restoration of mir-34a expression by 5-azacytidine augments alimta induced cell death in non-small lung cancer cells by downregulation of hmg b1, a2 and bcl-2 pathway

Objective: alimta (pemetrexed) as an antifolate drug has been approved for the treatment of lung cancer. the aim of the present study was to investigate the combination effect of 5-azacytidine (5-aza) and alimta on the mir-34a and its target genes expression and induction of apoptotic cell death in non-small lung cancer a549 cells. materials and methods: in this experimental study, lung cancer a549 cells were treated with various concentrations of alimta alone and combined with 5-aza. then, viability was assessed by trypan blue and mtt assays. mrna expressions were performed by real time-polymerase chain reaction (pcr) and western blot. flow cytometry used to detect apoptotic/ necrotic cells and cell cycle arrest. results: alimta alone reduced viability of the cells in a dose dependent manner with the half-maximal inhibitory concentration (ic50) value of 12 µm. pretreatment of the cells with 5-aza (5 µm) induced a synergistic cytotoxic effect with ic50 of 3 µm. sequential exposure of the cells to 5-aza and alimta enhanced mir-34a expression and significantly downregulated hmgb1, hmga2 and bcl-2 expressions. also, it was associated with reduction of nuclear hmgb1 and hmga2 content. caspase-3 activation, hmgb1 release into extracellular space and staining of the cells with annexine v/pi suggested that 5-aza reduced late apoptotic/necrotic cell death induced by alimta. in addition, combination of 5-aza and alimta arrested the cells at s and sub-g1 phases and inhibited colony formation. conclusion: 5-aza synergistically enhances alimta induced apoptotic cell death through hmg proteins regulation, mir34a gene expression and intrinsic apoptosis mechanism, providing a promising combination therapy in clinical lung cancer therapy.