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W. Alton Jones Cell Science Center (K.V., L.F., M.T., J.W.), Lake Placid, New York 12946; Clarkson University (K.V.), Potsdam, New York 13699 University College Dublin (L.F.), Belfield, Ireland; and Leo Pharmaceutical Products (L.B.), Ballerup, Denmark
Address all correspondence and requests for reprints to: Dr. JoEllen Welsh, W. Alton Jones Cell Science Center, 10 Old Barn Road, Lake Placid, New York 12946. E-mail: jwelsh{at}cell-science.org
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3], the biologically active form of vitamin D3 (cholecalciferol), is a potent negative growth regulator of both estrogen-dependent and -independent breast cancer cells in vitro (3, 4, 5, 6, 7). The vitamin D3 receptor, like the estrogen receptor, is a member of the steroid/thyroid/retinoic acid family of nuclear receptors. Although the estrogen receptor is present in only two thirds of breast tumors, the vitamin D3 receptor is present in over 80% of tumors and does not necessarily colocalize with the estrogen receptor (4, 8). Vitamin D3-based therapeutics thus offer promise as either adjunctive agents for estrogen-dependent tumors or alternative agents for estrogen-independent tumors.
Although it is clear that vitamin D3 compounds inhibit the growth of both estrogen receptor-positive and estrogen receptor-negative breast cancer cells (3, 4, 5, 6, 7), the precise mechanism of its effects is unclear. We initially demonstrated that 1,25-(OH)2D3 induces characteristic features of apoptosis, such as chromatin condensation, nuclear matrix degradation, and DNA fragmentation, in MCF-7 cells in vitro (9, 10). Subsequently, we and others reported that apoptosis in breast cancer cells treated with 1,25-(OH)2D3 or its synthetic analogs is associated with up-regulation of proteins linked to apoptosis in the mammary gland (such as clusterin and cathepsin B) and down-regulation of Bcl-2, an antiapoptotic protein (11, 12, 13, 14).
Although 1,25-(OH)2D3 exerts potent antiproliferative effects in vivo, chronic administration induces undesirable hypercalcemic side-effects (4, 15). For this reason, synthetic vitamin D3 compounds have been developed that mimic the antiproliferative effects of 1,25-(OH)2D3 with less calcemic activity (12, 13, 16, 17, 18). Previous studies have demonstrated the efficacy of the synthetic vitamin D3 analog EB1089 (Leo Pharmaceuticals, Ballerup, Denmark) in reducing the growth of breast cancer cells and tumors in vitro and in vivo (12, 17, 18, 19). Among several vitamin D3 analogs investigated, EB1089 exhibited the best profile for inhibition of nitrosomethylurea-induced rat mammary tumors in the absence of hypercalcemia (20). The first objective of the studies described here was to determine whether EB1089 could modulate the growth of human breast tumors using a nude mice xenograft model. Our second objective was to determine whether the antitumor effects of EB1089 in human breast tumors involve activation of apoptosis. Our data demonstrate that EB1089 significantly reduces the growth of established human breast tumors by enhancing apoptosis and reducing proliferation of tumor epithelial cells. These data emphasize the potential effectiveness of vitamin D3-based therapeutics for induction of apoptosis in human breast cancer.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MEM containing
5% FBS (Life Technologies, Grand Island, NY). Both cell lines were
grown in T-150 flasks and yielded 5–10 x 106
cells/flask depending on confluence. For inoculation into nude mice,
cells were washed with PBS, trypsinized, resuspended in MEM, and
pooled. After centrifugation, cells were resuspended in Matrigel
(Collaborative Biomedical, Waltham, MA)-MEM (4:1).
Nude mouse xenograft model
Three series of studies were conducted to examine the effects of
EB1089 on growth and apoptosis of MCF-7 xenografts. In all studies,
ovariectomized Ncr nu/nu mice (Taconic Farms, Germantown,
NY) were implanted sc with 17ß-estradiol-sustained release pellets
(Innovative Research, Sarasota, FL). The mice were fed a low calcium
(0.1%), purified rodent chow (Purina Test Diets, Richmond, IN) for the
duration of the study to minimize the calcemic effects of vitamin
D3 analog treatment. Mice were inoculated sc with
approximately 5 x 106 MCF-7 or MCF-7D3Res
cells suspended in 0.3 ml Matrigel-MEM. The tumor take rate ranged
from 95–100%. Tumor volumes were monitored weekly by caliper
measurement of the length, width, and height and were calculated using
the formula for a semiellipsoid (4/3
r3/2). After 3
weeks, mice bearing tumors with volumes averaging approximately 200
mm3 were randomized for treatment. Because of the
variations in tumor take and initial tumor growth as well as the
removal of mice for analysis at various time points, the number of mice
at each time point varied from experiment to experiment. The number of
mice analyzed is reported in the text or figure legends.
