ANTICANCER RESEARCH 26: 2567-2572 (2006)
Vitamin D Metabolism in Human Prostate Cells: Implications
for Prostate Cancer Chemoprevention by Vitamin D
JOHN N. FLANAGAN, MICHAEL V. YOUNG, KELLY S. PERSONS, LILIN WANG,
JEFFREY S. MATHIEU, LYMAN W. WHITLATCH, MICHAEL F. HOLICK and TAI C. CHEN
Department of Medicine, Section of Endocrinology, Diabetes and Nutrition,
Boston University School of Medicine, Boston, MA 02118, U.S.A.
Abstract. Background: Prostate cells can produce 1·,25-
dihydroxyvitamin D3 (1·,25(OH)2D3) from 25-hydroxyvitamin
D3 (25(OH)D3) to regulate their own growth. Here, the questions
of whether prostate cells express vitamin D-25-hydroxylase (25-
OHase) and can convert vitamin D3 to 1·,25(OH)2D3 were
investigated. Materials and Methods: Protein and receptor
binding assays were used to determine 25(OH)D3 and
1·,25(OH)2D3, respectively. Measurements of proliferation by
3H-thymidine incorporation, and 1·,25(OH) D-responsive gene
2
expression by real-time qPCR and by Western blot were used as
functional assays for the presence of 25-OHase activity. Results:
Prostate cells metabolized vitamin D3 to 1·,25(OH)2D3. Vitamin
D3 up-regulated 25(OH)D-24R-hydroxylase and IGFBP3, two
1·,25(OH)2D-responsive genes, in prostate cells. CYP2R1 was
the major form of 25-OHase expressed in normal and cancerous
prostate cells as determined by qPCR. Conclusion: The autocrine
synthesis of 1·,25(OH)2D3 from vitamin D3 suggests that
maintaining adequate levels of serum vitamin D could be a safe
and effective chemo-preventive measure to decrease the risk of
prostate cancer.
Vitamin D regulates over 60 genes by interacting with a
specific nuclear receptor, the vitamin D receptor (VDR) (1).
Abbreviations: 1·,25(OH)2D3, 1·,25-dihydroxyvitamin D3; 25-OHase,
vitamin D-25-hydroxylase; 25(OH)D3, 25-hydroxyvitamin D3;
1·-OHase, 25(OH)D-1·-hydroxylase; 24R-OHase, 25(OH)D-24R-
hydroxylase; IGFBP3, insulin-like growth factor binding protein-3;
vitamin D2, ergocalciferol; vitamin D3, cholecalciferol; 24,25(OH)2D,
24, 25-dihydroxyvitamin D; 1·,24,25(OH)3D, 1·,24,25-trihydroxy-
vitamin D; CYP, cytochrome; VDR, vitamin D receptor; DPPD, 1,
2-dianilinoethane; qPCR, quantitative PCR.
Correspondence to: Tai C. Chen, Rm M-1022, Boston University
Medical Center, 715 Albany Street, Boston, MA 02118, U.S.A. Tel:
(617) 638-4543, Fax: (617) 638-8898, e-mail: taichen@bu.edu
Key Words: Prostate cancer, vitamin D, hydroxylases, cytochrome
P450, chemoprevention.
0250-7005/2006 $2.00+.40
The genes include those associated with calcium homeostasis,
immune responses, cellular growth, differentiation, apoptosis,
and the enzymes involved in its own metabolism. Vitamin D
(which includes both vitamin D2 and vitamin D3) requires
two successive hydroxylation steps first in the liver by the
25-OHase and then in the kidneys by 1·-OHase (or CYP27B1)
to form the active metabolite, 1·,25(OH)2D (2). The kidneys
can also hydroxylate 25(OH)D and 1·,25(OH)2D at C-24 by
24R-OHase (or CYP24A1) to form 24, 25-dihydroxyvitamin D
(24,25(OH)2D) and 1·,24,25-trihydroxyvitamin D (1·,24,25
(OH)3D), respectively. The main function of CYP24A1 is to
regulate the circulating concentration of 1·,25(OH)2D (2).
Similarly to extra-renal 1·-hydroxylation, which occurs in
many tissues including skin, intestine, colon, breast and
prostate (3), a broad tissue distribution of extra-hepatic
25-OHase activity has been observed in adrenal, lung, spleen,
skin, kidney, colon and small intestine (4). At the present
time, there are at least six candidate 25-OHases, CYP27A1,
CYP2C11, CYP2D25, CYP2J3, CYP3A4 and CYP2R1. All
have been implicated in the conversion of vitamin D to
25(OH)D. Among them, only CYP27A1 is found in the
mitochondria, while the rest are located in the microsomal
fraction (5).
Previously, we have demonstrated that prostate cells are
capable of synthesizing 1·,25(OH)2D intracellularly from
25(OH)D (6), and that the antiproliferative effect of
25(OH)D was indistinguishable from that of 1·,25(OH)2D
(7). This finding has important implications for prostate
cancer chemoprevention because the risk of hypercalcemia
associated with the systemic administration of vitamin D and
25(OH)D is far lower than that for 1·,25(OH)2D (8). In this
study, we investigated whether prostate cells have the
capacity to convert vitamin D3 to 1·,25(OH)2D and
examined the potential of vitamin D3 to induce
1·,25(OH)2D3 biological activities in prostate cells. It was
demonstrated that prostate cells express 25-OHase mRNA,
and are capable of metabolizing vitamin D3 to
1·,25(OH)2D3, leading to the up-regulation of 24R-OHase
and IGFBP3, and the inhibition of prostate cell proliferation.
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 ANTICANCER RESEARCH 26: 2567-2572 (2006)
Table I. Sequence specific primers for real-time qPCR assays.
Gene Forward
CYP27A1 5’-CTA TTT GCT ACA TCC TGT TCG AGA AA-3’
CYP2R1 5’-TGA CCC ATC ATC TAC TTT CTC CA-3’
CYP3A4 5’-CGT GGC CCA ATC AAT TAT CTT T-3’
IGFBP3 5’-CAA GTT CCA CCC CCT CCA TT-3’
CYP24A 5’-GGC CTG GAT GTC GTA TTT GC-3’
Materials and Methods
Cell cultures. LNCaP and PC-3 cells (ATCC, Manassas, VA, USA)
were maintained in RPMI (Sigma, St Louis, MO, USA)
supplemented with 5% FBS (GibcoBRL, Gaithersburg, MD,
USA), whereas PZ-HPV-7 cells (ATCC) were grown in serum-free
keratinocyte growth medium (9).
Cell proliferation assay. Tritiated thymidine (Perkin-Elmer, Boston,
MA, USA) incorporation into DNA was used to determine the
effect of vitamin D3 on PZ-HPV-7 cell proliferation (9).
Determination of 25(OH)D and 1·,25(OH)2D. A competitive
protein binding assay and a radio-receptor binding assay (10,11)
were employed to measure the concentrations of 25(OH)D3 and
1·,25(OH)2D3, respectively. Briefly, cells, grown to 90%
confluence on 35-mm plates, were treated with 10-6 M vitamin D3
for 2 hours in the presence of the 10 mM 1,2-dianilinoethane
(DPPD). Lipid extracts were obtained with the addition of
methanol to monolayer cultures after the media had been removed.
The 25(OH)D and 1·,25(OH)2D fractions from the lipid extracts
were collected separately from C-18-OH cartridge chromatography,
and their concentrations were determined as described (10, 11).
