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Experimental Therapeutics, Preclinical Pharmacology |
,25-Dihydroxyvitamin D3 Down-Regulates Estrogen Receptor Abundance and Suppresses Estrogen Actions in MCF-7 Human Breast Cancer Cells1
Department of Medicine, Stanford University School of Medicine, Stanford, California 94305
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ABSTRACT |
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,25-Dihydroxyvitamin D3
[1,25(OH)2D3], the active metabolite of
vitamin D, is a potent inhibitor of breast cancer cell growth. Because
the estrogen receptor (ER) plays a key role in breast cancer
progression, we have studied the effects of
1,25(OH)2D3 on the regulation of ER in the
estrogen-responsive MCF-7 human breast cancer cell line, which is known
to predominantly express ER. 1,25(OH)2D3
causes significant inhibition of MCF-7 cell growth, and it also
decreases the growth-stimulatory effect of 17ß-estradiol
(E2). Treatment of MCF-7 cells with
1,25(OH)2D3 reduces ER levels in a
dose-dependent manner, as shown by ligand binding assays and Western
blot analysis. The 1,25(OH)2D3 analogues
EB-1089, KH-1060, Ro 27-0574, and Ro 23-7553 are more potent than
1,25(OH)2D3 in both their antiproliferative
actions as well as ER down-regulation. There is a striking correlation
(R2 = 0.98) between the
growth-inhibitory actions of 1,25(OH)2D3 or
analogues and their ability to down-regulate ER levels. Treatment with
1,25(OH)2D3 shows that the reduction in ER is
accompanied by a significant decrease in the steady-state levels of ER
mRNA. The decrease in ER mRNA is not abolished by the protein synthesis
inhibitor cycloheximide. Inhibition of mRNA synthesis with actinomycin
D reveals no significant differences between ER mRNA half-life in
control and 1,25(OH)2D3-treated cells. Nuclear
run-on experiments demonstrate significant decreases in
ER gene transcription at the end of 17 h of
treatment with 1,25(OH)2D3. These findings
indicate that 1,25(OH)2D3 exerts a direct
negative effect on ER gene transcription. Coincident
with the decrease in ER levels there is an attenuation of
E2-mediated bioresponses after
1,25(OH)2D3 treatment. Induction of
progesterone receptor by E2 is suppressed by
1,25(OH)2D3, and the E2-mediated
increase in breast cancer susceptibility gene (BRCA1) protein is
reduced by 1,25(OH)2D3 treatment. Overall,
these results suggest that the antiproliferative effects of
1,25(OH)2D3 and its analogues on MCF-7 cells
could partially be mediated through their action to down-regulate ER
levels and thereby attenuate estrogenic bioresponses, including breast
cancer cell growth. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNote Added in ProofREFERENCES |
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1,25(OH)2D3, the
biologically active form of vitamin D, is a major regulator of calcium
and phosphate homeostasis in the body (4
, 5)
. The
regulatory effects of
1,25(OH)2D3 are mediated
via the VDR, which is also a member of the steroid/thyroid/retinoid
receptor superfamily (4, 5, 6)
. In addition to its effects on
calcium and phosphate homeostasis,
1,25(OH)2D3 is an important
modulator of cellular proliferation and differentiation in a number of
normal and malignant cells (4
, 7, 8, 9)
. In breast cancer
cells, 1,25(OH)2D3 has
potent growth-inhibitory actions (10, 11, 12, 13)
. Although the
growth-inhibitory effects of
1,25(OH)2D3 on breast
cancer cells have been well established, the effects of
1,25(OH)2D3 on ER
expression are less well documented. Studies on ER in human breast
cancer cell lines have reported minor decreases (14)
or no
change (15)
in ER expression with
1,25(OH)2D3 treatment. A
more recent study reported significant decreases in ER protein levels
in the MCF-7 cells treated with EB-1089, a potent analogue of
1,25(OH)2D3
(13)
. Studies have also been conducted to indicate that
the E2-mediated bioresponses are attenuated by
1,25(OH)2D3 treatment
(16
, 17)
. Although the above-mentioned reports have
suggested potential cross-talk between
1,25(OH)2D3 and estrogen
signaling pathways, the extent of the interaction and the mechanism by
which 1,25(OH)2D3
causes down-regulation of ER are still not clarified.
