Wedelolactone Induces Growth of Breast Cancer Cells by Stimulation of Estrogen Receptor Signalling

Journal of Steroid Biochemistry & Molecular Biology 152 (2015) 76–83

Contents lists available at ScienceDirect

Journal of Steroid Biochemistry & Molecular Biology

journa l homepage: www.elsevier.com/locate/jsbmb

Wedelolactone induces growth of breast cancer cells by stimulation of estrogen receptor signalling

a a,b c,d c,d a,d,

Tereza Nehybova , Jan Smarda , Lukas Daniel , Jan Brezovsky , Petr Benes *

Laboratory of Cellular Differentiation, Department of Experimental Biology, Faculty of Science, Masaryk University, Kamenice 5/A36, 625 00 Brno, Czech Republic

Masaryk Memorial Cancer Institute, RECAMO, Zluty kopec 7, 656 53 Brno, Czech Republic

Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Faculty of Science,

Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech Republic

International Clinical Research Center, Center for Biological and Cellular Engineering, St. Anne’s University Hospital, Pekarska 53, 656 91 Brno, Czech

Republic

A R T I C L E I N F O A B S T R A C T

Article history: Wedelolactone, a plant coumestan, was shown to act as anti-cancer agent for breast and prostate

Received 17 December 2014

carcinomas in vitro and in vivo targeting multiple cellular proteins including androgen receptors,

Received in revised form 9 April 2015

5-lipoxygenase and topoisomerase IIa. It is cytotoxic to breast, prostate, pituitary and myeloma cancer

Accepted 26 April 2015

cell lines in vitro at mM concentrations. In this study, however, a novel biological activity of nM dose of

Available online 28 April 2015

wedelolactone was demonstrated. Wedelolactone acts as agonist of estrogen receptors (ER) a and b as

demonstrated by transactivation of estrogen response element (ERE) in cells transiently expressing either

Keywords:

ERa or ERb and by molecular docking of this coumestan into ligand binding pocket of both ERa and ERb.

Breast cancer

In breast cancer cells, wedelolactone stimulates growth of estrogen receptor-positive cells, expression of

Estrogen receptor

Phytoestrogen estrogen-responsive genes and activates rapid non-genomic estrogen signalling. All these effects can be

Wedelolactone inhibited by pretreatment with pure ER antagonist ICI 182,780 and they are not observed in ER-negative

breast cancer cells. We conclude that wedelolactone acts as phytoestrogen in breast cancer cells by

stimulating ER genomic and non-genomic signalling pathways.

1. Introduction believed to provide beneﬁts to human health although this is still

matter of intense debate [2].

Steroidal estrogens are important signalling molecules that act Wedelolactone is naturally occurring coumestan isolated from

via genomic and non-genomic transduction mechanisms. Phytoes- Eclipta alba and Wedelia calendulacea [3]. Traditional Asian and

trogens are plant-derived compounds that structurally and/or South American medicine uses these plants to treat hepatitis,

functionally mimic mammalian estrogens by direct interaction snake venom poisoning and viral infection [3,4]. Compounds

with nuclear and extranuclear estrogen receptors (ERs) [1]. extracted from Wedelia chinensis synergistically suppress growth

Phytoestrogens belong to diverse classes of compounds and are of prostate cancer cell lines both in vitro and in vivo [5,6].

Furthermore, the growth inhibitory and proapoptotic effects of

wedelolactone itself were documented in various cancer cell lines

including breast, colon, hepatocellular, pituitary and neuroblasto-

Abbreviations: AP-1, activator protein 1; CHFCS, charcoal stripped fetal bovine

serum; CCND1, cyclin D1; CTSD, cathepsin D; DMEM, Dulbecco’s modiﬁed Eagle’s ma [7–11]. The anti-cancer effects of wedelolactone have been

medium; DMSO, dimethyl sulfoxide; ECL, enhanced chemiluminescence; ER,

attributed to the inhibition of various protein kinases (IKK, FAK,

estrogen receptor; ERE, estrogen-responsive element; ERK, extracellular receptor

ERK, PKC), androgen receptor, DNA topoisomerase IIa, and

kinase; FCS, fetal calf serum; HEK-293, human embryonic kidney 293 cell line; JNK,

5-lipoxygenase [5–13].

c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinases; PDB, protein

data bank; PBS, phosphate buffered saline; PI3K, phosphatidylinositol-4,5-bispho- Wedelolactone is structurally related to another natural coume-

sphate 3-kinase; RLU, relative light units; RPMI, Roswell Park Memorial Institute stan, a coumestrol (Fig. 1). Coumestrol is a phytoestrogen acting as

medium; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

agonist of both ERa and ERb [14,15]. Although wedelolactone was

* Corresponding author at: Department of Experimental Biology, Faculty of

suggested to act as phytoestrogen [16,17], any directevidence for this

Science, Masaryk University, Kotlarska 2, 611 37 Brno, Czech Republic.

Tel.: +420 54949 3125; fax: +420 54949 5533. statement has not been published yet. Interestingly, Lim et al.

E-mail address: [email protected] (P. Benes). reported the absence of the luciferase reporter gene expression from

http://dx.doi.org/10.1016/j.jsbmb.2015.04.019

T. Nehybova et al. / Journal of Steroid Biochemistry & Molecular Biology 152 (2015) 76–83 77

Fig. 1. Chemical structures of coumestans, 17b-estradiol and genistein.

