Journal of Pharmacological Sciences 128 (2015) 97e107

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ERa down-regulation plays a key role in silibinin-induced autophagy

and apoptosis in human breast cancer MCF-7 cells
Nan Zheng a, Ping Zhang a, Huai Huang a, Weiwei Liu a, Toshihiko Hayashi a, Linghe Zang a,
Ye Zhang a, Lu Liu a, Mingyu Xia a, Shin-ichi Tashiro b, Satoshi Onodera c,
Takashi Ikejima a, *
a
ChinaeJapan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016, China
b
Institute for Clinical and Biomedical Sciences, Kyoto 603-8072, Japan
c
Department of Clinical and Biomedical Sciences, Showa Pharmaceutical University, Tokyo 194-8543, Japan

a r t i c l e i n f o a b s t r a c t

Article history: The estrogen receptor alpha (ERa) has been proven to be one of the most important therapeutic targets
Received 24 November 2014 in breast cancer over the last 30 years. Previous studies pointed out that a natural flavonoid, silibinin,
Received in revised form induced apoptosis in human breast cancer MCF-7 cells. In the present study we report that exposure of
10 March 2015
MCF-7 cells to silibinin led to cell death through the down-regulation of ERa expression. Silibinin-
Accepted 1 May 2015
Available online 12 May 2015
induced apoptosis of MCF-7 cells through up-regulation of caspase 6 due to ERa signalling repression
was further boosted by ERa antagonist. Moreover, up-regulation of autophagy induced by silibinin
accounted for apoptotic exacerbation, being further enhanced by ERa inhibition. Upon ERa activation,
Keywords:
Silibinin
series of downstream signalling pathways can be activated. We found that silibinin reduced the ex-
Estrogen receptor alpha (ERa) pressions of Akt/mTOR and extracellular-signal-related kinase (ERK), which respectively accounted for
MPP dihydrochloride hydrate (MPP) the induction of autophagy and apoptosis. These effects were further augmented by co-treatment with
Apoptosis ERa inhibitor. We conclude that the treatment with silibinin of ERa-positive MCF-7 cells down-regulates
Autophagy the expression of ERa, and subsequently mTOR and ERK signaling pathways, ERa downstream, finally
resulting in induction of autophagy and apoptosis.
© 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological
Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).

1. Introduction
In the modern world, breast cancer is one of the most common
malignances in women. It has been known more than a century (1)
that breast cancer is associated with cell-surface estrogen receptor
(ER). Human ER exists as two subtypes, ERa and ERb, which regu-
late the transcription of various target genes upon binding to es-
trogen response elements (ERE) present within the regulatory
region of the genes. In most ERa-positive breast cancers, especially
in early stages, the expression level of ERa is considerably higher
than that in normal breast epithelium (2). Therefore, targeting the
ERa signalling pathway has already become a focal point in the
development of breast cancer therapy.
Silibinin (Fig. 1A), a natural polyphenolic flavonoid, is a major
bioactive component of silymarin isolated from the plant milk
thistle Silybum marianum (L.) Gaertn. Silibinin has been extensively
used for its hepatoprotective effectiveness in Asia and Europe (3,4).
According to the reports, flavonoids have been shown to be cyto-
toxic to cancer cells (5,6). It has been reported that the estrogen
receptor is required for flavonoid-induced cytotoxicity in breast
cancer cell lines (7,8). Silibinin induces a loss of cell viability in MCF-
7 cells. However, underlying mechanism has not been completely
elucidated. It is not known if the estradiol-like effect of silibinin
contributes to its anti-cancer potency. Therefore, we focused on
ERa regulation in the cytotoxicity of silibinin in MCF-7 cells that
inherently express high levels of ERa.
2. Materials and methods
2.1. Reagents

* Corresponding author. Tel./fax: þ86 24 23844463.

