞的抑制率分别为32.48%、59.34%、66.42%、77.06%、81.05%（P<0.05）。流式细胞术分析表明，60和120 μmol/L DADS处理K562细胞24 h和48 h后，G/M期细胞百分比分别升高至18.6%和34.4%、17.5%和28.5%（P<0.05）。沉默MCL-1后G/M期细胞百分比显著升高（P<0.05）。与单独使用DADS或MCL-1相比，MCL-1+DADS对细胞的沉默作用增强更显著（P<0.05）。蛋白质免疫印迹显示，DADS可显著下调MCL-1、PCNA和CDK1的表达（P<0.05）。免疫共沉淀显示，MCL-1与PCNA和CDK1结合形成异二聚体，DADS处理K562细胞8 h后，其表达分别比对照组更显著下调（P<0.05）。
DADS可通过下调MCL-1抑制K562细胞增殖并诱导其G/M期阻滞，进而降低人白血病K562细胞中PCNA和CDK1的表达。此外，沉默MCL-1可增强DADS的作用效果。
