MELATONIN SENSITIZES HUMAN BREAST CANCER CELLS TO IONIZING RADIATION

DEPARTMENT OF PHYSIOLOGY AND PHARMACOLOGY UNIVERSITY OF CANTABRIA, SANTANDER, SPAIN

Carolina Alonso-González

FIBROBLASTS

ADIPOCYTES

EPITHELIAL CELLS

MAMMARY TUMOR

ESTROGENS

ESTROGENS

PERIPHERAL TISSUES

ANDROGENS

ADRENAL

ANDROGENS

ESTROGENS

Adipose tissue, skin, osteoblasts, condrocites, vascular endothelium, aortic soft muscle, brain (hypothalamus, tonsil)

Biologically inactive

estrogens

ANDROGENS

x Inhibition of estrogen

actions

SERMs ANTIESTROGENS

x Inhibition of

estrogen synthesis

SEEMs AROMATASE INHIBITORS

MELATONIN

Malignant epithelial cells

MCF-7

IRRADIATION (dose ranging 4-12 Gy)

Melatonin 1 mM

Melatonin 10 µM

Melatonin 1 nM 7 Days

Cell proliferation

Cell cycle phase distribution

RAD51 and DNA-PKs gene expression

3-6 days

1 mM 0

50

100

150

Melatonin

a

1 nM 10 µM Control

Ce

ll p

rolif

era

tio

n

(% o

f co

ntr

ol)

Alonso-González et al., J Pineal Res 58:189-197, 2015.

EFFECTS OF MELATONIN ON BREAST CANCER CELL PROLIFERATION

a

a b c 75

25

125

0 3 6

Days

1 mM 10 µM

1 nM Ce

ll p

rolif

era

tio

n

(% o

f co

ntr

ol)

0

50

100

150

75

25

125

a, p<0.001 vs Control b, p<0.001 vs Melatonin 1 mM c, p<0.01 vs Melatonin 10 µM

EFFECTS OF IONIZING RADIATION ON BREAST CANCER CELL PROLIFERATION (MCF-7)

0 3 6

Days

% o

f ce

ll p

rolif

era

tio

n w

ith

re

spe

ct

to c

on

tro

l no

n−

rad

iate

d c

ells

0

20

40

60

80

100

120

0 Gy

4 Gy

8 Gy

12 Gy

Alonso-González et al., J Pineal Res 58:189-197, 2015

0

25

50

75

100

125

Control Melatonin 1 mM Melatonin 10 µM Melatonin 1 nM

Ce

ll P

rolif

era

tio

n

(% o

f co

ntr

ol r

adia

ted

ce

lls)

a

a

a b c

a

a

Before irradiation

Before and after

irradiation

Before irradiation

Before and after

irradiation

Before irradiation

Before and after

irradiation

a b c

POTENTIATION OF RADIATION-INDUCED GROWTH INHIBITION BY MELATONIN

Alonso-González et al., J Pineal Res 58:189-197, 2015

a, p<0.001 vs Control b, p<0.001 vs Melatonin 1 mM c, p<0.01 vs Melatonin 10 µM

0

25

50

75

100

125 C

ell

cycl

e d

istr

ibu

tio

n

G1 S G2-M

a, p<0.001 vs Non-radiated cells b, p<0.001 vs Radiated cells

CELL CYCLE DISTRIBUTION IN MCF-7 CELLS AFTER TREATMENT WITH MELATONIN AND IONIZING RADIATION

0

25

50

75

100

125

a

Ce

ll cy

cle

dis

trib

uti

on

a

G1 S G2-M

Non-radiated cells Melatonin pretreated cells

a

Alonso-González et al., J Pineal Res 58:189-197, 2015

0

25

50

75

100

125

a

Ce

ll cy

cle

dis

trib

uti

on

a

G1 S G2-M

Radiated cells

a

0

25

50

75

100

125

b

b

G1 S G2-M

Melatonin Pretreated and Radiated cells

b C

ell

cycl

e d

istr

ibu

tio

n

Speidel, Arch Toxicol 89:501-517, 2015

DNA DAMAGE

IR

DOUBLE STRAND BREAKS (DSBs)

ATM MRN COMPLEX

P53 CHK2

• Apoptosis • Senescence

• Cell cycle arrest • DNA damage repair

P53 CHK2

Genomic Integrity Restored

SURVIVAL

M

G1 G2

S

NHEJ HR

NHEJ HR REPAIR OF DNA DOUBLE STRAND

BREAKS (DSBs)

RA

D5

1

(%

of

con

tro

l no

n-r

adia

ted

ce

lls)

