ARTICLES
Prevention of Preneoplastic Mammary Lesion
Development by a Novel Vitamin D Analogue,
1a-Hydroxyvitamin D5
Rajendra G. Mehta, Robert M. Moriarty, Rajeshwari R. Mehta,
Raju Penmasta, Gianluca Lazzaro, Andreas Constantinou, Liang Guo*
Background: The form of vitamin D (vitamin D3) in fortified
milk and the provitamin D produced by the body undergo
metabolic activation to a biologically active form, 1a,25dihydroxyvitamin D3 [1a,25(OH)2D3]. This compound can
induce cell differentiation and can prevent proliferation of
cancer cells. However, because 1a,25(OH)2D3 is hypercalcemic (effective in increasing serum calcium level), it is not
suitable for use in cancer prevention or cancer therapy trials.
Purpose: We synthesized a vitamin D5 series analogue, 1ahydroxy, 24-ethyl-cholecalciferol, or 1a-hydroxyvitamin D5
[1a(OH)D5], and evaluated its chemopreventive activity in
carcinogen-treated mammary glands in organ culture experiments. Methods: The analogue 1a(OH)D5 was synthesized from sitosterol acetate and was characterized by
nuclear magnetic resonance. Its purity was evaluated by
high-pressure liquid chromatography. The calcemic activities of vitamin D3 and D5 analogues were determined in vitamin D-deficient Sprague-Dawley rats. Mammary glands of
BALB/c mice were placed in organ culture and treated with
the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) to
induce preneoplastic lesions. Vitamin D analogues were
added to the culture medium at four different concentrations, and formation of mammary lesions was evaluated. The
effects of 1a(OH)D5 and 1a,25(OH)2D3 on the expression of
vitamin D receptors (VDRs) and transforming growth factor-b1 (TGF-b1) were studied by immunohistochemistry.
Statistical significance was determined by the chi-squared
test. All reported P values were two-sided. Results:
1a,25(OH)2D3 was fourfold more calcemic than 1a(OH)D5
at a dose of 0.042 µg/kg per day in rats. Both 1a,25(OH)2D3
and 1a(OH)D5 inhibited the development of DMBA-induced
preneoplastic lesions in mouse mammary glands compared
with untreated glands. The effect of the vitamin D3 analogue
was observed at a much lower concentration (0.01 µM).
Treatment with 1a(OH)D5 resulted in a dose-related (0.0110.0 µM) inhibition without any toxicity, whereas the vitamin
D3 analogue was highly potent but toxic at concentrations of
1.0 µM or higher. Normal mouse mammary glands poorly
express VDR and TGF-b1; incubation with 1a(OH)D5 or
1a,25(OH)2D3 dramatically induced their expression. Conclusions: This is the first report showing the possibility of
212 ARTICLES
chemoprevention by a vitamin D5 series compound. We conclude that 1a(OH)D5 is less calcemic than 1a,25(OH)2D3. It
is nontoxic at a wide range of concentrations, but it is potent
in inhibiting the development of preneoplastic lesions in
mammary glands in organ culture. In addition, we show for
the first time the induction of TGF-b1 in normal mammary
tissues by a chemopreventive agent. Implications: 1a(OH)D5
is a good candidate for in vivo chemoprevention studies. It
may mediate its action by inducing expression of VDR and of
TGF-b1, as is seen in other systems. [J Natl Cancer Inst
1997;89:212-8]
Vitamin D is a secosteroid and is classified as a hormone
within a steroid hormone family (1,2). It has been separated on
the basis of its chemical structure into different series, e.g., D2,
D3, D4, D5, and D6. To date, attention has been focused almost
exclusively on the vitamin D3 series of compounds. In its biologic form, vitamin D3 is inactive unless it is metabolized to
1a,25-dihydroxyvitamin D3 [1a,25(OH)2D3]. The inactive 24hydroxy form of the hormone is excreted from the body (3,4).
