Volume 15, Issue 7, Pages 1259-1263 (July 2007) Role of Acid Ceramidase in Resistance to FasL: Therapeutic Approaches Based on Acid Ceramidase Inhibitors and FasL Gene Therapy  Saeed Elojeimy, Xiang Liu, John C Mckillop, Ahmed M El-Zawahry, David H Holman, Jonathan Y Cheng, William D Meacham, Ayman EM Mahdy, Antonio F Saad, Lorianne S Turner, Joseph Cheng, Terrence A Day, Jian-Yun Dong, Alicja Bielawska, Yusuf A Hannun, James Scott Norris  Molecular Therapy  Volume 15, Issue 7, Pages 1259-1263 (July 2007) DOI: 10.1038/sj.mt.6300167 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 Acid ceramidase (AC) is over-expressed in head and neck cancer tumors. (a) Nineteen pairs of primary head and neck tumors (T) and matched normal samples (N) were obtained from the Hollings Cancer Center Tumor Bank and analyzed for AC expression by western blotting. (b) Immunoblot densitometric analysis of AC normalized by actin levels, and presented in the graph in comparison with 100% control. Molecular Therapy 2007 15, 1259-1263DOI: (10.1038/sj.mt.6300167) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 Inverse correlations between acid ceramidase (AC) levels and sensitivity to C-6-ceramide. (a) Western blot densitometric analysis showing endogenous AC levels in three head and neck cancer cell lines: SCC-1, SCC-12, and SCC-14a. (b) Sensitivity of SCC cell lines to C-6-ceramide. SCC cell lines were treated for 24 hours with 30 μmol/l of C-6-ceramide. Cell death was quantified using the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymeyhoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] cytotoxicity assay. Molecular Therapy 2007 15, 1259-1263DOI: (10.1038/sj.mt.6300167) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 Acid ceramidase (AC) over-expression confers resistance to FasL-induced cell killing. (a) AC protein levels were determined using western blots in the head and neck cancer cell line SCC-1 in AC–green fluorescent protein (AC-GFP) stably transfected cells and GFP stably transfected SCC-1 control cells. (b) Percentage cytotoxicity of SCC-1 AC-over-expressing cells and SCC-1 control GFP cells after 24 hours' treatment with AdGFPFasLTET at the indicated multiplicity of infection (MOI). Data are representative of two different experiments performed in triplicate. Molecular Therapy 2007 15, 1259-1263DOI: (10.1038/sj.mt.6300167) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 Acid ceramidase (AC) down-regulation by small interfering RNA (siRNA) sensitizes SCC-1 to CH11-induced cell killing. (a) AC protein levels were determined using western blots in the head and neck cancer cell line SCC-1 after transfection with AC-specific siRNA or scrambled siRNA for 48 hours. (b) Percentage cytotoxicity after 24 hours' exposure to CH11 (0.5 μg/ml and 1 μg/ml) of SCC-1 cells transfected with AC siRNA or scrambled siRNA for 48 hours. Data are representative of two different experiments performed in triplicate. Molecular Therapy 2007 15, 1259-1263DOI: (10.1038/sj.mt.6300167) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 LCL 204 sensitizes SCC-14a to CH11 treatment. (a) Cells were pre-treated for 12 hours with the indicated LCL 204 concentrations followed by 24 hours' treatment with either vehicle control or 0.5 μg/ml CH11. Cell death was quantified using the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymeyhoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] cytotoxicity assay. (b) Cells were pre-treated for 12 hours with 4 μmol/l LCL 204 concentrations followed by 12 hours' treatment with either vehicle control or 0.5 μg/ml CH11. After treatment, cell lysates were subjected to western blot assay to detect caspase 3 and caspase 8 cleavages, acid ceramidase (AC), and survivin. CTRL, control. Molecular Therapy 2007 15, 1259-1263DOI: (10.1038/sj.mt.6300167) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions