Methyl-donor depletion of head and neck cancer cells in vitro establishes a less aggressive tumour cell phenotype

Vanessa Hearnden, Hilary J. Powers, Abeir Elmogassabi, Rosanna Lowe, Craig Murdoch

DNA methylation plays a fundamental role in the epigenetic control of carcinogenesis and is, in part, influenced by the availability of methyl donors obtained from the diet. In this study, we developed an in-vitro model to investigate whether methyl donor depletion affects the phenotype and gene expression in head and neck squamous cell carcinoma (HNSCC) cells. HNSCC cell lines (UD-SCC2 and UPCI-SCC72) were cultured in medium deficient in methionine, folate, and choline or methyl donor complete medium. Cell doubling-time, proliferation, migration, and apoptosis were analysed. The effects of methyl donor depletion on enzymes controlling DNA methylation and the pro-apoptotic factors death-associated protein kinase-1 (DAPK1) and p53 upregulated modulator of apoptosis (PUMA) were examined by quantitative-PCR or immunoblotting. HNSCC cells cultured in methyl donor deplete conditions showed significantly increased cell doubling times, reduced cell proliferation, impaired cell migration, and a dose-dependent increase in apoptosis when compared to cells cultured in complete medium. Methyl donor depletion significantly increased the gene expression of DNMT3a and TET-1, an effect that was reversed upon methyl donor repletion in UD-SCC2 cells. In addition, expression of DAPK1 and PUMA was increased in UD-SCC2 cells cultured in methyl donor deplete compared to complete medium, possibly explaining the observed increase in apoptosis in these cells. Taken together, these data show that depleting HNSCC cells of methyl donors reduces the growth and mobility of HNSCC cells, while increasing rates of apoptosis, suggesting that a methyl donor depleted diet may significantly affect the growth of established HNSCC.