mt program meeting seminar on 01-15-2014 v2
TRANSCRIPT
(-)-Epicatechin Selectively Sensitizes Cancer Cells to Radiation and Stimulates
Mitochondrial Activity in Cancer Cells
Hosam A. Elbaz, Ph.D.
January 15th, 2014Department of Radiation Oncology,
Center for Molecular Medicine and Genetics, Wayne State University
Detroit, MI 48201.
Overview
• Introduction
• What is (-)-Epicatechin?
• Experimental methods
• (-)-Epicatechin and Mitochondrial Respiration
• (-)-Epicatechin and Radiosensitization
• Potential Mechanisms
• Conclusion and Future Directions
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.
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.
(-)-Epicatechin stimulated mitochondrial respiration and ETC protein expression
Hüttemann H et al. FASEB J. 2012; vol. 26 no. 4 1413-1422
Short term treatment of 40 µM (-)-Epicatechin enhances cytochrome c oxidase activity
p < 0.05;
* and ** compared to control
0 5 10 15 20 25 30 350
20
40
60
80
100
120
140
160 Control 5 min
20 min
Cytc [µM]
CcO
Spe
cific
Acti
vity
[O
2 nm
ol/m
in/m
g of
tota
l pro
tein
]
**
*
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
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
(-)-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)
O2
cons
umpt
ion
(nm
ol/m
in/m
g pr
otei
n)
0 20 50 100 2000
10
20
30 **
*
COX activity for NHDF Cells
(-)-Epicatechin (M)
O2
cons
umpt
ion
(nm
ol/m
in/m
g pr
otei
n)
0 1000
5
10
15
A B
(-)-Epicatechin sensitizes cancer cell lines to irradiation
Panc-1
IR dose (Gy)
Surv
ival
frac
tion
0 2 4 6 80.01
0.1
1 Control(-)-Epicatechin (20 M)
U87
IR dose (Gy)
Surv
ival
frac
tion
0 2 4 6 80.01
0.1
1 Control(-)-Epicatechin (20 M)
MIA PaCa-2
IR dose (Gy)
Surv
ival
frac
tion
0 2 4 6 80.01
0.1
1 Control(-)-Epicatechin (20 M)
A B C
D E(-)-Epicatechin alone
(-)-Epicatechin (M)
Surv
ival
frac
tion
0 25 50 100 2000.0
0.2
0.4
0.6
0.8
**
(-)-Epicatechin + 6 Gy
(-)-Epicatechin (M)
Surv
ival
frac
tion
0 25 50 100 2000.00
0.02
0.04
0.06
0.08
** *
(-)-Epicatechin does not sensitize NHDF cells to radiation.
NHDF Cells
IR dose (Gy)
Surv
ival
frac
tion
0 2 4 6 80.01
0.1
1 Control(-)-Epicatechin (20 M)
A B CNHDF Survival with EC and 6Gy
(-)-Epicatechin (M)
Surv
ival
frac
tion
0 1000.000
0.001
0.002
0.003 *
NHDF Survival with EC alone
(-)-Epicatechin (M)
Surv
ival
frac
tion
0 200.00
0.02
0.04
0.06
0.08
(-)-Epicatechin and irradiation modestly manipulate ETC protein expression
Rel
ativ
e ex
pres
sion
Com 1- N
DUFP6
Com 2- 70
kDa
Com 3- co
re 1
COX-1 1
Com 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
Rel
ativ
e ex
pres
sion
Com 1- NDUFP6
Com 2- 70
kDa
Com 3- co
re 1
COX-1 1
Com 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
Hypothesis: Activating cytochrome c oxidase increases mitochondrial membrane potential m and free radical
production. O
2•– g
ener
ation
[nm
ol/m
g/m
in]
H 2O2 g
ener
ation
[nm
ol/m
g/m
in]
[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]
(-)-Epicatechin and irradiation stimulate Chk2 phosphorylation (Thr68) and p21 expression in Panc-1 Cells.
p21 in 6 Gy Exposed Cells
(-)-Epicatechin (M)R
elat
ive
expr
essi
on0 20
0.0
0.5
1.0
1.5
2.0
*
A B 0 Gy 6 Gy
- + - +
IR
(-)-Epicatechin 20 µM
p21GAPDH
0 Gy 6 Gy
- + - +
IR
(-)-Epicatechin 20 µM
P-Chk2Chk2GAPDH
P-Chk2 in 6 Gy Exposed Cells
(-)-Epicatechin (M)
Rel
ativ
e ex
pres
sion
0 200.0
0.5
1.0
1.5
2.0
*
(-)-Epicatechin and irradiation do not stimulate Chk2 phosphorylation or p21 expression in NHDF cells
A B
(-)-Epicatechin stimulates caspase 3 cleavage in Panc-1 cells.
Pro-caspase 3
Cleaved caspase 3
β-actin
- + - +(-)-Epicatechin
IR 0 Gy 6 Gy
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
(-)-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
EGF reverses (-)-epicatechin’s inhibition of Panc-1 clonogenic survival
Surv
ival
frac
tion
Control EC EGF EC+EGF0.0
0.5
1.0
1.5
2.0
2.5
*
**
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 on Panc-1 clonogenesis.
(-)-Epicatechin
↑ COX activity↑ Mitochondrial respiration in cancer cells
↓ Warburg metabolism
↓Cell proliferation↑ Cellular stress
↓ MAPK signaling
↑ Sensitivity to cell death
Future Directions
• Is MAPK signaling 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 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?
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
Questions?