Evaluation of an aerosolized selective COX-2 inhibitor as a potentiator of doxorubicin in a non-small-cell lung cancer cell line

Purpose. To evaluate the in vitro effects of an aerosolized cyclooxygenase-2 (COX-2) inhibitor, nimesulide, on the cytotoxicity and apoptotic response of doxorubicin against the human lung adenocarcinoma cell line A549. Methods. Nimesulide was formulated into a metered dose inhaler (MDI) formulation and characterized for aerodynamic particle size and medication delivery. The in vitro cytotoxicity of nimesulide-MDI in the presence or absence of doxorubicin was assessed by using the six-stage viable impactor by an already standardized method. Induction of apoptosis in A549 cells by nimesulide (nonaerosolized or aerosolized) in combination with doxorubicin was evaluated by established techniques such as caspase-3 estimation and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining. Finally, to understand the mechanism of action, the influence of different treatments on the expression of COX-2 and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in A549 cells was studied by immunoblotting. Results. The nimesulide-MDI formulation had a mass median aerodynamic diameter (MMAD) of 1.1 mum, (GSD = 2.8) and a medication delivery of 51 mug/shot. Nimesulide-MDI ( 40 shots) in combination with doxorubicin (0.01 mug/ml) had a cell kill of more than 60% as determined by in vitro cytotoxicity assay. The specific caspase-3 activity in A549 cells treated with nimesulide (40 mug/ml) and doxorubicin (0.25 mug/ml) in combination was 3 and 5 times higher than doxorubicin and nimesulide, respectively. Further, TUNEL staining showed apoptosis in over 30% of A549 cells treated with aerosolized nimesulide and doxorubicin combination vs. negligible as seen in cells treated individually. The expression of COX-2 was not altered in control or treatments, whereas PPAR-gamma was expressed only in the combination treatment. Conclusion. Our results indicate that aerosolized nimesulide significantly enhances doxorubicin activity against A549 cells, and the enhanced cytotoxicity was probably mediated via a COX-2 independent mechanism.