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Modulation of Sphingolipid Metabolism Enhances Apoptin’s Cytotoxicity in Prostate Cancer

Modulation of Sphingolipid Metabolism Enhances Apoptin’s Cytotoxicity in Prostate Cancer. Joseph C. Cheng Laboratory of Dr. James S. Norris Department of Microbiology & Immunology Medical University of South Carolina October 31, 2007. Apoptin. Chicken Anemia Virus (CAV):. VP1. VP3. VP2.

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Modulation of Sphingolipid Metabolism Enhances Apoptin’s Cytotoxicity in Prostate Cancer

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  1. Modulation of Sphingolipid Metabolism Enhances Apoptin’s Cytotoxicity in Prostate Cancer Joseph C. ChengLaboratory of Dr. James S. Norris Department of Microbiology & ImmunologyMedical University of South Carolina October 31, 2007

  2. Apoptin Chicken Anemia Virus (CAV): VP1 VP3 VP2 Apoptosis induction VP1 No VP2 Weak VP3 Strong Noteborn, MH et al. (1994) JVI 68:346-351.

  3. How Apoptin Works in Cells ShieldingAggregationUbquitinationDegradation Apoptin Crm1 Apoptin Mitochondria Bcl-2 Nur77 Nur77 Nur77 P Nur77 Nur77 Nur77 Nur77 Cytochrome C P Caspase 9 Apoptin DEDAF Caspase 3 APC-1 Apoptosis Hippi

  4. Prostate Cancer • 217,000 new cases per year in U.S., (670,000 worldwide) • 27,000 deaths per year in U.S. • 2nd leading cause of cancer death in American men. • Improved methodologies of diagnosis and treatment have led to higher cure rate. • Cancer-related deaths are due to advanced disease by aggressive and resistant cancers.

  5. E3 E4 VP16 VP16 rTETR rTETR AdGFPApoptinTET Vector E1 tTA  TRE GFPApoptin TETR + VP16 SV40 poly A SV40 poly A CMV Promoter ITR ITR 5' 3' VP16 rTETR + - tetracycline or doxycycline

  6. FLIP L FLIP S Endogenous Gene Expression in Prostate Cancer Cells DU145 LNCaP LNCaP PC-3 DU145 PC-3 Survivin cIAP-1 XIAP Bcl-2 Bcl-xL Bax  tubulin DU145 (p53mt/mt), LNCaP (p53wt/wt), and PC-3 (p53null) Liu et al. (2006) Mol Ther. 14:637-46.

  7. Apoptin Causes Caspase 3 Dependent Apoptosis in Prostate Cancer Cells DU145 LNcap PC3 Ad-GFP Ad-Apop Ad-GFP Ad-Apop Ad-GFP Ad-Apop 32KD Caspase 3 17KD 12KD Bak Bax P-p53 Actin Liu et al. (2006) Mol Ther. 14:637-46.

  8. Prostate Cancer Cell Lines Show Similar Sensitivity to Ad-Apoptin Liu et al. (2006) Mol Ther. 14:637-46.

  9. Apoptin Radiation Stress (growth factor withdrawal, hypoxia, hyperthermia, DNA damage) Chemotherapy FasL/AdGFPFasL Sphingosine Kinase Ceramidases Sphingosine S1P Ceramide S1PP (Anti-apoptotic phenotype) (Pro-apoptotic phenotype) Growth inhibition (cell cycle arrest) Cell proliferation Apoptosis Transformation Angiogenesis Differentiation Modulation of telomerase activity (telomere length) Cell motility (endothelial) Senescence

  10. Apoptin Causes Sphingolipids Changes in DU145 Cells 200 Ceramide Sphingomyelin 180 Sphingosine 160 140 120 % Control 100 80 60 40 20 0 0 10 20 30 40 50 60 Hours post-infection Liu et al. (2006) Mol Ther. 14:627-36.

  11. Apoptin De novo Synthesis Sphingomyelin SMase Ceramide Synthase Sphingosine Kinase Ceramidases Ceramide Sphingosine S1P S1PP

  12. The importance of sphingomyelin hydrolysis in apoptosis • Lymphoblasts derived from patients with acid SMase deficiency (NPD), failed to undergo apoptosis in response to irradiation or CD95 ligation. • Radiation exposure of thymocytes from acid SMase knockout mice did not undergo apoptosis. • Ceramide generation induced by addition of exogenous acid SMase augmented apoptosis in human leukemic and prostate cancer cells. Santana, P. et al. (1996) Cell 86:189.De Maria, R. et al. (1998) J. Exp. Med. 187:897.Monney, L. et al. Eur. J. Biochem. 251:295.Condorelli, F. et al. (1999) Br. J. Pharmacol. 127:75.

