热休克蛋白90抑制剂对白血病细胞分化凋亡信号通路的作用

Study on signal transduction pathway in differentiation and apoptosis of leukemia cells induced by heat shock protein inhibitor

目的探讨热休克蛋白(HSP)90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、分化和凋亡作用的信号传导途径。方法采用不同剂量的17AAG 处理Kasumi-1细胞,Western blot 检测处理前后 kit 蛋白(CD117)及其下游信号分子 AKT、STAT3水平;免疫共沉淀法检测处理前后 HSP 与 kit 蛋白的结合状态变化。结果 17AAG 降低突变 kit 蛋白及其活性水平,但并不影响 c-kit mRNA 水平;17AAG 能降低组成性活化 kit 下游信号分子 AKT 及其磷酸化水平,同时磷酸化 STAT3水平亦降低,但总 STAT3水平没有变化;免疫共沉淀法检查结果显示经17AAG 处理1 h 后,与 kit 结合的 HSP90明显减少,同时结合的 HSPT0增加。结论在诱导白血病细胞分化凋亡过程中,17AAG 能降低 Kasumi-1细胞内 Asn822Lys 突变 kit 蛋白及其磷酸化水平,同时下调 kit 蛋白下游增殖及存活信号途径的信号分子;AKT 也是 HSP90的客户蛋白之一;17AAG 所导致的 kit 蛋白与HSP90及 HSP70结合状态的变化,符合分子伴侣复合物的循环模型。

Objective To explore the signal transduction pathway in the differentiation and apoptosis of leukemia cells induced by heat shock protein 90 （HSP90） inhibitor 17-Allylamide-17-demethoxygeldanamycin （17AAG）. Methods Kasumi-1 cells were treated with increasing concentrations or exposure time of 17AAG. The total kit protein （CD117 ）, phosphorylated kit protein and its downstream signaling molecules were measured by Western blot analysis. Mutated kit protein from control and 17AAG-treated Kasumi-1 cells was immunoprecipitated and immumoblotted for associated chaperones. Results Total kit protein and kit activity were decreased in 17AAG treated cells, but c-kit mRNA level was not. Total AKT protein and phospho- AKT, as well as phospho-STAT3 were rapidly down-regnlated in Kasumi-1 cell after treatment with 17AAG. There was no change in total STAT3 protein. Immunoprecipitation showed that 1 μM 17AAG treatment for 1 hour caused kit associated HSP90 decrease and HSP70 increase. Conclusion 17AAG-induced apoptosis of Kasumi-1 cells is associated with a decline in Asn822Lys mutated kit protein level and phosphorylated kit, and with a dowrrregnlation in its downstream activated signaling molecules involved in proliferation. AKT is a client protein of HSP90. The changes of kit associated HSP90 and HSP70 satisfy the circulation mode of molecular chaperone complex.