Ciglitazone Induces Bladder Tumor Cells Apoptosis Figure 1. Ciglitazone causes a G2/M cell cycle arrest in RT4 cells. Cells were treated for 24 h with or without ciglitazone at the indicated concentrations. (A) Percentage of cells showing the cell cycle distribution. (C) Hypodiploid DNA content (sub-G1 peak). (B), (D) After treatment, cells were harvested and total protein extracts were subjected to immunoblotting for detection of procaspase 3, PARP, p53, p21Cip1/Waf1, p27Kip1 and cyclin B1. b-actin was used as an internal loading control. Data shown are representative of 3 independent experiments performed in triplicates. doi:10.1371/journal.pone.0028354.g001 apoptotic pathways, the death receptor and pathways, seem to be activated in T24 cells. mitochondrial Ciglitazone did not act through PPARc transcriptional activation To determine whether PPARc transactivation was required for ciglitazone-promoted cell cycle arrest or apoptosis, RT4 and T24 cells were treated for 24 h by 40 mM of drug alone or in combination with 80 mM of GW9662, an irreversible potent inhibitor of PPARc. The action of ciglitazone on G2/M cell cycle arrest in RT4 cells was not inhibited (Figure 3A, left panel). As expected, the increased expression level of p27Kip1, observed with ciglitazone, was not affected when GW9662 was associated with the drug (Figure 3A, right panel). The impact of ciglitazone on T24 cell death (Figure 3B, left panel) and caspase 3 cleavage (Figure 3B, right panel) was not reversed in the presence of the PPARc antagonist. Thus, GW9662 had no inhibitory effect but yet it was effective since in T24 cells, it blocked the overexpression of the A-FABP PPARc target upon ciglitazone treatment (Figure 3C) as previously reported [24]. Taken together, this set of experiments established that in T24 bladder cancer cells, ciglitazone induced apoptosis through PPARc activation-indepen- dent signalling pathways. Ciglitazone up-regulates soluble and membrane-bound TRAIL in T24 cells We investigated whether ciglitazone can modulate the expres- sion of TRAIL in T24 and RT4 cells. Upon 40 mM ciglitazone exposure, we observed an increase of TRAIL ligand level in T24 whole cell extracts as assessed by western-blotting, an increase of membrane-bound TRAIL as evidenced by flow cytometry analysis with specific PE-conjugated TRAIL antibody, as well as an increase of TRAIL level in T24 cell conditioned media determined by ELISA (Figure 4A). On the other hand, ciglitazone, which did not induce apoptosis in RT4 cells, did not raise TRAIL in whole cell extracts as well as at the cell surface and in conditioned media (Figure 4B). We thus believe that ciglitzone could cause apoptosis of high grade T24 bladder cancer cells by increasing TRAIL expression. Ciglitazone did not affect DR4 and DR5 expression in T24 cells In T24 cells, we have shown a caspase 8 cleavage and the increase of TRAIL by ciglitazone. To analyse whether ciglitazone- induced cell death is dependent on modification of death receptor expression, cells were treated with 40 or 60 mM drug for 24 h. We demonstrated that ciglitazone did not modify DR4 and DR5 expression level as assessed in whole cell lysates by western-blotting (Figure 4C) as well as their cell surface expression as evidenced by flow cytometry analysis with specific PE-conjugated antibodies (Figure 4D). To investigate whether ciglitazone-triggered apoptosis was a function of death receptor signalling amplification, DR4 and DR5 stimulation was blocked by pre-incubation of cells with blocking anti-DR4 or anti-DR5 antibodies before drug treatment. The inactivation of both DR4 and DR5 significantly decreased the ciglitazone-promoted apoptosis (Figure 4E). To validate the death receptor blocking antibodies efficiency at the concentrations used, we exposed TRAIL-sensitive RT4 cells to TRAIL as a positive control (Figure 4E, insert). As expected, inactivating each of the receptors completely inhibited TRAIL-induced apoptosis. These data display that ciglitazone activates the extrinsic apoptotic pathway through DR4 and DR5. Ciglitazone inhibits bladder tumor xenograft development in nude mice To investigate the antitumor effect of ciglitazone in vivo (Figure 5), T24 and RT4 cells as well were subcutaneously injected in 6-week-old-female athymic nude mice. When palpable PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e28354