The active metabolite 1a,25(OH)2D3 has been shown to suppress the growth in vitro of many neoplastic cells, including
breast cancer cells (5,6). In addition, treatment of colon cancer
cells and leukemia cells with 1a,25(OH)2D3 results in a reduction in the growth rate of these cells (7,8). One of the limiting
factors in the successful use of vitamin D3 in cancer prevention
or cancer therapy is its calcemic activity (9). The concentrations
of vitamin D3 required to suppress growth of neoplastic cells
cause hypercalcemia and death. Therefore, in recent years, numerous analogues of vitamin D have been synthesized that can
reduce calcemic activity without compromising its antiproliferative activity (9,10). The differences in structures of these new
compounds arise mostly from modifications in the C and D rings
*Affiliations of authors: R. G. Mehta, R. R. Mehta, G. Lazzaro, A. Constantinou (Department of Surgical Oncology, College of Medicine), R. M. Moriarty,
R. Penmasta, L. Guo (Department of Chemistry), University of Illinois, Chicago.
Correspondence to: Rajendra G. Mehta, Ph.D., Department of Surgical Oncology, College of Medicine, University of Illinois, 840 S. Wood St. (M/C 820),
Chicago, IL 60612.
See ‘‘Notes’’ following ‘‘References.’’
Journal of the National Cancer Institute, Vol. 89, No. 3, February 5, 1997
and side chain of the vitamin. No attempt, however, has been
directed toward evaluating vitamin D compounds in other series.
More work needs to be directed toward evaluating such compounds in other series.
The effectiveness of a variety of chemopreventive agents has
been evaluated by organ culture of the mouse mammary gland
(11-13). In organ culture, mammary glands from mice respond to
a short stimulation with a carcinogen in the presence of appropriate hormones by developing preneoplastic lesions (14). When
implanted in syngeneic hosts, mammary cells prepared from
these lesions give rise to adenocarcinomas (15). Effective chemopreventive agents (e.g., certain retinoids, selenium, oltipraz,
and limonene) inhibit the formation of these lesions. The relative
activity of chemopreventive agents in vitro correlates well with
their activity in in vivo carcinogenesis experiments (13,16). Using this model system, we have evaluated the efficacy of 1ahydroxyvitamin D 5 [1a(OH)D 5 ] in preventing 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary lesion
formation in a mouse mammary gland organ culture model.
The mechanism of the vitamin D action is not completely
understood. Nuclear vitamin D receptor (VDR) protein binding
to 1a,25(OH)2D3 has been identified and is shown to be present
in a variety of tissues, including normal mammary glands and
mammary tumors, as well as in breast cancer cells (17,18). In the
cytosol of target organs or cells, [3H]1,25(OH)2D3 binds specifically to receptors with a dissociation constant (Kd) ranging from
1 × 10−10 M to 6 × 10−10 M. An increased nuclear VDR concentration has been found to be associated with an enhanced
expression of messenger RNA for vitamin D3 receptors (19,20).
The VDR gene has been cloned, and the molecular structure of
the receptor protein has been determined. The results have demonstrated that the VDR belongs to the steroid-, thyroid-, and
retinoid-receptor superfamily (21). All of these receptors act as
ligand-dependent transcription factors that bind to specific DNA
sequences. Two classes of response elements have been identified that are activated either by VDR alone or by heterodimers of
VDRs and retinoid X receptor (RXR) alpha (22,23).
In recent years, considerable attention has been given to the
regulation of cell growth by autocrine antiproliferative factors
[e.g., (24)]. Inhibition of cancer cell growth is often related to
enhanced production of transforming growth factor-b (TGF-b)
(25,26). TGF-b is further subclassified into the following three
isoforms of polypeptides: TGF-b1, TGF-b2, and TGF-b3 (27).
These isoforms are present in mammalian cells, including breast
cancer cells. The isoforms of TGF-b are regulated differentially
by steroid and protein hormones. In one report (28), a hexafluoro
analogue of vitamin D3, 1a,25-dihydroxy-16-ene-23yne-26,27hexafluorocholecalciferol (Ro24-5531), induced expression in
HL-60 human leukemia cells of TGF-b1 and its type 2 receptors.
These results suggest a possible interaction between the function
of VDR and TGF-b regulation. Induction of TGF-b, however, is
often reported only in transformed cells. Although the growthinhibitory role of TGF-b has been reported in the normal mammary gland (29), induction of TGF-b in response to chemopreventive agents in this tissue has not been reported previously.
In this study, we compare the chemopreventive action of vitamin D5 with that of the active metabolite of vitamin D3 and
attempt to determine the possible mechanism of such action by
studying the expression of VDRs and TGF-b1 in normal mammary epithelial cells.
