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Division of Hematology/Oncology (M.J.C., S.P., H.P.K.), Cedars-Sinai Medical Center/University of California, Los Angeles School of Medicine, Los Angeles, California 90048; Hoffman La Roche (M.R.U.), Nutley, New Jersey 07110; and SRI International (M.I.D.), Menlo Park, California 94025
Address all correspondence and requests for reprints to: Moray J. Campbell, Ph.D., Department of Immunology, Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,25(OH)2-16ene-23-yne-26,27,F6-19nor-D3,
code name LH] that were potent inhibitors of prostate
cancer cell growth at low, physiologically safer doses.
Using a panel of prostate cancer cell lines representing progressively
more transformed phenotypes, we found that the LNCaP cell line (least
transformed) was either additively or synergistically inhibited in its
clonal growth by LH and various naturally occurring and
receptor-selective retinoids, the most potent combination being with a
retinoic acid receptor (RAR)ß
-selective retinoid (SR11262). The
effect was not found with either PC-3 (intermediate transformation) or
DU-145 (most transformed). We also undertook RT-PCR to examine the
subtypes of RARs present, and we found that PC-3 and DU-145 did not
express RARß. Stable expression of RARß into the RARß-negative
PC-3 cells resulted in increased sensitivity to SR11262 and
LH proportional to the amount of RARß expressed.
This study indicates that RARß may play an important role in synergistically controlling cell proliferation, and expression is lost with increased prostate cancer cell transformation. Simultaneous administration of a potent vitamin D3 analog and receptor-selective retinoids may have therapeutic potential for the treatment of androgen-dependent and -independent prostate cancer.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Biological modifiers that have been investigated include the
physiologically active metabolites of both vitamins A and D, namely
all-trans retinoic acid (ATRA) and its isomer
9-cis retinoic acid (9cRA) and 1,25-dihydroxyvitamin
D3 [1,25(OH)2D3],
respectively. These compounds can inhibit the in vitro
growth of cancer cells derived from several tissues (6, 7, 8, 9, 10, 11), including
primary malignant prostate tissue (12) and cell lines (13, 14).
Vitamin D3 and retinoids mediate their activities by
binding to specific nuclear hormone receptors that function as
ligand-induced transcription factors, for instance:
1,25(OH)2D3 interacts exclusively with the
vitamin D3 receptor (VDR), ATRA and 9cRA bind to the
retinoic acid receptor (RAR), and 9cRA also binds to the retinoid
x receptor (RXR). The RARs and RXRs have three subtypes: , ß, and
(15). Evidence suggests that these transcription factors may
naturally act in concert. The VDRs and RARs show strong homology and
readily dimerize, principally with the RXR, which thus plays a pivotal
role as a cofactor in regulation of target gene transcription (16, 17).
Other studies have shown synergistic up-regulation of the murine
osteopontin promoter reporter gene by the combination of 9cRA and
1,25-(OH)2D3 (18). Cooperation between these
two receptor signaling pathways has been the basis for the
investigation of combinational therapy. We and others have previously
demonstrated that a potent vitamin D3 analog, in
combination with 9cRA, can synergistically inhibit proliferation of
human myeloid leukemic cells and MCF-7 breast cancer cells (19, 20).
One mechanism by which retinoids and vitamin D3 might
additively or synergistically control target genes includes the
targeting of multiple response elements in the target genes, for
example: the gene encoding p21(waf1/cip1), a
cyclin-dependent kinase inhibitor, contains both a vitamin
D3 response element and a retinoic acid response element
(RARE) within its promoter/enhancer region (21, 22). Not all
interactions between these two pathways necessitate activation through
RXR. Up-regulation of the human osteocalcin gene has been shown to
occur in an RXR-independent manner, possibly through VDR homodimers
(23, 24), or heterodimerization with other steroid hormone receptors,
such as RAR (25). However, the biological significance of these latter
two receptor-dimers is, as yet, unclear.
