Role of reactive oxygen species in brucein D-mediated p38-mitogen-activated protein kinase and nuclear factor-kappaB 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.


CONCLUSIONS
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.
Lau
S T
ST
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.
Lin
Z X
ZX
Leung
P S
PS
D000230:Adenocarcinoma / Q000188:drug therapy* / Q000378:metabolism; D000818:Animals; D045744:Cell Line, Tumor; D024202:Electrophoretic Mobility Shift Assay; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D019255:NADPH Oxidases / Q000502:physiology; D016328:NF-kappa B / Q000502:physiology*; D010190:Pancreatic Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D036702:Quassins / Q000494:pharmacology* / Q000627:therapeutic use; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013481:Superoxides / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays; D048051:p38 Mitogen-Activated Protein Kinases / Q000502:physiology*
D016328:NF-kappa B; D036702:Quassins; D017382:Reactive Oxygen Species; D013481:Superoxides; C030412:bruceine D; D019255:NADPH Oxidases; D048051:p38 Mitogen-Activated Protein Kinases; D005978:Glutathione