MS-275阻断STAT3和NF-κB信号通路并诱导U266细胞凋亡机制的探讨

Mechanism of MS-275 blocking STAT3 and NF-κB signaling,inducing apoptosis in U266 cells

目的:探讨组蛋白去乙酰化酶抑制剂MS-275诱导人多发性骨髓瘤细胞株U266凋亡的分子机制。方法:将不同浓度的MS-275以不同时间作用于U266细胞,用台盼蓝拒染法观察药物对细胞活力的影响;瑞氏-姬姆萨染色观察药物对细胞形态学变化;流式细胞仪分析细胞周期;通过蛋白质印迹法测定U266的聚ADP核糖聚合酶(PARP)裂解情况及信号蛋白(STAT3,IκB-α)的表达。结果:MS-275可以呈剂量时间依赖性作用抑制U266细胞的生长;经2μmol/LMS-275处理12～36h,U266细胞周期被阻滞于G0/G1期;瑞氏-姬姆萨染色显示,细胞发生明显变化;蛋白质印迹法检测表明,MS-275作用U266细胞后,PARP发生剪切,导致细胞凋亡;对细胞凋亡相关信号通路STAT3及NF-κB检测结果表明,STAT3,IκB-α发生磷酸化水平受到明显抑制。结论:MS-275对U266细胞的杀伤作用主要是通过诱导细胞凋亡实现,与细胞增殖相关的STAT3及NF-κB信号通路被阻断是凋亡发生的机制之一。

OBJECTIVE： To investigate the molecular mechanisms of histone deacetylase inhibitor MS-275 on apoptosis of human myeloma U266 cell line. METHODS： Cultured U266 cells were exposed to different concentrations of MS-275 for different duration. The cell viability was evaluated by the trypanblue exclusion assay and cell count. The cell morphologic changes were observed with Wright Giemsa staining. The cell cycle was analyzed by flow cytometry, the expression of the protein of poly （ADP-ribose） polymerase （PARP）, STAT3 and IκB-α were detected by Western blot. RESULTS： M8-275 arrested the proliferation of U266 cell cycle in a time and dose-dependent manner. After 2μmol/L MS-275 exposure for 12--36 hours, the cell cycle was arrested at G0/G1 phase and the morphological visible changes of U266 were comfirmed with Wright-Giemsa staining. The proteins were abstracted from the treated cells, and cleavage of PARP was examined by Western blot using a specific antibody that recognized cleaved PARP, respectively. The activation of two important survival signal pathways （STAT3 and NF-κB） was detected after the MS-275 treatment. The phosphorylation of STAT3 and IκB-α was remarkably downregulated. CONCLUSION： MS-275-induced apoptosis of U266 cells is mediated by the inactivation of two important survival signal pathways （STAT3 and NF-κB）.