0

25

50

75

100

125

Non-Radiated cells

Radiated cells

p<0.001

EFFECTS OF RADIATION AND MELATONIN ON RAD51 mRNA EXPRESSION IN MCF-7 CELLS

1 mM 0

50

100

150

Melatonin pretreatment before radiation

a

1 nM 10 µM Control

b c

b d e 75

25

125

RA

D5

1

(%

of

con

tro

l rad

iate

d c

ells

)

RADIATION a, p<0.001 vs Control b, p<0.001 vs Melatonin 1 mM c, p<0.001 vs Melatonin 10 µM

Alonso-González et al., J Pineal Res 58:189-197, 2015

DN

A-P

Kcs

(

% o

f co

ntr

ol n

on

-rad

iate

d c

ells

)

0

25

50

75

100

125

Non-Radiated cells

Radiated cells

p<0.001

EFFECTS OF RADIATION AND MELATONIN ON DNA-PKcs mRNA EXPRESSION IN MCF-7 CELLS

1 mM 0

50

100

150

Melatonin pretreatment before radiation

a

1 nM 10 µM Control

a

a b c 75

25

125

DN

A-P

Kcs

(

% o

f co

ntr

ol r

adia

ted

ce

lls)

RADIATION

a, p<0.01 vs Control b, p<0.001 vs Control c, p<0.01 vs Melatonin 1 mM d, p<0.001 vs Melatonin 1 mM e, p<0.01 vs Melatonin 10 µM

Alonso-González et al., J Pineal Res 58:189-197, 2015

Gatz and Wiesmüller, Cell Death Differ 13:1003-1016, 2006

1 mM 0

100

200

300

Melatonin pretreatment before radiation

a

1 nM 10 µM

a b

a b c d

150

50

250

RADIATION

Control Non-

Radiated cells

Control radiated

cells

a

(% o

f co

ntr

ol n

on

-rad

iate

d)

a, p<0.001 vs Control non-radiated cells b, p<0.001 vs Control radiated cells

c, p<0.001 vs Melatonin 1 mM d, p<0.001 vs Melatonin 10 µM

EFFECTS OF IONIZING RADIATION AND MELATONIN ON p53 mRNA EXPRESSION IN BREAST CANCER CELLS

400

350

Decreasing cell proliferation.

Inducing cell cycle arrest.

Down-regulating RAD-51 and also

DNA-PKc. Two proteins

involved in double-strand DNA break

repair mechanisms.

MELATONIN PRETREATMENT

BEFORE RADIATION SENSITIZES BREAST CANCER CELLS TO

THE IONIZING EFFECTS OF RADIATION

p53

RESEARCH GROUP CAROLINA ALONSO-GONZÁLEZ

ALICIA GONZÁLEZ CABEZA

CARLOS MARTÍNEZ CAMPA

JAVIER MENÉNDEZ MENÉNDEZ

SAMUEL COS CORRAL

Technical assistance

José Antonio de Cos Cossío

Gema Viar Ruíz

DHEA

ANDROSTENEDIONE

ESTRONE

P450 Aromatase

ANDROSTENEDIOL

TESTOSTERONE

ESTRADIOL

P450 Aromatase

17ß-Hydroxysteroid dehydrogenase

17ß-Hydroxysteroid dehydrogenase

17ß-Hydroxysteroid dehydrogenase

Oxidative forms

Reductant forms

blood

ESTRONE SULFATE

ESTRADIOL SULFATE

ANDROSTENEDIONE

DHEA

Estrone sulfotransferase

Estrone sulfatase

DHEA-S

Estradiol sulfotransferase

Estradiol sulfatase

17β-HSD type 1

Breast carcinoma epithelial cell

AROMATASEACTIVITY

AROMATASE mRNA EXPRESSION

EFFECTS OF IONIZING RADIATION AND MELATONIN ON AROMATASE ACTIVITY AND mRNA EXPRESSION IN

HUMAN BREAST CANCER CELLS

1 mM 0

50

100

150

Melatonin pretreatment before radiation

a b

1 nM 10 µM

a c a b

75

25

125

(% o

f co

ntro

l no

n-ra

diate

d c

ells)

Radiation

Control Non-

Radiated cells

Control radiated

cells

1 mM 0

50

100

150

Melatonin pretreatment before radiation

a b

1 nM 10 µM

a c

a b

75

25

125

(% o

f co

ntro

l no

n-ra

diate

d c

ells)

Radiation

Control Non-

Radiated cells

Control radiated

cells

a a

a, p<0.001 vs Control non-radiated cells b, p<0.001 vs Control radiated cells c, p<0.001 vs Melatonin 1 mM

1 mM 0

50

100

150

Melatonin pretreatment before radiation

a

1 nM 10 µM

a b d a c d e

75

25

125

Radiation

Control Non-

Radiated cells

Control radiated

cells

a

(% o

f co

ntro

l no

n-ra

diate

d)

