1. MELATONIN SENSITIZES HUMAN BREAST CANCER CELLS TO IONIZING RADIATION DEPARTMENT OF PHYSIOLOGY AND PHARMACOLOGY UNIVERSITY OF CANTABRIA, SANTANDER, SPAIN Carolina Alonso-González

2. FIBROBLASTS ADIPOCYTES EPITHELIAL CELLS MAMMARY TUMOR 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

3. RADIOTHERAPY ENDOCRINE THERAPY CHEMOTHERAPY MELATONIN SEEM SERM ¿?

4. 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-PKcs mRNA gene expression

5. Alonso-González et al., J Pineal Res 58:189-197, 2015. EFFECTS OF MELATONIN ON BREAST CANCER CELL PROLIFERATION (MCF-7) 036 Days 1 mM 10 µM 1 nM Cell proliferation (% of control) 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 1 mM 0 50 100 150 Melatonin a 1 nM10 µMControl Cell proliferation (% of control) a a b c 75 25 125 4 Days

6. EFFECTS OF IONIZING RADIATION ON BREAST CANCER CELL PROLIFERATION (MCF-7) 0 36 Days Post-Radiation Cell proliferation (% of control non−radiated cells) 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.

7. 0 25 50 75 100 125 Control Melatonin 1 mMMelatonin 10 µM Melatonin 1 nM Cell Proliferation (% of control radiated cells) 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 6 Days Post-Radiation

8. 0 25 50 75 100 125 Cell cycle distribution G1G1 SG 2 -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 (1 nM) AND IONIZING RADIATION 0 25 50 75 100 125 a Cell cycle distribution a G1G1 SG 2 -M Non-radiated cells Melatonin pretreated cells (1 nM) a Alonso-González et al., J Pineal Res 58:189-197, 2015. 0 25 50 75 100 125 a Cell cycle distribution a G1G1 SG 2 -M Radiated cells a 0 25 50 75 100 125 b b G1G1 SG 2 -M Melatonin Pretreated (1 nM) and Radiated cells b Cell cycle distribution

9. DNA DAMAGE IR DOUBLE STRAND BREAKS (DSBs) ATM MRN COMPLEX Apoptosis Senescence Cell cycle arrest DNA damage repair Genomic Integrity Restored SURVIVAL P53 CHK2

10. M G1G1 G2G2 S NHEJ HR NHEJ HR REPAIR OF DNA DOUBLE STRAND BREAKS (DSBs) Hoeijmakers JH, Nature 411: 366-374, 2001.

11. RAD51 (% of control non-radiated cells) 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 nM10 µMControl b c b d e 75 25 125 RAD51 (% of control radiated cells) 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.

12. M G1G1 G2G2 S NHEJ HR NHEJ HR REPAIR OF DNA DOUBLE STRAND BREAKS (DSBs) Hoeijmakers JH, Nature 411: 366-374, 2001.

13. DNA-PKcs (% of control non-radiated cells) 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 nM10 µMControl a a b c 75 25 125 DNA-PKcs (% of control radiated cells) 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.

14. 1 mM 0 100 200 300 Melatonin pretreatment before radiation a 1 nM10 µM a b a b c d 150 50 250 RADIATION Control Non- Radiated cells Control radiated cells a P53 (% of control non-radiated cells) 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

15. Decreasing cell proliferation. Inducing G1 cell cycle arrest. Down-regulating RAD-51 and also DNA-PKcs. Two proteins involved in DSBs repair mechanisms. MELATONIN PRETREATMENT SENSITIZES BREAST CANCER CELLS TO THE EFFECTS OF IONIZING RADIATION p53

16. CAROLINA ALONSO-GONZÁLEZ ALICIA GONZÁLEZ CABEZA CARLOS MARTÍNEZ CAMPA JAVIER MENÉNDEZ MENÉNDEZ ÁNGELA GARCÍA VIDAL SAMUEL COS CORRAL Technical assistance José Antonio de Cos Cossío Gema Viar Ruíz RESEARCH GROUP