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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 investigate the effect of 7-acetyl-lushan rubescensin A (ARA) on cell proliferation and cell cycle in human acute myeloid leukemia HL-60 cells and its mechanism of action Discussion. Methods MTT assay was used to detect the effect of ARA on the proliferation of HL-60 cells. Flow cytometry was used to detect cell cycle changes after HL-60 cells were treated with ARA for different time. Western blotting was used to detect the expression of Rb, p (phospho) -Rb (ser795 ), P-Rb (ser807 / 811), cyclin D1, cyclin D3, cyclin E2, CDK4 and CDK6, respectively. The level of reactive oxygen species (ROS) was detected by flow cytometry. Results ARA inhibited the proliferation of HL-60 cells in a dose-dependent manner. The IC50 of 48h was (1.96 ± 0.08) μmol / L. ARA induced significant cell cycle G0 / G1 arrest in HL-60 cells in a time and concentration dependent manner. Mechanistic studies have shown that ARA causes a total of Rb total protein bands and inhibits p-Rb (ser795) phosphorylation, significantly inhibit CDK4 protein expression, and partially reduce the expression of cyclin D3 and cyclin E2, but cyclin D1 and The role of CDK6 is not obvious. In addition, ARA significantly increased the level of ROS in HL-60 cells, while antioxidant N-acetylcysteine (NAC) blocked ARA-induced ROS elevation and cell cycle arrest. Conclusions ARA can significantly inhibit leukemic cell proliferation and induce cell cycle arrest. The mechanism may be related to inhibiting Rb activation and decreasing the expression of cyclin D3, cyclin E2 and CDK4, and ROS may play an important role in ARA-mediated biological effects effect.