1 mM 0

50

100

150

Melatonin pretreatment before radiation

a b

1 nM 10 µM

a d a b d 75

25

125

Radiation

Control Non-

Radiated cells

Control radiated

cells

a

(% o

f co

ntro

l no

n-ra

diate

d)

a, p<0.001 vs Control non-radiated cells b, p<0.001 vs Control radiated cells

c, p<0.01 vs Control radiated cells d, p<0.001 vs Melatonin 1 mM e, p<0.001 vs Melatonin 10 µM

EFFECTS OF IONIZING RADIATION AND MELATONIN ON THE TWO MAJOR PROMOTERS (pI.3 AND pII) DRIVING

AROMATASE EXPRESSION IN BREAST CANCER

Promoter II Promoter I.3

1 mM 0

50

100

150

Melatonin pretreatment before radiation

a c

1 nM 10 µM

a b d a c e 75

25

125

Radiation

Control Non-

Radiated cells

Control radiated

cells

a (%

of

cont

rol no

n-ra

diate

d)

a, p<0.001 vs Control non-radiated cells b, p<0.001 vs Control radiated cells

c, p<0.01 vs Control radiated cells d, p<0.01 vs Melatonin 1 mM e, p<0.01 vs Melatonin 10 µM

EFFECTS OF IONIZING RADIATION AND MELATONIN ON AROMATASE PROMOTER I.7 mRNA EXPRESSION IN BREAST

CANCER CELLS

Promoter I.7

SULFATASEACTIVITY

SULFATASE mRNA EXPRESSION

EFFECTS OF IONIZING RADIATION AND MELATONIN ON SULFATASE ACTIVITY AND mRNA EXPRESSION IN

HUMAN BREAST CANCER CELLS

1 mM 0

50

100

150

Melatonin pretreatment before radiation

a b

1 nM 10 µM

a b c a b c d

75

25

125

(% o

f co

ntro

l no

n-ra

diate

d c

ells)

Radiation

Control Non-

Radiated cells

Control radiated

cells

1 mM 0

50

100

150

Melatonin pretreatment before radiation

a b

1 nM 10 µM

a b c

a b d

75

25

125

(% o

f co

ntro

l no

n-ra

diate

d c

ells)

Radiation

Control Non-

Radiated cells

Control radiated

cells

a a

a, p<0.001 vs Non-radiated cells b, p<0.001 vs Control radiated cells c, p<0.01 vs Melatonin 1 mM d, p<0.001 vs Melatonin 10 µM

Control Melatonin

1 mM 10 µM 1 nM

0 h

G1 51 ± 0.8 67 ± 0.6 * 65.3 ± 1.3 * 69 ± 0.3 *

S 28 ± 1.1 19 ± 1.2 * 19.9 ± 0.9 * 18 ± 0.8 *

G2/M 21 ± 1.1 14 ± 0.8 * 14.3 ± 1.1 * 13 ± 0.2 *

6 h after treatment

G1 25.8 ± 0.7 42 ± 0.6 * 31.2 ± 0.6 31.9 ± 0.5

S 45.8 ± 0.5 38 ± 1.2 43.4 ± 0.5 42.2 ± 0.9

G2/M 28.4 ± 0.6 20 ± 0.8 * 25.3 ± 1.2 25.8 ± 0.4

12 h after treatment

G1 58.4 ± 0.4 58.1 ± 0.6 52.3 ± 0.6 51.6 ± 0.5

S 7.1 ± 0.1 6.1 ± 1.2 6.3 ± 0.5 6.2 ± 0.9

G2/M 33.6 ± 0.5 36 ± 0.8 41.3 ± 1.2 41.5 ± 0.2

24 h after treatment

G1 55.5 ± 0.6 68.6 ± 0.7 * 56.6 ± 0.6 65.4 ± 0.4 *

S 13.8 ± 0.5 7 ± 1.2 * 12.9 ± 0.4 9.7 ± 0.8 *

G2/M 30.6 ± 0.1 24 ± 0.4 * 29.9 ± 0.2 25.6 ± 0.1 *

CELL CYCLE DISTRIBUTION IN MCF-7 CELLS AFTER TREATMENT WITH MELATONIN AND IONIZING RADIATION

Decreasing tumoral estrogen production:

inhibition of aromatase and sulfatase activity and

expression.

Decreasing cell proliferation.

Inducing cell cycle arrest.

Down-regulating RAD-51 and DNA-PKcs, two proteins

involved in double-strand DNA break repair.

MELATONIN PRETREATMENT

BEFORE RADIATION SENSITIZES BREAST CANCER CELLS TO

THE IONIZING EFFECTS OF RADIATION

p53