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,25-Dihydroxyvitamin D3 Actions in LNCaP Human Prostate Cancer Cells Are Androgen-Dependent1
Departments of Medicine and Urology (D.M.P.), Stanford University School of Medicine, Stanford, California 94305
Address all correspondence and requests for reprints to: David Feldman, M.D., Division of Endocrinology, Gerontology and Metabolism, Stanford University Medical Center, Room S005, Stanford, California 94305-5103. E-mail address: feldman{at}cmgm.stanford.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-dihydroxyvitamin
D3 [1,25-(OH)2D3] significantly
inhibits cell proliferation and increases secretion of
prostate-specific antigen (PSA) in LNCaP cells, an androgen-responsive
human prostate cancer cell line. The present study was designed to
investigate the possible interactions between
1,25-(OH)2D3 and androgens in the regulation of
LNCaP cellular function. LNCaP cell growth was dose-dependently
inhibited by 1,25-(OH)2D3 (60% inhibition at
10 nM) when cells were cultured in medium supplemented with
FBS (FBS medium). 1,25-(OH)2D3-treated cells
showed a 5-fold increase in PSA secretion, similar to the increase seen
in dihydrotestosterone (DHT)-treated cells. In combination,
1,25-(OH)2D3 and DHT synergistically enhanced
PSA secretion 22-fold. This synergistic effect was even greater when
cells were cultured in medium supplemented with charcoal-stripped serum
(CSS medium), where endogenous steroids are substantially depleted.
Under these conditions, 1,25-(OH)2D3 and DHT
together stimulated PSA secretion up to 50-fold over the untreated
control. Radioligand binding assays and Western blot analyses showed
that the androgen receptor (AR) content was increased significantly by
1,25-(OH)2D3 at 48 h. Furthermore, the
steady-state mRNA level of AR was up-regulated approximately 2-fold by
1,25-(OH)2D3 at 24 h. When cells were
grown in CSS medium, 1,25-(OH)2D3 alone no
longer inhibited cell growth or induced PSA secretion. Titration
experiments revealed that the addition of DHT at 1 nM to
the medium restored the antiproliferative activity of
1,25-(OH)2D3. Conversely, an antiandrogen,
Casodex, completely blocked 1,25-(OH)2D3
antiproliferative and PSA stimulation activities when cells were
cultured in FBS medium. In conclusion, these results demonstrate that
the antiproliferative and PSA induction activities of
1,25-(OH)2D3 in LNCaP cells are dependent upon
androgen action and that AR up-regulation by
1,25-(OH)2D3 likely contributes to the
synergistic actions of 1,25-(OH)2D3 and DHT in
these cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hormonal actions of both 1,25-(OH)2D3 and DHT are mediated by their cognate cellular receptor proteins, the vitamin D receptor (VDR) and the androgen receptor (AR), respectively (13, 14, 15). These receptors are ligand-dependent transcriptional regulatory proteins which belong to the nuclear receptor superfamily (16, 17). Once bound to a specific ligand, the receptor activates gene transcription by binding to the hormone response element in the promoter region of a target gene. A notable androgen-responsive gene, the gene encoding prostate-specific antigen (PSA), contains a functional androgen response element in its promoter region (18, 19).
Our studies have been carried out in LNCaP cells, the first characterized human prostate cancer cell line which retains many of the characteristics of prostatic epithelial cells (20). Like normal prostatic cells, LNCaP cells express AR and respond to androgen stimulation. However, androgen action in LNCaP cells is biphasic with low concentrations of androgen stimulating cell growth whereas high concentrations of androgen lead to inhibition of cell proliferation (21). Futhermore, the AR in LNCaP cells contains a mutation in the ligand-binding domain which alters ligand specificity (22). The mutated AR can be activated not only by androgen but also progestins and estrogens as well as some antiandrogens (e.g. cyproterone acetate, hydroxyflutamide, nilutamide). Nevertheless, the antiandrogen Casodex shows antiandrogenic properties in growth studies of LNCaP cells and does not activate the mutated AR (22). In these cells, Casodex acts as an androgen antagonist by inhibiting both dissociation of the heteromeric complex of the AR with heat-shock proteins and the subsequent high-affinity binding of the receptor to the nucleus (23).