We previously discovered that the potent analog of vitamin
D3 (code name LH)
[1,25(OH)2-16ene-23-yne-26,27,-F6-19nor-D3]
inhibited clonal proliferation of LNCaP, PC-3, and DU-145 prostate
cancer cells, with 100 times higher potency than
1,25(OH)2D3 (13). Unfortunately, the
in vivo application of LH is limited because of
lethal hypercalcemia when administered at high doses, a common side
effect of vitamin D3 analogs (26). The potential may
therefore exist to improve the clinical potential of differentiation
therapy by combining the administration of a potent vitamin
D3 analog and retinoid.
In the present study, we evaluated combinations of LH and either natural or receptor-selective synthetic retinoids to determine whether such combinations would be effective, at a low physiologically safer doses, against prostate cancer cells. To determine the appropriate receptor subtype-selective retinoid for these studies, the RAR subtype transcripts in these cancer cell lines were identified. Inhibitory effects were determined in an extremely sensitive clonal growth assay. Apoptosis plays a significant role in the regulation of cell number in the normal prostate gland, and previous studies have shown that retinoids and/or vitamin D3 can initiate apoptosis in a variety of cancer cells. We were therefore interested in investigating whether any potent combinations of retinoids and LH were able to induce apoptosis. Finally, PC-3 prostate cancer cells, which did not express RAR-ß, were stably transfected with a RARß expression vector to examine the effects of reexpression of this receptor on their growth and apoptosis. The current study identifies expression of RARß as a critical receptor for potentiating the cooperative inhibition between vitamin D3 analogs and retinoids.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Vitamin D3 analogs and retinoids
The vitamin D3 analog (LH) and the
retinoids were kept in ethanol (10-3 M) at
-20 C in the dark. For experimental use, the analog solutions were
diluted in normal media. Their names and receptor affinities are
described in Table 1
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,
RARß, and RAR mRNAs was detected by PCR amplification with the
primer pairs as follows; RAR was a 171-bp fragment generated with
sense primer 5'-TCT GAC CAC TCT CCA GCA CCA GCT-3', antisense primer
5'-CTG AGG ACT TGT CCT GAC AGA CAA-3', and detected with an internal
oligonucleotide 5'-ATT GAC ACC CAG AGC AGC AGT TCT GAA GAG-3'. RARß
was a 204-bp fragment generated with sense primer 5'-ACG TCT GCC TGG
TTT CAC TGG CTT-3', antisense primer 5'-ACG TGA ACA CAA GGT CAG TCA
GAG-3', and detected with an internal oligonucleotide 5'-TTG CAC CAG
CTA TAC CCC AGA CAC AGA CAC-3'. RAR was a 226-bp fragment generated
with a sense primer 5'-ACA GAG CAC CAG CTC AGA GGA CAG-3', antisense
primer 5'-ATT CCT GGT CAC CTT GTT GAT GAT-3', and detected with an
internal oligonucleotide 5'-TGC AAT GAC AAG TCC TCT GGC TAC CAC TAT-3'.
GAPDH, as a control, was a 195-bp fragment generated by a sense primer
5'-CCA TGG AGA AGG CTG CGC-3', antisense primer 5'-CAA AGT TGT CAT GGA
TGA CC-3', and detected with an internal oligonucleotide 5'-ATG TTC GTC
ATG GGT GTG AAC CAT GAG AAG-3'.
For each reaction, 2 µl of template was amplified in the presence of
100 mM Tris-HCl, pH 9.0, 500 mM KCl, 5
mM MgCl2, 0.2 mM 2'-deoxynucleoside
5'-triphosphate, 0.3 µM primer, and 1.5 U TAQ polymerase
(Promega) in a final vol of 30 µl. The mixture was overlaid with
mineral oil and amplified in a thermal cycler with PCR cycle conditions
as follows: 94 C for 1 min, 55 C for 36 sec, 72 C for 36 sec for 35
cycles, then a final extension at 72 C for 7 min. As a positive
control, GAPDH primers were used under the same conditions. The
products (Table 2) of PCR (20 µl) were
electrophoresed on a 3% agarose gel and stained with ethidium bromide.
