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ONCOLOGY REPORTS
Abstract. Flavonoids are structurally similar to steroid
hormones, particularly estrogens, and therefore have been
studied for their potential effects on hormone-dependent
cancers. Baicalein is the primary avonoid derived from the
root of Scutellaria baicalensis Georgi. In the present study,
we investigated the effects of baicalein on 17β-estradiol
(E2)-induced migration, adhesion and invasion of MCF-7
and SK-BR-3 breast cancer cells. The results demonstrated
that baicalein suppressed E2-stimulated wound-healing
migration and cell-Matrigel adhesion, and ameliorated
E2-promoted invasion across a Matrigel-coated Transwell
membrane. Furthermore, baicalein interfered with E2-induced
novel G protein-coupled estrogen receptor (GPR30)-related
signaling, including a decrease in tyrosine phosphorylation
of epidermal growth factor receptor (EGFR) as well as phos-
phorylation of extracellular signal-regulated kinase (ERK) and
serine/threonine kinase Akt, without affecting GPR30 expres-
sion. The results also showed that baicalein suppressed the
expression of GPR30 target genes, cysteine-rich 61 (CYR61)
and connective tissue growth factor (CTGF) induced by E2.
Furthermore, baicalein prevented GPR30-related signaling
activation and upregulation of CYR61 and CTGF mRNA levels
induced by G1, a specic GPR 30 agonist. The results suggest
that baicalein inhibits E2-induced migration, adhesion and
invasion through interfering with GPR30 signaling pathway
activation, which indicates that it may act as a therapeutic
candidate for the treatment of GPR30-positive breast cancer
metastasis.
Introduction
Breast cancer is the most common malignancy affecting
women and is the leading cause of cancer-related mortality
among females worldwide (1). Despite advances in the diag-
nosis and treatment of breast cancer, approximately a third of
patients develop metastatic disease, with a median survival
time of less than 3 years (2). Preventing metastasis is impor-
tant for effectively overcoming breast cancer. Estrogen plays
an important role, not only in the initiation and proliferation
of breast cancer, but also in cancer metastasis (3). Most of the
biological effects of estrogen are mediated by binding and
activating classic estrogen receptors (ERs), ERα and ERβ,
to stimulate the transcription of target genes via genomic
and non-genomic mechanisms (4). Based on this knowledge,
hormonal therapies that interfere with ERα function a re
currently applied in patients with ERα-positive breast cancer,
and beneficial effects have been achieved. Although there
is general success with ERα expression and responsiveness
to ERα-targeted agents, some patients do not respond to
endocrine therapy, and most patients appear to have acquired
resistance (5). In addition, some types of ERα-negative breast
cancer cells remain estrogen responsive, indicating the exis-
tence of an alternative receptor for estrogen.
G protein-coupled estrogen receptor (GPR30), as a member
of the 7-transmembrane GPR family, mediates both rapid
signaling and transcriptional events in response to estrogen.
It mediates estrogenic-rapid non-genomic actions, through
the Gβγ subunit downstream, release of heparin-bound EGF
(HB-EGF) and metalloproteinase (MMP)-dependent transac-
tivation of epidermal growth factor receptors (EGFRs), as well
as activation of mitogen-activated protein kinases (MAPKs),
adenyl cyclase, and phosphoinositide 3-kinase (PI3K) (6).
