Transcript No Slide Title

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
Synthesis of vitamin D3
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
Metabolism of vitamin D
Actions of 1,25(OH)2D3 on
intestine
• Stimulates active calcium transport
• Induces expression of calbindin 9K
(calcium binding protein)
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
1,25(OH)2D3 and osteoclast formation
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
Calcium absorption and osteoporosis
Bone deformities in post-menopausal osteoporosis
1,25-dihydroxyvitamin D3 treatment of
osteoporosis
Bone disease and renal failure
The analogue 1a hydroxyvitamin D3 is used
to treat renal osteodystrophy
H
H
HO
OH
1 alpha hydroxyvitamin D3
Mechanism of action of vitamin D
DNA
1,25(OH)2D3
cytoplasm
VDR
nucleus
cell
Enhanced mRNA synthesis
From vitamin D responsive
genes
Homology between nuclear hormone receptors
Interaction of VDR with VDREs
Heterodimerization with RXR
VDR
RXR
3
5’-AGGTCA
AGGTCA- 3’
DR 3 VDRE
in promotor region of
target gene
Distribution of VDR in normal
human tissues
Tissue
Liver
Kidney
Thyroid
Adrenal
Stomach
Duodenum
Jejunum
Colon
Skin
Breast epithelium
Skeletal muscle
Immunocytochemical staining
+/++
++/+++
++/+++
+/++
+/++
++
++
+++
++
++
-
(+) weak, (++) moderate, (+++) strong, (-) negative
Vitamin D regulated genes/gene
products
Function
Gene product
Tissue/cells
Mineral homeostasis calbindin 9K
osteocalcin
intestinal mucosa
osteoblast
Peptide hormones
TSH
PTH
rat pituitary cells
rat parathyroid
Growth factors/
receptors
TGF-b
TNF-a
rat calvarial cells
leukaemia cells
Oncogenes
c-myc
breast cancer cells
Non-classical actions of vitamin D
• A wide variety of tissues and cells contain
the VDR and respond to 1,25dihydroxyvitamin D3
• Immunomodulatory actions and effects on
cells growth and differentiation identified
• Development of analogues capable of
modulating ‘non-classical’ actions of
vitamin D
Immunomodulatory actions of
1,25-dihydroxyvitamin D3
• Activates monocytes and promotes
differentiation of myeloid stem cells
• Suppresses lymphocyte proliferation,
immunoglobulin production and
cytokine synthesis
Differentiation of leukaemia cells by 1,25(OH)2D3
Activity profile of calcitriol and
its synthetic analogues
• Calcium mobilizing actions
• Anti-tumour/promotion of
differentiation
• Immunosuppressive effects
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
Vitamin D and psoriasis
• 1983 - 1,25-dihydroxyvitamin D3 shown
to promote keratinocyte differentiation
• 1985 - a patient with osteoporosis
receiving 1a hydroxyvitamin D3 showed
a dramatic improvement in her severe
psoriasis
• Development of vitamin D analogues for
topical treatment of psoriasis
Treatment of psoriasis with vitamin D
OH
OH
HO
OH
HO
OH
1,25-a-vit. D3
MC 903
O
OH
OH
HO
OH
HO
CB 1093
OH
EB 1089
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
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
Topical calcipotriol treatment in
advanced breast cancer
chest wall of patient treated with calcipotriol ointment
at start of treatment
after 5 weeks
Vitamin D analogues: profile of
activity
analogue
inhibition of
cell growth
calcaemic activity
__________________________________________________________________
1,25(OH)2D3
MC 903
CB 966
EB 1089
CB 1093
KH 1060
1
2
6
60
160
1000
1
0.05
0.2
0.5
0.27
1.3
Effects of vitamin D analogues on
progression NMU-induced rat
mammary tumours
CB 1093 1ug/kg
control
EB 1089 1ug/kg
control
200
% of initial tumor volume
% of initial tumor volume
300
2.54+/0.04mmol/l
***
***
100
200
150
2.67+/0.02mmol/l
100
**
50
**
2.98+/0.07mmol/l
3.06+/0.07mmol/l
0
0
0
1
2
3
week
4
5
**
0
1
2
3
week
4
5
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
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
Polymorphisms in the VDR Gene
exon
Ia Ib Ic
II
III
IV V VI
VII VIII IX
BsmI (B/b)
FokI start codon polymorphism
F = VDR (424 amino acids)
f = VDR (427 amino acids)
ApaI (A/a)
Long/Short
(L/S) poly (A)
microsatellite
TaqI (T/t)
Linkage disequilibrium
b=a=T=L
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
VDR polymorphisms and
breast cancer
Vitamin D appears to be protective against breast and prostate
cancer. It can inhibit growth and induce apoptosis in in vitro
models of both.
