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*
Department of Biochemistry, School of Medicine and the
Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02111-1837
2To whom correspondence should be addressed at Silico Insights, Incorporated, 400 W. Cummings Park, Suite 6475, Woburn, MA 01801. E-mail: pankaj{at}silicoinsights.com.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and 
(RAR, ) in MCF-7 cells and RAR, ß
and in Hs578T cells were not induced by all-t-RA
treatment at either the protein or mRNA level. Hs578T cells treated
with all-t-RA had significantly more cells in the G0/G1
stage of the cell cycle, but the same was not observed for MCF-7 cells.
All-t-RA induced a dose-dependent cell death in
MCF-7 cells, which may be a necrotic phenomenon. These results
demonstrate that ER status is an important, although not essential
factor for breast cancer cell response to carotenoid and retinoid
treatments, and the mode of action of all-t-RA in MCF-7
and Hs578T cells is not through the induction of RAR. Other mechanistic
pathways that are either followed by or concomitant with growth
inhibition are possible.
KEY WORDS: • ß-carotene • retinoic acid • breast cancer cells • cell cycle • retinoic acid receptors
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The offspring of
emigrants from countries with low breast cancer incidence to countries
with high breast cancer incidence acquire rates close to those of the
new country, suggesting that environmental and lifestyle influences are
important in the etiology of breast cancer (McMichael and Giles 1988
). This could be the result of noninherited factors
including, among other possibilities, diet (Trichopoulos and Willett 1996
).
Epidemiologic and laboratory investigations exploring the relationship
between diet and disease suggest an inverse correlation between
consumption of fruits and vegetables rich in carotenoids and certain
cancer incidence rates (Block et al. 1992
,
Ziegler 1989
). In a review of possible environmental
determinants of cancer, it was estimated that 35% of cancers in the
United States might be attributable to dietary factors (Doll and Peto 1981
). Several studies have shown that low levels of
either dietary intake or plasma carotenoids are associated with an
increased risk of breast cancer (Comstock et al. 1992
,
Potischman et al. 1990
, Weisburger 1991
).
Carotenoids may act as chemoprotective agents through biological
activities such as metabolism to retinoids, antioxidation,
immunoenhancement, protection against cellular mutagenesis and
malignant transformation, and inhibition of tumorigenesis
(Bendich 1989
, Krinsky 1989 and 1993
,
Shultz et al. 1992
). Carotenoids also function as
radical-trapping antioxidants in vitro and may have the same
activity in vivo (Burton and Ingold 1984
, Krinsky 1989
). Retinoids, on the other hand, exert a variety of effects
on basic biological processes such as growth, differentiation,
development and malignant transformation, in addition to
receptor-induced signal transduction. Knowledge of the effect of
these compounds has led to the hypothesis that retinoids may act as
chemopreventive agents as well as inhibitors of the growth of
established tumors (Hill and Grubbs 1992
, Peto et al. 1981
).
This study was designed to determine the effects of carotenoids and retinoids on the growth of human breast cancer cells. Briefly, the growth of estrogen-receptor positive (ER+)3 MCF-7 and estrogen-receptor negative (ER-) Hs578T and MDA-MB-231 human breast cancer cells was determined after treatment with a provitamin A carotenoid, ß-carotene, and the nonprovitamin A carotenoids, canthaxanthin and lycopene, and the retinoids, all-trans-, 9-cis-, and 13-cis-retinoic acid and all-trans-retinol. The mechanism underlying growth inhibition caused by all-trans-retinoic acid (all-t-RA) in MCF-7 and Hs578T cells was further studied by determining the retinoic acid receptor (RAR) expression, cell cycle arrest and apoptosis induction in these cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Growth media and other growth regulators were purchased from Gibco Life Technologies (Gaithersburg, MD). Crystalline ß-carotene, canthaxanthin, all-trans-, 9-cis-, and 13-cis-retinoic acid and all-trans-retinol were purchased from Sigma Chemical (St. Louis, MO). Crystalline lycopene was kindly provided by LycoRed, Beer-Shiva, Israel. Tetrahydrofuran (THF) stabilized with BHT and acetone was purchased from Fisher Scientific, Pittsburgh, PA. The retinoid receptor cDNA probes were obtained as a kind gift from the laboratory of Dr. Monica Peacocke, Columbia University, New York, NY.
