the role of vitamin d in breast cancer
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The effects of radiation on vitamin D actions in normal mammary cells, by Natalia L. MartinezTRANSCRIPT
The Role of Vitamin D in Breast Cancer :
The effects of radiation on vitamin D actions in normal mammary cells
Department of Chemistry, The College of New Rochelle Natalia L. Martinez
Mentor: Elvira Longordo
University at Albany Summer Research ProgramGEN*NY*Sis Center for Excellence in Cancer Genomics
JoEllen Welsh and Erika LaPorta
Breast Cancer
• Breast Cancer is the uncontrolled growth of breast cells, in the lobules or the ducts
Normal Cancer
Cellular Origin of Cancer
What Causes Cancer?
Intrinsic Factors• Heredity
• Diet• Hormones
Extrinsic Factors• Chemicals• Radiation
DNA Damage
• Chemicals and Radiation can damage DNA
• Cells can sense and repair damage– p53- acts in the damage response
mechanism – tumor suppressor
• There is now a focus on identifying additional agents that enhance DNA repair
• http://www.haemophilia.org.nz/DBimages/FCK/Image/dna.jpg
The link between Cancer & vitamin D
• Recent research has shown a link between vitamin D and the prevention and treatment of Breast Cancer
• Role of Vitamin D– Regulates Calcium and Phosphorus levels in
the blood– Prevention of Rickets
How is Vitamin D obtained?
• Food Sources– Fatty Fish
– Eggs– Fortified Foods
• Sunlight Exposure– UVB light
From Vitamin D to 1α,25(OH)2D3
(hv) UVB light
7-dehydrocholesterol found in the skin
cholecalciferol
Or vitamin D3.
vitamin D3 is hydroxylated
forming 25- hydroxycholecalciferol
hydroxylated once more
Producing the active form of vitamin D3,
1,25-dihydroxycholecalciferol.
UV Stimulated Synthesis of Calcitriol
Biochemistry Fifth Ed. Berg, Tymoczo and Stryer. W.H. Freeman, 2002.
The vitamin D receptor (VDR)
• It is found in the nucleus of different tissues through the body
• VDR is a nuclear hormone receptor
• VDR is the major mediator of 1,25D actions
1,25D & VDR
When the active form of vitamin D binds to its nuclear receptor VDR it regulates gene expression which influences cellular proliferation, differentiation and apoptosis
In My Project:
• Study the effects of radiation on vitamin D actions in normal human mammary cells (HME)
Hypothesis:• DNA damage caused by ionizing radiation will up-
regulate p53 and VDR expressionWestern Blot
• Protection of the cells can be achieved by the ligand binding of 1,25D3 to VDR in damaged cells
Crystal violet assay & immunoflurescent microscopy
Investigated the effects of radiation on vitamin D
receptor (VDR) expression
Procedure• Cell Culture
-HME cells (9 plates)• Ionizing Radiation
DOSES OF EXPOSURE Incubation Period.
Sham-0KV-0mA; 1min. 3 plates –analyzed after 1hr.
2Gy- 130KV- 2mA; 1min.
3 plates- analyzed after 3hr.
8Gy- 130KV- 7.7mA; 1 min
3 plates- analyzed after 6hr.
http://www.instapstudycenter.net/images/faxitron.GIF
Detection of p53 and VDR by Western Blot
Lamin A/C
Lamin A/C
8G
y
2G
y
Sh
am
.
8G
y.
2G
y
Sh
am
Sh
am
2G
y
8G
y
p53
VDR
6hrs. 3hrs. 1hr.
Use immunofluorescence microscopy to monitor expression and sub-cellular localization of VDR after
radiation exposure in the presence and absence of 1,25D
Detection of VDR by Confocal Microscopy
www.ion.ucl.ac.uk/.../neuropath/techniques.htm
Results1,25DEtOH
Sham 3hr
2Gy 3hr
8Gy 3hr
Results – 1hr
Sham EtOH
Sham
VitD
2Gy EtO
H
2Gy VitD
Results – 3hr
Sham EtOH
2Gy EtO
H
2Gy VitD
Sham V
itD
Examine whether treatment with 1,25D
alters HME cell response to radiation.
Procedure
• Plated cells in 6 well plates
• Treated with 100nM Vitamin D – 48hr. Prior to radiation exposure– Just before radiation exposure
• Cultured for 4 days
• Quantified cell density using Crystal Violet Assay
Morphology Sham-EtOH
Sham-VitD
2Gy-EtOH
2Gy-VitD
Results
Pretreated
EtOH Sham EtOH 2Gy VitD Sham VitD 2Gy0
1
2
3
* *
Treatment
Ab
sorb
ance
590
Cotreated
EtOH Sham EtOH 2Gy VitD Sham VitD 2Gy0
1
2
3
* *
Treatment
Ab
sorb
ance
590
Conclusion
• HME cells express VDR & p53 proteins
• Radiation may increase VDR & p53 expression
• VDR localization is altered by radiation treatment
• Vitamin D inhibits growth of HME cells
Acknowledgments
• Dr. JoEllen Welsh• Erika LaPorta
• Dr. Meggan Keith• Dr. Judy Narvaez
• Dr. Christopher Fernando• Deanna Morehead
• UASRP Staff and Scholars