Subsequently, it also triggers transcriptional responses by acti-
vating various transcription factors, such as FOS, JUN, EGR1
Baicalein suppresses 17-β-estradiol-induced migration, adhesion
and invasion of breast cancer cells via the G protein-coupled
receptor 30 signaling pathway
DANDAN SHANG1, ZHENG LI1, ZHUXIA ZHU1, HUAMEI CHEN1, LUJUN ZHAO2,3,
XUDONG WANG1 and YAN CHEN1-3
1Department of Physiology/Cancer Research Group, Guiyang Medical University School of Basic Medicine;
2Department of Pharmacology of Chinese Material Medica and 3Key Laboratory of Optimal Utilization
of Natural Medicine Resources, Guiyang Medical University, Guiyang, Guizhou 550004, P.R. China
Received October 4, 2014; Accepted January 26, 2015
DOI: 10.3892/or.2015.3786
Correspondence to: Dr Yan Chen, Department of Pharmacology
of Chinese Material Medica, Guiyang Medical University, 9 Beijing
Road, Guiyang, Guizhou 550004, P.R. China
E-mail: s0710189@sina.com
Professor Xudong Wang, Department of Physiology, Guiyang Medical
University, 9 Beijing Road, Guiyang, Guizhou 550004, P.R. China
E-ma il: xdwang@gmc.edu.cn
Key word s: baicalein, estrogen, G protein-coupled receptor 30,
migration, adhesion, invasion, breast cancer
SHANG et al: BAICALEIN INHI BITS ESTROGEN-INDUCED MIGRATION, ADHESION AN D INVASION
2
and ATF3 following the protein kinase signaling cascade (7).
GPR30 expression is positively associated with the features
of breast tumor progression, including tumor size, HER2/neu
status, and metastases. Yet, ERα is inversely associated with
HER2/neu and tumor size (8). ERα in breast cancer generally
indicates good prognosis and treatment responsiveness with
anti-estrogen agents (9); yet, GPR30 is overexpressed in inva-
sive breast cancer and is positively associated with a metastatic
phenotype (10). GPR30 is implicated in breast cancer metas-
tasis and it may provide a new potential target for endocrine
th e r a py.
The incidence of breast cancer is much higher in Western
countries than in Asian ones. Epidemiological studies
contribute these differences to dietary factors, particularly
soy consumption. Flavonoids are considered to have a
signicant potential in breast cancer chemoprevention (11).
Their polyphenolic ring is structurally similar to the steroid
nucleus of 17β-estradiol (Fig. 1A) and may have estrogenic
or anti-estrogenic activity (12). Baicalein is the primary
avonoid derived from the root of Scutellaria baicalensis
Georgi and is also found in soybeans and fruit, which bears
the three-ring structure of the avone backbone with phenolic
hydroxyl at the 5', 6', and 7' position (Fig. 1A). It possesses
several health benets including anti-inammatory, antioxi-
dant, antiviral, neuroprotective, and anticancer effects (13).
It has also been shown to have antitumor effects in various
hormone-dependent cancers such as breast, colon, and
prostate cancer. Previous studies have found that baicalein
suppresses 17β-estradiol (E2)-induced ER transactivation in
MCF-7 cells (14) and displaces >85% of estradiol binding in
mouse uterine cytosol (15). Additionally, baicalein exhibits
effective inhibitory activity against E2/insulin-like growth
factor 1 (IGF-1)-induced proliferative events in breast cancer
cells (16). These investigations propose that baicalein may
exert anti-estrogenic activity.
The present study investigated the inhibitory activity of
baicalein on E2-induced migration, adhesion, and invasion
of MCF-7 and SK-BR-3 breast cancer cells. Furthermore, we
investigated its effect on GPR30-related signaling, including
phosphorylation of EGFR as well as extracellular signal-regu-
lated kinase (ERK), serine/threonine kinase Akt activation,
and GPR30-mediated gene expression.
Materials and methods
Reagents and antibodies. Baicalein (purity >98%) was
provided by Professor Zhiyu Li (China Pharmaceutical
University, Jiangsu, China). It was dissolved in dimethyl
sulfoxide (DMSO) as a stock solution at 0.1 M and stored at
-20˚C. E2 and G1 from Sigma-Aldrich (St. Louis, MO, USA)
were dissolved in DMSO as a stock solution at 10-1 M and
stored at 4˚C. Matrigel was obtained from BD Biosciences
(Bedford, MA, USA). Bovine serum albumin (BSA) was
purchased from Beijing Solarbio Science and Technology
Co., Ltd. (Beijing, China). 3-(4,5-Dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) and cytosine
β-D-arabinofuranoside hydrochloride were purchased from
Sigma-Aldrich. The GPR30 (N-15)-R antibody (sc-48525-R,
polyclonal antibody, rabbit anti-human, 1:800) was purchased
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).
