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,25-Dihydroxyvitamin D3 Inhibits Prostate Cancer Cell Growth by Androgen-Dependent and Androgen-Independent Mechanisms1
Departments of Medicine (X.-Y.Z., D.F.) and Urology (D.M.P.), Stanford University School of Medicine, Stanford, California 94305; and Department of GU Medical Oncology, University of Texas M. D. Anderson Cancer Center (N.M.N.), Houston, Texas 77030
Address all correspondence and requests for reprints to: David Feldman, M.D., Division of Endocrinology, Room S-005, Stanford University Medical Center, Stanford, California 94305-5103. E-mail: feldman{at}cmgm.stanford.edu
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Abstract |
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,25-dihydroxyvitamin D3
[1,25-(OH)2D3] inhibits the growth of the
LNCaP human prostate cancer cell line by an androgen-dependent
mechanism. In the present study we examined the actions and
interactions of 1,25-(OH)2D3 and the androgen
5-dihydrotestosterone (DHT) on two new human prostate cancer cell
lines (MDA), MDA PCa 2a and MDA PCa 2b. Scatchard analyses revealed
that both cell lines express high affinity vitamin D receptors (VDRs)
with a binding affinity (Kd) for
[3H]1,25-(OH)2D3 of 0.1
nM. However, the MDA cell lines contain low affinity
androgen receptors (ARs) with a Kd of 25 nM for
[3H]DHT binding. This is 50-fold lower than the AR in
LNCaP cells (Kd = 0.5 nM). Their response
to DHT is greatly reduced; 2a cells do not respond to 100
nM DHT, and 2b cells show a modest response at that high
concentration. 1,25-(OH)2D3 causes significant
growth inhibition in both MDA cell lines, greater (for 2b cells) or
lesser (for 2a cells) than that in the LNCaP cell line. Moreover,
1,25-(OH)2D3 significantly up-regulates AR
messenger RNA in all three cell lines, as shown by Northern blot
analysis. The growth inhibitory effect of
1,25-(OH)2D3 on LNCaP cells is blocked by the
pure antiandrogen, Casodex, as we previously reported. However, Casodex
(at 1 µM) did not block the antiproliferative activity of
1,25-(OH)2D3 in MDA cells. In conclusion, the
growth inhibitory action of 1,25-(OH)2D3 in the
MDA cell lines appears to be androgen independent, whereas the
actions of 1,25-(OH)2D3 in LNCaP cells are
androgen dependent. Most importantly, the MDA cell lines, derived from
a bone metastasis of human prostate carcinoma, remain sensitive to
1,25-(OH)2D3, a finding relevant to the
therapeutic application of vitamin D and its low calcemic analogs in
the treatment of advanced prostate cancer. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-DIHYDROXYVITAMIN D3
[1,25-(OH)2D3], the hormonal form of vitamin
D, modulates cellular proliferation and differentiation in a broad
range of cell types (1, 2, 3), in addition to its classical role of
maintaining mineral homeostasis (4, 5). The hormone exerts its actions
via a specific nuclear vitamin D receptor (VDR), a ligand-inducible
transcription factor (6). Recently, the presence of VDR has been
demonstrated in prostate epithelial cells (7, 8, 9). Moreover, we (8, 9, 10, 11, 12, 13, 14)
and others (15, 16, 17, 18, 19, 20, 21, 22, 23, 24) have shown that
1,25-(OH)2D3 and its analogs significantly
inhibit the growth of primary cultures derived from human prostatic
tissues as well as several established human prostate cancer cell
lines. Among the commonly used cell lines, the LNCaP cell line exhibits the greatest sensitivity to growth inhibition by 1,25-(OH)2D3 (8, 15). LNCaP cells express the androgen receptor (AR) and respond to androgen stimulation (25). However, androgen action in LNCaP cells is biphasic, in that low concentrations of androgen stimulate cell growth, whereas high concentrations of androgen lead to inhibition of cell proliferation (26). We showed that 1,25-(OH)2D3 increases AR abundance and enhances cellular responses to androgen in these cells (10). Growth inhibition of LNCaP cells by 1,25-(OH)2D3 is mediated by an androgen-dependent mechanism and is preceded by the induction of AR gene expression (12). Furthermore, growth inhibition by androgens has been reported in LNCaP sublines that express high basal levels of AR and in other AR-containing cells (27, 28, 29).
