New Advances in Multimodality Treatment and Biological Research on Diffuse Malignant Peritoneal Mesothelioma Figure 5. Catalytic activity of caspase-9 as determined by hydrolysis of the fluorogenic substrate LEHD-AFC in control cells exposed to oligofectamine alone (unfilled column) and transfected with the control siRNA (gray column) or the antisurvivin siRNA (black column). Data are expressed as relative fluorescence units and represent mean values ± SD of 3 independent experiments (*P<0.01; Student’s t-test). Figure 4. Effects of RNAi-mediated survivin down-regulation on in vitro growth of STO cells. Data are expressed as percentage cell growth in cells transfected with the control siRNA (gray column) or the antisurvivin siRNA (black column) compared to control cells exposed to oligofectamine alone (unfilled column). Data represent mean values ± SD of 3 independent experiments (*P<0.05; **P<0.01; Student’s t-test). At the molecular level, RNAi-mediated inhibition of survivin expression in STO cells coincided with significantly increased catalytic activity of caspase-9, while no appreciable difference in caspase-9 activation was found for cells transfected with the control siRNA when compared to oligofectamine-exposed cells (Figure 5). A number of in vitro and in vivo studies have indicated that survivin down-regulation is able to sensitize human tumor cells of different histologic origins to conventional chemotherapeutic drugs with distinct mechanisms of action, as well as to ionizing radiation.51–53 To test whether survivin plays a role in the in vitro sensitivity of DMPM cells to anticancer drugs, we examined the effect of survivin down‑regulation on the apoptotic response to cisplatin and doxorubicin 72 h after treatment with antisurvivin siRNA. Exposure to cisplatin resulted in a concentration-dependent increase in the percentage of apoptotic cells that was significantly (P<0.01) higher in cells exposed to antisurvivin siRNA than in those transfected with control siRNA or treated with oligofectamine, accounting for 30.5% to 85.9% of the overall cell population (Figure 6). A concentrationdependent increase in caspase-9 catalytic activity was also observed following cisplatin exposure; this enhancement was significantly (P<0.02) greater in cells transfected with antisurvivin siRNA than in those exposed to the control siRNA or oligofectamine. Results obtained after a 1 h exposure to doxorubicin showed a significantly (P<0.05) greater induction of apoptosis (ranging from 9.9% to 63.2% as a function of drug concentration) in cells transfected with the antisurvivin siRNA (Figure 6). Concentration-dependent www.slm-oncology.com 09-0009_Deraco.indd 61 Figure 6. Effects of RNAi-mediated survivin down-regulation on the apoptotic response of STO cells to cisplatin and doxorubicin. The percentage of STO cells with apoptotic morphology with respect to the overall population as assessed by fluorescence microscopy after exposure to oligofectamine alone (unfilled column) or the control siRNA (gray column) or antisurvivin siRNA (black column) in the absence or presence of different cisplatin or doxorubicin concentrations. Data represent mean values ± SD of 3 independent experiments (*P<0.05; **P<0.01; ***P<0.001; Student’s t-test). activation of caspase-9 was also consistently observed, and the extent of this activation was significantly (P<0.02) greater in cells transfected with antisurvivin siRNA than in those exposed to the control siRNA or oligofectamine. These findings demonstrate that the level of survivin expression influences the in vitro response of DMPM cells to cisplatin and doxorubicin. Such results could 61 APJOH 2009; 1: (1). March 2009 3/18/09 3:36:12 PM