Abstract
Troglitazone (TGZ) is a group of potent PPARγ (peroxisome proliferator-activated receptor γ) agonists known to inhibit proliferation, alter cell cycle regulation, and induce apoptosis in various cancer cell types. TGZ is also an oral anti-type II diabetes drug that can reverse insulin resistance. Aspirin (ASA), a nonselective cyclooxygenase (COX) inhibitor, has been successfully used as an anti-inflammatory drug. It has been observed that ASA’s anti-tumor effect can be attributed to inhibition of cell cycle progression, induction of apoptosis, and inhibition of angiogenesis. Methotrexate (MTX) has been widely used for the treatment of cancer. MTX was also applied to be a potent and effective therapy for rheumatoid arthritis (RA). Above all, we demonstrate for the first time that, when administered in combination, TGZ and ASA can produce a strong synergistic effect in growth inhibition and G1 arrest in lung cancer CL1-0 and A549 cells. Whereas we observed that ASA concomitance decreased the MTX-mediated S phase arrest and, surprisingly, increased the proportion of cells at the G1 phase in MTX+ASA. Examination by colony formation assay revealed an even more profound synergy in combination treatment of TGZ+ASA. On the other hand, marked antagonistic effects were observed in both CL1-0 and A549 cells when the two drugs of MTX and ASA were combined. In Western blot, combined TGZ and ASA also could down-regulate Cdk2, E2F-1, cyclin B1, cyclin D3 protein, and the ratio of phospho-Rb/Rb. Importantly, apoptosis was 4 synergistically induced by the combination treatment, as evidenced by caspase-3 activation and PARP cleavage. The involvements of PI3K/Akt inhibition and p27 up-regulation, as well as hypophosphorylation of Rac1 at ser71, were demonstrated. Additionally, our investigation proved that ASA regulates cell cycle progression and Bcl-2 expression, as well as subsequently antagonizes MTX efficiency. Taken together, these results suggest that clinically achievable concentrations of TGZ and ASA used in combination may produce a strong anticancer synergy that warrants further investigation for its clinical applications. However, the results also suggest that ASA may have adverse consequences in patients who are receiving MTX therapy.