To study the interactions of 1,25-(OH)2D3 with androgens in other prostate cancer cells beside the LNCaP model, we used two new AR-positive cell lines, MDA PCa 2a and MDA PCa 2b (30). Both cell lines were derived from a single bone metastasis of prostate carcinoma in a patient who failed androgen ablation therapy. These two cell lines have different genetic features (karyotype) and different phenotypes (morphology and growth rate), reflecting the genetic heterogeneity of the tumor (30). These cells retain two important characteristics of cells of prostate origin: the expression of both AR and prostate-specific antigen (PSA). We recently identified two mutations in the ligand-binding domain (L701H and T877A) of the AR in the MDA PCa 2a cell line (13). Both mutations were also found in the AR gene of MDA PCa 2b cells (30A ). The single T877A mutation is present in the AR gene of the LNCaP cell line (31).
In the current study we first characterized the VDR and AR in MDA PCa
2a and MDA PCa 2b cell lines. We then examined the actions and
interactions of vitamin D and the androgen 5-dihydrotestosterone
(DHT) in these cells and compared the results to those in LNCaP cells.
We found that 1,25-(OH)2D3 induced AR gene
expression in all three cell lines. DHT had small effects on cell
growth and PSA secretion when MDA cells were cultured in FBS-containing
medium. MDA PCa 2a and MDA PCa 2b cells, in contrast to LNCaP,
maintained a response to 1,25-(OH)2D3 in the
presence of the pure antiandrogen, Casodex. Thus,
1,25-(OH)2D3 inhibits the growth of these cells
by an androgen-independent mechanism, whereas growth inhibition of
LNCaP cells by 1,25-(OH)2D3 is mediated by an
androgen-dependent mechanism.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dihydro-[1,2-3H]testosterone (SA,
40–70 Ci/mmol) were obtained from Amersham Pharmacia Biotech (Arlington Heights, IL). Nonradioactive DHT was obtained
from Steraloids, Inc. (Wilton, NH). The human AR complementary DNA
(cDNA) was a gift from Dr. M. McPhaul (University of Texas Southwestern
Medical Center, Dallas, TX). Tissue culture medium RPMI 1640 and
BRFF-HPC-1 were obtained from Mediatech (Herndon, VA) and Biological
Research Faculty Facility, Inc. (Ijamsville, MD), respectively. FBS was
obtained from Life Technologies, Inc. (Gaithersburg, MD).
Charcoal-stripped FBS was purchased from HyClone Laboratories, Inc. (Logan, UT). Aprotinin, pepstatin, and
soybean trypsin inhibitor were purchased from Roche Molecular Biochemicals (Indianapolis, IN). All other
reagents, except where indicated, were purchased from
Sigma (St. Louis, MO).
Cell culture and hormone treatment
The LNCaP human prostate carcinoma cell line was obtained from
American Type Culture Collection (Rockville, MD). LNCaP
cells were routinely cultured in RPMI 1640 medium supplemented with 5%
FBS and antibiotics at 37 C in a humidified atmosphere of 5%
CO2. The human prostate cancer cell lines MDA PCa
2a and 2b (30) were maintained in BRFF-HPC-1 medium, supplemented with
20% FBS and gentamicin.
Hormone stocks [1,25-(OH)2D3, DHT, and Casodex] were prepared in 100% ethanol at a concentration 1000-fold higher than the working concentrations. Fresh culture media were premixed with hormone stock and then added to triplicate wells. Medium and hormone were replenished every 3 days. Controls received ethanol vehicle at a concentration equal to that in hormone-treated cells.
