骨髓瘤细胞系对硼替佐米敏感性与β-连环素表达水平关系的研究

目的 探讨β-连环素(β-catenin)与骨髓瘤细胞系对硼替佐米(Bortezomib)敏感性的关系.方法 以骨髓瘤细胞系RPMI8226、CZ-1、NCI-H929细胞为研究对象,采用锥虫蓝拒染法及改良四呷基偶氮唑盐比色(MTT)法检测药物对细胞的增殖抑制,Annexin V及PI染色流式细胞术检测药物对细胞凋亡的影响,RT-PCR及Western blot法比较硼替佐米、2-甲氧基雌二醇(2-methoxyestradiol,2ME2)单药或联合处理后,不同细胞系胞质内β-连环素的表达及变化,分析β-连环素表达水平与骨髓瘤细胞系对硼替佐米敏感性的关系.结果 β-连环素蛋白在不同骨髓瘤细胞系的基础表达水平由高到低依次为RPMI8226、CZ-1、NCI-H929细胞,对应的硼替佐米半数抑制浓度(IC50)分别为(49.8±0.6)、(24.7±0.4)、(8.4±0.2)nmol/L;硼替佐米(0、1、5、10 nmol/L)单药处理后,各细胞系β-连环素蛋白水平呈时间、剂量依赖性升高,而相应mRNA水平无明显变化;与硼替佐米(5 nmol/L)单药处理组相比,硼替佐米(5 nmol/L)联合2ME2(1 μmol/L)处理后,RPMI8226、CZ-1及NCI-H929细胞胞质β-连环素蛋白水平分别降低64.03%、52.56%和51.48%,凋亡细胞比例分别增加8.00、1.86、1.19倍.结论 β-连环素表达水平较高的骨髓瘤细胞系对硼替佐米敏感性较低;小剂量2ME2联合硼替佐米可降低β-连环素水平,增强骨髓瘤细胞系对硼替佐米的敏感性.

Study on the relationship of β-catenin level and sensitivity to Bortezomib of myeloma cell lines

Objective To explore the relationship of β-catenin and sensitivity to Bortezomib of myeloma cell lines.Methods Myeloma cell lines RPMI8226,CZ-1 and NCI-H929 were treated with Bortezomib and 2ME2,alone or in combination.Typan blue dye exclusion and modified MTT were used to assess the cell viability with or without treatment.Annexin V-FITC and PI staining was performed to detect apoptosis rate.RT-PCR was used to detect β-catenin mRNA and western blot to analyze β-catenin protein.Results The basic expression level of β-catenin was different in tested myeloma cell lines：RPMI8226 was the most while NCI-H929 the least and CZ-1 the intermediate.IC50 of RPMI8226、CZ-1 and NCI-H929 were(49.8±0.6),(24.7±0.4)and(8.4±0.2)nmol/L,respectively.After the treatment oi Bortezomib(at 0,1,5,10 nmoL/L),β-catenin level of tested cell lines accumulated in a time and dose dependant manner for western blot,while no significant change was observed in the result of RT-PCR.The β-catenin protein levels in the Bortezomib(5 nmol/L)and 2ME2(1 μmol/L)treated cell group were much lower than that in Bortezomib(5 nmol/L)group,the decrease of the gray scale of β-catenin/β-actin was 64.03%for RPMI8226、52.56%for CZ-1,51.48%for NCI-H929,and the apoptosis rates were 8.00,1.86 and 1.19 times increase compared to untreated group.Conclusion Myeloma cell lines with higher β-catenin level are less sensitive to Bortezomib,and combination treatment of low dose 2ME2 and Bortezomib can reduce β-catenin accumulation and enhance the sensitivity to Bortezomib.