vitamin d fat soluble ‘vitamin’ synthesised in skin, food sources include fish oils...
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Vitamin D• Fat soluble ‘vitamin’
• Synthesised in skin, food sources include fish oils
• Physiological forms - vitamin D3 (cholecalciferol)
- vitamin D2 (ergocalciferol)
hormonal form synthesised in kidney
• Functions - intestine: calcium absorption
- bone: promotes mineralization
• Deficiency syndromes - children: rickets
- adults: osteomalacia
• Mechanism of action - receptor mediated
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Synthesis of vitamin D3
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Sunlight as a source of vitamin D
• Adequate supplies of vitamin D3 can be synthesized with sufficient exposure to solar ultraviolet B radiation
• Depends on latitude and season
• Summer sunlight in Cape Town = 2500 IU vitamin D3 daily
• Melanin, clothing or sunscreens that absorb UVB will reduce cutaneous production of vitamin D3
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Metabolism of vitamin D
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Actions of 1,25(OH)2D3 on intestine
• Stimulates active calcium transport
• Induces expression of calbindin 9K (calcium binding protein)
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Actions of 1,25(OH)2D3 on bone
• Osteoblasts:
– Simulates synthesis of collagen, osteocalcin and other bone matrix proteins
• Osteoclasts:
– Stimulates osteoclastic bone resorption (indirect action)
– Stimulates osteoclast recruitment
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1,25(OH)2D3 and osteoclast formation
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Abnormalities in vitamin D metabolism/action
• Vitamin D deficiency– Inadequate intake– Limited sunlight exposure
• Acquired or inherited disorders of vitamin D metabolism
• Inherited resistance to the actions of vitamin D
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Calcium absorption and osteoporosis
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Bone deformities in post-menopausal osteoporosis
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1,25-dihydroxyvitamin D3 treatment of osteoporosis
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The analogue 1 hydroxyvitamin D3 is used to treat renal osteodystrophy
Bone disease and renal failure
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OH OH
H
H
1 alpha hydroxyvitamin D3
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VDR
Enhanced mRNA synthesisFrom vitamin D responsivegenes
1,25(OH)2D3
cellcytoplasm nucleus
DNA
Mechanism of action of vitamin D
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Homology between nuclear hormone receptors
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Interaction of VDR with VDREs
RXR VDR
35’-AGGTCA AGGTCA- 3’
Heterodimerization with RXR
DR 3 VDREin promotor region oftarget gene
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Distribution of VDR in normal human tissues
Tissue Immunocytochemical stainingLiver +/++Kidney ++/+++Thyroid ++/+++Adrenal +/++Stomach +/++Duodenum ++Jejunum ++Colon +++Skin ++Breast epithelium ++Skeletal muscle - (+) weak, (++) moderate, (+++) strong, (-) negative
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Vitamin D regulated genes/gene products
Function Gene product Tissue/cells
Mineral homeostasis calbindin 9K intestinal mucosaosteocalcin osteoblast
Peptide hormones TSH rat pituitary cellsPTH rat parathyroid
Growth factors/ TGF- rat calvarial cellsreceptors TNF- leukaemia cells
Oncogenes c-myc breast cancer cells
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Non-classical actions of vitamin D
• A wide variety of tissues and cells contain the VDR and respond to 1,25-dihydroxyvitamin D3
• Immunomodulatory actions and effects on cells growth and differentiation identified
• Development of analogues capable of modulating ‘non-classical’ actions of vitamin D
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Immunomodulatory actions of 1,25-dihydroxyvitamin D3
• Activates monocytes and promotes differentiation of myeloid stem cells
• Suppresses lymphocyte proliferation, immunoglobulin production and cytokine synthesis
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Differentiation of leukaemia cells by 1,25(OH)2D3
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Activity profile of calcitriol and its synthetic analogues
• Calcium mobilizing actions
