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UMich meeting seminar on 03-12-2014 v1.pptx [Autosaved]

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UMich meeting seminar on 03-12-2014 v1.pptx [Autosaved]

  1. 1. (-)-Epicatechin Selectively Radiosensitizes Cancer Cells and Stimulates Mitochondrial Activity in Cancer Cells by Inhibiting MAPK Pathway Hosam A. Elbaz, Ph.D. March 12th, 2014 Department of Radiation Oncology, Center for Molecular Medicine and Genetics, Wayne State University Detroit, MI 48201.
  2. 2. Overview • Introduction • What is (-)-Epicatechin? • Experimental methods • (-)-Epicatechin and Mitochondrial Respiration • (-)-Epicatechin and Radiosensitization • Potential Mechanisms • Conclusion and Future Directions
  3. 3. Introduction • Radiotherapy is ideal for most solid tumors. • Radiation resistance and adverse effects are frequently encountered. • Radiosensitizing agents would improve therapeutic outcome. • Most cancer types exhibit 40-60% reduction in mitochondrial activity and rely on aerobic glycolysis for energy (Warburg effect). • Drugs that increase mitochondrial respiration would interfere with the Warburg effect and could sensitize cancer cells to conventional therapy.
  4. 4. What is (-)-Epicatechin? • A natural polyphenolic flavanol – Flavonoid family – Natural ingredient in cacao, green tea and dark chocolate – Antioxidant properties • Molecular Weight: 290.268097 Da • IUPAC name: (2R,3R)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol • It has a number of beneficial health effects.
  5. 5. (-)-Epicatechin stimulated mitochondrial respiration and ETC protein expression Hüttemann H et al. FASEB J. 2012; vol. 26 no. 4 1413-1422
  6. 6. Short term treatment of 40 µM (-)-Epicatechin enhances cytochrome c oxidase activity p < 0.05; * and ** compared to control 0 20 40 60 80 100 120 140 160 180 0 5 10 15 20 25 30 35 CcOSpecificActivity [O2nmol/min/mgoftotalprotein] Cytc [µM] Control 5 min 20 min ** *
  7. 7. General hypothesis ↑ COX activity ↑ Mitochondrial respiration in cancer cells ↓ The Warburg metabolism and ↑ ROS production in cancer cells ↑ Cellular stress ↑ Sensitivity to cell death (-)-Epicatechin
  8. 8. Experimental methods • Cytochrome c oxidase activity • Cell lines: • Panc-1 • MIA PaCa-2 • U87 • Normal human dermal fibroblasts (NHDF) • Clonogenic survival assay • Enzymes for checkpoint and OXPHOS complexes • P-Chk2: phosphorylated checkpoint kinase protein 2 at Thr68 • p21. • P-Erk2: phosphorylated extracellular kinase 2 • OXPHOS complexes: • Complex I: NDUFB6 subunit • Complex II: 70kDa subunit • Complex III: Core 1 subunit • Complex IV: subunit 1 • Complex V: α subunit
  9. 9. (-)-Epicatechin increases mitochondrial activity in Panc-1 cells but not in normal human dermal fibroblast (NHDF) cells COX activity for Panc-1 Cells (-)-Epicatechin (M) O2consumption (nmol/min/mgprotein) 0 20 50 100 200 0 10 20 30 * * * COX activity for NHDF Cells (-)-Epicatechin (M) O2consumption (nmol/min/mgprotein) 0 100 0 5 10 15 A B
  10. 10. (-)-Epicatechin sensitizes cancer cell lines to irradiation Panc-1 IR dose (Gy) Survivalfraction 0 2 4 6 8 0.01 0.1 1 Control (-)-Epicatechin (20 M) U87 IR dose (Gy) Survivalfraction 0 2 4 6 8 0.01 0.1 1 Control (-)-Epicatechin (20 M) MIA PaCa-2 IR dose (Gy) Survivalfraction 0 2 4 6 8 0.01 0.1 1 Control (-)-Epicatechin (20 M) A B C D E(-)-Epicatechin alone (-)-Epicatechin (M) Survivalfraction 0 25 50 100 200 0.0 0.2 0.4 0.6 0.8 * * (-)-Epicatechin + 6 Gy (-)-Epicatechin (M) Survivalfraction 0 25 50 100 200 0.00 0.02 0.04 0.06 0.08 * * *
  11. 11. (-)-Epicatechin does not sensitize NHDF cells to radiation. NHDF Cells IR dose (Gy) Survivalfraction 0 2 4 6 8 0.01 0.1 1 Control (-)-Epicatechin (20 M) A B C NHDF Survival with EC and 6Gy (-)-Epicatechin (M) Survivalfraction 0 100 0.000 0.001 0.002 0.003 * NHDF Survival with EC alone (-)-Epicatechin (M) Survivalfraction 0 20 0.00 0.02 0.04 0.06 0.08
