Fig. 5.

The effect of superoxide anion and hydrogen peroxide on COX-2 expression in pancreatic cancer cells. BxPC-3 cells were treated for 12 or 24 h with a combination of X 1 mM and various concentrations of the enzyme XO or p-NO-ASA 20 μM. (A) After BxPC-3 cells were treated for 90 min with either X plus XO or vehicle control, ROS levels were determined using either DHE or DCFDA fluorescent probes, as in Materials and Methods. The corresponding intensity of fluorescence was determined by flow cytometry. (B) The growth of BxPC-3 cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after 24 h of treatment with either a combination of X 1 mM and various concentrations of the enzyme XO or p-NO-ASA 20 μM. The number after XO in the abscissa indicates U/ml. (C) Immunoblots showing the induction of COX-2 by X plus XO, which is dependent upon the amount of XO used and is analogous to that obtained by NO-ASA. α-Tubulin was the loading control. Each lane has been quantified by densitometry and values have been normalized to the corresponding α-tubulin (loading control). The results for each immunoblot are shown in the graphs; letters in the abscissa represent the corresponding immunoblot lanes. (D) BxPC-3 cells were treated for 3 h with various concentrations of H₂O₂ and the expression of COX-2 was determined by immunoblotting. Loading control, β-actin.