Effects of a prenyl-baicalein derivative on ER (+) MCF-7 and ER (-) MDA-MB-231 breast tumor cell lines

Article abstract of DOI:10.1007/s00044-011-9848-1

The effect of a prenyl-baicalein derivative in the DNA synthesis of the estrogen-dependent ER (+) MCF-7 and the estrogen-independent ER (-) MDA-MB- 231 cell lines was evaluated using both complete and steroid- free medium. The results revealed a biphasic

Full text of DOI:10.1007/s00044-011-9848-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3154–3160
DOI 10.1007/s00044-011-9848-1
ORIGINAL RESEARCH
Effects of a prenyl-baicalein derivative on ER (+) MCF-7
and ER (2) MDA-MB-231 breast tumor cell lines
•
•
•
´
Marta Perro Neves Ana Camo˜es Maria de Sa˜o Jose Nascimento
•
Honorina Cidade Maria Emılia Sousa Madalena Maria Magalhaes Pinto
•
´
˜
Received: 2 June 2011 / Accepted: 25 October 2011 / Published online: 11 November 2011
Ó Springer Science+Business Media, LLC 2011
Abstract The effect of a prenyl-baicalein derivative in
the DNA synthesis of the estrogen-dependent ER (?)
MCF-7 and the estrogen-independent ER (-) MDA-MB-
231 cell lines was evaluated using both complete and ste-
roid-free medium. The results revealed a biphasic effect in
DNA synthesis of ER (?) MCF-7 cells. Moreover, this
prenylflavone seemed to reduce the estrogenic effect of
17b-estradiol and to increase the antiestrogenic effect of
4-hydroxytamoxifen and fluvestrant on the ER (?) MCF-7
cells.
Introduction
Estrogens have shown to exert a variety of beneficial
effects in men and women providing protection against
osteoporosis, menopausal symptoms, cardiovascular dis-
eases, and potentially in some neurodegenerative patholo-
gies. In spite of all these benefits, estrogens are also one of
the most important risk factors for breast cancer (Acker-
man and Carr, 2002). For this reason, the search for
estrogen-like compounds has been an attractive field for the
scientific community because of their possible role as
health-promoting and disease-preventing agents (Collins-
Burow et al., 2000; Milligan et al., 1999; Oh et al., 2006;
Osborne et al. 2000; Vaya and Tamir, 2004). Phytoestro-
gens are non-steroidal plant-derived compounds that
functionally mimic mammalian estrogens and therefore are
considered to play an important role in the prevention of
cancers, heart disease, menopausal symptoms, and osteo-
porosis (Dixon, 2004; Ososki and Kennelly, 2003). This
biologic diversity of phytoestrogens is due in part to their
ability to act as estrogen agonists and/or antagonists, like
selective estrogen receptor modulators (SERM). As estro-
gen agonists, phytoestrogens mimic endogenous estrogens
causing estrogenic effects and as estrogen antagonists, they
may block or alter estrogen receptors (ER) preventing
estrogenic activity. Consequently, they cause antiestro-
genic effects, being not surprising to consider the potential
of phytoestrogens as efficient inhibitors of estrogen-medi-
ated growth of human breast cancer (Brzezinski and Debi,
1999; Ososki and Kennelly, 2003; Wang and Kurzer, 1997;
Zhao and Mu, 2011).