Silibinin with a purity of 99% was purchased from Jurong Best
E-mail address: ikejimat@vip.sina.com (T. Ikejima). Medicine Material (Zhenjiang, Jiangsu, China). The reagent was
Peer review under responsibility of Japanese Pharmacological Society. dissolved in dimethylsulfoxide (DMSO) to make a stock solution.

http://dx.doi.org/10.1016/j.jphs.2015.05.001
1347-8613/© 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
98 N. Zheng et al. / Journal of Pharmacological Sciences 128 (2015) 97e107

Fig. 1. The cytotoxicity of silibinin in MCF-7 cells is correlated with the down-regulation of estrogen receptor a (ERa). (A) The chemical structure of silibinin. (B) The cells were
treated with various doses of silibinin for 12, 24, 36 or 48 h. Viability was measured by MTT assay. (C) Western blot analysis of ERa levels after treatment with 100, 200 and 300 mM
silibinin for 24 h b-actin was used as an equal loading control. (D) The cells were pre-treated with 10, 20 and 40 mM ICI 182,780 for 1 h prior to silibinin (200 mM) administration for
24 h, and the cell viability was measured by MTT assay, n ¼ 3, mean ± S.E.M, *p < 0.05, compared with silibinin -treated group. (E) The different suppression of silibinin in ERa-high
expressive MCF-7 and ERa-low expressing MDA-MB231 (MB231) human breast cancer cells. Compare between the two strains of human breast cancers at the specific concentration
of silibinin, **p < 0.01. ERa levels were examined by western blot analysis. (F) Cells were cultured for 24 h and then incubated with different concentrations of MPP (a specific ERa
antagonist MPP dihydrochloride) for 1 h, and then silibinin 200 mM was added and cultured for 24 h. The viability was determined by the MTT assay, n ¼ 3, mean ± S.E.M, **p < 0.01,
compared with silibinin-treated group. (G) The cells were transfected with control or ERa-targeting siRNA for 48 h, and then incubated with different concentrations of silibinin for
24 h. The viability was measured by MTT assay, n ¼ 3, mean ± S.E.M, *p < 0.05, **p < 0.01, si-ERa compared with si-con group.
 N. Zheng et al. / Journal of Pharmacological Sciences 128 (2015) 97e107
MEM and DMEM complete media were used for dilution and the
final concentration of DMSO was kept below 0.1% in cell culture,
which had no detectable effects on cells. Methylthiazolyldiphenyl-
tetrazolium bromide (MTT), propidiumiodide (PI), RNase A, mon-
odansyl cadaverine (MDC), rhodamine 123, estrogen receptor (ER)
antagonist Fulvestrant (ICI 182,780), estrogen receptor alpha (ERa)-
specific antagonist MPP 1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-
(2-piperidinylethoxy)phenol]-1H-pyrazole-dihydro- chloride, and
primary antibody against LC3 were purchased from Sigma Chemi-
cal (St. Louis, MO, USA). Polyclonal antibody against ERa was pur-
chased from Proteintech Group (Chicago, IL, USA). Primary
antibodies against p62, caspase-6, caspase-8, Bid, Akt1/2, p-Akt1/2,
Bcl-2, Bax, cytochrome c, ERK1, p-ERK 1/2, GRB2, Raf, SOS, inhibitor
of caspase-activated DNase (ICAD), poly-ADP-ribose polymerase
(PARP) and b-actin, as well as horseradish peroxidase-conjugated
secondary antibodies, were purchased from Santa Cruz Biotech-
nology (Santa Cruz, CA, USA). The SuperSignal West Pico Chemi-
luminescent Substrate® used in conjunction with the horseradish
peroxidase (HRP) enzyme was purchased from Thermo Scientific
(Rockford, IL, USA).
2.2. Cells culture
Human breast cancer MCF-7, MDA-MB231 cells and human
hepatoma G2 (HepG2) were obtained from American Type Culture
Collection (ATCC) (Manassas, VA, USA). MCF-7 cells were cultured
in Minimum Essential Medium (MEM, Gibco, Grand Island, NY,
USA), while MDA-MB231 and HepG2 cells were in Dulbecco's
Modified Eagle medium (DMEM, Gibco, Grand Island, NY, USA). All
media were supplemented with 10% fetal bovine serum (FBS)
(Beijing Yuanheng Shengma Research Institution of Biotechnology,
Beijing, China), penicillin (100 U/ml) and streptomycin (100 mg/
ml). Cells were incubated at 37  C with 5% CO2 in a humidified
atmosphere. All experiments were performed on logarithmically
growing cells.
2.3. Fluorescent microscopy of MDC staining
Autofluorescent compound MDC stains acidic lysosomes and
thus it is widely used in studies of autophagy together with the
measurement of other parameters (9). The cells were seeded into
24-well cell culture plates (Corning, NY, USA) at a density of 2  104