In the first series of studies, MCF-7 tumor-bearing mice were treated with EB1089 at a dose of 60 pmol/day. This dose was chosen based on preliminary studies in nontumor-bearing BALB/c mice, which indicated that doses up to 90 pmol/day could be tolerated with little weight loss or elevation of serum calcium when animals were fed the low calcium diet (data not shown). EB1089 was suspended in 80% propylene glycol-20% PBS and administered daily via sc injection. Control mice received daily injections of the vehicle alone. In a third group of tumor-bearing mice, which served as a positive control, estradiol pellets were removed to induce apoptotic tumor regression, as reported by Kyprianou et al. (22). Body weights and tumor volumes were monitored weekly, and mice were killed after 2–5 weeks of treatment. At the termination of treatment, mice were anesthetized with sodium pentobarbital, and blood was collected by cardiac puncture for serum calcium determination. Tumors were removed, weighed, and fixed in 4% formalin for histological analysis.
In the second series of studies, the dose of EB1089 was lowered to 45 pmol/day, because mice given 60 pmol EB1089/day experienced weight loss and hypercalcemia. In this experiment, mice bearing MCF-7 tumors were randomized into control (n = 14), EB1089-treated (n = 16), and estradiol withdrawal (n = 3) groups. To determine whether EB1089 induced nonspecific or indirect effects on tumor growth kinetics, tumors were also established from a vitamin D3-resistant variant of MCF-7 cells, termed MCF-7D3Res cells. We have previously demonstrated that MCF-7D3Res cells are resistant to the growth inhibitory effects of EB1089 in vitro (21). Mice bearing MCF-7D3Res tumors were randomized into control (n = 8) and EB1089-treated (n = 6) groups in one experiment and into control (n = 3) and estradiol withdrawal (n = 3) groups in another trial. The experimental designs for these studies were otherwise identical to those described for studies employing the 60 pmol/day dose.
To investigate whether EB1089 could be administered via sustained release pellets similar to those used for estrogen supplementation, a preliminary study was conducted with custom-made pellets (Innovative Research) designed to continuously release 30, 45, or 60 pmol EB1089/day for 5 weeks. MCF-7 xenografts were established from MCF-7 cells, and EB1089 or placebo pellets were implanted ip under sodium pentobarbital anesthesia 3 weeks after inoculation. Tumor volumes were measured, and treatment was terminated after 5 weeks. Due to the small numbers of mice in each group (EB1089 pellets, n = 3 for 30 pmol and n = 1 each for 45 or 60 pmol; control, n = 4), the data for all three doses were combined in the analysis.
Histological analysis of tumors
Tumors were embedded in paraffin, sectioned at 5 µm, and
stained with hematoxylin (Gill’s formulation 3, Fisher Scientific,
Fairlawn, NJ) and eosin Y (Sigma Chemical Co., St. Louis, MO). The
epithelial nature of the tumors was verified by immunostaining with
antibodies directed against epithelia-specific antigen and cytokeratin
18 (data not shown). The mitotic index and apoptotic index were
assessed by quantitative morphometric analysis of proliferating cell
nuclear antigen (PCNA) expression and in situ terminal
transferase-mediated fluorescein deoxy-UTP nick end labeling (TUNEL),
two established markers of proliferation and apoptosis. For PCNA
localization, formalin-fixed, paraffin-embedded sections were incubated
for 30 min with a mouse monoclonal anti-PCNA (Nova Castra Laboratories,
Newcastle Upon Tyne, UK) at a 1:100 dilution in 1% BSA-PBS. A
biotin-conjugated antibody to mouse IgG (Vector Laboratories,
Burlingame, CA) was applied at a 1:200 dilution for 30 min in 1%
BSA-PBS. The ABC technique was used (avidin and biotinylated
horseradish peroxidase complex, Vector) followed by diaminobenzidine
(Sigma) to localize peroxidase in the sections, and the sections were
counterstained with hematoxylin (Harris modified, Fisher). DNA
fragmentation was assessed by TUNEL, using the commercially
available assay according to manufacturer’s directions (Boehringer
Mannheim, Indianapolis, IN). In these sections, nuclei were
counterstained with Hoechst 33258 dye (Sigma).