Cells and cell culture conditions.
MCF-7 human breast cancer cells were obtained from the Michigan Cancer
Foundation (Detroit, MI). Hs578T and MDA-MB-231 human breast cancer
cells were obtained from American Type Culture Collection (Rockville,
MD). MCF-7 cells were maintained in -MEM medium containing HEPES,
MEM nonessential amino acids, sodium pyruvate, L-glutamine,
insulin, gentamycin and 10% fetal bovine serum (5% serum for the
experiments). Hs578T cells were maintained in Dulbecco’s modified
Eagle’s medium with 4.5 g/L glucose, 10 mg/L insulin and 10% fetal
bovine serum. MDA-MB-231 cells were maintained in a 1:1 mixture of
Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium containing
2 mmol/L L-glutamine, and 10% fetal bovine serum. Cells
were grown in 100-mm culture dishes and incubated at 37°C in a
humidified atmosphere of 5% CO2 in air. Cells were seeded
at a concentration of 
5000 cells/cm2 for the
experiments, unless otherwise indicated. The trypan blue exclusion
method using a hemocytometer was used to distinguish viable from dead
cells.
Preparation of carotenoid and retinoid solutions.
Solutions of ß-carotene, canthaxanthin and lycopene were prepared in THF containing 0.25 g/L BHT as a preservative. Fresh solutions of the carotenoids were prepared in a nitrogen environment in a plastic glove bag (Aldrich, Milwaukee, WI) on each day of the experiment as needed. The required amount of THF was withdrawn from the sealed reagent bottle by a syringe purged with nitrogen to prevent oxidation of the solvent. Crystalline synthetic ß-carotene, canthaxanthin and lycopene were stored in the dark at -70°C between experiments to minimize oxidation and decay. Stock solutions of the carotenoids were prepared to result in concentrations of 1, 3, 7, 10 and 20 mmol/L. The appropriate stock solution (1 µL) was added to each milliliter of the media to result in a final carotenoid concentration of 1, 3, 7, 10 and 20 µmol/L in the media. In this way, the concentration of THF in the media was 0.1%, which did not cause any toxicity to the cells as determined by cell growth (data not shown). All-trans-, 9-cis- and 13-cis-retinoic acid and all-trans-retinol were dissolved in dimethyl sulfoxide to produce stock solutions of 0.1 mol/L. Further dilutions were made in acetone. Retinoids were then dissolved in the growth media to reach final concentrations of 10 nmol/L, 100 nmol/L and 1 µmol/L at a 0.25% concentration of acetone that was not toxic to the cells (data not shown). The purity of the carotenoid and retinoid stocks was determined by HPLC and the concentration was determined by spectrophotometric analysis. Red light was used during preparation of carotenoid and retinoid solutions to prevent photodamage to these compounds.
Treatment of cells.
Treatments were applied 24 h after cultures had been seeded to ensure proper attachment of the cells to the plastic culture plates or dishes. MCF-7, Hs578T and MDA-MB-231 cells were incubated with different carotenoids or retinoids for the periods mentioned in the respective experiments. Control cells received medium supplemented with THF or acetone only. Control and treatment media were changed every other day.
Cell proliferation.
For the determination of cell proliferation, MCF-7, Hs578T and MDA-MB-231 cells were incubated with ß-carotene, canthaxanthin, lycopene, all-trans-, 9-cis- and 13-cis-retinoic acid and all-trans-retinol solutions for up to 9 d. Proliferating viable cells attached to the plastic wells were harvested and their numbers counted on d 2, 5 and 9 (data are shown for d 9 only, i.e., when the cells reached confluency). For cell harvesting, the medium was aspirated from the wells; 0.5 mL of 0.25% trypsin solution was added to each well and incubated at 37°C for 5, 3 and 2 min, for MCF-7, MDA-MB-231 and Hs578T cells, respectively. The reaction was stopped with 1.5 mL of the growth medium at room temperature. The cells were collected and counted in duplicate using an electronic Coulter Counter (model Z1; Coulter, Miami, FL). Cell morphology was monitored by periodic evaluation of the cells under a phase contrast microscope throughout the experiments.
