格列卫激活caspase-3诱导K562细胞凋亡

Gleevec induces apoptosis in K562 cells through activating caspase-3

本文主要探讨格列卫诱导慢性粒细胞白血病K562细胞凋亡的分子机制。采用流式细胞术检测格列卫对K562细胞凋亡和细胞周期的影响、caspase泛阻断剂Z-VAD-FMK及沉默PDCD4表达对格列卫诱导K562细胞凋亡的影响;Western blotting分析si-PDCD4对PDCD4蛋白的沉默效果,以及格列卫对caspase-3和PARP蛋白的活化及PDCD4蛋白表达的影响。结果显示,格列卫能显著诱导K562细胞发生凋亡和阻滞于G0/G1期,促进caspase-3和PARP蛋白的活化,上调PDCD4蛋白的表达;Z-VAD-FMK抑制剂显著抑制格列卫诱导的细胞凋亡(47.97%±10.56%vs 31.05%±9.206%,P<0.05);si-PDCD4能有效抑制PDCD4蛋白的表达;si-PDCD4沉默PDCD4蛋白表达能部分抑制格列卫诱导的细胞凋亡(46.97%±14.32%vs 42.8%±11.43%)。综上所述,格列卫通过激活caspase-3诱导K562细胞凋亡。

The present study is to elucidate the mechanisms underlying Gleevec-induced apoptosis of chronic myeloid leukemia （CML） K562 cells in vitro. The apoptotic cell death and cell cycle distribution after Gleevec treatment and the effect of PDCD4 siRNA on Gleevec-induced apoptosis of K562 cells were analyzed by flow cytometry. The effect of Gleevec on p-Crkl, caspase-3, PARP and PDCD4 protein levels, and the knockdown efficacy of PDCD4 siRNA were detected by Western blotting. The results showed that Gleevec dramatically suppressed the phosphorylation level of Crkl in a dose-dependent manner and induced significant apoptosis and Go/Gl cell cycle arrest of K562 cells in time- and dose-dependent manners. In addition, Gleevec activated caspase-3 and its downstream substrates PARP, and the caspase pan inhibitor Z-VAD-FMK （50 μmol·L^-1） markedly reduced Gleevec-induced apoptosis from 47.97% ± 10.56% to 31.05% ± 9.206% （P 〈 0.05）. Moreover, Gleevec significantly increased the protein expression of programmed cell death 4 （PDCD4）. PDCD4 knockdown by siRNA reduced Gleevec-induced apoptosis from 46.97% ± 14.32% to 42.8%±11.43%. In summary, Gleevec induced apoptosis in K562 cells via caspase-3 activation.