cells per well for 24 h. The cells in culture for another 24 h by the
treatments with different reagents were examined. The cells
treated were rinsed twice with ice-cold PBS, stained with 0.05 mM
MDC solution in the dark at 37  C for 30 min. They were observed
with a fluorescence microscope (Olympus, Tokyo, Japan).
2.4. Flow cytometry of MDC positive cells
Cells subjected to the indicated treatments were collected
(5  105 cells per sample), incubated with 0.05 mM MDC solution in
the dark at 37  C for 30 min and analysed using a FACScan flow
cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
2.5. Cytotoxicity assay
MCF-7 cells were seeded into 96-well cell culture plates
(Corning, NY, USA) at a density of 5  103 cells/well and cultured for
24 h. Then the cells were subjected to the indicated treatments for
24 h. The cells were rinsed twice with ice-cold PBS and incubated
with 100 mL of 0.5 mg/mL MTT solution at 37  C for 3 h. Then the
supernatant was discarded, and the residual cell layer was dis-
solved with 150 mL of DMSO. Thereafter, the optical absorbance (A
value) was measured at 490 nm wavelength using a microplate
99
reader (Thermo Scientific Multiskan MK3, Shanghai, China). Cell
growth inhibitory ratio was calculated using the following
equation:
Cell growth inhibitory ratio (%) ¼ 100  (A490, controlA490, sam-
ple)/(A490, controlA490, blank)
2.6. Flow cytometric analysis of dying cells
Propidium iodide (PI), a fluorescent dye that specifically binds to
cellular DNA, was used to quantify DNA content. Cells subjected to
the indicated treatments were collected (5  105 cells per sample),
fixed with 70% (v/v) ethanol at 4  C overnight, rinsed with ice-cold
PBS twice and incubated with 1 mL of PI solution (PI 50 mg/L and
RNase A1g/L) in the dark for 30 min. The cellular DNA content was
next analyzed using a FACScan flow cytometer. Cells with DNA
content less than normal diploid (at subG0/G1 phase) are taken as
dead cells.
2.7. Determination of mitochondrial membrane potential
The mitochondrial membrane potential was examined using the
mitochondrial dye rhodamine 123. After incubation with indicated
treatments, MCF-7 cells were collected and stained with 1 mg/mL
rhodamine 123 at 37  C for 30 min. The fluorescence intensity of
the cells was analysed by FACScan flow cytometry.
2.8. Transfection of siRNA
MCF-7 cells were transfected with negative control siRNA (NC-
siRNA) or siRNA targeting ERa (ERa-siRNA) at a final concentration
of 10 nM using siRNA-Mate (GenePharma, Shanghai, China) ac-
cording to the manufacturer's instructions. The sequences of the
ERa-siRNA duplex were as follows: sense strand, 50 -GAGGGA-
GAAUGUUGAAACATT-30 ; antisense strand, 50 -UGUUUCAACAUU-
CUCCCUCTT-30 . The sequences of NC-siRNA duplex were: sense
strand, 50 -UUCUCCGAACGUGUCACGUTT-30 ; antisense strand, 50 -
ACGUGACACGUUCGGAGAATT-30 . The transfected cells were main-
tained for another 48 h before subsequent experiments.
2.9. Western blot analysis
After the indicated treatments, both adherent and floating cells
were collected at the predetermined time points and lysed with
RIPA lysis buffer (Beyotime, Haimen, Jiangsu, China) supplemented
with PMSF (1 mM) for 30 min. After centrifugation at 12,000  g for
10 min, the supernatant was collected and the protein concentra-
tion was determined with the Bio-Rad protein assay reagent (Bio-
Rad, Hercules, CA, USA). The lysates that were adjusted with the
total amount of proteins equal were separated on 10e13% SDS-
PAGE. The separated protein bands were transferred onto Milli-
pore Immobilon®-P Transfer Membrane (Millipore Corporation,
Billerica, MA, USA). After blocking with 5% skimmed milk at room
temperature for 2 h, the membranes were incubated with primary
antibodies at 4  C overnight and then with corresponding HRP
conjugated secondary antibodies at room temperature for 2 h. After
that, the blots were visualized using the SuperSignal West Pico
Chemiluminescent Substrate® purchased from Thermo Scientific
(Rockford, IL, USA).
2.10. Statistical analysis
All the data and results obtained by at least three independent
experiments were expressed as mean ± S.D. Comparisons between
groups were determined using Student's t-test. One-tailed p-values
are significant when p < 0.05.
100 N. Zheng et al. / Journal of Pharmacological Sciences 128 (2015) 97e107

