丙咪嗪对结肠癌HT-29细胞增殖、凋亡的影响及其机制

Effect of imipramine on HT-29 cells’ proliferation, cell cycle arrest and apoptosis and its mechanism

目的:探讨丙咪嗪对结肠癌HT-29细胞的增殖、细胞周期和凋亡的影响及其作用机制.方法:按ACTT方法常规培养HT-29细胞,并以不同浓度(0、1、5、10、25、50和100μmol/L)丙咪嗪干预,MTT法检测各浓度干预24、48和72h后的细胞增殖能力;丙咪嗪以各浓度(0、1、10、50和100μmol/L)干预HT-29细胞24h,PI染色后流式细胞仪检测细胞周期分布,AnnexinV/PI双染后流式细胞仪和DNA片段梯试验检测细胞凋亡,Western blot检测Eag1蛋白的变化,逆转录PCR检测p21、p27、CyclinE1和CDK2mRNA表达水平的变化.结果:各浓度丙咪嗪干预HT-29细胞24、48和72h后,IC50分别为43、32、22μmol/L;丙咪嗪以剂量依赖方式诱导HT-29细胞在细胞周期G0/G1期停滞,随丙咪嗪干预的浓度增加,HT-29细胞的凋亡指数逐渐升高(P<0.01),HT-29细胞中Eag1蛋白的表达水平逐渐下降(P<0.05),p27和p21mRNA表达水平逐渐增强(P<0.05),CyclinE1和CDK2mRNA的表达水平逐渐减弱(P<0.05).结论:丙咪嗪可抑制HT-29细胞增殖,阻滞HT-29细胞周期进展,诱导细胞凋亡.其作用机制可能与抑制Eag1钾通道的活性,上调p27和p21表达、下调CyclinE1和CDK2表达有关.

AIM： To investigate the effect of imipramine on cell growth, cell cycle and apoptosis of HT-29 colon cancer cells, and to elucidate its molecular mechanism. METHODS： Human colon cancer HT-29 cells were grown with routine cell cultivation and cells were treated with different concentrations of imiprmine. Cell survival was determined us-ing MTT assay at 24 h, 48 h and 72 h, respective-ly; cell cycle distribution was assessed by FACS flow cytometery after propidium iodide stain-ing; apoptosis of HT-29 cells was detected using Annexin V/PI methods and DNA ladder assay.Expression level of Eag1 protein was detected by Western blot, and mRNA expressions of p21, p27, CyclinE1 and CDK2 were determined by reverse transcription-polymerase chain reaction. RESULTS： After treatment with imipramine in HT-29 cells at 24, 48 and 72 h, IC50 were 43, 32 and 22 μmol/L, respectively. Cell viability decreased dose-dependently and time-depend-ently after treatment with imiprmince in HT 29 cells. Cell cycle arrested during the G0/G1 phase accompanied by the induction of apoptosis in a dose-dependent manner. With imipramine increasing, HT-29 cells apoptosis index gradu-ally increased （P 〈 0.01）. Expression level of Eag1 protein was decreased in a dose-dependent manner （P 〈 0.05）. The p21 mRNA and p27 mRNA were up-regulated （P 〈 0.05）, and CDK2 mRNA and CyclinE1 mRNA were suppressed in imipramine-treated HT-29 cells in a dose-dependent manner （P 〈 0.05）. CONCLUSION： Imipramine, a non-specific in-hibitor of Eag1 potassium channel, induces cell growth inhibition, cell-cycle arrest and apopto-sis in HT-29 cells through up-regulation of p27 and/or p21.