39: The essential role of reactive oxygen species in cisplatin-induced sensitization of malignant pleural mesothelioma cells to Fas ligand mediated apoptosis

Abstract

Background: Despite adequately expressing Fas, the majority of cultured malignant pleural mesothelioma (MPM) cells are refractory to the cytotoxic effect of soluble Fas ligand (sFasL). Pre-treating FasL-resistant cells with sublethal concentrations of cisplatin (CDDP) sensitizes them to subsequent exposure to FasL. The goal of this study is to evaluate the role of reactive oxygen species (ROS) in CDDP-mediated enhancement of sFasL cytotoxicity. Methods: Nine Fas-positive MPM cells were treated with sFasL (5 to 100 ng/ml × 48 hours) with or without pretreatment with CDDP (0.5 to 4 microg/ml × 24 hours). Cell viability and apoptosis were determined by MTT and TUNEL-based ApoBrdU assays. Generation of ROS in control and treated cells were quantified using H2DCFDA dye and flow cytometry. Mitochondrial inner membrane potential was determined by JC-1 staining and flow cytometry. Activated Bax was detected by immunoprecipitation and immunoblotting. Specific caspase proteolytic activity was quantified by colorimetric assays. Results: Three of nine MPM cells were sensitive to sFasL (IC50 values <100 ng/ml). CDDP significantly sensitized MPM cells, regardless of their intrinsic susceptibility to sFasL cytotoxicity, to this apoptosis-inducing ligand with 2- to > 10-fold reduction of sFasL IC50’s. While CDDP (0.5 or 1.0 microg/ml) or sFasL (50 ng/ml) mediated <20% apoptosis, 80% to 95% of combination-treated cells were apoptotic. ROS generation in CDDP/sFasL-treated MPM cells (detected by 2-fold increase of DHDFA fluorescence) played an important role in combination treatment-induced cytotoxicity as the anti-oxidant N-acetylcysteine not only eliminated ROS production but also completely abrogated the cytotoxic effect of this drug combination. Moreover, cleavage of BID and release of cytochrome c from mitochondria, collapse of mitochondria inner membrane potential as well as activation of caspase 8, 9 and 3 in CDDP/sFasL-treated MPM cells were all suppressed by N-acetylcysteine. Moreover, treating MPM cells with 100 microM of H2O2 for 2 hours increased the sensitivity of MPM cells to sFasL. Finally, over-expression of Bcl2 or concurrent exposure to selective caspase 9 inhibitor also abrogated CDDP/sFasL- or H2O2/sFasL-mediated cytotoxicity. Conclusion: Cisplatin sensitizes MPM cells to sFasL-mediated cytotoxicity and apoptosis via molecular pathway(s) that depend(s) on ROS and mitochondria-associated death signaling cascade.