Northern blot analysis of RAR(, ß and ) mRNA expression.
MCF-7 and Hs578T cells were incubated with all-t-RA for
2, 6, 8, 24 and 72 h. RAR, ß and expression was examined
at the transcription level in the control and treatment cells by
Northern blot analysis of total cellular RNA, which was extracted from
the cells using the cesium chloride gradient method (Tsou et al. 1994
). RNA from each sample (30 µg) was
subjected to electrophoresis on a 1.2% agarose gel containing 5%
formaldehyde. The gel was transferred to a Nytran Plus nylon membrane
using a Turboblotter system (Schleicher & Schuell, Keene, NH).
The membrane(s) were subjected to UV crosslinking and then hybridized
to [-32P]-dCTP-labeled retinoid receptor cDNA probes
(Tsou et al. 1994
). pHE7 was used as a control gene to
monitor equivalent loading of RNA in each lane because the levels of
pHE7 are not affected by retinoic acid (Tsou et al. 1994
). After high stringency washing (0.1X standard saline
citrate at 60°C), the membranes were exposed to photographic
film using an intensifying screen at -80°C for various lengths of
time (between 1 and 7 d). The films were developed using an RG II
film processor (Fuji, Elmsford, NY).
Semiquantitative evaluation of Northern blots.
The films of Northern blot analysis were quantified by densitometric scanning using a PD model densitometer (Molecular Dynamics, Sunnyvale, CA) and ImageQuant software. The pixel value obtained for each individual band was normalized to the value of its relative pHE7 expression. The values obtained after normalization were used to compare the expression of retinoid receptors against their respective controls in the cell lines at different time points. Data are expressed as fold induction, considering the value of the control to be 1.
Flow cytometry.
MCF-7 and Hs578T cells were incubated with all-t-RA for 6 and 9 d. After exposure to experimental conditions, cells were collected and washed twice with cold PBS and suspended again in PBS. Absolute ethanol was added dropwise with vortexing at low speed to bring the ethanol concentration to 70%; this suspension was fixed at 4°C for 30 min. Cells were again washed twice with PBS after removal of ethanol and incubated with 10 mg/L RNase solution in PBS at 37°C for 30 min, with mixing several times during incubation. After being washed twice with PBS, cells were suspended in propidium iodide solution at 1 x 106 cells/0.5 mL concentration. Cells were then analyzed for their cell cycle distribution using a Flow cytometer (Beckton Dickinson, Franklin Lakes, NJ) and ModFit LT 2.0 software (Beckton Dickinson) for data analysis.
Morphological analysis.
MCF-7 cells were incubated with all-t-RA for 7 d.
Cells were grown on chamber slides for the morphologic analysis. After
exposure to experimental conditions, cells were washed with PBS and
stained with acridine orange (25 mg/L) and propidium iodide (25 mg/L)
for 15 min in the dark. Cells were then washed in PBS, fixed in 10%
formalin for 30 min in the dark and washed again in PBS.
Dual-stained cells were viewed using a fluorescence microscope.
Cells were coded as either early apoptotic (bright green, highly
condensed chromatin), late apoptotic (bright orange, highly condensed
chromatin) or necrotic cells (bright orange nucleus without condensed
chromatin) (Broaddus et al. 1996
).
Statistics.