Steroid receptor ligand binding and Scatchard analysis
Cell monolayers were harvested, and high salt nuclear extracts
were made as previously described (10, 11, 12, 13). The protein concentration
of the extract was determined by the method of Bradford (32). In a
typical binding assay, 200 µl soluble extract (0.5–1 mg protein/ml)
were incubated with the indicated concentrations of
[3H]DHT or
[3H]1,25-(OH)2D3
for 16–20 h at 0 C. Bound and free hormones were separated by
hydroxylapatite. Specific binding was calculated by subtracting
nonspecific binding obtained in the presence of a 250-fold excess of
radioinert steroid from total binding measured in the absence of
radioinert steroid. Data were expressed as femtomoles of
[3H]DHT or
[3H]1,25-(OH)2D3
bound per mg protein.
Assay of cell proliferation
Cell proliferation was assessed by measurement of attained cell
mass using an assay of DNA content. As previously described (10), cells
were seeded in six-well tissue culture plates (Becton Dickinson and Co., Lincoln Park, NJ) at a density of 50,000–200,000
cells/well in 3 ml medium containing 5% FBS. After incubation for
24 h, the medium was replaced with fresh medium containing 5%
FBS. Cells were treated with vehicle (ethanol; final concentration,
0.1%), 1,25-(OH)2D3, DHT,
or Casodex. On the sixth day, cell monolayers were processed for DNA
assay using the method of Burton (33). The DNA content of each
treatment was derived from the mean value of triplicate wells in an
experiment. Each experiment was repeated three times.
Assay of PSA secretion
The conditioned medium collected in cell proliferation assays was
subjected to a low speed centrifugation to remove cell debris. PSA
values in the supernatant were determined by the TOSOH assay, an
automated immunoenzymometric assay system (TOSOH Medics, Inc., Foster
City, CA), as previously described (10). Results were expressed as
nanograms of PSA per µg DNA.
Northern blot analysis
The method has been described previously (12). In brief,
semiconfluent monolayer cells were treated with
1,25-(OH)2D3 in RPMI medium
containing 5% charcoal-stripped FBS or 5% FBS for 24 h before
isolation of total RNA. Ten micrograms of total RNA were denatured,
fractionated by electrophoresis, and transferred to Hybond-N nylon
membrane (Amersham Pharmacia Biotech). The bound RNA was
immobilized and hybridized with a random primed
32P-labeled 1.1-kb
HindIII-EcoRI fragment of the human AR cDNA at 60
C (34). To control for RNA sample loading and transfer, Northern blots
were also hybridized with a 32P-labeled 0.9-kb
EcoRI fragment of the human cDNA for the ribosomal protein
gene L7 (7). The silver grain pixel intensity of each AR and L7 band
was scanned by a densitometer, and the data were integrated by scanner
software and indexed to the corresponding levels of L7 messenger RNA
(mRNA).
Statistical analysis
ANOVA was used to assess the statistical significance of
difference. P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The inset illustrates
dose-response saturation plots obtained after correction for
nonspecific binding. Linear regression analysis of Scatchard plots
revealed a single class of specific and high affinity receptors with an
apparent dissociation constant (Kd) of 0.1
± 0.02 (n = 3) and 0.18 ± 0.03 (n = 3)
nM for MDA PCa 2a and 2b cells, respectively.
These binding affinities are similar to values in LNCaP cells (7)
(Kd = 0.14 nM) and other
classical vitamin D target tissue (35). The VDR content was higher in
MDA PCa 2a (51 ± 3 fmol/mg protein; n = 3) than in 2b cells
(33 ± 2 fmol/mg protein; n = 3) or LNCaP cells (31 fmol/mg
protein) (7), and this difference is statistically significant
(P < 0.05). Thus, these two new MDA cell lines express
VDR with affinity and abundance similar to those of other established
vitamin D target cells.