ERE in wedelolactone-treated breast cancer MCF-7 cells [18]. counted daily using Casy RT cell counter (Roche-Innovatis, Basel,

Moreover, Xu et al. recently reported anti-estrogenic properties of Switzerland). To determine EC50 values, MCF-7 and T47D cells

3-butoxy-1,8,9-trihydroxy-6H-benzofuro[3,2-c]benzopyran-6-one, were exposed to various concentrations of wedelolactone for three

the new wedelolactone derivative [19]. Therefore, the aim of this days and counted. EC50 values were calculated using GraphPad

study is to analyze the estrogenic properties of wedelolactone in PRISM 6 software (GraphPad-San Diego, CA, USA).

breast cancer cells in detail. We show that wedelolactone acts as

phytoestrogen indeed by stimulating genomic and non-genomic 2.4. Determination of ER and AP-1 activities by the luciferase reporter

signalling from estrogen receptors. assay

2. Material and methods To test transactivation rates of ER and AP-1, 5 Â10 cells were

seeded in 2 ml of regular growth medium in 6-well plates and

2.1. Chemicals and plasmids cultured for 24 h. Transfection was performed using 8 ml of the

Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) with a

Wedelolactone, 17b-estradiol, and ICI 182,780 were all pur- mixture containing 1 mg of reporter plasmid (ERE-luc or p3TP-lux)

chased from Sigma–Aldrich (Sigma, St. Louis, MI). The ERE-luc and 1 mg of CMVbgal reference plasmid. Six hours later, the

plasmid was obtained from Panomics, Inc. (Panomics, Inc., medium was replaced with the steroid-depleted medium with/

Fremont, CA); pCDNA3.1-nv5-ERb, pEGFP-C1-ERa described by without 100 nM ICI 182,780 for 4 h. Then, 10 nM wedelolactone,

Stenoien et al. and Wittmann et al. were obtained from Addgene 10 nM 17b-estradiol or DMSO were added. Cells were incubated for

(Addgene, USA) [20,21], CMV-bgal and p3TP-lux were described 24 h, harvested and processed for luciferase and b-galactosidase

previously [22]. assays as described elsewhere [22]. The luciferase activity of each

sample was normalized for transfection efﬁciency according to the

2.2. Cell culture b-galactosidase activity.

The human breast cancer cell lines MDA-MB-231 (often 2.5. Immunoblotting

referred as ER-negative but expressing low amount of ERb),

MCF-7 and T47D (both expressing ERa and ERb) were cultured in Cells were seeded at the density 5 Â10 cells/well in growth

RPMI 1640 medium supplemented with 10% FCS, 2 mM L- medium in 6-well plates. Next day, the cells were washed with PBS

glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin in a and exposed to 100 nM ICI 182,780 or solvent in steroid depleted

humidiﬁed atmosphere of 5% CO2 at 37 C. Human embryonic medium for 4 h. Then, the cells were treated with 10 nM

kidney 293 cell line (HEK-293) was grown in DMEM supplemented wedelolactone, 10 nM 17b-estradiol or DMSO for 30 min (analysis

with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ of rapid non-genomic ER signalling) or 24 h (analysis of genomic ER

ml streptomycin. Both wedelolactone and 17b-estradiol were used signalling). Cells were harvested and lysed by boiling in a buffer

in 10 nM concentrations. To inhibit ER activation, cells were containing 0.1 M Tris (pH 6.8),16% v/v glycerol, 3.2% w/v SDS,10% v/

pretreated with 100 nM ICI 182,780 for 4 h. v b-mercaptoethanol, and 0.005% w/v bromophenol blue for 5 min.

Cell lysates were subjected to SDS–PAGE and immunoblotted.

2.3. Cell proliferation assay Sample loading was normalized according to the protein

concentration determined by DC protein assay (Biorad, Hercules,

To test proliferation rates, 1 Â10 MDA-MB-231, MCF-7 and CA). Blots were probed with anti-p-c-Jun (Ser 73), anti-c-Jun

T47D cells were seeded in 2 ml of growth medium in 6-well plates. (6OA8, Cell Signalling Technology, Inc., Beverly, MA), anti-cyclin

Next day, the cells were washed with PBS and growth medium was D1, anti-c-Myc, anti-ERK 1 (sc-246, sc-42 and sc-93, Santa Cruz

replaced with RPMI 1640 lacking phenol red supplemented with Biotechnology Inc., Heidelberg, Germany), anti-cathepsin D

2% charcoal stripped fetal bovine serum (CHFCS), 2 mM L-glutamin, (610801, BD Transduction Laboratories, San José, CA), or anti-p-

100 U/ml penicillin, and 100 mg/ml streptomycin (steroid depleted p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (4370, Cell Signalling

medium). Next day, the cells were pretreated with either Technology, Inc., Beverly, MA) antibodies. To control for sample

antiestrogen ICI 182,780 or solvent (DMSO) in fresh steroid loading, blots were probed with a a-tubulin speciﬁc antibody

depleted medium. Then, the cells were exposed to wedelolactone, (T9026, Sigma, St. Louis, MI). The blots were then probed with

17b-estradiol or DMSO as a solvent for three days. Cells were secondary antibodies conjugated with peroxidase (Sigma, St. Louis,

78 T. Nehybova et al. / Journal of Steroid Biochemistry & Molecular Biology 152 (2015) 76–83

MI) and developed by standard ECL procedure using Immobilon 3. Results

Western Chemiluminescent HRP Substrate (Millipore, Billerica,

MA). Intensity of the bands was quantiﬁed by scanning densitom- 3.1. Wedelolactone stimulates proliferation of the ER-positive human

etry using the ImageJ image analysis software (NIH, Bethesda, MD) breast cancer cells

and normalized according to the a-tubulin controls.

To examine the effects of wedelolactone on proliferation of

breast cancer cells, ER-positive (T47D, MCF-7) and ER-negative

2.6. Molecular docking

(MDA-MB-231) cells were cultured for 72 h in estrogen-free

medium with/without 10 nM wedelolactone and enumerated

The three-dimensional structures of 17b-estradiol (ZINC ID:

daily. 17b-estradiol-treated cells were used as positive controls.

ZINC13520815) and wedelolactone (ZINC ID: ZINC6483512) were

To assess the involvement of ER in the wedelolactone-controlled

downloaded from the ZINC database [23]. The output ﬁles in Sybyl

growth of breast cancer cells, we pretreated the cells with the ER

mol2 format were converted into AutoDock Vina [24] compliant

inhibitor ICI 182,780 for 4 h. The growth curve proﬁles documented

pdbqt format by MGLTools [25]. The crystal structure of human

that wedelolactone signiﬁcantly stimulated proliferation

estrogen receptors alfa (PDB ID: 2QSE chain B) and beta (PDB ID:

ER-positive but not ER-negative cells and that growth-stimulatory

1QKM) were used as the targets for the molecular docking. All

effect of wedelolactone was abolished by pretreatment with ICI

ligands and water molecules were removed from both structures.

