Withania somnifera Root Extract Inhibits Mammary Cancer Metastasis and Epithelial to Mesenchymal Transition

Abstract

Though clinicians can predict which patients are at risk for developing metastases, traditional therapies often prove ineffective and metastatic disease is the primary cause of cancer patient death; therefore, there is a need to develop anti-metastatic therapies that can be administered over long durations to specifically inhibit the motility of cancer cells. Withaniasomnifera root extracts (WRE) have anti-proliferative activity and the active component, Withaferin A, inhibits the pro-metastatic protein, vimentin. Vimentin is an intermediate filament protein and is part of the epithelial to mesenchymal transition (EMT) program to promote metastasis. Here, we determined whether WRE standardized to Withaferin A (sWRE) possesses anti-metastatic activity and whether it inhibits cancer motility via inhibition of vimentin and the EMT program. Several formulations of sWRE were created to enrich for Withaferin A and a stock solution of sWRE in EtOH could recover over 90% of the Withaferin A found in the original extract powder. This sWRE formulation inhibited breast cancer cell motility and invasion at concentrations less than 1µM while having negligible cytotoxicity at this dose. sWRE treatment disrupted vimentin morphology in cell lines, confirming its vimentin inhibitory activity. To determine if sWRE inhibited EMT, TGF-β was used to induce EMT in MCF10A human mammary epithelial cells. In this case, sWRE prevented EMT induction and inhibited 3-D spheroid invasion. These studies were taken into a human xenograft and mouse mammary carcinoma model. In both models, sWRE and Withaferin A showed dose-dependent inhibition of tumor growth and metastatic lung nodule formation with minimal systemic toxicity. Taken together, these data support the hypothesis that low concentrations of sWRE inhibit cancer metastasis potentially through EMT inhibition. Moreover, these doses of sWRE have nearly no toxicity in normal mouse organs, suggesting the potential for clinical use of orally administered WRE capsules.

Full text

Withania somnifera Root Extract Inhibits Mammary
Cancer Metastasis and Epithelial to Mesenchymal
Transition
Zhen Yang1, Anapatricia Garcia2, Songli Xu1, Doris R. Powell3, Paula M. Vertino3, Shivendra Singh4, Adam
I. Marcus1*
1 Department of Hematology and Medical Oncology, Winship Cancer Institute of Emory University, Emory University School of Medicine, Atlanta, Georgia,
United States of America, 2 Yerkes National Primate Research Center, Emory University School of Medicine, Atlanta, Georgia, United States of America,
3 Department of Radiation Oncology, Winship Cancer Institute of Emory University, Emory University School of Medicine, Atlanta, Georgia, United States of
America, 4 Department of Pharmacology & Chemical Biology, and University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine,
Pittsburgh, Pennsylvania, United States of America
Abstract
Though clinicians can predict which patients are at risk for developing metastases, traditional therapies often prove
ineffective and metastatic disease is the primary cause of cancer patient death; therefore, there is a need to develop
anti-metastatic therapies that can be administered over long durations to specifically inhibit the motility of cancer
cells. Withania somnifera root extracts (WRE) have anti-proliferative activity and the active component, Withaferin A,
inhibits the pro-metastatic protein, vimentin. Vimentin is an intermediate filament protein and is part of the epithelial to
mesenchymal transition (EMT) program to promote metastasis. Here, we determined whether WRE standardized to
Withaferin A (sWRE) possesses anti-metastatic activity and whether it inhibits cancer motility via inhibition of vimentin
and the EMT program. Several formulations of sWRE were created to enrich for Withaferin A and a stock solution of
sWRE in EtOH could recover over 90% of the Withaferin A found in the original extract powder. This sWRE
formulation inhibited breast cancer cell motility and invasion at concentrations less than 1µM while having negligible
cytotoxicity at this dose. sWRE treatment disrupted vimentin morphology in cell lines, confirming its vimentin
inhibitory activity. To determine if sWRE inhibited EMT, TGF-β was used to induce EMT in MCF10A human
mammary epithelial cells. In this case, sWRE prevented EMT induction and inhibited 3-D spheroid invasion. These
studies were taken into a human xenograft and mouse mammary carcinoma model. In both models, sWRE and
Withaferin A showed dose-dependent inhibition of tumor growth and metastatic lung nodule formation with minimal
systemic toxicity. Taken together, these data support the hypothesis that low concentrations of sWRE inhibit cancer
metastasis potentially through EMT inhibition. Moreover, these doses of sWRE have nearly no toxicity in normal
mouse organs, suggesting the potential for clinical use of orally administered WRE capsules.