Vitamin D exerts its cellular actions by binding to a specific
intracellular receptor, the VDR. The ligand/receptor complex then
acts as a TF leading to increased or decreased transcription of
sensitive genes.
The human VDR gene contains a number of SNPs that may alter the
activity or function of the VDR
SNPs in the VDR gene have previously been associated with
disorders of bone metabolism eg. osteoporosis and altered bone
mineral density
We investigated whether SNPs in the VDR gene were associated
with risk of breast cancer
Geography of Prostate Cancer in USA
Prostate cancer mortality amongst
white men per 100 000
Calculated UV radiation
19.8 - 20.1
20.4 - 20.7
<29.4
29.4 - 31.4
20.1 -20.4
20.7 - 21.0
31.4 - 33.4
>33.4
From Hanchette CL et al. Cancer 1992; 70(12); 2861-9
Studies of association between VDR
genotype and prostate cancer
STUDY
PATIENTS
POLY
OR
Ingles et al
57 non hispanic whites
Poly A
4.61
Taylor et al
108 men - radical prostatectomy Taq I
(Black and White)
0.32
Ma J et al
327 (questionairre identified)
Bsm
Taq I
Not significant
Not significant
Correa-Cerro
132
Poly A
Taq I
Not significant
Not significant
Kibel et al
41 men - metastatic disease
Poly A
Taq I
Not significant
Not significant
p<0.01
Project Aim
To determine VDR polymorphism
frequencies and their association with breast
cancer risk*
*Bretherton-Watt et al 2001. Br J Cancer vol 85,
171-175,
Volunteers
Controls (n=241, median age 55.2 years,
range 51-79 years).
Breast cancer patients (n=181, median age
61.2 years, range 29-91, median time since
diagnosis 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 obtained
prior to collection of a blood sample.
Identification of Bsm I SNP
The polymorphism constitutes a single nucleotide
change (G to T) which deletes a Bsm I
endonuclease restriction site
CGCATTC
Restriction site present (b)
CTCATTC
Restriction site absent (B)
Genotyping SNPs: the traditional way
Step
Time
Genomic DNA extracted from whole blood
PCR amplification of the polymorphism
performed using VDR specific primers.
PCR product is digested 65oC with BsmI
restriction enzyme
Digest is analysed by agarose gel
electrophoresis.
40 mins
2.5 hours
1 hour
2 hours
Presence/absence of restriction site is
indicated by b/B
Therefore:
bb – homozygous, restriction site
BB – homozygous, no restriction site
Bb – heterozygote
bb
Bb
BB
Results
There was a significant difference between
BsmI/Poly A genotype frequencies in cancer
patients compared to healthy controls.
This difference was such that the odds of breast
cancer for a woman of genotype bb/LL are twice
those for a woman of genotype BB/SS.
There was no association with the FokI
polymorphism and breast cancer. However, FokI
polymorphism appeared to modulate the risk
associated with BsmI/Poly (A) genotype.
Possession of FF genotype reduced the risk
associated with genotype bb/LL.
VDR polymorphisms in control and
breast cancer patients
patients
controls
BsmI
2 analysis
p< 0.01
16.2%
p> 0.2
bb
Bb
BB
55.2%
FokI
2 analysis
28.6%
10.5%
43.1%
46.4%
15.5%
16.2%
35.7%
48.1%
ff
Ff
FF
39.8%
44.8%
1,25(OH)2D3
bb/LL
high risk
VDR
BB/SS
More/less VDR produced?
Different/less stable VDR produced?
Altered ability to switch on/regulate genes?
Altered sensitivity to hormones and environmental factors?
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