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) revealed low affinity binding
receptors with apparent Kd values of 25 ± 4
(n = 3) and 23 ± 3 (n = 3) nM for MDA PCa
2a and 2b cells, respectively. These binding affinities were
significantly lower (higher Kd) than the AR in
LNCaP cells (Kd = 0.5 nM; Fig. 2B).
MDA cells expressed a higher abundance of AR [685 ± 149 fmol/mg
protein for 2a (n = 6) and 840 ± 32 fmol/mg protein for 2b
(n = 3)] than LNCaP cells (457 fmol/mg protein). Therefore, these
two new cell lines express AR with low affinities (23–25
nM), and their AR contents were in the range of 600–900
fmol/mg protein.
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. Both MDA PCa 2a and 2b cells were
similar to LNCaP cells in their response to
1,25-(OH)2D3 treatment
(Fig. 3, B and C).
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, B and C). A higher
concentration of DHT (100 nM) did not affect the growth of
MDA PCa 2a cells, but caused growth inhibition (60–70%) in MDA PCa 2b
cells. Combination treatment with
1,25-(OH)2D3 and DHT did
not result in a greater inhibition of the growth of these cells than
treatment with 1,25-(OH)2D3
alone. These data suggest that MDA PCa 2a and 2b cells differ from
LNCaP cells in exhibiting reduced sensitivity to androgens. Moreover,
the enhanced response to the
1,25-(OH)2D3/androgen
combination as seen in LNCaP cells was not detected in these cells.
Effect of
1,25-(OH)2D3 and
androgen on cell growth in the presence of Casodex
We previously demonstrated that
1,25-(OH)2D3 elicits its
antiproliferative effect on LNCaP cells by an androgendependent
mechanism (10, 12). Here we investigated the mechanism of the
1,25-(OH)2D3/androgen
interaction in MDA PCa 2a and 2b cell lines using the antiandrogen,
Casodex. Casodex alone has an inhibitory effect on the growth of LNCaP
and MDA PCa 2b cells, but not on MDA PCa 2a cells. Treatment of these
cells with 1 µM Casodex in FBS-containing medium for 6
days resulted in 31%, 38%, and 0.8% growth inhibition for LNCaP, 2b,
and 2a cells, respectively.
As shown in Fig. 4A, compared with
Fig. 3A, Casodex completely blocked the inhibition of LNCaP cell growth
by 1,25-(OH)2D3. Casodex at
a concentration of 1 µM reversed the effect of 1
nM DHT on LNCaP cell growth, but not the effect of 10
nM DHT. Furthermore, in the presence of 1 µM
Casodex, no growth inhibition was seen with the combined treatment of
both hormones at 1 nM. A high concentration of DHT (10
nM) overcame the blockade by 1 µM Casodex.
Therefore, Casodex antagonized the inhibitory effect of
1,25-(OH)2D3 or a low dose
of DHT on LNCaP cell growth.
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) and MDA PCa 2b cells (Fig. 4C) was unaffected by 1
µM Casodex. These results suggest that the
antiproliferative actions of
1,25-(OH)2D3 in these cells
are androgen independent. Interestingly, MDA PCa 2a cells remained
unresponsive to DHT in the presence of Casodex (Fig. 4B), whereas MDA
PCa 2b cells showed a response to DHT at 10 nM (Fig. 4C).
DHT at 10 nM and Casodex at 1 µM produced a
50% increase in cell growth of MDA PCa 2b cells (Fig. 4C), similar to
the effect of DHT at 1 nM and Casodex at 1 µM
on LNCaP (Fig. 4A). This finding is consistent with the data in Fig. 3
showing that MDA PCa 2b cells responded to 100 nM DHT in
the absence of Casodex (60–70% growth inhibition). Hence, MDA PCa 2b
cells are more responsive to DHT than MDA PCa 2a cells, but both are
less responsive than LNCaP cells.