Although both the VDR and the AR are present in LNCaP cells, relatively little data are available on the interrelationship of 1,25-(OH)2D3 and DHT effects in these cells. We (5) and Miller et al. (8) reported that LNCaP cells responded differently to 1,25-(OH)2D3 treatment when cultured either in medium containing FBS (FBS medium) or in medium containing charcoal-stripped serum (CSS medium) where endogenous steroids are substantially depleted. LNCaP cells grow slowly when they are cultured in CSS medium and 1,25-(OH)2D3 does not inhibit cell proliferation. Furthermore, we found that these cells were more responsive to DHT in the presence of 1,25-(OH)2D3 than in the absence of 1,25-(OH)2D3. On the basis of these observations, we proposed that cross-talk between 1,25-(OH)2D3 and DHT signaling may exist, and that possible interactions between these two important hormones might modulate the cellular response of LNCaP cells.
In this report, we present evidence for cooperative interactions of 1,25-(OH)2D3 and DHT on cellular function of LNCaP cells under several experimental conditions. Two parameters of cellular function, cell growth and PSA secretion, were evaluated. Our results demonstrate that androgen action was required for both 1,25-(OH)2D3-induced effects. We postulate that 1,25-(OH)2D3-induced up-regulation of AR is the basis for the antiproliferative activity of 1,25-(OH)2D3 in LNCaP cells as well as the synergistic stimulation of PSA secretion following combined 1,25-(OH)2D3 and DHT treatment.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dihydro-[1,
2-3H]-testosterone (specific activity, 40–70 Ci/mmol)
were obtained from Amersham Chemical Co. (Arlington Heights, IL).
Nonradioactive 1,25-(OH)2D3 was the generous
gift of Dr. M. Uskokovic (Hoffmann La-Roche Co., Nutley, NJ).
Nonradioactive DHT was obtained from Steraloids Inc. (Wilton, NH).
Bicalutamide (Casodex or ICI 17,334) was a gift from Zeneca
Pharmaceuticals (Macclesfield, Cheshire, UK). Aprotinin, pepstatin, and
soybean trypsin inhibitor were purchased from Boehringer Mannheim
Biochemicals (Indianapolis, IN). Tissue culture media were purchased
from Mediatech (Herndon, VA). All other reagents, except where
indicated, were purchased from Sigma Chemical Co. (St. Louis, MO). The
anti-AR monoclonal antibody F39.4 and the rat AR cDNA were generous
gifts from Dr. T. H. Van der Kwast (Erasmus University, Rotterdam,
Netherlands) and Dr. S. Liao (Univ. of Chicago, Chicago, IL),
respectively. FBS was obtained from GIBCO BRL. Charcoal-stripped FBS
was purchased from Sigma Chemical Co. (St. Louis, MO).
Cell culture and hormone treatment
The LNCaP human prostate carcinoma cell line was obtained from
the American Type Culture Collection (Rockville, MD). Cells were
routinely cultured in RPMI-1640 medium supplemented with 5% FBS and
antibiotics, at 37 C in a humidified atmosphere of 5% CO2.
For experiments, LNCaP cells were trypsinized and seeded at an
appropriate density and hormonal treatments were initiated one day
after plating.
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. Media and hormone were replenished every 2 days. Controls received ethanol vehicle at a concentration equal to that in hormone-treated cells.
Assay of cell proliferation
Cell proliferation was assessed by measurement of attained cell
mass using an assay of DNA content. As previously described (5), LNCaP
cells were seeded in six-well tissue culture plates (Becton Dickinson &
Co., Lincoln Park, NJ) at a density of 50,000 cells per well in 3 ml of
RPMI-1640 containing 5% FBS. After incubation for 24 h, the
medium was replaced with fresh medium containing 5% FBS (FBS medium)
or 5% CSS (CSS medium). Cells were treated with vehicle (ethanol,
final concentration 0.1%), 1,25-(OH)2D3, or
DHT at concentrations of 0, 1, and 10 nM. On the 6th day,
conditioned media were collected for PSA analysis and cell monolayers
were processed for DNA assay using the method of Burton (24). 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 LNCaP cell line is known to express PSA which was assayed as
a marker of cellular differentiation. The conditioned media collected
in cell proliferation assays were subjected to 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 (5).
