0013-7227/98/$03.00/0
Endocrinology
Copyright © 1998 by The Endocrine Society
Vol. 139, No. 4
Printed in U.S.A.
Expression of Retinoic Acid Receptor-b Sensitizes
Prostate Cancer Cells to Growth Inhibition Mediated by
Combinations of Retinoids and a 19-nor Hexafluoride
Vitamin D3 Analog*
MORAY J. CAMPBELL, SUSAN PARK, MILAN R. USKOKOVIC,
MARCIA I. DAWSON, AND H. PHILLIP KOEFFLER†
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
ABSTRACT
Retinoids and analogs of vitamin D3 may achieve greater in vivo
applications if the toxic side effects encountered at pharmacologically
active doses could be alleviated. These seco-steroid hormones often act
in concert, and therefore, we attempted to dissect these interactions
by isolating combinations of receptor-selective retinoids and a potent
vitamin D3 analog [1a,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
P
ROSTATE cancer is the most frequently diagnosed nonskin cancer among American men and the second leading cause of cancer mortality among this group (1). Despite
the increase in the incidence of this disease, no successful
long-term therapies exist once the cancer progresses beyond
the prostate capsule. Blockade of androgen stimulation often
leads to a reduction of tumor volume and a partial or full
remission, but within a few years, the disease will reemerge
in a poorly differentiated, androgen-independent form,
which is usually fatal. The lack of curative therapies for this
disease has resulted in the impetus to develop nonandrogenbased therapies. One area of intensive research is growth
regulation to retard proliferation (2), promote cell death (3),
and/or induce differentiation of cells to a terminally mature,
nondividing stage (4, 5).
Biological modifiers that have been investigated include
the physiologically active metabolites of both vitamins A and
Received October 2, 1997.
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.
* This manuscript was supported by NIH Grants CA-43277, CA42710, CA-70675– 01, and CA-26038; a United States Army Grant for
Breast Cancer Research; and also, in part, by the Concern Foundation
and the Parker Hughes Trust.
† 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.
a retinoic acid receptor (RAR)bg-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 RARb. Stable expression of RARb into the RARbnegative PC-3 cells resulted in increased sensitivity to SR11262 and
LH proportional to the amount of RARb expressed.
This study indicates that RARb 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. (Endocrinology
139: 1972–1980, 1998)
D, namely all-trans retinoic acid (ATRA) and its isomer 9-cis
retinoic acid (9cRA) and 1a,25-dihydroxyvitamin D3
[1a,25(OH)2D3], respectively. These compounds can inhibit
the in vitro growth of cancer cells derived from several tissues
(6 –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:
1a,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 3 receptor (RXR). The RARs and RXRs have three subtypes: a, b,
and g (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
1a,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
1972
RARb CONTROLS PROSTATE CANCER CELL GROWTH
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) [1a,25(OH)2-16ene-23-yne-26,27,-F619nor-D3] inhibited clonal proliferation of LNCaP, PC-3, and
DU-145 prostate cancer cells, with 100 times higher potency
than 1a,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-b, were
stably transfected with a RARb expression vector to examine
the effects of reexpression of this receptor on their growth
and apoptosis. The current study identifies expression of
RARb as a critical receptor for potentiating the cooperative
inhibition between vitamin D3 analogs and retinoids.
Materials and Methods
Cells
Cell lines were obtained from ATCC (Rockville, MD) and maintained
as recommended. LNCaP was established from a metastatic lesion in the
supraclavicular lymph node of a patient with prostate cancer; PC-3 was
derived from a primary adenocarcinoma of the prostate; and DU-145
was established from prostate cancer metastatic to the brain. LNCaP was
maintained in RPMI 1640 medium with 10% FCS; PC-3 and DU-145 were
maintained in DMEM with 10% FCS.
Vitamin D3 analogs and retinoids
The vitamin D3 analog (LH) and the retinoids were kept in ethanol
(1023 m) at 220 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.
