Investigation of the effects of melatonin on granulosa cell proliferation and DNA methylation

Melatonin, a pineal hormone with antioxidant and regulatory functions, has emerged as a key modulator of ovarian physiology. Its presence in fol licular fluid suggests important roles in granulosa cell function, follicle devel opment, and reproductive outcomes. However, its effects on granulosa cell tumour (GCT) biology and epigenetic regulation remain insufficiently defined. This study aimed to investigate the effects of melatonin on proliferation and global DNA methylation in human granulosa tumour cells (COV434) com pared with healthy endothelial controls (HUVECs). COV434 and HUVEC cells were treated with melatonin at 1, 10, 100, and 1000 µM. Cell viabil ity and proliferation were assessed using the MTT [3-(4.5-dimethylthiazol-2- yl)-diphenyl tetrazolium bromide] (Cambridge, UK) assay and xCELLigence RTCA system (Roche), while DNA methylation was quantified with a 5-mC ELISA kit (Epigentek Group Inc, USA). Experimental groups included nega tive, sham, melatonin-treated, and positive controls. Melatonin showed a cell type-dependent effect. In COV434 cells, proliferation was significantly inhib ited, with an IC50 of 10.55 µM, whereas HUVECs displayed increased prolifer ation at higher doses. DNA methylation levels decreased in both cell types in a dose-dependent manner, reaching the highest significance in COV434 cells at 1000 µM (p < 0.001). In conclusion, melatonin demonstrated a dose-dependent inhibitory effect on COV434 cell proliferation while simultaneously reducing global DNA methylation levels.