Ombination of ZOL and CDDP were attributable to increased apoptotic cell death.Combinatory effects of ZOL and CDDP in vivoWe investigated anti-tumor effects of ZOL in combination with CDDP in an orthotopic animal model (Fig. 3). Nude mice injected with MSTO-211H cells in the pleural cavity received ZOL intrapleurally and/or CDDP intraperitoneally. All the tumors were found in the pleural cavity without any detectable extrapleural metastatic foci. ZOL or CDDP administration inhibited the tumor growth compared with PBS-injected group. A combinatory administration of ZOL and CDDP further decreased tumor weights, demonstrating that the combination produced greater CB5083 therapeutic effects than the case treated with a single agent. We did not notice body weight loss in the combinatory group, indicating that the combination was 
not toxic to the tested animals.Figure 3. Combinatory effects with ZOL and CDDP in an orthotopic animal model. MSTO-211H cells (16106) were inoculated into the pleural cavity of BALB/c nu/nu mice (n = 6) (day 1), and then ZOL (25 mg, day 3) was administrated into the pleural cavity and/or CDDP (100 mg, day 5) into the peritoneal cavity (CDDP). PBS was used as a control. Tumor weights were measured on day 24. The SE bars are also shown. * P,0.05, ** P,0.01. doi:10.1371/journal.pone.0060297.gZOL order CP21 induced p53 activationWe examined whether p53 activation was involved in the ZOLmediated cytotoxicity since the p53 pathways play a key role in apoptosis induction. Firstly, we tested possible p53 activation in wild-type p53 mesothelioma with CDDP (Fig. 4A). CDDP-treated MSTO-211H and EHMES-10 cells induced phosphorylation of p53 at the Ser 15 residue, a hallmark of p53 activation, and upregulated p53 protein levels. We then examined influence of ZOL on p53 expressions and found that ZOL treatments phosphorylated p53 at Ser 15 and 
augmented p53 protein levels in both cells (Fig. 4B). These data showed that ZOL induced p53 activation and subsequently raised a possibility that the ZOL-mediated cytotoxicity was caused by p53 activation. We also investigated the combinatory effects of CDDP and ZOL on the p53 phosphorylation at Ser 15 (Fig. 4C). The phosphorylation level in cells treated with both agents was greater than that in cells treated with either CDDP or ZOL, suggesting that both agents cooperatively activated the p53 pathways.EHMES-10 cells (Fig. 4E). Control siRNA treatments unexpectedly increased the cytotoxicity in MSTO-211H cells at high ZOL 15755315 concentrations. These data suggested that the ZOL-mediated cytotoxicity was independent of p53 activation. We also analyzed cell cycle changes in ZOL-treated MSTO-211H cells after they were transfected with p53-siRNA (Fig. 4F, Table 2). Cell cycle distributions showed that p53-siRNA treatments marginally influenced the ZOL-mediated increase of sub-G1 phase populations. The decreased level of sub-G1 phase fractions due to the p53-siRNA treatment was disproportionately lower than that of the p53 protein expression after transfection with siRNA. In contract, the p53-siRNA treatment increased S and G2/M phase and decreased G0/G1 phase fractions, showing that downregulated p53 promoted cell cycle progression. These data demonstrated that decreased p53 levels influenced the cell cycle but little affected the ZOL-mediated cytotoxicity, and confirmed that the ZOL-induced p53 activation was irrelevant to the ZOLmediated cytotoxicity. Control-siRNA treated cells increased subG1 phase fractions, whic.Ombination of ZOL and CDDP were attributable to increased apoptotic cell death.Combinatory effects of ZOL and CDDP in vivoWe investigated anti-tumor effects of ZOL in combination with CDDP in an orthotopic animal model (Fig. 3). Nude mice injected with MSTO-211H cells in the pleural cavity received ZOL intrapleurally and/or CDDP intraperitoneally. All the tumors were found in the pleural cavity without any detectable extrapleural metastatic foci. ZOL or CDDP administration inhibited the tumor growth compared with PBS-injected group. A combinatory administration of ZOL and CDDP further decreased tumor weights, demonstrating that the combination produced greater therapeutic effects than the case treated with a single agent. We did not notice body weight loss in the combinatory group, indicating that the combination was not toxic to the tested animals.Figure 3. Combinatory effects with ZOL and CDDP in an orthotopic animal model. MSTO-211H cells (16106) were inoculated into the pleural cavity of BALB/c nu/nu mice (n = 6) (day 1), and then ZOL (25 mg, day 3) was administrated into the pleural cavity and/or CDDP (100 mg, day 5) into the peritoneal cavity (CDDP). PBS was used as a control. Tumor weights were measured on day 24. The SE bars are also shown. * P,0.05, ** P,0.01. doi:10.1371/journal.pone.0060297.gZOL induced p53 activationWe examined whether p53 activation was involved in the ZOLmediated cytotoxicity since the p53 pathways play a key role in apoptosis induction. Firstly, we tested possible p53 activation in wild-type p53 mesothelioma with CDDP (Fig. 4A). CDDP-treated MSTO-211H and EHMES-10 cells induced phosphorylation of p53 at the Ser 15 residue, a hallmark of p53 activation, and upregulated p53 protein levels. We then examined influence of ZOL on p53 expressions and found that ZOL treatments phosphorylated p53 at Ser 15 and augmented p53 protein levels in both cells (Fig. 4B). These data showed that ZOL induced p53 activation and subsequently raised a possibility that the ZOL-mediated cytotoxicity was caused by p53 activation. We also investigated the combinatory effects of CDDP and ZOL on the p53 phosphorylation at Ser 15 (Fig. 4C). The phosphorylation level in cells treated with both agents was greater than that in cells treated with either CDDP or ZOL, suggesting that both agents cooperatively activated the p53 pathways.EHMES-10 cells (Fig. 4E). Control siRNA treatments unexpectedly increased the cytotoxicity in MSTO-211H cells at high ZOL 15755315 concentrations. These data suggested that the ZOL-mediated cytotoxicity was independent of p53 activation. We also analyzed cell cycle changes in ZOL-treated MSTO-211H cells after they were transfected with p53-siRNA (Fig. 4F, Table 2). Cell cycle distributions showed that p53-siRNA treatments marginally influenced the ZOL-mediated increase of sub-G1 phase populations. The decreased level of sub-G1 phase fractions due to the p53-siRNA treatment was disproportionately lower than that of the p53 protein expression after transfection with siRNA. In contract, the p53-siRNA treatment increased S and G2/M phase and decreased G0/G1 phase fractions, showing that downregulated p53 promoted cell cycle progression. These data demonstrated that decreased p53 levels influenced the cell cycle but little affected the ZOL-mediated cytotoxicity, and confirmed that the ZOL-induced p53 activation was irrelevant to the ZOLmediated cytotoxicity. Control-siRNA treated cells increased subG1 phase fractions, whic.