title: Acid-induced p16 hypermethylation contributes to development of esophageal adenocarcinoma via activation of NADPH oxidase NOX5-S.
abstract: Inactivation of tumor suppressor gene p16 may play an important role in the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA). Hypermethylation of p16 gene promoter is an important mechanism inactivating p16. However, the mechanisms of p16 hypermethylation in EA are not known. Therefore, we examined whether acid increases methylation of p16 gene promoter and whether NADPH oxidase NOX5-S mediates acid-induced p16 hypermethylation in a Barrett's cell line BAR-T and an EA cell line OE33. We found that NOX5-S was present in BAR-T and OE33 cells. Acid-induced increase in H(2)O(2) production and cell proliferation was significantly reduced by knockdown of NOX5-S. Exogenous H(2)O(2) remarkably increased p16 promoter methylation and cell proliferation. In addition, acid treatment significantly increased p16 promoter methylation and decreased p16 mRNA level. Knockdown of NOX5-S significantly increased p16 mRNA, inhibited acid-induced downregulation of p16 mRNA, and blocked acid-induced increase in p16 methylation and cell proliferation. Conversely, overexpression of NOX5-S significantly decreased p16 mRNA and increased p16 methylation and cell proliferation. In conclusion, NOX5-S is present in BAR-T cells and OE33 cells and mediates acid-induced H(2)O(2) production and cell proliferation. NOX5-S is also involved in acid-induced hypermethylation of p16 gene promoter and downregulation of p16 mRNA. It is possible that acid reflux present in BE patients may activate NOX5-S and increase production of reactive oxygen species, which in turn increase p16 promoter methylation, downregulate p16 expression, and increase cell proliferation, thereby contributing to the progression from BE to EA.
authors.affiliation: Department of Medicine, Rhode Island Hospital and Warren Alpert Medical School of Brown University, Providence, 02903, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Hong
authors.forename: Murray
authors.initials: M
authors.lastname: Resnick
authors.forename: Jose
authors.initials: J
authors.lastname: Behar
authors.forename: Li Juan
authors.initials: LJ
authors.lastname: Wang
authors.forename: Jack
authors.initials: J
authors.lastname: Wands
authors.forename: Ronald A
authors.initials: RA
authors.lastname: DeLellis
authors.forename: Rhonda F
authors.initials: RF
authors.lastname: Souza
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Spechler
authors.forename: Weibiao
authors.initials: W
authors.lastname: Cao
mesh_terms: D000230:Adenocarcinoma / Q000378:metabolism*; D002460:Cell Line; D004938:Esophageal Neoplasms / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology*; D019942:Genes, p16 / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008745:Methylation; D000074664:NADPH Oxidase 5; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D017382:Reactive Oxygen Species
chemical_list: D008565:Membrane Proteins; D017382:Reactive Oxygen Species; D000074664:NADPH Oxidase 5; D019255:NADPH Oxidases; C431571:NOX5 protein, human