Quantitation of apoptosis and proliferation
PCNA expression and TUNEL were quantitated by viewing and
photographing random fields of each tissue section on a Nikon
Optiphot-2 microscope and Nikon Microflex UFX-IIA photomicrographic
attachment (Nikon Corp., Melville, NY), using a x40 objective. The
photographs were scanned and analyzed with the University of Texas
Health Science Center at San Antonio Image Tool program, and the
percentage of cells staining positively for PCNA or TUNEL was
calculated. For both TUNEL and PCNA, 2–6 fields of view (containing at
least 250 cells) were quantitated on each section, with 4–8 samples
evaluated for each treatment per time point.
Statistical analyses
Statistical comparisons were performed using Student’s unpaired
t test (for two groups) or one-way ANOVA for more than two
groups. Data are expressed as the mean ± SE, and
differences between means were considered significant at
P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. In control mice,
average tumor volume increased rapidly over the 3-week treatment
period. In EB1089-treated mice, tumor volume increased during the first
week of treatment and then plateaued, with no further increase in tumor
volume between weeks 2–3. In control mice, the mean change in tumor
volume between the first and third weeks was 272.3 ± 113.2
mm3 (n = 4) compared with 3.6 ± 15.6
mm3 (n = 5) for tumors from EB1089-treated mice
(P < 0.05). Tumor volume after 2 weeks was
significantly lower in the EB1089 group (351.2 ± 80.4
mm3; n = 10) than in the control group (631.1 ±
65.9 mm3; n = 7). In mice subjected to removal of the
estradiol supplementation, tumors regressed rapidly, becoming
undetectable within 3 weeks, demonstrating the estrogen dependence of
the MCF-7 xenografts.
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).
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Temporal changes in tumor volume for animals bearing MCF-7 tumors and
treated with 45 pmol/day EB1089 or vehicle are shown in Fig. 2. This graph shows mean tumor volumes for all animals
that completed the 5-week study protocol (i.e. not including
tumors that were removed for histological analysis at various times).
Consistent with the data shown in Fig. 1
for the 60 pmol/day dose, the
growth of MCF-7 tumors in mice treated with 45 pmol/day EB1089 was
slower than that of tumors in vehicle-treated control animals from 1
week on. The mean change in tumor volume between the first and fifth
weeks was 366.6 ± 53.6 mm3 (n = 6) in control
mice compared with 53.2 ± 56.9 mm3 (n = 6) in
EB1089-treated mice (P < 0.01). Tumor volume after 5
weeks was significantly (P < 0.01) lower in the
EB1089-treated group (428.6 ± 274.0 mm3, n = 6)
than in the control group (1716.0 ± 217.7 mm3, n
= 6). Final tumor weight was significantly (P < 0.01)
lower in EB1089-treated mice (0.43 ± 0.27 g; n = 6)
than in control mice (1.52 ± 0.19 g; n = 6). These data
indicate a good correlation between tumor volume assessed by caliper
measurement in live mice and actual tumor size measured after death.
The antitumor effect of EB1089 persisted over the entire 5-week
experiment. In addition, although not readily evident from the graph
(which shows mean tumor volume), two MCF-7 tumors completely regressed
in response to EB1089 treatment. Tumors that regressed were monitored
for up to 2 months in the absence of EB1089 treatment, and no regrowth
was observed (data not shown). In contrast, spontaneous regression was
never observed in tumors from control mice.
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). There were no significant differences in
MCF-7D3Res tumor volumes of control or EB1089-treated mice
at any time point throughout the 5-week experiment. As demonstrated in
Fig. 3b, MCF-7D3Res tumors underwent rapid regression in
response to estradiol withdrawal. Thus, despite resistance to the
antitumor effects of EB1089, tumors derived from the
MCF-7D3Res cells retained the ability to undergo regression
in response to estradiol deprivation, which is known to induce
apoptosis in MCF-7 tumors (22).