Primary antibodies against p-ERK (T202/Y204) (AP0484,
polyclonal antibody, rabbit anti-human, 1:1,000), extracel-
lular regulated protein kinase 1/2 (ERK1/2) (L352) (BS1112,
polyclonal antibody, rabbit anti-human, 1:1,000), and GAPDH
(1A6) (MB001, monoclonal antibody, mouse anti-human,
1:1,000) were purchased from Bio-World (Dublin, OH, USA).
Primary antibodies for p-Akt (Ser473) (193H12) (#4058,
monoclonal antibody, rabbit anti-human, 1:1,000), Akt (11E7)
(#4685, monoclonal antibody, rabbit anti-human, 1:1,000),
p-EGFR (Tyr1173) (53A5) (#4407, monoclonal antibody,
rabbit anti-human, 1:1,000) and EGFR (D38B1) (#4267, mono-
clonal antibody, rabbit anti-human, 1:1,000) were purchased
from Cell Signaling Technology (Beverly, MA, USA). The
anti-mouse or anti-rabbit IgG horseradish peroxidase-conju-
gated secondary antibodies (1:3,000) were purchased from
Santa Cruz Biotechnology, Inc.
Cell lines and culture. Human breast cancer cell lines MCF-7
and SK-BR-3 were obtained from the Kunming Cell Bank of
the Chinese Academy of Sciences (Yunnan, China). MCF-7
cells were maintained in Dulbecco's modied Eagle's medium
(DMEM) with 10% FBS, and SK-BR-3 cells were maintained
in RPMI-1640 (all from Hyclone, Logan, UT, USA) with 10%
FBS (Gibco-BRL, Carlsbad, CA, USA), both supplemented
with 100 U/ml penicillin and 100 µg/ml streptomycin (both
from Sigma-Aldrich). These cells were incubated at 37˚C in
humidied air with 5% CO2.
Cell treatment. Before the indicated treatments, cells were
cultured in phenol red (PR)-free medium without serum for
24 h. Cells were then treated with or without 10 nM of E2
(or 1 µM G1) and different concentrations of baicalein (5, 10,
and 15 µM) in PR-free medium without serum for a speci-
ed period of time. Control cells were incubated in DMSO
(0. 01%, v / v).
MTT assay. The 96-well microculture plates were individu-
ally seeded with 100 µl of the respective cell suspension and
100 µl of treatment medium. After incubation at 37˚C for
24 h, 20 µl of MTT (5 mg/ml) was added, and the plates were
incubated for an additional 4 h at 37˚C. The medium was then
removed, and 100 µl DMSO was added and mixed thoroughly.
Spectrometric absorbance values at 570 nm were measured
using a microplate reader (Bio-Rad, Lincoln, NE, USA). The
results are expressed as inhibitory rates with respect to the
controls, calculated using the following formula:
Inhibitory rate (%) = [(Acontrol - Atreated)/Acontrol] x100.
Wound-healing assay. Cells were seeded into 12-well plates
at 90% conuency. Monolayers were wounded with a micro-
pipette tip and rinsed with phosphate-buffered saline (PBS)
3 times to remove any oating cells. Then the cells were
incubated in the absence or presence of E2 (10 nM) with or
without various concentrations of baicalein (5, 10, and 15 µM)
for 24 h. Cytosine β-D-arabinofuranoside hydrochloride
(10 µM) was added 1 h before the test treatment to inhibit cell
proliferation (17). Images were captured at 0 and 24 h after
wound application. The level of cell migration into the wound
was quantied as the migration rate compared against the cells
at 0 h for each group.
ONCOLOGY REPORTS 3
Cell adhesion assay. The cell adhesion assay was performed
as described (18). Briefly, 96-well plates were coated with
Matrigel (50 µg/well) at 37˚C for 1 h and blocked with 0.5%
BSA (v/v) at 4˚C for 4 h. After treatment with E2 (10 nM) or
E2 (10 nM) plus baicalein (5, 10, and 15 µM) for 24 h, the cells
were harvested and suspended in serum-free medium. Cells
at a density of 2x105 cells/ml (100 µl) were added into the
plates and then incubated at 37˚C for 1 h. After incubating for
adhesion, the medium was carefully suctioned out. The wells
were then washed with PBS to remove non-adherent cells. The
colorimetric MTT assay was employed to analyze the number
of adherent cells. Spectrometric absorbance was measured at
570 nm by using a microplate reader (Bio-Rad). The results
are expressed as adhesive cells with respect to the control as
a percentage.