Steroid receptor ligand-binding assay
LNCaP cells were seeded at a density of 150,000 cells per 100-mm
dish in 10-ml FBS medium. After 6 days of incubation in CSS medium with
a hormone at 0, 1, or 10 nM, cells were harvested and high
salt nuclear extracts were made as previously described (25). Protein
concentrations of the extracts were determined by the method of
Bradford (26). In a typical binding experiment, 200 µl of soluble
extract (1–2 mg protein/ml) were incubated with 10 nM of
[3H]1,25-(OH)2D3 or
[3H]DHT for 16 -20 h at 4 C. Bound and free hormone were
separated by the hydroxylapatite method (25) and specific binding
was calculated as reported (25). Data were expressed as fmoles of
[3H]1,25-(OH)2D3 or
[3H]DHT bound per mg protein.
Western blot analysis
Cells were treated with ethanol or
1,25-(OH)2D3 (10 nM) in CSS medium
for 6 days. They were harvested at 24 h, 48 h, 72 h, and
144 h and sonicated extracts were prepared as described for
ligand-binding studies. Aliquots of 100 µg of protein were heated in
SDS sample buffer at 95 C for 5 min before electrophoresis in an 8%
SDS-polyacrylamide gel. After electrophoresis, the gels were
transferred and processed as previously described (25). After transfer,
the blots were incubated with anti-AR monoclonal antibody F39.4 (1: 100
dilution) for 1 h at room temperature with gentle shaking. The
blots were washed and then incubated with the horseradish
peroxidase-conjugated rabbit antimouse IgG (1:1000 dilution) for 1
h at room temperature. Blots were rewashed and developed with the ECL
system according to the manufacturer’s instructions (Amersham Chemical
Co.).
Northern blot analysis
The method has been previously described (5, 6). Semiconfluent
LNCaP cells were treated with graded concentrations of
1,25-(OH)2D3, or 10 mM dibutyryl
cAMP, in CSS medium 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)
as previously described (5, 6). The bound RNA was immobilized and
hybridized with a random primed [32P]-labeled 1.8 kb
NruI-PstI fragment of the rat AR cDNA at 55 C
(14). 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 (5, 6). The silver grain pixel intensity of each AR and L7 band
was scanned by a densitometer and the data integrated by scanner
software and indexed to the corresponding levels of L7 mRNA.
Statistical analysis
ANOVA was used to assess 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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, either 1,25-(OH)2D3 or DHT
inhibited cell growth up to 40% at a concentration of 1
nM. Cell proliferation was inhibited more than 60% by each
individual hormone at 10 nM. A combination of both hormones
at 1 nM resulted in enhanced inhibition (70%) of cell
growth. The cellular proliferation of LNCaP cells was further decreased
in the presence of both hormones, each at 10 nM. These
results suggest that these two hormones interact cooperatively in the
inhibition of cell proliferation.
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), the quantity of PSA was expressed as ng of PSA
per µg of DNA. As shown in Fig. 1B, either
1,25-(OH)2D3 or DHT alone enhanced the
secretion of PSA in a dose-dependent manner.
1,25-(OH)2D3-treated cells showed a 5-fold
increase in PSA secretion [from 5 to 27 ng of PSA per µg of DNA with
10 nM of 1,25-(OH)2D3], similar to
the increase seen in DHT-treated cells. In combination,
1,25-(OH)2D3 and DHT synergistically augmented
PSA secretion up to 22-fold (from 5 to 112 ng of PSA per µg of DNA
with 10 nM of each hormone). These data indicate that
1,25-(OH)2D3 and DHT also interact
cooperatively in regulating PSA secretion by LNCaP cells.
Lack of antiproliferative activity of
1,25-(OH)2D3 in CSS medium
In previous studies, we (5) and Miller et al. (8) had
observed that LNCaP cells responded differently to hormonal treatment
when cultured in FBS medium vs. CSS medium where endogenous
steroids are substantially depleted. In the next set of experiments, we
examined the interaction of 1,25-(OH)2D3 and
DHT when cells were cultured in CSS medium. Consistent with previous
observation (5, 8), LNCaP cells grew much slower in CSS medium than in
FBS medium. The doubling time of LNCaP cells was 4-fold slower than
that in FBS medium (6 days vs. 1.5 days).