Keywords Antiproliferative ꢀ Breast adenocarcinoma ꢀ
Estrogen receptor ꢀ Prenylflavones
´
M. P. Neves ꢀ A. Camo˜es ꢀ M. de Sa˜o Jose Nascimento ꢀ
H. Cidade ꢀ M. E. Sousa ꢀ M. M. M. Pinto (&)
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Centro de Quımica Medicinal da Universidade do Porto
(CEQUIMED-UP), Laboratorio de Quımica Organica e
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Farmaceutica, Departamento de Ciencias Quımicas, Faculdade
de Farmacia, Universidade do Porto, Rua Anıbal Cunha, 164,
ˆ
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4050-047 Porto, Portugal
e-mail: madalena@ff.up.pt
M. P. Neves ꢀ H. Cidade ꢀ M. E. Sousa ꢀ M. M. M. Pinto
´
Faculdade de Farmacia, Laboratorio de Quımica Organica e
Farmaceutica, Departamento de Ciencias Quımicas,
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ˆ
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Universidade do Porto, Rua Anıbal Cunha, 164, 4050-047 Porto,
Portugal
Flavonoids are an outstanding family of compounds
which have displayed a variety of biological activities
´
M. de Sa˜o Jose Nascimento
Faculdade de Farmacia, Laboratorio de Microbiologia,
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(Brahmachari and Gorai, 2006; Lopez-Lazaro et al., 2002;
ˆ
Departamento de Ciencias Biologicas, Universidade do Porto,
Rua Anıbal Cunha 164, 4050-047 Porto, Portugal
´
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Middleton et al., 2000). They represent one of the largest
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Med Chem Res (2012) 21:3154–3160
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groups of phytoestrogens with a chemical structure which
resembles 17b-estradiol (1, Fig. 1) and also the ability to
mimic estrogen activity (Oh et al., 2006; Ososki and
Kennelly, 2003; Vaya and Tamir, 2004; Wang and Kurzer,
1997). Baicalein (2, Fig. 1), a natural occurring flavone,
has been reported to have antiestrogenic activity, by
inducing MCF-7 cell death making this compound a
potential alternative for ER (?) breast cancer prevention
and treatment (Po et al., 2002). Among flavonoids,
prenylated derivatives have long been recognized for their
myriad biological properties, being the antitumor effect
one of the most reported in the literature (Botta et al., 2005;
Cidade et al., 2009). In fact, it has been demonstrated that
isoprenylation of flavonoids significantly improved their
growth inhibitory effect on human tumor cell lines (Botta
et al., 2005; Cidade et al., 2009; Daskiewicz et al. 2005;
Neves et al., 2011). Moreover, prenylflavonoids have also
been reported as phytoestrogens, especially 8-prenylna-
ringenin (3, Fig. 1) which was found to be more estrogenic
than any other known phytoestrogens (Milligan et al. 1999;
Schaefer et al., 2003). Artelastin (4, Fig. 1), isolated from
Artocarpus elasticus by our research group (Kijjoa et al.,
1996), is another example of a prenylflavonoid resembling
the effect of phytoestrogens (Pedro et al., 2005; Pedro
et al., 2006). Previously, we have reported that compound
4 exhibited a biphasic effect in the DNA synthesis of ER
(?) MCF-7 breast tumor cell line which was stimulatory at
low concentrations and inhibitory at high concentrations.
During our research work on the search for potential
antitumor compounds, several baicalein (2) derivatives
were synthesized and evaluated for their inhibitory activity
against the in vitro growth of several human tumor cell
lines (Neves et al., 2011). In particular, a baicalein (2)
investigated the effect of this flavone (5) on the prolifera-
tion of two breast tumor cell lines (ER (?) MCF-7 and ER
(-) MDA-MB-231) in both complete and steroid-free
medium (SFM). We further evaluate the effect of this
compound in the estrogenic activity of 17b-estradiol (1),
and in the antiestrogenic activity of 4-hydroxytamoxifen
(6) and fulvestrant (7) in ER (?) MCF-7 cell line, using
SFM.
Materials and methods
Chemistry
1H and 13C NMR spectra were taken in CDCl3 at room
temperature, on Bruker Avance 300 instruments
1
(300.13 MHz for H and 75.47 MHz for 13C). Chemical
shifts are expressed in d (ppm) values relative to tetra-
methylsilane (TMS) as an internal reference; HRMS mass
spectra were recorded as ESI (electrospray ionization) mode
at C. A. C. T. I.-University of Vigo, Spain. Prenyl bromide
and baicalein (2) were purchased from Sigma Aldrich.
Compound 5 was synthesized from baicalein (2) according
to the previously described procedure (Neves et al., 2011).
