三氧化二砷与硼替佐米联合应用对多发性骨髓瘤KM3细胞凋亡的作用及其机制研究

摘要

本研究探讨硼替佐米(Bor)单用及与三氧化二砷(As2O3)联合应用对多发性骨髓瘤(MM)细胞株KM3细胞凋亡的影响及其相关机制.采用MTT法检测Bor单用及与As2O3联合应用对KM3细胞增殖的抑制作用,计算其IC50值.采用Annexin V-FITC凋亡试剂盒检测各药物组KM3细胞早期及晚期凋亡率,流式细胞术测定各药物组KM3细胞跨膜电位的变化.采用RT-PCR检测各药物组KM3细胞Caspase-3、Bim、Bcl-xL mRNA水平的变化.结果表明,Bor联合As2O3对KM3细胞生长抑制明显高于Bor单用,处理72 h作用最明显[(27.64±0.81)％vs(21.67±2.20)％,P＜0.05].联合用药组KM3细胞早期凋亡率及晚期凋亡率均较Bor单药组明显增多,前者在作用48 h时效果最明显[(53.20 +3.70)％vs (35.40 +2.58)％,P＜0.01],后者在作用72 h时效果最明显[ (63.96±2.97)％vs(54.08±3.76)％,P＜0.01].联合用药组KM3细胞线粒体跨膜电位较Bor单药组明显下降,作用48 h时下降最明显.与Bor单药组比较,联合用药组KM3细胞Caspase-3 mRNA、Bim mRNA均升高,Bcl-xLmRNA下降.结论:在体外As2O3可增强Bor对KM3细胞增殖的抑制,增加KM3细胞的凋亡,并且可能通过抑制Bcl-xL mRNA表达,诱导Caspase-3和BIM mRNA表达的途径增强凋亡作用.%This study was purposed to investigate the effect of bortezomib (Bor) and arsenic trioxide ( As2 O3 ) combination on multiple myeloma cell line KM3 and its mechanisms.KM3 cells were cultured with different concentration of Bor or As2 O3 as well as both for a certain time.The cell proliferation was analysed by MTT assay and the concentration of 50％ proliferation inhibition (IC50) was calculated.Early apoptosis and late apoptosis of KM3 cells were detected by Annexin-V-FITC Kit,and the change of transmembrane potential was measured by flow cytometry.mRNA of Caspase-3,Bim and Bcl-xL were detected by RT-PCR.The results showed that the proliferation inhibitory rate of KM3 cells treated by Bor plus As2O3 was much higher than that of KM3 cells treated by Bor only for 72 h [ ( 27.64 ± 0.81 ) ％ vs (21.67 ±2.20)％,P ＜0.05].There were more KM3 cells treated by Bor plus As2O3 in early apoptosis at 48 h and late apoptosis at 72 h than that of KM3 cells treated only by Bor [ (53.20 + 3.70) ％ vs (35.40 + 2.58 ) ％,P ＜ 0.01 ;(63.96 ± 2.97 ) ％ vs (54.08 + 3.76 ) ％,P ＜ 0.01].Transmembrane potential (△ψm) of KM3 cells treated by Bor plus As2O3 decreased more at 48 h,as compared with Bor alone.The expression levels of caspase-3 mRNA and Bim mRNA in KM3 cells treated with Bor plus As2O3 were higher than that in KM3 cells treated with Bor alone.But the expression level of Bcl-xL mRNA was lower than that in KM3 cells treated with Bor alone.It is concluded that As2O3 can enhance the apoptosis-inducing effect of Bor on multiple myeloma cell line KM3,which is associated with decreasing the expression of Bcl-xl mRNA and increasing the expression of Caspase-3 and Bim mRNA.