182,780 (Fig. 2). To assess EC50 values, MCF-7 and T47D cells were

The hydrogen atoms were added to the targets with H++ server

exposed to various concentrations of wedelolactone for three days

[26] at pH 6.8. The Gasteiger charges and AutoDock atom types

and counted. This analysis revealed that MCF-7 cells are more

were assigned to targets by MGLTools. The ligand binding domains

sensitive to lower concentrations of wedelolactone than T47D cells

of both receptors were selected as target regions for the docking

(EC50—9.52 nM for MCF-7, 18.28 nM for T47D, Fig. 3).

performed by AutoDock Vina. The region selected for docking was

represented by the box of 20 Å Â 16.25 Å Â 15 Å centered at ERa

3.2. Wedelolactone activates nuclear ERs in breast cancer cells

residues A350, L346 and L384. The coordinates of the grid center

were subsequently transferred to the aligned structure of ERb. The

To determine whether wedelolactone regulates activities of

interaction of ligands with the active site residues was determined

nuclear estrogen receptors, we measured expression of ER-speciﬁc

by PoseView [27].

luciferase reporter (ERE-luc) in transiently transfected ER-positive

(T47D, MCF-7) and ER-negative (MDA-MB-231) cells upon

2.7. Statistical analysis treatment with wedelolactone or 17b-estradiol. We found that

wedelolactone signiﬁcantly activated transcription from ERE in

Data are presented as mean SD. Statistical comparisons ER-positive but not ER-negative cells and that this effect was

’ <

between groups were performed by Student s t-test. p 0.05 inhibited by pretreatment with ICI 182,780. Interestingly, wede-

ﬁ

was considered to be statistically signi cant. lolactone signiﬁcantly reduced transcription from ERE in estradiol-

Fig. 2. The effect of wedelolactone on proliferation of ER-positive (T47D, MCF-7) and ER-negative (MDA-MB-231) cell lines. The cells were pretreated with 100 nM ICI

182,780 or solvent for 4 h and subsequently incubated with/without 10 nM wedelolactone (w), 10 nM 17b-estradiol (e2) or DMSO for 24, 48 and 72 h in steroid-depleted

medium. The data represent the mean values from three independent experiments. Error bars indicate the standard deviations. * indicates a signiﬁcant (p < 0.05) difference of

the number of wedelolactone or 17b estradiol -treated cells from the number of control DMSO-treated cells.

T. Nehybova et al. / Journal of Steroid Biochemistry & Molecular Biology 152 (2015) 76–83 79

Fig. 3. Determination of growth-response and EC50 values for wedelolactone-treated MCF-7 and T47D cell lines. The cells were treated with various concentrations of

wedelolactone (w) for three days, counted and EC50 values were determined using GraphPad PRISM 6 software (GraphPad-San Diego, CA, USA). The data represent the mean

values from three independent experiments.

treated ER-positive cells suggesting a competitive interaction of 3.4. Wedelolactone binds into the ligand binding pockets of both ERa

both compounds with ERs (Fig. 4). and ERb

3.3. Wedelolactone acts as agonist of both ERa and ERb The ability of AutoDock Vina to reproduce experimental binding

mode of the natural ligand 17b-estradiol complexed with ERa was

Previous studies have shown that naturally occurring steroidal tested prior predicting the binding mode of wedelolactone in both

compounds can interact with both ERa vs. ERb. This is also the case receptors. The root-mean-square deviation in position of all heavy

of coumestrol [14,15,28]. To determine transactivation activity of atoms between the experimental binding mode available in the

wedelolactone for ERa and ERb, we used similar procedure as crystal structure (PDB ID: 1ERE) and the best-scoring binding

described in chapter 3.2. Nevertheless, human embryonic kidney mode from docking was only 0.7 Å. Similarly, when the best-

cells (HEK-293) were used for transfection instead, as they express scoring binding mode of 17b-estradiol docked into ERb was

neither ERa nor ERb receptors [29]. The cells were co-transfected compared with 17b-estradiol taken from the aligned crystal

with reporter plasmids together with plasmid coding for either structure of hERa, the root-mean-square deviation was also only

ERa or ERb, pretreated with ICI 182,780 or solvent and exposed to 0.7 Å, conﬁrming the ability of AutoDock Vina to correctly

wedelolactone, 17b-estradiol or DMSO for 24 h. We found that reproduce the studied complexes.

transactivation activity from ERE increased in the presence of In total, three and four binding modes were obtained for

wedelolactone 3.1-fold in ERa-expressing cells and 4.3-fold in wedelolactone in ERa and ERb, respectively. The best binding

ERb-expressing cells. Again, this increase was inhibited by mode could be identiﬁed unambiguously for wedelolactone in ERb

pretreatment with ICI 182,780 (Fig. 5). When both ERa- or ERb- based on the binding energy. The binding energies of the ﬁrst two

expressing cells were treated simultaneously with wedelolactone binding modes of wedelolactone in ERa were comparable. Since

and 17b-estradiol, a signiﬁcantly lower induction of the ERE the most relevant binding modes of 17b-estradiol were: (i) similar

transactivation was observed when compared with cells treated in both ERa and ERb and (ii) accurately reproduced by the binding

with 17b-estradiol only. This ﬁnding suggests that wedelolactone energy derived by AutoDock Vina, the best-scoring binding mode

can interfere with interactions of 17b-estradiol with both ERa and of wedelolactone in ERa similar to mode identiﬁed in ERb was

ERb (Fig. 5). selected as the most relevant one (Table 1 and Fig. 6).