  12. 12. (-)-Epicatechin and irradiation modestly manipulate ETC protein expression RelativeexpressionC om 1-N D U FP6C om 2-70kD aC om 3-core 1 C O X-1 1 C om 5- 0.0 0.5 1.0 1.5 2.0 0 M / 0 Gy 20 M / 0 Gy 100 M / 0 Gy 200 M / 0 Gy Relativeexpression C om 1-N D U FP6C om 2-70kD aC om 3-core 1 C O X-1 1 C om 5- 0.0 0.5 1.0 1.5 2.0 0 M / 6 Gy 20 M / 6 Gy 100 M / 6 Gy 200 M / 6 Gy** B C
  13. 13. Hypothesis: Activating cytochrome c oxidase increases mitochondrial membrane potential m and free radical production. O2 •–generation [nmol/mg/min] H2O2generation [nmol/mg/min] [Reviewed in Liu, S. (1999) J Bioenerg Biomembr 31:367-76] [Reviewed in Bin-Bing S. Zhou & Jiri Bartek. (2004) Nature Reviews Cancer 4, 216-225]
  14. 14. (-)-Epicatechin and irradiation stimulate Chk2 phosphorylation (Thr68) and p21 expression in Panc-1 Cells. p21 in 6 Gy Exposed Cells (-)-Epicatechin (M)Relativeexpression 0 20 0.0 0.5 1.0 1.5 2.0 * A B 0 Gy 6 Gy - + - + IR (-)-Epicatechin 20 µM p21 GAPDH 0 Gy 6 Gy - + - + IR (-)-Epicatechin 20 µM P-Chk2 Chk2 GAPDH P-Chk2 in 6 Gy Exposed Cells (-)-Epicatechin (M) Relativeexpression 0 20 0.0 0.5 1.0 1.5 2.0 *
  15. 15. (-)-Epicatechin and irradiation do not stimulate Chk2 phosphorylation or p21 expression in NHDF cells A B
  16. 16. (-)-Epicatechin stimulates caspase 3 cleavage in Panc-1 cells. Pro-caspase 3 Cleaved caspase 3 β-actin - + - +(-)-Epicatechin IR 0 Gy 6 Gy
  17. 17. EGFR signalling inhibits mitochondrial function. Demory M L et al. J. Biol. Chem. 2009;284:36592-36604 Modified from Hüttemann H et al. Adv. Exp. Med. Biol. 2012;748:237-64 Receptor tyrosine kinase signaling ↑ EGFR translocation to the mitochondria COX activity ↓ Warburg metabolism ↑ ∞ Cell proliferation MAPK members interact with COX (-)-Epicatechin
  18. 18. (-)-Epicatechin and radiation inhibit Erk phosphorylation in Panc-1 cells A P-Erk1/2 Erk1/2 (-)-Epicatechin (µM) 0 20 50 100 200 GAPDH B 6Gy + (-)-Epicatechin (µM) 0 20 50 P- Erk1/2 Erk1/2 GAPDH Erk2 phosphorylation in Panc-1 cells (-)-Epicatechin (M) Relativeexpression 0 20 50 100 200 0.00 0.25 0.50 0.75 1.00 1.25 * * * P-Erkin6GyExposedPanc-1Cells (-)-Epicatechin(M) Relativeexpression 0 20 50 0.0 0.2 0.4 0.6 0.8 1.0 *
  19. 19. EGF reverses (-)-epicatechin’s stimulation of mitochondrial respiration in Panc-1 cells O2consumption (nmol/min/mgprotein) Control EC EGF EC+EGF 0 5 10 15 20 25 * *
  20. 20. EGF reverses (-)-epicatechin’s inhibition of Panc-1 clonogenic survival Survivalfraction Control EC EGF EC+EGF 0.0 0.5 1.0 1.5 2.0 2.5 * * *
  21. 21. Discussion and Conclusion • (-)-Epicatechin – Increases COX activity and potentially reverses Warburg effect. – “selectively” sensitizes cancer cells to irradiation. • (-)-Epicatechin and radiation – increased P-Chk2 and p21 levels in Panc-1 cells – No effect in NHDF cells. • (-)-Epicatechin with or without radiation. – reduced Erk phosphorylation • (-)-Epicatechin reduced EGF’s effect mitochondrial activity and Panc-1 clonogenesis. (-)-Epicatechin ↑ COX activity ↑ Mitochondrial respiration in cancer cells ↓ Warburg metabolism ↓Cell proliferation ↑ Cellular stress ↓ MAPK signaling ↑ Sensitivity to cell death
  22. 22. Future Directions • How does (-)-epicatechin regulate mitochondrial respiration via MAPK signaling in cancer cells? • Is (-)-epicatechin’s radiosensitization mediated by reduced MAPK signaling? • How does (-)-epicatechin’s effect on MAPK signaling cause radiosensitization in cancer cells? • Can (-)-epicatechin be used as a radiosensitizer in vivo? • Animal model examination • Efficacy vs. safety • Can (-)-epicatechin sensitize cancer cells to chemotherapy?
  23. 23. Acknowledgements • Principle investigators – Steven Zielske – Maik Hüttemann • Angelika Burger Shared Postdoctoral Award, Karmanos Cancer Institute • Department of Radiation Oncology • Center for Molecular Medicine and Genetics • WSU Office for Vice President for Research • Zielske lab – Current members • Morgan Laney – Former members • Ethan Brock • Aisha Fasih • Deborah Antwih • Kristina Gabbarah • Hüttemann lab – Icksoo Lee – Jenney Liu – Joseph Shay – Gargi Mahapatra
  24. 24. Questions?

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