Materials and Methods
Synthesis of 1a(OH)D5. As a first step, b-sitosterol acetate was converted to
7-dehydro-b-sitosterol acetate by allelic bromination (using dibromantin and
sodium bicarbonate in hexane and refluxing for 30 minutes) and dehydrobromination (using tetrabutylammonium fluoride, s-collidine, and tetrahydrofurane and
refluxing for 6 hours). 7-Dehydro-b-sitosterol was reduced to 7-dehydro-3bsitosterol by refluxing for 6 hours with lithium aluminum hydride and tetrahydrofurane. The reaction mixture was subjected to photolysis and then to thermolysis (by ethanol reflux) to yield vitamin D5. We converted vitamin D5 to
1a(OH)D5 by following the Paaren–DeLuca hydroxylation sequence (30).
1a(OH)D5 was crystallized, and the crystalline product was fully characterized
by 1H nuclear magnetic resonance (NMR) at 400 Hz, mass spectroscopy (chemical ionization), and infrared (IR) and ultraviolet spectroscopy. The purity was
determined by high-pressure liquid chromatography (HPLC). The following
properties were recorded: melting point—150 °C-152 °C; IR (KBr) cm−1—3349
(−OH stretching) and 2955 (−CH stretching); 1H NMR—d6.35 (doublet [d],
C-6H), 6.03 (d, C-7H), 5.33 (singlet [s], C-19H), 4.44 (multiple [m], C-1H), 4.24
(m, C-3H), and 0.55 (s, C-18H3); mass spectrum (CI)—429 (M + 1, 72%), 411
(M + 1 − H2O, 100%), and 393 (M + 1 − 2H2O, 14%); and UV—l max, 265 nm
(molar extinction coefficient [e] 4 18 000).
HPLC analysis of vitamin D analogues. Vitamin D analogues were dissolved in acetonitrile at a final concentration of 0.2 mg/mL. Aliquots (10 mL)
were injected on a Suplex PKB-100 HPLC column at ambient temperature. The
HPLC was carried out with the use of an Hitachi L-6000 pump, an L-4200
UV-VIS detector, and an AS-2000 autosampler (Hitachi Instruments, Inc., Naperville, IL). Vitamin D analogues were eluted with the mobile phase of acetonitrile–methanol–water (52:30:18, vol/vol) with the flow rate at 1 mL/minute,
and the elution profile was monitored at 254 nm.
Measurement of calcemic activity. Three-week-old Sprague-Dawley male
rats were obtained from the Holtzman Laboratory, Madison, WI. Up to three rats
were housed together in a polycarbonate cage. The animal cages were kept under
yellow light. The rats (eight to 10 per group per concentration of both vitamin D
analogues used) were fed vitamin D-free diet containing 0.47 g/100 g calcium
and 0.3 g/100 g phosphorus. After the rats had consumed this diet for 3 weeks,
their plasma calcium levels were measured. Rats exhibiting plasma calcium
levels of less than 6.0 mg/dL were considered to be vitamin D deficient. Such rats
were administered appropriate vitamin D analogues intragastrically for 14 days.
At the end of this period, the plasma calcium levels were again measured.
Induction of preneoplastic lesions in mammary glands and their prevention by vitamin D3 and D5 analogues. Young, virgin BALB/c female mice, 3-4
weeks of age, were obtained from Charles River Laboratories, Wilmington, MA.
The entire culture procedure was described in detail previously (12-14). Briefly,
the mice were pretreated for 9 days with 17b-estradiol (1 mg in 0.1 mL saline per
animal) and progesterone (1 mg in 0.1 mL saline per animal). They were then
killed by cervical dislocation, and the thoracic pair of mammary glands was
dissected out on silk rafts and incubated for 10 days in Waymouth MB752
medium (Life Technologies, Inc. [GIBCO BRL], Gaithersburg, MD) containing
the following growth-promoting hormones: insulin (5 mg/mL), prolactin (5 mg/
mL), aldosterone (1 mg/mL), and hydrocortisone (1 mg/mL). The carcinogen
7,12-dimethylbenz[a]anthracene (DMBA) at a dose of 2 mg/mL was added to the
medium on day 3 for 24 hours to induce mammary lesions. The DMBAcontaining medium was then removed, and the mammary glands were incubated
for an additional 14 days with medium containing only insulin. This procedure
allowed the normal glands to undergo structural regression in which all the
normal alveolar structures were disintegrated. However, the alveolar lesions in
the carcinogen-treated glands behaved differently. They had acquired altered
hormone responsiveness, and these structures did not regress. These structures
were termed ‘‘mammary lesions.’’ The vitamin D analogues (ranging in concentration from 0.01 mM to 10.0 mM) were included in the medium during the
first 10 days of the in vitro culture to determine if they lowered the incidence of
mammary lesion formation. Throughout the culture period, the glands were
maintained at 37 °C in an environment of 95% air and 5% CO2. The procedure
is presented schematically in Fig. 1. At the end of the culture period, the glands
were fixed in formalin, stained in alum-carmine solution, and evaluated for the
presence or absence of mammary lesions. All hormones and chemicals were
purchased from the Sigma Chemical Co., St. Louis, MO.