Effect of
1,25-(OH)2D3 and
androgens on PSA secretion
All three cell lines express PSA (10, 30). As shown in Fig. 5, treatment with
1,25-(OH)2D3 resulted in
the dose-dependent stimulation of PSA secretion by LNCaP cells as well
as by MDA PCa 2a and 2b cells. Because cell number changed with
hormonal treatment (Fig. 3), PSA levels are expressed as nanograms of
PSA per µg DNA.
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, both
1,25-(OH)2D3 and DHT alone
enhanced the secretion of PSA by LNCaP cells in a dose-dependent
manner.
1,25-(OH)2D3-treated cells
showed a 5-fold increase in PSA secretion (from 5 to 27 ng PSA/µg
DNA), similar to the increase seen in the DHT-treated cells. In
combination, 1,25-(OH)2D3
and DHT synergistically augmented PSA secretion up to 22-fold. These
data indicate that
1,25-(OH)2D3 and DHT
interact cooperatively in regulating PSA secretion by LNCaP cells, as
shown previously (10).
MDA PCa 2a and 2b cells express high basal levels of PSA, as
previously reported (30). The baseline PSA levels in these cells range
between 50–60 ng/µg DNA, which are 10-fold higher than values in
LNCaP cells. DHT had a minimal effect on PSA secretion by the MDA cell
lines (Fig. 5, B and C).
1,25-(OH)2D3-treated cells
showed 2- and 4-fold increases in PSA secretion in MDA PCa 2a and 2b
cells, respectively. In combination, DHT did not significantly enhance
the effect of 1,25-(OH)2D3
on PSA secretion. Therefore,
1,25-(OH)2D3 and DHT did
not interact cooperatively in regulating PSA secretion by these cells,
in contrast to LNCaP cells.
Effect of
1,25-(OH)2D3 and
androgen on PSA secretion in the presence of Casodex
1,25-(OH)2D3 at 1 or
10 nM in the presence of 1 µM Casodex no
longer induced PSA in LNCaP cells, as shown in Fig. 5A. Casodex
completely blocked the action of DHT at a low concentration (1
nM). The induction of PSA by DHT at 10 nM was
also partially inhibited by Casodex. Moreover, in the presence of
Casodex, the combined treatment with
1,25-(OH)2D3 and DHT at 1
nM did not increase the PSA level. Administration of both
hormones (10 nM each) to LNCaP cells appeared to overcome
the blockade by Casodex (from 1.7–67 ng PSA/µg DNA). These data
suggest that the antiandrogen blocked the
1,25-(OH)2D3 action to
stimulate PSA in LNCaP cells. In contrast, MDA PCa 2a and 2b cells
responded to 1,25-(OH)2D3
in the presence of Casodex (Fig. 5, B and C). The high baseline PSA
levels in these cell lines were unaffected by Casodex. Moreover, the
antiandrogen did not modify the effect of
1,25-(OH)2D3, DHT, or both
hormones on these cells, in contrast to the result seen with LNCaP
cells (Fig. 5A).