The DNA was transferred to Hybond membranes (Amersham, Little Chalfont,
Buckinghamshire, UK) for Southern blot analysis using a
32P-labeled 30-mer oligonucleotide internal to the
amplified regions.
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Stable transfection of RARß
The RARß expression construct was a generous gift of Dr.
Elizabeth Allegretto (Ligand Pharmaceuticals, San Diego, CA). The
RARß and neoresistance plasmids were stably transfected, at a 5:1
ratio, into PC-3 cells using Lipofectamine (Gibco BRL) in a weight
ratio of lipofectamine:total plasmid of 3:1 (wt/wt), according to the
manufacturers’ instructions. Single clones were isolated in the
presence of 500 µg G418, in which untransfected cells died. The
expression of exogenous RARß was determined by semiquantitative
RT-PCR.
Colony formation in soft agar
The potency of LH and retinoids was determined by
single-dose (10-9 M) combination studies in
soft agar. Trypsinized and washed single-cell suspensions of LNCaP,
PC-3, and DU-145 prostate cancer cells from 80% confluent cultures
were counted and plated onto 24-well flat-bottomed plates using a
two-layer soft agar system with 1 x 103 cells in 400
µl of media per well, as described previously (27). The cells were
maintained in either RPMI 1640 medium or DMEM. The feeder layer was
prepared with agar (1%) equilibrated at 42 C. Before addition of this
layer to the plate, the vitamin D3 analog and/or retinoids
(final concentration 10-9 M) were pipetted
into the wells. Stock solutions of vitamin D3 analog and
retinoids and experimental plates were kept in the dark to minimize
UV-catalyzed degradation. After 14 days of incubation, the colonies
(
50 cells) were counted using an inverted microscope. All experiments
were done at least three times in triplicate per experimental
point.
Measurement of apoptosis
Combinations of LH and a retinoid that demonstrated
significant inhibition of clonal proliferation were investigated for
their capacity to induce apoptosis of LNCaP, PC-3, and DU-145 cells.
These cells were exposed to either LH or retinoid alone or
in combination at 10-7 M for 4 days, with
fresh analogs added at day 2. Total cells, both in the media and those
adhering to the plastic, were harvested and fixed in 1% methanol-free
formaldehyde for 15 min and washed in PBS (28). The cell concentration
was corrected to 1 x 106 cells/ml and fixed in 5 ml
of 70% ethanol. Single- and double-stranded DNA breaks were labeled
with bromodeoxyuridine triphosphate (BrD-UTP) for 40 min at 37 C with
terminal transferase (Boehringer Mannheim, Indianapolis, IN). The cells
were permeabilized with a 0.3% Triton-X 100 and 0.5% BSA in PBS, and
DNA breaks were tagged by bromodeoxyuridine and then identified using a
fluorescein isothiocyanate-conjugated antibromodeoxyuridine antibody.
Cells were stained with propidium iodide for 30 min, and green
fluorescence was measured by fluorescence-activated cell sorter
analysis at 510–550 nm. As a positive control, each cell line was
exposed to 50 µg/ml etoposide for 4 days.
Statistical analysis
The interactions of two compounds were assessed by measuring the
mean of either LH or retinoid acting alone (±
SEM). The combination of the mean clonal inhibition for
each compound acting alone was the predicted combined effect. The mean
observed combined clonal inhibition was then compared with this value,
using the Student’s t test. Classification of the
inhibitory effects was as follows: synergistic effects were those with
an experimental value significantly greater than the predicted value,
additive effects were those where the experimental value did not
significantly differ from the predicted value, subadditive effects were
those where the experimental value was significantly less than the
predicated value, and squelching effects were those where the
experimental value was significantly lower than either agent acting
alone.