The purpose of the present investigation is to study the effects of
1,25(OH)2D3 on ER and
E2-mediated effects in MCF-7 breast cancer cells.
As discussed below, the MCF-7 cells used in this study do not
express ERß, as measured by reverse transcription-PCR. Therefore, in
these studies the effect is limited to ER. For simplicity, we refer
to the ER in these cells as ER. To achieve our goal of investigating
the effect of 1,25(OH)2D3
on breast cancer cells, we have studied the relationship between
changes in MCF-7 growth rate, levels of ER protein, steady-state ER
mRNA, and gene transcription in cells treated with
1,25(OH)2D3 or its
analogues. We have also assessed the correlation between the effects of
1,25(OH)2D3 and its
analogues KH-1060, EB-1089, Ro 27-0574, and Ro 23-7553 on the growth of
MCF-7 cells and the changes elicited in ER levels. Furthermore, we have
investigated how changes in ER abundance induced by
1,25(OH)2D3 and its
analogues alter the functional responses to E2 in
MCF-7 cells. We have established that the
1,25(OH)2D3-mediated effect
on ER gene expression is at the transcriptional level.
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MATERIALS AND METHODS |
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,25-Dihydroxy-22,24-diene-24,26,27-trihomovitamin
D3 (EB-1089) and
1,25-dihydroxy-20-epi-22-ene-24,26,27-trihomovitamin
D3 (KH-1060) were generous gifts from Dr. L.
Binderup (Leo Pharmaceuticals, Ballerup, Denmark). MCF-7 cells were
obtained from American Type Culture Collection (Rockville, MD). Culture
media and other supplements were purchased from Mediatech (Herndon,
VA). All other chemicals and reagents, including anti-actin antibody,
were obtained from Sigma Chemical Co. (St. Louis, MO).
Cell Culture.
MCF-7 cells were routinely cultured in T-75 flasks at 37°C under an
atmosphere of 5% CO2. They were maintained in
RPMI 1640 supplemented with 10% calf serum, 100 units/ml penicillin,
and 100 µg/ml streptomycin. At confluence, the flasks contained
approximately 1.5–2 x 107 cells and were
routinely subcultured every 7–10 days. For experiments, the growth
medium was replaced with phenol red-free RPMI 1640 supplemented with
10% calf serum (CSS), twice stripped of endogenous hormones using
charcoal and Dextran T-70. We used medium containing CSS in most of our
studies to minimize the effects of E2, which is
known to down-regulate its own receptor (18)
. The medium
containing CSS (treatment medium) and hormones, as indicated, was
introduced to the cells 24 h after subculture, and fresh medium
and hormones were replenished every 2 days. Stock solutions of steroid
hormones were made in 100% ethanol and added to the treatment medium.
All controls received ethanol vehicle at a concentration equal to that
in the hormone-treated cells (0.1% v/v).
Cell Proliferation Assay.
DNA content or attained cell mass was used as a measure of cell
proliferation. MCF-7 cells were seeded in six-well tissue culture
plates (Becton Dickinson, Lincoln Park, NJ) at a density of 50,000
cells/well in 3 ml of RPMI 1640 containing 10% calf serum. Twenty-four
h later, fresh medium was added. Cells were grown in RPMI 1640 medium
with 10% calf serum or CSS and were treated with various doses of
1,25(OH)2D3 or its
analogues in the presence or absence of 10 nM
E2. Fresh medium and hormones were added every
other day. At the end of 6 days, cell monolayers were processed as
described earlier (19)
, and DNA contents were determined
by the method of Burton (20)
.
Ligand Binding Assays.
