Melatonin Differentially Modulates Temozolomide Responses in Glioblastoma and Endothelial Cells: Associations with Global DNA Methylation and Cortistatin Levels

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults and remains highly resistant to current therapeutic strategies. Temozolomide (TMZ) is a standard chemotherapeutic agent used in GBM treatment; however, intrinsic and acquired resistance limits its efficacy. This study investigated treatment-associated response patterns to TMZ and melatonin in U87MG glioblastoma cells and human umbilical vein endothelial cells (HUVECs). Relative MTT metabolic activity was assessed using the MTT assay, and cell-cycle distribution was analyzed by flow cytometry. Global DNA methylation was evaluated using a 5-methylcytosine assay, with calibration-derived values expressed as %5-mC. CORT was assessed semi-quantitatively by ELISA, with background-corrected OD values normalized to the corresponding untreated control within each experimental run and expressed as relative CORT levels (% of control). Linear mixed-effects models were used to evaluate TMZ concentration, melatonin exposure, and the TMZ × melatonin interaction, with biological experiment included as a random effect. TMZ and melatonin produced treatment-condition- and cell-type-associated response patterns. In HUVECs, relative MTT metabolic activity remained comparatively high across most TMZ concentrations, whereas a more pronounced decrease was observed at 100 µM TMZ during melatonin co-treatment. U87MG cells showed a non-linear MTT response across TMZ concentrations, and the magnitude and direction of the melatonin-associated effect varied according to TMZ dose. Significant TMZ × melatonin interactions were identified for relative MTT metabolic activity in both cell types. Global DNA methylation-related measurements also showed treatment-associated differences, with distinct response patterns between HUVEC and U87MG cells; calibration-derived %5-mC values were used for quantitative presentation of global DNA methylation. Relative CORT levels likewise varied across treatment conditions, with larger relative changes observed in U87MG cells under several combination conditions. The observed findings should be interpreted as exploratory and hypothesis-generating. The present data do not establish therapeutic selectivity, pharmacological synergy, a causal epigenetic mechanism, or a CORT-mediated stress-response pathway. Further validation using additional glioblastoma models, non-malignant neural controls, fully quantitative and appropriately normalized CORT measurements, and targeted functional pathway analyses is required before mechanistic or clinical conclusions can be drawn.