5-氮杂胞苷体外抗骨髓瘤细胞增殖及诱导凋亡的初步研究(英文)

Preliminary Study on 5-Azacytidine Anti-myeloma Activity In Vitro

本研究探讨5-氮杂胞苷对骨髓瘤细胞株中XAF1基因表达的影响及体外抗骨髓瘤细胞增殖效率。采用逆转录PCR方法检测骨髓瘤细胞株RPMI8226和XG-7中XAF1基因的表达。采用甲基化特异性PCR(MSP)方法检测XAF1基因CpG岛甲基化状态。采用0-5μmol/L5-氮杂胞苷处理骨髓瘤细胞株。采用CCK-8比色法检测5-氮杂胞苷处理对骨髓瘤细胞增殖抑制作用,应用Graphpad5.0软件分析5-氮杂胞苷对骨髓瘤细胞的生长抑制作用。采用Annexin V/7-AAD染色流式细胞仪检测细胞凋亡。结果表明:XG-7细胞不表达XAF1 mRNA,RPMI8226细胞表达XAF1 mRNA转录本1和2。XG-7和RPMI8226细胞株XAF1基因启动子CpG岛均存在过甲基化。XG-7和RPMI8226细胞株经2.5μmol/L5-氮杂胞苷处理72小时后仅表达XAF1 mRNA转录本1,并且XAF1基因启动子CpG岛甲基化程度降低。5-氮杂胞苷抗骨髓瘤作用呈时间和浓度依赖性。5-氮杂胞苷处理48小时抑制XG-7骨髓瘤细胞株的IC50值为2.6μmol/L。1.0、2.0、2.5、5.0μmol/L浓度的5-氮杂胞苷处理XG-7细胞48小时后诱导细胞凋亡率分别为(34.3±8.0)%,(54.8±3.1)%,(64.1±3.4)%,(81.0±4.1)%。1.0-4.0μmol/L5-氮杂胞苷与1.0-4.0μmol/L亚砷酸联合应用具有协同抗骨髓瘤细胞作用,联合指数均小于1.0。结论:骨髓瘤细胞中XAF1表达缺失与启动子CpG岛过甲基化有关。5-氮杂胞苷在临床上能达到的药物浓度下具有抗骨髓瘤作用,其作用机制是诱导骨髓瘤细胞凋亡,与亚砷酸具有协同抗骨髓瘤作用。

This study was aimed to investigate the effect of 5-azacytidine （5-AZA） on XAF1 expression in myeloma cells and efficacy of 5-AZA treatment for myeloma in vitro. XAFI expression was analyzed by semi-quantitative PCR. Methylation-specific PCR （MSP） was used to detect the methylation status of XAF1 promoter CpG islands. RPMI 8226 and XG-7 cells were treated with 0 - 5 μmol/L of 5-AZA. Expression of XAF1 mRNA variants was confirmed by gel electrophoresis. The results indicated that the untreated RPMI 8226 cell expressed XAF1 mRNA transcript 1 and transcript 2, untreated XG-7 cells did not express XAFI mRNA. Hypermethylation of XAF1 promoter CpG islands could be detected in both cell lines. Both cell lines expressed full-length XAF1 transcript after being treated with 2.5 μmol/L of 5-AZA for 72 hours. 5-AZA treatment led XAF1 promoter CpG island to hypomethylation in both cell lines. 5-AZA exerted anti-myeloma activity in a time- and concentration-dependent manner. The ICso value of XG-7 cells treated with 5- AZA for 48 hours was 2.6 μmol/L. 1.0, 2.0, 2.5 and 5.0 μmol/L of 5-AZA treatment for 48 hours induced （34.3 ± 8.0）%, （54.8 ±3.1）%, （64.1 ±3.4）%, （81.0 ±4.1）% apoptosis in XG-7 cell line respectively. The combination of 1.0 - 4.0 μmol/L of 5-AZA with 1.0 - 4.0 μmol/L of arsenic trioxide （ATO） exhibited synergistic toxicity in myeloma cells with all CI values less than 1.0. It is concluded that lack of XAF1 expression and abnormal expression of XAF1 in myeloma cell lines are associated with the hypermethylation of XAF1 gene promoter CpG island. 5-AZA treat- ment can induce the expression of XAF1 mRNA and protein in myeloma. 5-AZA exerts anti-myeloma activity via apoptosis at clinically achievable concentrations. The findings suggested that 5-AZA and ATO may be an effective combination in the therapy of patients with multiple myeloma.