Folic Acid Oversupplementation Alters Global DNA Methylation and Circadian Clock Gene Expression in FaDu Head and Neck Squamous Cell Carcinoma Cells: An Exploratory Study

Background/Objectives: Folic acid (FA), the synthetic form of folate (vitamin B9), is a vitamin involved in one-carbon metabolism, DNA synthesis, and DNA methylation. Because folate metabolism influences methyl donor availability, FA availability may affect circadian clock gene regulation through methylation-related or other metabolic mechanisms in head and neck squamous cell carcinoma (HNSCC). This exploratory study aims to determine whether FA oversupplementation influences cell viability and alters global DNA methylation, as well as the expression and methylation status of circadian clock genes in HNSCC cells. Methods: FaDu HNSCC cells were treated with 20 or 80 μM FA and compared with a no-added-FA (NF) reference condition. Cell viability was assessed using a CCK-8 assay after 48 and 72 h. For methylation and gene expression analyses, cells were synchronized with forskolin and treated with FA (20 or 80 μM) for 7 days. Global DNA methylation was measured using a 5-methylcytosine colorimetric assay. Following the NF and 80 μM FA treatment, cytosine-phosphate-guanine (CpG) island methylation in the BMAL1 and PER2 promoters, RNA expression, and protein levels were analyzed by bisulfite conversion followed by Sanger sequencing, qPCR, and ELISA. Results: FA treatment significantly reduced cell viability at 48 h (20 μM, p < 0.05; 80 μM, p < 0.01) and 72 h (both concentrations p < 0.01). Global DNA methylation was modestly reduced, reaching statistical significance only in the 80 μM FA group (p < 0.05). No qualitative differences were observed in the promoter methylation of the analyzed CpG regions of BMAL1 and PER2 following 80 μM FA treatment. However, BMAL1 and PER2 mRNA expressions were significantly increased (p < 0.05), while protein levels showed non-significant upward trends following 80 μM FA treatment. Conclusions: These exploratory findings suggest that the observed transcriptional changes are unlikely to be explained by methylation changes within the analyzed promoter regions of BMAL1 and PER2. The mechanisms underlying these changes remain unclear and require further investigation.