1,25-(OH)2D3
up-regulation of AR in three prostate cancer cell lines
We previously reported that
1,25-(OH)2D3 increased AR
gene expression in LNCaP cells (12). Here we examined
1,25-(OH)2D3 regulation of
AR in MDA PCa 2a and 2b cells. Cells were treated with
1,25-(OH)2D3 for 24 h,
and the effect on steady state AR mRNA levels was assessed by Northern
blot analysis. As shown in Fig. 6, AR
mRNA transcripts were increased by
1,25-(OH)2D3 in all three
cell lines in both charcoal-stripped FBS-containing medium (Fig. 6A)
and FBS-containing medium (Fig. 6C). The levels of AR mRNA were
quantitatively determined by densitometric scanning of the
autoradiographs, with correction for the L7 mRNA signal (Fig. 6, B and
D). At 10 nM
1,25-(OH)2D3, AR mRNA was
up-regulated 8-fold in LNCaP, 5-fold in MDA PCa 2b, and 20-fold in MDA
PCa 2a cells when cells were cultured in charcoal-stripped
FBS-containing medium (Fig. 6A). Similarly, in FBS-containing medium,
25 nM
1,25-(OH)2D3 increased AR
mRNA 2-fold in LNCaP, 3-fold in MDA PCa 2b, and 2-fold in MDA PCa 2a
cells (Fig. 6C). The positive effect of
1,25-(OH)2D3 on AR mRNA was
greater (5-fold over the control untreated value) when MDA cells were
treated with 1,25-(OH)2D3
for 48 h than for 24 h in FBS-containing medium, consistent
with our previous report on LNCaP cells (12). Therefore,
1,25-(OH)2D3-mediated
up-regulation of AR is a general phenomenon among the AR-positive
prostate cancer cells that we tested.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The limited number of established human prostate cancer cell lines available for investigation has hindered prostate cancer research. The three commonly used cell lines representing progressively more transformed phenotypes are LNCaP (least transformed), PC-3 (intermediate transformation), and DU 145 (most transformed). They were derived from different metastases of prostate carcinoma: LNCaP from a lymph node metastasis (25), PC-3 from a bone metastasis (36), and DU 145 from a brain metastasis (37). The newly established human prostate cancer cell lines MDA PCa 2a and 2b were derived from a bone metastasis (30), similar to PC-3 cells. However, unlike PC-3 cells, which express extremely low or undetectable level of AR, these cells, like LNCaP cells, express abundant AR as well as inducible PSA, features typical of prostate cancer cells (13, 30). The MDA PCa 2a and 2b cells represent new cell line models for advanced prostate cancer. We were therefore interested in exploring the hormonal responses of these cells.
Regulation of cell growth and gene expression by steroid hormones depends upon the presence of functional receptors. We first characterized the VDR and AR in these cells and then compared them with those in the LNCaP cell line. The affinities for [3H]1,25-(OH)2D3 and abundance of VDRs are similar in all three cell lines: LNCaP, MDA PCa 2a, and MDA PCa 2b. Correspondingly, their responses to 1,25-(OH)2D3 are qualitatively similar, but the antiproliferative effect is greater in MDA PCa 2b than in 2a or LNCaP cells or PC-3 cells (data not shown), although PC-3 cells have twice as many VDRs as MDA PCa 2b or LNCaP cells (8). This finding supports the idea that 1,25-(OH)2D3 responses require VDR, but the VDR content does not necessarily correlate with the magnitude of the hormonal response (8, 24). The growth inhibitory effect of 1,25-(OH)2D3 on all of the cell lines that we tested can be ranked in the following sequence: 2b>LNCaP>2a>PC-3>>DU 145. Other factors besides the VDR content contribute to the amplitude of the 1,25-(OH)2D3 response. For example, inhibition of 24-hydroxylase, the 1,25-(OH)2D3-inducible enzyme that initiates the 1,25-(OH)2D3 inactivation pathway, increases the sensitivity of DU145 cells to 1,25-(OH)2D3-induced growth inhibition (14).
We observed that the ARs in MDA PCa 2a and 2b cells exhibited low affinity for [3H]DHT. This is probably responsible for the decreased androgen sensitivity observed in these cells. We recently demonstrated that the AR genes in both 2a and 2b cell lines contain double mutations (L701H and T877A) (13). One of the mutations, T877A, is present in the AR gene of LNCaP cells (31). The second mutation L701, or possibly the interaction of the two mutations, may be responsible for differences in androgen responsiveness between these cells and LNCaP cells. The androgen responses of the cell lines that we tested can be ranked in the following sequence: LNCaP>>2b>2a. As the AR affinity for DHT binding is similar in 2a and 2b cells, other factors must contribute to the differences in androgen sensitivity.
