The Antidiabetic Drug Ciglitazone Induces High Grade Bladder Cancer Cells Apoptosis through the Up- Regulation of TRAIL Marie-Laure Plissonnier1., Sylvie Fauconnet1,2., Hugues Bittard2, Isabelle Lascombe1* 1 Laboratoire de Biologie Cellulaire et Mole´culaire, Equipe d’Accueil 3181 – Institut Fe´de´ratif de Recherche Nu133, Universite´ de Franche – Comte´, Faculte´ des Sciences Me´dicales et Pharmaceutiques, Besanc¸on, France, 2 CHRU (Centre Hospitalier Re´gional Universitaire) de Besanc¸on, Service d’Urologie et d’Andrologie, Besanc¸on, France Abstract Background: Ciglitazone belongs to the thiazolidinediones class of antidiabetic drug family and is a high-affinity ligand for the Peroxisome Proliferator-Activated Receptor c (PPARc). Apart from its antidiabetic activity, this molecule shows antineoplastic effectiveness in numerous cancer cell lines. Methodology/Principal Findings: Using RT4 (derived from a well differentiated grade I papillary tumor) and T24 (derived from an undifferentiated grade III carcinoma) bladder cancer cells, we investigated the potential of ciglitazone to induce apoptotic cell death and characterized the molecular mechanisms involved. In RT4 cells, the drug induced G2/M cell cycle arrest characterized by an overexpression of p53, p21waf1/CIP1 and p27Kip1 in concomitance with a decrease of cyclin B1. On the contrary, in T24 cells, it triggered apoptosis via extrinsic and intrinsic pathways. Cell cycle arrest and induction of apoptosis occurred at high concentrations through PPARc activation-independent pathways. We show that in vivo treatment of nude mice by ciglitazone inhibits high grade bladder cancer xenograft development. We identified a novel mechanism by which ciglitazone kills cancer cells. Ciglitazone up-regulated soluble and membrane-bound TRAIL and let TRAIL-resistant T24 cells to respond to TRAIL through caspase activation, death receptor signalling pathway and Bid cleavage. We provided evidence that TRAIL-induced apoptosis is partially driven by ciglitazone-mediated down-regulation of c-FLIP and survivin protein levels through a proteasome-dependent degradation mechanism. Conclusions/Significance: Therefore, ciglitazone could be clinically relevant as chemopreventive or therapeutic agent for the treatment of TRAIL-refractory high grade urothelial cancers. Citation: Plissonnier M-L, Fauconnet S, Bittard H, Lascombe I (2011) The Antidiabetic Drug Ciglitazone Induces High Grade Bladder Cancer Cells Apoptosis through the Up-Regulation of TRAIL. PLoS ONE 6(12): e28354. doi:10.1371/journal.pone.0028354 Editor: Laszlo Buday, Hungarian Academy of Sciences, Hungary Received August 16, 2011; Accepted November 7, 2011; Published December 12, 2011 Copyright: 2011 Plissonnier et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by research grants from Ligue Nationale Contre le Cancer (Comite´ du Doubs). M.L. Plissonnier was supported by a fellowship from the Cance´ropoˆ le du Grand–Est. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: isabelle.lascombe@univ-fcomte.fr . These authors contributed equally to this work. Introduction Urothelial carcinoma of the bladder accounts for ,5% of all cancer deaths in humans. The majority of bladder tumors (75%) are non muscle-invasive at diagnosis and after local surgical therapy, have a high risk of recurrence and a propensity to progress in grade or stage [1]. Muscle invasive tumors (25%) have a poorer prognosis [2] since 50% of patients will relapse with metastatic disease within 2 years of treatment. Despite the advances with surgery and intravesical immuno- and chemother- apy in the management of patients and although new chemother- apeutic agents (tyrosine kinase inhibitors, anti-angiogenic agents…) are used, no improvement in survival has been observed. Thus, developing novel effective chemotherapeutic regimens with fewer side effects are definitely needed to decrease the morbidity and the mortality of urothelial carcinoma. Thiazolidinediones (TZD), including rosiglitazone, troglitazone, pioglitazone and ciglitazone are a class of insulin-sensitizing drugs. Besides, rosiglitazone and pioglitazone are currently in clinical use for the treatment of type II diabetes for million of patients. These TZD are high-affinity chemically synthesized ligands of Peroxi- some Proliferator-Activated Receptor c (PPARc) [3,4]. PPARc is a ligand-activated transcription factor of the nuclear hormone receptor superfamily. In addition to its known role in regulation of metabolism and inflammation, PPARc has also been implicated in carcinogenesis based on studies showing its ability to modulate cellular differentiation, proliferation, and apoptosis. Apart from their antidiabetic activity, TZD elicit growth inhibitory effects in vitro on cancer cells of diverse tissue origins and in vivo [5]. Few studies indicate that pioglitazone or troglitazone exhibited antiproliferative or pro-apoptotic activities against bladder cancer cell lines [6–9] and in rat urothelium [10]. Only one study reported that ciglitazone exhibited inhibitory effects on cell number in a series of cell lines modelling metastatic transitional cell carcinoma of the bladder (TSU-Pr1, TSU-Pr1-B1 and TSU- Pr1-B2) [6]. PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e28354