Journal of the National Cancer Institute, Vol. 89, No. 3, February 5, 1997
ARTICLES 213
Fig. 1. Protocol for mammary gland organ culture. Thoracic pairs of the mammary glands were removed from female BALB/c mice pretreated for 9 days with
estrogen at a dose of 1 mg per animal plus progesterone at a dose of 1 mg per
animal (E + P). Glands were incubated with IPAH hormones (i.e., insulin [I] at
a dose of 5 mg/mL, prolactin [P] at 5 mg/mL, aldosterone [A] at 1 mg/mL, and
hydrocortisone [H] at 1 mg/mL for 10 days and with 7,12-dimethylbenz[a]anthracene (DMBA) at a dose of 2 mg/mL for 24 hours on the 3rd day in the
presence or absence of the vitamin D3 and the vitamin D5 analogues as chemopreventive agents (see ‘‘Materials and Methods’’ section). Glands respond to the
presence of hormones by developing alveolar structures. These normal alveolar
structures disintegrate when the hormones are withdrawn from the medium.
However, if the glands are exposed to DMBA on the 3rd day for 24 hours, as
shown in the diagram, they do not disintegrate completely in the absence of
hormones. These nonregressed alveolar structures are preneoplastic and are
called ‘‘mammary lesions.’’ The growth of these structures does not require the
presence of hormones.
Immunohistochemistry of VDRs and TGF-b1. Normal mouse mammary
glands were dissected and incubated with growth-promoting hormones either
alone or in the presence of vitamin D analogues for only 3 days. In this experiment, the glands were not exposed to DMBA (see protocol described in the
previous paragraph). The glands were fixed in buffered formalin, and 5-mm-thick
sections were prepared for histopathologic evaluations. The sections were
mounted on adhesive-coated slides (Superfast; Fisher Scientific Co., Itasca, IL),
dried at 60 °C for 1 hour, deparaffinized in xylene, dehydrated in a series of
alcohol, and finally washed with phosphate-buffered saline (PBS). To block the
nonspecific antibody reactions, we treated the tissue sections with 5% dried skim
milk for 10 minutes and then incubated them with primary mouse antibody
(either against VDR or against TGF-b1; both obtained from BioGenex Laboratories, San Ramon, CA) overnight at 0-4 °C. The tissues were rinsed in PBS and
incubated with biotinylated rabbit anti-mouse antibody (Dako Corp., Carpenteria, CA) for 10 minutes; the remaining steps were followed according to the
manufacturer-specified protocol; i.e., the reaction was stopped by rinsing the
sections with PBS, which was followed by 10 minutes’ incubation with peroxidase-conjugated streptavidin, three 10-minute rinses with PBS, and a 5-minute
incubation in a substrate, 3,38-diaminobenzidine tetrachloride. The tissues were
counterstained with hematoxylin–eosin, dehydrated through graded series of alcohol and xylene, and finally mounted in Permount (Fisher Scientific Co.). Slides
were evaluated for the presence or absence of the VDR or TGF-b1 and for the
intensity of staining in the positively stained samples.
Statistical analysis. Statistical significance was determined by the chi-squared
test. All reported P values were obtained from two-sided tests.
Results
We synthesized highly purified 1a(OH)D5. The chemical
structures of 1a,25(OH)2D3 and 1a(OH)D5 are shown in Fig. 2.
The major differences between these two molecules are the lack
of hydroxylation at the C-25 position and the presence of ethyl
group at the C-24 position in the newly synthesized vitamin D5
analogue. The purity of these compounds was evaluated by
HPLC analysis. The purity specifications are described in the
‘‘Materials and Methods’’ section. As shown in Fig. 2,
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Fig. 2. Chemical structures and high-pressure liquid chromatography (HPLC)
profiles of vitamin D analogues. The agents were dissolved in acetonitrile (200
mg/mL), and 10-mL aliquots were injected on a Suplex PKB-100 HPLC column.
