Increase in oxidative stress and activation of NOX in
–transformed pancreatic cancer cells. A, comparison of basal superoxide levels in parental (E6E7) and K-ras–transformed E6E7 cells (K-ras). K-ras–transformed cells exhibit a 2-fold increase in superoxide generation detected by fluorescence probe HET. B, comparison of basal hydrogen peroxide levels in parental and K-ras–transformed E6E7 cells. K-ras–transformed cells exhibit a 3-fold increase in hydrogen peroxide generation detected by fluorescence probe DCF-DA. C, the levels of CuZnSOD (SOD1) and MnSOD (SOD2) were both up-regulated, as assessed by Western blotting analysis. D, total mRNA levels of NOX2 and NOXA1 measured by real-time polymerase chain reaction (PCR). E, the levels of p22phox and phosphorylated p40phox were both up-regulated, as assessed by Western blotting analysis. F, NOX activity was measured in the presence of 100 μmol/L NADPH or NADH by lucigenin-derived chemiluminescence in 50 μg of membrane fraction from parental and K-ras–transformed E6E7 cells. The values are shown as the mean ± standard deviation (SD). *, P < 0.05; **, P < 0.01. NOX, NADPH oxidase; HET, hydroethidine.