1973
TABLE 1. Receptor-selective retinoids
Retinoids
ATRA all-trans-retinoic acid
9 cRA 9-cis-retinoic acid
Am 580 [4-(5,6,7,8-Tetrahydro-5,5,8,8tetramethyl-2-naphthalenyl)
carboxamido]benzoic acid
SR11262 (E)-3-[4-(5,6,7,8-Tetrahydro-3,5,5,8,8pentamethyl-2-naphthalenyl)penyl]propenoic
acid
SR11364 6-[3-(1-Adamantyl)-5-hydroxyphenyl]2-naphthalenecarboxylic acid
LG1069 [4-[1-(5,6,7,8-Tetrahydro-3,5,5,8,8pentamethyl-2-naphthalenyl)-1ethenyl]benzoic acid
SR11237 [2-(5,6,7,8-Tetrahydro-5,5,8,8tetramethyl-2-naphthalenyl)-2-(4carboxyphenyl)]-1,3-dioxolane
Selectivity(ref. no.)
RAR
RAR and RXR
RARa(47)
RARbg
RARg(48)
RXR(49)
RXR(50)
RT-PCR
Total RNA from each cell line was extracted with TRIZOL reagent
(GIBCO BRL, Grand Island, NY) and was treated for 30 min at 37 C with
RQ1 ribonuclease-free deoxyribonuclease (Promega, Madison, WI) in
100 mm Tris-HCl, pH 9.0, 500 mm KCl, and 2 mm MgCl2. The complementary DNA was prepared from deoxyribonuclease-treated, phenolchloroform-extracted messenger RNA (mRNA) (1 mg) by reverse transcription with Moloney murine leukemia virus reverse transcriptase
(Promega) at 42 C for 60 min in the presence of 100 mm Tris-HCl, pH
9.0, 500 mm KCl and 2 mm MgCl2, 100 pm random hexamers (Pharmacia,
Piscataway, NJ), 2 mm 29-deoxynucleoside 59-triphosphates, and 20 U
RNasin ribonuclease inhibitor (Promega) in a 20-ml reaction vol. Expression of RARa, RARb, and RARg mRNAs was detected by PCR
amplification with the primer pairs as follows; RARa was a 171-bp
fragment generated with sense primer 59-TCT GAC CAC TCT CCA
GCA CCA GCT-39, antisense primer 59-CTG AGG ACT TGT CCT GAC
AGA CAA-39, and detected with an internal oligonucleotide 59-ATT
GAC ACC CAG AGC AGC AGT TCT GAA GAG-39. RARb was a 204-bp
fragment generated with sense primer 59-ACG TCT GCC TGG TTT CAC
TGG CTT-39, antisense primer 59-ACG TGA ACA CAA GGT CAG TCA
GAG-39, and detected with an internal oligonucleotide 59-TTG CAC
CAG CTA TAC CCC AGA CAC AGA CAC-39. RARg was a 226-bp
fragment generated with a sense primer 59-ACA GAG CAC CAG CTC
AGA GGA CAG-39, antisense primer 59-ATT CCT GGT CAC CTT GTT
GAT GAT-39, and detected with an internal oligonucleotide 59-TGC
AAT GAC AAG TCC TCT GGC TAC CAC TAT-39. GAPDH, as a control,
was a 195-bp fragment generated by a sense primer 59-CCA TGG AGA
AGG CTG CGC-39, antisense primer 59-CAA AGT TGT CAT GGA TGA
CC-39, and detected with an internal oligonucleotide 59-ATG TTC GTC
ATG GGT GTG AAC CAT GAG AAG-39.
For each reaction, 2 ml of template was amplified in the presence of
100 mm Tris-HCl, pH 9.0, 500 mm KCl, 5 mm MgCl2, 0.2 mm 29-deoxynucleoside 59-triphosphate, 0.3 mm primer, and 1.5 U TAQ polymerase (Promega) in a final vol of 30 ml. 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 ml) 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.
