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.
Keywords: 5-Azacytidine; Alimta; Apoptosis; Lung Cancer Cell; miR-34a.
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