Apoptosis occurs via two separate yet interlinked signaling mechanisms: the extrinsic death receptor-induced pathway activated by proapoptotic receptor signals at the cellular surface, and the intrinsic mitochondria-mediated pathway activated by mitochondrial signals from within the cell [11]. The key mediators of apoptosis are caspases, a family of cysteine proteases that cleave a critical set of cellular proteins near specific aspartic acid residues. Among potential effective anticancer treatments, TRAIL (TNFa-related apoptosis inducing ligand) is a promising anti- neoplastic agent because it induces apoptosis in cancer cells with only negligible effect on normal cells [12]. TRAIL triggers caspase cascade via the extrinsic apoptotic pathway through interaction with TRAIL-responsive death receptors (DR), such as DR4 and DR5, leading to the formation of the death-inducing signalling complex (DISC), the recruitment and rapid activation of caspase 8. In type I cells, TRAIL-R-induced caspase-8 activation directly results in downstream caspase activation and apoptosis, whereas in type II cells caspase-8 activation is ineffective and the signal must be amplified through caspase-8-cleavage of the pro-apoptotic Bid protein and the activation of mitochondrial apoptotic pathway [13] through caspase 9 activation. Both caspases 8 and 9 activate the executioner caspase 3, which is the primary activator of apoptotic DNA fragmentation and leads to cancer cell apoptosis [14]. Survivin and cellular FLICE-inhibitory protein (c-FLIP) are downstream and upstream anti-apoptotic regulators of the apoptotic signaling cascade, respectively. Like other inhibitors of apoptosis (IAP), survivin acts down- stream of mitochondria to prevent processing or activation of initiator caspase-9, leading to inhibition of the activity of the effector caspases. However, the role of survivin is not limited to apoptosis inhibition, but also involves the regulation of cell division [15]. C-FLIP is the major protein that prevents caspase 8 from activation by death receptors through binding to Fas-associated death domain and caspase 8 at the DISC. Although multiple splicing isoforms of c-FLIP mRNA have been described, only two of them, c-FLIPS and c-FLIPL, have been significantly studied at the protein level. Both proteins can be recruited to the death- inducing signaling complex and inhibit death receptor-mediated apoptosis [16]. Both c-FLIPL and c-FLIPS are quick turnover proteins; thus, their levels are subject to regulation by ubiquitin/ proteasome-mediated degradation [17,18]. Results from preclinical studies with recombinant TRAIL have demonstrated its significant anti-tumor activities, indicating the potential of utilizing TRAIL as an anticancer agent [19]. Despite this proapoptotic role of TRAIL in the transformed cells, many cancer cells develop resistance towards TRAIL-induced apoptosis. Thus, it has been reported that bladder cancer cell lines, such as T24 and HT1376, are TRAIL-resistant [20]. Identification of drugs or agents that can overcome TRAIL resistance and sensitize cancer cells towards TRAIL-induced apoptosis is thus critically important for targeting TRAIL-resistant cancer cells. The combination of TRAIL with radiation and some chemotherapeu- tic agents [21,22] have provided limited success, because this combination also resulted in an increase in systemic toxicity. The present study reported the effects of ciglitazone on RT4 (derived from a well differentiated grade I papillary tumor) and T24 (derived from an undifferentiated grade III carcinoma) bladder cancer cell lines. The choice of these particularly bladder cancer cell lines is relevant since in RT4 cells, P53 is wild type and the mesenchymal marker N-cadherin is not expressed whereas the epithelial marker E-cadherin is detected. Inversely, in T24 cells, P53 is mutated and E-cadherin is absent and replaced by N- cadherin [23]. Thus, depending on differentiated state of the cells, Ciglitazone Induces Bladder Tumor Cells Apoptosis we showed that ciglitazone individual treatment induced G2/M phase cell cycle arrest but triggered apoptosis only in T24 high grade bladder cancer cells through PPARc activation-independent mechanisms. In addition, for the first time to our knowledge, the treatment of nude mice by ciglitazone significantly impairs the growth of high grade bladder tumor xenografts confirming our in vitro results. Most interestingly, the combination of ciglitazone and TRAIL displayed that ciglitazone let TRAIL-refractory T24 cells to respond to TRAIL. For the first time in bladder cancer cells, we revealed that ciglitazone-mediated up-regulation of TRAIL and a marked decrease of c-FLIP and survivin mediated in part through a proteosomal degradation process contribute to ciglitazone- induced cell death and to the sensitizing effect of this TZD on TRAIL-induced apoptosis. Results Ciglitazone blocks RT4 (low grade) and T24 (high grade) cells in G2/M phase but induces apoptosis only in T24 cells through caspase activation To investigate the effect of ciglitazone on RT4 and T24 bladder cancer cells apoptosis, cells were treated with varying concentra- tions of drug (5–60 mM) for 24 h and assessed for propidium iodide stained DNA content using flow cytometric analysis. As shown in Figure 1A, in RT4 cells, the drug induced a dose- dependent increase of cells in the G2/M phase of the cell cycle in association with an increase of expression level of p53, p21Cip1/ Waf1 and p27Kip1 and a decline of cyclin B1 protein level (Figure 1B). But there was no significant increase of the sub-G1 population compared to untreated control cells (Figure 1C) indicating that a 24 h-ciglitazone treatment did not induce apoptosis of these cells even at a high concentration of 60 mM drug. Besides, ciglitazone did not induce cleavage of caspase 3 and its substrate PARP (Figure 1D). As observed for RT4 cells, there was a dose-dependent increase of G2/M fraction of T24 cells (data not shown) but unlike RT4 cells, the analysis of nuclear DNA distribution in T24 cells showed a dose-dependent increase of sub-G1 population with ,40% cell death from 40 mM concentration (Figure 2A). To characterize the cell death pathway triggered by ciglitazone, the cleavage of caspases 9, 8, 3 was determined by western blotting analysis. From a 40 mM concentration of drug, a considerable enhanced processing of both caspase 9 and caspase 8 to their active fragments was detected (Figure 2B). Caspase 3 which represents the classical downstream execution enzyme of caspase 8 and 9 was cleaved into its active form (20/18 kDa). Activation of caspase 3 was confirmed by the cleavage of the pro-form PARP (116 kDa) to the inactive form (85 kDa) (Figure 2B). As shown in Figure 2C, while p53 and Bax expression levels were unchanged, Bcl-2 was drastically decreased and Bid was truncated. These results indicated that T24 cells could amplify the apoptotic signal through the mitochondria and behave like type II cells after ciglitazone exposure. The caspase cascade activation is a key event for apoptotic process. As expected, the incubation with caspase 8 (Z-IETD- FMK) or 9 (Z-LEHD-FMK) specific inhibitors blocked caspases 8 or 9 processing (Figure 2D, left panel) and decreased the ciglitazone-induced apoptosis as evidenced by a significant decrease of sub-G1 population (Figure 2D, right panel) associated with a less caspase 3 cleavage (Figure 2D, left panel). These data indicate that RT4 and T24 cells did not have a similar response to ciglitazone treatment. Indeed, ciglitazone-induced apoptosis was restricted to T24 cells even though it blocked T24 and RT4 cells at the G2/M phase of the cell cycle. In addition, the two main PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e28354