Real-time qPCR analysis. Total RNA from normal human liver
(Cat#-64099-1) and normal prostate (Cat#-64108-1) tissue were
purchased from BD Biosciences, Mountain View, CA, USA. Total
RNA from prostatic cell lines was isolated using the SV Total RNA
kit (Promega, Madison WI, USA). For each sample, cDNA was
generated with 2 mg of total RNA using Superscript RNAase H-
(Invitrogen, Carlsbad, CA, USA) with random hexamer primers.
Sequence specific primers were designed against coding sequences
of human CYP27A1 (414120), CYP2R1 (33591221), CYP3A4
(13904851), IGFBP3 (40675391) and CYP24A (306703) with
Primer Express software (Applied Biosystems, Forest City, CA,
USA) (Table I).
For each real-time qPCR reaction, 20 ng of single-stranded
cDNA was mixed with 2X SYBR Green PCR Master Mix (Applied
Biosystems) and an optimal concentration of sequence specific
primers. Samples were analyzed on an ABI Prism 7700 sequence
detection system. To normalize the amount of sample cDNA added
to the reaction, Taqman PDAR eukaryotic 18S rRNA, (Applied
Biosystems) was used as the endogenous control. Relative
quantitation of gene expression was calculated using the DDCt
method (User Bulletin publication #2, Applied Biosystems).
Western blot analysis. Cells lysates in a buffer containing 100 mM
Tris (pH 8.5), 250 mM NaCl, 1% NP40, 1 mM EDTA and 5 mM
2568
Reverse
5’-CTG GAA CAT TAA CCC GAT GGA-3’
5’-TTA GGA TAA AGG GCC ATG AAA AGA-3’
5’-GCA TCA ATT TCC TCC TGC AGT T-3’
5’-TCT CTA CGG CAG GGA CCA TAT T-3’
5’-ACA ATC CAA CAA AGA GCC AAA TGC AGT TGA A-3’
DTT were sonicated, boiled and centrifuged at 14,000 rpm for 10
minutes to remove particulate matter. A sample of 50 Ìg of protein
extracts was separated on a 10% SDS-PAGE gel, transferred to
nitrocellulose paper and probed with a rabbit polyclonal antibody
against human IGFBP3 (sc-9028, Santa Cruz Biotechnology, Santa
Cruz, C∞, USA), followed by a secondary antibody using goat anti-
rabbit IgG conjugated to horseradish peroxidase. The paper was
then developed with chemiluminescent reagents (Amersham
Biosciences, Piscataway, NJ, USA).
Results
Inhibition of cellular proliferation by vitamin D3 in normal
prostate cells, an indication of prostatic 25-OHase activity. In
order to determine if prostate cells have 25-OHase activity
and the ability to hydroxylate vitamin D to 25(OH)D before
being activated to 1·,25(OH)2D, we investigated whether
vitamin D3 would inhibit the growth of PZ-HPV-7 cells (9).
We found that cells treated with vitamin D3 at 10–6 M for
18 hours reduced 3H-thymidine incorporation by 40%
compared to the ethanol vehicle control. For a positive
control, PZ-HPV-7 cells were treated with 1·,25(OH)2D3
at 10–6 M for 18 hours which reduced the cell proliferation
by 80% of the control, similar to previously published
studies (7). These results indicate that vitamin D3 elicits a
similar biological effect as 1·,25(OH)2D3 on prostate cell
growth, which may reflect that vitamin D3 has been
metabolized to 25(OH)D3 by a prostatic 25-OHase.
Conversion of vitamin D3 to 1·,25(OH)2D3 in normal
prostate cells. To further investigate the presence of
25-OHase in PZ-HPV-7 cells, we measured 25(OH)D3 and
1·,25(OH)2D3 formation in prostate cells treated with
vitamin D3 (10, 11). Although we were unable to detect
25(OH)D3 in the lipid extracts of PZ-HPV-7 cells incubated
for 2 hours with 10–6 M of vitamin D3, which could result
from the high endogenous 1·-OHase activity, we detected
50±3 fmols of 1·,25(OH)2D3 in the same cultures (Figure 1).