5,6-Dihydroxy-7-(3-methylbut-2-enyloxy)-2-phenyl-4H-
chromen-4-one (5)
1
Yield: 75%; H NMR (CDCl3, 300 MHz): d 12.49 (1H, s,
5-OH), 7.91–7.88 (2H, m, H-20,60), 7.55–7.51 (3H, m,
H-30,40,50), 6.69 (1H, s, H-3), 6.62 (1H, s, H-8), 5.56 (1H,
brt, J = 6.8, H-200), 5.44 (1H, s, 6-OH), 4.70 (2H, d,
J = 6.8, H-100), 1.83 (3H, s, H-400), 1.79 (3H, s, H-500); 13C
NMR (CDCl3, 75 MHz): 182.7 (C-4) 164.0 (C-2), 152.1
(C-7), 150.6 (C-8a), 145.6 (C-5), 139.8 (C-300), 131.5 (C-
10), 131.2 (C-40), 129.8 (C-6), 129.1 (C-30, 50), 126.2 (C-20,
60), 118.3 (C-200), 106.0 (C-4a), 105.4 (C-3), 91.3 (C-8),
66.2 (C-100), 25.8 (C-400), 18.3 (C-500); ESI-HRMS (?) m/z:
Anal. calc. for C20H19O5 (M ? H)?: 339.1227; found:
339.1235.
derivative,
5,6-dihydroxy-7-(3-methylbut-2-enyloxy)-2-
phenyl-4H-chromen-4-one (5), revealed to have a potent
growth inhibitory effect and to induce cell cycle arrest in
G1 phase (Neves et al., 2011). Since the antiproliferative
effect of flavonoids has already been correlated to ER-
dependent mechanisms (Le Bail et al., 1998; So et al.,
1997; Davis et al., 2008), in the present study, we
Fig. 1 Structures of 17b-estradiol (1) and three natural flavonoids (2–4) with antiproliferative properties
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Med Chem Res (2012) 21:3154–3160
Biological activity
Attached cells were exposed for 48 h to five serial dilutions
of compound 5 (1.85, 5.56, 16.67, 50.00, and 150.00 lM).
Following this incubation period, adherent cells were fixed
in situ with TCA, washed and stained with SRB. The
bound stain was solubilized and the absorbance was mea-
sured at 492 nm in a plate reader (Biotek Instruments Inc.
PowerWave XS, Winooski, USA). A dose–response curve
was obtained for each cell line and the concentration that
caused cell growth inhibition of 50% (GI50, corresponding
to the concentration of compound that inhibited 50% of the
net cell growth), was determined as described elsewhere
(Monks et al., 1991). The effect of the vehicle solvent
(DMSO) on the growth of these cell lines was evaluated in
preliminary experiments, by exposing untreated control
cells to the maximum concentration of DMSO used in each
assay (0.25%). Final concentrations of DMSO did not
interfere with the biological activity tested (data not
shown). Doxorrubicin was used as positive control and
tested in the same manner.
Reagents and stock solutions of compounds
Fetal bovine serum (FBS), L-glutamine, phosphate buffer
saline (PBS), and trypsin were from Gibco Invitrogen Co.
(Scotland, UK). RPMI-1640 with and without phenol red
were from Cambrex (New Jersey, USA). Activated carbon,
dextran, dimethylsulfoxide (DMSO), doxorubicin, 17b-
estradiol (1), ethylenediaminetetracetic acid (EDTA),
4-hydroxytamoxifen (6), fulvestrant (7), penicillin, strep-
tomycin, sulforhodamine B (SRB), and trypan blue were
from SigmaChemical Co. (Saint Louis, MO, USA). Tri-
chloroacetic acid (TCA) and Tris were purchased from
Merck (Darmstadt, Germany). [3H] Thymidine was from
Amersham (Illinois, USA). The scintillation liquid was
from ICN radiochemicals (Irvine, CA, USA). Charcoal-
stripped FBS was obtained according to the procedure
described elsewhere (Gritzapis et al., 2003). Stock solu-
tions of compounds were prepared in DMSO and stored at
-20°C. Appropriate dilutions of the compounds were
freshly prepared just prior to the assays.
