热休克蛋白90抑制剂诱导Kasumi-1细胞凋亡和分化的机制探讨

The heat shock protein 90 inhibitor induces apoptosis and differentiation of Kasumi-1 and its mechanisms

目的探讨热休克蛋白90(HSP90)抑制剂17-丙烯胺-17-去甲氧格尔德霉素(17AAG)诱导白血病细胞生长抑制、分化和凋亡作用。方法应用MTT比色法观察17AAG对白血病细胞系Kasumi-1细胞的生长抑制作用,用流式细胞术分析细胞周期和分化抗原的表达,用Annexin V标记、DNA凝胶电泳和流式细胞术分析细胞凋亡,Western blot检测干细胞因子受体(KIT)蛋白水平,RT-PCR测定c-kit mRNA水平。结果17AAG明显抑制Kasumi-1细胞的生长,半数抑制浓度(IC50)为0.62μmol/L;17AAG诱导Kasumi-1细胞凋亡呈时间、剂量依赖性,17AAG处理24h早期凋亡细胞增加,48h晚期凋亡细胞增加,早期凋亡细胞减少。17AAG可诱导Kasumi-1细胞髓系分化抗原CD11b及CD15表达增加,并呈剂量和时间依赖性。经17AAG作用后细胞阻滞于G0/G1期,伴有S期细胞减少。Western blot检测发现17AAG可降低KIT水平,处理2h开始减少,处理20h后KIT基本消失,但c-kit mRNA水平并未受影响。结论HSP90抑制剂17AAG通过降解KIT蛋白抑制Kasumi-1细胞生长,使细胞阻滞于G0/G1期,诱导其发生凋亡及部分分化。

Objective To explore the effect of 17-allylamide-17-demethoxygeldanamycln（17AAG） , a heat shock protein 90 （HSP90） inhibitor , on the growth, differentiathm and apoptosis of leukemic Kasumi-1 cells. Methods Kasumi-1 cells were lreated with 17AAG at differenl concentrations in suspension culture. Cell proliferation was analysed by MTT assay, expression of myeloid-specific differentiation antigen and c.ell cycle by flow cytometr？ , cell apoptosis by annexin V staining , agrose gel electrophoresis and flow eytometry. KIT protein was analysed bv Western blot and c-kit mRNA by RT-PCIL Results 17AAG treatment caused a dose-dependent inhibition of the cell proliferation with the IC50 of 0.62 μmol/L. A dose-dependent increase in early apoptosis occured at 24 hours treatment and in late apoptosis at 48 hours treatment. 17AAG induced a lime- and dose-dependent increase in expression of myeloid cell surface protein CD11b and CD15, a progressive decline in S-phase cell fraction and an increase in G0/G1 cells . When Kasumi-1 cells were incubated with 1 μmol/L of 17AAG, KIT protein began to decrease at 2 hours and KIT protein could hardly be detected at 20 hours, but c-kit mRNA was not decreased. Conclusion 17AAG treatment of Kasu- mi-1 cells eouht lower KIT protein expression, inhibil cell proliferation, induce cell partial differentiation, apoptosis and accumulation in G0/G1 phase .