Figure 3

Celecoxib induced down-regulation of pAkt, increase in Bax, and caspase 3/7 in MDA-MB-231 cells. (a) Total Akt and phosphorylated Akt (pAkt). Western blot analysis of cell lysates prepared from vehicle and celecoxib (20–60 μmol/l) treated cells. SDS-PAGE electrophoresis was performed using 10% resolving gel. Protein was loaded at 100 μg per lane and the protein of interest was detected using specific antibodies. Celecoxib treatment at 40 and 60 μmol/l caused decreases in the levels of pAkt in MDA-MB-231 cells, with no change in MDA-MB-468 cells. Numbers below each lane represents percentage of protein expression compared with vehicle-treated cell lysate, which was set to equivalent to 100%, as determined by densitometric analysis. Control cell extracts from Jurkats were used as positive control for Akt and pAkt. (b) Average densitometric values of three separate experiments showing reduction in pAkt with celecoxib treatment. There was a significant decrease (P = 0.002) in the levels of pAkt with 60 μmol/l celecoxib treatment. (c) Western blot analysis of BAX. Increased expression of BAX protein was observed with increasing concentrations of celecoxib in MDA-MB-231 cells but not in MDA-MB-468 cells. The experiment was repeated three times with similar results. A β-actin blot is included to show equal loading. (d) Spectrofluorometric analysis of lysates prepared from vehicle and celecoxib (20–60 μmol/l) treated cells at 48 hours. Activity of caspases 3 and 7 was monitored by enzymatic cleavage using a fluorescence microplate reader with excitation at 485 ± 10 nm and emission detection at 530 ± 12.5 nm. In MDA-MB-231 cells, activities of caspases 3 and 7 were increased significantly at 40 μmol/l and 60 μmol/l drug concentrations. No increase in caspase activity was evident in the MDA-MB-468 cells. Mean values from three experiments ± standard deviation is shown. P values represent significant differences between vehicle control and celecoxib treatment.