Role of reactive oxygen species in brucein D-mediated p38-mitogen-activated protein kinase and nuclear factor-κB signalling pathways in human pancreatic adenocarcinoma cells

BACKGROUND: In human pancreatic adenocarcinoma, nuclear factor-kappa-B (NF-kappaB) transcription factor is constitutively activated that contributes to the resistance of the tumour cells to induced apoptosis. In our earlier studies, we have shown that brucein D (BD) mediated apoptosis through activation of the p38-mitogen-activated protein kinase (MAPK) signalling pathway in pancreatic cancer cells. This study investigated the function of reactive oxygen species (ROS) in BD-mediated p38-MAPK and NF-kappaB signalling pathways in PANC-1 cells. METHODS: Glutathione and dihydroethidium assays were used to measure the antioxidant and superoxide levels, respectively. The protein expression of p22(phox), p67(phox) and p38-MAPK were examined by western blot. The NF-kappaB activity was evaluated by electrophoretic mobility shift assay. RESULTS: Treatment with BD depleted the intracellular glutathione levels in PANC-1 cells. Brucein D triggered the activation of NADPH oxidase isoforms, p22(phox) and p67(phox) while enhancing the generation of superoxide. Increases in both intracellular ROS and NADPH oxidase activity were inhibited by an antioxidant, N-acetylcysteine (NAC). Brucein D-mediated activation of p38-MAPK was also inhibited by NAC. However, inhibition of NF-kappaB activity in BD-treated cells was independent of ROS. In vivo studies showed that BD treatment effectively reduced the rate of xenograft human pancreatic tumour in nude mice with no significant toxicity. CONCLUSION: These data suggest that BD is an apoptogenic agent for pancreatic cancer cells through activation of the redox-sensitive p38-MAPK pathway and inhibition of NF-kappaB anti-apoptotic activity in pancreatic cancer cells.