Fig. 4. Wedelolactone transactivates the ERE-driven reporter gene in ER-positive Fig. 5. Wedelolactone induces transactivation functions of both ERa and ERb in

breast cancer cells. The T47D, MCF-7 and MDA-MB 231 cells were transiently HEK-293 cells. The cells were transiently transfected with ERE-luc and CMVb-gal

transfected with ERE-luc and CMVb-gal plasmids, pretreated with 100 nM ICI plasmids together with plasmid coding for either ERa or ERb, pretreated with

182,780 or solvent and subsequently treated with 10 nM wedelolactone (w), 10 nM 100 nM ICI 182,780 or solvent and subsequently treated with 10 nM wedelolactone

17b-estradiol (e2) or DMSO for 24 h. The cells were harvested and assayed for (w), 10 nM 17b-estradiol (e2) or DMSO for 24 h. The cells were harvested and

luciferase and b-galactosidase activities. Normalized luciferase activity is indicated assayed for luciferase and b-galactosidase activities. Normalized luciferase activity

in arbitrary light units. The data represent the mean values from three independent is indicated in arbitrary light units. The data represent the mean values from three

experiments. Error bars indicate the standard deviations. * indicates a signiﬁcant independent experiments. Error bars indicate the standard deviations. * indicates a

(p < 0.05) difference in the relative luciferase activities between wedelolactone or signiﬁcant (p < 0.05) difference in the relative luciferase activities between

17b-estradiol-treated cells and the control DMSO-treated cells. wedelolactone or 17b-estradiol-treated cells and the control DMSO-treated cells.

80 T. Nehybova et al. / Journal of Steroid Biochemistry & Molecular Biology 152 (2015) 76–83

Table 1

17b-estradiol was stabilized by the same speciﬁc interactions as

Binding energies of protein–ligand complexes.

wedelolactone with one extra hydrogen bond formed to E353 in ERa

Predicted binding energy (kcal/mol) and E305 in ERb, respectively (data not shown). This additional

interaction could be responsible for predicted higher afﬁnity of this

Binding mode 17b-estradiol Wedelolactone

ligand compared to wedelolactone. A computational molecular

hERa hERb hERa hERb

modelling therefore conﬁrmed the ability of wedelolactone to bind

1 À10.1 À10.5 À7.3 À7.4

into the ligand binding pockets of both ERa and ERb.

2 À9.0 À8.8 À7.2 À6.6

3 À8.8 À8.8 À5.5 À6.5

3.5. Wedelolactone activates expression of ER-regulated genes in

4 À8.5 À8.4 N/A À6.4

5 N/A À7.6 N/A N/A ER-positive breast cancer cells

N/A—the binding mode not available, the relevant binding modes are in bold.

To further conﬁrm that wedelolactone activates genomic

ER-signalling pathway, we analyzed expression of selected

representatives of ER-regulated genes (CCND1, MYC, CATHD) in

breast cancer cells upon treatment with wedelolactone or 17b-

estradiol by immunoblotting. We found that wedelolactone and

17b-estradiol increased production of proteins encoded by all

studied genes in ER-positive but not ER-negative breast cancer cells

and that this increase was inhibited by pretreatment with ICI

182,780 (Fig. 7).

3.6. Wedelolactone activates the non-genomic ER signalling in breast

cancer cells

Besides functioning via nuclear ERs (genomic ER signalling),

estrogens have also been shown to act more rapidly via

extranuclear ERs resulting in activation of various kinase signalling

pathways (non-genomic ER signalling) [30]. To analyze the effect of

wedelolactone on non-genomic ER signalling, breast cancer cells

were exposed to 10 nM wedelolactone for 30 min and the amount

of phosphorylated forms of ERK and c-Jun proteins were

determined by immunoblotting. We observed that phosphoryla-

tion of both ERK and c-Jun proteins increased upon treatment with

wedelolactone or 17b-estradiol and that this increases was

prevented by pretreatment with ICI 182,780 (Fig. 8).

3.7. Wedelolactone stimulates transactivation by AP-1

The c-Jun protein homodimerizes or heterodimerizes with Fos

or other Jun-related proteins to form transcription factor complex

AP-1 [31]. The wedelolactone—induced increase of the c-Jun

phosphorylation (Fig. 8) suggests that this coumestan might be

Fig. 6. Comparison of the most relevant binding modes of wedelolactone in human

capable of modulating activity of AP-1. To investigate whether

ERa (A) and ERb (B). The ligand binding cavities of both receptors are represented

wedelolactone regulates activity of AP-1, we measured the

by the gray surface. The residues providing key interactions with wedelolactone are

luciferase expression from p3TP-lux plasmid in transiently

represented by cyan sticks.

transfected T47D, MCF-7 and MDA-MB 231 cells treated with

wedelolactone, 17b-estradiol or DMSO in steroid-depleted media

for 24 h. We found that wedelolactone signiﬁcantly activated

ﬁ

Besides the hydrophobic contacts, wedelolactone could form transcription from the AP-1-speci c reporter in ER-positive T47D

B-stacking interaction with F404 (hERa) and F356 (hERb) and three and MCF-7 cells but not ER-negative MDA-MB-231 cells and that

hydrogen bonds with following residues: H524/H475, R394/346 and this activation was abolished by pretreatment with ICI 182,780

G521/G472 of hERa/hERb, respectively (Fig. 6). The natural ligand (Fig. 9).

Fig. 7. Wedelolactone increases expression of CCND1, MYC and CTSD in breast cancer cells in ER-dependent fashion. T47D, MCF-7 and MDA-MB-231 cells were pretreated

with 100 nM ICI 182,780 or solvent and then exposed to 10 nM wedelolactone (w), 10 nM 17b-estradiol (e2) or DMSO for 24 h. Cells were harvested and extracted proteins

were resolved by SDS–PAGE and analyzed by immunoblotting. To control for sample loading, the blots were probed with a a-tubulin-speciﬁc antibody.

