蛋白酶体抑制剂硼替佐米诱导髓系白血病细胞株 HL60凋亡的机制研究

Apoptosis of myeioid leukemia cell line HL60 induced by Bortezomib,a proteasome inhibitor

目的探讨蛋白酶体抑制剂硼替佐米诱导髓系白血病细胞 HL60凋亡的机制。方法四甲基偶氮唑盐(MTT)法、Hoechst33342染色形态学观察及流式细胞仪证实细胞凋亡,Western 印迹检测 B 细胞淋巴瘤/白血病-2(Bcl-2)、Caspase-9、Caspase-3、多聚 ADP-核糖聚合酶(PARP)蛋白表达。结果硼替佐米对 HL60细胞的增殖抑制作用呈浓度和时间依赖关系,30 nmol/L 的硼替佐米作用24 h 能明显抑制 HL60细胞增殖,抑制率为76％,形态学观察可见明显的胞核凝聚、固缩及碎裂;流式细胞仪检测可见明显的凋亡峰凋亡率为62.6％;Western 印迹检测显示 Bcl-2表达下降,Caspase-9、Caspase-3、PARP 蛋白均裂解激活。结论硼替佐米能诱导髓系白血病细胞 HL60凋亡,其机制与Bcl-2蛋白的抑制及 Caspase 凋亡信号途径的激活有关。

Objective To explore the mechanism of apoptosis of myeloid leukemia cells induced by Bortezomib, a proteasome inhibitor. Methods Human acute myeloid leukemia cells of the line HL60 were cultured and treated with Bortezomib of the concentrations of 0, 10, 20, 30, and 40μmol/L for 24 hours. MTr assay and flow cytometry were used to detect the proliferation inhibition and apoptosis. Hoechst33342 staining was used to observe the morphology of the cells. Western blotting was used to detect the protein expression of Bcl-2, Caspase-9, Caspase-3, and poly ADP-ribose polymerase （PARP）. Results Bortezomib could induce HL60 cell apoptosis dose- and time- dependently. After treated for 24 hours by 30 nmol/L Bortezomib the HL60 cells＇ proliferation was significantly inhibited, the inhibition rate was 76%, and the cell nuclei became progressively pyknotic and were extensively fragmented. FCM showed apoptosis peaks 24 hours after treatment of Bortezomib of the concentrations of were 10 and 20 nmol/L, the apoptosis rate was 62.6%. Bcl-2 protein expression was down-regulated and the protein expressions of Caspase-9, Caspase-3, and PARP were all up-regulated. Conclusion The mechanism of Bortezomib to induce apoptosis of myeloid leukemia cells is associated with down-regulation of Bcl-2 protein expression and cleaved activation of Caspase-9, Caspase-3 and PARP proteins.