组蛋白去乙酰化酶抑制剂MS-275对人骨髓瘤U266细胞生长的影响及其可能机制

Effects of histone deacetylase inhibitor MS-275 on expression of survivin and NF-κB in the human myeioma cell line U266 cells

目的 探讨组蛋白去乙酰化酶抑制剂（HDACi）MS-275对人骨髓瘤细胞U266的凋亡抑制基因生存素（survivin）和核因子-κB（NF—κB）表达的影响。方法台盼蓝拒染法观察MS-275对细胞活力的影响；瑞氏-姬姆萨染色观察细胞形态学变化；流式细胞仪分析细胞周期；Western blot检测survivin、p21、细胞周期依赖激酶4（CDK4）和NF—KB的抑制蛋白（IKB—α）等的表达，以及凋亡信号通路中caspase-3活化及蛋白聚ADP核糖聚合酶（PARP）裂解情况。结果MS-275抑制U266细胞增殖，阻断细胞周期于G0／G1期，呈时间-剂量依赖性。MS-275作用U266细胞48h的IC50为1．39μmol／L。2μmol／LMS-275作用U266细胞24h后，G0／G1期细胞所占比例为（64．57±4．09）％；作用36h后，G0／G1期细胞所占比例为（87．20±2．83）％；瑞氏-姬姆萨染色显示，U266细胞形态发生明显变化。Western blot检测表明，MS-275作用U266细胞后，survivin和CDK4表达下降，p21表达增加，IκB—α磷酸化水平受到明显抑制，caspase-3被裂解活化，其底物蛋白PARP发生剪切，细胞发生凋亡。结论MS-275可诱导人骨髓瘤细胞系U266凋亡，NF-KB信号通路阻断是凋亡发生的机制之一。

Objective To study the effects of HDACi on the expression of survivin and NF-κB in human myeloma cell line U266 cells. Methods U266 cells were cultured in RPMI 1640 in the presence of MS-275, and the cell viability was evaluated by trypan blue exclusion assay and cell count. The cell morphological changes were observed with Wright-Giemsa staining. The cell cycle was analyzed by flow cytometry, and the proteins of poly （ADP-ribose） polymerase （PARP）, caspase-3, survivin, p21 ,CDK4 and IκB-α were detected by Western blotting. Results MS-275 inhibited the growth of U266 cells in a dose- and time-dependent manner. The cell cycle was arrested at G0/G1 phase. After exposure at 1.39 μmol/L MS-275 for 48 hours, the cell viability was decreased to 50%. The cell ratios of G0/G1 phase were increased to （ 64.57 ± 4.09 ） % for 24 h and （ 87.20 ±2.83 ） % for 36 h after 2 μmol,/L MS-275 treatment. The visible morphological changes of U266 cells were confirmed with Wright-Giemsa staining. Cleaved- PARP, increased expression of p21, downregulation of expression of survivin, CDK4 and the phosphorylation of IκB-α was found by Western blot in MS-275 treated U266 cells. Conclusion MS-275-induced apoptosis of U266 cells is mediated by downregulation of expression of survivin and inactivation of NF-κB survival signalling pathways.