Results are expressed as means ± SD of three determinations. Comparisons of mean values of control and treatment cells were made using ANOVA on the whole population followed by two-sample Student’s t test between control and each treatment group. The statistical significance of difference (P < 0.05) for the treatment groups was determined relative to their respective control group.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The effects of ß-carotene at 1, 3 and 10 µmol/L and
lycopene at 7, 10 and 20 µmol/L, dissolved in THF (0.1%),
on the growth of MCF-7, Hs578T and MDA-MB-231 cells are shown in
Figure 1
. ß-Carotene significantly inhibited the growth of MCF-7 and Hs578T
cells on d 9 (P < 0.05) at concentrations 
3
µmol/L, whereas it significantly increased the growth of MDA-MB-231
cells at 1 µmol/L (Fig. 1A
). Lycopene significantly
inhibited the growth of MCF-7 at 10 and 20 µmol/L and
MDA-MB-231 cells at 20 µmol/L on d 9 (P < 0.05; Fig. 1B
). Canthaxanthin, at concentrations up to 20
µmol/L, did not affect cell growth (data not shown).
|
The effects of all-trans-, 9-cis- and
13-cis-retinoic acid, dissolved in acetone (0.25%), on the
growth of Hs578T cells are shown in Figure 2
. All-trans- and 9-cis-retinoic acid significantly
inhibited the growth of Hs578T cells at 100 nmol/L and 1
µmol/L on d 9 (P < 0.05), whereas
13-cis-retinoic acid had no effect on cell growth.
All-trans-retinol at 100 nmol/L and 1 µmol/L
significantly inhibited the growth of MCF-7 cells, stimulated the
growth of MDA-MB-231 cells and had no effect on Hs578T cells on d 9 of
treatment (P < 0.05; Fig. 3
). Significant effects (P < 0.05) of
all-trans-, 9-cis- and 13-cis-retinoic
acid were observed on the growth inhibition of MCF-7 cells and growth
stimulation of MDA-MB-231 cells. These data are not represented here
because similar results have previously been published elsewhere
(Marth et al. 1993
, Van heusden et al. 1998
).
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Several of the growth experiments were also conducted at high and low
initial densities of MCF-7 cells. Figure 4A
shows the growth of the cells treated with ß-carotene and
all-t-RA, when grown at a high initial density (15,000
cells/cm2). ß-Carotene and all-t-RA
at 10 and 1 µmol/L, respectively, did not inhibit growth under these
conditions, whereas these concentrations significantly reduced the cell
growth when the initial cell density was 5000
cells/cm2 (Figs. 1
, 4)
.
|
8 x 105 cells/well on d 5 to 5
x 106 cells/well on d 9 (not shown). To
determine whether the growth inhibitory effect of all-t-RA
was due to the rapid cell doubling between these two time points, one
set of cells was treated with 1 µmol/L all-t-RA
from d 5 onward and compared with another set of cells supplemented
with 1 µmol/L all-t-RA from d 1 onward. The
cell numbers in the group supplemented from d 5 onward were not
different from the control group (Fig. 4B
), indicating that
an accumulation of retinoic acid in the cells before this time point is
necessary for the retinoid to be effective. The other possibility could
be that the cell density on d 5 was too large for the retinoid to have
an effect. Effect of all-t-RA on the expression of RAR.
The expression of RAR and in MCF-7 cells as a result of
all-t-RA treatment is shown in Figure 5
. Cells were treated with 1 µmol/L all-t-RA for
2, 6, 8, 24 or 72 h and investigated for the induction of retinoid
receptors at the mRNA level using Northern blot analysis. The bands
were compared using densitometric scanning after normalizing with the
pHE7 control to determine the fold induction under retinoid treatment
(Table 1
). Small inductions of RAR and were observed at some time points,
but these inductions were not consistent. Similar results of Northern
blot analysis were observed in Hs578T cells (Fig. 5
and Table 1
) using
similar treatments and duration. RAR(, ß and ) were not found
to be induced consistently. Similarly, no induction of retinoic acid
receptors was found at the protein level in MCF-7 and Hs578T cells
treated with 1 µmol/L all-t-RA using Western
blot analysis (data not shown).