When comparing LNCaP with MDA PCa 2b cells, two lines of evidence demonstrated that MDA PCa 2b cells have decreased sensitivity to DHT. First, in FBS-containing medium, both LNCaP and MDA PCa 2b cells were growth inhibited by DHT, at 1 nM for LNCaP and at 100 nM for 2b cells. Second, in the presence of Casodex (1 µM) and FBS, the response of LNCaP cells to 1 nM DHT was identical to the response of MDA PCa 2b cells to 10 nM DHT.
As the AR in MDA cells has a 50-fold lower affinity for DHT binding than the AR in LNCaP cells, it is not surprising that the low affinity AR of MDA cells has a decreased affinity for Casodex. We tried competitive binding analysis to evaluate the ligand specificity of the mutant AR in MDA cells using [3H]DHT as a ligand and various agents, including Casodex as cold competitors. Unfortunately, we were unable to obtain useful data due to the very low affinity of the mutant receptor for [3H]DHT and the high levels of nonspecific binding. As Casodex usually requires a 1000-fold excess concentration to inhibit DHT binding, this approach was not successful. We are in the process of recreating the mutant AR in vitro that will be used to test the antagonist activity of Casodex in more sensitive assays than the competitive binding analysis.
The high basal level of PSA seen in MDA PCa 2a and 2b cells is worth noting. It is not clear whether the mutated AR or other mechanisms unrelated to the AR are responsible for the constitutive production of PSA in these cells. Furthermore, this high basal PSA was unaffected by Casodex, whereas in LNCaP cells, the low basal level of PSA was decreased by Casodex.
DHT has a minimal effect on PSA secretion by MDA PCa 2a and 2b cells in FBS-containing medium. In contrast, 1,25-(OH)2D3 increased PSA in these cells. We have previously shown that 1,25-(OH)2D3 increases PSA by AR signaling in LNCaP cells (10). It is possible that 1,25-(OH)2D3 regulates PSA in MDA PCa 2a and 2b cells by other mechanisms. For example, 1,25-(OH)2D3 may induce a more differentiated phenotype that secretes more PSA per cell. Evidence that 1,25-(OH)2D3 stimulates prostate cell differentiation includes increased expression of both PSA and E-cadherin (22), a cell adhesion protein that may act as a putative tumor suppressor, in LNCaP cells. E-Cadherin is also increased by 1,25-(OH)2D3 in PC-3 cells (22). In accord with mechanisms for 1,25-(OH)2D3-induced PSA, transforming growth factor-ß1, a known differentiating factor, up-regulates PSA production in MDA PCa 2a cells (38).
AR up-regulation by 1,25-(OH)2D3 appears to be common in all three AR-positive cell lines that we tested. Hence, 1,25-(OH)2D3 as a differentiating agent may alter the androgen sensitivity of prostate cancer cells. Interestingly, 1,25-(OH)2D3 does not regulate AR gene expression in the human breast cancer cell line T47D (data not shown), indicating cell type specificity.
Collectively, our data indicate that
1,25-(OH)2D3 causes
prostate cell growth inhibition by two different pathways: an
androgen-dependent and an androgen-independent mechanism (Fig. 7). The androgen-dependent mechanism of
1,25-(OH)2D3 action has
been demonstrated in LNCaP cells (10). Androgens are powerful
regulators of prostate cell growth and gene expression. When both AR
and VDR signaling pathways coexist, as in LNCaP cells, the
1,25-(OH)2D3
antiproliferative actions are AR dependent. On the other hand, examples
of the androgen-independent mechanism of vitamin D action include MDA
PCa 2a and MDA PCa 2b cells (low affinity ARs due to mutations), PC-3
cells (low or undetectable levels of AR), and primary cultures of
prostate epithelial cells (lack of AR expression), as well as
ARnegative DU 145 cells cotreated with liarozole (14). Two
findings, 1) the minimal response of MDA cells to DHT and 2) the lack
of Casodex inhibition of
1,25-(OH)2D3
antiproliferative action, both suggest that
1,25-(OH)2D3 acts by an
androgen-independent mechanism in these cells. Hence, when the AR
signaling pathway is absent or negligible,
1,25-(OH)2D3 acts via an
androgen-independent pathway.