When cells were cultured in CSS medium, no growth inhibition was
observed in the presence of either 1,25-(OH)2D3
or DHT (Fig. 2A). Either hormone individually stimulated
cell proliferation under these conditions (from 200% to 284% for
1,25-(OH)2D3 or 364% for DHT). However, in the
presence of added DHT (10 nM),
1,25-(OH)2D3 (10 nM) significantly
(P < 0.05) inhibited cell growth up to 50% (from
359% to 168%). The data indicate that in the presence of DHT, the
antiproliferative action of 1,25-(OH)2D3 could
again be detected. Under these CSS conditions (Fig. 2B),
1,25-(OH)2D3 alone did not affect the PSA
level, whereas DHT alone (10 nM) caused an 11-fold increase
in PSA secretion (from 0.7 to 7.9 ng of PSA per µg DNA). The amount
of PSA secretion was elevated 51-fold (from 0.7 to 36 ng per µg of
DNA) in the presence of both hormones at 10 nM each. Thus,
the cooperative effect of both hormones on stimulation of PSA was much
greater when cells were cultured in CSS medium than in FBS medium.
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), as has been
reported by others (21). Addition of DHT to the CSS medium at low
concentrations (from 0.01 to 1 nM) led to stimulation of
cellular proliferation in a dose-dependent manner, whereas a further
increase in DHT concentration greater than 10 nM caused
inhibition of growth. The DHT stimulation of cell growth could be
detected at a concentration of 0.1 nM with significant
stimulation at 1 nM. The DNA content in treated cells was
increased 2-fold over the untreated control by DHT at 1
nM.
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).
LNCaP cells became more sensitive to DHT treatment when
1,25-(OH)2D3 was included in the culture
medium. Now 0.1 nM of DHT was the concentration that
achieved significant stimulation, an order of magnitude lower than the
DHT concentration in Fig. 3A. In combination with 10 nM of
1,25-(OH)2D3, DHT at concentrations greater
than 1 nM inhibited cell growth. DHT at 10 nM
caused the same effect as 100 nM in Fig. 3A, again a shift
of 10-fold. On the other hand, addition of DHT up to 1 nM
did potentiate the antiproliferative action of
1,25(OH)2D3 when cells were cultured in CSS
medium (Figs. 2A and 3B). Therefore, the presence of DHT at an
appropriate concentration was necessary for the antiproliferative
function of 1,25-(OH)2D3 when cells were
cultured in CSS medium.
1,25-(OH)2D3 up-regulation of the AR
content: [3H]DHT binding
We were next interested in studying the molecular mechanisms for
the augmentation of DHT action by 1,25-(OH)2D3
in LNCaP cells. Because steroid hormones act via specific receptors in
target cells and the abundance of receptor determines the magnitude of
response (27, 28), we evaluated the possibility of heterologous
regulation of AR by 1,25-(OH)2D3 and of VDR by
DHT in these cells. LNCaP cells were treated with graded concentrations
of 1,25-(OH)2D3 at 0, 1, and 10 nM
for 6 days in CSS medium. Nuclear extracts were prepared from cells
treated with hormones and control. Radioligand binding assays showed
that 1,25-(OH)2D3 augmented
[3H]DHT binding in a dose-dependent manner, as depicted
in Fig. 4. Cells treated with 1 nM of
1,25-(OH)2D3 showed a more than 2-fold increase
in DHT-binding (from 197 to 430 fmol/mg protein). Addition of 10
nM of 1,25-(OH)2D3 further
up-regulated the AR content (from 197 to 532 fmol/mg protein). On the
other hand, DHT treatment caused only a slight increase in
[3H]1,25-(OH)2D3 binding (data
not shown).
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). A 2-fold increase in AR abundance was detected at
48 h after 1,25-(OH)2D3 treatment which
persisted for 144 h.
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). The data indicate that
1,25-(OH)2D3 is an inducer of AR mRNA as potent
as dibutyryl-cAMP, a known regulator of the AR mRNA (30).
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, Casodex totally blocked
1,25-(OH)2D3 action when cells were cultured in
FBS medium as compared with Fig. 1A. In the presence of 1
µM Casodex, addition of
1,25-(OH)2D3 at concentrations of 1 or 10
nM to the culture medium no longer inhibited cell growth.
