Simvastatin enhances irinotecan-induced apoptosis in human non-small cell lung cancer cells by inhibition of proteasome activity

Simvastatin, a potent inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA) is known to stimulate apoptotic cell death and induce cell cycle arrest through inhibition of proteasome. The purpose of this study is to investigate whether simvastatin would be synergistic with irinotecan against human non-small cell lung cancer (NSCLC) cells. Antitumor effect was measured by growth inhibition of cells and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cytotoxic interaction between irinotecan and simvastatin was assessed using the combination index. Effects on cell cycle distribution and apoptosis were determined by flow cytometry and DNA fragmentation. Proteasome activity was measured by ELISA quantification of 20S proteasome. NF-kappa B activation was determined using TransAM (TM) NF-kappa B p65 Transcription Factor Assay Kit. I kappa B-alpha was measured by immunoblot. A combination of irinotecan with simvastatin showed significantly enhanced cell growth inhibition compared with irinotecan alone, which resulted in a synergistic cytotoxicity. Irinotecan and simvastatin combination treatment of A549 and H460 cells increased G(1) phase arrest, which was associated with up-regulation of p21(WAF1/CIP) and p53 compared with irinotecan alone. In addition, simvastatin combination treatment increased irinotecan-related apoptosis as determined by fluorescence microscopy and flow cytometric analysis. We also found that combination therapy showed superior proteasome inhibitory activity leading to effectively suppress NF-kappa B transcription factor activation. Consistently, this effect was associated with up-regulation of I kappa B-alpha. These findings suggest that simvastatin enhances irinotecan-induced apoptosis in human NSCLC cells through inhibition of proteasome activity.