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Figure 6
shows the cell cycle distribution of Hs578T cells treated with 1
µmol/L all-t-RA for 6 and 9 d. A
significant increase in cell number in the G0/G1 stage of the cell
cycle after treatment with all-t-RA was observed for d 6 and
9. No increase was observed for MCF-7 cells under similar conditions
(data not shown).
|
Chromatin condensation was used as the criterion to determine the
induction of apoptosis in the cells as a result of all-t-RA
treatment. MCF-7 cells did not contain fragmented nuclei, a marker of
apoptosis, when treated with 1 and 10 µmol/L
all-t-RA for 7 d and stained with acridine orange and
propidium iodide for fluorescent microscopy (data not shown). However,
all-t-RA treatment induced necrosis in the cells on the
basis of orange-red staining of the cells. Orange cells with no
nuclear fragmentation were considered dead and counted against live
green cells as a marker for necrosis. All-t-RA–induced
necrosis was significant (P < 0.05) and dose dependent
in MCF-7 cells (Table 2
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The results of both
case-control and prospective studies have shown a risk reduction
associated with diets high in vitamin A. Retinoids including
all-trans-, 9-cis- and 13-cis retinoic
acid have been studied by various investigators using cancer cells in
culture as model systems of breast cancer (Prakash et al. 2000
). Few investigators have tried to study the role of other
retinoids, and to an even lesser extent, carotenoids, for their effects
on breast cancer cell growth in culture. The growth assays conducted in this study were based on the time required for control cultures to reach confluency (d 9) at an initial seeding density of 5000 cells/cm2. Until d 9, cells demonstrated normal cell morphology as determined by phase contrast microscopy. Beyond this point, a change in the cell morphology and a rapid increase in cell detachment were observed. Therefore, d 9 was determined to be the point of termination of these growth studies. The cells were counted in both control and treatment cultures on d 2, 5 and 9. The cells that die during culture detach from the plastic of the culture plates and are automatically removed when the growth medium is changed. Hence, the cells counted at these time points were actually the proliferating viable cells attached to the plates. The results demonstrate that the number of these proliferating viable cells was altered in treatment groups compared with control groups on d 9 of treatment. The inhibitory treatments led to more cell detachment from the plastic and hence a lower cell count. Growth inhibition was observed to be maximal at d 9 of treatment; therefore the data are shown for this time point only.
Tetrahydrofuran (0.1%) and acetone (0.25%) were used in this study to
deliver carotenoids and retinoids to the cells and were not found to be
toxic to the cells (data not shown). Carotenoids and retinoids were
supplied to the cells every other day in this study. Other
investigations, however, have used from 1 (Zhu et al. 1997
) to 4 d (Shultz et al. 1992
) as the
time period between feeding the cells. Similarly, the entire duration
of treatment before determining the effects on cell growth also differs
among studies. The reported duration of treatment has varied from 3
(Kaleagasioglu et al. 1993
) to 13 d (Shultz et al. 1992
).
ß-Carotene at 10 µmol/L is similar to the concentration
found in the sera of humans supplemented with ß-carotene
(Nierenberg et al. 1991
). In general, concentrations of
1 and 10 µmol/L for retinoids and carotenoids,
respectively, resulted in maximum inhibition of cell growth in this
study. This is consistent with other studies in which retinoids have
been used at varying levels, with the 1 µmol/L dose
resulting in maximum growth cessation (Chen et al. 1997
,
Fanjul et al. 1996
, Marth et al. 1993
,
Van heusden et al. 1998
, Zhao et al. 1995
, Zhu et al. 1997
). The growth inhibitory
effect of lycopene in MCF-7 cells (at 10 µmol/L) observed
in this study, however, was markedly less than that observed by
Levy et al. (1995)
(0.3 µmol/L), possibly
due to the different culture conditions and the assay used.
