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

Abstract

Pancreatitis is associated with release of pro-inflammatory cytokines and reactive oxygen species and plays an important role in the development of pancreatic cancer. We recently demonstrated that dual oxidase 2 (Duox2), an NADPH oxidase essential for ROS-related, gastrointestinal host defense, is regulated by IFN-γ-mediated Stat1 binding to the Duox2 promoter in pancreatic tumor lines. Because lipopolysaccharide (LPS) enhances the development and invasiveness of pancreatic cancer in vivo following Toll-like receptor 4 (TLR4)-related activation of NF-κB, we examined whether LPS, alone or combined with IFN-γ, regulated Duox2. We found that upregulation of TLR4 by IFN-γ in BxPC-3 and CFPAC-1 pancreatic cancer cells was augmented by LPS, resulting in activation of NF-κB, accumulation of NF-κB (p65) in the nucleus, and increased binding of p65 to the Duox2 promoter. TLR4 silencing with siRNAs, and two independent NF-κB inhibitors, attenuated LPS- and IFN-γ–mediated Duox2 upregulation in BxPC-3 cells. Induction of Duox2 expression by IFN-γ and LPS may result from IFN-γ-related activation of Stat1, acting in concert with NF-κB-related upregulation of Duox2. Sustained extracellular accumulation of H2O2 generated by exposure to both LPS and IFN-γ was responsible for an ≈ 50% decrease in BxPC-3 cell proliferation associated with a G1 cell cycle block, apoptosis, and DNA damage. We also demonstrated up-regulation 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.

Reciprocal activation between IL-6/STAT3 and NOX4/Akt signalings promotes proliferation and survival of non-small cell lung cancer cells

“…However, the mechanisms remain largely unknown. This study combined with another report showing IFN-γ and LPS can synergistically induce Duox2/DuoxA2 expression and ROS production in human pancreatic cancer cells [6], strongly supports that NOXs are the major source of ROS production during inflammatory stimuli and act as a critical modulator of the inflammatory response in the context of tumorigenesis.…”

“…Alternatively, cytokines can also stimulate reactive oxidative species (ROS) accumulation in neighboring epithelial cells. For example, IFN-γ and LPS synergistically increase ROS production in human pancreatic cancer cell lines [6]. ROS have been reported to promote tumor cell survival and growth through activation of various signal pathways in cancer cells [7, 8].…”

NADPH Oxidases: A Perspective on Reactive Oxygen Species Production in Tumor Biology

“…DUOX2 expression was also markedly upregulated in BxPC-3 cell xenografts in the absence of cytokine treatment, presumably as a result of the proinflammatory milieu of the tumor microenvironment. Patients with chronic pancreatitis were also found to have increased DUOX expression adjacent to areas of inflammatory cell infiltrates by immunohistochemistry, further supporting a role for inflammatory stimuli in the regulation of DUOX2 expression (147).…”

“…The role of Toll-like receptor 4 (TLR4), a known target of bacterial lipopolysaccharide (LPS), in the upregulation of DUOX2 in pancreatic cancer cells was evaluated in a subsequent study (147). Exposure to both IFN-c and LPS synergistically upregulated the expression of DUOX2 and DUOXA2 in BxPC-3 cells, and increased intra-and extracellular H 2 O 2 production by way of TLR4.…”

Hypothyroidism-associated missense mutation impairs NADPH oxidase activity and intracellular trafficking of Duox2

“…Besides the beneficial proinflammatory effects of Duox2, it is becoming widely accepted that excessive ROS production can lead to pathologic cell functions. According to this concept elevated Duox2 expression was found in patients with chronic pancreatitis, ulcerative colitis, Crohn's disease and in many human cancers [23, 26, 28]. In other systems, Duox enzymes also play an important role in extracellular matrix cross-linking [29, 30]; the produced H 2 O 2 can serve as paracrine mediator in zebrafish [31], whereas in human lung epithelial cells ROS-based signals can accelerate cell migration [8, 32].…”