Other statistical analyses were preformed using the Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows the expression of the
different RAR mRNAs. The three cell lines expressed RAR and
RAR. LNCaP, but not PC-3 or DU-145, expressed RARß mRNA. Exposure
of various cell types to ATRA often induces increased RARß expression
(29). Treatment of PC-3 cells with ATRA (10-7
M) failed to induce expression of RARß (Fig. 2a). Because DU-145 is a more transformed
cell line than PC-3 (30, 31, 32, 33, 34), we chose to stably transfect PC-3 with
RARß, because this would possibly yield a more meaningful comparison
with LNCaP cells. Total RNA isolated from the five stable RARß
transfectants of PC-3 and wild-type LNCaP cells was examined for the
expression of RARß, using semiquantitative PCR (Fig. 2a). Levels were
normalized to GAPDH and expressed as a percent expression by LNCaP
(Fig. 2b). We were interested in obtaining the widest spectrum of
phenotypic effects, and therefore, we chose to study the biology of the
clones with the lowest and highest expression of RARß; these were
clone 4 (25% expression, relative to LNCaP) and clone 5 (220%
expression, relative to LNCaP) and the mock-transfected, neoresistant
clone.
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. Expression of
RARß, in either LNCaP or the stable PC-3 transfectants, correlated
with their decreased growth in liquid culture and decreased cloning
efficiency. This relationship holds true, except for PC-3-ß1 (which,
although it has approximately the same level of expression of RARß as
LNCaP, it has a growth rate in liquid culture that is slower than
stable transfectants with approximately double the level of RARß
expression). The general trend is true, given that a small level of
expression of RAR-ß (PC-3-ß4 has 25% expression of LNCaP) only
slightly reduces growth; and when this level is equal to or greater
than LNCaP, growth is further reduced. For example, in liquid culture
for 4 days, LNCaP and PC-3-ß5 cells had a significantly lower mean
fold increase in cell number [2.6 ± 0.3 and 2.0 ± 0.2 (±
SE), respectively] than wild-type PC-3 and DU-145
(5.3 ± 0.7 and 8.0 ± 1.0, respectively). The mean cloning
efficiency of LNCaP was lower than those of wild-type PC-3 and DU-145;
and the cloning efficiency of PC-3-ß5 (RARß stable transfectant)
was significantly lower than those of wild-type PC-3 and DU-145. The
statistical significance for these relationships is indicated by the
Student’s t test (P values given in Table 2).
Effects of receptor-selective retinoids and LH combinations on
clonal growth in soft agar
Figure 3, a and b, shows the effects
of a single concentration of LH and various concentrations
of retinoids on the growth of the three prostate cancer cell lines and
three PC-3 stable transformants.
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). LH (10-9 M)
inhibited 22 ± 3% (mean ± SE) clonal growth of
LNCaP cells, and ATRA and 9cRA (10-9 M)
inhibited 14 ± 4% and 30 ± 2% colony formation,
respectively. Together, LH and either ATRA or 9cRA
(10-9 M) inhibited a mean 56 ± 4% and
58 ± 4% colonies, respectively, compared with the nontreated
control. The effect of LH with 9cRA (10-9
M) was additive; and the combination of LH plus
ATRA (10-9 M) mediated a synergistic
inhibition of LNCaP cells (Table 3).
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, ß, ; and 9cRA can also
mediate its effects through similar pathways, as well as by a
RXR-responsive route. However, it is problematic to dissect the
signaling pathways involved in cellular inhibition, because ATRA and
9cRA can coisomerize in culture. Therefore, we used analogs that were
selective for RAR (Am 580), RARß, and (SR11262), RAR
(SR11364), and RXR (LG1069 and SR11237). The Am 580 (10-9
M) was most inhibitory (29 ± 2%), and SR11262 was
least inhibitory (10 ± 2%). These selective analogs, when
combined with LH, showed additive inhibitory activity (Fig 3a). A dramatic exception to this was the synergistic inhibition
observed with LH plus SR11262 (10-9
M), which resulted in the greatest level of inhibition for
any of the combinations (mean 71% ± 2.3%), whereas the mean
predicated inhibition was 37%. The RXR-selective analogs LG1069 and
SR11237 displayed additive inhibitory effects when combined with
LH.
