摘要
目的:探讨唯BH3域(BH3 only domain,BH3)模拟物3-硫吗啉基-8-氧-8H-苊并[1,2-b]吡咯-9-腈(S1)诱导急性髓系白血病(AML)原代细胞凋亡的分子标志物。方法:分离27例人初发AML的单个核细胞,使用流式细胞术检测细胞凋亡。通过四氮唑盐(XTT)法检测S1作用下AML细胞的半数抑制浓度(IC50)。应用Western blot测定AM L细胞中BCL-2家族蛋白及磷酸化BCL-2(p BCL-2)蛋白表达量,并通过光密度法进行半定量分析。通过XTT法及免疫共沉淀法检测S1与特异性信号调节激酶MEK/ERK抑制剂PD98059联合作用下的AML细胞活性及其BCL-2家族蛋白之间的相互作用。结果:S1能够诱导AM L原代细胞凋亡。根据AM L原代细胞对S1的敏感度不同,将27例AML原代细胞分为3组:1敏感组12例,IC50<15μmol/L;2中度敏感组8例,14μmol<IC50<30μmol/L;3耐药组7例,IC50>30μmol/L。p BCL-2/(BCL-2+M CL-1)的比值与IC50呈现良好的线性相关性(R2=0.71,P<0.0001)。在PD98059的协同作用下,p BCL-2水平下调,S1诱导AM L原代耐药组细胞的凋亡率由9.8%显著提升至64.5%(联合指数CI=0.4),并且促进了AM L原代耐药细胞BCL-2家族蛋白之间形成异源二聚体的解离。结论:S1与PD98059的联合作用能够降低AML患者p BCL-2水平,干扰BCL-2与促凋亡蛋白的相互作用。
Objective：This study was to investigate the molecular biomarkers of apoptosis induced by BH3 mimetic S1 in human primary AML cells. Methods ：Mononuclear cells were isolated from 27 newly diagnosed AML samples. Apoptosis was analyzed by flow cytometry. IC50 value of S1 on these samples was determined by XTF assay. The expression level of BCL-2 family members and phosphorylated BCL-2 were assessed by Western blot with subsequent semi-quantitatively densitometric analysis. XTT assay was performed to determine the cell viability of the combined use of S1 and MEK/ERK inhibitor PD98059. The interactions between BCL-2 and pro-apoptosis proteins were tested by cimmunoprecipitation. Results： The flow-cytometry detection showed that S1 induced the apoptosis of primary AML cells. Based on the responses, 27 primary samples could be classified into three groups ： （ 1 ） a sensitive group （ 12 of 27 cases） with IC50 〈14 μmol/L, （2） an intermediate group （8 of 27 cases） with IC50 of 14 -30 μmol/L and （3） a resistant group （7 of 27cases） with ICs0 〉 30 μmol/L. The ratio of pBCL-2/（ BCL-2 ＋ MCL-1 ） showed a good linear correlation with the IC50 values. （ R2 = 0.71, P 〈 0. 0001 ）. PD98059 suppressed BCL-2 phosphorylation. When PD98059 suppressed BCL-2 phosphorylation, the apoptotic rate of drug-resistant cells induced by S1 increased from 9. 8% to 64. 5% （combination index, CI = 0. 4 ）, accompanied by more dissociation of BCL-2 heterodimers. Conclusion： The combination of S1 with PD98059 decrease pBCL-2 level of AML patients and inhibits of the antiapoptotic function of BCL-2 through enhancing the dissociation of BCL-2 heterodimers.
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2015年第1期39-44,共6页
Journal of Experimental Hematology
