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University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
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University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
University Health Network High-Throughput Screens for ...
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University Health Network High-Throughput Screens for ...

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  • FaDu tumours
  • Once CTAB is concentrated into the tumor mitochondria, the H + -gradient across the inner mitochondrial membrane may begin to dissipate the consequent   M decrease will be sensed by the mitochondrial permeability transition pore (PTP) Opening of the PTP causes mitochondrial outer membrane permeabilization (MOMP), a pivotal event in the intrinsic apoptotic pathway, leading to the disruption of essential mitochondrial functions and the release of apoptogenic factors, such as cytochrome c
  • Forward transfection = plate cells --> 24h later add the RNAi/lipofectamine Reverse transfection = Add RNAi/lipofectamine into well then put cells directly on top
  • Flourescence image obtained using the INCell Analyzer 1000 at 4x objective of FaDu cells stained with Cell Tracker TM Orange CMTMR and Hoescht 33342. Colonies are defined using the Developer Toolbox software as an adjacent set of cytoplasmic signals (blue outline) containing stained nuclei (green outline). C) The readout from Developer Toolbox is an excel worksheet that lists all colonies with their corresponding number of nuclei within a specific well. D) A custom software program was created to convert the list output into a matrix containing the number of colonies per well on the original plate, filtered to include only colonies with ≥ 6 and ≤ 350 nuclei. E) The matrix data are then plotted as a clonogenic survival curve.
  • mRNA qRT-PCR at 24 hrs post-transfection
  • 48 hours post-transfection using 40 nM UROD siRNA.
  • Cells were transfected x 48 hrs, then RT, and then immediately seeded for CFA; stain & count on 13 days after seeding.
  • 48 hours post-transfection using 40 nM UROD siRNA.
  • Transcript

    • 1. High-Throughput Screens for Identifying Novel Anti-cancer Agents F-F Liu MD, FRCPC Radiation Oncologist/Senior Scientist University Health Network
    • 2. Outline
      • Introduction
      • Forward Chemical Screen
      • Reverse Screen
      • Conclusions
    • 3. HTS
      • Test large number of molecules simultaneously via miniaturization and automation of assay protocols
      • 1. Drug discoveries
      • 2. Understand biological processes
    • 4. Approaches to HTS
      • 1. Reverse screens
      • 2. Forward screens
    • 5. Reverse Genetics Reverse Classical Genetics Reverse Chemical Genetics Mutate Gene Insert in vivo Look for phenotype Screen for chemical binding Add compound in vivo Look for phenotype
    • 6. Reverse Chemical Genetics Target Compound Disease Bcr-Abl Imatinib CML, GIST Src/Abl Dasatinib Advanced CML Her1/EGFR Erlonitib NSCLC Proteasome Bortezomib Multiple Myeloma
    • 7. Forward Genetics Forward Classical Genetics Random mutagenesis Select mutant with phenotype Identified mutated gene
    • 8. Forward Genetics Forward Chemical Genetics Forward Classical Genetics Identified mutated gene Random mutagenesis Select mutant with phenotype Add 1 compound/well Plate Cells Identify protein target Select compound that produces phenotype
    • 9. Forward Chemical Genetics Target Identification 1. Biotin labeling 2. Y3H 3. Micro-array
    • 10. Forward Chemical Genetics
    • 11. Outline
      • Introduction
      • Forward Chemical Screen
      • Current Reverse Screen
      • Conclusions
    • 12. Forward HTS for Head & Neck Cancer Therapeutics
    • 13. Screening Procedure Day 1 (Seed Cells)
      • Choose cells that double every 21 h
        • reduce bias
      • Screen cancer vs . normal
        • choose “hit” criteria with caution
      • Lower dynamic range than other assays, but less manipulation
      Day 4 (MTS - viability) Day 2 (Add Compounds) (1/well)
    • 14. Screening Procedure LOPAC Library (1280 compounds) Prestwick Library (1120 compounds) 6 compounds Anti-microbial 23 compounds NO YES 64 hits screened by FaDu cell viability 3 novel 1 studied 29 compounds 35 compounds NIH/3T3 viability DECREASE
    • 15. Screening Procedure 6 compounds 3 novel 1 studied 5757 viability 1 compound 5 compounds DECREASE 4 compounds C666-1 viability 1 compound DECREASE
    • 16.