To undertake semiquantitative RT-PCR, we predetermined the linear
amplification conditions to be 27 cycles and amplified RARb and
GAPDH (control) in LNCaP and RARb stable transfectants of PC-3.
After Southern blot analysis, using a 32P-labeled 30-mer oligonucleotide
internal to the amplified regions, relative band intensities were determined by densitometry.
RARb CONTROLS PROSTATE CANCER CELL GROWTH
1974
TABLE 2. RARb mRNA expression levels and growth
characteristics of prostate cancer cells lines and PC-3 stable
RARb-transfectants
Cell line
Level of expression of
RARb mRNA (%
compared with
LNCaP expression)a
Fold increase in
cell number in
liquid cultureb
Soft agar cloning
efficiency (%)c
LNCaP
PC-3
PC-3-Neo
PC-3-b1
PC-3-b2
PC-3-b4
PC-3-b5
DU-145
100
0
0
95
195
25
220
0
2.6 6 0.3d
5.3 6 0.7
5.0 6 0.3
1.5 6 0.1
2.4 6 0.2
3.9 6 0.1
2.0 6 0.2f
8.0 6 1.0
12.5 6 0.6e
15.4 6 0.5
15.3 6 0.8
nd
nd
5.4 6 0.4
3.2 6 0.2g
19.8 6 0.6
Endo • 1998
Vol 139 • No 4
induce apoptosis of LNCaP, PC-3, and DU-145 cells. These cells were
exposed to either LH or retinoid alone or in combination at 1027 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 3 106 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 mg/ml etoposide for 4 days.
a
Levels were compared with those of LNCaP cells.
To determine growth in liquid culture, 2 3 105 cells were plated
into triplicate 10-cm dishes and grown for 4 days, viable cells were
counted, and the mean 6 SE increase in cell number was expressed as
fold-increase of the initial inoculum. Results represent the mean 6 SE
of triplicate plates in duplicate.
c
To assess cloning efficiency, 1 3 103 cells were plated into soft
agar, according to Materials and Methods. After 14 days, colonies
were enumerated, and the cloning efficiency was calculated as the
mean colony count expressed as a percentage of the initial cell inoculum. Results represent the mean 6 SE of triplicate dishes in duplicate experiments.
d
The fold-increase in cell number of LNCaP cells was significantly
less than that of PC-3 and DU-145 cells (P , 0.02 and P , 0.002,
respectively).
e
The cloning efficiency of LNCaP cells was significantly less than
that of DU-145 cells (P , 0.002).
f
The fold-increase in cell number of PC-3-b-5 (but not PC-3-b-4)
cells was significantly less than that of PC-3, DU-145, and PC-3-neo
cells (P , 0.005, P , 0.005, and P , 0.005, respectively).
g
The cloning efficiency of PC-3-b-5 cells was significantly less than
that of PC-3, DU-145, and PC-3-neo cells (P , 0.005, P , 0.0001, and
P , 0.005, respectively).
nd, Not determined.
b
Stable transfection of RARb
The RARb expression construct was a generous gift of Dr. Elizabeth
Allegretto (Ligand Pharmaceuticals, San Diego, CA). The RARb 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 mg G418, in
which untransfected cells died. The expression of exogenous RARb was
determined by semiquantitative RT-PCR.
Statistical analysis
The interactions of two compounds were assessed by measuring the
mean of either LH or retinoid acting alone (6 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.
Results
RAR subtypes expressed in LNCaP, PC-3, DU-145, and
stable transfectants
Figure 1 shows the expression of the different RAR
mRNAs. The three cell lines expressed RARa and RARg.
LNCaP, but not PC-3 or DU-145, expressed RARb mRNA.
Exposure of various cell types to ATRA often induces increased RARb expression (29). Treatment of PC-3 cells with
ATRA (1027 m) failed to induce expression of RARb (Fig. 2a).
