Ciglitazone Induces Bladder Tumor Cells Apoptosis Figure 3. Ciglitazone action is PPARc activation-independent. RT4 and T24 cells were treated for 24 h by 40 mM of ciglitazone alone or in combination with 80 mM of GW9662, an irreversible potent inhibitor of PPARc. (A, left) Accumulation of RT4 cells in G2/M phase of the cell cycle was analysed by flow cytometry analysis ; (A, right) After treatment, whole cell lysates were prepared and 20 mg of total protein extract were subjected to immunoblotting for detection of p27Kip1. (B, left) The percentage of cells showing hypodiploid DNA content (sub-G1 peak) was evaluated by flow cytometry analysis ; (B, right) After treatment, whole cell lysates were prepared and 15 mg of total protein extract were subjected to immunoblotting for detection of procaspase 3. (C) Assessment of the PPARc antagonist GW9662 efficiency on Adipocyte-Fatty Acid Binding Protein (A-FABP), a known PPARc target in T24 cells by western-blotting analysis. b-actin was used as an internal loading control. Data are means 6 SD of 3 independent experiments performed in triplicates. *P,0.05, significant differences compared with untreated cells with the use of two-tailed unpaired Student’s t test. doi:10.1371/journal.pone.0028354.g003 indicate, for the first time to our knowledge, that the significant reduction of tumor volume observed in ciglitazone-treated mice (xenografted with T24 bladder cancer cells) requires the inhibition of proliferation as well as the induction of apoptosis. Ciglitazone and TRAIL combined treatment induces synergistic apoptosis through death receptor signalling pathway, caspase activation and Bid cleavage T24 cells are known to be highly resistant to TRAIL after exposure to increasing concentrations of exogenous soluble recombinant human TRAIL ranging from 10 to 200 ng/ml [25]. According to our results demonstrating that ciglitazone up- regulates TRAIL expression, we postulated that ciglitazone kills T24 cells by restoring their sensitivity to TRAIL-induced apoptosis. To validate this hypothesis, cells were treated with vehicle or 40 mM ciglitazone for 24 h or 50 ng/ml recombinant TRAIL for the last 12 h alone or in combination. As previously reported, we showed also that T24 cells were refractory to TRAIL- induced apoptosis. More interestingly, there was a significant increase of cell death in ciglitazone and TRAIL-cotreated cells compared to ciglitazone alone-treated cells (Figure 6A, top). The TRAIL-sensitizing action of ciglitazone resulted in pro-caspase 8 and 3 processing and PARP proteolysis but involved also the mitochondria amplification loop as indicated by caspase 9 and Bid cleavage (Figure 6A, bottom). In the presence of caspase 8 (Z- IETD-FMK) or caspase 9 (Z-LEHD-FMK) specific inhibitors, we observed an inhibition of either caspase 8 or caspase 9 processing and the absence of Bid cleavage as well, in ciglitazone and TRAIL cotreated cells (Figure 6B, bottom). This was associated with a decrease of cell death in the presence of caspase inhibitors as evidenced by a significant decrease of DNA fragmentation (Figure 6B, top). To confirm the involvement of death receptor activation, DR4 and DR5 stimulation was inhibited by pre- incubation of cells with blocking anti-DR4 or anti-DR5 antibodies before treatment (Figure 6C). Inactivating each of the receptors upon TRAIL and ciglitazone cotreatment efficiently blocked cell death confirming that apoptosis was mediated through specific interactions between TRAIL and its receptors. Ciglitazone down-regulates c-FLIP and survivin through a proteasome-dependent degradation We further investigated the effect of ciglitazone and the association of ciglitazone with TRAIL on the expression of apoptosis inhibitors such as survivin and c-FLIP known to be involved in the regulation of both intrinsic and extrinsic apoptotic pathways. As presented in Figure 6D, ciglitazone, as individual treatment or in combination with TRAIL, reduced both c-FLIPS and survivin protein levels to the same extent and dramatically down-regulated c-FLIPL. On the contrary, TRAIL single treatment did not affect considerably c-FLIP and survivin protein levels. These findings indicate that down-regulation of c- FLIP and survivin likely contributes to ciglitazone-triggered apoptosis and to ciglitazone-mediated sensitization to TRAIL- induced apoptosis. PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e28354