Casodex at a concentration of 1 µM reversed the effect of
DHT at a low concentration (1 nM), but not the effect of 10
nM of DHT. Instead, a 1000-fold excess of Casodex (10
µM) was found to be required to reverse the effect of 10
nM of DHT (data not shown). Moreover, in the presence of
Casodex at a concentration of 1 µM, no growth inhibition
was seen with the combined treatment of both
1,25-(OH)2D3 and DHT each at 1 nM.
A high concentration of both hormones (10 nM each) appeared
to overcome the blockade by Casodex. These data suggest that androgen
action is required for the antiproliferative effect of
1,25-(OH)2D3 on LNCaP cells.
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), as compared with Fig. 1B. As
expected, Casodex completely blocked the action of DHT at a low
concentration (1 nM). The induction of PSA by DHT at a
concentration of 10 nM was also attenuated by Casodex.
Moreover, in the presence of Casodex, an increase in the PSA level was
not induced by the combined treatment of both
1,25-(OH)2D3 and DHT at 1 nM.
Administration of both hormones (10 nM each) to LNCaP cells
appeared to overcome the blockade by Casodex (from 1.7 to 67 ng of PSA
per µg of DNA). These data indicate that the antiandrogen is capable
of blocking the actions of 1,25-(OH)2D3 to
inhibit cell growth and stimulate PSA in LNCaP cells. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When cells were cultured in FBS medium, cell proliferation was substantially inhibited by the addition of either 1,25-(OH)2D3 or DHT. Combined 1,25-(OH)2D3 and DHT treatment gave a response that exceeded the sum of the individual actions on cell growth. However, the growth inhibitory effects of hormones were not seen when cells were grown in CSS medium. Under these conditions, cells proliferated at a slow rate. The doubling time of LNCaP cells was 6 days in CSS medium, whereas it was 1.5 days in FBS medium. The treatment of serum with charcoal not only depletes endogenous steroids but also removes growth factors which support rapid cell division. Moreover, activation of AR by growth factors has been reported in LNCaP cells (31). CSS has been widely used in studies of steroids on cell growth. However, the experimental results derived from such studies have to be carefully evaluated because hormonal effects on cell growth are complex under these conditions and the biphasic response to androgens makes data even more difficult to interpret. Therefore, a second functional response, PSA induction, was indispensable for our studies.
In FBS medium, LNCaP cells showed a synergistic response to combined
1,25-(OH)2D3 and DHT treatment on PSA secretion
(Fig. 1B). In CSS medium, 1,25-(OH)2D3 no
longer inhibited cell growth or induced PSA secretion; however, it did
potentiate the cellular responses to DHT (Fig. 2). DHT in the presence
of 1,25-(OH)2D3 induced PSA secretion 50-fold
over control, as compared with 10-fold in the absence of
1,25-(OH)2D3. These results clearly indicate
cooperative activity of 1,25-(OH)2D3 and DHT
which caused increased responsiveness of LNCaP cells to either
hormone.
PSA induction by DHT was observed in both FBS and CSS medium (Figs. 1B and 2B). On the other hand, 1,25-(OH)2D3 failed
to induce PSA secretion in CSS medium. We considered two possible
interpretations of these findings. First,
1,25-(OH)2D3 action may be dependent upon the
rate of cellular proliferation. Because VDR abundance correlates well
with the rate of cell growth (28), the lack of PSA induction by
1,25-(OH)2D3 may be explained by the low levels
of VDR expression when cells were cultured in CSS medium (data not
shown). Second, 1,25-(OH)2D3 action to induce
PSA may be dependent upon androgen activity present in the serum
when cells were cultured in FBS medium. The following observations
point to the latter possibility. First, when cells were cultured in CSS
medium, DHT at 10 nM did not significantly change the VDR
abundance (data not shown), but the DHT-induced PSA levels were 5-fold
higher in the presence of 1,25-(OH)2D3 than in
the absence of 1,25-(OH)2D3. Second, when cells
were cultured in FBS medium, although
1,25-(OH)2D3 treatment caused growth arrest,
the total amount of PSA secretion detected in the culture medium
increased substantially. Thus, 1,25-(OH)2D3
action on PSA secretion was not dependent upon cellular
proliferation.