A significant effect (P < 0.05) of
all-trans- and 9-cis-retinoic acid was observed
on the growth inhibition of Hs578T cells (Fig. 2)
. Similarly, there was
a significant effect (P < 0.05) of
all-trans-, 9-cis- and 13-cis-retinoic
acid on the growth inhibition of MCF-7 cells and growth stimulation of
MDA-MB-231 cells. These data are not presented here, however, because
similar results have previously been published elsewhere (Marth et al. 1993
, Van heusden et al. 1998
). On the
basis of a report by Repa et al. (1993)
that indicated
that all-trans-retinol is a ligand for the RAR and only
four- to sevenfold less potent than all-t-RA in binding to
RAR(, ß and ) proteins in competitive binding assays, we set
out to test the efficacy of this compound on the growth of MCF-7,
Hs578T and MDA-MB-231 cell lines. We found that a 1 µmol/L
dose of all-trans-retinol induced growth inhibition almost
similar to that observed with the same dose of all-t-RA in
both MCF-7 (not shown) and Hs578T cells.
The initial cell density appears to be of paramount importance in
determining the effects of carotenoids and retinoids on cell
proliferation. In the series of growth experiments conducted in our
laboratory, we found that the cells must be at an appropriate density
to be sensitive to retinoid and carotenoid treatments. When the
starting cell numbers of MCF-7 cells were increased to 15,000
cells/cm2 (Fig. 4)
, neither ß-carotene nor
all-t-RA inhibited growth at concentrations of 10 and 1
µmol/L, respectively. However, significant growth
inhibition was achieved using the same levels of these compounds when
the starting cell numbers were reduced to almost one third (Figs. 1
, 4)
. We also observed that an uptake of all-t-RA by the cells
in the early phase of cell culture is essential for this retinoid to
inhibit growth. Cells treated with retinoic acid from the middle phase
of culture, i.e., when the cells achieved a count of 98,000
cells/cm2, showed no growth inhibition (Fig. 4)
.
Among patients with breast cancer, tumors that express estrogen
receptors (ER) are associated with improved survival and better
response to hormone therapy than those not expressing these receptors
(Rutqvist 1990
). Despite the general observation that
ER(-) breast cancer cells are not sensitive to retinoids
(Lacroix and Lippman 1980
, van den Burg et al. 1993
), the growth of ER(-) Hs578T cells was inhibited by
all-trans- and 9-cis-retinoic acid as well as by
ß-carotene in this investigation. Thus, breast cancer cells may be
sensitive to carotenoid and retinoid treatments irrespective of their
ER status.
Retinoids exert their modulatory effects on cell growth by binding to
the nuclear retinoid receptor proteins, of which there are two classes,
the RAR and the retinoid X receptor (RXR), each of which has three
subtypes (, ß and ). Retinoic acid is thought to first bind to
its nuclear receptors; this ligand-receptor complex in the form of
homodimers or heterodimers then binds to its respective response
elements (retinoic acid response elements) in the promoters of a
variety of retinoic acid–responsive genes. Binding of the natural
retinoids all-t-RA and 9-cis-retinoic acid to the
retinoid receptors enhances receptor responses (Allenby et al. 1993
, Heyman et al. 1992
, Levin et al. 1992
). The RAR bind to both all-t-RA and
9-cis-retinoic acid with high affinity, whereas RXR bind to
9-cis-retinoic acid only (Heyman et al. 1992
,
Levin et al. 1992
). RXR play an extremely important role
in mediating retinoic acid and vitamin D effects at the level of gene
expression by forming heterodimers with RAR and vitamin D receptor,
respectively (Zhao et al. 1995
).
The Northern blot analyses revealed that all three cell lines used in
this study express basal levels of RAR and , whereas only Hs578T
cells express RARß. The level of the basal expression of these
receptor subtypes was also variable in the three cell lines. We did not
observe a consistent induction of any of the RAR subtypes in MCF-7 and
Hs578T cells either at the mRNA level using Northern blot analysis
(Fig. 5)
or at the protein level using Western blot analysis (data not
shown).
Several studies have tried to correlate the retinoid receptors to the
retinoid sensitivity, and a majority of these studies have used
different receptor-selective retinoids for this purpose.