T. Nehybova et al. / Journal of Steroid Biochemistry & Molecular Biology 152 (2015) 76–83 81

Fig. 8. Wedelolactone increases phosphorylation of ERK and c-Jun proteins. Cells were pretreated with 100 nM ICI 182,780 or solvent for 4 h and subsequently exposed to

10 nM wedelolactone (w), 10 nM 17b-estradiol (e2) or DMSO for 30 min. Cells were harvested and extracted proteins were resolved by SDS–PAGE and analyzed by

immunoblotting. To control for sample loading, the blots were probed with a a-tubulin-speciﬁc antibody.

4. Discussion describe that wedelolactone may act in similar manner. It

stimulates proliferation of ER-positive breast cancer cells, enhan-

In the 1980s, epidemiological studies correlated lower inci- ces transcription from ERE, induces expression of endogenous

dence of breast cancer in Asian women with their signiﬁcantly ER-responsive genes and activates non-genomic ER-signalling

higher intake of phytoestrogens when compared to women in pathway. All these effects can be inhibited by pretreatment with a

Europe and North America [32,33]. Moreover, the increase in pure antiestrogen, ICI 182,780 and were not observed in

breast cancer incidence has been demonstrated in the descendants ER-negative breast cancer cells. We found wedelolactone to be

of women, who have migrated from countries with low breast more potent stimulator of MCF-7 than T47D cell proliferation.

cancer incidence, as they adopt new dietary and lifestyle habits Moreover, MCF-7 cells exhibited lower EC50 value in proliferation

[34]. These observations initiated an extensive research on the assay. This documents that MCF-7 cells are more sensitive to the

relation between phytoestrogen intake and breast cancer preven- growth-promoting effect of nanomolar wedelolactone. Superior

tion. However, data collected from animal and human studies to sensitivity of MCF-7 cells to growth-stimulatory effects of other

date have not provided clear conclusion to recommend high phytoestrogen, genistein, was reported previously [41–43] and

phytoestrogen intake to reduce breast cancer risk. Unanswered attributed to higher ERa/ERb ratio detected in MCF-7 cells.

questions also remain regarding the effects of phytoestrogens to Moreover, we observed stimulation of AP-1 transactivation

women at high risk of breast cancer and those already diagnosed activity in ER-positive breast cancer cells by wedelolactone as well.

with breast cancer, the interaction of these compounds with The ability of estrogen/phytoestrogen to regulate activity of the

hormonal therapies in cancer patients, their effects on menopausal AP-1 transcription factor by genomic (e.g., interaction of ERs and

symptoms and their action as endocrine disruptors [2,35,36]. AP-1 on the AP-1-speciﬁc DNA-binding sites, recruitment of

Wedelolactone has been recently identiﬁed as anti-cancer coactivators, up-regulation of expression of the AP-1 components)

compound in breast cancer models both in vitro and in vivo and non-genomic mechanisms (e.g., activation of MAPKs) was

[7,10,13]. It belongs to the group of coumestans, plant polycyclic established previously [44,45]. Notably, Sarveswaran et al.

aromatic secondary metabolites, with coumestrol being the most reported that apoptosis induced by 10–30 mM wedelolactone in

studied of this group. Both genomic and non-genomic mechanisms prostate cancer cells is dependent on its ability to activate JNK [12].

have been proposed to mediate phytoestrogenic effects of We observed that wedelolactone acts as agonist of both ERa

coumestrol [37,38]. Coumestrol binds to nuclear ERs and induces and ERb in the ERE-driven transactivation assay as well as the

expression of speciﬁc estrogen-responsive genes [37] but it also ability of wedelolactone to bind into the ligand binding pockets of

initiates signalling from the extranuclear ERs by activation of both ERa and ERb in the molecular docking analysis. Interestingly,

ERK1/2, JNK, PI3K and changes in Ca ﬂuxes [38,39]. Recently, when both estradiol and wedelolactone were administered

another coumestan—psoralidin—has been identiﬁed as ERa and simultaneously, a lower induction of ERE transactivation was

ERb agonist and regulator of endogenous ER-responsive genes in observed when compared with the cells treated with estradiol

breast and endometrial cancer cell lines [40]. In this study, we only. This ﬁnding suggests that wedelolactone partially antago-

nizes the stimulative effect of estradiol on both receptors.

Previously, the phytoestrogenic properties of wedelolactone

were suggested due to its structural similarity to coumestrol and

estrogenic activity of Wedelia plant extract but this hypothesis has

not been experimentally proved so far [16,17]. Moreover, recent

study by Lim et al. reported no effect of pure wedelolactone on the

ERE transactivation in MCF-7 cells which seems to be contradictory

to our results [18]. Comparing the experimental methodologies

between our study and the study by Lim et al., we found that while

we tested estrogenic effects of wedelolatone in steroid-depleted

media (no phenol red, 2% charcoal stripped bovine serum), Lim

Fig. 9. Wedelolactone stimulates transactivation by AP-1 in ER-positive breast

et al. used regular media containing phenol red and 10% FCS [18].

cancer cells. T47D, MCF-7 and MDA-MB 231 cells were transiently transfected with

Elimination of estrogenic compounds from tissue culture media

p3TP-lux and CMVb-gal plasmids, pretreated with 100 nM ICI 182,780 or solvent

(phenol red, steroids presented in FCS) was reported to be

and subsequently treated with 10 nM wedelolactone (w), 10 nM 17b-estradiol (e2)

or DMSO for 24 h. The cells were harvested and assayed for luciferase and important for evaluation of estrogenic and anti-estrogenic effects

-galactosidase activities. Normalized luciferase activity is indicated in arbitrary

b in estradiol-responsive breast cancer cell models [46,47]. When we

light units. The data represent the mean values from three independent

tested the ERE transactivation using the same experimental setup

experiments. Error bars indicate the standard deviations. * indicates a signiﬁcant

as described by Lim et al. (media with phenol red and 10% FCS) [18],

(p < 0.05) difference in the relative luciferase activities between wedelolactone or

ﬁ

17b-estradiol-treated cells and the control DMSO-treated cells. no signi cant effect of wedelolactone on ERE transactivation was

82 T. Nehybova et al. / Journal of Steroid Biochemistry & Molecular Biology 152 (2015) 76–83

observed (3.40 Æ 0.52 RLU in DMSO-treated cells vs. (CZ.1.07/2.3.00/20.0183) and RECAMO (CZ.1.05/2.1.00/03.0101) via

4.37 Æ 0.13 RLU in wedelolactone-treated cells, n = 3, p = 0.11), thus the human resources project “IntegRECAMO: Intelectual Anchor”

conﬁrming our hypothesis. (CZ.1.07/2.3.00/20.0097).

