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