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Radiotherapy & Resistance

Radiotherapy & Resistance. Thierry M Muanza, MD Radiation Oncology Jewish General Hospital Segal Cancer Centre McGill University. 2 E Conférence Québécoise Sur La Résistance Thérapeutique du Cancer Q-CROC Montréal, November 5-6, 2010. Potential Conflict of Interest. Dr. Thierry Muanza

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Radiotherapy & Resistance

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  1. Radiotherapy & Resistance Thierry M Muanza, MD Radiation Oncology Jewish General Hospital Segal Cancer Centre McGill University 2E Conférence Québécoise Sur La Résistance Thérapeutique du Cancer Q-CROC Montréal, November 5-6, 2010

  2. Potential Conflict of Interest • Dr. Thierry Muanza • None

  3. Radiotherapy • Along with surgery and chemotherapy, radiotherapy is a mainstay of cancer treatment • 75% cancer patients in the course of their disease will undergo radiotherapy

  4. Radiobiology • IR local release of large amount of energy • ~ 33eV dissipated / ionizing event, enough to break strong chemical bond • energy associated C=C bond is 4.9 eV • Types: • Electromagnetic • particulate

  5. IR: mechanism of action .

  6. Radiation Induced DNA Damage http://www.radiation-scott.org/radsource

  7. Chromosomal Damage Apoptosis Reproductive death Necrosis

  8. pH2XA Radiation Induced DNA Damage Repair G1 arrest E2F G1 arrest E2F Apoptosis ATM G2 arrest pRb pRb P53 P53 CyclineB BAX cdK /Cycline D Cycline E p21 WAF1/ CIPI N Cycline-B-p34 cdc2 Cytochrome c G2 arrest Apaf1 Caspase 9 Smac/ Diablo Caspase 3 Apoptosis Survivin Caspase 6

  9. DNA Repair Mechanisms RNA Pol II or XPC HR Rad51 /Rad52 NER XPD / XPB pH2aX Rad52/MRE11/NBSI ERCC1 / XPG SSB BRAC1/BRAC2 ATM DSB DNA Pol  / ligase BER NHEJ Completion of DSB repair DNA-PKcs / Ku80/70 Completion of repair XRCC1 MRE11/Rad51/NBSI DNA Pol  / DNA ligase III DNA ligase IV, XRCC4

  10. Cell Survival Curves

  11. CA: RT sensitivity

  12. Cell cycle: RT sensitivity

  13. Tumor Oxygenation

  14. Re-oxygenation

  15. Effect of Oxygen

  16. RT resistance Ma et al. JCO 21, 2003

  17. Radiosensitizers

  18. Radiosensitizers • Nonhypoxic • Halogenated pyrimidines • DNA • Cycling tissues • Hypoxic • Free radical process (fixed) • Misonidazole (toxicity) • Overgaard metaanalyisis: 4.6% LC, 2.8% OS • Hypoxic cytotoxins • Bioreductive Rx • Tirapazamine (nitroxide) • Mitomycin C

  19. Radiosensitizers

  20. Radiosensitizers

  21. Cell Proliferation Assay (MTT) MDA-MB-468 cells Heravi, Muanza et al. ACD 20, 2009

  22. Cell Proliferation Assay (MTT) **: P<0.01

  23. Colony Forming Assay

  24. γH2AX Immunofluorescent Staining 0.5 Gy Control 1 Gy

  25. γH2AX Immunofluorescent Staining Control ZRBA1 9uM ZRBA1 18uM

  26. γH2AX Immunofluorescent Staining 0.5 Gy Control ZRBA1 9uM 0.5 Gy and ZRBA1 9uM

  27. γH2AX Immunofluorescent Staining 1 Gy Control ZRBA1 9uM 1 Gy and ZRBA1 9uM

  28. γH2AX Immunofluorescent Staining

  29. FACS Analysis 1 hrs post treatment 24 hrs post treatment

  30. Comet Assay

  31. Comet Assay ZRBA1 Control ZRBA1 and radiation Radiation

  32. EGFR Inhibitory Activity (ZRBA1)

  33. EGFR Signaling Pathway RAF TGF MEK EGFR HER2 RAS ERK1/2 P TK Serine112 PI3K AKT P BAD P Serine136 Apoptosis P Caspase 9 Caspase 3 • Over-expression of EGFR associates with activation of the AKT pathway. Survival

  34. Conclusions • Significant cell killing in cells exposed to combination of ionizing radiation with ZRBA1. • Higher G2M arrest in cells exposed to the combined treatment. • The most effective schedule for this combination is administration of drug before and / or concurrent with radiation. • ZRBA1 potentiates the effect of radiation in MDA-MB-468 cells. • The combination of IR and ZRBA1 induces the formation of γH2AX foci.

  35. Future Plans • Better understanding of the molecular mechanism induced by this novel therapeutic approach. • Evaluate the status of DNA repair proteins. • Evaluate the expression levels of antiapoptotic and proapoptotic proteins. • Investigation for other possible modes of cell death. • In-vivo correlative studies.

  36. Cancer Stem Cells • Self-renewing cell • Pluripotent cell: recapitulate its tumor in SCID mice

  37. CSC: mechanisms of resistance

  38. CSC: RT resistance • CSC enrichment • Chk1/Chk2 inhibitor S. Bao Nature 444, 2006

  39. CSC: RT resistance • apoptosis Cell cycle arrest

  40. CSC: DNA damage repair kinetics

  41. Future directions • Clinical models for identification of RT resistance • Rectal cancer • CNS tumors • Zevalin resistant lymphoma

  42. Acknowledgments • Dr. Thierry Muanza - Mitra Heravi - Lillian Lee - Azusa Maeda - Ava Schlisser • Dr. Danuta Radzioch • Dr. Bertrand Jean-Claude (Cancer Drug Research Laboratory) - Dr. Zakaria Rachid - Margarita Todorova

  43. Thank You!

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