Figure 5
Cyclooxygenase-2 (Cox-2) expression protects cells against daunomycin-induced apoptosis. (a) Increased daunomycin-induced apoptosis in HCT116 in the presence of Cox-2 inhibitors. Left, HCT116 cells were treated with the indicated amount of nimesulid before exposure to daunomycin (2 μ M, 24 h). Survival, assessed by WST-1 colorimetric assay, was expressed as percentage of the average value observed with untreated cells. Right, HCT116 cells were pre-treated with nimesulid (50 μ M), celecoxib (10 μ M) or rofecoxib (2 μ M) before being exposed or not exposed (Ctrl) to daunomycin treatment (2 μ M, 24 h). Cell extracts were incubated with a fluorogenic caspase-3 substrate, and relative caspase activation was expressed by normalizing fluorescence to the level measured in untreated cells. Bottom: Detection of PARP cleavage product in HCT116 treated with nimesulid before exposure to daunomycin (2 μ M, 24 h). Cell lysates were analysed by Western blot with anti-cleaved PARP antibody. (b) Enhanced proapoptotic effects of Cox-2 inhibitors in TE-1 cells cultured at 32°C. TE-1 cells were grown at 37°C or 32°C, treated with celecoxib (10 μ M) or rofecoxib (2 μ M) before exposure to daunomycin (1 μ M, 24 h). Results were expressed by scoring the percentage of increase (positive values) or decrease (negative values) in global cell mass as measured using the sulforhodamine B assay, with 0 representing no change in global cell mass over the duration of treatment (30 h), positive values representing net cell growth, and negative values indicating a net decrease in cell mass owing to massive cell death.
