Investigation of the Effects of Melatonin on Granulosa Cell Proliferation and DNA Methylation

Abstract

Melatonin, a pineal hormone with antioxidant and regulatory functions, has emerged as a key modulator of ovarian physiology. Its presence in follicular fluid suggests important roles in granulosa cell function, follicle development, 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) compared with healthy endothelial controls (HUVECs). COV434 and HUVEC cells were treated with melatonin at 1, 10, 100, and 1000 µM. Cell viability 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 negative, sham, melatonin-treated, and positive controls. Melatonin showed a cell type-dependent effect. In COV434 cells, proliferation was significantly inhibited, with an IC₅₀ of 10.55 µM, whereas HUVECs displayed increased proliferation 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.