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Department of Cell Biology, Baylor College of Medicine (S.E.B., N.L.W.), Houston, Texas 77030; Ligand Pharmaceuticals (E.A.A.) San Diego, California 92121; and the Department of Molecular and Cellular Physiology, University of Cincinnati (J.W.P.), Cincinnati, Ohio 45267
Address all correspondence and requests for reprints to: Dr. Nancy L. Weigel, Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030. E-mail: nweigel{at}bcm.tmc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, ß, and 
are encoded by separate genes
and are activated by all-trans-retinoic acid or
9-cis-retinoic acid (4, 7). In contrast, the retinoid X
receptors , ß, and only bind 9-cis-retinoic acid
(7). Although the RXRs form homodimers, they are believed to act more
commonly as heterodimer partners for other receptors, including both
the RARs and the VDR. In addition to the reports of growth inhibition by 1,25-dihdroxyvitamin D3, other studies have demonstrated that retinoic acid can inhibit the growth of prostate cells (8). Because of the potential for interaction between retinoid- and vitamin D3-mediated pathways, we used natural and synthetic ligands for VDR, RARs, and RXRs to examine the effects of these compounds on the growth of LNCaP prostate cancer cells and to begin to determine the mechanism by which VDR inhibits the growth of these cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-Dihydroxyvitamin D3 was obtained from Solvay
DuPhar (Weesp, The Netherlands). All-trans-retinoic acid,
5-bromo-2'-deoxyuridine (BrdU), and propidium iodide (PI) were
purchased from Sigma Chemical Co. (St. Louis, MO).
9-Cis-retinoic acid, LG100268
(6-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronapthalen-2-yl)cyclo-propyl]nicotinic
acid; a RXR-specific ligand) (9), and TTNPB
(4-[(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-napthyl)2-propylene]benzoic
acid) (10) were gifts from Drs. Elizabeth Allegretto and J. Wesley Pike
(Ligand Pharmaceuticals, San Diego, CA). EB1089 (11)
[1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-1'(E),3'(E)-dien-1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene]
was kindly provided by Leo Pharmaceutical Products (Ballerup,
Denmark). All hormones were dissolved in ethanol to form stock
solutions and stored at -20 C in the dark. Coulter Counter model ZF,
Zapoglobin II, the Profile I flow cytometer, and Isoton were obtained
from Coulter Cytometry (Hialeah, FL). Hanks’ Balanced Salt Solution
without calcium or magnesium and 0.05% trypsin-EDTA were obtained from
Life Technologies (Gaithersburg, MD). All other chemicals were reagent
grade unless otherwise stated.
Cell cultures
The LNCaP prostate cancer cell line was obtained from the
American Type Culture Collection (Rockville, MD). The cells were
cultured in RPMI 1640 medium (Life Technologies) supplemented with 10%
Rehatuin FBS (Intergen Co., Purchase, NY) at 37 C in a humidified
atmosphere of 5% CO2.
Cell growth assays
LNCaP cells were seeded at 13,000 cells/well in 3 ml medium in
6-well culture dishes. After allowing cells to attach for 24 h,
cells were treated with vehicle (ethanol; final concentration, 0.1%)
or the indicated concentrations of hormones. The medium containing
vehicle and/or hormones was changed every 3 days. After 9 days, cells
were washed with Hanks’ Balanced Salt Solution without calcium or
magnesium and removed from the plate by incubation with 0.5 ml 0.05%
trypsin-EDTA, and the reaction was stopped with an equal volume of
medium containing serum. Cell suspension (0.5 ml) was diluted in 10 ml
isoton and treated with 3 drops of Zapoglobin II to lyse the cells, and
each sample was counted twice in a Coulter counter. All samples were
tested in triplicate, and statistical significance was determined by
one-way ANOVA using the SigmaStat program (Jandel Scientific, San
Rafael, CA). Each experiment was performed a minimum of three times,
and a representative experiment is shown.
Assay of retinoid receptor levels
LNCaP cells were grown to confluency in RPMI medium supplemented
with 10% Rehatuin FBS and then harvested. Cell pellets were stored at
-80 C. Pellets were thawed and resuspended in cold buffer containing
0.4 M KCl, 10 mM Tris-HCl (pH 7.5), 1
mM dithiothreitol, 0.5 mM PMSF, and 1 µg/ml
aprotinin. Cells were sonicated on ice for 15 sec and then centrifuged
at 4 C at 15,000 rpm in a microfuge for 45 min. Supernatants were
collected, and protein concentrations were determined. Total soluble
protein extracts (500 µg/assay point) were incubated with 20
nM [3H]9-cis-retinoic acid (Ligand
Pharmaceuticals; 59 Ci/mmol) overnight at 4 C protected from light, as
described previously (12). Incubation with each of the
subtype-selective antiretinoid receptor antibodies or with nonspecific
rabbit or mouse IgG was conducted as described previously (12).