MCF-7 cells growing in CSS-containing medium were treated with either
E2 (10 nM) or
1,25(OH)2D3 or analogues
(1, 10, or 100 nM) for 2 days. Cells were then harvested,
and high salt cell extracts were made as described previously
(21)
. The protein concentration of the extracts was
measured by the method of Bradford (22)
. Aliquots of the
extracts were incubated overnight at 4°C with either 10
nM [3
H]E2 or
10 nM [3
H]progesterone for ER and
PR measurements, respectively. Two hundred-fold excess of
nonradioactive hormone was used to correct for nonspecific binding.
Bound and free hormones were separated using hydroxylapatite, and
specific binding was calculated as described earlier (21)
.
Western Blot Analysis.
Aliquots of cell extracts prepared as described above were mixed with
3x SDS sample buffer, boiled for 5 min, and subjected to 10%
SDS-PAGE. After transfer to nitrocellulose membranes, immunoblotting
with either the antimouse monoclonal antibody to human ER (H222; 1:500
dilution in 1% Carnation nonfat milk; a gift from Abbott laboratories)
or antimouse monoclonal antibody to the human BRCA1 protein (C-20; 2
µg/ml in 1% Carnation nonfat milk; from Santa Cruz Biotechnology)
was carried out as described previously (19)
. The blots
were then probed with a horseradish peroxidase-conjugated antimouse
secondary antibody, and the immunoreactive bands were detected using an
enhanced chemiluminescence (ECL) kit obtained from Amersham (Arlington
Heights, IL). High molecular weight markers from Life Technologies,
Inc. (Grand Island, NY) were used to estimate the sizes of the
immunoreactive bands.
Northern Blot Analysis.
Total RNA was isolated from cells treated with either ethanol vehicle
or 1,25(OH)2D3 as described
previously (23
, 24)
. Changes in ER mRNA levels
attributable to 1,25(OH)2D3
treatment were detected by Northern blot analysis. A 2.1-kb
EcoRI fragment of the human ER cDNA was subjected
to random prime labeling using [32P]dCTP and
the Rediprime labeling kit (Amersham), and the labeled fragment was
used to probe the blots. To control for differences in RNA sample
loading and transfer, the blots were also hybridized with a
32P-labeled, 0.9-kb EcoRI fragment of
the gene encoding the human L7 ribosomal protein. The membranes were
exposed to X-ray films (Hyperfilm MP) for about 17 h at -80°C.
Autoradiograms were scanned using a Molecular Dynamics Computing
densitometer (model 300A; Molecular Devices, Menlo Park, CA), and the
ER mRNA levels were indexed to the corresponding L7 mRNA levels.
Measurement of ER mRNA Half-Life.
To determine the half-life of ER mRNA, MCF-7 cells were grown as
described earlier and treated with ethanol (control) or 100
nM 1,25(OH)2D3
for 24 h. At the end of 24 h, transcription was terminated by
the addition of 4 µM actinomycin D. Because the reported
half-life of ER mRNA is 
4 h, total RNA was extracted at regular time
intervals up to 6 h after actinomycin treatment. Twenty µg of
total RNA were used for Northern blot analysis as described earlier.
Transcriptional Run-On Assay.
Nuclei were isolated from MCF-7 cells treated with 100 nM
1,25(OH)2D3 for various
time intervals according to the procedure described by Stott
(25)
. Briefly, MCF-7 cells treated with
1,25(OH)2D3 or ethanol
vehicle were harvested at various time intervals and resuspended in
ice-cold nuclei isolation buffer [10 mM Tris-HCl (pH 7.4),
10 mM NaCl, 5 mM MgCl2,
and 1 mM DTT] containing 0.5% NP40. The intact nuclei
were then pelleted by centrifugation at 2000 rpm for 5 min. The pellets
were resuspended in nuclei freezing buffer [50 nM Tris-HCl
(pH 8.5), 50% w/v glycerol, 5 mM
MgCl2, and 0.1 mM EDTA] in aliquots
of 108 nuclei/ml and frozen as aliquots of 210
µl at -70°C until needed. RNA elongation was carried out as
described earlier (25)
. Frozen aliquots of nuclei were
thawed on ice and incubated with [32P]UTP and
unlabeled ATP, CTP, and GTP at 30°C for 45 min. The radiolabeled
nascent RNA transcripts were isolated using TRIzol reagent, followed by
chloroform extraction and ethanol precipitation. Isolated RNA was then
hybridized to nitrocellulose filters containing cDNAs for ERand L7 for 72 h at 65°C. Filters were then
washed and exposed to X-ray film. Autoradiographs were scanned, and
results were normalized by comparison to the transcriptional level of
L7.