Invasion assay. Invasive ability of the cells was measured by
assay using a Transwell chamber (Millipore, Billerica, MA,
USA) containing membranes with an 8-µm pore size and,
coated with Matrigel as previously described (19). The cells
were exposed to E2 (10 nM) with or without different concen-
trations of baicalein (5, 10, and 15 µM) for 24 h. The cells
were then trypsinized and suspended at a nal concentration
of 5x105 cells/ml in serum-free medium. The cell suspension
was added into each 10-mm upper chamber, and medium with
10% FBS was added into the bottom chamber as a chemoat-
tractant. After incubating for 24 h, the upper surfaces of the
membranes were swabbed to remove non-invaded cells, and
the cells that attached to the lower surface were fixed in
100% methanol, stained with hematoxylin and eosin (H&E)
(Beyotime Institute of Biotechnology, Jiangsu, China), and
counted under a microscope. Five randomly chosen elds were
analyzed for each group.
Western blot analysis. Cells were collected after E2 (10 nM)
or G1 (1 µM) with or without baicalein (5, 10, and 15 µM)
treatment for 1 h, and lysed in lysis buffer (Beyotime Institute
of Biotechnology). The lysates were claried by centrifugation
at 4˚C for 15 min at 13,000 x g. The concentration of protein
in the supernatants was measured using bicinchoninic acid
(BCA) assay kit (Beyotime Institute of Biotechnology) with a
Varioskan Multimode microplate spectrophotometer (Thermo
Waltham, MA, USA). Total proteins (30 µg/lane) were sepa-
rated on a 12 or 8% SDS-PAGE gel, and transferred to a PVDF
membrane (Millipore, Bedford, MA, USA). The membrane
was blocked with 5% non-fat milk and subsequently incubated
with the indicated antibodies. Blots were visualized by using
an enhanced chemiluminescence kit (Millipore, Billerica,
MA, USA). Digital images of the blots were produced by a
Syngene Gel Imaging System and quantied with GeneSnap
(Syngene, Frederick, MD, USA) software.
Real-time reverse transcription-polymerase chain reaction
(RT-PC R). Total RNA was extracted after a 1-h treatment with
10 nM E2 or G1 (1 µM) in the presence or absence of baical-
cein (5, 10 and 15 µM) by using an RNA extraction kit (code
no. 9767; Takara, Dalian, China) according to the manufac-
turer's instructions. Complementary DNA (cDNA) was
synthesized using a Strand Complementary DNA Synthesis kit
(code no. 6210A; Takara). Real time PCR was performed on an
iCycle iQ (Bio-Rad) using 20 ng of cDNA template and 10 µM
primers. Cysteine-rich 61 (CYR61) primers (forward, 5'-ACT
Figure 1. Effects of baicalein on cell viability. (A) Chemical structure of 17β-estradiol (E2) and baicalein. (B) Inhibitory effects of baicalein on the cell viability
of MCF-7 and SK-BR-3 cells at 24 h as detected by MTT assay. Cells were treated with various concentrations of baicalein for 24 h. The result is represented
by the inhibitory rate. (C) Cells were incubated with the indicated doses of baicalein with or without E2 (10 nM) after 24 h, and cellular viability was assessed
in (a) MCF-7 and (b) SK-BR-3 cells. Values are mean ± SEM. #P<0.05 compared with control group, *P<0.05 compared with the E2-treated group.