Little is known about the molecular mechanisms underlying the antiproliferative action of 1,25-(OH)2D3 or the stimulatory effect of androgen in LNCaP cells. Previous reports show that the biphasic growth response of LNCaP cells to androgen is dependent upon the concentration of the hormone (21). Most of the published studies on DHT have been performed with CSS medium. Our study evaluated the effects of two hormones in FBS medium vs. CSS medium in the hope that we might dissect the activity of each hormone. We believe that two factors are important for hormone action. One is the overall concentration of androgen in the culture medium. The other is the abundance of AR that determines the magnitude of the hormonal response. In the absence of androgen action (CSS medium or addition of Casodex), 1,25-(OH)2D3 itself had a marginal effect on cell growth and PSA production in LNCaP cells. In the presence of androgen (endogenous from FBS or exogenous by DHT administration), 1,25-(OH)2D3 exhibited antiproliferative effects and increased PSA secretion. We believe these 1,25-(OH)2D3 actions are mediated by up-regulating the AR content.
Heterologous up-regulation of AR by 1,25-(OH)2D3 in LNCaP cells provides a possible mechanism for the synergistic stimulation of PSA secretion following combined 1,25-(OH)2D3 and DHT treatment. The concentration of functional AR is central to the magnitude of androgenic response in cells. The basal level of AR was significantly lower when cells were cultured in CSS medium than that in FBS medium. 1,25-(OH)2D3 augmented [3H]DHT-binding from 197 to 532 fmol/mg protein in CSS medium. 1,25-(OH)2D3 slightly increased AR level when cells were grown in FBS medium (from 378 to 436 fmol/mg protein), indicating that other factors besides AR may contribute to the synergistic effect of 1,25-(OH)2D3 and DHT when cells are cultured in FBS medium. During the preparation of this manuscript, two other research groups also reported that 1,25-(OH)2D3 provokes a 2-fold increase in AR content in LNCaP cells (32, 33).
Three lines of evidence support our conclusion that androgen action is required for the antiproliferative activities of 1,25-(OH)2D3. First, the antiandrogen, Casodex, blocked 1,25-(OH)2D3 actions on cell growth and PSA induction in FBS medium. The doubling time of LNCaP cells in FBS medium increased from 1.5 days to 3 days in the presence of Casodex. The basal level of PSA secretion by LNCaP cells in the presence of Casodex was much lower than in the absence of Casodex (1.7 vs. 5 ng of PSA per ug of DNA). The elevated DNA level and PSA secretion in the absence of Casodex may result from the action of endogenous androgens present in FBS medium. Second, there were striking similarities in cellular response to 1,25-(OH)2D3 when cells were cultured in CSS medium and in FBS medium plus Casodex, given the fact that under the former conditions a low level of androgen was present and under the latter conditions androgen action was blocked. No growth inhibition or PSA induction by 1,25-(OH)2D3 was observed under either condition. The basal levels of DNA content and PSA secretion in either case were lower than when cells were cultured in FBS medium. Third, DHT dose-response studies in CSS medium revealed that 0.1 nM of DHT [the concentration reported to be present in FBS medium (34)] restored the antiproliferative activity of 1,25-(OH)2D3. Taken together, our data show that androgen action is important for both growth inhibition and PSA induction by 1,25-(OH)2D3 in LNCaP cells.
Whereas LNCaP cells secrete PSA, no PSA mRNA was detectable in two other established human prostate cancer cell lines, PC-3 and DU 145, and levels did not change with exposure to 1,25-(OH)2D3 (data not shown). The levels of VDR were approximately 2- to 3-fold higher in PC-3 cells (78 fmol/mg protein) than the amount in the LNCaP (26 fmol/mg) and DU 145 (31 fmol/mg) cells. Unlike the LNCaP cells, there was no detectable AR protein in these two cell lines. As previously reported, both PC-3 and DU 145 cells exhibit a response to 1,25-(OH)2D3 by the induction of 24-hydroxylase mRNA (7), suggesting that androgen receptor is not required for 1,25-(OH)2D3 action in these cells. Also, primary cultures of human prostate cells do not express AR but are growth-inhibited by 1,25-(OH)2D3 (6), indicating that androgen is not necessary for 1,25-(OH)2D3 action in these cells as well. In contrast to LNCaP cells, mechanisms other than androgen signaling are responsible for the antiproliferative effect of 1,25-(OH)2D3 on these primary cells. Therefore, our finding that androgen mediates the antiproliferative activity of 1,25-(OH)2D3 in LNCaP cells is not the situation in all prostate cancer cells.