Fig. 2. Treatment with a specific ERa antagonist MPP dihydrochloride (MPP) enhances the apoptosis of MCF-7 cells induced by silibinin. (A) Morphologic changes of apoptotic MCF-
7 cells were observed at 24 h (a, 200  magnification, bar ¼ 20 mm), Con: control, Sili: silibinin. (B) Flow cytometric analysis of apoptotic cell ratios after PI staining (SubG1 cells). (a)
The data are presented as the mean ± S.E.M. of the results for three independent experiments. (b) **p < 0.01 compared with silibinin-treated group. (C) The cells were incubated
with 200 mM silibinin with/without 10 mM MPP for 24 h. The rhodamine-123 fluorescent intensity was analysed by flow cytometry. #p < 0.05 versus control group, *p < 0.05 versus
silibinin group.
 N. Zheng et al. / Journal of Pharmacological Sciences 128 (2015) 97e107 101

Fig. 3. Inhibition of ERa enhances the MCF-7 cell death induced by silibinin through extrinsic and mitochondrial-apoptosis pathways. The cells were incubated with or without
10 mM MPP for 1 h. Then 200 mM silibinin was added to the cells which were cultured for 24 h. Cell lysates were separated by 11% SDS-PAGE, and the levels of (A) caspase 8, bid, (B)
Bax (mitochondria), Bcl-2 (mitochondria), cytochrome c (cytoplasm), (D) caspase 6, PARP, ICAD were detected by Western blot analysis. Band density of the specific protein was
analyzed with Quantity One image software and the results were expressed as relative density to b-actin. (C) The expression of caspase 3 was detected in the MCF-7 cells treated
with silibinin and/or MPP. The expression in HepG2 cells was used as a positive control.
102 N. Zheng et al. / Journal of Pharmacological Sciences 128 (2015) 97e107

Fig. 4. Inhibition of ERa reinforces silibinin-induced MCF-7 cell autophagy. (A) The cells were incubated with or without 10 mM MPP for 1 h. Following the addition of 200 mM
silibinin, the cells were cultured for 24 h. Changes of fluoresce intensity were observed by fluorescence microscopy with MDC staining (  400 magnification, bar ¼ 20 mm). (B) MCF-
7 cells were cultured in the presence of 0, 100, 200 and 300 mM silibinin for 12 and 24 h. The MDC positive ratio was determined by flow cytometric analysis. (C) The positive ratio of
MDC staining was detected by flow cytometric analysis, n ¼ 3, mean ± S.E.M, **p < 0.01, compared with silibinin-treated group. (D) MCF-7 cells were lysed after the treatment with
100, 200 or 300 mM silibinin for 24 h, and the protein levels of p62 and LC3 were detected by western blot analysis. (E) The protein levels of p62 and LC3 were detected by western
blot analysis. Band density of the specific protein was analysed with Quantity One image software. (n ¼ 3, means ± SD; #p < 0.05 vs control group).
 N. Zheng et al. / Journal of Pharmacological Sciences 128 (2015) 97e107 103

