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| Profound Cytotoxicity of the Histone Deacetylase Inhibitor SAHA (Suberoylanilide Hydroxamic Acid) and TRAIL (Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand) Combination in Malignant Pleural Mesothelioma |
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Javier T. Varona Santos1, Medhi Wangpaichitr2, Min You2, Niramol Savaraj2, Dao M. Nguyen1; 1Division of Cardiothoracic Surgery, Department of Surgery, MIller School of Medicine, University of Miami, Miami, FL; 2Division of Medical Oncology, Department of Medicine, VA Medical Center, University of Miami, Miami, FL Objective: The majority of solid malignancies including malignant pleural mesothelioma (MPM) are refractory to TRAIL-mediated cytotoxicity. SAHA has mild antitumor activity against MPM in a phase I clinical trial. This study is designed to evaluate the cytotoxicity of the SAHA+TRAIL combination on a panel of 10 MPM cells and two normal cells (primary fibroblasts and endothelial cells) in vitro. Methods: Cytotoxicity and apoptosis mediated by TRAIL (0 - 100 ng/ml), SAHA (0.5 - 5.0 microM) or concurrent SAHA+TRAIL combinations are evaluated by MTT assay and AnnexinV-FITC. NF-kB transcriptional activity is measured by luciferase-based reporter plasmid and lucinometer. Protein expression is evaluated by western blot. Results: MPM cells exhibit a wide range of intrinsic susceptibility to TRAIL (2/10 sensitive: IC50 <50 ng/ml, 5/10 mildly sensitive: IC50: 150 to 2500 ng/ml, 3/10 resistant: IC50: infinity). Profound enhancement of cytotoxicity is observed following treatment with SAHA+TRAIL combinations in 7 MPM cells that exhibit some degrees of TRAIL sensitivity as indicated by 2-fold to >100-fold reduction of TRAIL IC50’s (Figure 1a). Supra-additive induction of apoptosis is observed in representative MPM cells treated with SAHA+TRAIL (Figure 1b). Combination-induced cytotoxicity and apoptosis is equally abrogated by either selective caspase 8 or caspase 9 inhibitor and thus implicating the essential role of the mitochondria-driven death signaling cascade. SAHA-dependent strong activation of NF-kB is completely abrogated by TRAIL and this is paralleled with caspase 8-mediated cleavage of RIP (receptor-interacting protein) to form the NF-kB dominant negative cRIP fragment in combination-treated cells. Overexpression of the caspase 8 resistant/uncleavable RIP mutant results in maintenance of high NF-kB transcriptional activity and less cytotoxicity in the H513 MPM cells treated with SAHA+TRAIL. Most importantly, this combination is not toxic to normal cells. Conclusion: SAHA+TRAIL combination induces profound cytotocidal effect in MPM cells that is dependent on the intrinsic death signaling pathway, caspase 8-mediated RIP processing and down-modulation of NF-kB transcriptional activity. Further investigation is ongoing to develop molecular strategy to promote SAHA+TRAIL cytotoxicity in TRAIL-resistant MPM cells. 
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