Statistical Analysis.
Data are presented as mean ± SD of three to four individual
measurements. Statistical analysis was done by Student’s t
test or ANOVA using the Statview 4.5 software (Abacus Concepts,
Berkeley, CA). P <0.05 is considered significant.
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RESULTS |
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demonstrates the effect of
1,25(OH)2D3 and its
analogues on the growth of MCF-7 cells cultured in medium containing
10% calf serum.
1,25(OH)2D3 and each of the
analogues tested caused a dose-dependent decrease in the cell growth.
The analogues were more potent inhibitors of growth than
1,25(OH)2D3, with the order
of potency being Ro 27-0574 > KH-1060 > EB-1089 > Ro 23-7553 >
1,25(OH)2D3. The calculated
IC50 values are shown in Table 1
.
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shows the effect of
1,25(OH)2D3 and its
analogues on growth of MCF-7 cells cultured in phenol red-free RPMI
containing 10% CSS in the absence or presence of 10 nM
E2. The total DNA content in these controls was
slightly less than that observed with the controls grown in medium
containing 10% calf serum (31 ± 6.6 versus 43 ±
0.42 µg DNA/well).
1,25(OH)2D3 and its
analogues demonstrated a higher degree of growth inhibition in medium
containing serum (Fig. 1)
than medium containing CSS (Fig. 2)
. From
Fig. 2
, E2 treatment caused a 2-fold increase in
growth (P <0.001) when compared with controls. Significant
decreases in DNA content were seen after cotreatment with
E2 and
1,25(OH)2D3 or its
analogues when compared with E2 treatment alone.
1,25(OH)2D3 and its
analogues were able to partially or completely counteract the
E2-mediated increase in growth, with the
analogues being more potent than
1,25(OH)2D3.
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, %1,25(OH)2D3 induced a
dose-dependent decrease in ER levels, which was modest at 1 and 10
nM and higher (50%) at 100 nM
(P < 0.001). As in the proliferation experiments, the
analogues were more potent than
1,25(OH)2D3, with the order
of potency being Ro 27-0574 > KH-1060 > EB-1089 > Ro
23-7553 >
1,25(OH)2D3.
IC50 values for ER down-regulation are shown in Table 1
.
Data in Table 1
demonstrate that the antiproliferative effect of
1,25(OH)2D3 or a given
analogue correlated very well (R2 =
0.98) with ER suppression.
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A). At this time point, the ER levels declined by 50%
(from 479 ± 41 to 213 ± 37 fmol/mg protein). This decrease
was transient, because by day 4, the decrease in ER was less
pronounced, and by day 6, ER levels were back to that seen in control
cells. Scatchard analysis (data not shown) of
[3
H]E2 binding revealed
that 1,25(OH)2D3 treatment
for 2 days caused a decrease in ER abundance (Nmax) from 492
fmol/mg protein to 251 fmol/mg protein, with no change in the affinity
for E2 (Kd =
0.70 nM in controls versus 0.62
nM in
1,25(OH)2D3-treated cells).
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B) revealed a pattern of
changes similar to that observed with ligand binding studies (Fig. 4
A). A 66-kDa band representing the immunoreactive ER
protein could be detected in both treated and untreated groups. A
significant decrease (50%) in the ER protein levels was seen at the
end of 2 days in the
1,25(OH)2D3-treated cells.
The ER protein levels remained suppressed (30%) at day 4 and
gradually returned to control levels by the end of 6 days. The
experiment was repeated three times to confirm the pattern of changes
and to rule out possible differences attributable to loading.