Evaluation of the breast tumor cell lines proliferation
in a SFM
Cell culture
The effect of compound 5 on the proliferation of breast ER
(?) MCF-7 and ER (-) MDA-MB-231 tumor cells was
performed by both SRB and [3H] thymidine incorporation
assays. In brief, ER (?) MCF-7 and ER (-) MDA-MB-231
cells were plated in 96-well plates at appropriate densities
to ensure exponential growth throughout the experimental
period (1.5 9 105 cells/ml), and then allowed to adhere
overnight. Attached cells were treated with a range of
concentrations of compound 5 (0.10, 0.20, 0.39, 0.78, 1.56,
3.13, 6.25, 12.50, 25.00, 50.00, and 100.00 lM) prepared
by serial dilution using RPMI-SFM for 72 h. In the SRB
assay, adherent cells were fixed in situ with TCA, washed
and stained with SRB (Monks et al., 1991). The bound
stain was solubilized and the absorbance was measured at
492 nm in a plate reader (Biotek Instruments Inc. Power-
Wave XS, Winooski, USA). The percentage of cellular
protein content was calculated comparing the absorbance
of treated cells with that of untreated control cells. In the
[3H] thymidine incorporation assay, 5 lCi/ml of [3H]
thymidine was added and cells were incubated for further
4 h at 37°C. Pulsed cells were then harvested on a glass
filter 102 9 256 mm (Skatron, Norway) using a semiau-
tomatic cell harvester (Skatron Instruments, Norway) and
allowed to dry. Incorporation of radioactive thymidine was
determined by liquid scintillation counter LS 6500 (Beck-
man Instruments, CA, USA) and defined by comparing the
arithmetic mean of counts per minute (c.p.m.) of treated
cells with that of the untreated control cells. In both
methods, 4-hydroxytamoxifen (6, 10-1 lM in RPMI-SFM)
and fulvestrant (7, 10-1 lM in RPMI-SFM) were used as
Two human tumor cell lines were used: MCF-7 (estrogen-
dependent breast adenocarcinoma, ECACC, UK) and
MDA-MB-231 (estrogen-independent breast adenocarci-
noma, ECACC, UK). The human tumor cell lines were
grown as monolayer and routinely maintained in RPMI-
1640 medium supplemented with 5% (MCF-7) or 10%
(MDA-MB-231) heat-inactivated FBS, 2 mM glutamine,
100 U/ml of penicillin, and 100 lg/ml of streptomycin at
37°C in a humidified atmosphere containing 5% CO2. For
the experiments in SFM, cells were grown as monolayer
and routinely maintained in RPMI-1640 medium without
phenol red supplemented with 5 or 10% charcoal-stripped
fetal bovine serum, 2 mM glutamine, 100 U/ml of peni-
cillin, and 100 lg/ml of streptomycin at 37°C in a
humidified atmosphere containing 5% CO2 (designed
thereafter as RPMI-SFM).
Evaluation of the breast tumor cell lines growth inhibition
by SRB assay in a complete medium
The effect of compound 5 on the in vitro growth of two
human tumor cell lines, ER (?) MCF-7 and ER (-) MDA-
MB-231, were evaluated according to the procedure that
uses the protein binding dye SRB to assess cell growth
(Skehan et al., 1990; Monks et al., 1991). Cells were plated
in 96-well plates at appropriate densities to ensure expo-
nential growth throughout the experimental period
(1.5 9 105 cells/ml), and then allowed to adhere overnight.
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Med Chem Res (2012) 21:3154–3160
3157
120
100
80
positive control for antiestrogenic activity and 17b-estra-
diol (1, 10-1 lM in RPMI-SFM) was used as positive
control for estrogenic activity. The effect of the vehicle
solvent (DMSO) on the growth of these cell lines was
evaluated in preliminary experiments (blank), by exposing
untreated control cells to the maximum concentration of
DMSO used in each assay (0.25%).
ER (+) MCF-7
*
ER (–) MDA-MB-231
60
40
20
0
-20
-40
Statistical analysis
0
25
50
75
100
125
150
175
All experimental data are presented as means SEM from
at least three independent experiments (most of them per-
formed in duplicate). Statistical analysis was carried out
using a paired Student’s t test.
Concentration of compound 5 (µM)
Fig. 3 Effect of compound 5 on the growth of ER (?) MCF-7 and
ER (-) MDA-MB-231 cell lines. Cells were treated with several
concentrations of compound 5, with complete medium (blank) or with
DMSO control for 48 h. Results represent means SEM from at
least three independent experiments performed in duplicate. Doxor-
rubicin was used as positive control (GI50 (MCF-7) = 42.8
8.2 nM; GI50 (MDA-MB-231) = 10.9 1.3 nM). *P \ 0.01 (which
means that growth inhibitory activity in ER (?) MCF-7 was
significantly higher than in ER (-) MCF-7)
Results and discussion
Chemistry
Considering that this kind of behavior was already
observed for many phytoestrogens (Davis et al., 2008; Le
Bail et al., 1998; Pedro et al., 2006), we decided to
investigate the possible involvement of the estrogenic
receptor (ER) in the observed growth inhibitory effect.