PC-3 prostate cancer cell line. The PC-3 cells were less
sensitive to clonal inhibition than were LNCaP cells to the effects of
either 10-9 M ATRA (7% ± 2%) or 9cRA (29%
± 3%) or were either when combined with LH (38% ± 1.5%
and 42% ± 2.3, respectively). No combinations displayed synergism.
Significantly, the natural retinoid (9cRA) and two of the selective
retinoids (SR11262 and SR11237, 10-9 M), in
combination with LH, displayed only subadditive or
squelching effects. Equally notable, the RXR-selective retinoid LG1069,
in combination with LH, retained the additive effects
observed with LNCaP cells (Fig 3a).
DU-145 prostate cancer cell line. Many of the retinoids were
weakly inhibitory and were either subadditively inhibitory, when
combined with LH, or squelched the inhibitory effect of
LH. For example, the combination of LH with
either ATRA, SR11262, or LG1069, displayed a squelching effect.
Significantly, LH with either 9cRA or the RXR-selective
retinoid SR11237 was additive in inhibition. For example, SR11237
(10-9 M) was potent on its own (22% ± 3.1);
and when combined with LH, it mediated an additive
inhibition (47 ± 1.5) (Fig. 3a).
PC-3-neo, PC-3-ß-4, and PC-3-ß-5. The effects of
LH and retinoids on the clonal growth of 2 RARß stable
transfectant clones of PC-3 and a mock-transfected, neoresistant
control clone are shown in Fig. 3b. None of the clones were
significantly altered in their growth in the presence of LH.
The clonal growth of the neoresistant control clone (PC-3-neo) in the
presence of either ATRA or SR11262 (RARß-selective), or when
either were combined with LH, was unaltered, compared with
wild-type PC-3 (Fig. 3, a and b). The 2 clones stably expressing
transfected RARß [PC-3-ß-4 (low expression) and PC-3-ß-5 (high
expression)] were approximately equally inhibited by either ATRA or
SR11262 alone (Fig. 3b). However, the low RARß-expressing clone
PC-3-ß-4 was less sensitive to clonal growth inhibition than the high
RARß-expressing PC-3-ß-5 clone in the presence of LH
plus the RARß-selective retinoid (SR11262). Both wild-type PC-3
and PC-3-neo displayed a squelching inhibition by the combination of
LH and SR11262; however, the same combination displayed
subadditive inhibition against PC-3-ß-4 and additive inhibition
against PC-3-ß-5 (Fig. 3b and Table 3). Inhibition by the combination
of LH plus SR11262 was greatest in PC-3-ß-5 (60 ±
4.6%), less in PC-3-ß-4 (40% ± 1.2%), and least in PC-3-neo
(25 ± 1.4%). Inhibition by LH and SR11262 in
PC-3-ß-5 cells was the third most potent of all of the 27
combinations of compounds, whereas this same pair was the third least
inhibitory combination against wild-type PC-3 cells. The stably induced
expression of RARß receptor seemed to reverse the squelching effect
of LH and SR11262 noted to occur with the wild-type PC-3
cells, and instead, it produced an additive inhibition of
PC-3-ß-5.
The results of Figs. 2a and 3, a and b, are summarized in Table 3.