We also incubated heat-inactivated cells with either vitamin
D3 (10–6 M) or 1·,25(OH)2D3 (10–8 M) for 2 hours in the
presence of DPPD as controls. We were able to detect
1·,25(OH)2D3 in heat-inactivated cells treated with
 Flanagan et al: Vitamin D Metabolism and Prostate Cancer

Figure 1. Production of 1·,25(OH)2D3 from vitamin D3 in prostate cells.

PZ-HPV-7 cells were incubated with and without vitamin D3 (10–6 M) in
the presence of DPPD for 2 hours. In addition, PZ-HPV-7 cells were
inactivated with heat and then incubated with either vitamin D3 (10–6 M)
or 1·,25(OH)2D3 (10–8 M) under the same incubation conditions.
Histograms represent mean±SEM of three experiments.

1·,25(OH)2D3, but not those treated with vitamin D3

(Figure 1). This indicates that the 1·,25(OH)2D3 detected
in live PZ-HPV-7 cells treated with vitamin D3 was derived
from the enzymatic hydroxylation of vitamin D3 to
25(OH)D3 catalyzed by endogenous 25-OHase prior to the
hydroxylation by 1·-OHase.

Up-regulation of 1·,25(OH)2D3-inducible genes by vitamin D3

in PZ-HPV-7 cells, indicating prostate cells contain all the
necessary hydroxylases involved in activation of vitamin D.
Prostate cells express 24R-OHase (12) and IGFBP3 (13),
which are inducible by 1·,25(OH)2D3 but not vitamin D3 or
25(OH)D3 per se (14). Therefore, we examined the up-
regulation of 24R-OHase and IGFBP3 expression in
response to vitamin D3 as indicators of the two-step
activation of vitamin D3 via 25-hydroxylation and
subsequent 1·-hydroxylation to form 1·,25(OH)2D3 in PZ-
HPV-7 cells. PZ-HPV-7 cells at 90% confluence were
treated with vitamin D3 (10–8, 10–7 and 10–6 M) or
1·,25(OH)2D3 (10–9, 10–8 and 10–7 M) for 24 hours. As a
positive control, cells were also treated with 1·,25(OH)2D3.
The expression of 24R-OHase and IGFBP3 mRNAs were
analyzed by real-time qPCR, using total RNA prepared
from the treated cells. Figure 2A demonstrates that
24R-OHase expression was up-regulated in a dose-
dependent manner by vitamin D3 (32-, 241- and 1688-fold)
and 1·,25(OH)2D3 (2-, 69- and 930-fold) versus controls, at
the indicated doses. Similarly, IGFBP3 expression was up-
Figure 2. The expression of (A) 24R-OHase mRNA, (B) IGFBP3 mRNA
and (C) IGFBP3 protein in PZ-HPV-7 cells treated with vitamin D3 or
1·,25(OH)2D3. Prostate cells were treated with vehicle control (0.01%
EtOH),vitamin D3, or 1·,25(OH)2D3 for 24 hours. The expression of
24R-OHase (panel A) and IGFBP3 (panel B) mRNAs were analyzed by
real-time qPCR. The data are presented as relative mRNA fold change
versus vehicle (0.01% EtOH). The data were calculated according to the
¢¢Ct method and standardized to an endogenous control, S18.
Histograms are the mean±SD of two independent experiments with six
determinations each. For Western blot analysis of IGFBP3 (panel C), total
protein extracts were separated by SDS-PAGE, transferred to nitrocellulose
membranes and blotted with an anti-human IGFBP3 antibody.