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, although serum
calcium was elevated in mice treated for 5 weeks with 45 pmol/day
EB1089 compared with that in control mice, the levels were not as high
as those in mice receiving 60 pmol/day (Table 1). Furthermore, the
significant loss of body weight that was evident with the 60 pmol/day
treatment was not observed when EB1089 was given at the 45 pmol/day
dose. Corrected final body weight (mouse body weight minus tumor
weight) was similar for EB1089-treated and control mice. There were no
significant differences in body weight or serum calcium in mice bearing
tumors derived from MCF-7 cells compared with those derived from
MCF-7D3Res cells regardless of treatment with EB1089 (data
not shown). As tumors derived from MCF-7D3Res cells did not
respond to EB1089 treatment, the antitumor effect of EB1089 in MCF-7
tumors is probably not due to indirect effects of the vitamin
D3 analog on body weight or calcium homeostasis.
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. Tumors from vehicle-treated mice
were primarily composed of tumor epithelial cells, with small amounts
of mouse-derived stroma and frequent blood vessels. The majority of
epithelial cells in control tumors were quiescent, although mitotic
figures were visible in most sections. Tumors from mice treated with 60
pmol/day EB1089 were composed primarily of quiescent epithelial cells,
with few mitotic figures. In many areas, epithelial cells with classic
apoptotic morphology (condensed cells with pyknotic nuclei) were
frequent. Many EB1089-treated tumors displayed large areas of stroma
where deletion of epithelial cells had occurred. The extent of
vascularization appeared equivalent in tumors from EB1089-treated and
control mice.
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. Nuclei of epithelial cells in
MCF-7 tumors from control mice exhibited normal morphology and were
generally negative for DNA fragmentation. In contrast, sections of
tumors from EB1089-treated mice exhibited a high prevalence of
condensed, irregularly shaped nuclei that were positive for DNA
fragmentation. Tumors from mice that had been subjected to estradiol
withdrawal also exhibited irregularly shaped, condensed nuclei that
were positive for DNA fragmentation. The morphology of tumors from
estradiol-deprived mice was similar to that of tumors from
EB1089-treated mice, although the extent of apoptosis was higher after
estradiol deprivation than after EB1089 treatment (Fig. 5). This
finding is consistent with the more pronounced tumor regression in mice
subjected to estradiol withdrawal compared with that in mice given
EB1089 treatment (
Figs. 1–3). Quantitative analysis indicated an
increase in the percentage of TUNEL-positive cells in tumors from
EB1089-treated mice compared with that in tumors from control mice at
all time points examined (Table 3). After
2 weeks of treatment, the higher dose of EB1089 (60 pmol/day) was
associated with a more pronounced increase in apoptotic index than the
45 pmol/day dose. After 5 weeks of treatment, however, both doses of
EB1089 were equivalent, with tumors from treated mice exhibiting a
6-fold increase in the percentage of cells positive for DNA
fragmentation compared with that in control tumors.
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). In control tumors, PCNA expression was detected in
the nucleus of approximately half of all epithelial cells. A decrease
in PCNA expression was detected in tumors from EB1089-treated mice at
all time points examined. Quantitation of PCNA expression after 2 weeks
indicated a 2.5-fold reduction in PCNA staining in EB1089-treated
tumors compared with control tumors (Table 3). Treatments with 45 and
60 pmol/day EB1089 were equally effective in down-regulation of PCNA
expression at both time points. The reduced expression of PCNA in
EB1089-treated tumors compared with that in control tumors was
maintained throughout the 5 weeks of treatment.