Because the growth-inhibitory effects of
1,25(OH)2D3 and its
analogues were more evident in medium containing serum (Fig. 1)
, it was
important to establish the down-regulation of ER in serum containing
medium. Fig. 4
C demonstrates the effect of
1,25(OH)2D3 and its
analogues on ER levels in the presence of medium containing 10% calf
serum. A significant fall in ER levels could be seen with
1,25(OH)2D3 and all of the
analogues tested. Expression of actin, which was used as a control, did
not change. Thus, ER down-regulation by
1,25(OH)2D3 or its
analogues could be seen in medium containing CSS as well as regular
serum. These results correlate with the observations from ligand
binding studies (Fig. 4
A) and growth assays (Fig. 2)
,
confirming that the analogues were more potent than
1,25(OH)2D3 in both assays
(Table 1)
.
Effect of 1,25(OH)2D3 on ER mRNA.
To determine whether the changes in ER attributable to
1,25(OH)2D3 treatment
occurred at the mRNA level, we examined the steady-state levels of ER
mRNA by Northern blot analysis. Fig. 5
A is a representative Northern blot and Fig. 5
B
its corresponding densitometric scan. All experiments
(n = 4) were conducted in the presence of CSS to
minimize the effects of E2 present in the medium.
1,25(OH)2D3 decreased ER
mRNA (60%; P < 0.001) only at 24 h. The
decreased levels of ER mRNA were transient, with ER mRNA returning to
near normal levels by 48 h. Data from Northern blot analysis
correlate with the observations from Western blot analysis and ligand
binding studies, which showed an 50% drop in the ER protein levels
after 48 h of treatment.
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, inhibition of protein synthesis by cycloheximide did not prevent the
1,25(OH)2D3-mediated
decrease in steady-state levels of ER mRNA compared with either the
ethanol-treated cells or the cells treated with only cycloheximide. An
75% decrease in ER mRNA was seen 24 h after treatment with
1,25(OH)2D3 (Fig. 6
,
Lanes 2 and 4), suggesting that ongoing protein
synthesis is not necessary for the down-regulation of ER mediated by
1,25(OH)2D3.
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that the half-life of ER mRNA did not decrease with
1,25(OH)2D3 treatment;
rather, a small increase was seen. The half-life was 4.5 h in the
controls versus 6 h in the
1,25(OH)2D3-treated cells,
a difference that was not statistically significant, suggesting that
1,25(OH)2D3 had no effect
on ER mRNA turnover.
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demonstrates a decrease in the level of ER gene
transcription with
1,25(OH)2D3 treatment.
ER transcription decreased at 6 h (50% inhibition) and
reached a nadir at 17 h (80% inhibition) before rising back to
control levels at 48 h. The decrease was specific for ERtranscription because L7 did not change with
1,25(OH)2D3 treatment.
These data suggest that
1,25(OH)2D3 down-regulates
ER gene expression at the transcriptional level.
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Fig. 9
A shows the changes in PR induction by
E2 in response to
1,25(OH)2D3 treatment. Cell
extracts were made at the end of 2 days of treatment with
E2,
1,25(OH)2D3, or a
combination of both; and PR levels were determined.
1,25(OH)2D3 by itself did
not have any effect on PR levels. A 6-fold increase in PR levels was
seen at the end of 2 days of E2 treatment when
compared with controls. E2 failed to induce a
measurable increase in PR in the presence of
1,25(OH)2D3, suggesting
that this functional response of ER was suppressed in the presence
1,25(OH)2D3.
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B). Detectable
increases in BRCA1 levels were seen at the end of 2 days of
E2 treatment (Lane 3).
1,25(OH)2D3, on its own,
did not have any effect on the levels of BRCA1 protein (Lane
2). However,
1,25(OH)2D3 suppressed the
estrogen-mediated rise in BRCA1 levels (Lane 4). No changes
could be seen in the levels of actin used to control for loading
differences. |
DISCUSSION |
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(28)
.