The phytoestrogen effect on cancer cells is reported to be

biphasic. Multiple studies described that phytoestrogens stimu- References

lated growth of ER-positive but not ER-negative breast cancer cell

– –

lines at low concentrations (0.01 10 mM) [48 51]. In contrast, [1] R. Baber, Phytoestrogens and post reproductive health, Maturitas 66 (2010)

344–349.

phytoestrogens inhibited growth and induced cell death in both

[2] H.B. Patisaul, W. Jefferson, The pros and cons of phytoestrogens, Front.

ER-positive and ER-negative cells at high concentrations (20–

Neuroendocrinol. 31 (2010) 400–419.

100 mM) suggesting that their growth-inhibitory effect does not [3] H. Wagner, B. Geyer, Y. Kiso, H. Hikino, G.S. Rao, Coumestans as the main active

principles of the liver drugs Eclipta alba and Wedelia calendulacea, Planta Med.

depend on their estrogenic properties [48,50,51]. The ER-indepen-

5 (1986) 370–374.

dent growth-inhibitory, pro-apoptotic and anti-cancer effects of

[4] M. Kobori, Z. Yang, D. Gong, V. Heissmeyer, H. Zhu, Y.K. Jung, M.A. Gakidis, A.

phytoestrogens were attributed to inhibition of protein kinases, Rao, T. Sekine, F. Ikegami, C. Yuan, J. Yuan, Wedelolactone suppresses LPS-

cell cycle progression, DNA topoisomerases, angiogenesis and/or induced caspase-11 expression by directly inhibiting the IKK complex, Cell

Death Differ. 11 (2004) 123–130.

their redox properties [39,50].

[5] F.M. Lin, L.R. Chen, E.H. Lin, F.C. Ke, H.Y. Chen, M.J. Tsai, P.W. Hsiao, Compounds

The biphasic effect of wedelolactone on cancer cells was

from Wedelia chinensis synergistically suppress androgen activity and growth

–

demonstrated as well. While in the previous studies, we and others in prostate cancer cells, Carcinogenesis 28 (2007) 2521 2529.

[6] C.H. Tsai, F.M. Lin, Y. Yang, M.T. Lee, T.L. Cha, G.J. Wu, S.C. Hsieh, P.W. Hsiao,

have reportedvarious growth-inhibitoryand pro-apoptoticeffectsof

Herbal extract of Wedelia chinensis attenuates androgen receptor activity and

mM wedelolactone in both ER-positive and ER-negative cells

orthotopic growth of prostate cancer in nude mice, Clin. Cancer Res. 15 (2009)

[7,10,13,52], the presented study demonstrates its ER-dependent 5435–5444.

[7] P. Benes, L. Knopfova, F. Trcka, A. Nemajerova, D. Pinheiro, K. Soucek, M. Fojta, J.

growth-stimulatoryeffect in nM concentrations. The mechanisms of

Smarda, Inhibition of topoisomerase IIa: novel function of wedelolactone,

anti-cancer effects of wedelolactone are presumably similar to those

Cancer Lett. 303 (2011) 29–38.

used by other phytoestrogens as recently reviewed by us [39]. [8] Z. Chen, X. Sun, S. Shen, H. Zhang, X. Ma, J. Liu, S. Kuang, Q. Yu, Wedelolactone a

naturally occurring coumestan, enhances interferon-g signaling through

Only limited data are available about bioavailability of

inhibiting STAT1 protein dephosphorylation, J. Biol. Chem. 288 (2013)

wedelolactone in vivo. Tsai et al. reported that the plasma 14417–14427.

concentration of wedelolactone was equal to 50 nM/l in mice [9] J.R. Vender, M.D. Laird, K.M. Dhandapani, Inhibition of NFkappaB reduces

cellular viability in GH3 pituitary adenoma cells, Neurosurgery 62 (2008) feeded daily with Wedelia chinensis extract for four weeks (4 mg/

1122–1127.

kg) 24 h after the last feeding. However, when mice were fed with

[10] A.I. Idris, H. Libouban, H. Nyangoga, E. Landao-Bassonga, D. Chappard, S.H.

wedelolactone alone for three weeks (2 mg/kg daily), wedelolac- Ralston, Pharmacologic inhibitors of IkappaB kinase suppress growth and

tone was undetectable in plasma 6 h after the last oral dose [6]. migration of mammary carcinosarcoma cells in vitro and prevent osteolytic

bone metastasis in vivo, Mol. Cancer Ther. 8 (2009) 2339–2347.

These data demonstrate poor bioavailability of wedelolactone.

[11] S. Sukumari-Ramesh, J.N. Bentley, M.D. Laird, N. Singh, J.R. Vender, K.M.

Similarly, low bioavailability was also reported for coumestrol as

Dhandapani, Dietary phytochemicals induce p53- and caspase-independent

compared with other phytoestrogens including daidzein and cell death in human neuroblastoma cells, Int. J. Dev. Neurosci. 29 (2011)

701–710.

genistein [53]. Mallis et al. reported coumestrol Cmax value equal

[12] S. Sarveswaran, S.C. Gautam, J. Ghosh, Wedelolactone a medicinal plant-

to 70 nM/l in plasma of rats 4 h after single oral dose (3 mg/kg) but

derived coumestan, induces caspase-dependent apoptosis in prostate cancer

the concentration of coumestrol dropped under 15 nM/l 8 h after cells via downregulation of PKCe without inhibiting Akt, Int. J. Oncol. 41 (2012)

2191–2199.

feeding [53]. As poor bioavailability complicates the use of

[13] Y.J. Lee, W.L. Lin, N.F. Chen, S.K. Chuang, T.H. Tseng, Demethylwedelolactone

wedelolactone in pre-clinical studies, pharmaceutical formula-

derivatives inhibit invasive growth in vitro and lung metastasis of MDA-MB-

tions of wedelolactone aiming to improve its bioavailability are 231 breast cancer cells in nude mice, Eur. J. Med. Chem. 56 (2012) 361–367.