In summary, we have demonstrated that 1,25-(OH)2D3 and DHT cooperatively regulate cellular functions of LNCaP cells. AR up-regulation by 1,25-(OH)2D3 contributes to the synergistic stimulation of PSA secretion following combined 1,25-(OH)2D3 and DHT treatment when cells were cultured in CSS medium. Other factors besides AR may play an important role in the synergistic effect when cells are cultured in FBS medium. Moreover, androgen action is essential for 1,25-(OH)2D3 to mediate an antiproliferative action in LNCaP cells.
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Acknowledgments |
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Footnotes |
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Received January 9, 1997.
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References |
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 A. J. James, I. U. Agoulnik, J. M. Harris, G. Buchanan, W. D. Tilley, M. Marcelli, D. J. Lamb, and N. L. Weigel A Novel Androgen Receptor Mutant, A748T, Exhibits Hormone Concentration-Dependent Defects in Nuclear Accumulation and Activity Despite Normal Hormone-Binding Affinity Mol. Endocrinol., December 1, 2002; 16(12): 2692 - 2705. [Abstract] [Full Text] [PDF]
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 C.-L. Hsieh, L. Yang, L. Miao, F. Yeung, C. Kao, H. Yang, H. E. Zhau, and L. W. K. Chung A Novel Targeting Modality to Enhance Adenoviral Replication by Vitamin D3 in Androgen-independent Human Prostate Cancer Cells and Tumors Cancer Res., June 1, 2002; 62(11): 3084 - 3092. [Abstract] [Full Text] [PDF]
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A. V. Krishnan, X.-Y. Zhao, S. Swami, L. Brive, D. M. Peehl, K. R. Ely, and D. Feldman A Glucocorticoid-Responsive Mutant Androgen Receptor Exhibits Unique Ligand Specificity: Therapeutic Implications for Androgen-Independent Prostate Cancer Endocrinology, May 1, 2002; 143(5): 1889 - 1900. [Abstract] [Full Text] [PDF]
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K. A. Moffatt, W. U. Johannes, T. E. Hedlund, and G. J. Miller Growth Inhibitory Effects of 1{alpha}, 25-Dihydroxyvitamin D3 Are Mediated by Increased Levels of p21 in the Prostatic Carcinoma Cell Line ALVA-31 Cancer Res., October 1, 2001; 61(19): 7122 - 7129. [Abstract] [Full Text] [PDF]
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 S. C. Dixon, K. B. Knopf, and W. D. Figg The Control of Prostate-Specific Antigen Expression and Gene Regulation by Pharmacological Agents Pharmacol. Rev., March 1, 2001; 53(1): 73 - 92. [Abstract] [Full Text] [PDF]
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M. D. Sadar and M. E. Gleave Ligand-independent Activation of the Androgen Receptor by the Differentiation Agent Butyrate in Human Prostate Cancer Cells Cancer Res., October 1, 2000; 60(20): 5825 - 5831. [Abstract] [Full Text]
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J. L. Martin and S. L. Pattison Insulin-Like Growth Factor Binding Protein-3 Is Regulated by Dihydrotestosterone and Stimulates Deoxyribonucleic Acid Synthesis and Cell Proliferation in LNCaP Prostate Carcinoma Cells Endocrinology, July 1, 2000; 141(7): 2401 - 2409. [Abstract] [Full Text] [PDF]
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X.-Y. Zhao, D. M. Peehl, N. M. Navone, and D. Feldman 1{alpha},25-Dihydroxyvitamin D3 Inhibits Prostate Cancer Cell Growth by Androgen-Dependent and Androgen-Independent Mechanisms Endocrinology, July 1, 2000; 141(7): 2548 - 2556. [Abstract] [Full Text] [PDF]
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L. R. Qadan, C. M. Perez-Stable, R. H. Schwall, K. L. Burnstein, R. C. Ostenson, G. A. Howard, and B. A. Roos Hepatocyte Growth Factor and Vitamin D Cooperatively Inhibit Androgen-Unresponsive Prostate Cancer Cell Lines Endocrinology, July 1, 2000; 141(7): 2567 - 2573. [Abstract] [Full Text] [PDF]
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