NF-κB抑制剂PDTC对人骨髓瘤U266细胞增殖和凋亡的影响

目的:探讨NF-κB特异性抑制剂PDTC对多发性骨髓瘤U266细胞增殖和凋亡的影响及可能的作用机制。方法:采用不同浓度PDTC(25、50、100和200μmol/L)处理U266细胞,CCK-8法和活细胞计数检测U266细胞的增殖活性;流式细胞术检测细胞凋亡率及细胞周期;RT-qPCR及Western blot检测PDTC处理前后DNA甲基转移酶1(DNMT1)mRNA和蛋白的表达;Western blot检测NF-κB(P65)、DNMT1、Bcl-2、cyclin D1、cleaved caspase-3和cleaved caspase-8的蛋白水平。结果:PDTC处理U266细胞48 h后,NF-κB(P65)的蛋白水平被抑制;PDTC抑制U266细胞增殖,作用呈浓度及时间依赖性;PDTC作用48 h后,与对照组比较,细胞凋亡率呈浓度依赖性增高(P0.05),细胞被阻滞在G2期;DNMT1的mRNA及蛋白水平均降低;Western blot结果显示PDTC可通过抑制NF-κB下调Bcl-2的表达,使cyclin D1、cleaved caspase-3和cleaved caspase-8蛋白水平增加。结论:PDTC抑制NF-κB信号通路,通过诱导U266细胞凋亡从而抑制细胞增殖,其机制可能与抑制DNMT1的表达、阻滞细胞周期和启动凋亡途径有关。

Effect of NF-κB inhibitor pyrrolidine dithiocarbamate on proliferation and apoptosis of human multiple myeloma U266 cells

AIM:To explore the effect of pyrrolidine dithiocarbamate (PDTC),an NF-κB inhibitor,on the proliferation and apoptosis of human multiple myeloma U266 cells and its mechanisms.METHODS:The U266 cells were treated with PDTC at different concentrations (0,25,50,100 and 200 μmol/L)in vitro.The growth inhibitory rate of the U266 cells was detected by CCK-8 assay and cell counting.The cell cycle of the U266 cells was determined by flow cyto-metry,and the apoptosis was examined by flow cytometry with Annexin V-FITC/PI staining.The effect of PDTC on the expression of DNA methyltransferase 1(DNMT1) at mRNA and protein levels was measured by RT-qPCR and Western blot,respectively.The effects of PDTC on the protein levels of NF-κB (P65),DNMT1,Bcl-2,cyclin D1,cleaved caspase-3 and cleaved caspase-8 were determined by Western blot.RESULTS:The protein level of NF-κB (P65) was decreased after treatment with PDTC for 48 h or 72 h.PDTC inhibited the proliferation of U266 cells in both dose-and time-dependent manners.After treatment with PDTC for 48 h,the percentage of U266 cells in G₂ phase increased compared with control group (P<0.05).PDTC induced the apoptosis of U266 cells in a dose-dependent manner.The expression of DNMT1 at mRNA and protein levels decreased (P<0.05).The results of Western blot showed that the expression of Bcl-2 in PDTC groups decreased,while the protein levels of cyclin D1,cleaved caspase-3 and cleaved caspase-8 were higher than those in control group (P<0.05).CONCLUSION:The NF-κB inhibitor PDTC inhibits the proliferation of U266 cells by inducing cell apoptosis.It may be related to the down-regulated expression of DNMT1,cell cycle arrest and activation of the apoptotic pathways.