SHANG et al: BAICALEIN INHI BITS ESTROGEN-INDUCED MIGRATION, ADHESION AN D INVASION
4
TCA TGG TCC CAG TGC TC-3' and reverse, 5'-AAA TCC
GGG TTT CTT TCA CA-3') (20), connective tissue growth
factor (CTGF) primers (forward, 5'-ACC TGT GGG ATG
GGC ATC T-3' and reverse, 5'-CAG GCG GCT CTG CTT
CTC TA-3') (21) and β-actin primers (forward, 5'-AGT TGC
GTT ACA CCC TTT C-3' and reverse, 5'-CCT TCA CCG
TTC CAG TTT-3') (22) were synthesized by Sangon Biotech
(Shanghai, China). The mRNA levels were measured using
SYBR Premix Ex Taq™ (code no. RR037A; Takara). The
reactions were performed for 30 sec at 95˚C, followed by
40 cycles at 95˚C for 5 sec and 60˚C for 30 sec. The melting
curve was analyzed at 55 to 95˚C to detect a single gene-
specic peak and to verify the absence of primer dimer peaks.
The CYR61 or CTGF mRNA level was quantied in relation to
β-actin mRNA level by using the 2-ΔΔCt method.
Statistical analysis. Statistically signicant differences were
calculated by one-way ANOVA followed by the Bonferroni
post-hoc test for multiple-group comparisons. A P<0.05 was
considered to indicate a signicant difference, and data are
expressed as mean ± standard error of the mean (SEM). The
data shown in this test were obtained from 3 independent
experiments.
Results
Cytotoxic effect of baicalein on MCF-7 and SK-BR-3 cells.
To investigate the effect of baicalein on cell viability, MCF-7
and SK-BR-3 cells were treated with various concentrations of
baicalein for 24 h, and viability was detected by MTT assay.
The IC50 values of baicalein in the MCF-7 and SK-BR-3 cells
after 24 h were 66.3±5.9 and 103.5±9.4 µM, respectively
(Fig. 1B). According to the results, baicalein at 5, 10 and 15 µM
had no obvious cytotoxic effect on the MCF-7 or SK-BR-3
cells in the presence or absence of E2 (Fig. 1C), and were used
in following study.
Figure 2. Effects of baicalein on the E2-induced migration of MCF-7 and SK-BR-3 breast cancer cells. (A) Conuent monolayers were scratched and incubated
in the absence or presence of E2 (10 nM) with or without various concentrations of baicalein (5, 10, and 15 µM) for 24 h. The images were captured at 0
and 24 h after wounding (magnication, x100). These gures are representative of 3 separate experiments. (B) The inhibition of E2-stimulated migration by
baicalein. Values are mea n ± SEM. *P<0.05 compared with the E2-treated group. E2, 17β-estradiol.
ONCOLOGY REPORTS 5
Baicalein inhibits E2-induced cell migration. After treatment
with E2, MCF-7 and SK-BR-3 cells were found to have signi-
cantly enhanced migration into the wound, while cells treated
with baicalein had less migratory ability (Fig. 2A). E2 stimula-
tion for 24 h signicantly increased the migration rate from
13±2 to 52±3% in the MCF-7 cells, and from 10±4 to 40±5%
in the SK-BR-3 cells, respectively. After treatment with 15 µM
baicalein for 24 h, the migration rate decreased to 17±2% in
the MCF-7 cells and 13±4% in the SK-BR-3 cells, respectively
(Fig. 2B). These results demonstrated that baicalein suppressed
E2-induced cell migration.
Baicalein suppresses E2-promoted cell adhesion to Matrigel.
Adhesion of tumor cells to extracellular matrix (ECM) is
an essential step for tumor invasion and metastasis. We next
examined the effect of baicalein on E2-promoted cell adhesion
Figure 4. Effect of baicalein on E2-stimulated cell invasion in MCF-7 and SK-BR-3 cells. (A) Cells were treated with or without E2 (10 nM) and di fferent
concentrations of baicalein (5, 10 and 15 µM) for 24 h. Invasive cells that passed through the membrane were evaluated using H&E staining (magnication,
x200). The images a re representative of 3 separate experiments. (B) The results are expressed as invasive cells with respect to the cont rol (as 100%). Values
are mean ± SEM. *P<0.05 indicates signicant difference from the E2-treated group. E2, 17β-estr adiol.