Because DU-145 is a more transformed cell line than PC-3
(30 –34), we chose to stably transfect PC-3 with RARb, because this would possibly yield a more meaningful comparison with LNCaP cells. Total RNA isolated from the five
Colony formation in soft agar
The potency of LH and retinoids was determined by single-dose (1029
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 3 103 cells in 400 ml 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 1029
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
FIG. 1. Expression of RAR mRNA subtypes in prostate cancer cell
lines. Total mRNA was isolated from subconfluent cells, reversetranscribed, and amplified, as described in Materials and Methods.
The resolved amplification products were transferred to membrane
and probed with 32P-labeled specific oligonucleotides. Blots were exposed to film for 6 h at 280 C.
RARb CONTROLS PROSTATE CANCER CELL GROWTH
1975
LNCaP, it has a growth rate in liquid culture that is slower
than stable transfectants with approximately double the level
of RARb expression). The general trend is true, given that a
small level of expression of RAR-b (PC-3-b4 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-b5 cells had a significantly lower mean fold increase in cell number [2.6 6 0.3 and 2.0 6 0.2 (6 se), respectively] than wild-type PC-3 and DU-145 (5.3 6 0.7 and 8.0 6
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-b5 (RARb 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.
FIG. 2. RARb mRNA levels in stably transfected PC-3 cells. Total
mRNA from subconfluent cells was reverse-transcribed and amplified
with a low number of reactions, as described in Materials and Methods. A, Resolved amplification products were transferred to membrane and probed with specific oligonucleotides. RAR-b levels in PC3-b-1 (lane 1), PC-3-b-2 (lane 2), PC-3-b-3 (lane 3), PC-3-b-1 (lane 4),
PC-3-b-5 (lane 5), PC-3-neo (lane 6), PC-3 (lane 7), PC-3 1 ATRA (1027
M) (lane 8), and LNCaP (lane 9). Blots were exposed to film for 6 h at
280 C. B, Quantitation of relative RARb mRNA in the PC-3 stable
transfectants, compared with levels in LNCaP cells. The RARb specific bands were first normalized to GAPDH mRNA control and then
compared with levels of LNCaP RARb mRNA.
stable RARb transfectants of PC-3 and wild-type LNCaP cells
was examined for the expression of RARb, 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 RARb; these were clone 4 (25% expression, relative to
LNCaP) and clone 5 (220% expression, relative to LNCaP)
and the mock-transfected, neoresistant clone.
Liquid and soft-agar growth characteristics of LNCaP,
PC-3, and DU-145 cells and PC-3-b clones
Growth features in liquid culture and soft agar of the
prostate cancer cell lines and the RARb containing clones in
the absence of a retinoid and/or LH are shown in Table 2.
Expression of RARb, 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-b1 (which, although it has
approximately the same level of expression of RARb as
LNCaP prostate cancer cell line. All of the combinations displayed additive inhibitory effects except those with ATRA
and SR11262, which displayed synergistic ability to inhibit
clonal growth of LNCaP (Fig 3a). LH (1029 m) inhibited 22 6
3% (mean 6 se) clonal growth of LNCaP cells, and ATRA and
9cRA (1029 m) inhibited 14 6 4% and 30 6 2% colony formation, respectively. Together, LH and either ATRA or 9cRA
(1029 m) inhibited a mean 56 6 4% and 58 6 4% colonies,
respectively, compared with the nontreated control. The effect of LH with 9cRA (1029 m) was additive; and the combination of LH plus ATRA (1029 m) mediated a synergistic
inhibition of LNCaP cells (Table 3).