      • Forward small molecule HTS: 3 existing antimicrobials with novel anticancer properties:
        • 1. Benzethonium Chloride
          • Yip et al, Clin Cancer Res 15, 5557, 2006
        • 2. Alexidine Dihydrochloride
          • Yip et al, Mol Cancer Ther 5, 2234, 2006
        • 3. Cetrimonium Bromide
          • Ito et al, (manuscript under review)
      HNC Therapeutics
    • 17. Cetrimonium Bromide (CTAB)
      • Quaternary ammonium compound
      • Delocalized lipophilic cation
        • Lipophilic; delocalized positive charge
        • Penetrates hydrophobic cellular membranes
        • Accumulates in mitochondria due to negative   M
      • Interacts with mitochondrial H + -ATP synthase
    • 18. Anti-Cancer Specificity
    • 19. Combination Therapy
    • 20. CTAB Induces Apoptosis
    • 21. CTAB Induces Apoptosis
    • 22. CTAB Induces Apoptosis FaDu: Hypopharyngeal SCC C666-1: NPC GM05757: Primary normal fibroblast
    • 23. CTAB Induces Apoptosis
    • 24. CTAB Inhibits Mitochondrial ATP Synthase Activity
    • 25. CTAB Reduced Intracellular ATP Levels
    • 26.   M of Cancer vs . Normal Cells
    • 27. Anti-Cancer Specificity
    • 28. CTAB Ablates In Vivo Tumour-Forming Capacity
    • 29. CTAB Reduces Growth of Established Tumours
    • 30. Proposed mode of action -   M   P
      • CTAB is concentrated
      • in tumor mitochondria
      • due to    M
        •  CTAB-ATP synthase
        • interactions
      - + +
    • 31. - Matrix Outer Mitochondrial Membrane Inner membrane Adapted from Wikipedia
      • H + -gradient across IMM
      • dissipates
        •    M is sensed by PTP
         M
      • PTP opening induces MOMP
        • Mitochondrial dysfunction
        • Apoptogenic factors released
      • Caspase activation
        • Apoptosis
      MOMP MOMP Apoptosis Proposed mode of action +
    • 32. Conclusions
      • 1. Identified a novel mitochondria- mediated apoptogenic anti- cancer agent using a forward HTS.
      • 2. Selective in vitro and in vivo efficacy against HNC models
        • Rooted at the mitochondria
        •   M differences between cancer vs . normal cells
    • 33. Outline
      • Introduction
      • Forward Chemical Screen
      • Current Reverse Screen
      • Conclusions
    • 34. Objective
      • Design HTS to identify novel genes that can selectively sensitize cancer cells to radiation when suppressed
    • 35. Proposed Modifications
      • 1. Target-driven siRNA-based approach
      • 2. Incorporate RT into screen
      • 3. Utilize a more appropriate readout for determining radiosensitization
      Adapted from Nature Rev Genetics 7:373, 2006 Readout? RT +
    • 36. Kassner; Comb Chem & HTS ; 11:175, 2008
    • 37. Korn & Krause; 11:503, 2007 The multidisciplinary approach of high-content screening (HCS) requires a combination of different expertise.