Ligand-receptor-antibody complexes were then precipitated with protein
A-Sepharose (Pharmacia, Piscataway, NJ) and analyzed by scintillation
counting to quantify receptor levels.
Cell cycle experiments
LNCaP cells were plated in 10-cm dishes at 250,000 cells/dish
and treated with the indicated concentrations of hormone or vehicle
(0.1% ethanol) for 6 days. Before harvesting, the cells were pulse
labeled with 10 µM BrdU for 15 h. Cells were scraped
into medium, washed once with PBS, and fixed according to the
recommendations of Boehringer Mannheim (Indianapolis, IN). Briefly,
ice-cold 70% ethanol was added dropwise while vortexing, and cells
were fixed overnight at 4 C. After centrifuging for 5 min at 800
x g, cells were resuspended in ice-cold 0.1 M
HCl-0.5% Triton X-100 and incubated on ice for 10 min. After adding 5
ml double distilled H2O, the cells were centrifuged as
described above, and the supernatant was removed. Cell pellets were
resuspended in 2 ml double distilled H2O and boiled for 10
min, after which they were quickly transferred to ice for 5 min. Five
milliliters of PBS-0.5% Triton X-100 were added to each sample, and
cell pellets were recovered by centrifugation as described above.
Pellets were resuspended in 100 µl PBS-0.1% BSA with 20 µl
anti-BrdU antibody (Becton Dickinson, San Jose, CA) and incubated for
1 h at room temperature. Five milliliters of PBS were added, and
cell pellets were recovered by centrifugation. Pellets were resuspended
in 50 µl PBS-0.1% BSA with 2.5 µl fluorescein isothiocyanate
(FITC)-labeled goat antimouse IgG (Zymed Laboratories, San Francisco,
CA) for 1 h. Five milliliters of PBS were added, and cell pellets
were recovered by centrifugation. Pellets were resuspended in 500 µl
PBS with 5 µg/ml PI and 200 µg/ml ribonuclease. Cells were stored
at 4 C for 1 h before processing. Samples were analyzed using a
Profile I flow cytometer (Coulter Electronics, Hialeah, FL). At least
5000 forward scatter gated events were collected per specimen. PI
fluorescence was collected using linear amplification with doublet
discrimination, and BrdU-FITC fluorescence was collected using
logarithmic amplification. The emissions were split using a 550 long
pass dichroic filter (Coulter Electronics, Hialeah, FL). FITC emissions
were collected using a 525 band pass filter, and PI emissions were
collected using a 575 band pass filter. Controls included cell
populations stained only for PI or FITC to adjust spectral overlap.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows that whereas as little as
10-8 M 1,25-dihydroxyvitamin D3
inhibits the growth of LNCaP cells, 10-5 M
all-trans-retinoic acid is required to comparably inhibit
cell growth. The finding that high concentrations of
all-trans-retinoic acid are necessary to elicit a response
is consistent with previous reports (8).
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shows the levels of RAR,
-ß, and - and RXR, -ß, and - measured by the ability to
immunoprecipitate radiolabeled receptor with form-specific antibodies.
In contrast to MCF-7 breast cancer cells, which do not express RARß,
RXRß, or RXR (detection limit, 5 fmol/mg) (12), all six forms are
present in LNCaP cells and are thus targets for retinoid action. The
levels range from 9–34 fmol/mg. Interestingly, this range is similar
to the expression level of VDR in LNCaP cells (
30 fmol/mg) (3)
(Blutt, S., data not shown).
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, we found that although 10-8
M 9-cis-retinoic acid alone appears to be
slightly growth stimulatory, the combination of
9-cis-retinoic acid and 1,25-dihydroxyvitamin D3
synergistically inhibits the growth of LNCaP cells.
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, low concentrations of all-trans-retinoic acid
are growth stimulatory, as reported previously (8) (see also Fig. 1);
the combination of 1,25-dihydroxyvitamin D3 and
all-trans-retinoic acid was no more effective than
1,25-dihydroxyvitamin D3 alone.
