Figure 3

Effect of the Celecoxib analogues on apoptosis induction. To follow the fate of the cells upon Akt inhibition, indicators of apoptosis were temporally measured. Poly(ADP-ribose) polymerase (PARP) cleavage and nucleosomal fragmentation were measured after 12 and 24 hours. The cells were treated with Ly294002 (30 μmol/l), Celecoxib (50 or 100 μmol/l), OSU03012 (5, 7.5, or 10 μmol/l), or OSU03013 (5, 7.5, or 10 μmol/l) and then the cell pellets were split for PARP and nucleosomal cleavage. (a) There was a dose dependent increase in PARP cleavage on treatment with OSU03012 and OSU03013 at both 12 and 24 hours. LY294002 similarly induced PARP cleavage but to a lesser extent. Celecoxib at 50 μmol/l did not have sustained effects on PARP cleavage, whereas the high dose of celecoxib (100 μmol/l) did. (b) The induction of apoptosis was secondarily analyzed by nucleosomal fragmentation. There was a dose dependent increase in nucleosomal fragmentation upon treatment with increasing concentration of either OSU03012 or OSU03013. Likewise, Ly294002 induced fragmentation of the nucleosomes at both time points. Celecoxib at 50 μmol/l did not have such an effect. In contrast, high dose celecoxib did stimulate apoptosis. Each treatment was conducted in replicates of six on two difference occasions. (c) Impact of signal transduction inhibitors on cell survival. The MDA-MB-453 cells were exposed to Ly294002 (30 μmol/l), celecoxib, OSU03012, and OSU03013 at the indicated concentrations and cell viability was assessed 24 hours later. OSU03012 and OSU03013 killed more than 90% of the cells with 10 μmol/l of the respective inhibitors. Celecoxib at 100 μmol/l had a similar effect, but celecoxib at 50 μmol/l was not effective at reducing cell viability. This screen was conducted in replicates of four in three separate experiments. (d) The T47D cells similarly responded to the celecoxib analogues based on cell survival. Each experiment was performed in replicates of six on three separate occasions. (e) MDA-MB-453 cells were treated for 2, 4, 6, or 24 hours with Ly294002 and P-Akt was measured (top panel). A comparison was made with the MDA-MB-453 cells exposed to OSU03012 (10 μmol/l) for 2, 4, or 6 hours. Total Akt was measured as a control for sample input.