• Anti-tumour/promotion of differentiation
• Immunosuppressive effects
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Pathogenesis of psoriasis
• Chronic or chronically relapsing skin disease
• ? Susceptibility heritable
• Results form epidermal stem cell growth, initiated by lymphokines released from activated T cells
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Vitamin D and psoriasis
• 1983 - 1,25-dihydroxyvitamin D3 shown to promote keratinocyte differentiation
• 1985 - a patient with osteoporosis receiving 1 hydroxyvitamin D3 showed a dramatic improvement in her severe psoriasis
• Development of vitamin D analogues for topical treatment of psoriasis
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Treatment of psoriasis with vitamin D
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OHHO
OH
1,25--vit. D3
OHHO
OH
EB 1089CB 1093
OHHO
OH
O
OHHO
OH
MC 903
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Vitamin D and breast cancer
• Risk of breast cancer inversely related to intensity of local sunlight and 1,25-D levels
• Low serum 1,25-D levels correlated with disease progression and development of bone metastases
• >80% breast tumour specimens VDR positive and presence of receptor is associated with increased disease free survival
• 1,25-D and its analogues inhibit growth and promote apoptosis in vitro and in vivo
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Trial of topical calcipotriol (MC903) therapy in advanced breast cancer
• 19 patients with locally advance or metastatic breast cancer and evaluable cutaneous deposits were treated daily with one gram calcipotriol (MC903) ointment
• All patients were normocalcaemic at entry
• 14 patients completed 6 weeks of treatment. 3 showed a partial and one a minimal response
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Topical calcipotriol treatment in advanced breast cancer
chest wall of patient treated with calcipotriol ointment
at start of treatment after 5 weeks
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Vitamin D analogues: profile of activity
analogue inhibition of calcaemic activity cell growth
__________________________________________________________________
1,25(OH)2D3 1 1
MC 903 2 0.05
CB 966 6 0.2
EB 1089 60 0.5
CB 1093 160 0.27
KH 1060 1000 1.3
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Effects of vitamin D analogues on progression NMU-induced rat
mammary tumours
5432100
100
200
300
EB 1089 1ug/kg
control
week
% o
f in
itia
l tum
or v
olum
e
******
2.54+/-0.04mmol/l
3.06+/-0.07mmol/l
5432100
50
100
150
200
CB 1093 1ug/kgcontrol
week
% o
f in
itia
l tum
or v
olum
e
**
****
2.67+/-0.02mmol/l
2.98+/-0.07mmol/l
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Processes involved in the tumour suppressive activity of vitamin D
analogues
• Inhibition of cell proliferation
• Induction of apoptosis
• Promotion of cell differentiation
• Inhibition of angiogenesis
• Altered elaboration or response to growth factors
• Inhibition of metastasis
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Polymorphisms in the VDR gene
• Vitamin D receptor gene polymorphisms have been identified
• These include single base change mutations in the 3’ UTR region
• These SNPs have been reported to be associated with altered risk for certain diseases
• It is not yet known in what way these differences in the gene may affect the activity of the translated receptor protein
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Ia Ib Ic II III IV V VI VII VIII IX
FokI start codon polymorphismF = VDR (424 amino acids)f = VDR (427 amino acids)
BsmI (B/b)
ApaI (A/a)
TaqI (T/t)
Long/Short (L/S) poly (A) microsatellite
Linkage disequilibriumb = a = T = L
exon
Polymorphisms in the VDR Gene
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Association between VDR polymorphisms and disease
• Bone mineral density
• Early postnatal growth
• Diabetes mellitus
• Psoriasis
• TB and hepatitis B virus infection
• Primary hyperparathyroidism
• Prostatic and breast cancer
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VDR polymorphisms and breast cancer
Vitamin D appears to be protective against breast and prostatecancer. It can inhibit growth and induce apoptosis in in vitromodels of both.
Vitamin D exerts its cellular actions by binding to a specificintracellular receptor, the VDR. The ligand/receptor complex thenacts as a TF leading to increased or decreased transcription ofsensitive genes.