Citation: Yang Z, Garcia A, Xu S, Powell DR, Vertino PM, et al. (2013) Withania somnifera Root Extract Inhibits Mammary Cancer Metastasis and
Epithelial to Mesenchymal Transition. PLoS ONE 8(9): e75069. doi:10.1371/journal.pone.0075069
Editor: Michael Klymkowsky, University of Colorado, Boulder, United States of America
Received March 1, 2013; Accepted August 9, 2013; Published September 12, 2013
Copyright: © 2013 Yang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by an R21 from the National Center for Complementary and Alternative Medicine (1R21AT005231) awarded to AIM,
the Godfrey Charitable Trust, and Golfer's Against Cancer. This work was also supported by a P30 CCSG awarded to the Winship Cancer Institute
(3P30CA138292). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: aimarcu@emory.edu
Introduction
Breast cancer is one of the most common cancers among
women in the United States and the second leading cause of
female cancer death [1]. Greater than 90% of breast cancer
patient deaths are attributed to metastatic disease where the
primary tumor has invaded distant sites. Since these metastatic
cells are often highly aggressive, difficult to detect, and
chemoresistant [2], one therapeutic strategy could be to
prevent metastatic disease rather than treat it once it occurs.
The metastatic process can be categorized into three stages-
tumor cell invasion into surrounding tissue, intravasation into
blood or lymphatic vessels, and extravasation into a new host
environment [3-5]. In many cases, metastatic cells undergo
epithelial-to-mesenchymal transition (EMT), where genetic and
epigenetic events cause a polarized epithelial cell to become
migratory and invasive [6]. At the molecular level, EMT causes
a gain of vimentin and fibronectin expression, and loss of E-
cadherin at the cell membrane [7,8]. Accumulating evidence
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shows that EMT is a major mechanism driving breast cancer
progression and metastasis [9-13].
Withania somnifera (Ashwagandha) plants are widely used in
East Indian medicine. The W. somnifera root extract (WRE) is
composed of 14 compounds known as withanolides, with
Withaferin A being the most prominent [14-16]. Withaferin A is
a steroidal lactone that induces apoptosis and inhibits tumor
growth by targeting signaling proteins such as P53, FOXO3A,
Notch-1, Hsp90 and STAT3 [17-22]. Withaferin A treatment
leads to cell cycle arrest, increased reactive oxidative stress
[23-26], inhibition of the JAK/STAT pathway [27], inhibition of
angiogenesis [28], and modified cell shape and behavior [29].
Withaferin A also possesses potent anti-invasive activity, which
could potentially be due to its interaction with the pro-migratory
protein vimentin.
Vimentin is type III intermediate filament and classical EMT
protein marker [30]. Though vimentin functions in maintaining
cell structure, it is also a highly dynamic polymer that
assembles and dissembles in a motile cell. When cells undergo
EMT, vimentin expression is increased and this is thought to
provide cells with a more mesenchymal, pro-motile phenotype
[31]. The precise role of vimentin during EMT and development
is unclear, since a vimentin mouse knockout model did not
show severe developmental defects [32]. Additional studies
however did go on to show that these mice had impaired
fibroblast wound healing, and a reduced capacity to contract a
collagen network [33].
Withaferin A is proposed to bind to vimentin through a
covalent modification of cysteine 328 [34], leading to changes
in vimentin morphology and phosphorylation [29]; however,
other data show that mutation of cysteine 328 does not impact
Withaferin A-induced vimentin inhibition [29]. When given intra-
peritoneally (IP), Withaferin A effectively inhibits breast cancer
metastasis and has nearly no observable toxicity [35].
Less is known about the anti-tumor activity of the Withaferin
A parent root extract, WRE. Similar effects are observed on
tumor growth, cell cycle, and angiogenesis with WRE treatment
[36-40] along with immunomodulatory effects in colon and lung
cancer [41,42]. Nevertheless, studies directly comparing WRE
to Withaferin A have not been performed, and its anti-
metastatic activity has not been well studied. Furthermore,
WRE possesses several advantages over Withaferin A, since it
can be given orally in a capsule and the active withanolides
could have pharmacological synergy; therefore, we wanted to
determine the anti-metastatic efficacy of WRE standardized
(sWRE) to the pure active component, Withaferin A, in breast
cancer. We showed that sWRE can inhibit human breast
cancer cell invasion in vitro and metastasis in both allograft and
xenograft breast cancer mouse models, similar to the pure
small molecule Withaferin A. sWRE induces vimentin
reorganization and morphologic cellular changes in human
breast cancer cells and, importantly, inhibits EMT induction in
normal human mammary epithelial cells. Our results shed light
on the potential of sWRE as a novel complementary alternative
medicine used as an anti-metastatic preventative therapy in
high-risk breast cancer patients.