Although there have been reports indicating that ERß may also be
expressed in MCF-7 cells (29
, 30)
, we could not detect any
ERß mRNA by reverse transcription-PCR in our cultures of MCF-7 cells.
Hence, we have interpreted our observations on the assumption that the
MCF-7 cells used in this study express only ER. Further
investigations are needed to ascertain whether these observations can
be extrapolated to other breast cancer cell lines. It will also be of
great interest to study the effects of
1,25(OH)2D3 on ER
regulation in cells that express both the and ß isoforms.
In our studies with MCF-7 cells,
1,25(OH)2D3 and its
structural analogues clearly demonstrate dose-dependent
antiproliferative properties (Fig. 2)
. These results confirm earlier
observations (10
, 11
, 13
, 14
, 31, 32, 33, 34, 35)
and show that the
analogues, in addition to being less calcemic (36
, 37)
,
are more potent than
1,25(OH)2D3 in their
ability to inhibit growth of MCF-7 cells. The growth-inhibitory effects
are more evident in serum containing medium than medium containing CSS.
Furthermore, 1,25(OH)2D3
and its analogues are antiproliferative, even in the presence of added
E2. Although
1,25(OH)2D3 and Ro 23-7553
could only partially counteract the E2-mediated
growth of MCF-7 cells, the other analogues are clearly more potent
because they are able to completely abolish the stimulatory effects of
E2.
We next investigated whether the antiproliferative effects of 1,25(OH)2D3 on the MCF-7 cells could be attributable to its action to down-regulate ER levels. Although previous studies have shown that 1,25(OH)2D3 down-regulates ER protein, the results in different studies have been variable (13 , 14) . Using ligand binding studies and Western blot analysis, we have demonstrated significant down-regulation of ER levels in medium containing CSS as well as in the presence of complete serum. There is a high degree of correlation (R2 = 0.98) between the ability of the different 1,25(OH)2D3 analogues to inhibit cell growth and their ability to decrease ER levels. These results suggest that the actions of 1,25(OH)2D3 and its analogues to inhibit cell growth might be, in part, attributable to their ability to down-regulate ER levels. However, because 1,25(OH)2D3 has also been shown to inhibit the growth of ER-negative breast cancer cell lines (35) , we emphasize that ER down-regulation is only one of several pathways through which 1,25(OH)2D3 and its analogues act to inhibit breast cancer cell growth.
In an attempt to elucidate the mechanism of ER regulation, we studied
the effect of 1,25(OH)2D3
on the steady-state levels of ER mRNA and ER gene transcription. The
steady-state levels of ER mRNA are decreased 60–80% (Figs. 5
A and 6) by
1,25(OH)2D3, indicating
that the regulation occurs at the mRNA level.
1,25(OH)2D3 does not
significantly alter the half-life of ER mRNA, and the decrease in ER
mRNA is not dependent on new protein synthesis, suggesting that the
1,25(OH)2D3 effects on ER
are transcriptional rather than posttranscriptional. This hypothesis is
confirmed by nuclear run-on assays, which demonstrate significant
decreases in ER gene transcription after
1,25(OH)2D3 treatment. The
magnitude of changes in transcription rate is more pronounced, and they
seem to occur earlier than those observed with the changes in
steady-state ER mRNA levels. The trends, however, are similar. Taken
together, these data suggest that the predominant mechanism of
1,25(OH)2D3-mediated
down-regulation of ER gene expression is at the
transcriptional level.
Evidence from our study supports the hypothesis that the 1,25(OH)2D3-bound VDR interacts directly with a nVDRE in the ER gene promoter inhibiting its transcription. Further support for this hypothesis will require the identification of an nVDRE in the ER gene promoter. nVDRE sequences have been described in other genes (38 , 39) .