regulated by both vitamin D3 and 1·,25(OH)2D3 as
indicated in Figure 2B. We also evaluated the IGFBP3
protein by Western blot analysis in vitamin D3 and
1·,25(OH)2D3-treated PZ-HPV-7 cells. As demonstrated in
Figure 2C, the IGFBP3 protein was up-regulated by
pretreatment of PZ-HPV-7 cells with vitamin D3 (10–6 M)
and 1·,25(OH)2D3 (10–7 M). These results demonstrate that

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 ANTICANCER RESEARCH 26: 2567-2572 (2006)
Figure 3. The mRNA expression profiles of CYP27A, CYP2R1 and
CYP3A4 in normal liver and prostate tissues, and in cultured prostate
cells. The data are presented as the relative mRNA fold difference in
reference to liver. The data was calculated according to the DDCt method
and standardized to an endogenous control, S18. The histograms are the
mean±SD of two experiments with six determinations each.
N.D.: not determined.
vitamin D3 modulated VDR responsive genes, and reflect
the two-step activation of vitamin D3 via 25-hydroxylation
then 1·-hydroxylation to form 1·,25(OH)2D3.
Expression profile of candidate 25-OHases – CYP27A,
CYP2R1 and CYP3A4 in human liver and non-cancerous and
cancerous prostate cells. Three 25-OHases, including the
mitochondrial CYP27A1 and the two microsomal enzymes,
CYP3A4 and CYP2R1, have been found in several species
2570
including humans (4,5). In order to determine which p450s
might be responsible for the observed 25-OHase activity in
prostate cells, the expression of these three candidate 25-
OHases were investigated by real-time qPCR. Figure 3
shows that CYP2R1 expression was two-fold greater in
normal human prostate tissue, three-fold greater in PZ-HPV-7
cells and six-fold greater in LNCaP prostate cancer cells
than in normal liver tissue, whereas CYP27A1 was
expressed ten-fold more in normal liver tissue than in
normal prostate tissue. Very little or no expression of
CYP27A1 was found in the PZ-HPV-7, LNCaP and PC-3
cells. PC-3 cells also expressed very little or no CYP2R1.
CYP3A4 was expressed only in normal liver tissue and not
in any prostate cells examined. The data suggest that
CYP2R1 represents the major 25-OHase in prostate cells,
and is probably responsible for the conversion of vitamin D3
to 25(OH)D3.
Discussion
Although the existence of extra-hepatic 25-OHases has been
known for some times, its physiological significance is
currently not entirely clear. The best studied extra-hepatic
25-OHase system is the one described in human
keratinocytes (15-17). Using 3H-vitamin D3 as substrate,
Schuessler et al. (15) observed continuous slow formation of
3H-25(OH)D in cultured human keratinocytes, following a
3
linear time course and yielding only 1.5% of the product
after 4 hours of incubation. This finding is in agreement with
the low but constant levels of CYP27A1 mRNA expression
found in human keratinocytes by qPCR (15, 16). The low but
constant formation of 3H-25(OH)D3 resulted in a sustained
up-regulation of 24R-OHase after the addition of
physiological doses of vitamin D3. Since both vitamin D3 and
25(OH)D3 lack intrinsic 24-OHase-inducing capacity (18),
up-regulation of 24-OHase has to be the consequence of the
two-step activation process via 25-hydroxylation and
subsequent 1·-hydroxylation. The data, therefore, suggest
that the local cutaneous reservoir of vitamin D3 together
with 25-hydroxylation at the rate limiting step of the vitamin
D3 cascade would provide the low, but constant levels of
1·,25(OH)2D3 that could be sufficient to exert hormonal
activity in the target cells. In this report, evidence was
provided that 1·,25(OH)2D3 was detected by the thymus
receptor binding assay after the addition of 10–6 M vitamin
D3 into the PZ-HPV-7 cells (Figure 1), suggesting the
presence of 25-OHase, in addition to 1·-OHase, in
PZ-HPV-7 prostate cells. Its presence was further confirmed
by three functional assays. First, treatment of PZ-HPV-7
cells with vitamin D3 at 10–6 M caused a 40% inhibition of
3H-thymidine incorporation into DNA. Second, the addition
of vitamin D3 to PZ-HPV-7 cells caused a dose-dependent
up-regulation of 24-OHase (CYP24A1) (Figure 2A). Third,
 Flanagan et al: Vitamin D Metabolism and Prostate Cancer
the addition of vitamin D3 to PZ-HPV-7 cells caused a dose-
dependent up-regulation of IGFBP3 mRNA expression
(Figure 2B). CYP24A1 and IGFBP3 are two genes known to
be sensitive to 1·,25(OH)2D3 regulation in prostate cells (12,
13). The vitamin D3-induced IGFBP3 expression was further
supported by the detection of IGFBP3 protein using
Western blot analysis (Figure 2C). Thus, the data indicate
that vitamin D3 was first converted to 25(OH)D3 and then
to 1·,25(OH)2D3 before exerting biological actions in
prostate cells. It is likely that prostate cells work similarly to
keratinocytes, producing sufficient and constant levels of
1·,25(OH)2D to exert hormonal activity in the cells from the
local pool of vitamin D3, together with 25-hydroxylation at
the rate limiting step of the vitamin D3 cascade.