3. Results 3.3. Treatment with MPP enhances the cytotoxicity of silibinin

through both extrinsic and intrinsic-apoptosis pathways
3.1. ERa down-regulation was involved in silibinin-induced
cytotoxicity of MCF-7 cells Western blot assay was carried out to further determine the
features of silibinin and MPP-induced MCF-7 cell death. The
To test the cytotoxicity of silibinin on MCF-7 cells, the cells were treatment with silibinin resulted in that extrinsic apoptosis-related
cultured with 100e300 mM silibinin for 12, 24, 36 and 48 h. Silibinin protein pro-caspase-8 was cleaved into active caspase-8, as well as
was found to decrease the proportion of viable cells in a time and Bid was cleaved. MPP further augmented this effect (Fig. 3A). As for
dose-dependent manner with an IC50 (at 24 h) of 200 mM (Fig. 1B). the intrinsic apoptosis pathway, translocation of the anti-apoptotic
As a flavonoid, silibinin might affect the estrogen receptor signal- protein Bcl-2 to mitochondria was markedly decreased in the
ling pathways (5). Therefore, we investigated the levels of ERa. silibinin-treated group, while the pro-apoptotic translocation of
Western blot analysis showed that treatment with various con- Bax to mitochondria and the release of cytochrome c to cytoplasm
centrations of silibinin for 24 h reduced ERa expression in a were increased. MPP also augmentedthese processes furthermore
concentration-dependent manner (Fig. 1C). Fulvestrant (ICI (Fig. 3B). The results indicate that the intrinsic apoptosis pathway
182,780), an estrogen receptor antagonist which blocks both ERa was also upregulated.
and ERb signalling, was applied with silibinin to examine the effect Caspase 3 is the downstream executor of apoptosis in many
of estrogen receptor signalling pathway on MCF-7 cell growth. The cells. However, MCF-7 cells were reported to be negative in the
results showed that ICI 182,780 augmented silibinin-induced MCF- expression of caspase-3 (13,14). Accordingly, the expressions of
7 cell growth inhibition (Fig. 1D). To further elucidate the role of pro-caspase 3 and activated caspase 3 were not detected in
ERa, we examined the cytotoxicity of silibinin on two different silibinin-treated MCF-7 cells (Fig. 3C). It was reported that silibinin
strains of human breast cancer cells: one is MCF-7 cells with an
inherently high expression of ERa and the other is MDA-MB-231
cell with a rather low expression of ERa. MDA-MB-231cells are
often utilized as a negative control for the examination of ERa
involvement (10). Western blot analysis of the ERa levels of MCF-7
and MDA-MB-231 indicated, in accordance with the previous re-
ports, that the ERa expression in MCF-7 cells was high, but low in
MDA-MB-231 cells (Fig. 1E (a)).
MTT assay showed that the growth inhibitory effect of silibinin
on MCF-7 cells was higher than that on the MDA-MB-231 cells
(Fig. 1E (b)), evidencing the positive correlation between ERa
down-regulation and cytotoxicity. The specific ERa-antagonist,
MPP (11), dose-dependently augmented silibinin-induced MCF-7
cell growth inhibition (Fig. 1F), even to a larger extent than ICI
182,780 (Fig. 1D). Since high dose of MPP (20 mM) alone also
inhibited cell proliferation, we chose a lower dose (10 mM) of MPP
in the following experiments. To further confirm the cytotoxicity of
silibinin in combination with repression of ERa on MCF-7 cells, we
transfected the siRNA targeting ERa (si-ERa) into cells (Fig. 1G (a)).
We found that silencing of ERa markedly enhanced the cytotoxic
effect of silibinin (Fig. 1G (b)). Therefore, silibinin exerted anti-
cancer effects on MCF-7 cells through the inhibition of ERa
pathways.

3.2. Treatment with MPP enhances silibinin-induced apoptosis of

MCF-7 cells

To determine the features of MCF-7 cell growth inhibition, the

morphologic changes were examined. Obvious changes of cell
nuclei and cell number were observed in the cells treated with
200 mM silibinin plus 10 mM MPP for 24 h when compared with the
cells treated with silibinin alone, (Fig. 2A). PI staining was applied
to analysis of the changes of DNA contents: silibinin increased the
ratio of cells at subG1 phase in cell cycle at 24 h, while co-treatment
with MPP increased the ratio furthermore (Fig. 2B). These results
suggested that apoptosis might be involved in this growth inhibi-
tion. It was well documented that many anticancer drugs induced
apoptosis following mitochondrial dysfunctions (12). Therefore, we
examined intactness of mitochondrial membranes by rhodamine
123 staining. Decrease in rodamine-123 fluorescence intensity re-
flects the reduction in mitochondrial transmembrane potential Fig. 5. Autophagy promotes silibinin- and MPP-induced apoptosis. (A) The cells were
(Djm). Compared with silibinin-treated group, co-treatment with treated with 10 mM MPP for 1 h, and then 200 mM silibinin was added and cultured for
24 h in the presence or absence of rapamycin (10 nM) or 3 MA (2 mM), and the cell
MPP resulted in a further reduction in Djm (Fig. 2C), indicating that viability was measured by MTT assay, n ¼ 3, mean ± S.E.M, *p < 0.05, **p < 0.01,
inhibition of ERa markedly enhances the cytotoxicity of silibinin on compared with silibinin and MPP-treated group. (B) The protein levels of caspase 6,
MCF-7 cells through mitochondrial dysfunction. PARP and ICAD were detected by western blot analysis.
104 N. Zheng et al. / Journal of Pharmacological Sciences 128 (2015) 97e107

