Mechanisms whereby acid reflux may accelerate the progression from Barrett esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. We have previously shown that NADPH oxidase NOX5-S generates reactive oxygen species (ROS) when Barrett's metaplastic cells are exposed to acid. Besides metaplastic cells, other H2O2-producing cells (e.g. inflammatory cells) present in BE mucosa may produce additional ROS which may also affect metaplastic cells contributing to esophageal tumorigenesis. In this study we investigate whether exogenous H2O2 stimulates cell proliferation by increasing NOX5-S expression. Low dose (10-13 M) of H2O2 significantly increased thymidine incorporation, NOX5-S mRNA and protein expression in a Barrett's EA cell line FLO. H2O2-induced increase in NOX5-S expression was significantly inhibited by knockdown of NF-κB1 p50 with p50 siRNA in EA cell lines FLO and OE33. H2O2 significantly increased p65 phosphorylation and the luciferase activity in FLO cells transfected with a NF-κB activation reporter plasmid pNF-κB-Luc. H2O2-induced increase in luciferase activity in FLO cells was significantly decreased by knockdown of ERK2 MAP kinase. Overexpression of p50 and p65 remarkably increased the luciferase activity in FLO cells transfected with a NOX5-S reporter plasmid NOX5-LP. In addition, H2O2-induced thymidine incorporation in FLO cells was significantly decreased by the MEK1/2 inhibitor PD98059 and ERK2 siRNA, but not by ERK1 siRNA. Similarly, H2O2-induced increase in NOX5-S expression was significantly decreased by ERK2 siRNA in FLO and OE33 cells. We conclude that low dose of H2O2 increases cell proliferation. H2O2-induced increase in cell proliferation may depend on sequential activation of ERK2 MAP kinase, NF-κB1, p50 and NOX5-S.
- Received March 29, 2011.
- Revision received July 9, 2011.
- Accepted July 11, 2011.
- The American Society for Pharmacology and Experimental Therapeutics