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. Overview
• Introduction
• What is (-)-Epicatechin?
• Experimental methods
• (-)-Epicatechin and Mitochondrial Respiration
• (-)-Epicatechin and Radiosensitization
• Potential Mechanisms
• Conclusion and Future Directions

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. 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. (-)-Epicatechin stimulated mitochondrial respiration and ETC
protein expression
Hüttemann H et al. FASEB J. 2012; vol. 26 no. 4 1413-1422

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. 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. 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. (-)-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. (-)-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. (-)-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. (-)-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. 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. (-)-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. (-)-Epicatechin and irradiation do not stimulate Chk2
phosphorylation or p21 expression in NHDF cells
A B

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. 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. (-)-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. 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. 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. 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. 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. 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. Questions?