Fig. 1.

NO-ASA induces the expression of COX-2 in colon and pancreatic cancer cell lines. (A) HT-29 colon cancer cells were treated for 24 h with o-, m- or p-NO-ASA or ASA 5 mM and their total cell protein lysates were assessed for COX-2 or COX-1 expression by immunoblotting. α-Tubulin was the loading control. NO-ASA induces the expression of COX-2 concentration-dependently. Bottom panel: HT-29 cells were treated with p-NO-ASA 30 μM or vehicle for 24 h and examined by immunofluorescence microscopy as in Materials and Methods. (a) Control cells; primary antibody = non-specific IgG. (b) Vehicle-treated cells; primary antibody = anti-COX-2 mAb. (c) NO-ASA-treated cells; primary antibody: anti-COX-2 mAb. Note that the last lane in the first, third and fourth immunoblot is from cells treated with conventional ASA. (B) BxPC-3 pancreatic cancer cells were treated for 24 h with o-, m- or p-NO-ASA at the indicated concentrations or ASA 5 mM and their total cell lysates were assessed for COX-2 or COX-1 expression by immunoblotting, showing concentration-dependent induction of COX-2. α-Tubulin was the loading control. Bottom panel: BxPC-3 cells treated with p-NO-ASA 30 μM or vehicle for 24 h were examined by immunofluorescence microscopy as in Materials and Methods. (a) Cells treated with NO-ASA; primary antibody: non-specific IgG. (b) Cells treated with vehicle; primary antibody: anti-COX-2 mAb. (c) Cells treated with NO-ASA; primary antibody: anti-COX-2 mAb. (C) The growth of BxPC-3 cells plated as in Materials and Methods and treated with p-NO-ASA 20 μM for the indicated time periods was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. At each time point, cells treated in parallel with p-NO-ASA 20 μM (+) or vehicle (−) were assayed by immunoblotting for the expression of COX-2 or COX-1. Loading control, α-tubulin. (D) BxPC-3 cells were treated with various concentrations of p-NO-ASA for 6 or 24 h and PGE₂ levels were assayed in the culture medium as in Materials and Methods.