Ciglitazone Induces Bladder Tumor Cells Apoptosis Figure 5. Antitumor activity of ciglitazone administered as monotherapy in athymic mice bearing T24 and RT4 bladder cancer xenografts. Mice were inoculated subcutaneously with exponentially growing T24 and RT4 bladder cancer cells (56106 cells). When tumor size reached 40 mm3 in volume, mice were randomly divided into control and treated groups (n = 10). Intraperitoneal injections of ciglitazone were weekly administered at a dose of 15 mg/kg during three weeks. Control animals received only saline vehicle following an identical schedule. (A) The growth tumor curves were determined by measuring the tumor volume. *P,0.05, significant differences compared with untreated animals with the use of two-way ANOVA test (evaluation of the tumor volume development over time) ; **P,0.05, significant differences between control and treated mice at each post-graft time with the use of two-tailed unpaired Student’s t test. (B) Immunohistochemical staining of representative paraffin- embedded sections of tumors from untreated or ciglitazone-treated mice. Sections were fixed and stained for Ki-67 and active caspase 3. Each panel is representative of 5 sections for each of ten tumors from control and ciglitazone-treated mice. Original magnification, 620. doi:10.1371/journal.pone.0028354.g005 The ubiquitine-proteasome pathway plays a central role in the regulation of cell cycle control and apoptosis [26]. Several anti- apoptotic proteins such as c-FLIP and survivin represent prominent targets of the proteasome. To determine whether the ciglitazone-induced down-regulation of such apoptosis negative regulators is mediated through this process, we investigated the effects of the proteasome inhibitor MG132. MG132 restored the levels of survivin and c-FLIP (Figure 6E, bottom) indicating that down-regulation of both apoptosis inhibitors was proteasome dependent. As shown in the Figure 6E (top) the sub-G1 cell population level in cotreated cells was significantly decreased in the presence of MG132. Thus, the recovery of c-FLIP and survivin, that significantly attenuated ciglitazone and TRAIL combination-induced apoptosis, was required but not sufficient to overcome cotreatment-promoted apoptosis in T24 cells. PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e28354