Ciglitazone Induces Bladder Tumor Cells Apoptosis (E) Cells were pretreated with 25 mM proteasome inhibitor MG132 for 30 min and stimulated with 40 mM ciglitazone for 12 h ; top, the percentage of cells showing hypodiploid DNA content was evaluated by flow cytometry analysis ; bottom, immunoblotting detection of c-FLIP and survivin. b-actin was used as an internal loading control. (B, C, E) Data are means 6 SD of 2 independent experiments performed in triplicates. *P,0.05, significant differences compared with untreated cells ; **, P,0.05, significant differences compared with ciglitazone and TRAIL cotreated cells with the use of two-tailed unpaired Student’s t test. doi:10.1371/journal.pone.0028354.g006 Discussion The present report demonstrated that ciglitazone induced cell growth inhibition through cell cycle course control or induction of apoptosis according to the tumor grade of bladder cancer-derived urothelial cells. Apoptotic effects occurred at high concentrations (40–60 mM) independently of PPARc activation as already reported in other cellular models [27]. Several potential PPARc- activation independent mechanisms that may induce apoptosis have been proposed. They include the generation of reactive oxygen species [28], the induction of cellular acidosis [29], the involvement of early growth response-1 and NSAID-activated- gene 1 in colon cancer [30]. For the first time in malignant urothelial cells, we present evidence that ciglitazone induced G2/M cell cycle arrest and changes in cell cycle regulators. The major regulator of the G2 to M transition is the MPF (M-phase promoting factor), which comprises the catalytic subunit Cdc2 and the regulatory subunit cyclin B1. Several observations suggest that p21Cip1/Waf1 plays an important role in G2/M checkpoint control. P21Cip1/Waf1 inhibits either Cdc2 kinase activity or prevent its activation by directly binding to Cdc2, thereby blocking the interaction of cyclin B1- Cdc2 complexes with their substrates [31]. Our results show that in low grade papillary bladder tumor-derived RT4 cells, ciglitazone led to the down-regulation of cyclin B1 protein level and a marked increase in p21Cip1/Waf1 and p27Kip1 expression associated with a raise of p53 protein level. P21 is normally induced by p53. P53 has been identified as a direct target of PPARc [32] and is a potent inhibitor of cellular proliferation. Its expression leads to G1 phase arrest only [33] but some studies reported that p53 hinders G2/M progression in the rat cell line REF52 [34], in a human ovarian cancer cell line [35] and in skin cells [36]. A recent study demonstrated that a prolonged exposure of ciglitazone (72 h) increased p27Kip1 protein level through both induction of p27Kip1 gene transcription as well as inhibition of p27Kip1 protein proteasome-mediated degradation [37]. No PPRE or similar elements have been identified in the promoter. Besides, in RT4 cells, p27Kip1 up-regulation was not abolished by PPARc specific antagonist arguing for the involvement of a PPARc activation-independent mechanism. Ciglitazone could regulate p27Kip1 gene transcription through binding of Sp1 transcription factor on Sp1 binding elements located in the promoter region as described for 1,25 vitamin D3 in U937 cells [38]. Ciglitazone induced G2/M cell cycle arrest in both RT4 and T24 cells but triggered apoptotic cell death only in T24 cells through a PPARc-activation independent pathway. Very few studies are available on the effect of ciglitazone in bladder cancer cells. Chaffer et al. (2006) showed that this antidiabetic drug caused a small decrease in cell number of TSU-Pr1/TSU-Pr1-B1/TSU- Pr1-B2 cell lines after 24 h exposure, which subsided to a modest 10–15% inhibition over days 3 to 9. [39] reported that ciglitazone induced cell death in HT-1376 and 5637 bladder cancer cell lines. Here, we deepened and revealed the underlying mechanisms involved in ciglitazone-induced apoptosis in high grade T24 bladder cancer cells. Our data support the involvement of both intrinsic and extrinsic apoptotic signalling pathways as evidenced by cleavage of caspases 9, 8 and 3 as well as the caspase substrate PARP and the caspase processing blockage with the caspase 8 and 9 specific inhibitors. The cleavage of Bid protein in T24 cells suggests that processing/activation of procaspase 8 drives the caspase cascade with the requirement of the loop amplification from the mitochondria. Most importantly, our study provides first in vitro evidence of a novel mechanism by which ciglitazone induces apoptosis through soluble and membrane-bound TRAIL expression up-regulation leading to activation of DR4 and DR5 since their knockdown by specific blocking antibodies significantly decreased ciglitazone-triggered apoptosis demonstrating their critical role in this process. Only one study, carried out in animal cells and more particularly in rat GH-secreting (GH3) cells, revealed that ciglitazone-induced apoptosis was associated with an increased expression of TRAIL assessed by western-blotting analysis in whole cell extracts [40]. Malignant urothelial cells showed differential apoptotic suscep- tibilities or resistance to TRAIL-induced apoptosis. We showed that T24 cells were resistant to TRAIL as already reported [20,25]. Oka et al. (2006) revealed that this protection from TRAIL-induced apoptosis was related to a high expression of constitutively active Akt. Here, we demonstrated that ciglitazone sensitized cells to exogenous TRAIL. This result could have an important impact for bladder cancer treatment. Indeed, TRAIL could play a role in BCG-induced antitumor effects [41]. BCG endovesical instillations are effective against bladder tumor recurrence. According to some randomized controlled trials, only BCG can curb progression. It has been postulated that BCG stimulates the immune system by activating macrophages, T lymphocytes, B lymphocytes, thus augmenting the host response to tumor surveillance [42]. Functional TRAIL has been detected in the urine of patients undergoing BCG therapy and increased urine levels in those patients who responded to BCG immunotherapy compared with non responders [41]. Thus, ciglitazone could improve and potentiate BCG therapy in responders and sensitize to BCG therapy in non responders. The underlying mechanisms explaining how thiazolidinediones sensitize cells to TRAIL-induced cell death remain poorly understood but could involve the down-regulation of inhibitors of apoptosis proteins [43], the ROS-mediated up-regulation of DR5 [36] or the caspases 3 and 8-mediated b-catenin cleavage [44]. It has been documented that some TZD decrease c-FLIP [17] or survivin expression [45]. Interestingly, the present findings propose that a down-regulation of c-FLIP was associated with apoptosis induced by ciglitazone combined with TRAIL and the reduction in survivin level could be partly responsible for the action of ciglitazone on the TRAIL-promoted apoptosis in T24 cells. Survivin is expressed at high levels in many tumors and has been described as a prognostic marker for bladder cancer progression [46]. Survivin inhibits mitochondrial apoptosis but is able to block TRAIL-induced cell death. This can be explained by cross-links between extrinsic and intrinsic death pathways through the Bid protein cleavage as detected upon ciglitazone treatment in T24 cells. Survivin is a bifunctional protein. In addition to act as an apoptosis inhibitor, survivin is a key regulator of mitosis and is involved in G2/M transition. Besides, siRNA mediated down- regulation of survivin in bladder cancer cell lines, such as RT4 and T24, was associated with a specific G2/M arrest [47,48]. Now, we showed that ciglitazone increased the fraction of RT4 and T24 PLoS ONE | www.plosone.org 9 December 2011 | Volume 6 | Issue 12 | e28354