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, administration of EB1089 via
sustained release pellets elicited a similar antitumor response, as
observed in Figs. 1 and 2, for 60 and 45 pmol sc injections. Tumors
from mice implanted with EB1089 pellets grew at a slower rate than
tumors from mice implanted with placebo pellets from 1 week on. The
mean change in tumor volume between the first and fifth weeks was
1594.3 ± 418.7 (n = 4) in mice bearing placebo pellets
compared with 285.8 ± 164.3 (n = 5) in mice bearing EB1089
pellets (P < 0.01), including one EB1089-treated tumor
that regressed completely. Final tumor volume at 5 weeks was
significantly (P < 0.05) lower in the EB1089 pellet
group (619.7 ± 256.0 mm3; n = 5) than in the
placebo group (2189.6 ± 554.8 mm3; n = 4). There
were no significant differences in body weights [control, 24.6 ±
0.2 g (n = 5); EB1089 pellets, 23.6 ± 1.5 g (n = 4)]
or serum calcium [control, 9.5 ± 0.2 (mg/dl) (n = 5);
EB1089 pellets, 9.6 ± 0.2 (mg/dl) (n = 4)] between
mice bearing placebo or EB1089 pellets after 5 weeks.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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0.8 µg/kg BW) were 4-fold less
than those of vehicle-treated mice. At this dose of EB1089, serum
calcium was minimally elevated, and no weight loss was observed. These
findings complement those of Colston’s group (17, 18, 19, 20), who
demonstrated that oral administration of EB1089 at doses up to 1
µg/kg BW daily slowed the growth of established
nitrosomethylurea-induced rat mammary tumors without induction of
hypercalcemia. In our nude mice studies, treatment with 60
pmol/mouse·day EB1089 had a more pronounced antitumor effect than
treatment with 45 pmol/day, but the higher dose was associated with
hypercalcemia, weight loss, and mortality. Preliminary studies using
sustained release pellets designed to continuously release EB1089 at
doses up to 60 pmol/day indicated that this mode of administration
induced antitumor effects similar to those achieved with daily
injections. These preliminary data indicated that pellet delivery of
EB1089 for 5 weeks was not associated with hypercalcemia or weight
loss. As delivery of EB1089 via pellets offers obvious advantages over
daily injections (especially when working with nude mice), additional
studies to document the actual release rate of EB1089 from pellets and
directly assess the efficacy of pellet administration relative to sc
injections are warranted. Histological examination indicated that the decreased size of tumors from EB1089-treated mice, compared with that in control mice, was associated with a reduction in the epithelial component and an increase in the stroma. The 4-fold reduction of tumor volume in mice treated with 45 pmol/day EB1089 for 5 weeks could reflect a decreased rate of cell proliferation, an increased rate of cell death, or both. Our analyses confirmed that EB1089 mediates tumor regression by modulation of both apoptosis and proliferation of tumor epithelial cells.
Quantitative morphometric analysis of DNA fragmentation indicated that tumors from EB1089-treated mice exhibited apoptotic morphology and a 6-fold increase in the percentage of TUNEL-positive cells compared with tumors from control mice. Our studies also demonstrated that MCF-7 tumor regression and DNA fragmentation induced by EB1089 were morphologically similar to tumor regression resulting from estradiol withdrawal, which is known to induce apoptosis in estrogen-dependent MCF-7 tumors (22). Our data demonstrating induction of apoptosis in MCF-7 tumors in vivo correlate with earlier findings that demonstrated that 1,25-(OH)2D3 and its structural analogs induce apoptosis in MCF-7 cells in vitro (3, 4, 9, 10, 11, 12). In addition to induction of apoptosis, EB1089-treated tumors exhibited a significant decrease in proliferation, as measured by PCNA expression, at all time points examined. These data are consistent with flow cytometric studies of MCF-7 cells in vitro, which indicated that 1,25-(OH)2D3 and EB1089 increase the percentage of cells in G0/G1 and reduce the percentage of cells in S phase (12). Thus, our in vivo results with EB1089 correlate well with the in vitro reports that vitamin D3 compounds induce both growth arrest and apoptosis in estrogen-dependent breast cancer cells (5, 11, 12, 14).
The quantitative data indicate that effect of EB1089 on tumor cell
proliferation (2- to 3-fold decrease) was less than the effect of
EB1089 on apoptosis (4- to 8-fold increase). Although actual mean tumor
volumes plateau rather than decrease in EB1089-treated mice,
histological examination indicated a reduction in epithelial cells and
replacement by stromal tissue in EB1089-treated tumors, supporting the
concept that the epithelial cell compartment has regressed by
apoptosis. An effect of EB1089 on tumor cell apoptosis is consistent
with our observation in two studies that some EB1089-treated tumors
regressed completely. As tumors that underwent complete regression in
response to EB1089 were not available for analysis, the apoptotic index
in some tumors treated with EB1089 may be even higher than that
indicated by the quantitative data presented in Table 3. Our data
support the hypothesis that EB1089 has a predominant effect on the
apoptotic cell death pathway in vivo.
