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

Abstract

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 using MTT assay at 24 h, 48 h and 72 h, respectively; cell cycle distribution was assessed by FACS flow cytometery after propidium iodide staining; 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-dependently 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 gradually 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 inhibitor of Eag1 potassium channel, induces cell growth inhibition, cell-cycle arrest and apoptosis in HT-29 cells through up-regulation of p27 and/or p21.