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.     

划痕损伤对大鼠脑皮层星形胶质细胞NF-κB表达及继发性死亡的影响

目的观察划痕损伤对大鼠脑皮层星形胶质细胞NF-B表达及自噬、凋亡和坏死的影响。方法建立SD大鼠脑皮层星形胶质细胞划痕损伤模型,用NF-B抑制剂吡咯醛二硫氨基甲酸（PDTC）干预48 h,分别用流式细胞术、免疫荧光法、Western blot检测不同时间点（1、6、12、24、48 h）星形胶质细胞凋亡、坏死及NF-B p65蛋白、自噬蛋白LC3Ⅱ表达。结果星形胶质细胞NF-B蛋白表达在划痕损伤后1 h开始增多,24 h达高峰,凋亡、坏死及LC3Ⅱ表达均随时间推移有不同程度升高（P〈0.01）。PDTC处理后,上述变化均有不同程度的抑制（P〈0.01）。结论划痕损伤后可激活脑星形胶质细胞NF-B表达、自噬、凋亡和坏死。     

NF-κB抑制剂PDTC对卵巢癌细胞HO8910增殖、凋亡的影响

应用四甲基偶氮唑盐比色(MTT)法检测不同浓度吡咯烷二硫代氨基甲酸(PDTC)、顺铂(DDP)和联合应用PDTC与DDP对HO8910细胞生长抑制的影响;采用流式细胞仪检测不同浓度PDTC作用HO8910后的细胞凋亡率.结果表明: ① PDTC浓度为25、50、100和200 μmol·L-1时,作用12、24、36 h均明显抑制HO8910细胞的增殖,与对照组比较差异极显著(P<0.01),并具有时间、剂量依赖性.②顺铂联合应用PDTC时能显著提高顺铂杀伤卵巢癌细胞作用.③ 25、50、100 μmol·L-1 PDTC诱导卵巢癌HO8910细胞凋亡.PDTC能有效抑制卵巢癌HO8910细胞增殖并能够增强顺铂化疗作用,提高疗效.     

PDTC和顺铂对人鼻咽癌细胞株CNE1、CNE-GL、CNE2Z的联合作用

目的观察PDTC和顺铂（DD）对人鼻咽癌细胞株CNE1、CNE1-GL、CNE2Z的联合抑制效应。方法单独及联合应用PDTC和DDP处理后,采用MTT法测定CNE1、CNE1-GL、CNE2Z细胞的生长抑制效应。采用直线回归法求出中效浓度,利用中效原理计算联合用药指数。结果在实验剂量范围内,单独及联合应用PDTC和DDP的生长抑制效应具有剂量依赖性。对CNE1细胞,药物效应（抑制率）大于85%时,具有协同作用（CI〈l）;对CNE1-GL、CNE2Z细胞,两药合用具有拮抗作用。结论单独及联合应用PDTC和DDP对CNE1、CNE1-GL、CNE2Z具有生长抑制作用,两药联用对CNE1细胞具有协同作用。     

7S β-伴大豆球蛋白通过NF-κB信号通路引起IPEC-J2细胞的炎性反应

采用细胞体外培养技术,研究不同浓度7Sβ-伴大豆球蛋白对猪小肠上皮细胞(IPEC-J2细胞)的影响。试验分为A、B、C、D、E、F 6组,其中A为对照组,其余各组中分别添加1、5、10、5、5 mg·mL^-1的β-伴大豆球蛋白,并且在E和F组分别添加1μmol·L^-1 NF-κB(PDTC)和iNOS(L-NAME)抑制剂。用CCK-8法检测各组细胞存活率,用ELISA法检测细胞NO、DAO、5-HT、IL-6和IL-10含量,用Western blot法检测细胞p-NF-κB p65、iNOS、COX-2的蛋白表达量,用qPCR法检测细胞NF-κB p65、IKKβ、iNOS、IKKα、COX-2 mRNA的相对转录量。结果显示:β-伴大豆球蛋白降低IPEC-J2细胞存活率,添加PDTC和L-NAME增高IPEC-J2细胞存活率,促进NO、DAO、5-HT和IL-6的分泌,并降低IL-10的分泌,同时增加p-NF-κB p65、iNOS、COX-2的蛋白表达量及NF-κB p65、IKKβ、iNOS、IKKα、COX-2 mRNA的相对转录量,添加PDTC和L-NAME后抑制了β-伴大豆球蛋白的作用。结果表明,β-伴大豆球蛋白引起IPEC-J2细胞损伤,随浓度增大损伤增加,PDTC和L-NAME可以降低β-伴大豆球蛋白对细胞的作用。