Figure 5

COX-2 expression is regulated by p38 in papilloma cells but not normal cells; p38 contributes to the viability of papilloma cells. (A) Cells cultured in serum-free KGM with EGF and insulin were treated for 48 h with 10 µM SB202190, a specific inhibitor of p38 which also inhibits p38 phosphorylation, and analyzed by Western blot for expression of COX-2 and for phosphorylation of Erk and Akt. A representative blot is shown. The bar graph shows the mean ± SD relative level of COX-2, normalized to β-actin and expressed relative to levels in control normal cells (*P < 0.05 compared with control cells of each type). (B) Dose-responsive inhibition of COX-2 expression and PGE₂ synthesis by papilloma cells treated with SB202190. Cells cultured in KGM with EGF and insulin were treated with 0, 5, 10, or 15 µM SB202190 for 48 h, extracted and analyzed by Western blot for COX-2. Bar graph shows the mean ± SD of COX-2 levels in three experiments, normalized to β-actin, and expressed relative to levels in cells treated with DMSO, the solvent for the inhibitor (*P < 0.05, **P < 0.01). Culture supernatants from three experiments were analyzed for PGE₂ by EIA (*P < 0.05, **P < 0.01 compared with controls). (C) Dose-responsive reduction in cell number and increased apoptosis in papilloma cells treated with SB202190. Papilloma cells cultured in KGM with EGF and insulin were incubated for 48 h with increasing concentrations of SB202190. Control cells received vehicle. Metabolic reduction of MTT was used as a surrogate measure of cell number, apoptosis measured by release of nucleosome fragments. Results are means ± SD from three separate experiments. SB202190 significantly reduced papilloma cell number at doses of 10 and 15 µM (*P < 0.01), and increased apoptosis even at the low concentration of 5 µM compared with control cells (*P < 0.01).