[14] C. Chandsawangbhuwana, M.E. Baker, 3D models of human ERa and ERb

under testing [54].

complexed with coumestrol, Steroids 80 (2014) 37–43.

[15] D.M. Harris, E. Besselink, S.M. Henning, V.L. Go, D. Heber, Phytoestrogens

induce differential estrogen receptor alpha- or beta-mediated responses in

5. Conclusions

transfected breast cancer cells, Exp. Biol. Med. 230 (2005) 558–568.

[16] A. Shirwaikar, R.G. Prabhu, S. Malini, Activity of Wedelia calendulacea Less. in

In this study, we demonstrate that the natural coumestan post-menopausal osteoporosis, Phytomedicine 13 (2006) 43–48.

[17] N. Kaushik-Basu, A. Bopda-Waffo, T.T. Talele, A. Basu, P.R. Costa, A.J. da Silva, S.

wedelolactone acts as phytoestrogen in nanomolar concentrations

G. Saraﬁanos, F. Noël, Identiﬁcation and characterization of coumestans as

by stimulating ER genomic and non-genomic signalling in breast

novel HCV NS5B polymerase inhibitors, Nucleic Acids Res. 36 (2008)

cancer cells. The estrogenic effects of wedelolactone should be 1482–1496.

[18] S. Lim, H.J. Jang, E.H. Park, J.K. Kim, J.M. Kim, E.K. Kim, K. Yea, Y.H. Kim, W. Lee-

taken into account when evaluating its anti-cancer properties.

Kwon, S.H. Ryu, P.G. Suh, Wedelolactone inhibits adipogenesis through the ERK

pathway in human adipose tissue-derived mesenchymal stem cells, J. Cell.

Biochem. 113 (2012) 3436–3445.

Conﬂict of interest

[19] D. Xu, T.H. Lin, C.R. Yeh, M.A. Cheng, L.M. Chen, C. Chang, S. Yeh, The

wedelolactone derivative inhibits estrogen receptor-mediated breast,

ﬂ

The authors declare that they have no con ict of interest. endometrial, and ovarian cancer cells growth, Biomed. Res. Int. 2014 (2014)

713263.

[20] D.L. Stenoien, M.G. Mancini, K. Patel, E.A. Allegretto, C.L. Smith, M.A. Mancini,

Declaration about the role of funding sources

Subnuclear trafﬁcking of estrogen receptor-alpha and steroid receptor

coactivator-1, Mol. Endocrinol. 14 (2000) 518–534.

ﬁ

The authors declare that neither of sponsors did participate in [21] B.M. Wittmann, A. Sherk, D.P. McDonnell, De nition of functionally important

mechanistic differences among selective estrogen receptor down-regulators,

study design; in the collection, analysis and interpretation of data;

Cancer Res. 67 (2007) 9549–9560.

in the writing of the manuscript; or in the decision to submit the

[22] S. Sevcíková, K. Soucek, L. Kubala, V. Bryja, J. Smarda, Differential effects of v-

article for publication. Jun and c-Jun proteins on v-myb-transformed monoblasts, Cell. Mol. Life Sci.

59 (2002) 1690–1705.

[23] J.J. Irwin, B.K. Shoichet, ZINC—a free database of commercially available

Acknowledgements

compounds for virtual screening, J. Chem. Inf. Model. 45 (2005) 177–182.

[24] O. Trott, A.J. Olson, AutoDock Vina: improving the speed and accuracy of

docking with a new scoring function, efﬁcient optimization, and

This work was funded by grants NT13441-4/2012 (IGA, Ministry

multithreading, J. Comput. Chem. 31 (2010) 455–461.

of Health of the Czech Republic), by European Regional Develop-

[25] M. Sanner, Python: a programming language for software integration and

—

ment Fund projects FNUSA-ICRC (CZ.1.05/1.1.00/02.0123), CEB development, J. Mol. Graph Model. 17 (1999) 57–61.

T. Nehybova et al. / Journal of Steroid Biochemistry & Molecular Biology 152 (2015) 76–83 83

[26] J.B. Gordon, T. Myers, V. Folta, L.S. Shoja, A, H++: a server for estimating pKas estrogen receptor signaling molecule isolated from Psoralea corylifolia, Bioorg.

and adding missing hydrogens to macromolecules, Nucleic Acids Res. 33 Med. Chem. Lett. 24 (2014) 1403–1406.

(2005) 368–371. [41] D.G. Pons, M. Nadal-Serrano, M.M. Blanquer-Rossello, J. Sastre-Serra, J. Oliver,

[27] K. Stierand, P.C. Maass, M. Rarey, Molecular complexes at a glance: automated P. Roca, Genistein modulates proliferation and mitochondrial functionality in

generation of two-dimensional complex diagrams, Bioinformatics 22 (2006) breast cancer cells depending on ERalpha/ERbeta ratio, J. Cell. Biochem. 115

1710–1716. (2014) 949–958.

[28] S.O. Mueller, S. Simon, K. Chae, M. Metzler, K.S. Korach, Phytoestrogens and [42] H.S. Seo, D.G. DeNardo, Y. Jacquot, I. Laïos, D.S. Vidal, C.R. Zambrana, G.

their human metabolites show distinct agonistic and antagonistic properties Leclercq, P.H. Brown, Stimulatory effect of genistein and apigenin on the

on estrogen receptor alpha (ERalpha) and ERbeta in human cells, Toxicol. Sci. growth of breast cancer cells correlates with their ability to activate ER alpha,

80 (2004) 14–25. Breast Cancer Res. Treat. 99 (2006) 121–134.