The HPLC conditions are described in the ‘‘Materials and Methods’’ section.
The retention time for 1a,25-dihydroxyvitamin D3 [1a,25(OH)2D3] was about 5
minutes, whereas that for 1a-hydroxyvitamin D5 [1a(OH)D5] was about 34
minutes. Two plots were created with different chart speeds. Since the retention
time was much longer for 1a(OH)D5, the chart speed was reduced compared
with that required for 1a,25(OH)2D3.
1a,25(OH)2D3 and 1a(OH)D5 were eluted with retention times
of approximately 5 and 34 minutes, respectively. Both of these
vitamin D analogues exhibited about 98% purity. Stability studies have suggested that both can be stored in powder form for a
year at −20 °C, whereas in solution they are stable for 1 month
at the same temperature. For all the experiments described in this
article, stock solutions of vitamin D compounds were prepared
fresh each time.
One of the primary reasons to synthesize new vitamin D
agents is to prepare analogues that have reduced calcemic activity compared with that of 1a,25(OH)2D3, but without compromising the antiproliferative activity. We measured the calcemic
activity of both of these vitamin D analogues. As shown in Table
1, the vehicle-treated control rats showed a plasma calcium concentration of 5.4 ± 0.3 mg/dL (mean ± standard deviation).
When the rats were treated with vitamin D analogues at a dose
of 0.042 mg/kg per day, the following plasma calcium concentrations were observed: 6.0 ± 0.63 mg/dL for 1a(OH)D5-treated
rats (11% increase over that of the vehicle-treated control group,
Journal of the National Cancer Institute, Vol. 89, No. 3, February 5, 1997
Table 1. Effects of vitamin D analogues on plasma calcium levels in vitamin
D-deficient rats
Treatment*
None
No. of
rats
8
Dose,
mg/kg
per day
Plasma calcium,
mg/dL†
0.0
5.4 ± 0.28
1a(OH)D5
8
10
0.042
0.25
6.0 ± 0.63
7.9 ± 1.5
1a,25(OH)2D3
8
10
0.042
0.25
P‡
.121
.002
8.1 ± 1.2
10.1 ± 1.8
.001
<.0001
*1a(OH)D 5 4 1a-hydroxyvitamin D 5 ; 1a,25(OH) 2 D 3 4 1a,25dihydroxyvitamin D3.
†Values 4 means ± standard deviation.
‡Two-sided test.
P 4 .121, not statistically significant when compared with that
of the control group) and 8.1 ± 1.2 mg/dL for 1a,25(OH)2D3treated rats (50% increase over that of the control group, P 4
.001, statistically significant difference when compared with that
of the control group). At a higher concentration of vitamin D
analogues (0.25 mg/kg per day), 1a(OH)D5 treatment resulted in
a plasma calcium concentration of 7.9 ± 1.5 mg/dL, compared
with 10.1 ± 1.8 mg/dL for 1a,25(OH)2D3 treatment. Although
both analogues at this concentration increased the plasma calcium levels in comparison with those in vehicle-treated control
rats, these results showed that 1a(OH)D5 had overall lower calcemic effects than 1a,25(OH)2D3. 1a,25(OH)2D3 treatment resulted in an 87% increase in the plasma calcium level in rats
when compared with the vehicle-treated rats. On the other hand,
in animals treated with a higher concentration of 1a(OH)D5,
there was only a 50% increase in the plasma calcium concentration compared with that in the control animals. These results suggest that 1a(OH)D5 is much less calcemic than
1a,25(OH)2D3.
To evaluate the efficacy of the newly synthesized vitamin D5
analogue in preventing mammary lesion formation, we incubated
15 mammary glands per group (135 glands in total) from
BALB/c mice with appropriate hormones and exposed the
glands to DMBA on day 3 for 24 hours (see ‘‘Materials and
Methods’’ section). The mammary glands were incubated for 10
days with the vitamin D analogues in concentrations ranging
from 0.01 mM to 10.0 mM. The incidence of mammary lesions
was calculated for each group and was reported as the ratio of the
number of mammary glands showing lesions to the total number
of mammary glands at risk. Table 2 shows the incidence of
mammary lesions in the various groups treated with vitamin D
analogues. There was a dose-related decrease in the number of
glands exhibiting lesions in the vitamin D5-treated group. In
contrast, in the group treated with 0.01 mM vitamin D3, only two
of 14 glands developed lesions. At higher concentrations of this
analogue, no mammary lesions were observed. We calculated
the percent inhibition of formation of lesions in each treatment
group by comparing the incidences of lesions between the control group and the treatment group. As shown in Fig. 3, both
1a(OH)D5 and 1a,25(OH)2D3 inhibited the formation of mammary lesions by nearly 100%. At a concentration of 0.01 mM, the
vitamin D3 analogue inhibited mammary alveolar lesion formation by 75%; incubation of glands with concentrations of 0.1 mM
and higher showed 100% inhibition. In contrast, the vitamin D5
analogue inhibited the lesion formation in a dose-dependent
manner, reaching 100% inhibition at a concentration of 10.0 mM.
