Astaxanthin Increases Tumor Suppressor Gene Expression and Affects Cellular Biological Behavior in Oral Dysplastic Keratinocytes by Regulating DNA Methylation.

Abstract

The inactivation of tumor suppressor genes (TSGs) caused by abnormal DNA methylation is confirmed to be widely present in oral potential malignant diseases (OPMDs). Carotenoids like lycopene and astaxanthin can regulate DNA methylation and exert anticancer effects. Therapeutic effect of astaxanthin in OPMDs and oral squamous cell carcinoma (OSCC) models is confirmed, but the relationship between the anti-cancer ability of astaxanthin and its DNA methylation regulation ability remains unclear. Whole-genome bisulfite sequencing (WGBS) were used to provide biological information associated with DNA methylation. Methylation specific PCR was used to detect the methylation level of specific sites. Related markers were evaluated by qRT-PCR and western blot. CCK8 assay, cell scratch assay, flow cytometric analysis were performed to investigate the cell viability, migration, cell cycle, and apoptosis after treated with concentrations of astaxanthin. WGBS revealed that HOXA3 and SOX1 were the TSGs with significant differences in promoter CpG methylation of oral dysplastic keratinocytes (DOK) cells. After treatment with 8 μM astaxanthin, the promoter CpG methylation levels of the TSGs were significantly reduced, resulting in the increase in gene expression. The overall effect of astaxanthin on DOK cells is inhibiting cell viability, reducing cell migration, leading to cell cycle G0/G1 arrest, and promoting apoptosis. This study confirmed significant differences in DNA methylation patterns among oral normal, dysplastic, and cancerous cells. Astaxanthin can reduce the promoter CpG methylation level of TSGs by reducing DNA methyltransferase 1 protein expression level, upregulating mRNA and protein expression, and subsequently modulating the biological behavior of DOK.