We are interested to know which form of 25-OHase is
predominately expressed in prostate cells. The mRNAs for
CYP27A1, CYP3A4 and CYP2R1 have previously been
found in some human tissues. We therefore compared
their expression in prostate cells to that in the liver by
qPCR. Our qPCR data demonstrated that essentially no
expression of CYP27A1 and CYP3A4 was found in
prostate cells. On the contrary, CYP27A1 and CYP3A4
were expressed at least 10-fold greater in normal liver
tissue than in normal prostate tissue and cells. This is in
agreement with the finding that the function of CYP3A4
is mainly for the metabolism and detoxification of
xenobiotics and carcinogens rather than hydroxylation of
vitamin D (19). Our qPCR data also show that CYP2R1
was expressed to a much greater extent in normal human
prostate tissue and cultured prostate cells than in normal
liver tissue. It has been reported that CYP2R1 is an
evolutionarily conserved enzyme and its mRNA was more
highly expressed in the testis than the liver (20). Most
importantly, a homozygous mutation in exon 2 of the
CYP2R1 gene on chromosome 11p15.2, which causes the
substitution of a proline for an evolutionarily conserved
leucine at amino acid 99 in the CYP2R1 protein, has been
identified to be responsible for the elimination of 25-OHase
activity in a patient with the classic symptoms of vitamin
D deficiency with low circulating levels of 25(OH)D (21).
It was therefore concluded that CYP2R1 is a biologically
relevant vitamin D 25-OHase in humans. Our data
demonstrating that CYP2R1 is the major form of
25-OHase expressed in prostate tissue and cultured
prostate cells strongly support this conclusion.
Despite the encouraging data of 1·,25(OH)2D as a potential
chemotherapeutic agent for prostate cancer (22, 23), it is not
suitable for clinical use because systemic administration of
1·,25(OH)2D can cause hypercalcemia and hypercalciuria (22,
23). Thus, in order for vitamin D metabolites to be useful in
prostate cancer chemoprevention, a method must be devised
to safely expose prostate cells to 1·,25(OH)2D without
systemic side-effects. The presence of the two activating
enzymes (CYP2R1/25OHase and 1·-OHase) and the catabolic
enzyme (24R-OHase) for the vitamin D system in prostate
cells suggest that local production of 1·,25(OH)2D3 could
provide an important cell growth regulatory mechanism. In
conclusion, our results suggest that maintaining adequate levels
of serum vitamin D or 25(OH)D by oral supplementation or
sun exposure can be a safe and effective chemo-preventive
measure to decrease the risk of prostate cancer.
Acknowledgements
This work was supported in part by grants 4118PP1017 and
41211159016 from The Commonwealth of Massachusetts, and US
Army DAMD17-01-0025.
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Received December 29, 2005
Accepted January 9, 2006