7-乙酰基-鲁山冬凌草甲素诱导HL-60细胞周期阻滞及其机制研究  

HL-60 cell cycle arrest induced by 7-acetyl-lushanrubescensin A and its mechanisms

作　　者：刘高媛[1] 余祖胤[2] 善亚君[2] 柳晓兰[2] 赵振虎[2] 赵勤实[3] 陈丁丁[1] 从玉文[2] 

机构地区：[1]中国药科大学临床药学教研室,南京210009 [2]军事医学科学院放射与辐射医学研究所,北京100850 [3]中国科学院昆明植物研究所,昆明650204

出　　处：《军事医学科学院院刊》2010年第2期156-159,共4页Bulletin of the Academy of Military Medical Sciences

摘　　要：目的研究7-乙酰基-鲁山冬凌草甲素(7-acetyl-lushanrubescensin A,ARA)对人急性髓系白血病细胞株HL-60的细胞增殖和细胞周期的影响,并对其作用机制进行探讨。方法MTT法检测ARA对HL-60细胞增殖的影响;在ARA作用HL-60细胞不同时间后,流式细胞仪检测细胞周期变化;Western印迹检测周期相关蛋白Rb、p(phospho)-Rb(ser795)、p-Rb(ser807/811),细胞周期蛋白(cyclin)D1、cyclin D3、cyclin E2、CDK4及CDK6的蛋白表达;流式细胞仪检测细胞内活性氧(ROS)水平。结果ARA剂量依赖性地抑制HL-60细胞增殖,48h的IC50为(1.96±0.08)μmol/L。ARA诱导HL-60细胞发生明显的细胞周期G0/G1阻滞,具有时间和浓度依赖性。机制研究显示ARA引起Rb总蛋白出现移行条带,并抑制p-Rb(ser795)位点磷酸化,显著抑制CDK4的蛋白表达,并部分降低cyclin D3和cyclin E2的表达水平,但对cyclin D1和CDK6的作用则不明显。此外,ARA显著增加HL-60细胞内ROS水平,而抗氧化剂N-乙酰半胱氨酸(NAC)能够阻断ARA诱导的ROS升高及细胞周期阻滞。结论ARA能够明显抑制白血病细胞增殖,诱导细胞周期阻滞,其机制可能与抑制Rb活化和降低cyclin D3、cyclin E2和CDK4的表达水平相关,并且ROS在ARA介导的生物学效应中可能有重要作用。Objective To explore the effect of 7-acetyl-lushanrubescesin A（ARA）on cell proliferation and cell cycle of HL-60 myeloid leukemia cell line,and to further investigate the mechanism of action.Methods MTT assay was used to assess the proliferation of HL-60 cells treated with ARA.After treatment of HL-60 cells with ARA of different durations,cell cycle changes were determined by flow cytometry.The protein expression of Rb,p（phospho）-Rb（ser795）,p-Rb（ser807/811）,cyclin D1,cyclin D3,cyclin E2,CDK4 and CDK6 were detected by Western blot.The intracellular reactive oxygen species（ROS）level was measured by flow cytometry.Results ARA inhibited the proliferation of HL-60 cells in a concentration-dependent manner,with an IC50 of（1.96±0.08）μmol/L at 48 h.ARA induced significant G0/G1 phase arrest of HL-60 cells in a time-and concentration-dependent manner.Studies on the mechanisms underlying these effects indicated that treatment with ARA induced the rapidly migrating band of total Rb protein,and reduced the pRb phosphorylation on ser795.Furthermore,ARA markedly inhibited the protein expression of CDK4,and partially decreased expression of cyclin D3 and cyclin E2,but had no effect on cyclin D1 and CDK6.In addition,ARA significantly increased the intracellular ROS level of HL-60 cells,whereas antioxidant NAC blocked the ROS production and cell cycle arrest induced by ARA.Conclusion ARA can significantly inhibit proliferation and induce cell cycle arrest of leukemia cells.The mechanisms underlying these effects may be related to inhibition of pRb phosphorylation and down-regulation of cyclin D3,cyclin E2,and CDK4 protein expression.Furthermore,ROS production may play an important role in the biological effects of ARA.

关 键 词：7-乙酰基-鲁山冬凌草甲素 HL-60细胞 细胞增殖 细胞周期阻滞 活性氧 白血病 

分 类 号：R733.7[医药卫生—肿瘤]