Methods
Reagents and antibodies
WFA was purchased from Chromadex (Irvine, CA) and WRE
was provided by Verdure Sciences (Noblesville, IN) with the
certificate of analysis stating that it is free of heavy metals,
bacteria, and fungus. The antibody against vimentin was
purchased from Sigma (St. Louis, MO), E-cadherin from BD
Biosciences (Bedford, MA), fibronectin from Abcam
(Cambridge, MA), and GAPDH from Cell Signaling (Beverly,
MA).
sWRE preparation
100% ethanol was heated to 60°C and then mixed with WRE
to the concentration of 250mg/ml for 30 minutes in a glass
beaker. Distilled H2O was then slowly added to lower the
ethanol concentration to 90%, and stirring continued for
another 30 minutes. The mixture was then spun at 4000rpm in
a centrifuge for 15 minutes at room temperature and the
supernatant was collected. The supernatant was then passed a
0.22µm filter, aliquoted, and kept at -80°C for future use.
Cell lines and culture conditions
Human MDA-MB-231 (ATCC # HTB-26), MCF-7 (# HTB-22)
and T47D (# HTB-133) breast cancer cell lines were purchased
from the American Type Culture Collection (ATCC, Manassas,
VA). Hs578-T, HCC1806 and MDA-MB-468 human breast
cancer cell lines were provided by Dr. O, Regan (Emory
University [45]). Human MCF10A mammary epithelial cell line
was provided by Dr. Vertino (Emory University [45]). Murine
breast carcinoma 4T1 cells were provided by Dr. Dewhirst
(Duke University [35,45]). T47D and HCC1806 were grown in
RPMI 1640 with 10% FBS. MDA-MB-231, MCF-7, Hs578-T,
MDA-MB-468 and 4T1 were grown in DMEM 10% FBS.
MCF10A cells were grown in DMEM/F12 supplemented with
5% FBS, 20ng/ml EGF, 0.5µg/ml Hydrocortisone, 100ng/ml
cholera toxin, and 10µg/ml insulin. All cell lines were
maintained in a humidified incubator at 37°C in a 5% CO2
atmosphere.
Cytotoxicity assay
The Promega CellTiter 96® AQueous Non-Radioactive Cell
Proliferation Assay (MTS) was performed for determining the in
vitro cytotoxicity of sWRE. Briefly, cells were cultured in 96-well
plates overnight and then treated with sWRE at the indicated
concentration for 72 hours. Cell viability was assessed by
determining the absorbance at 490nm as described by the
manufacturer (Promega, Madison, WI). Cell viability was
expressed as: Aexp group /Acontrol X 100.
In vitro wound healing assay
A cell migration wounding assay was performed as
described previously [43]. Cells were cultured in 6 wells plate to
100% confluency. After wounding with a pipette tip, cells were
washed with PBS and new media with the respective
concentration of sWRE was added. Cells then were allowed to
migrate for 24 hours at 37°C. Images of cells were taken at
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time 0 and 24 hours with an Olympus IX51 widefield
microscope (Center Valley, PA) at 5X magnification with a
Hamamatsu Orca ER CCD camera.
Matrigel invasion assay
Cell invasion was assayed using the Roche xCelligence
RTCA DP (Real-Time Cell Analyzer Dual Plate) Instrument
(Indianapolis, IN). DMEM with 10% FBS was added into the
bottom chamber of a CIM-Plate 16. 250µg/milliliter Matrigel (BD
Biosciences) was polymerized in the wells of the top chamber
for 1 hour at 37°C. Serum free media was added to the top
chamber, incubated for 30 minutes at 37°C, and a background
measurement was taken. Cells were pretreated with different
concentrations of sWRE in serum free media over night. These
cells were then added to the top chamber and the plate was
incubated in 37°C with the xCelligence plate reader.