    • 38. Specific Aims
      • 1. Define experimental parameters for target-driven siRNA-based HTS
      • 2. Conduct the screen using siRNA libraries to identify novel radiosensitizing targets
      • 3. Validate and characterize hits
    • 39. Specific Aims
      • 1. Define experimental parameters for target-driven siRNA-based HTS
      • 2. Conduct the screen using siRNA libraries to identify novel radiosensitizing targets
      • 3. Validate and characterize hits
    • 40. siRNA Transfection Parameters Parameters for 96-well Format Optimal Condition Cancer model A549 Forward vs. reverse transfection Reverse Cell number (50  7500 cells/well) 750 cells Transfection reagent volume (0.01  0.33  l/well) 0.08  l siRNA concentration (10  100 nM/well) 40 nM Transfection time (24  48 h) No difference
    • 41. Katz et al ; Biotechniques ; 44:9, 2008
    • 42. Katz et al ; Biotechniques ; 44:9, 2008
    • 43. Automated Clonogenic Assay
      • Conclusions:
      • Effective in measuring effects of cytotoxic agents on cancer cells in vitro
      • Limitations
          • Low cell # reduces dynamic range
          • Unable to identify sensitization
      Katz et al ; Biotechniques ; 44:9, 2008
    • 44. BrdU Cell Proliferation Assay
      • Bromodeoxyuridine (BrdU)
        • Thymidine analogue
        • Incorporated into newly synthesized DNA strands of actively proliferating cells (S phase)
      • Non-radioactive alternative to [ 3 H]-thymidine incorporation
      • Examines cellular effects with long-term kinetics that are more reflective of therapeutic response
    • 45. Positive siRNA Control DNA Ligase IV siRNA Scrambled siRNA Day 1 Day 2 Day 3 Day 5 Day 6 Seed cells + siRNA Transfection Radiation Treatment … Add BrdU Readout
    • 46. Specific Aims
      • Define experimental parameters for target-driven siRNA-based HTS
      • Conduct screen using siRNA libraries to identify novel radiosensitizing targets
      • Validate and characterize hits
    • 47. siRNA Libraries
          • Dharmacon human si ARRAY libraries :
        • 4 siRNAs pooled/gene
        • Protein Kinase siRNA library
              • 800 genes
        • Druggable siRNA library
              • 6080 genes
    • 48. siRNA Screens
    • 49. Specific Aims
      • Define experimental parameters for target-driven siRNA-based HTS
      • Conduct screen using siRNA libraries to identify novel radiosensitizing targets
      • Validate and characterize hits
    • 50. Validation of siRNA Hits siRNA library (6880 siRNAs) 137 siRNAs Eliminate Transfect ± RT (0 vs . 2 Gy)
          • 188 siRNAs (2 Gy)
      51 confirmed hits Top 15 hits 0 Gy 2 Gy Decreased surviving fraction Increased surviving fraction No effect
    • 51. Potential Radiosensitizing Targets 1 2 3 4 5 6 7 8 9 10 11 12 13 0 2 Gy Scrambled siRNA ATM STK6
    • 52. Potential Radiosensitizing Targets Genes #1-15
    • 53. Caveat
          • Off-target Effects (OTE)
          • Up to 30% of sequences can cause phenotypic change, which do not correlate with gene silencing effect.
          • Hence, critical to confirm “hits” using a variety of different approaches.
    • 54. Anti-Cancer Specificity Criteria:  ≥ 40%  survival fraction compared to 0 Gy  Radiosensitization observed with ≥2 siRNAs UTSCC 8a UTSCC 42a
          • FaDu
      3-4 siRNAs/ gene 15 hits (A549)
    • 55. mRNA Knockdown #1 #3 Scrambled Scrambled
    • 56. Confirmed Radiosensitizing Targets
      • FaDu, UTSCC 8a, and UTSCC 42a:
        • Integrin, alpha V (ITGAV)
        • Gene #2
        • Gene #3
    • 57. mRNA Knockdown Kinetics in FaDu
    • 58. Western for Protein k/d in FaDu Cells (48 hrs) 40 KDa GAPDH Lipo Neg siRNA Protein #3
    • 59. Clonogenic Assay in FaDu cells
    • 60. RT
    • 61. Outline
      • Introduction
      • Forward Chemical Screen
      • Current Reverse Screen
      • Conclusions
    • 62. Conclusions
          • HTS enable rapid discovery of novel anti-cancer agents.
          • Centrimonium bromide is one such novel mitochondrial-mediated apoptogenic compound.
    • 63. Conclusions
          • 3. Experimental parameters need to be carefully defined to conduct RNAi-based radiosensitization screens
          • 4. Identified novel genes with potential radiosensitizing properties
    • 64. Dr. Mariano Elia Chair in Head & Neck Cancer Research SLRI Robotics Facility

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