Coincident with decreases in ER levels, the functional responses to
E2 are also attenuated because of
1,25(OH)2D3 treatment (Fig. 9)
. It was demonstrated earlier (17)
that EB-1089, a
potent analogue of vitamin D, down-regulates ER expression in MCF-7
cells and limits E2 responsiveness measured as
the induction of PR protein and pS2 mRNA. In our study, the
E2 induction of PR protein is completely
inhibited by 1,25(OH)2D3,
although there is only a 50% decrease in ER levels. Thus, the
attenuation of the ER functional response is greater than that of ER
down-regulation. One possible explanation for this finding is that
1,25(OH)2D3 might act at
multiple sites in the E2-mediated pathway.
Demirpence et al. (16)
have demonstrated that
1,25(OH)2D3 decreases ER
binding to an ERE element, suggesting that in addition to ER
regulation, 1,25(OH)2D3 has
other sites of action in the ER pathway to inhibit
E2-mediated transactivation of target genes.
We also studied the effect of
1,25(OH)2D3 on BRCA1
expression, which has been implicated in the development and/or
progression of hereditary breast cancer (40)
. In addition
to its role as a tumor suppression gene (41
, 42)
,
BRCA1 has been shown to be regulated by
E2 (43
, 44)
and also linked to cell
cycle events (45)
. Marks et al.
(46)
have suggested that the increased expression of BRCA1
with E2 treatment is attributable to an increase
in the percentage of cells undergoing DNA synthesis. If this is indeed
the case, a decrease in BRCA1 levels would be expected with
1,25(OH)2D3 treatment,
because 1,25(OH)2D3
inhibits MCF-7 cell proliferation (Fig. 1)
. However, Campbell et
al. (47)
have shown that vitamin D analogues increase
the expression of BRCA1 in MCF-7 cells. In our experiments, BRCA1
expression remained unchanged with
1,25(OH)2D3 treatment
alone. However, 1,25(OH)2D3
caused a significant reduction of the E2-mediated
increase in BRCA1 protein expression, probably because of the
down-regulation of ER.
The ability of 1,25(OH)2D3 and its analogues to down-regulate ER levels and suppress E2 actions may have important clinical ramifications. 1,25(OH)2D3 and its analogues have received increasing attention as potential therapeutic agents in the treatment and/or prevention of cancers in a number of organs including breast, colon, and prostate. However, 1,25(OH)2D3 given in pharmacological doses routinely induces hypercalcemia, thus limiting its use in a clinical setting (48) . Because the analogues are more potent in addition to being less calcemic, it is hoped that they will exhibit improved efficacy with reduced side effects as anticancer agents.
In conclusion, our evaluation of the effects of 1,25(OH)2D3 on MCF-7 cells is based on two different actions: (a) its effect to down-regulate ER expression; and (b) its effect to inhibit growth. Our data show a high degree of correlation between the antiproliferative properties of 1,25(OH)2D3 and its analogues and their ability to decrease ER expression. On the basis of these observations, we speculate that some of the growth-inhibitory actions of 1,25(OH)2D3 and its analogues in ER(+) cells are linked to their ability to down-regulate ER levels. The major mechanism of this effect is a suppression of the ER gene transcription. It is likely that 1,25(OH)2D3 acts at several points along the estrogen response pathway, affecting the levels of ER as well as their ability to function as enhancers of transactivation. This study adds down-regulation of ER to the many postulated mechanisms by which 1,25(OH)2D3 inhibits breast cancer cell growth.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grant DK42482 and Department of
the Army Grant DAMD 17-98-8556. 
2 To whom requests for reprints should be
addressed, at Division of Endocrinology, SUMC, Room S-005, Stanford, CA
94305-5103. Phone: (650) 725-2910; Fax: (650) 725-7085. E-mail: feldman{at}cmgm.stanford.edu
3 The abbreviations used are: ER, estrogen
receptor; E2, 17ß-estradiol;
1,25(OH)2D3, 1,25-dihydroxyvitamin
D3; VDR, vitamin D receptor; nVDRE, negative vitamin D
response element; ERE, estrogen response element; CSS,
charcoal-stripped serum; PR, progesterone receptor.
Received 2/14/00; revised 5/ 5/00; accepted 5/11/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNote Added in ProofREFERENCES |
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