ATRA can mediate activities through RARa, b, g; 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
RARa (Am 580), RARb, and g (SR11262), RARg (SR11364),
and RXR (LG1069 and SR11237). The Am 580 (1029 m) was
most inhibitory (29 6 2%), and SR11262 was least inhibitory
(10 6 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 (1029 m), which resulted in the greatest level
of inhibition for any of the combinations (mean 71% 6 2.3%),
whereas the mean predicated inhibition was 37%. The RXRselective 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 1029 m ATRA (7% 6 2%) or 9cRA (29% 6 3%) or were
either when combined with LH (38% 6 1.5% and 42% 6 2.3,
respectively). No combinations displayed synergism. Significantly, the natural retinoid (9cRA) and two of the selective
1976
RARb CONTROLS PROSTATE CANCER CELL GROWTH
Endo • 1998
Vol 139 • No 4
FIG. 3. Inhibition of clonal growth of prostate cancer cells exposed to either LH and/or retinoids (1029 M). Results expressed as percent (mean 6
SEM) of nontreated control cells. Each point represents a mean of at least three experiments with triplicate dishes. A, LNCaP, PC-3, and DU-145;
B, PC-3-neo, PC-b-4, and PC-3-b-5.
retinoids (SR11262 and SR11237, 1029 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
(1029 m) was potent on its own (22% 6 3.1); and when
combined with LH, it mediated an additive inhibition (47 6
1.5) (Fig. 3a).
PC-3-neo, PC-3-b-4, and PC-3-b-5. The effects of LH and retinoids on the clonal growth of 2 RARb 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 (RARbg-selective),
RARb CONTROLS PROSTATE CANCER CELL GROWTH
1977
TABLE 3. Summary of VDR and RAR Expression in prostate cancer cell lines and their sensitivity to clonal inhibition by combinations of
LH and various retinoids
Receptor expression
[Ref. no.]
VDR(51)
RARa
RARb
RARg
Prostate cancer cell line
LNCaP
Yes
Yes
Yes
Yes
Type of inhibition mediated by the combination
ATRA 1 LH
Synab
9cRA 1 LH
Add
AM 580 1 LH
Add
SR11262 1 LH
Syne
SR11364 1 LH
Add
LG1069 1 LH
Add
SR11237 1 LH
Add
PC-3
Yes
Yes
No
Yes
PC-3-neo
Yes
Yes
No
Yes
of a retinoid with LH
Add
Add
Subd
nd
Add
nd
Squea
Squea
Add
nd
Add
nd
a
Sub
nd
PC-3-b4
PC-3-b5
DU-145
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Suba
nd
nd
Suba
nd
nd
nd
Add
nd
nd
Add
nd
nd
nd
Squec
Add
Subd
Squec
Add
Squea
Add
Sque, Squelching effects; Sub, subadditive effects; Add, additive effects; Syn, Synergistic effects; nd, not determined; LH, 1a,25(OH)2-16ene-23-yne-26,27,-F6-19nor-D3; VDR, Vitamin D receptor; PC-3-neo, cells stably transfected with the neo resistance gene; PC-3-b-4, PC-3 cells
stably transfected with RARb expression vector; PC-3-b-5, PC-3 cells stably transfected with RARb expression vector.
a
P , 0.05.
b
The interactions of two compounds were assessed by measuring the mean of either LH or retinoid acting alone (6 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 to 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, sub-additive 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.
c
P , 0.01.
d
P , 0.001.
e
P , 0.0001.
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 RARb [PC-3-b-4 (low expression) and PC-3-b-5 (high expression)] were approximately
equally inhibited by either ATRA or SR11262 alone (Fig. 3b).
However, the low RARb-expressing clone PC-3-b-4 was less
sensitive to clonal growth inhibition than the high RARbexpressing PC-3-b-5 clone in the presence of LH plus the
RARbg-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-b-4 and additive inhibition against PC-3-b-5 (Fig. 3b and Table 3). Inhibition by the combination of LH plus SR11262 was greatest
in PC-3-b-5 (60 6 4.6%), less in PC-3-b-4 (40% 6 1.2%), and
least in PC-3-neo (25 6 1.4%). Inhibition by LH and SR11262
in PC-3-b-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 RARb 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-b-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 RARb 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 con-
FIG. 4. Induction of apoptosis. LNCaP cells were exposed to either
LH and/or retinoids (1027 M, 4 days), or to 50 mg/ml etoposide. Apoptosis was measured as described in Materials and Methods.
