Hsp90抑制剂新生霉素诱导HL-60细胞凋亡及其机制

目的研究Hsp90抑制剂新生霉素(novobiocin,NB)诱导HL-60细胞凋亡的作用,探讨该作用与线粒体凋亡途径的关系,并进一步研究NB对Hsp90客户蛋白(clientprotein)AKT和ERK2功能的影响。方法细胞用NB处理后,采用AO/EB染色后荧光显微镜观察凋亡形态,采用AnnexinV-FITC/PI双染后流式细胞仪检测细胞凋亡率,分光光度法检测Caspase-9和Caspase-3的活性,用蛋白免疫印迹法检测细胞色素C的含量,以及procaspase-3、p-AKT(Ser473)和p-ERK2的蛋白水平。结果NB能明显抑制HL-60细胞增殖,IC50是0.3546mmol.L-1;NB能促进细胞色素C释放入胞质,激活Caspase-3/9的活性,触发HL-60细胞凋亡;NB能抑制AKT和ERK的功能,使细胞内p-AKT(Ser473)和p-ERK2的蛋白含量减少。结论NB可通过线粒体途径诱导HL-60细胞凋亡,还可干扰Hsp90伴侣功能阻断增殖信号通路,抑制HL-60细胞生长。

The mechanisms of apoptosis in HL-60 cells induced by Hsp90 inhibitor novobiocin

Aim To confirm the effects of Hsp90 inhibitor NB on HL-60 cells,and reveal the relationship between NB-induced apoptosis and mitochondrion pathway;to further clarify the mechanism of growth inhibition,and the functions of Hsp90 client protein AKT and ERK2 were also measured.Methods When HL-60 cells were treated with NB,the typical morphological changes of apoptosis were observed by AO/EB examined by fluorescence microscopy and Annexin V-FITC/PI examined by flow cytometer.The activities of Caspase-9 and Caspase-3 were analyzed by spectrophotometry.The amounts of cytochrome C in cytosolic and S-100 fraction,procaspase-3,p-ERK2,and p-AKT(Ser473)were tested by Western blot.Results NB was a potent inhibitor of proliferation of HL-60 cells with IC50 values of 0.3546 mmol·L-1;NB induced cytosolic accumulation of cytochrome C and activities of Caspase-3/9,triggering apoptosis of HL-60 cells.NB inhibited the function of AKT and ERK through downregulation of p-ERK2 and p-AKT(Ser473).Conclusion NB was able to induce the apoptosis of HL-60 cells by mitochondrion pathway.The HL-60 cells growth inhibition was also involved in the disruption of Hsp90 chaperon function.