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Epicatechin Stimulates Mitochondrial
Activity and Selectively Sensitizes Cancer
Cells to Radiation
Hosam A. Elbaz, Ph.D.
N...
Overview
• Introduction
• What is (-)-Epicatechin?
• Experimental methods
• (-)-Epicatechin and Mitochondrial Respiration
...
Introduction
• Radiotherapy is ideal for most solid tumors.
• Radiation resistance and adverse effects are frequently enco...
What is (-)-Epicatechin?
• A natural polyphenolic flavanol
– Flavonoid family
– Natural ingredient in cacao, green tea and...
(-)-Epicatechin stimulated mitochondrial respiration and ETC
protein expression
Hüttemann H et al. FASEB J. 2012; vol. 26 ...
Short term treatment of 40 µM (-)-Epicatechin enhances
cytochrome c oxidase activity
p < 0.05;
* and ** compared to
contro...
General hypothesis
(-)-Epicatechin
↑ COX activity
↑ Mitochondrial respiration in cancer cells
↓ The Warburg metabolism and...
Experimental methods
• Cytochrome c oxidase activity
• Cell lines:
• Panc-1
• MIA PaCa-2
• U87
• Normal human dermal fibro...
(-)-Epicatechin increases mitochondrial activity in Panc-1
cells but not in normal human dermal fibroblast (NHDF) cells
CO...
(-)-Epicatechin sensitizes cancer cell lines to irradiation
Panc-1
IR dose (Gy)
Survivalfraction
0 2 4 6 8
0.01
0.1
1
Cont...
(-)-Epicatechin does not sensitize NHDF cells to radiation.
NHDF Cells
IR dose (Gy)
Survivalfraction
0 2 4 6 8
0.01
0.1
1 ...
(-)-Epicatechin and irradiation modestly manipulate ETC
protein expression
RelativeexpressionC
om
1-N
D
U
FP6C
om
2-70kD
a...
Activating cytochrome c oxidase increases mitochondrial
membrane potential m and free radical production.
O2
•–generatio...
(-)-Epicatechin and irradiation stimulate Chk2
phosphorylation (Thr68) and p21 expression in Panc-1 Cells.
p21 in 6 Gy Exp...
(-)-Epicatechin and irradiation do not stimulate p21
expression or Chk2 phosphorylation in NHDF cells
EGFR signaling inhibits mitochondrial function.
Demory M L et al. J. Biol. Chem. 2009;284:36592-
36604
Modified from Hütte...
(-)-Epicatechin inhibits Erk2 phosphorylation in Panc-1 cells
A B
Erk2 phosphorylation in Panc-1 cells
(-)-Epicatechin (M...
(-)-Epicatechin and radiation reduce Erk2 phosphorylation in
Panc-1 cells
P-Erk in 6 Gy Exposed Panc-1 Cells
(-)-Epicatech...
EGF reverses (-)-epicatechin’s inhibition of Panc-1 clonogenic
survival
Survivalfraction
Control EC EGF EC+EGF
0.0
0.5
1.0...
Discussion and Conclusion
• (-)-Epicatechin potentially reverses Warburg effect;
• (-)-Epicatechin “selectively” sensitize...
Future Directions
• Is MAPK activity involved with (-)-epicatechin’s increased mitochondrial respiration
in cancer?
• How ...
Acknowledgements
• Principle investigators
– Steven Zielske
– Maik Hüttemann
• Department of Radiation
Oncology
• Center f...
Questions?
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CMMG seminar on 11072013 11-06-2013

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CMMG seminar on 11072013 11-06-2013

  1. 1. Epicatechin Stimulates Mitochondrial Activity and Selectively Sensitizes Cancer Cells to Radiation Hosam A. Elbaz, Ph.D. November 07th, 2013 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. • Most cancer types exhibit 40-60% reduction in mitochondrial activity and rely on aerobic glycolysis for energy (Warburg effect). • Radiosensitizing agents would improve therapeutic outcome.
  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 (-)-Epicatechin ↑ COX activity ↑ Mitochondrial respiration in cancer cells ↓ The Warburg metabolism and ↑ ROS production in cancer cells ↑ Cellular stress ↑ Sensitivity to cell death
  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. 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 p21 expression or Chk2 phosphorylation in NHDF cells
  16. 16. EGFR signaling 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
  17. 17. (-)-Epicatechin inhibits Erk2 phosphorylation in Panc-1 cells A B 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-Erk1/2 Erk1/2 (-)-Epicatechin (µM) 0 20 50 100 200 GAPDH Panc-1
  18. 18. (-)-Epicatechin and radiation reduce Erk2 phosphorylation in Panc-1 cells P-Erk in 6 Gy Exposed Panc-1 Cells (-)-Epicatechin (M) Relativeexpression 0 20 50 0.0 0.2 0.4 0.6 0.8 1.0 * 6Gy + (-)-Epicatechin (µM) 0 20 50 P- Erk1/2 Erk1/2 GAPDH A B
  19. 19. 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 * * *
  20. 20. Discussion and Conclusion • (-)-Epicatechin potentially reverses Warburg effect; • (-)-Epicatechin “selectively” sensitizes cancer cells to irradiation; • P-Chk2 and p21 levels increased significantly in response to (-)- epicatechin and IR in Panc-1 cells but not NHDF cells; • (-)-Epicatechin reduced Erk2 phosphorylation with or without radiation. • EGF rescued Panc-1 cells from (-)-epicatechin’s inhibitory effect on Panc-1 clonogenesis.
  21. 21. Future Directions • Is MAPK activity involved with (-)-epicatechin’s increased mitochondrial respiration in cancer? • How does (-)-epicatechin regulate mitochondrial respiration via MAPK signaling in cancer cells? • Is (-)-epicatechin’s radiosensitization mediated by reduced MAPK activity? • 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?
  22. 22. Acknowledgements • Principle investigators – Steven Zielske – Maik Hüttemann • Department of Radiation Oncology • Center for Molecular Medicine and Genetics • Karmanos Cancer Institute • WSU Office for Vice President for Research • Zielske lab – Current members • Morgan Laney • Ethan Brock – Former members • Aisha Fasih • Deborah Antwih • Kristina Gabbarah • Hüttemann lab – Icksoo Lee – Jenney Liu – Joseph Shay – Gargi Mahapatra
  23. 23. Questions?

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