First, we have studied the influence of the composition of
the culture medium on the growth inhibitory effect of ER
(?) MCF-7 and ER (-) MDA-MB-231 cells, since the
presence of estrogen-like compounds in RPMI medium has
already been described to disclose estrogenic effects of
several compounds on breast tumor cell line (Jensen et al.,
2003; Journe et al., 2004; Paiva et al., 2011). Therefore,
ER (?) MCF-7 and ER (-) MDA-MB-231 cells were
cultured in complete medium (RPMI) and also in steroid-
free medium (RPMI-SFM), and then treated with a range of
the highest concentrations tested (3.13–100.00 lM) of
compound 5. After treatment, cellular growth was assessed
using two different methodologies: SRB and [3H] thymi-
dine incorporation assays (data not shown). In the ER (?)
MCF-7 cell line, it is possible to observe that compound 5
exhibited a significant more potent effect (P \ 0.05) in
steroid-free conditions (Fig. 4b) than in complete medium
(Fig. 4a), suggesting that the growth inhibitory effect of
The synthetic approach used to synthesize the prenylated
baicalein derivative 5 was by microwave-assisted organic
synthesis (MAOS). Therefore, the starting material 2 was
submitted to microwave irradiation (200 W, 60°C) for 2 h
in presence of prenyl bromide and anhydrous potassium
carbonate (Fig. 2) (Neves et al., 2011).
Effect of compound 5 on ER (?) MCF-7 and ER (-)
MDA-MB-231 human breast tumor cell lines
The effect of the synthesized prenylated flavone 5 on the in
vitro growth of ER (-) MDA-MB-231 (estrogen-inde-
pendent breast adenocarcinoma cell line) and ER (?)
MCF-7 (estrogen-dependent breast adenocarcinoma) was
determined according to the procedure that uses the protein
binding dye SRB to assess cell growth (Monks et al., 1991;
Skehan et al., 1990). Results showed that compound 5
caused a dose-dependent inhibitory effect in both human
breast tumor cells (Fig. 3). Noteworthy, compound 5 was
significantly (P \ 0.01) more active in the ER (?) MCF-7
than in ER (-) MDA-MB-231 cells, showing GI50 values
of 11.2 2.0 lM for ER (?) MCF-7 and of 29.7
2.1 lM for ER (-) MDA-MB-231 cells.
Fig. 2 Synthesis of compound
5. MW Microwaves
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Med Chem Res (2012) 21:3154–3160
compound 5 could be influenced by the presence of
estrogen-like compounds. By contrast, in the ER (-)
MDA-MB-231 cells, no significant differences were
observed on the cell growth, independent of the medium
used (RPMI or RPMI-SFM) (Fig. 4c, d).
stimulatory/inhibitory effect of compound 5 could be
mediated via ER. Compound 5 was further evaluated in the
presence of 17b-estradiol (1) to determine if flavonoid 5
could inhibit estradiol-induced cell proliferation on MCF-7
cells. ER (?) MCF-7 cells were concomitantly treated with
both compounds 1 and 5, and subsequently evaluated using
the [3H] thymidine incorporation assay (Fig. 6). These in
vitro experiments were also conducted in SFM to allow for
the assessment of estrogenic responses. The results pre-
sented in Fig. 6 showed that when ER (?) MCF-7 cells
were treated with both 10-1 lM of 17b-estradiol (1) and
In order to investigate if the effects on ER (?) MCF-7
and ER (-) MDA-MB-231 cell lines could be influenced
by the concentration of compound 5, exponentially grow-
ing cells were treated with a broad concentration range
(0.1–100 lM) of compound 5 for 72 h and DNA synthesis
was quantified by thymidine incorporation. To ensure that
the observed effects were only due to the tested compound,
this assay was carried out using SFM (Fig. 5).
compound
5 (0.1–0.78 lM), a significant reduction
(P \ 0.05) of the proliferation effect of 17b-estradiol (1)
alone or compound 5 alone was observed, suggesting a
competition between both compounds for the ER (Fig. 6).