1a,25(OH)2D3 was toxic to the glands at concentrations of 1.0
mM or higher. Dilation of ducts and disintegration of the morphologic structures were observed. Treatment of mammary
glands with 1a(OH)D5 did not result in any toxicity to the
glands.
To determine the effects of vitamin D analogues on the structural differentiation as well as their toxic effects on mammary
glands, we incubated mammary glands with growth-promoting
hormones for 3 days either alone or in the presence of 0.1 mM or
1.0 mM vitamin D analogues. The histopathologic characteristics
of the treated and untreated glands are shown in Fig. 4. The
control mammary gland structure was represented by normal
alveolar and ductal structures (Fig. 4, A and B, respectively).
1a,25(OH)2D3 at a concentration of 0.1 mM did not show toxicity (Fig. 4, C). Mammary glands displayed normal ductal and
alveolar structures. At a concentration of 1.0 mM, vitamin D3
analogue treatment resulted in disintegration of ducts and structural toxicity to the glands (Fig. 4, D). In contrast, treatment with
the vitamin D5 analogue at a concentration of 1.0 mM retained
the healthy structural characteristics, as seen in the untreated
glands. In fact, some secretion was obvious in the lumen of the
ducts (Fig. 4, E).
Since the role of chemopreventive agents (including analogues of vitamin D3 and vitamin D5) on the induction of TGF-b
in normal mammary epithelial cells has not been studied, the
histologic sections of normal mammary glands treated with either only hormones (insulin, progesterone, aldosterone, and hydrocortisone) or hormones plus vitamin D analogues were processed immunohistochemically to investigate the effects of
Table 2. Effects of vitamin D analogues on incidence of 7,12-dimethylbenz[a]anthracene-induced lesions in BALB/c mouse mammary glands in organ culture
1a,25-Dihydroxyvitamin D3
1a-Hydroxyvitamin D5
Concentration, mM
None (control)
0.01
0.1
1.0
10.0
No. of glands
with lesions/total
No. of glands treated
% incidence*
9/15
6/16
4/16
2/14
0/15
60.0
37.5
25.0
14.3
0.0
P†
No. of glands
with lesions/total
No. of glands treated
% incidence*
P†
.21
.048
.011
.003
9/15
2/14
0/15
0/15
0/15
60.0
14.3
0.0
0.0
0.0
.011
.003
.003
.003
*(Number of glands with lesions/total number of glands treated) × 100.
†Two-sided test. Statistical significance when compared with control group.
Journal of the National Cancer Institute, Vol. 89, No. 3, February 5, 1997
ARTICLES 215
Fig. 3. Inhibition of 7,12-dimethylbenz[a]anthracene-induced mammary lesions
in BALB/c mouse mammary gland organ culture. Mammary glands were incubated with increasing concentrations of vitamin D3 and vitamin D5 analogues for
the first 10 days of a 24-day culture. After the 24-day culture, glands were fixed,
stained, and scored for alveolar lesions. Percent inhibition was calculated by the
formula [1 − (% incidence in the treated group/% incidence in the control group)
× 100]. Closed circles 4 1a-hydroxyvitamin D5; closed squares 4 1a,25dihydroxyvitamin D3.
vitamin D analogues on the induction and localization of VDRs
and TGF-b1. As shown in Fig. 5, VDRs were localized in the
nuclei of mammary epithelial cells. There was no selective localization of VDR in ductal or alveolar cells. Treatment with
either 1.0 mM 1a(OH)D5 or 0.1 mM 1a,25(OH)2D3 induced
expression of VDRs detectable in the nuclei of both ductal and
alveolar cells (Fig. 5, B and C, respectively). This induction was
dependent on the concentration of the analogue; VDR induction
was much less at the lower concentration of the vitamin D5
analogue (data not shown). For the vitamin D3 analogue, intense
staining was evident at a lower concentration (0.1 mM) (Fig. 5,
C). However, at a concentration of 1.0 mM, reduced or absent
staining was observed as a result of apparent toxicity (Fig. 5, D).