Effects of RXR- and RAR-specific compounds on LNCaP cell growth
To determine whether the effects of 9-cis-retinoic acid
on growth are mediated by the RXR receptors or the RAR receptors, a
RXR-specific ligand, LG100268 (9), was administered in combination with
1,25-dihydroxyvitamin D3. As shown in Fig. 3A, this compound alone exhibits some growth inhibitory
properties, but the combination of 1,25-dihydroxyvitamin D3
and LG100268 is more effective than either alone. For comparison, an
RAR-specific ligand, TTNPB (10), was tested (Fig. 3B). This compound
stimulated growth as did all-trans-retinoic acid and had no
effect on the response to 1,25-dihydroxyvitamin D3. Taken
together, these studies suggest that activation of RXR can inhibit
growth somewhat, whereas activation of RAR stimulates growth unless the
concentration of ligand is extremely high (see Fig. 1). Moreover, the
combination of 1,25-dihydroxyvitamin D3 and an RXR ligand
is more effective in inhibiting growth than either alone.
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compares the relative responsiveness of LNCaP cells
to EB1089 and 1,25-dihydroxyvitamin D3 under these growth
conditions. Similar to the results of others (15), we found that EB1089
is growth inhibitory at lower concentrations than 1,25-dihydroxyvitamin
D3. Moreover, as depicted in Fig. 4B, the combination of a
low concentration of EB1089 (10-10 M) and
9-cis-retinoic acid is more effective than EB1089 alone.
Note that there appears to be a maximal inhibitory response, and
9-cis-retinoic acid does not further reduce growth when
used in combination with higher concentrations of EB1089.
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shows a
typical plot of BrdU incorporation plotted as log green fluorescence
from the FITC antibody (x-axis) vs. cell count.
The left panel shows control cells with large populations of
cells in G1 (essentially no fluorescence) as well as a
large population of cells that have incorporated BrdU and are in
S/G2/M. In contrast, the right panel shows cells
treated with 10-7 M 1,25-dihydroxyvitamin
D3. Many fewer cells have entered the S phase or passed
through to G2/M. As shown in Table 2, those
treatments that inhibit growth cause accumulation of cells in the
G1 phase of the cell cycle; treatments that are more growth
inhibitory resulted in a higher proportion of cells accumulating in the
G1 phase. For example, whereas 10-8
M 1,25-dihydroxyvitamin D3 caused 61% of the
cells to accumulate in G1, 10-8 M
EB1089 resulted in 76% of the cells in G1. Similarly,
whereas 61% of the cells were in G1 after treatment with
10-8 M 1,25-dihydroxyvitamin D3,
this number increased to 75% when 9-cis-retinoic acid was
combined with 10-8 M 1,25-dihydroxyvitamin
D3. These data indicate that the growth inhibitory action
of 1,25-dihydroxyvitamin D3 is at least in part due to a
slowing of the progression of cells into the S phase.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Compounds that have the potential for inhibiting the growth of prostate cancer cells in vivo are of great interest due to the paucity of alternatives to androgen ablation therapy, which is effective initially, but ultimately fails. Although all-trans-retinoic acid inhibits LNCaP cell growth at high concentrations, the requirement for high concentrations combined with the finding that low concentrations are growth stimulatory (8) suggest that this compound is unlikely to be useful clinically. However, Peehl et al. (24), using primary human prostate cell strains grown in a defined serum-free medium, obtained quite different results. They found that 3 nM all-trans-retinoic acid and 0.25 nM 1,25-dihydroxyvitamin D3 each slightly inhibited the growth of a cell strain derived from normal prostate, and the combination of the two synergistically inhibited growth. In contrast, 0.3 nM all-trans-retinoic acid inhibited the growth of a tumor-derived strain by 60%, 0.25 nM 1,25-dihydroxyvitamin D3 inhibited growth by 20%, and the combination was additive, resulting in a growth inhibition of 84%. In addition to potential differences in responses of individual cell lines, the key difference between the studies of Peehl et al. (25) and those reported here is the use of serum in the LNCaP cell studies. Serum contains proteins that bind and transport the retinoids and 1,25-dihydroxyvitamin D3, thus reducing their free concentrations. In fact, Peehl et al. (24) have shown that the addition of as little as 0.5% charcoal-stripped serum to their medium was sufficient to increase the amount of 1,25-dihydroxyvitamin D3 required to effect 50% growth inhibition 10-fold (from 0.25 to 2.5 nM). Hence, the requirement for high concentrations of all-trans-retinoic acid to inhibit the growth of LNCaP cells may be due to the necessity of using serum to grow these cells. However, these compounds are bound to serum proteins in vivo, so that the results obtained with the LNCaP cells may more closely approximate the levels required in vivo to reduce cell growth.
