title: Methyl-donor depletion of head and neck cancer cells in vitro establishes a less aggressive tumour cell phenotype.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Human Nutrition Unit, Department of Oncology, University of Sheffield, Sheffield, S10 2RX, UK.|School of Clinical Dentistry, University of Sheffield, Sheffield, S10 2TA, UK.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Hearnden
authors.affiliation: Human Nutrition Unit, Department of Oncology, University of Sheffield, Sheffield, S10 2RX, UK.
authors.forename: Hilary J
authors.initials: HJ
authors.lastname: Powers
authors.affiliation: Human Nutrition Unit, Department of Oncology, University of Sheffield, Sheffield, S10 2RX, UK.
authors.forename: Abeir
authors.initials: A
authors.lastname: Elmogassabi
authors.affiliation: Human Nutrition Unit, Department of Oncology, University of Sheffield, Sheffield, S10 2RX, UK.
authors.forename: Rosanna
authors.initials: R
authors.lastname: Lowe
authors.affiliation: School of Clinical Dentistry, University of Sheffield, Sheffield, S10 2TA, UK. c.murdoch@sheffield.ac.uk.
authors.forename: Craig
authors.identifier: http://orcid.org/0000-0001-9724-122X
authors.initials: C
authors.lastname: Murdoch
mesh_terms: D017209:Apoptosis; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D019175:DNA Methylation; D006258:Head and Neck Neoplasms / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D010641:Phenotype
keywords: Apoptosis; DAPK; DNA methylation; Head and neck cancer; Methyl donor