Impedance measurements were taken in the bottom well and
the impedance increase correlates to increasing numbers of
migrated cells. Changes in impedance, which is reflected by
the cell invasion index values, were monitored for at least 24
hours.
Western blotting
Protein levels in cell lysates were measure using a standard
BCA assay. Cell lysates in sample buffer were separated on a
10% SDS-PAGE gels, and were transferred to PVDF
membranes (Bio-Rad, Hercules, CA). After blocking with 5%
fat-free milk-containing TBST, the membranes were separately
incubated with antibodies to vimentin, E-cadherin, fibronectin,
and GAPDH at room temperature for 2 hours. Membranes
were then washed and incubated with horseradish peroxidase
conjugated secondary antibodies for 1 hour. The bands were
detected using Amersham ECL Plus reagents and then
exposed to film.
Confocal imaging
Cells were cultured on glass cover slips and treated with
sWRE for 24 hours. Cells were then fixed and processed for
immunofluorescence microscopy as previously described [35].
Cells were stained using a primary anti-vimentin antibody and
secondary Alexa 488-conjugated goat anti-mouse IgG.
Coverslips were mounted onto slides and imaged using Zeiss
LSM510 META confocal microscope with a 40X Plan-
NEOFLUAR oil objective (NA 1.3).
Real-time PCR
Total RNA was isolated from cells using the RNeasy Mini kit
(Qiagen, Valencia, CA) and pretreated with DNase I. cDNA
was then synthesized using random hexamer primers and
MMLV- reverse transcriptase. Target-specific primers were
used to amplify cDNA in triplicate using a reaction mixture that
contained 1µl of the appropriately diluted cDNA sample,
0.2µmol/l primers and 12.5µl of IQ SYBR Green supermix (Bio-
Rad). 18S rRNA was amplified from the same samples as an
internal control. The reaction was subjected to a hot start for 3
min at 95°C and 40 cycles of 95°C, 10 s; 55°C (18S) or 63°C
(vimentin), 30 sec. Primers for real-time PCR analysis were for
vimentin: 5’ AATGGCTCGTCACCTTCGTGAA3’ and 5’
CAGATTAGTTTCCCTCAGGTTCAG3’ and for 18S, 5’
GAGGGAGCCTGAGAAACG G3’ and 5’
GTCGGGAGTGGGTAATTT GC3’
Three dimensional spheroid invasion assay
Agarose-coated plates were made by loading each well with
0.75% agarose in DMEM with 10% FBS. After gelation,
MCF10A cells were added into the wells and incubated at
37°C. Cell spheroids formed within 3-5 days. 5-10 spheroids
were mixed with Matrigel in DMEM or DMEM plus sWRE. The
mixture was placed in the middle of a 35 mm imaging petri dish
with a #1.5 coverslip (MatTek Corporation) and then
sandwiched on top with an additional cover slip. The dish was
then placed in a 37 °C incubator for 30 min to allow for Matrigel
polymerization, then 2ml of DMEM with 10% FBS was added.
The dish was transferred to the live cell PerkinElmer Ultraview
ERS spinning disk confocal microscope [44] and images were
acquired using 20X Zeiss objective (NA 0.75) every 20 minutes
for 24 hours with a Hamamatsu Orca ER camera.
In vivo sWRE toxicity study
Eight week old female Balb/c mice were purchased from
Harlan and housed in the Winship Cancer Animal Models
facility according to our approved IACUC protocol. Mice were
kept in groups of five per cage and fed with AIN76A control diet
and water ad libitum. The mice were randomized into 3 groups
of 3 mice per group and treated by oral gavage with either
vehicle (9% ethanol) or vehicle containing sWRE at 4, and
8mg/kg body weight, 3 times a week for 4 weeks. Mice body
weight was recorded each time after oral gavage. After 4
weeks of treatment, mice were sacrificed and organs (heart,
lung, liver, spleen and kidney) were collected and sent for
pathological analysis. Toxic effects were evaluated by a
blinded veterinarian pathologist based upon the presence of
inflammation, necrosis and fibrosis using a scale from normal
(1+) to moderate (3+).
