(data not shown) cells in G2/M phase of the cell cycle. CDK1/ cyclin B1 is responsible for regulating survivin through phosphor- ylation of Thr34 [49]. Thus, we could hypothesize that ciglitazone led to the survivin down-regulation by acting on CDK1/cyclin B1 complex. The decrease of cyclin B1 associated with the RT4 and T24 cells accumulation in G2/M phase of the cell cycle could prevent the phosphorylation and accumulation of survivin, removing a viability checkpoint through which it leads to apoptosis in the case of T24 cells. Further experiments are needed to clarify this point. Several mechanisms explain TZD-mediated down-regulation of downstream (survivin) and upstream (c-FLIP) anti-apoptotic regulators of the apoptotic signalling cascade namely, ubiquitina- tion and proteasome-dependent degradation with or without transcriptional events [43,50]. Here, despite the recovery of survivin and c-FLIP protein level after MG132 pretreatment, T24 cells did not completely rescue from cell death. The down- regulation of both proteins is mediated partially through proteasomal degradation because in the presence of MG132, the protein levels did not reach those of untreated cells and this was not sufficient to prevent ciglitazone-induced apoptosis. Further investigations are needed to better understand the molecular mechanisms involved in this process. We have evaluated the potential anti-urothelial cancer activity of ciglitazone in a mouse model of bladder cancer xenografts. Importantly, our results showed that ciglitazone significantly reduced the development of bladder tumors in mice xenografted with high grade T24 cells but not in mice xenografted with low grade RT4 cells. In addition, ciglitazone did not appear to induce toxicity since the body weight and overall appearance of mice given the drug were not different from those of controls (data not shown). These data demonstrate that ciglitazone has a strong anti- urothelial cancer activity in vivo. The immunohistochemical analysis of caspase 3 activation suggests that in vivo ciglitazone effects are cytotoxic and correlated with the induction of apoptosis. So, to observe a significant reduction of bladder tumor development, both inhibition of proliferation and induction of apoptosis are needed. Collectively, this work has identified a novel pathway for the antitumor properties of ciglitazone in human bladder cancer cells. We demonstrate for the first time that ciglitazone is able to reduce bladder cancer development in an in vivo model. According to our study, it is needed to elucidate in bladder cancer the distinct PPARc-independent mechanisms of ciglitazone action prior to clinical exploration. Furthermore, animal studies are warranted in combination of ciglitazone and TRAIL to strengthen our in vitro results. We suggest that endovesical instillations of ciglitazone could be used as individual treatment or in combination with TRAIL or BCG therapy. This could be a potential efficient treatment tool for bladder cancer. Materials and Methods Reagents The PPARc agonist Ciglitazone and the PPARc antagonist GW9662 were provided from Cayman Chemical. Soluble recombinant TRAIL, proteasome inhibitor MG132, caspase 9 (Z-LEHD-FMK) specific inhibitor and propidium iodide (PI) were purchased from Sigma. Caspase 8 (Z-IETD-FMK) inhibitor was from Bachem (Heidelberg, Germany). The final concentration of organic solvent (dimethylsulfoxide) was less than 0.1% which had no effect on cell viability. DR4 and DR5 blocking antibodies were from Alexis Biochemicals (Lausanne, Switzerland). Ciglitazone Induces Bladder Tumor Cells Apoptosis Cell lines, culture conditions, and treatment The human bladder tumor cell lines RT4 and T24 were obtained from ATCC. Cells were maintained in Mc COY’s 5a medium supplemented with 10% fetal calf serum (FCS) (Invitro- gen, Cergy Pontoise, France), 1% antibiotic antimycotic mixture (10 mg/ml streptomycin, 10 000 units/ml penicillin, 25 mg/ml amphotericin B), 2 mM glutamine and 15 mM Hepes (Sigma) at 37uC in a humidified 5% CO2, 95% O2 air incubator. Cells were tested for the absence of mycoplasma before the beginning of experiments. RT4 cells (4.26104 cells/cm2) and T24 cells (5.26103 cells/cm2) were plated in 12-well plates for flow cytometry analysis and in 6-well plates for protein extraction and cultured in 5% FCS culture medium. After 24 h attachment, cells were washed with serum-free culture medium and then treated either with vehicle or with ciglitazone (5 to 60 mM) for 24 h or with TRAIL (20, 50, 100 ng/ml) for 12 h, alone or in combination for 24 h in a serum-free culture medium. In that case, TRAIL was added the last 12 h in the culture medium. DNA fragmentation and cell cycle analyses The distribution of cells in the sub-G1, G0/G1, S and G2/M cell cycle phases was determined by flow cytometry. After ciglitazone treatment, RT4 and T24 cells were detached with trypsin/EDTA, washed in phosphate-buffered saline (PBS) and centrifuged at 1 200 rpm for 10 min. Total population of cells, including floating and adherent cells, was fixed overnight at 4uC by cold absolute ethanol. After fixation followed by washes with PBS/5 mM EDTA, cells were stained with 1 mg/ml propidium iodide in 50 ml PBS/5 mM EDTA complemented with 1 mg/ml RNAse A DNAse-free (Sigma) and incubated at room temperature for 15 min. The DNA content was analysed by flow cytometry using an excitation wavelength set at 488 nm and emission at 610 nm (FC 500, Beckman Coulter). Twenty thousand events were analysed per sample and apoptosis was determined from the Sub-G1 DNA content with CXP software (Beckman Coulter). The percentages of nuclei in the G0/G1, S, G2/M phases of the cell cycle were quantified using Multicycle analysis software (Beckman Coulter). Gating was set to exclude cell debris, doublets, and clumps. Western blotting After ciglitazone treatment, cells were washed with cold PBS and scraped in 120 ml of lysis buffer RIPA (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P40, 0.5% sodium desoxycholate) supplemented with protease inhibitors (Roche, Meylan, France). Then, cell lysates were sonicated and centrifuged at 10 000 rpm for 10 min at 4uC. Protein concentration was estimated using the Bio-Rad protein assay (Bio-Rad, Marnes-la- Coquette, France). Protein extracts (20 mg for RT4 cells or 15 mg for T24 cells) were solved in Laemmli buffer (Bio-Rad) and separated by a 7.5–15% SDS-PAGE. Proteins were transferred onto PVDF membranes (GE Healthcare, Buckinghamshire, UK) and non specific binding was blocked in TBS-Tween 20 buffer (0.5 mM Tris-HCl, 45 mM NaCl, 0.05% Tween 20, pH 7.4) containing 5% nonfat milk. Membranes were incubated for 1 h with the following appropriate primary antibodies: mouse monoclonal anti-caspase 8 (clone 3-1-9, 1:1 000), anti-p21Cip1/Waf1 (6B6, 1:500), anti-p27kip1 (G173-524, 1:500), anti-p53 (DO-1, 1:500), anti-cyclin D1 (G124- 326, 1:1 000) and anti-PARP (4C10-5, 1:1 000) were obtained from BD Pharmingen. Anti-mouse A-FABP (#AF1443, 1:1 000) was from R&D Systems (Abingdon, UK). Mouse monoclonal cyclin B1 (1:1 000) was kindly gifted by Dr. Marchini (DKFZ, Heidelberg). Rabbit polyclonal anti-caspase 9 (1:1 000), mouse monoclonal anti- survivin (1:500) and mouse monoclonal Bid (1:500) were from Cell PLoS ONE | www.plosone.org 10 December 2011 | Volume 6 | Issue 12 | e28354