Aminoguanidine (AG), a diamine oxidase and a nitric oxide synthase inhibitor, was used in diabetes, thyroid follicular carcinoma, hepatocellular carcinoma, pancreatic cancer xenografts and in breast cancer research. The effects of AG on these pathologic conditions may be related to its regulatory effects on cell proliferation, angiogenesis, and expression of antioxidant enzymes. However, its role as pro and/or anti-oxidant affecting signaling and function in pancreatic tumor cell lines has not been studied. The current study tested the hypothesis that exposure of AR42J cells to aminoguanidine will induce pro-oxidant effects that may lead to increased proliferation and growth of these cells.Methods.
AR42J cells were grown in F-12 nutrient medium in 5% CO2 at 37°C to attain over 90% confluency before being treated with 20 uM hydrogen peroxide (H2O2) for 20 min and 100 uM AG for 30 min separately and in combination. Cell lysates collected from these experiments were measured for formation of lipid peroxides by malondialdehyde (MDA) assay and for activation of phospho-ERK 1/2 signal transduction by Western blotting. The activation of ERK signaling was further confirmed by immunohistochemical analysis. Effect of ERK1/2 on cell proliferation in response to AG and H2O2 was evaluated by MTT assay while the functional status of AR42J cells was determined by release of amylase following CCK-8 stimulation.Results.
MDA concentration in cells treated with AG was not different from untreated cells. However, treatment with H2O2 either alone or in combination with AG increased MDA significantly (p<0.05). AG treatment alone induced 3.5 fold activation of pERK-1/2, as compared to 2.5 fold increase with H2O2 alone (p<0.05) as compared to untreated control. The results of ERK activation were confirmed further by its co-localization employing FITC-conjugated ERK antibody. AG -induced maximal cell proliferation occurred at 48 hr. incubation (p<0.05); these values were not significantly different from that of H2O2 treated and control cells. Cell function (CCK-stimulated amylase release) was significantly enhanced by AG (p<0.05).Conclusion.
These data suggest that in an in-vitro system, AG acts as a pro-oxidant on AR42J cell proliferation and possibly affects the resulting function.