Fig. 6. Changes in the expressions of ERa downstream proteins in silibinin- and MPP-treated MCF-7 cells. (A) The cells were incubated with or without 10 mM MPP for 1 h, then
200 mM silibinin was added and cultured for 24 h, the levels of (A) GRB2, SOS, PI3K, AKT, mTOR, (B) MAPK-related proteins Raf, ERK, p-ERK were detected by Western blot analysis.
b-actin was used as an equal loading control. Band density of the specific protein was analyzed with Quantity One image software. (C) The cells were incubated with different
concentrations of PD98059 for 1 h. Following the addition of 200 mM silibinin, and the cells were cultured for 24 h. The viability was measured by MTT assay, n ¼ 3, mean ± S.E.M,
*p < 0.05, compared with silibinin-treated group. (E) The cells were treated with 10 mM MPP for 1 h. Following the addition of 200 mM silibinin, the cells were cultured for 24 h in the
presence or absence of PD98059 (30 mM). The cell viability was measured by MTT assay, n ¼ 3, mean ± S.E.M, *p < 0.05, compared with silibinin and MPP-treated group. (D) The
protein levels of p62 and LC3 and (F) EGFR were detected by western blot analysis.

activated caspase 3 in HepG2 cells (15). Therefore, silibinin-treated silibinin induced the activation of caspase 6. MPP treatment
HepG2 cells were used as a positive control for the detection of augmented the cleavage of pro-caspase-6 to caspase-6 further-
caspase 3 (Fig. 3C). We examined the expression of another com- more. The substrates of caspase-6, ICAD and PARP, were cleaved
mon downstream executor of apoptosis, caspase-6, and found that accordingly (Fig. 3D). In conclusion, MPP pre-treatment enhanced
 N. Zheng et al. / Journal of Pharmacological Sciences 128 (2015) 97e107
both intrinsic and extrinsic apoptotic pathways in silibinin-treated
MCF-7 cells.
3.4. Inhibition of ERa augmented silibinin-induced MCF-7 cell
autophagy
Autophagy is a fundamental cellular pathway by which cyto-
plasmic materials, including soluble macromolecules and organ-
elles, are delivered into lysosomes for degradation (15). Autophagy
plays an important role in the cellular responses towards stresses
such as starvation, hypoxia and DNA damage. The morphologic
changes were observed in the MDC-stained cells by fluorescence
microscopy. Combined treatment with silibinin and MPP caused an
obvious increase in the number of MDC-labelled autophagolyso-
somes compared with the silibinin alone group (Fig. 4A). The
quantitative analysis of autophagy by MDC flow cytometric analysis
showed the consistent results (Fig. 4B and C). Microtubule-
associated protein light chain 3 (LC3) is now widely used to
monitor autophagy. The levels of LC3 conversion (from cytosolic
LC3-I to membrane-bound lipidated LC3-II) or the amounts of LC3-
II were examined by immunoblotting analysis (16). An alternative
method for detecting the autophagic flux is the measurement of
p62 degradation, since p62 can bind LC3, thus serving as a selective
substrate of autophagy (16). Dose-dependent up-regulation of the
conversion from LC3-I to LC3-II and down-regulation of p62 caused
by silibinin treatment were observed (Fig. 4D). Combined use of
silibinin with MPP enhanced expressions of LC3-II and conversion
from LC3-I to LC3-II significantly increased, while p62 amount was
further reduced (Fig. 4E).
3.5. Autophagy promoted apoptosis in MCF-7 cells by the co-
treatment with silibinin and MPP
To investigate the crosstalk of apoptosis and autophagy in sili-
binin and MPP-treated MCF-7 cells, 3 MA, a specific autophagic
inhibitor, and rapamycin, an autophagic inducer, were employed.
As shown in Fig. 5A, silibinin and MPP-induced cell growth inhi-
bition was further augmented by rapamycin treatment but partially
reversed by 3 MA. Western blot analysis showed that the activation
of caspase-6 and the cleavage of caspase substrates, PARP and ICAD,
caused by cotreatment with MPP and silibinin were reversed by
3 MA treatment (Fig. 5B). These findings indicated that the auto-
phagy induced by silibinin and MPP co-treatment intensified the
apoptotic process in MCF-7 cells.
3.6. Combination of silibinin and MPP treatments repressed the
downstream signals of ERa
The pathways through Shc-Grb2-Sos (Src homology-growth
factor receptor binding protein 2-son of sevenless) for activation
of MAPK and are involved in the downsteam of activated ERa (17).
One of them is RAS/ERK pathway, which can induce oncogenic gene
expression programs, being often activated in cancer cells. Another
is PI3K/Akt pathway which is related with the mammalian target of
rapamycin (mTOR). Hou X. and co-workers reported that ERa raised
