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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