(-)-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?