Figure 3. Effects of baicalein on E2-promoted cell adhesion to Matrigel in (A) MCF-7 and (B) SK-BR-3 cells. Cells were treated with or without E2 (10 nM)
or E2 (10 nM) plus baicalein (5, 10 and 15 µM) for 24 h. The results are expressed as adhesive cells with respect to the control (as 100%). *P<0.05 i ndicates
signicant difference from the E2-treated group. Values are mean ± SEM. E2, 17β-estr adiol.
SHANG et al: BAICALEIN INHI BITS ESTROGEN-INDUCED MIGRATION, ADHESION AN D INVASION
6
to Matrigel, a reconstituted basement membrane (an impor-
tant component of ECM). E2 greatly increased cell-Matrigel
adhesion by 195.0±27% in the MCF-7 and by 161±8% in
the SK-BR-3 cells, when compared with the control (Fig. 3).
Treatment with 5, 10, and 15 µM baicalein reduced the adhe-
sive cells to 151±16, 126±10, and 119±13% in the MCF-7 cells
(Fig. 3A); and 152±5, 103±15, and 89±9% in the SK-BR-3
cells (Fig. 3B), respectively. The data showed that baicalein
effectively suppressed E2-promoted cell adhesion to Matrigel.
Baicalein reduces E2-stimulated cell invasion. T he
Matrigel-coated Transwell chamber system was used to inves-
tigate the effects of baicalein on E2-enhanced cell invasion.
After incubation with E2 for 24 h, the invaded cells across
the Matrigel-coated membrane were increased in both cell
lines (Fig. 4A). In the MCF-7 cell line, the invaded cells were
increased to 176±18% in the E2 treatment group compared
with the control, but decreased to 132±6 and 106±5% in
the 10 and 15 µM baicalein treatment groups, respectively
(Fig. 4B). Similar results were observed in the SK-BR-3 cells.
After SK-BR-3 cells were treated with baicalein, the invaded
cells in the E2-treated group decreased from 240±34 to
231±28%, 165±10% and 114±4% in the 5, 10 and 15 µM treat-
ment groups, respectively (Fig. 4B). Baicalein was evidently
able to suppress E2-enhanced cell invasion.
Baicalein prevents E2-induced GPR30 signal transduction.
The transmembrane receptor, GPR30, is able to mediate
estrogen responsiveness in ER-positive or ER-negative breast
cancer cells. Here, we aimed to determine whether or not
baicalein has inhibitory effects on GPR30 expression or its
signal transduction. We observed that GPR30 expression
remained unchanged after exposure to E2 with or without
baicalein in both the MCF-7 and SK-BR-3 cells (Fig. 5A).
Previous studies have indicated that GPR30 activation triggers
EGFR transactivation by tyrosine phosphorylation in breast
cancer cells (23,24), thus we sought to evaluate the inuence
of baicalein in EGFR phosphorylation upon E2 stimulation.
When cells were treated with baicalein in the presence of E2,
there was a signicant decrease in EGFR phosphorylation
(Tyr 1173) at 10 and 15 µM (Fig. 5B). GPR30 triggers EGFR
transactivation leading to the phosphorylation of ERK1/2 (24)
Figure 5. Effect of baicalein on E2-stimulated activation of the GPR30 signaling pathway. The cells were harvested after E2 (10 nM) with or without baicalein
(5, 10 and 15 µM) treatment for 1 h. (A) Western blot assay was used to examine the expression of GPR30. GAPDH was used as a loading control. (B) The total
and phosphorylated protein levels of EGFR, ERK1/2 and Akt. *P<0.05, signicantly different from the E2-treated group. (C) The total and phosphorylated
protein levels of EGFR, ER K1/2 and Akt in the cells treated with G1 (1 µM) in the presence or absence of baicalein (15 µM) treatment for 1 h. *P<0. 05 vs.
the G1-treated group. Densitometric analysis of the relative ratios of GPR30/GAPDH, phosphor ylated EGFR (Try 1173)/total EGF R, phosphorylated ERK1/2
(Thr 202/Tyr 204)/total ER K1/2 and phosphorylated Akt (Ser473)/total Akt. Values are mean ± SEM. E2, 17β-estradiol; GPR30, G protein-coupled estrogen
receptor; ER K, extracellular signal-regulated kinase; EGFR, epidermal growth factor receptor.