This behavior was already described for some flavonoids
(Le Bail et al., 1998; So et al., 1997).
Results presented in Fig. 5 reveal that in the ER (?)
MCF-7 cells (Fig. 5a), the prenylated derivative 5 pre-
sented a biphasic effect, that is, a growth stimulatory effect
at low concentrations (0.10–0.20 lM) and a growth
inhibitory effect at high concentrations (3.13–100 lM). In
accordance with the results presented with the SRB assay
(Fig. 4b), at high concentrations (3.13–100 lM), the tested
compound (5) exhibited a more potent antiproliferative
effect on the ER (?) than on ER (-) cells, as measured
by the [3H] thymidine incorporation assay (Fig. 5a, b).
These results are in agreement with those reported for
phytoestrogens (Pedro et al., 2006), suggesting that the
In addition, the effect of baicalein derivative 5 in the
antiestrogenic activity of the partial antagonist 4-hy-
droxytamoxifen (6) and of the pure antiestrogen fluvestrant
(7) was investigated using the [3H] thymidine incorporation
assay to assess DNA synthesis (Fig. 7). The results showed
that when ER (?) MCF-7 cells were treated concomitantly
with 4-hydroxytamoxifen (6) and compound 5 (3.13
and 6.25 lM), a significant increase (P \ 0.05) of the
Fig. 4 Effect of compound 5 on the growth of ER (?) MCF-7 (a,
b) and ER (-) MDA-MB-231 (c, d) cell lines determined by the SRB
assay using complete medium (RPMI, a, c) or steroid-free medium
(RPMI-SFM, b, d). Results are presented as the % relative to
untreated control cells and represent means SEM from at least
three independent experiments performed in duplicate. *P \ 0.05
(which means that growth inhibitory activity in RPMI-SFM was
significantly higher than in RPMI)
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Med Chem Res (2012) 21:3154–3160
3159
Fig. 5 Effect of compound 5 in the synthesis of DNA of two breast
tumor cell lines: ER (?) MCF-7 (a) and ER (-) MDA-MB-231 (b),
determined by the [3H] thymidine incorporation assay using RPMI-
SFM. Results are presented as the % relative to untreated control cells
and represent means SEM from at least three independent exper-
iments performed in duplicate. *P \ 0.05 (which means that growth
stimulatory/inhibitory effects were significantly different in ER (?)
MCF-7 and MDA-MD-231 cell lines)
Fig. 7 Effect of compound 5 in the synthesis of DNA of ER (?)
MCF-7 cell line in the presence of 4-hydroxytamoxifen (6) or
fluvestrant (7), determined by the [3H] thymidine incorporation assay
using RPMI-SFM. Results are presented as the % relative to untreated
control cells and represent means SEM from at least three
independent experiments performed in duplicate. *P \ 0.05 (which
means that the antiproliferative effect was significantly lower than
with 6 or 7 alone)
Fig. 6 Effect of compound 5 in the synthesis of DNA of ER (?)
MCF-7 cell line in the presence of 17b-estradiol (1, 10-1 lM)
determined by the [3H] thymidine incorporation assay using RPMI-
SFM. Results are presented as the % relative to untreated control cells
and represent means SEM from at least three independent exper-
iments performed in duplicate. *P \ 0.05 (which means that growth
stimulatory effect was significantly lower than with 17b-estradiol (1)
or compound 5 alone)
cell lines was significantly different, suggesting a possible
involvement of the estrogen receptor. This prenylated ba-
icalein derivative 5 revealed to act as an estrogen agonist at
low concentrations, whereas it produces estrogen antago-
nist effects at higher concentrations, being these data
reported here for the first time. In addition, compound 5
showed an antiproliferative effect in the presence of both
estradiol and estrogen antagonists, 4-hydroxytamoxifen
(6), and fulvestrant (7). Hence, the prenylflavone 5
deserved to be further explored as an antiestrogenic agent
given the importance of this effect on the combinatory
therapy for ER (?) dependent breast tumors.
antiestrogenic effect of 4-hydroxytamoxifen (6), which was
dependent on the concentration of flavone 5, was observed
(Fig. 7). Likewise, a significant increase (P \ 0.05) of the
antiestrogenic effect of fluvestrant (7) was observed
(Fig. 7). Since the antiestrogenic activity of both ER
antagonists (6 and 7) on the ER (?) MCF-7 cells was
increased in the presence of compound 5, it is possible to
infer that this prenylflavone may act by an additive/syn-
ergistic mechanism when in combination with antiestro-
gen-like compounds.