Induction of apoptosis
Only LNCaP cells (but not the other prostate cancer cell lines or
their stable RARß transfectants) displayed detectable apoptosis after
their exposure to either retinoids, LH, or a combination of
both (Fig. 4). LNCaP cells underwent a
low, but consistent, level of apoptosis under normal growth conditions
(approximately 3%). In the presence of LH, a small, but
statistically significant (P < 0.02), increase in
apoptosis occurred [mean 9.4% ± 0.6% (± SE)]. None of
the retinoids alone induced more than 9% apoptosis, but the
combinations of LH and selective retinoids produced a
striking enhancement of apoptosis. For example, LH or 9cRA
(10-7 M) alone resulted in a mean level of
apoptotic LNCaP cells of 9.1% ± 0.9% and 3.8% ± 0.7%,
respectively; but their combination (5 x 10-8
M of each analog) induced a mean 38.4% ± 3.6% apoptotic
cells. Of the RAR-selective retinoids, the RARß-selective retinoid
(SR11262) was additive with LH and the RAR-selective
retinoid (SR11364) was synergisitic with LH: the SR11262 and
LH together (5 x 10-8 M each)
resulted in a mean 18.5% ± 2.5% apoptosis, and SR11364 and
LH resulted in a mean 24% ± 5% apoptotic LNCaP cells
(P < 0.001, with respect to either LH or
SR11262 combined). Alone, SR11262 and SR11364 (10-7
M) induced 8% ± 0.4% and 3% ± 0.2% apoptosis,
respectively.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and , but RARß was not detected in either wild-type PC-3 or
DU-145. Various cancers have deregulated or nonfunctional RARß
expression as transformation occurs (29, 35, 36, 37, 38). For example, loss of
RARß expression in breast cancer cell lines correlated with their
inability to undergo apoptosis, to express estrogen receptors, and to
resist growth arrest by hormonal withdrawal (39). This same pattern of
loss of RARß expression with increased transformation was found also
in the current study. LNCaP cells express androgen receptors and
demonstrate growth stimulation by androgens (30). They also express
functional wild-type 53-kDa tumor suppressor protein and retinoblastoma
proteins. As prostate cancer progresses, androgen-dependence is
often lost. PC-3 and DU-145 cells do not express androgen receptors and
do grow independently of androgen. Both lines have 53-kDa tumor
suppressor protein mutations (31) and a compromised E-cadherin cell
adhesion/metastasis suppression system (32) (personal communication, J.
Schalken). DU-1 45 cells, which are the most transformed, also have
mutations of both the retinoblastoma and p16ink4a (16-kDa
member of the Ink4 tumor suppressor proteins) genes (33, 34). Thus, the
loss of RARß expression in the PC-3 and DU-145 cell lines correlates
with their more transformed phenotypes. Other studies have examined the ability of ATRA to induce RARß expression in cancer cell lines (40, 41). One study identified lung cancer cell lines that did not increase RARß expression in the presence of ATRA but were able to activate a transfected reporter gene construct containing the RARE from the RARß gene promoter (ßRARE) (42). The reasons for these abnormalities in the regulation of RARß expression were not clear to the investigators. Our current study reflected this pattern as we examined the response of PC-3 cells transiently transfected with a ßRARE-luciferase construct and found that these cells were able to increase luciferase activity in the presence of ATRA (data not shown). Thus, we conclude that PC-3 cells have a defect in the transactivation of the endogenous RARß gene.
Various studies have examined the effect of stable reintegration of
RARß in cancer cell lines. One study with ovarian cancer cell lines
found no significant difference in the retinoid responsiveness of cell
lines that do or do not express RARß (43). This reflects the current
study, where the effect of the RARß-selective ligand (SR11262)
alone was not significantly different between LNCaP, PC-3, and DU-145,
and only slightly elevated in PC-3-ß5. In another study with Calu6
lung cancer cell lines stably transfected with RARß, only a small
in vitro inhibitory effect with ATRA was noted in a
short-term culture assay; but, the cell line had a significantly
reduced tumorigenic potential in vivo (44). This may reflect
the sensitivity of these cells to synergistic clonal inhibition by
physiological levels of serum ATRA and
1,25(OH)2D3; this reflected the current
findings of synergistic inhibition by SR11262 and LH at low
doses in a 14-day clonal assay.