The human VDR gene contains a number of SNPs that may alter theactivity or function of the VDR
SNPs in the VDR gene have previously been associated withdisorders of bone metabolism eg. osteoporosis and altered bonemineral density
We investigated whether SNPs in the VDR gene were associatedwith risk of breast cancer
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20.7 - 21.020.1 -20.4
19.8 - 20.1 20.4 - 20.7
Prostate cancer mortality amongst white men per 100 000
>33.431.4 - 33.4
<29.4 29.4 - 31.4
Calculated UV radiation
From Hanchette CL et al. Cancer 1992; 70(12); 2861-9
Geography of Prostate Cancer in USA
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STUDY PATIENTS POLY OR Ingles et al 57 non hispanic whites Poly A 4.61 Taylor et al 108 men - radical prostatectomy Taq I 0.32 p<0.01 (Black and White) Ma J et al 327 (questionairre identified) Bsm Not significant Taq I Not significant Correa-Cerro 132 Poly A Not significant Taq I Not significant Kibel et al 41 men - metastatic disease Poly A Not significant Taq I Not significant
Studies of association between VDR genotype and prostate cancer
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To determine VDR polymorphism frequencies and their association with breast cancer risk*
*Bretherton-Watt et al 2001. Br J Cancer vol 85, 171-175,
Project Aim
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Controls (n=241, median age 55.2 years,range 51-79 years).Breast cancer patients (n=181, median age61.2 years, range 29-91, median time sincediagnosis 4.3 years, range 0.4-27.5 years).
Inclusion criteria to the study were:1) A recent mammogram and breast history;2) Caucasian (due to ethnic variations in
polymorphism distribution).Written informed consent was obtainedprior to collection of a blood sample.
Volunteers
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Identification of Bsm I SNP
The polymorphism constitutes a single nucleotide change (G to T) which deletes a Bsm I endonuclease restriction site
CGCATTC
CTCATTC
Restriction site present (b)
Restriction site absent (B)
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Step TimeGenomic DNA extracted from whole blood 40 minsPCR amplification of the polymorphismperformed using VDR specific primers.
2.5 hours
PCR product is digested 65oC with BsmIrestriction enzyme
1 hour
Digest is analysed by agarose gelelectrophoresis.
2 hours
Genotyping SNPs: the traditional way
} } }
bb Bb BB
Presence/absence of restriction site isindicated by b/BTherefore:bb – homozygous, restriction site BB – homozygous, no restriction site
Bb – heterozygote
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TThheerree wwaass aa ssiiggnniiffiiccaanntt ddiiffffeerreennccee bbeettwweeeennBBssmmII//PPoollyy AA ggeennoottyyppee ffrreeqquueenncciieess iinn ccaanncceerrppaattiieennttss ccoommppaarreedd ttoo hheeaalltthhyy ccoonnttrroollss..TThhiiss ddiiffffeerreennccee wwaass ssuucchh tthhaatt tthhee ooddddss ooff bbrreeaassttccaanncceerr ffoorr aa wwoommaann ooff ggeennoottyyppee bbbb//LLLL aarree ttwwiicceetthhoossee ffoorr aa wwoommaann ooff ggeennoottyyppee BBBB//SSSS..
TThheerree wwaass nnoo aassssoocciiaattiioonn wwiitthh tthhee FFookkIIppoollyymmoorrpphhiissmm aanndd bbrreeaasstt ccaanncceerr.. HHoowweevveerr,, FFookkIIppoollyymmoorrpphhiissmm aappppeeaarreedd ttoo mmoodduullaattee tthhee rriisskkaassssoocciiaatteedd wwiitthh BBssmmII//PPoollyy ((AA)) ggeennoottyyppee..PPoosssseessssiioonn ooff FFFF ggeennoottyyppee rreedduucceedd tthhee rriisskkaassssoocciiaatteedd wwiitthh ggeennoottyyppee bbbb//LLLL..
Results
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VDR polymorphisms in control and breast cancer patients
p< 0.01
patients
43.1%46.4%
10.5%bbBbBB
controls
28.6%
55.2%
16.2%2 analysis
p> 0.2
15.5%
44.8%
39.8%ffFfFF
16.2%
48.1%
35.7%
BsmI
2 analysis
FokI
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VDR
More/less VDR produced?Different/less stable VDR produced? Altered ability to switch on/regulate genes?Altered sensitivity to hormones and environmental factors?
1,25(OH)2D3
bb/LL
BB/SS
high risk
low risk
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Summary
• Recent research has identified the possibility of developing analogues capable of modulating non-classical actions of vitamin D
• Polymorphisms in the VDR gene may contribute to susceptibility to certain diseases