ditions (approximately 3%). In the presence of LH, a small,
but statistically significant (P , 0.02), increase in apoptosis
occurred [mean 9.4% 6 0.6% (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 (1027 m) alone
resulted in a mean level of apoptotic LNCaP cells of 9.1% 6
0.9% and 3.8% 6 0.7%, respectively; but their combination
(5 3 1028 m of each analog) induced a mean 38.4% 6 3.6%
apoptotic cells. Of the RAR-selective retinoids, the RARbgselective retinoid (SR11262) was additive with LH and the
RARg-selective retinoid (SR11364) was synergisitic with LH:
1978
RARb CONTROLS PROSTATE CANCER CELL GROWTH
the SR11262 and LH together (5 3 1028 m each) resulted in
a mean 18.5% 6 2.5% apoptosis, and SR11364 and LH resulted in a mean 24% 6 5% apoptotic LNCaP cells (P , 0.001,
with respect to either LH or SR11262 combined). Alone,
SR11262 and SR11364 (1027 m) induced 8% 6 0.4% and 3%
6 0.2% apoptosis, respectively.
The effects of LH and either ATRA or SR11262 on apoptosis
were examined in the five PC-3-b clones. No significant
apoptosis (,5%) was detected in the clones exposed to these
compounds, either individually or in combination (data not
shown).
Discussion
We investigated whether the combination of a potent analog of vitamin D3 (LH) with natural or receptor-selective
retinoids had enhanced antiproliferative effects on prostate
cancer cells. Furthermore, we identified the RAR subtypes
expressed in three prostate cancer lines (LNCaP, PC-3, and
DU-145) and stably transfected RARb into PC-3 cells. Each
line expressed RARa and g, but RARb was not detected in
either wild-type PC-3 or DU-145. Various cancers have deregulated or nonfunctional RARb expression as transformation occurs (29, 35–38). For example, loss of RARb 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 RARb 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 wildtype 53-kDa tumor suppressor protein and retinoblastoma
proteins. As prostate cancer progresses, androgendependence 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 RARb 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 RARb expression in cancer cell lines (40, 41). One study
identified lung cancer cell lines that did not increase RARb
expression in the presence of ATRA but were able to activate
a transfected reporter gene construct containing the RARE
from the RARb gene promoter (bRARE) (42). The reasons for
these abnormalities in the regulation of RARb 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 bRARE-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 RARb gene.
Various studies have examined the effect of stable reintegration of RARb in cancer cell lines. One study with ovar-
Endo • 1998
Vol 139 • No 4
ian cancer cell lines found no significant difference in the
retinoid responsiveness of cell lines that do or do not express
RARb (43). This reflects the current study, where the effect
of the RARbg-selective ligand (SR11262) alone was not significantly different between LNCaP, PC-3, and DU-145, and
only slightly elevated in PC-3-b5. In another study with
Calu6 lung cancer cell lines stably transfected with RARb,
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 RARbg-selective ligand
(SR11262), which was nearly inactive on its own at 1029 m;
however, in the presence of analog LH at the same combined
dose (1029 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 RARb
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 RARb expression in
PC-3 cells produced a prominent additive growth inhibition.
These data suggested that RARb 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 RARb reexpression in the PC-3
cells. In studies examining the role of RARb in breast cancer
cell lines, this receptor was lost in both estrogen receptorpositive and -negative lines (45, 46). On introduction of
RARb 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 RARb 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
RARb CONTROLS PROSTATE CANCER CELL GROWTH
intracellular coisomerization of these two natural retinoids.
However, LH plus the RARbg-selective retinoid (SR11262)
was synergistic in LNCaP and additive when the RAR-b
receptor was reexpressed. The RARg-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 RARb 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 RARb when in the presence of LH plus either
ATRA or SR11262. Loss of expression of RARb 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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