the phosphorylation levels of PI3K, Akt and mTOR in endometrial
carcinoma cells (18). We examined the activation of these two ER
downstream signaling pathways.
Western blot analysis showed that PI3K/Akt/mTOR pathway
was down-regulated by either silibinin alone or silibinin and MPP
co-treatment (Fig. 6A). As has been demonstrated in many studies,
inhibition of mTOR promotes autophagy (19,20). Therefore, silibi-
nin enhanced autophagy by down-regulating ERa downstream
PI3K/Akt/mTOR pathway.
105
As shown in Fig. 6B, treatment with MPP obviously reduced the
p-ERK expression compared with silibinin alone group. To examine
the role of ERK, we pre-treated the cells with ERK inhibitor
PD98059, and found that the cytotoxicity was obviously enhanced
by ERK inhibition (Fig. 6C and D). Since p62 degradation and from
LC3-I to LC3-II increased, we further confirmed significantly
increased autophagy after PD98059 exposure (Fig. 6E). These
findings indicated that apoptotic action of silibinin on MCF-7 cells
was mediated through ERa downstream RAS/ERK pathway. EGFR
signaling also leads to inhibition of ERK1/2. Crosstalk between
EGFR and ER signaling has been established and proved to have
significant impact on complex biological processes such as cell
growth and cell survival of breast cancer cells. To test the rela-
tionship between down-regulation of ERa and EGFR, we examined
the changes of expressed EGFR protein levels in MCF-7 cells treated
with silibinin with/without MPP. We found that inhibition of ERa
with silibinin or/and MPP, the expression levels of EGFR was down-
regulated as shown in (Fig. 6F). Thus, the effect of silibinin on ERK is
mediated by ERa that may have cross-talk with EGFR.
4. Discussion
Breast cancer is one of the most frequently diagnosed cancers
and the leading cause of death by cancer among females world-
wide. To develop breast cancer therapies, targeting of ERa, which is
highly expressed in approximately 70% of all breast tumors (21), is
taken as a targeting molecule.
ERa is a member of the steroid receptor superfamily that reg-
ulates processes such as growth and differentiation in various
target cells by affecting transcription. ERa not only plays an
important role in many human tissues and organs, such as mam-
mary gland, genital tract, and central nervous system (22), but also
it has a close correlation with gaining the peak bone mass and
maintaining the bone mass (23). Women who are menopausal or
postmenopausal usually produce less ERa, leading to bone loss (24).
However, in the majority of breast cancers, ERa is greatly up-
regulated than normal breast cells and its expression is a hall-
mark of hormone-dependent tumor growth (25). In addition, the
presence of elevated levels of ERa in benign breast epithelium
appears to indicate an increased risk of breast cancer, suggesting a
role for ERa in breast cancer initiation, as well as progression (26).
Silibinin, which has been used as a liver protectant, induced
apoptosis in human breast cancer MCF-7 cells (27). In this study, we
report the evidence for the mechanism of silibinin-induced
apoptosis in that downregulation of ERa expression in the MCF-7
cells exposed to silibinin underlies. Consistent with previous re-
ports, silimarin, which contains silibinin as a major component, has
been used in the clinical treatment of hepatic diseases and phar-
macologic studies have indicated that silymarin is not toxic even at
high doses. Besides, pretreatment with silibinin 500 mM signifi-
cantly inhibited UV-induced apoptosis in HaCaT cells after 9 h in-
cubation (28). Therefore we believe that silibinin appears to be
toxic for cancer cells, but relatively safe for normal ones. ERa re-
ceptors are known to exist in cytosol and also as membrane bound-
ER. However, in MCF-7 cells transfected with GFP-ERa, the fluo-
rescence of GFP-ERa was observed only in the nucleus (29).
Therefore, it seemed that ERa receptors are mainly found in
nucleus.
The mechanism we propose here is supported by the finding
that ERa-specific antagonist, MPP, combined with silibinin
enhanced all the effects of silibinin on MCF-7 cells. The enhance-
ment with MPP can be interpreted as inhibition of the activity of
ERa remaining after downregulation with silibinin. Apoptosis is
characterized by the sequential activation of caspase cascades.
Caspase 8 is believed to be one of the initiator caspases that can
106 N. Zheng et al. / Journal of Pharmacological Sciences 128 (2015) 97e107
activate downstream caspase-3 (30). However, MCF-7 cells do not
express caspase-3 (13,14). Instead, upregulation of the caspase-6