Mammary carcinoma model
All mouse studies were performed in accordance with our
approved Emory University Institutional Animal Care and Use
Committee protocol. Eight weeks old female Balb/c mice were
divided randomly into 4 groups with 10 mice in each group. 106
4T1 cells in PBS were injected subcutaneously into the
mammary fat pad of each mouse. One week after injection,
sWRE were given by oral gavage with either vehicle or vehicle
containing sWRE at 1, 4, and 8mg/kg body weight 3 times a
week for 4 weeks. Withaferin A was intraperitoneally injected
by dissolving in 10% DMSO, 20% Cremophor-EL and 50%
PBS at 1, 4, and 8mg/kg 3 times a week for 4 weeks. The
tumor volume was recorded before gavage using calipers with
volume=(width)2 x length/2. Mice were sacrificed after 4 weeks
of treatment and metastatic lung nodules were counted under a
dissecting microscope. For H&E staining, the lung and primary
tumor from vehicle and sWRE-treated mice were fixed in 10%
neutral-buffered formalin, processed, embedded in paraffin,
and sectioned at 5µm thickness. Representative tumor sections
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from vehicle control, sWRE-treated, and Withaferin A-treated
mice were processed for H&E staining.
Xenograft mouse breast cancer model
All mouse studies were performed in accordance with our
approved Emory University Institutional Animal Care and Use
Committee protocol. Eight week old female athymic nude
Foxn1nu mouse were purchased from Harlan and housed in
the Winship Cancer Animal Models facility. Mice were kept in
groups of five per cage and fed with AIN76A control diet and
water ad libitum. Mice were divided randomized into 7 groups
with 10 mice in each group and 106 MDA-MB-231 cells in PBS
were injected subcutaneously into the mammary fat pad of
each mouse. One week after injection, mice were treated with
vehicle (9% ethanol), vehicle containing sWRE at 1, 4, and
8mg/kg by oral gavage, or intraperitoneally injected with
Withaferin A dissolved in 10% DMSO, 20% Cremophor-EL and
50% PBS at 1, 4, and 8mg/kg 3 times a week for 4 weeks. The
tumor volume was recorded before gavage. Mice were
sacrificed after 4 weeks of treatment and metastatic lung
nodules were counted under a dissecting microscope. For H&E
staining, the lung and tumor from vehicle and sWRE-treated
mice were fixed in 10% neutral-buffered formalin, processed,
embedded in paraffin and sectioned at 5µm thickness.
Representative tumor sections from control and sWRE-treated
mice were processed for H&E staining.
Statistical Analysis
Data were analyzed statistically using GraphPad Prism for
Windows (version 5). One-way ANOVA was carried out to
compare the mean of lung nodules among the experimental
groups. Linear mixed effects model was used to compare the
mean tumor volumes among the groups. Values of p<0.05
were considered significant.
Results
WRE Standardization, Solubility, and Cytotoxicity
WRE powder was solubilized in H2O or 90% ethanol (EtOH)
at different concentrations to determine optimal conditions to
standardize WRE (sWRE) to the pure small molecule
Withaferin A. The concentration of Withaferin A in each sWRE
formulation was measured by HPLC (Figure 1A) and the
recovery rate of Withaferin A after solubulization was
calculated for each sample (Figure 1B). The content of
Withaferin A compared to other withanolides using HPLC is
shown in Table 1. When sWRE at 250mg/ml was dissolved in
water, the Withaferin A recovery rate was 4%. The largest
percent recovery of Withaferin A in water was 16%, observed
at a starting concentration of 10 mg/ml sWRE. In contrast,
when WRE powder was dissolved in 90% EtOH the Withaferin
A recovery rate ranged from 80-92%. These results show that
re-solubilization of sWRE in 90% EtOH is clearly superior to
that in H2O. To estimate the long-term stability of the WRE
stock solution in 90% EtOH, HPLC analysis was done on WRE
after 6 and 12 months of storage at -80°C. The results show
that about 90% of the initial Withaferin A can be detected after
6 months, and about 80% after 12 months (Figure 1C, D).