Conclusions
ˆ
Acknowledgments The authors thank Fundac¸a˜o para a Ciencia e a
Tecnologia under the project CEQUIMED - PEst-OE/SAU/UI4040/
2011, by FEDER funds through the COMPETE program under the
projects FCOMP-01-0124-FEDER-011057, and FCOMP-01-0124-
In conclusion, for the prenylated flavone 5, the growth
inhibitory effect against ER (?) and ER (-) breast tumor
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Med Chem Res (2012) 21:3154–3160
FEDER-015752, U. Porto and Santander-Totta for financial support.
Marta Perro Neves also thanks FCT for PhD grant (SFRH/BD/21770/
2005).
inflammation, heart disease, and cancer. Pharmacol Rev 52:
673–751
Milligan SR, Kalita JC, Heyerick A, Rong H, De Cooman L, De
Keukeleire D (1999) Identification of a potent phytoestrogen in
Hops (Humulus lupulus L.) and beer. J Clin Endocrinol Metab
84:2249–2252
References
Monks A, Scudiero D, Skehan P, Shoemaker R, Paul K, Vistica D,
Hose C, Langley J, Cronise P, Vaigro-wolf A, Gray-Goodrich M,
Campbell H, Mayo J, Boyd M (1991) Feasibility of a high-flux
anticancer drug screen using a diverse panel of cultured human
tumor cell lines. J Natl Cancer Inst 83:757–776
Neves MP, Cidade H, Pinto M, Silva AM, Gales L, Damas AM, Lima
RT, Vasconcelos H, Nascimento MSJ (2011) Prenylated deriv-
atives of baicalein and 3,7-dihydroxyflavone: synthesis and study
of their effects on tumor cell lines growth, cell cycle and
apoptosis. Eur J Med Chem 46:2562–2574
Ackerman GE, Carr BR (2002) Estrogens. Rev Endocr Metab Disord
3:225–230
Botta B, Vitali A, Menendez P, Misiti D, Delle Monache G (2005)
Prenylated flavonoids: pharmacology and biotechnology. Curr
Med Chem 12:713–739
Brahmachari G, Gorai D (2006) Progress in the research on naturally
occurring flavones and flavonols: an overview. Curr Org Chem
10:873–898
Oh SM, Kim YP, Chung KH (2006) Biphasic effects of kaempferol
on the estrogenicity in human breast cancer cells. Arch Pharm
Res 29:354–362
Brzezinski A, Debi A (1999) Phytoestrogens: the ‘‘natural’’ selective
estrogen receptor modulators? Eur J Obstet Gynecol Reprod Biol
85:47–51
Osborne CK, Zhao H, Fuqua SAW (2000) Selective estrogen receptor
modulators: structure, function, and clinical use. J Clin Oncol
18:3172–3186
Ososki AL, Kennelly EJ (2003) Phytoestrogens: a review of the
present state of research. Phytother Res 17:845–869
Paiva AM, Sousa ME, Camo˜es A, Nascimento MSJ, Pinto MM.
(2011) Prenylated xanthones: antiproliferative effects and
enhancement of the growth inhibitory action of 4-hydroxytam-
oxifen in estrogen receptor-positive breast cancer cell line. Med
Chem Res. doi:10.1007/s00044-011-9562-z
Pedro M, Ferreira MM, Cidade H, Kijjoa A, Bronze-da-Rocha E,
Nascimento MSJ (2005) Artelastin is a cytotoxic prenylated
flavone that disturbs microtubules and interferes with DNA
replication in MCF-7 human breast cancer cells. Life Sci
77:293–311
Pedro M, Lourenc¸o CF, Cidade H, Kijjoa A, Pinto M, Nascimento
MSJ (2006) Effects of natural prenylated flavones in the
phenotypical ER (?) MCF-7 and ER (-) MDA-MB-231 human
breast cancer cells. Toxicol Lett 164:24–36
Po LS, Chen Z-Y, Tsang DSC, Leung LK (2002) Baicalein and
genistein display differential actions on estrogen receptor (ER)
transactivation and apoptosis in MCF-7 cells. Cancer Lett
187:33–40
Schaefer O, Hu¨mpel M, Fritzemeier K-H, Bohlmann R, Schleuning
W-D (2003) 8-Prenyl naringenin is a potent ERa selective
phytoestrogen present in hops and beer. J Steroid Biochem Mol
Biol 84:359–360
Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D,
Warren JT, Bokesch H, Kenny S, Boyd MR (1990) New
colorimetric cytotoxicity assay for anticancer-drug screening.