Little growth inhibition of LNCaP, PC-3, and DU-145 cells occurred when
they were exposed to the RAR-selective retinoids alone; however, what
differentiated these cell lines was their disparate response to the
combination of a retinoid with LH. Only LNCaP cells
displayed a synergistic inhibition by either a natural retinoid (ATRA)
or a conformationally restricted ligand (SR11262) combined with
LH, suggesting a cooperation between these different
receptors. This potentiation was most noticeable with the
RARß-selective ligand (SR11262), which was nearly inactive on its
own at 10-9 M; however, in the presence of
analog LH at the same combined dose (10-9
M), this was the most potent combination, with an
approximate 7-fold increase in inhibition. The mechanism for this
interaction is unclear. It may involve liganded RARß recruiting RXR
from VDR-RXR heterodimers, thereby promoting VDR homodimers that
potentiate the effects of LH. In contrast, LH and
SR11262 displayed squelching with PC-3 and DU-145 cells, LH
alone being more potent than the combination. Reintroduction of
high-level RARß expression in PC-3 cells produced a prominent
additive growth inhibition. These data suggested that RARß was
important for allowing cooperative potent inhibitory effects between
retinoids and vitamin D3 compounds.
The LNCaP cells readily underwent apoptosis with combinations of retinoids and LH. Interestingly, the combinations that were most potent at inhibiting clonal growth were not those that resulted in the highest level of apoptosis. For example, the combination of LH and SR11262 was the most potent at inhibition of clonal growth, but it was only the fifth most potent initiator of apoptosis. Furthermore, induction of apoptosis in the other two cell lines was not observed and was not restored with stable RARß reexpression in the PC-3 cells. In studies examining the role of RARß in breast cancer cell lines, this receptor was lost in both estrogen receptor-positive and -negative lines (45, 46). On introduction of RARß into estrogen receptor-positive lines, growth inhibition and apoptosis were observed (46); but, on introduction of this receptor into estrogen-negative lines, only growth inhibition was observed. We also did not observe apoptosis in the androgen-negative PC-3 cell line, either before or after introduction of a RARß expression vector.
ATRA synergized with LH in inhibiting LNCaP (but not PC-3 or
DU-145) growth, although the combination of LH and either
ATRA or 9cRA was at least additive in all three cell lines. These data
are difficult to interpret because of the intracellular coisomerization
of these two natural retinoids. However, LH plus the
RARß-selective retinoid (SR11262) was synergistic in LNCaP and
additive when the RAR-ß receptor was reexpressed. The
RAR-selective retinoid (SR11364) was additively inhibitory in all
three cell lines, The RXR-selective retinoids displayed contradictory
behavior, as one of the synthetic RXR-selective retinoids (LG1069 or
SR11237) plus LH was additive in all three cell lines,
although LH plus SR11237 was subadditive with PC-3 and
LH plus LG1069 was squelching with DU-145. We previously
demonstrated that high concentrations of other, RXR-selective retinoids
alone were potent inhibitors of prostate cancer cell lines (14), and
presumably these effects are very ligand specific and therefore need
further investigation. These data suggest that RAR-mediated pathways
are important for inhibiting both androgen-dependent and -independent
prostate cancer cells.
We have demonstrated a role for RARß to allow high level inhibition of cancer cell proliferation by RAR-selective retinoids and a vitamin D3 analog. Apoptosis seems to be important for inhibiting cell proliferation of LNCaP cells by LH-retinoid combinations but not PC-3 or DU-145 cells. Interestingly, this capacity is not restored to PC-3 cells by expression of RARß when in the presence of LH plus either ATRA or SR11262. Loss of expression of RARß may be a useful marker for prostate cancer progression, and restoration of its expression could prove to be an attractive therapeutic goal.
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Footnotes |
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2 Member of the UCLA Jonsson Comprehensive Cancer Center and holder
of an endowed Mark Goodson Chair of Oncology Research at Cedars-Sinai
Medical Center/UCLA School of Medicine.