RAR-selective retinoid agonists inhibited the
anchorage-dependent growth of MCF-7 cells after a 7-d treatment,
suggesting that RAR is the retinoid receptor involved in the
inhibition of adherent cell growth by retinoids (Dawson et al. 1995
). In another study, retinoic acid strongly inhibited the
proliferation of ER(+) MCF-7 cells but not of ER(-) Hs578T and
MDA-MB-231 cells, and RAR mRNA was highly expressed in the
RA-responsive lines, but not in the unresponsive lines (van den Burg et al. 1993
). The authors suggested that the loss of
functional RAR may be a frequent event, leading to RA unresponsiveness
of ER(-) breast cancer cells. RARß-negative cell lines MCF-7 and
MDA-MB-231 underwent growth inhibition associated with G1 arrest when
treated with 1 µmol/L all-t-RA after a human
RARß gene was introduced into these cell lines using a retroviral
vector-mediated gene transduction (Seewaldt et al. 1995
). Similarly, RARß induction in MCF-7 cells by
all-t-RA was demonstrated using different
receptor-selective retinoids. By using an RAR-selective
antagonist (Ro 41–5253), RARß expression was induced by
all-t-RA through an RAR-dependent signaling pathway in
MCF-7 cells (Shang et al. 1999
). Receptor selective
retinoids induced apoptosis in MCF-7 cells, indicating that RAR,
RAR and RXR can mediate programmed cell death (Toma et al. 1998
). These observations, which have followed an indirect
approach of relating retinoid receptors to the cell growth inhibitory
mechanism, suggest the involvement of all three subtypes of RAR in the
growth inhibition of breast cancer cells. The results of our study,
which involved a direct method of determining the expression of RAR as
a result of treatment with their natural ligand all-t-RA,
indicate that altering RAR expression is not involved in the growth
inhibition of breast cancer cells caused by this retinoid. Similarly,
RAR selectively binding retinoids (CD2325, CD666 and CD437)
inhibited the proliferation of MCF-7 cells similar to
all-t-RA and no correlation was found between expression of
RAR and antiproliferative effects of the retinoids
(Widschwendter et al. 1997
).
The flow cytometry results of this investigation indicate an arrest of
Hs578T cells in the G0/G1 stage of the cell cycle as a result of
all-t-RA treatment (Fig. 6)
. The same effect was not
observed for MCF-7 cells. It is possible that all-t-RA
induces the expression of G0/G1-specific gene(s) in Hs578T cells, which
were not assessed in this study. Apoptotic induction as a result of
all-t-RA treatment was not observed in MCF-7 cells because
the morphological nuclear fragmentation did not occur. The use of more
sensitive techniques for the determination of early apoptosis, such as
Annexin V or BRDU staining methods, may provide greater insight into
retinoid-induced apoptosis in these cells. There was, however, a
dose-dependent necrotic death observed in MCF-7 cells induced by
all-t-RA (Table 2)
on the basis of the accumulation of
fluorescent dyes in the cells (Broaddus et al. 1996
) as
explained in Materials and Methods.
The overall results of our study demonstrate that carotenoids and retinoids inhibit the growth of both ER(+) and ER(-) breast cancer cell lines, indicating that estrogen receptor status is an important, although not essential factor for the responsiveness of breast cancer cells to carotenoid and retinoid treatments. Also, the mechanism behind growth inhibition of ER(+) MCF-7 and ER(-) Hs578T cells by all-t-RA does not involve transcriptional modulation of the RAR by this retinoid. We are convinced, however, that the mode of action of carotenoids and retinoids in breast cancer cells should not be considered to be limited to the enhancement of basal expression of retinoid receptors (both RAR and RXR series) as a result of treatment. Other mechanistic pathways that are either followed by or concomitant with growth inhibition are possible, including the induction of activation protein-1–responsive genes and the cell cycle–specific genes.
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FOOTNOTES |
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3 Abbreviations used: all-t-RA, all-trans-retinoic acid; ER(+), estrogen receptor positive; ER(-), estrogen receptor negative; RAR, retinoic acid receptor; RXR, retinoid X receptor; THF, tetrahydrofuran.
Manuscript received July 10, 2000. Initial review completed August 14, 2000. Revision accepted January 17, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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