The Synergistic Anticancer Effect of Troglitazone Combined With Aspirin Causes Cell Cycle Arrest and Apoptosis in Human Lung Cancer Cells Kun-Huang Yan, 1,2 Chih-Jung Yao, 2,5 Hwan-You Chang, 3 Gi-Ming Lai, 2,6 Ann-Lii Cheng, 4 and Shuang-En Chuang 2 * 1 Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, ROC 2 National Institute of Cancer Research, National Health Research Institutes, Miaoli County, Taiwan, ROC 3 Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan, ROC 4 Departments of Internal Medicine and Oncology, National Taiwan University Hospital, Taipei, Taiwan, ROC 5 Medical Education and Research Department, Taipei Medical University-Wan Fang Hospital, Taipei, Taiwan, ROC 6 Cancer Center, Taipei Medical University-Wan Fang Hospital, Taipei, Taiwan, ROC Troglitazone (TGZ) is a synthetic thiazolidinedione drug belonging to a group of potent peroxisome proliferator- activated receptor g (PPARg) agonists known to inhibit proliferation, alter cell cycle regulation, and induce apoptosis in various cancer cell types. TGZ is an oral anti-type II diabetes drug that can reverse insulin resistance. For more then 100 yr, aspirin, a nonselective cyclooxygenase (COX) inhibitor, has been successfully used as an anti-inflammatory drug. Recently, Aspirin (ASA) and some other nonsteroidal anti-inflammatory drugs (NSAIDs) have drawn much attention for their protective effects against colon cancer and cardiovascular disease; 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. In this report we demonstrate for the first time that, when administered in combination, TGZ and ASA can produce a strong synergistic effect in growth inhibition and G 1 arrest in lung cancer CL1-0 and A549 cells. Examination by colony formation assay revealed an even more profound synergy. In Western blot, combined TGZ and ASA also could downregulate Cdk2, E2F-1, cyclin B1, cyclin D3 protein, and the ratio of phospho-Rb/Rb. Importantly, apoptosis was synergistically induced by the combination treatment, as evidenced by caspase-3 activation and PARP cleavage. The involvement of PI3K/Akt inhibition and p27 upregulation, as well as hypophosphorylation of Rac1 at ser71, were demonstrated. 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. ß 2009 Wiley-Liss, Inc. Key words: PPARg; aspirin; NSAID; synergy; troglitazone INTRODUCTION Lung cancer is one of the leading causes of global cancer death. Development of novel chemothera- peutic strategies involving drug combination is of particular interest. Numerous in vitro studies have shown that peroxisome proliferator-activated recep- tor g (PPARg) ligands could exert synergistic anti- cancer effects in combination use with other therapeutics, such as statins and many nonsteroidal anti-inflammatory drugs (NSAIDs) [1]. Although the mechanisms underlying PPARg agonists’ anticancer effects remain elusive, many PPARg ligands may serve as potential therapeutic agents for NSCLC [2,3]. The synthetic thiazolidine- diones (TZDs) compounds are a group of potent PPARg agonists that include troglitazone (TGZ), ciglitazone (CGZ), pioglitazone (PIO), and rosiglita- zone (Rosi). The order of ligand potency for PPARg was Rosi > PIO > TGZ > CGZ, as measured by reporter assays [4]. Certain NSAIDs and the potent MOLECULAR CARCINOGENESIS 49:235–246 (2010) ß 2009 WILEY-LISS, INC. Additional Supporting Information may be found in the online version of this article. Abbreviations: NSAID, nonsteroidal anti-inflammatory drugs; TZD, thiazolidinedione; PG, prostaglandin; TGZ, troglitazone; CGZ, ciglitazone; PIO, pioglitazone; PPARg, peroxisome proliferator- activated receptor g; ASA, aspirin; CI, combination index. Novelty and impact: (i) The synergy between aspirin and PPARg agonists (e.g., troglitazone) reported in our paper is dramatic in amplitude, especially when evaluated by colony formation assay. Importantly, both drugs are within clinically achievable concen- trations. (ii) Although many scientists and clinicians have hoped PPARg agonists hold potential anticancer traits and have conducted various relevant basic research and clinical trials, aspirin’s potential in this area has rarely been explored. We are the first to combine the two types of drugs for anticancer synergy. With this dramatic combination effect of aspirin and troglitazone, a promising regimen of cancer therapy and/or tertiary chemoprevention may be materialized in the future. *Correspondence to: National Institute of Cancer Research, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli County 35053, Taiwan, ROC. Received 6 May 2009; Revised 11 September 2009; Accepted 23 September 2009 DOI 10.1002/mc.20593 Published online 11 November 2009 in Wiley InterScience (www.interscience.wiley.com)