Received October 2, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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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 J. M. Alfaro, B. Fraile, M. V. T. Lobo, M. Royuela, R. Paniagua, and M. I. Arenas Immunohistochemical Detection of the Retinoid X Receptors {alpha}, {beta}, and {gamma} in Human Prostate J Androl, January 1, 2003; 24(1): 113 - 119. [Abstract] [Full Text] [PDF]
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F. Richter, A. Joyce, F. Fromowitz, S. Wang, J. Watson, R. Watson, R. J. Irwin Jr, and H. F. S. Huang Immunohistochemical Localization of the Retinoic Acid Receptors in Human Prostate J Androl, November 1, 2002; 23(6): 830 - 838. [Abstract] [Full Text] [PDF]
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 J. Huang, W. C. Powell, A. C. Khodavirdi, J. Wu, T. Makita, R. D. Cardiff, M. B. Cohen, H. M. Sucov, and P. Roy-Burman Prostatic Intraepithelial Neoplasia in Mice with Conditional Disruption of the Retinoid X Receptor{alpha} Allele in the Prostate Epithelium Cancer Res., August 15, 2002; 62(16): 4812 - 4819. [Abstract] [Full Text] [PDF]
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L. Farhana, M. Dawson, A. K. Rishi, Y. Zhang, E. Van Buren, C. Trivedi, U. Reichert, G. Fang, M. W. Kirschner, and J. A. Fontana Cyclin B and E2F-1 Expression in Prostate Carcinoma Cells Treated with the Novel Retinoid CD437 Are Regulated by the Ubiquitin-mediated Pathway Cancer Res., July 1, 2002; 62(13): 3842 - 3849. [Abstract] [Full Text] [PDF]
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 B. Lefebvre, C. Brand, P. Lefebvre, and K. Ozato Chromosomal Integration of Retinoic Acid Response Elements Prevents Cooperative Transcriptional Activation by Retinoic Acid Receptor and Retinoid X Receptor Mol. Cell. Biol., March 1, 2002; 22(5): 1446 - 1459. [Abstract] [Full Text] [PDF]
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X. Guo, B. S. Knudsen, D. M. Peehl, A. Ruiz, D. Bok, R. R. Rando, J. S. Rhim, D. M. Nanus, and L. J. Gudas Retinol Metabolism and Lecithin:Retinol Acyltransferase Levels Are Reduced in Cultured Human Prostate Cancer Cells and Tissue Specimens Cancer Res., March 1, 2002; 62(6): 1654 - 1661. [Abstract] [Full Text] [PDF]
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F. Lin, D. Xiao, S. K. Kolluri, and X.-k. Zhang Unique Anti-Activator Protein-1 Activity of Retinoic Acid Receptor {beta} Cancer Res., June 1, 2000; 60(12): 3271 - 3280. [Abstract] [Full Text]
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B. Lin, G.-q. Chen, D. Xiao, S. K. Kolluri, X. Cao, H. Su, and X.-k. Zhang Orphan Receptor COUP-TF Is Required for Induction of Retinoic Acid Receptor beta , Growth Inhibition, and Apoptosis by Retinoic Acid in Cancer Cells Mol. Cell. Biol., February 1, 2000; 20(3): 957 - 970. [Abstract] [Full Text]
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 K. M. Sommer, L. I. Chen, P. M. Treuting, L. T. Smith, and K. Swisshelm Elevated retinoic acid receptor beta 4 protein in human breast tumor cells with nuclear and cytoplasmic localization PNAS, July 20, 1999; 96(15): 8651 - 8656. [Abstract] [Full Text] [PDF]
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 D. Pasquali, V. Rossi, D. Prezioso, V. Gentile, V. Colantuoni, T. Lotti, A. Bellastella, and A. A. Sinisi Changes in Tissue Transglutaminase Activity and Expression during Retinoic Acid-Induced Growth Arrest and Apoptosis in Primary Cultures of Human Epithelial Prostate Cells J. Clin. Endocrinol. Metab., April 1, 1999; 84(4): 1463 - 1469. [Abstract] [Full Text]
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