ONCOLOGY REPORTS 7
and activation of the PI3K/Akt transduction pathway (25).
Thus, it was essential to analyze the phosphorylation status
of ERK1/2 and Akt. Consequently, E2-induced phosphory-
lation of ERK1/2 at Thr 202/Tyr 204 and Akt at Ser 373
were repressed in the presence of baicalein at 10 and 15 µM
(Fig. 5B). In addition, GPR30 agonist G1 was used to further
conrm whether baicalein interferes with GPR30 signal trans-
duction. Indeed, treatment with baicalein at 15 µM prevented
G1-induced EGFR, ERK and Akt phosphorylation (Fig. 5C).
Baicalein suppresses E2-induced GPR30-mediated gene
expression. Previous reports have shown that CYR61 a nd CTGF
are the target genes which mediate the stimulatory effects
triggered by GPR30 activation (26). The potential of baicalein
to regulate GPR30-mediated expression of both target genes
was detected by real-time PCR. E2 upregulated CYR61 and
CTGF mRNA levels in the 2 cell lines. The downregulation
of the 2 genes in the MCF-7 and SK-BR-3 cells (Fig. 6A) was
observed after exposure to baicalein, when compared with
the E2-treated groups. In addition, the upregulation of CYR 61
and CTGF mRNA levels induced by G1 in the MCF-7 and
SK-BR-3 cells was also inhibited in the presence of baicalein
at 15 µM (Fig. 6B).
Discussion
Estrogens are essential for the growth and development of
mammary glands (27). However, prolonged exposure to
endogenous or exogenous estrogens is a major risk factor
for the development of breast cancer (28). Estrogens have
been confirmed to play a key role in breast cancer metas-
tasis (29,30). Tumor metastasis consists of a series of highly
relevant biological processes including disruption of cell-cell
and augmentation of cell-ECM adhesion capacity, induction of
cellular motility, invasiveness to the basement membrane and
ECM constituents, angiogenesis, and formation of a metastatic
lesion (31). In the present study, baicalein treatment inhibited
cell migration in response to E2 stimulation. Furthermore, it
suppressed E2-enhanced cell adhesion to Matrigel. Moreover,
it signicantly decreased E2-promoted invasive ability across
a Transwell membrane coated with Matrigel. These results
imply that baicalein may possess anti-estrogenic ability to
suppress E2-induced migration, adhesion and invasion of
MCF-7 and SK-BR-3 cells in vitro.
GPR30 is independent of the ER status in breast cancer
cells and tissue samples, and its action differs from the clas-
sical nuclear ERs, ERα and ERβ (7). Recent studies have
demonstrated that GPR30 mediates E2-enhanced cell migra-
tion and invasion in triple-negative or ERα-negative breast
cancer cells (32,33). Furthermore, we recently found that
GPR30 mediates E2-induced calpain activation, which is
correlated with the metastatic phenotype of breast cancer, in
both ERα-positive and -negative cells (34). We also showed
that tamoxifen, an ER antagonist but a GPR30 agonist, could
not suppress E2-promoted migration but enhanced migra-
tion in breast cancer cells and induced truncation of cyclin E
Figure 6. Effect of baicalein on E2-induced GPR30 target gene transcription. The CY R 61 and CTGF mRNA expression levels were detected by real-time
PCR. (A) The cells were treated with 10 nM E2 in the presence or absence of baicalcei n (5, 10 and 15 µM) for 1 h. Relative mRNA levels of (a) CY R61 and
(b) CTGF. *P<0.05 vs. E2-treated group. (B) The cells were treated with 1 µM G1 with or without 15 µM baicalcein for 1 h. Relative mRNA levels of (a) CYR61
and (b) CTGF. *P<0.05 vs. the G1-treated group. Results obtained from experiments performed in triplicate were norma lized to the β-actin mRNA level and
are shown as fold changes compared to the control cells. Values are mean ± SEM. E2, 17β-estradiol; GPR30, G protein-coupled estrogen receptor; CY R61,
cysteine-rich 61; CTGF, connective tissue growth factor.
