Activation of TLR4 Is Required for the Synergistic Induction of Dual Oxidase 2 and Dual Oxidase A2 by IFN-γ and Lipopolysaccharide in Human Pancreatic Cancer Cell Lines

Pancreatitis is associated with release of proinflammatory cytokines and reactive oxygen species and plays an important role in the development of pancreatic cancer. We recently demonstrated that dual oxidase (Duox) 2, an NADPH oxidase essential for reactive oxygen species-related, gastrointestinal host defense, is regulated by IFN-gamma-mediated Stat1 binding to the Duox2 promoter in pancreatic tumor lines. Because LPS enhances the development and invasiveness of pancreatic cancer in vivo following TLR4-related activation of NF-kappa B, we examined whether LPS, alone or combined with IFN-gamma, regulated Duox2. We found that upregulation of TLR4 by IFN-gamma in BxPC-3 and CFPAC-1 pancreatic cancer cells was augmented by LPS, resulting in activation of NF-kappa B, accumulation of NF-kappa B (p65) in the nucleus, and increased binding of p65 to the Duox2 promoter. TLR4 silencing with small interfering RNAs, as well as two independent NF-kappa B inhibitors, attenuated LPS- and IFN-gamma-mediated Duox2 upregulation in BxPC-3 cells. Induction of Duox2 expression by IFN-gamma and LPS may result from IFN-gamma-related activation of Stat1 acting in concert with NF-kappa B-related upregulation of Duox2. Sustained extracellular accumulation of H2O2 generated by exposure to both LPS and IFN-gamma was responsible for an similar to 50% decrease in BxPC-3 cell proliferation associated with a G(1) cell cycle block, apoptosis, and DNA damage. We also demonstrated upregulation of Duox expression in vivo in pancreatic cancer xenografts and in patients with chronic pancreatitis. These results suggest that inflammatory cytokines can interact to produce a Duox-dependent pro-oxidant milieu that could increase the pathologic potential of pancreatic inflammation and pancreatic cancer cells. The Journal of Immunology, 2013, 190: 1859-1872.