  13. RTPCR for Acid Sphingomyelinase (ASMase)/Acid Ceramidase (AC) Ad-GFP Ad-Apoptin Control 6hrs 16hrs 30hrs 48hrs Control 6hrs 16hrs 30hrs 48hrs ASMase AC Rig/S15

  14. Sphingomyelin ASMase Ceramide Acid Ceramidase Sphingosine Actin Western blot Ad-GFP Ad-GFPApoptin ASMase Acid Ceramidase 30 hours post-infection Liu et al. (2006) Mol Ther. 14:627-36.

  15. GFP (Green) ASMase (Red) Overlay Ad-GFP Ad-GFPApoptin Translocation of Acid SMase by Confocal Microscopy Detection 16 hours post-infection Liu et al. (2006) Mol Ther. 14:627-36.

  16. Ad-Apoptin Increases ASMase Activity MOI Liu et al. (2006) Mol Ther. 14:627-36.

  17. Desipramine Partly Delays Apoptin-induced Cell Death DU145 Co-treated with Ad-Apoptin and Desipramine (1uM and 2.5 uM) 90 Ad-GFPApoptin 80 Apoptin+ Desipramine (1 uM) Apoptin+Desipramine (2.5 uM) 70 60 * p<0.01 50 Cell Viability (%) * p<0.01 40 30 20 10 0 40 20 30 50 60 MOI of Ad-Apoptin Liu et al. (2006) Mol Ther. 14:627-36.

  18. Scrambled sequence siRNA PKC delta-siRNA Ad-GFPApoptin Ad-GFP

  19. Summary • Tumor-selective viral protein Apoptin induces apoptosis in prostate cancer cells. • There was no obvious correlation between Apoptin-induced cell death and the status of pro- and anti-apoptotic molecules. • Apoptin-mediated cell death involves modulation of the sphingomyelin-ceramide pathway. • Apoptin induces acid sphingomyelinase translocation and activation through PKC. • Inhibition of acid sphingomyelinase reduces the efficacy of apoptin-induced cell death.

  20. Ceramide/S1P Pathway Apoptin Acid Ceramidase Sphingosine Kinase Ceramide Sphingosine Sphingosine-1-P Angiogenesis Anti-apoptosis Pro- apoptosis >60% Gleason grades 5-6 tissues over-express AC. >80% Gleason grades 8-10 tissues over-express AC.

  21. 80 60 40 DU145-EGFP 20 DU145-ACEGFP#3 DU145-ACEGFP#7 0 20 40 60 80 100 150 MOI Over-expression of AC Protect Apoptin’s Killing Cytotoxicity of Ad-Apoptin in DU145 cells Over-expressing Acid Ceramidase Mock #3 #7 Cell Viability Liu et al. (2006) Mol Ther. 14:637-46.

  22. O H H O N O 2 . H N H C l LCL204: Acid Ceramidase Inhibitor LIPIDOMICS CORE

  23. Previous Studies: • AC inhibition by LCL204 results in ceramide accumulation and conversion from an anti-apoptotic phenotype to a pro-apoptotic phenotype. • LCL204 displays lysomotropic properties by causing rapid lysosomal membane permeabilization (LMP) resulting in translocation of the lysosomal proteases cathepsins B and D into the cytosol. • Apoptosis induced by LCL204 is dependent on Bak, suggesting that LMP induces a mitochondrial apoptotic pathway. • LCL204 significantly down-regulates anti-apoptotic genes Flip and Survivin. Holman et al. 2007 Cancer Chemother Pharmacol DOI: 10.1007/s00280-007-0465-0

  24. Acid Ceramidase Inhibitor LCL204 Enhanced Apoptin’s Effect DU145 Cells Treated with Ad-GFPApoptin (MOI 20)) and Followed by LCL204 (5 uM) 120 100 80 * # Cell Viability (%) 60 40 * # ^ 20 0 NT LCL 204 Ad-GFPApoptin Ad-GFPApoptin+LCL Treatments Liu et al. (2006) Mol Ther. 14:637-46.

  25. Design of in vivo experiments Tumors are treated with 5 intraperitoneal injections of LCL204 75 mg/kg (Q 3 days). Tumors are treated with 4intratumoralinjections of 2 X 109 PFU adenovirus (Q 3 days).

  26. 800 Control 700 LCL-204 Apoptin 600 Apoptin+LCL 500 * 400 (% of original) Relative tumor volume 300 * # 200 100 0 0 5 10 15 20 25 30 Days Animal Study Liu et al. (2006) Mol Ther. 14:637-46.

  27. 100 80 Control 60 LCL204 Apoptin Survival Rate (%) Apoptin+LCL 40 20 0 0 20 40 60 80 Days Animal Study Liu et al. (2006) Mol Ther. 14:637-46.

  28. Summary • Apoptin-mediated cell death involves modulation of the sphingomyelin-ceramide pathway. • Ceramide accumulates in response to Apoptin via increased biosynthesis (ASMase) and retention (AC). • Pretreatment of prostate cancer cells with AC inhibitor sensitizes tumors to Apoptin, indicating AC is a potential therapeutic target.

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