SHANG et al: BAICALEIN INHI BITS ESTROGEN-INDUCED MIGRATION, ADHESION AN D INVASION
8
via GPR30 (35). It appears that GPR30, regardless of ERα
expression, plays an important role in E2-enhanced migration
and invasion of breast cancer cells. Our results showed that
baicalein suppressed E2-promoted migration and invasion
in GPR30-positive/ERα-negative SK-BR-3 cells and also
in GPR30/ERα-positive MCF-7 cells. Although baicalein
had little effect on GRP30 expression, it markedly inhibited
the E2-induced GPR30 signal activation, by decreasing
EGFR phosphorylation and activation of ERK1/2 and Akt.
Meanwhile, baicalein suppressed G1-induced upregulation of
the phosphorylation of EGFR, ERK1/2 and Akt. The results
indicate that the inhibitive effect of baicalein on the response
of MCF-7 and SK-BR-3 cells to estrogen may derive partly
from interfering with GPR30 signal transduction.
CYR61 and CTGF, also known as CCN1 and CCN2, belong
to the CCN protein family, which exerts their biological func-
tions by binding and activating cell surface integrins to stimulate
cell growth, adhesion, matrix production and migration, and by
regulating angiogenesis and tumorigenesis (36). CYR61 and
CTGF expression is signicantly associated with tumor size and
lymph node metastasis, suggesting that they play a role in the
progression of breast cancer (20). CYR61 is a survival and pro-
angiogenic factor and induces metastasis and drug resistance
in breast cancer cells (37). CTGF enhances migratory/invasive
processes in human breast cancer by activating the integrin-
αvβ3-ERK1/2-dependent upregulation of S100 calcium-binding
protein A4 (S100A4) (38). The two genes related to breast cancer
metastasis are the target genes of GPR30. Here, we found that
baicalein signicantly in hibited E2- or G1-induced upregulation
of CTGF and CYR61 gene expression. These results suggest
that baicalein may prevent E2-induced migration, invasion
and adhesion of MCF-7 and SK-BR-3 cells also by suppressing
GPR30-dependent gene transcription.
Flavonoids possess a chemical structure similar to estrogen,
and some have been considered to exert an anti-estrogenic
effect and reduce the risk of breast cancer. Genistein, which is
the main ingredient responsible for the cancer chemopreven-
tive activity of soy-derived foods, has garnered much research.
However, it displays a biphasic effect on the proliferation and
death of MCF-7 cells and displays estrogenic activity to trans-
activate ERα at low concentrations. Although baicalein is an
isomer of genistein, it does not have a biphasic effect on ER,
which suggests that it could be a better chemopreventive agent
than genistein (14). Moreover, genistein can stimulate GPR30
to mediate c-fos upregulation in breast cancer cells (39)
and induce the proliferation of thyroid cancer cells (40). In
the present study, we found that baicalein suppressed E2 or
G1-induced GPR30 signal activation indicating a different
activity from genistein. The precise difference between the
two compounds with similar chemical structure remains to
be resolved in future studies. The relationship between the
structure of avonoids and the inhibition of E2 events warrants
further study. To fully investigate the mechanism, we need to
explain the detailed molecular mechanisms with which baica-
lein inuences GPR30 and downstream signaling, as well as
crosstalks with other receptors. In addition, whether baicalein
has dual effects on both ERα and GPR30 still needs further
investigation.
In conclusion, baicalein attenuates E2-induced migration,
adhesion and invasion of MCF-7 and SK-BR-3 breast cancer
cells through interference with GPR30 pathway activation.
This indicates that baicalein may be a promising agent for the
treatment of GPR30-positive breast cancer metastasis.
Acknowledgements
The present study was supported by the Natural Science
Foundation of Guizhou Province of China [grant no. QKH-
J(2014)2007], the National Natural Science Foundation of China
(grant nos. 81302804 and 31360252), Startup Foundation for
Doctors of Guiyang Medical University [grant no. (2013)09] and
the Foundation for Training Programs of Innovation and
Entrepreneurship for Undergraduates of Guiyang Medical
University (grant no. 201410660038).
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