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Acknowledgments |
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Footnotes |
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Received October 26, 1999.
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References |
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 J. Kaeding, J. Belanger, P. Caron, M. Verreault, A. Belanger, and O. Barbier Calcitrol (1{alpha},25-dihydroxyvitamin D3) inhibits androgen glucuronidation in prostate cancer cells Mol. Cancer Ther., February 1, 2008; 7(2): 380 - 390. [Abstract] [Full Text] [PDF]
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 S. Wedren, C. Magnusson, K. Humphreys, H. Melhus, A. Kindmark, F. Stiger, M. Branting, I. Persson, J. Baron, and E. Weiderpass Associations between Androgen and Vitamin D Receptor Microsatellites and Postmenopausal Breast Cancer Cancer Epidemiol. Biomarkers Prev., September 1, 2007; 16(9): 1775 - 1783. [Abstract] [Full Text] [PDF]
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 H.-J. Ting, B.-Y. Bao, J. E. Reeder, E. M. Messing, and Y.-F. Lee Increased Expression of Corepressors in Aggressive Androgen-Independent Prostate Cancer Cells Results in Loss of 1{alpha},25-Dihydroxyvitamin D3 Responsiveness Mol. Cancer Res., September 1, 2007; 5(9): 967 - 980. [Abstract] [Full Text] [PDF]
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 L. Peng, P. J. Malloy, J. Wang, and D. Feldman Growth Inhibitory Concentrations of Androgens Up-Regulate Insulin-Like Growth Factor Binding Protein-3 Expression via an Androgen Response Element in LNCaP Human Prostate Cancer Cells Endocrinology, October 1, 2006; 147(10): 4599 - 4607. [Abstract] [Full Text] [PDF]
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 S. Nagpal, S. Na, and R. Rathnachalam Noncalcemic Actions of Vitamin D Receptor Ligands Endocr. Rev., August 1, 2005; 26(5): 662 - 687. [Abstract] [Full Text] [PDF]
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 L.-C. Li, P. R. Carroll, and R. Dahiya Epigenetic Changes in Prostate Cancer: Implication for Diagnosis and Treatment J Natl Cancer Inst, January 19, 2005; 97(2): 103 - 115. [Abstract] [Full Text] [PDF]
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 C. Jung, R.-S. Kim, H.-J. Zhang, S.-J. Lee, and M.-H. Jeng HOXB13 Induces Growth Suppression of Prostate Cancer Cells as a Repressor of Hormone-Activated Androgen Receptor Signaling Cancer Res., December 15, 2004; 64(24): 9185 - 9192. [Abstract] [Full Text] [PDF]
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 M. F. McCarty Targeting Multiple Signaling Pathways as a Strategy for Managing Prostate Cancer: Multifocal Signal Modulation Therapy Integr Cancer Ther, December 1, 2004; 3(4): 349 - 380. [Abstract] [PDF]
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 L. V. Stewart and N. L. Weigel Vitamin D and Prostate Cancer Experimental Biology and Medicine, April 1, 2004; 229(4): 277 - 284. [Abstract] [Full Text] [PDF]
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D. M. Peehl, A. V. Krishnan, and D. Feldman Pathways Mediating the Growth-Inhibitory Actions of Vitamin D in Prostate Cancer J. Nutr., July 1, 2003; 133(7): 2461S - 2469. [Abstract] [Full Text] [PDF]
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 H. Chen, M. Hewison, B. Hu, and J. S. Adams Heterogeneous nuclear ribonucleoprotein (hnRNP) binding to hormone response elements: A cause of vitamin D resistance PNAS, May 13, 2003; 100(10): 6109 - 6114. [Abstract] [Full Text] [PDF]
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 M. E. Taplin and S.-M. Ho The Endocrinology of Prostate Cancer J. Clin. Endocrinol. Metab., August 1, 2001; 86(8): 3467 - 3477. [Full Text] [PDF]
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