The effects of the vitamin D analogues on the induction of TGFb1 were also evaluated. We studied tissue sections from the
mammary glands treated with the vitamin D analogues or those
from untreated control glands for the induction of TGF-b1. We
found extensive induction of TGF-b1 in the cytoplasm of mammary epithelial cells (Fig. 6). Again, the pattern of intensity was
comparable to that of induction of VDR. The extent of induction
of TGF-b1 after treatment with the vitamin D5 analogue at a
concentration of 1.0 mM (Fig. 6, B) was similar to that observed
with the vitamin D3 analogue at a concentration of 0.1 mM (Fig.
6, C). However, at a concentration of 1.0 mM of the vitamin D3
analogue, TGF-b1 expression was much reduced as a result of
toxicity (Fig. 6, D). These results indicate that the vitamin D5
216 ARTICLES
Fig. 4. Effects of vitamin D analogues on the structural differentiation of mammary
glands in vitro. Mammary glands were incubated with insulin, prolactin, aldosterone,
and hydrocortisone for 3 days either alone or with vitamin D analogues. The glands
were not treated with 7,12-dimethylbenz[a]anthracene and did not undergo regression by hormone withdrawal. The glands were fixed in formalin and processed for
histopathologic evaluation. The sections were stained with hematoxylin–eosin. Normal alveolar and ductal structures are shown in panels A and B, respectively. Panels
C and D represent treatment of the glands with vitamin D3 analogue at concentrations of 0.1 and 1.0 mM, respectively. (Note the dilation and disintegration of the
ducts in panel D, whereas no toxicity was observed at 0.1 mM 1a,25dihydroxyvitamin D3, as shown in panel C.) Panel E represents incubation of glands
with a concentration of 1.0 mM of 1a-hydroxyvitamin D5. No toxicity was observed
(original magnification for panels A-E ×40).
analogue is considerably less toxic than the vitamin D3 analogue.
Moreover, they indicate that this remarkable induction of TGFb1 in mammary epithelial cells by the vitamin D5 analogue may
be of importance in cancer chemoprevention.
Discussion
In recent years, considerable attention has been directed to the
process of preventing carcinogenesis. The term ‘‘chemopreven-
Journal of the National Cancer Institute, Vol. 89, No. 3, February 5, 1997
Fig. 5. Effects of vitamin D on the induction of vitamin D receptors (VDRs) in
mammary gland organ culture. Mammary glands were incubated with 1.0 mM
1a-hydroxyvitamin D5 [1a(OH)D5] or 0.1 and 1.0 mM 1a,25-dihydroxyvitamin
D3 [1a,25(OH)2D3] for 3 days. Glands were fixed and processed for histopathologic evaluation. Sections (5 mm) were processed for VDR immunohistochemistry as described in the ‘‘Materials and Methods’’ section. Increased VDR
expression in the nuclei of normal mammary alveolar epithelial cells was observed in both vitamin D3- and vitamin D5-treated glands. Control (A);
1a(OH)D5, 1.0 mM (B); 1a,25(OH)2D3, 0.1 mM (C); and 1a,25(OH)2D3, 1.0 mM
(D) (original magnification for panels A-D ×40).
Fig. 6. Effects of 1a-hydroxyvitamin D5 [1a(OH)D5] on the induction of transforming growth factor-b1 (TGF-b1) in mammary gland organ cultures. Mammary glands were incubated with vitamin D analogues and processed as described in Fig. 5 legend. The glands were incubated with anti-TGF-b1 antibodies
as described in the ‘‘Materials and Methods’’ section and were evaluated for
TGF-b1 expression. Increased cytoplasmic expression of TGF-b1 is seen in the
normal alveolar epithelial cells in vitamin D5-treated glands. Control (A);
1a(OH)D5, 1.0 mM (B); 1a,25-dihydroxyvitamin D3 [1a,25(OH)2D3], 0.1 mM
(C); and 1a,25(OH)2D3, 1.0 mM (D) (original magnification for panels A-D
×40).
