双硫仑/铜通过MAPK通路诱导凋亡逆转白血病细胞多柔比星耐药的研究

Disulfiram/Cu activated MAPK pathways and reverse drug resistance of adriamycin resistant leukemia cells

目的:探讨丝裂原激活的蛋白激酶(MAPK)通路诱导凋亡在双硫仑/铜逆转耐多柔比星的人急性白血病细胞株(HL60/ADM)耐药分子机制中的作用。方法:流式细胞仪检测加入MAPK通路抑制剂SP600125后多柔比星单药组、双硫仑/铜单药组及双硫仑/铜联合多柔比星组HL-60/ADM凋亡细胞比例的改变;倒置显微镜下观察上述各个处理组作用后HL60/ADM细胞24h后形态学变化;蛋白质印迹法检测上述各组作用HL60/ADM细胞后JNK、磷酸化JNK(p-JNK)、c-Jun、磷酸化c-Jun(p-c-Jun)和Bcl-2蛋白表达变化。结果:SP600125将双硫仑/铜联合多柔比星组凋亡细胞比例从(73.24±10.58)%降至(40.70±6.03)%,差异有统计学意义,t=6.218,P=0.025。形态学观察显示,多柔比星和双硫仑/铜单药组HL60/ADM细胞凋亡特征不明显,双硫仑/铜联合多柔比星组出现明显的细胞凋亡现象,且SP600125能减轻双硫仑/铜联合多柔比星组细胞的凋亡程度。蛋白质印迹法显示,双硫仑/铜联合多柔比星处理HL60/ADM细胞后MAPK通路中磷酸化-JNK及其下游分子c-Jun和磷酸化c-Jun表达较其他组显著增加并能被SP600125显著抑制;此外,双硫仑/铜联合多柔比星还能下调抗凋亡Bcl-2蛋白的表达。结论:双硫仑/铜可通过MAPK通路诱导凋亡逆转HL60/ADM对多柔比星耐药。

OBJECTIVE： To investigate the role of MAPK pathway in reversing drug resistance HL60/ADM by Disulfiram/ Cu（DS/Cu）. METHODS： HL60/ADM cells were firstly pretreated with MAPK pathway inhibitor SP600125 and then exposed to DS/Cu complex,ADM or DS/Cu plus ADM for 24 h. Flow cytometric analysis was adopted to detect apoptotic HL60/ADM cell population. The morphological alteration were observed under an inverted fluorescence microscope. Western bloting analysis was used to examined JNK,p-JNK,c-Jun,p-c-Jun and Bcl-2 protein expression in HL60/ADM. RESULTS： SP600125 obviously decrease DS/Cu ＋ ADM induced apoptosis of HL60/ADM cells from （73.24±10.58）% to （40.70 ±6.03）% （t=6. 218, P=0. 025）. Cells treated with DS/Cu or ADM alone did not show any visible apoptosis characters. Whereas,apoptotic morphology appeared under DS/Cu plus ADM treatment including chromatin condensation, membrane blebbing, nuclear breakdown, and the appearance of membraneassociated apoptotic bodies. Western blot results showed markedly increase in p-JNK,c-Jun and p-c-Jun in HL60/ADM cells treated with a combination of ADM and DS/Cu,while no obvious increase was noticed with ADM and DS/ Cu alone. As a control,SP600125 inhibited the ADM plus DS/Cu complex-induced p-JNK,and c-Jun and the increased expression of c-Jun. In additon,Bcl-2 expression was also inhibited in HL60/ADM cells treated with DS/Cu combined with ADM. CON- CLUSION： DS/Cu can reverse drug resistance of HL60/ADM cells through activating MAPK pathways as well as inhibiting anti apoptotic bcl-2 expression.