expression was obtained for the MCF-7 cells treated with silibinin
in accordance with the report by Kagawa S et al., showing that Bax-
induced levels of caspase-6 activation could be used as an evalua-
tion index of apoptosis when the cells did not express caspase-3
(31).
A number of studies showed relationship between ERa
expression and levels of apoptosis related proteins including Bcl-2
and Bax (32e34). The relationships between ERa and apoptosis
pathways in silibinin-treated cells observed in the present study
indicate that death rate of MCF-7 cells induced by silibinin through
the down-regulation of ERa involves both the extrinsic and
intrinsic apoptosis pathways.
Estradiol-bound ERa interacts with various signalling molecules
such as PI3K (35) and MAP kinases (36). The PI3K/Akt and MEK/ERK
signalling pathways are important downstream pathways of ERa.
The observations in the present study that silibinin reduced the
phosphorylation levels of both ERK and Akt and that additional
treatment with a specific inhibitor of ERK enhanced the cytotoxic
effects on MCF-7 cells treated with silibinin and MPP are rationally
interpreted, since they are downstream signaling pathways of ERa.
Our results are in accordance with the results of Melyssa and co-
workers, who reported that MCF-7 breast carcinoma cells pro-
moted survival through activation of PI3K/Akt crosstalk pathway
(37). We therefore conclude that silibinin shows cytotoxic effect
against MCF-7 cells through the down-regulation of ERa signalling
pathway.
Physiological levels of autophagy are essential for normal
cellular homeostasis. The absence of autophagy increases cell death
during metabolic stress and on treatment with cytotoxic chemo-
therapeutic agents. By contrast, excessive levels of autophagy
promotes cell death (38). It is postulated that autophagic cell death
induced by some anticancer agents underlines their potential as a
new cancer therapy modality (39). MPP in combination with sili-
binin activates autophagy which enhances apoptosis in MCF-7 cells.
The results are consistent with the notions in several studies in that
autophagy is triggered in some cancers in response to various
anticancer agents, including As2O3, tamoxifen and temozolomide
(40).
In conclusion, the present study demonstrates that down-
regulation of ERa expression by the treatment with silibinin of
MCF-7 cells plays a key role in leading the cells to follow ERa-MEK/
ERK and ERa-Akt/mTOR pathways to apoptosis. ERa inhibition with
ERa-specific antagonist, MPP, augments autophagy and apoptosis
by extrinsic-apoptosis and mitochondrial-apoptosis pathways.
Autophagy plays a pro-apoptotic role with the detailed mechanism
remaining to be cleared.
In the present study, we highlight just ERa signalling for the
silibinin effect. However, it is not known whether the antitumor
activity of ERb is involved. Estrogen receptors ERa and ERb share
considerable sequence homology yet exert opposite effects on
breast cancer cell proliferation. Bin et al. illustrated that modulation
of ERb-specific antitumor activity can be considered as a molecular
strategy for cancer therapy (41). It is suggested that the mecha-
nisms of silibinin's toxic effect on MCF-7 cells may also be associ-
ated with ERb. Up-regulation of ERb is another mechanism of toxic
effect on cancer cells. Studies also have shown that ERb exerts
antiproliferative effects in ERa expressing MCF7 cells, probably by
initiating degradation of ERa. Meanwhile, ERb-selective agonist
DPN inhibited cell growth and induced apoptosis. In addition, there
are reports which show that the proliferating MCF-7 cells express
both ERa and ERb. In order to examine whether the cytotoxic effect
of silibinin is related with ERa, we performed MTT experiments
using MDA-MB231 cells that have low expression of ERa and
positive ERb as a negative control. MTT assay showed that the
growth inhibitory effect of silibinin on MCF-7 cells was higher than
that on the MDA-MB-231 cells (Fig. 1E (b)), evidencing the positive
correlation between ERa down-regulation and cytotoxicity. We
assume that silibinin also dose-dependently induced cytotoxicity in
MDA-MB231 cells and ERb expression in MDA-MB231 cells was
increased. In order to prove this hypothesis, further experiments
are necessary. Thus, ERb could be another key mechanism of
silibinin-induced apoptosis and autophagy in many cell lines as
well as MCF-7 cell. Therefore, we are going to use ERb as a target
object for future research.
Conflicts of interest
The authors declare that there are no conflicts of interest.
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