5-Azacytidine upregulates melatonin MT1 receptor expression in rat C6 glioma cells: oncostatic implications.

The multiple physiological effects of the indoleamine melatonin, are mediated primarily by its two G protein-coupled MT1 and MT2 receptors. Treatment with histone deacetylase (HDAC) inhibitors, including valproic acid (VPA) and trichostatin A, upregulates melatonin receptors in cultured cells and the rat brain. VPA increases histone H3 acetylation of the MT1 gene promoter in rat C6 glioma cells, indicating that this epigenetic mechanism is involved in upregulation of MT1 expression. Since HDAC inhibitors can alter DNA methylation, the possible involvement of this other epigenetic mechanism, in the regulation of MT1 expression, was examined. RT-qPCR and western blotting studies confirmed that treatment with the DNA demethylating agent, 5-azacytidine (AZA; 10 or 20 µM) for 24 or 48 h, suppressed DNA methyltransferase 1 mRNA and protein expression in C6 cells. Subsequent treatment with AZA (1-25 µM) for 24 h, revealed a significant concentration-dependent upregulation of MT1 mRNA expression. Moreover, a combination of 5 µM AZA plus 3 mM VPA caused a synergistic upregulation of the MT1 receptor, which exceeded the sum of the independent effects of these drugs. These results show that DNA methylation plays a role in the regulation of the MT1 receptor, consistent with the established effects of this major epigenetic mechanism on gene transcription. Combinatorial epigenetic regulation of melatonin receptor expression could provide novel strategies for enhancing the oncostatic, neuroprotective and other therapeutic benefits of this pleiotropic indoleamine and its receptor agonists.