Studies with xenografts derived from MCF-7D3Res cells that display resistance to EB1089 in vitro (21) demonstrate that resistance to EB1089 is maintained in vivo. Although the basis for vitamin D3 resistance in these cells is unclear, MCF-7D3Res cells (20) and tumors (data not shown) express the vitamin D3 receptor protein at levels comparable to those in MCF-7 cells and tumors. The growth rates of tumors derived from MCF-7 and MCF-7D3Res cells in the absence of treatment were comparable, indicating that tumors selected for vitamin D3 resistance are unlikely to be more aggressive than tumors that are sensitive to vitamin D3. Tumors derived from MCF-7D3Res cells displayed comparable regression in response to estradiol withdrawal, suggesting a functional apoptotic pathway in these tumors that can be activated by other strategies that induce apoptosis. This finding is consistent with our in vitro work demonstrating that MCF-7D3Res cells are resistant to EB1089 but sensitive to antiestrogens such as tamoxifen (21). We are currently examining whether tumors derived from MCF-7 and MCF-7D3Res cells exhibit comparable sensitivity to antiestrogen-induced apoptosis in vivo to further test the hypothesis that antiestrogens and vitamin D3 compounds act independently to induce apoptosis in breast cancer cells. Support for this hypothesis would suggest that for patients with mixed tumors containing estrogen-dependent and estrogen-independent cells, a distinct therapeutic advantage might be achieved by combining agents that activate vitamin D3-mediated apoptosis with those that disrupt estrogen-mediated survival signals.
In summary, our studies demonstrate that the vitamin D3 analog EB1089 induces human breast tumor regression by a mechanism that involves both activation of apoptosis and inhibition of proliferation. Our work also indicates that the pathways involved in vitamin D3-mediated apoptosis of MCF-7 tumors are distinct from the pathways that trigger apoptosis in response to estradiol withdrawal. These results support further clinical studies on the therapeutic efficacy of vitamin D3 analogs such as EB1089 against human breast cancer.
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Acknowledgments |
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Footnotes |
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Received August 22, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Hydoxyvitamin D3, hypercalcemia, and growth
suppression of 7,12-dimethylbenz[a}anthracene-induced rat mammary
tumors. Breast Cancer Res Treat 22:133–140[CrossRef][Medline]
25(OH)2D3 and its analogs in breast cancer.
In: Norman AW, Bouillon R, Thomasett M (eds) Proceedings of the Ninth
Workshop on Vitamin D. de Gruyter, Berlin, pp 477–484
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I. M. Byrne, L. Flanagan, M. P. R. Tenniswood, and J. Welsh Identification of a Hormone-Responsive Promoter Immediately Upstream of Exon 1c in the Human Vitamin D Receptor Gene Endocrinology, August 1, 2000; 141(8): 2829 - 2836. [Abstract] [Full Text] [PDF]
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K. El Abdaimi, N. Dion, V. Papavasiliou, P.-E. Cardinal, L. Binderup, D. Goltzman, L.-G. Ste-Marie, and R. Kremer The Vitamin D Analogue EB 1089 Prevents Skeletal Metastasis and Prolongs Survival Time in Nude Mice Transplanted with Human Breast Cancer Cells Cancer Res., August 1, 2000; 60(16): 4412 - 4418. [Abstract] [Full Text]
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L. Verlinden, A. Verstuyf, M. Van Camp, S. Marcelis, K. Sabbe, X.-Y. Zhao, Pierre De Clercq, M. Vandewalle, and R. Bouillon Two Novel 14-Epi-Analogues of 1,25-Dihydroxyvitamin D3 Inhibit the Growth of Human Breast Cancer Cells in Vitro and in Vivo Cancer Res., May 1, 2000; 60(10): 2673 - 2679. [Abstract] [Full Text]
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S. E. Blutt, T. C. Polek, L. V. Stewart, M. W. Kattan, and N. L. Weigel A Calcitriol Analogue, EB1089, Inhibits the Growth of LNCaP Tumors in Nude Mice Cancer Res., February 1, 2000; 60(4): 779 - 782. [Abstract] [Full Text]
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