tion’’ was coined to denote the prevention of cancer by purified
and well-characterized chemical agents (31). Numerous chemopreventive agents have been identified that can arrest the process
of either initiation or progression of the disease. These agents
include retinoids, thiols, inhibitors of polyamine biosynthesis
and prostaglandin biosynthesis, and antihormones. An active
metabolite of vitamin D3, 1a,25(OH)2D3, inhibits the proliferation of many cell types (6-8); moreover, it is a potent inducer of
differentiation, as is seen in HL-60 cells (3). However, the use of
this analogue to treat cancer patients is precluded because of its
calcemic activity. It has been well established that vitamin D
treatment results in an increased concentration of calcium in
plasma of experimental animals (9). Numerous structural modifications have been reported to generate less calcemic vitamin D
analogues with increased antiproliferative or differentiating ability (10).
As this article demonstrates, we synthesized a novel vitamin
D5 analogue and determined its calcemic activity. We found that
1a(OH)D5 exhibited lower toxicity than 1a,25(OH)2D3. Nonetheless, 1a(OH)D5 is not completely devoid of calcemic activity.
There are at least two specific vitamin D3 analogues, Ro24-5531,
a hexafluoro derivative of vitamin D3 (28), and 22-oxa-calcitriol
(32), that have been shown to be effective against mammary
carcinogenesis. Ro24-5531 is non-calcemic and is capable of
inhibiting carcinogen-induced mammary lesions in organ culture; however, it was toxic at a concentration of 0.1 mM in organ
culture (data not shown), and the toxicity was observed when the
analogue was given in vivo at a dose of 10 mg/kg diet. Since
several hundred analogues of vitamin D3 have been evaluated
without much success (10), the logical step toward finding an
ideal compound was to evaluate the vitamin D analogues found
within the D4, D5, and D6 series.
Various reports (33,34) have shown that TGF-b1 is a negative
growth regulator for tumor cells. Increased expression of TGFb1 is directly related to a reduced rate of growth of cancer cells
in vitro. Several chemopreventive agents induce TGF-b1 in vitro
in transformed cells, including breast cancer cells (25). In a
report (28), a non-calcemic analogue of vitamin D3, Ro24-5531,
induced expression of TGF-b1 and its type 2 receptors. Similarly, chemopreventive agents have been shown to induce TGFb1 in breast cancer cells in culture (34). There is no report,
however, yet indicating induction of TGF-b1 in normal mammary epithelial cells. Induction of TGF-b1 by chemopreventive
agents in normal cells may be more relevant to chemoprevention
Journal of the National Cancer Institute, Vol. 89, No. 3, February 5, 1997
ARTICLES 217
than induction of TGF-b1 in transformed cells. In this study, we
have shown that TGF-b1 could be induced by 1a(OH)D5 in
mammary ductal cells and mammary alveolar cells.
Numerous studies have reported that the action of vitamin D
is mediated by nuclear VDRs. Our results are consistent with
those published (19-21,23). Our study shows that the analogue of
the vitamin D5 series induces nuclear VDRs in the mammary
glands in in vitro culture. Both 1a(OH)2D3 and 1a(OH)D5 induced VDRs in mammary epithelial cells. The induction of VDR
with 1.0 mM 1a(OH)D5 was much greater than with the nontoxic
concentration of 1a,25(OH)2D3. Since the mammary epithelial
cells (e.g., HBL100), which lack VDRs, also fail to respond to
1a(OH)D5 and do not show induction of TGF-b1 (data not
shown), the results presented in this article indicate that the
chemopreventive activity of the vitamin D analogue may be
mediated by induction of TGF-b1 in mammary tissue and that
the vitamin D action may require VDRs. These results also point
out that this remarkable induction of TGF-b1 in mammary epithelial cells by vitamin D analogues may be of significant importance in cancer chemoprevention.
Experiments presented here constitute the first step toward
our long-term goal of investigating the efficacy of chemoprevention by and the mechanism(s) of action of analogues of the
vitamin D5 series of compounds. Reduced calcemic activity and
lack of toxicity make 1a(OH)D5 an attractive candidate for in
vivo chemoprevention studies.
(15)
(16)
(17)
(18)
(19)
(20)
(21)
(22)
(23)
(24)
(25)
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Notes
Supported by an Illinois Department of Public Health research grant.
Manuscript received July 30, 1996; revised October 30, 1996; accepted December 3, 1996.
Journal of the National Cancer Institute, Vol. 89, No. 3, February 5, 1997