Our finding that low concentrations of all-trans-retinoic acid, 9-cis-retinoic acid, and TTNPB, the RAR-selective compound, stimulate growth, whereas the RXR-selective compound, LG100268, is somewhat growth inhibitory, suggests that partial activation of RAR stimulates growth, whereas activation of RXR alone may be somewhat growth inhibitory. The synergistic response to combination treatment with 9-cis-retinoic acid and 1,25-dihydroxyvitamin D3 suggests that the combination not only abrogates the stimulatory effect of 9-cis (which is probably a RAR-mediated response), but also enhances the ability of 1,25-dihydroxyvitamin D3 to inhibit growth. When the synthetic RXR ligand, LG100268, is used, there is no stimulation of growth, and in combination with 1,25-dihydroxyvitamin D3, growth is inhibited more effectively than with either alone.
Under growth conditions in which the LNCaP cells are actively growing, we and others found that 1,25-dihydroxyvitamin D3 is growth inhibitory even at rather low concentrations (3). Peehl et al. (24) also found that all concentrations of 1,25-dihydroxyvitamin D3 inhibited the growth of prostate cells in a defined medium. Other investigators, using conditions in which the cells grow very poorly (charcoal-stripped serum without supplementary growth factors or steroid), report that 1,25-dihydroxyvitamin D3 is slightly growth stimulatory (3, 25). However, a serum-containing medium with growth factors and steroids is probably more similar to an in vivo environment, suggesting that there is little likelihood that one would observe a biphasic growth effect with 1,25-dihydroxyvitamin D3in vivo.
The ability of EB1089 to act at lower concentrations than 1,25-dihydroxyvitamin D3 despite a similar affinity for the VDR is thought to be due to its decreased affinity for vitamin D-binding proteins, thus allowing more efficient cellular uptake (14). We have found EB1089 to be very effective in inhibiting the growth of LNCaP cells. The studies have shown that there are a number of other analogs with similar properties (13, 14, 26, 27) that are also promising candidates for use as growth inhibitors; development of optimal compounds is still ongoing. Our finding that EB1089 is effective at very low concentrations and that its action can be enhanced by 9-cis-retinoic acid suggest that EB1089 alone or in combination with 9-cis-retinoic acid might be useful clinically, and that an RXR-specific compound in combination with EB1089 might be most useful because the RXR-specific compound does not stimulate growth at any concentration tested.
The mechanism(s) by which 1,25-dihydroxyvitamin D3 inhibits growth of cancer cells is unknown. Studies by various investigators have reported that 1,25-dihydroxyvitamin D3 causes a G1 arrest of T47D breast cancer cells (28), whereas others have found that 1,25-dihydroxyvitamin D3 induces apoptosis in MCF7 breast cancer cells (29). We show here that treatment with 1,25-dihydroxyvitamin D3 causes LNCaP cells to accumulate in the G1 phase of the cell cycle. Although LNCaP and PC3 prostate cancer cells are responsive to 1,25-dihydroxyvitamin D3, DU145 cells, which also contain functional VDR, are not. These cells lack functional retinoblastoma protein (Rb), which is a critical regulator of the G1/S checkpoint. Gross et al. (30) have shown that prostate epithelial cells immortalized by expression of simian virus 40 (which inactivates Rb) are not responsive to 1,25-dihydroxyvitamin D3, suggesting that functional Rb is required for this response. However, they also found that expression of functional Rb in DU145 cells was insufficient to restore the response to 1,25-dihydroxyvitamin D3 (30). Whether another component of the Rb pathway is also nonfunctional in DU145 cells has yet to be determined. Our studies of the cell cycle distribution of treated LNCaP cells are consistent with the hypothesis that 1,25-dihydroxyvitamin D3 acts in the G1 phase of the cell cycle, slowing passage into the S phase, and that the more profound the growth inhibition, the greater the accumulation of cells in G1. Although treatment with 1,25-dihydroxyvitamin D3 may induce many genes, the finding that 1,25-dihydroxyvitamin D3 induces synthesis of messenger RNA for several of the cyclin-dependent kinase inhibitors in the myelomonocytic cell line, U937 (31), suggests that these are likely targets of 1,25-dihydroxyvitamin D3 action. It will be of particular interest to determine whether the induction of one or more of these genes is enhanced by the combination of 1,25-dihydroxyvitamin D3 and 9-cis-retinoic acid in prostate cells.
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Acknowledgments |
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Footnotes |
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Received August 26, 1996.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 1,25-dihydroxyvitamin D3; 
, EB1089. B: *,
P < 0.05 compared to ethanol control; **,
P < 0.05 compared to corresponding EB1089 sample.
Eth, Ethanol vehicle; 9C, 9-cis-retinoic acid; B, EB1089.