Figure 4. Caspase-3 inhibition limits the anti-proliferative effect of paclitaxel on tumor cells in a cell-dependent fashion.

Tumor cells cultured in 96-well plates were incubated for 10 min with a final concentration of 10 μM Ac-DEVD-CHO, a reversible caspase-3 inhibitor, then treated with varying concentrations of drug and allowed to grow for 48 h. Cell proliferation was then measured using CyQuant® Cell Proliferation assay, as previously described. Appropriate controls were performed, without inhibitor and with a nonspecific inhibitor and experiments done in triplicate. N=3. Data represent mean values ± S. D. The % change in cell proliferation was determined relative to inhibitor controls. (A) At 6.25 μM of paclitaxel, A549 cells showed the most gain with about 55% change in the number of proliferating cells, relative to controls without the inhibitor, while 4T1-luc cells showed a modest gain, with approximately 24% change in cell number, relative to controls. A427-cells benefited the least, with about 11% change in proliferation, relative to controls. This pattern was replicable at concentrations ≤ 12.5 μM, but not at concentrations ≥25 μM, perhaps due to loss of inhibition at higher drug doses. (B) To complement studies in (A), cells treated with 6.25 μM of drug, were allowed to grow for 48 h, after which drug was washed off with phosphate buffered saline and cells re-cultured for another 48 h in fresh medium. Appropriate controls with non-drug treated cells were performed and each experiment done in triplicate. N=3. % Restored Growth was computed by the formula: 100% [1- (treatment/control)]. A427, with the least effector caspase activity, showed the most favorable response, with approximately 18% growth restoration after drug withdrawal, followed by A549-cells, with 12% growth restoration, while 4T1-luc cells showed the least viability, with 6% restored growth.