J Natl Cancer Inst 82:1107–1112
So FV, Guthrie N, Chambers AF, Carrollb KK (1997) Inhibition of
proliferation of estrogen receptor-positive MCF-7 human breast
cancer cells by flavonoids in the presence and absence of excess
estrogen. Cancer Lett 112:127–133
Cidade H, Neves M, Kijjoa A (2009) Natural prenylated flavones:
chemistry and biological activities—an overview. In: Brahma-
chari G (ed) Natural products: chemistry, biochemistry and
pharmacology. Narosa Publishing House PVT. Ltd., New Dehli,
pp 463–519
Collins-Burow BM, Burow ME, Duong BN, McLachlan JA (2000)
Estrogenic and antiestrogenic activities of flavonoid phyto-
chemicals through estrogen receptor binding-dependent and -
independent mechanisms. Nutr Cancer 38:229–244
Daskiewicz J-B, Depeint F, Viornery L, Bayet C, Comte-Sarrazin G,
Comte G, Gee JM, Johnson IT, Ndjoko K, Hostettmann K,
Barron D (2005) Effects of flavonoids on cell proliferation and
caspase activation in a human colonic cell line HT29: an SAR
study. J Med Chem 48:2790–2804
Davis DD, Dıaz-Cruz ES, Landini S, Kim Y-W, Brueggemeier RW
(2008) Evaluation of synthetic isoflavones on cell proliferation,
estrogen receptor binding affinity, and apoptosis in human breast
cancer cells. J Steroid Biochem Mol Biol 108:23–31
Dixon RA (2004) Phytoestrogens. Annu Rev Plant Biol 55:225–261
Gritzapis AB, Baxevanis CN, Missitzis I, Katsanou ES, Alexis MN,
Yotis J, Papamichail M (2003) Quantitative fluorescence cyto-
metric measurement of estrogen and progesterone receptors:
correlation with the hormone binding assay. Breast Cancer Res
Treat 80:1–13
Jensen J, Kitlen JW, Briand P, Labrie F, Lykkesfeldt AE (2003)
Effect of antiestrogens and aromatase inhibitor on basal growth
of the human breast cancer cell line MCF-7 in serum-free
medium. J Steroid Biochem Mol Biol 84:469–478
Journe F, Chaboteaux C, Dumon JC, Leclercq G, Laurent G, Body JJ
(2004) Steroid-free medium discloses oestrogenic effects of the
bisphosphonate clodronate on breast cancer cells. Br J Cancer
91:1703–1710
Kijjoa A, Cidade HM, Pinto MMM, Gonzalez MJTG, Anantachoke
C, Gedris T, Herz W (1996) Prenylflavonoids from Artocarpus
elasticus. Phytochemistry 43:691–694
Vaya J, Tamir S (2004) The relation between the chemical structure
of flavonoids and their estrogen-like activities. Curr Med Chem
11:1333–1343
Le Bail JC, Varnat F, Nicolas JC, Habrioux G (1998) Estrogenic and
antiproliferative activities on MCF-7 human breast cancer cells
by flavonoids. Cancer Lett 130:209–216
´
´
Wang C, Kurzer MS (1997) Phytoestrogens concentration determines
effects on DNA synthesis in human breast cancer cells. Nutr
Cancer 28:236–247
Lopez-Lazaro M (2002) Flavonoids as anticancer agents: structure-
activity relationship study. Curr Med Chem Anticancer Agents
2:691–714
Zhao E, Mu Q (2011) Phytoestrogen biological actions on mamma-
lian reproductive system and cancer growth. Sci Pharm 79:1–20
Middleton E Jr., Kandaswami C, Theoharides TC (2000) The effects
of plant flavonoids on mammalian cells: implications for
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