[29] N.I. Chantzi, A.K. Meligova, E. Dhimolea, C.C. Petrou, D.J. Mitsiou, V. Magafa, A. [43] B. Yuan, L. Wang, Y. Jin, H. Zhen, P. Xu, Y. Xu, C. Li, H. Xu, Role of metabolism in

Pechtelidou, I. Florentin, E. Kitraki, P. Cordopatis, D.G. Tiniakos, M.N. Alexis, the effects of genistein and its phase II conjugates on the growth of human

Insights into ectopic estrogen receptor expression, nucleocytoplasmic breast cell lines, AAPS J. 14 (2012) 329–344.

distribution and interaction with chromatin obtained with new antibodies [44] P.J. Kushner, D.A. Agard, G.L. Greene, T.S. Scanlan, A.K. Shiau, R.M. Uht, P. Webb,

to estrogen receptors a and b, Steroids 76 (2011) 974–985. Estrogen receptor pathways to AP-1, J. Steroid Biochem. Mol. Biol. 74 (2000)

[30] S.R. Hammes, E.R. Levin, Minireview: recent advances in extranuclear steroid 311–317.

receptor actions, Endocrinology 152 (2011) 4489–4495. [45] L. Björnström, M. Sjöberg, Estrogen receptor-dependent activation of AP-1 via

[31] E. Shaulian, AP-1—The Jun proteins: oncogenes or tumor suppressors in non-genomic signalling, Nucl. Recept. 2 (2004) 3.

disguise? Cell Signal. 22 (2010) 894–899. [46] Y. Berthois, J.A. Katzenellenbogen, B.S. Katzenellenbogen, Phenol red in tissue

[32] H. Adlercreutz, Western diet and Western diseases: some hormonal and culture media is a weak estrogen: implications concerning the study of

biochemical mechanisms and associations, Scand. J. Clin. Lab. Invest. Suppl. estrogen-responsive cells in culture, Proc. Natl. Acad. Sci. U. S. A. 83 (1986)

201 (1990) 3–23. 2496–2500.

[33] H. Adlercreutz, T. Fotsis, C. Bannwart, K. Wähälä, T. Mäkelä, G. Brunow, T. Hase, [47] P. Darbre, J. Yates, S. Curtis, R.J. King, Effect of estradiol on human breast cancer

Determination of urinary lignans and phytoestrogen metabolites potential cells in culture, Cancer Res. 43 (1983) 349–354.

antiestrogens and anticarcinogens, in urine of women on various habitual [48] C.Y. Hsieh, R.C. Santell, S.Z. Haslam, W.G. Helferich, Estrogenic effects of

diets, J. Steroid Biochem. 25 (1986) 791–797. genistein on the growth of estrogen receptor-positive human breast cancer

[34] D. Deapen, L. Liu, C. Perkins, L. Bernstein, R.K. Ross, Rapidly rising breast cancer (MCF-7) cells in vitro and in vivo, Cancer Res. 58 (1998) 3833–3838.

incidence rates among Asian–American women, Int. J. Cancer 99 (2002) [49] D.H. Han, M.S. Denison, H. Tachibana, K. Yamada, Relationship between

747–750. estrogen receptor-binding and estrogenic activities of environmental

[35] H. Roberts, Safety of herbal medicinal products in women with breast cancer, estrogens and suppression by ﬂavonoids, Biosci. Biotechnol. Biochem. 66

Maturitas 66 (2010) 363–369. (2002) 1479–1487.

[36] I. Bilal, A. Chowdhury, J. Davidson, S. Whitehead, Phytoestrogens and [50] P.J. Magee, I.R. Rowland, Phyto-oestrogens, their mechanism of action: current

prevention of breast cancer: the contentious debate, World J. Clin. Oncol. 5 evidence for a role in breast and prostate cancer, Br. J. Nutr. 91 (2004) 513–531.

(2014) 705–712. [51] C. Wang, M.S. Kurzer, Phytoestrogen concentration determines effects on DNA

[37] R. Ise, D. Han, Y. Takahashi, S. Terasaka, A. Inoue, M. Tanji, R. Kiyama, synthesis in human breast cancer cells, Nutr. Cancer 28 (1997) 236–247.

Expression proﬁling of the estrogen responsive genes in response to [52] P. Benes, P. Alexova, L. Knopfova, A. Spanova, J. Smarda, Redox state alters anti-

phytoestrogens using a customized DNA microarray, FEBS Lett. 579 (2005) cancer effects of wedelolactone, Environ. Mol. Mutagen. 53 (2012) 515–524.

1732–1740. [53] L.M. Mallis, A.B. Sarkahian, H.A. Harris, M.Y. Zhang, O.J. McConnell,

[38] C.S. Watson, N.N. Bulayeva, A.L. Wozniak, R.A. Alyea, Xenoestrogens are potent Determination of rat oral bioavailability of soy-derived phytoestrogens

activators of nongenomic estrogenic responses, Steroids 72 (2007) 124–134. using an automated on-column extraction procedure and electrospray

[39] T. Nehybova, J. Smarda, P. Benes, Plant coumestans: recent advances and future tandem mass spectrometry, J. Chromatogr. B Analyt. Technol. Biomed. Life

perspectives in cancer therapy, Anticancer Agents Med. Chem. 14 (2014) Sci. 796 (2003) 71–86.

1351–1362. [54] K. Upadhyay, N.K. Gupta, V.K. Dixit, Development and characterization of

[40] X. Liu, J.W. Nam, Y.S. Song, A.N. Viswanath, A.N. Pae, Y.S. Kil, H.D. Kim, J.H. Park, phyto-vesicles of wedelolactone for hepatoprotective activity, Drug Dev. Ind.

E.K. Seo, M. Chang, Psoralidin a coumestan analogue, as a novel potent Pharm. 38 (2012) 1152–1158.