HSP90C-端抑制剂新生霉素与HSP90N-端抑制剂联合应用对白血病细胞的作用

目的体外研究HSP90 C-端抑制剂新生霉素(novobio-cin,NB)与HSP90 N-端抑制剂格尔德霉素(Geldnamycin,GA)的联合应用对Bcr-Abl-的HL-60细胞和Bcr-Abl+的HL-60/Bcr-Abl细胞增殖抑制及诱导凋亡的作用及该作用与细胞色素C释放、Caspase-9/3活性的关系,并且研究序贯应用NB和GA对HL-60细胞的作用。方法细胞用NB和GA联合处理后,采用AO/EB和AnnexinV/PI双染检测细胞凋亡,分光光度法检测Caspase-9/3的活性,用蛋白免疫印迹法检测细胞色素C和procaspase-3的含量。用MTT法检测NB→GA和GA→NB序贯处理对HL-60细胞增殖的抑制作用。结果NB和GA合用抑制HL-60细胞增殖有单独相加的作用;NB和GA合用激活HL-60和HL-60/Bcr-Abl细胞中Caspase-3/9的活性程度高于单独使用NB或GA,协同触发细胞凋亡;预先用NB处理可增强HL-60细胞对GA的敏感性,相比之下,预先用GA处理不影响HL-60细胞对NB的敏感性。结论NB和GA合用可协同抑制白血病细胞生长,诱导细胞凋亡;NB可增强GA对HL-60细胞的抑制能力,而GA不影响NB对HL-60细胞的抑制作用。

Effects of cotreatment or sequence treatment with HSP90 C-terminal inhibitor NB and HSP90 N-terminal inhibitor GA on leukemia cells

Aim To elucidate the effects of cotreatment with HSP90 C-terminal inhibitor NB and HSP90 N-terminal inhibitor GA on proliferation or induction of apoptosis of Bcr-Abl-HL-60 cells and Bcr-Abl+ HL-60/Bcr-Abl cells,and reveal the relationship between apoptosis and mitochondrion pathway;to further investigate the effect of sequence treatment with NB and GA on HL-60 cells.Methods When cells were treated with NB and GA,the typical morphological changes of apoptosis were examined by fluorescence microscopy after AO/EB staining or by flow cytometer with Annexin V/PI staining.The activities of Caspase-9 and Caspase-3 were analyzed by spectrophotometry.The level of cytochrome C in cytosolic and S-100 fraction,and that of procaspase-3 were tested by Western blot.MTT was used to analyze the sequence effects of NB and GA on HL-60 cells.Results Cotreatment with NB and GA exerted additive effect on proliferation inhibition and induction of apoptosis.Cotreatment with NB and GA induced more activation of Caspase-3/9 than treatment with NB or GA alone in HL-60 cells and HL-60/Bcr-Abl cells.Pretreatment with NB was able to sensitize HL-60 cells to GA;in contrast,pretreatment with GA had no influence on the effect of NB on HL-60 cells.Conclusion Combination of NB and GA had additive effects on proliferation inhibition and apoptosis induction of leukemia cells;NB was able to enhance the effect of GA on HL-60 cells,in contrast,GA had no influence on the effect of NB on HL-60 cells.