To assess cytotoxicity, breast cancer cell lines were treated
with increasing concentrations of Withaferin A-standardized
WRE (sWRE) for 24 and 72 hours (Figure 2A, B). Among the
six cell lines tested, four (MDA-MB-468, HCC 1806, Hs587-T,
and MDA-MB-231) are triple negative breast cancer cell lines
[45]. Since Withaferin A is a vimentin-targeting agent [30,34],
vimentin expression along with other EMT markers were
assessed in the cell lines. Two of the four triple negative cell
lines, (Hs578-T and MDA-MB-231) were vimentin-positive and
all other cell lines were vimentin-negative. These vimentin
positive cell lines also exhibited EMT induction since they
displayed E-cadherin loss and were fibronectin positive (Figure
2C). Interestingly, different sensitivity to sWRE was observed
across the six cell lines, where the two most sensitive cell lines,
Hs578-T and MDA-MB 231, were vimentin-positive with a 72
hour IC50 of 0.5µM and 0.4µM respectively (Figure 2B). The
vimentin-negative cell lines had IC50s that ranged from 1.2µM
to 4.0µM. To probe this, cytotoxicity assays were performed in
isogenic control and vimentin siRNA depleted MDA-MB 231
cells. In this case, vimentin depleted cells show a minor
Figure 1. Withaferin A recovery in sWRE. sWRE powder
was dissolved in different solvents and the concentration (A)
and recovery rate (B) of Withaferin A in sWRE was measured
by HPLC. The stability of Withaferin A in sWRE (90% EtOH
stock solution) over time is shown in a bar graph with the
absolute (C) and relative (D) Withaferin A concentration.
doi: 10.1371/journal.pone.0075069.g001
Table 1. Withanolides in WRE by HPLC.
Withanolides Concentration (mg/ml) Percentage (%)
Withaferin A 5.88 79.03
Withanoside V 0.356 4.78
12-Deoxywithastramonolide 1.06 14.25
Withanolide A 0.136 1.83
Withanolne 0.0137 0.18
Total Withanolides 7.44 100.00
doi: 10.1371/journal.pone.0075069.t001
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decrease in sWRE cytotoxicity compared to isogenic control
cells (Figure 2D).
sWRE inhibits breast cancer cell motility and invasion,
and disrupts vimentin morphology
We have previously shown the Withaferin A inhibits breast
cancer cell motility and metastasis [35]; therefore, we sought to
determine if sWRE shows anti-invasive efficacy. The effect of
sWRE on the cell motility of triple negative breast cancer cells
(human MDA-MB-231 and mouse 4T1) was tested using a
wound healing assay. WRE inhibits cell motility in a dose-
dependent manner after 24 hours of treatment at 0.5µM in both
cell lines (Figure 3A,B). An expanded experiment at lower
doses in MDA-MB 231 cells shows inhibition of cell motility at
0.25 µM as well (Figure S1). We then wanted to determine how
sWRE impacts cell motility when vimentin protein is absent.
This experiment was first attempted in MCF7 and MDA MB 468
breast carcinoma cells, both of which do not have detectable
vimentin by western blotting; however in these cases, cells
were also not motile so the experiment could not be performed
(data not shown). Instead, we used the lung epithelial cell line,
BEAS-2B, which lacks vimentin (Figure S1-B) and shows some
motility. sWRE had a 24 hr anti-proliferative IC50 of 2.9µM and
a 48 hr IC50 of 1.9 µM (Figure S1-C). In a wounding assay,
lower doses of sWRE (0.125 to 1 µM) below the IC50 had nearly
no impact on cell motility (Figure S1-D). It was not until
treatment with 2 µM sWRE which is near the 24 hr anti-
proliferative IC50, did we observe appreciable inhibition of
motility (Figure S1-D).
The anti-invasive activity of sWRE was tested using a real
time Matrigel invasion assay in a Boyden chamber. sWRE was
again able to significantly inhibit cell invasion in both cell lines
at doses as low as 0.25µM in 4T1 and 0.5µM in MDA-MB-231
cells (Figure 3C, D). These results show that sWRE inhibits cell
motility and is anti-invasive in triple negative breast cancer
cells.
Since Withaferin A inhibits vimentin [30,34], we explored
whether sWRE has similar vimentin-disrupting ability. Vimentin
immunofluorescence in control MDA-MB-231 cells show that
vimentin is networked throughout the spindle-shaped
cytoplasm; however, upon sWRE treatment (0.5µM and 1.0µM)
for 16 hours, cells were not as elongated and the vimentin
network was abolished (Figure 3E). Instead, vimentin
accumulated as a perinuclear bundle in most cells, which is
similar to the effect of pure Withaferin A [35]. Furthermore,
sWRE did not decrease total cellular protein levels until 48 hrs
of treatment (Figure S2), suggesting that this is not due to a
defect in total protein synthesis. Therefore, based upon these
observations we conclude that sWRE also possesses vimentin
inhibitory activity at low doses.
Figure 2. sWRE cytotoxicity in breast cancer cell lines. (A and B) Line graphs showing the % survival of six breast cancer cell
lines treated with increasing concentrations of sWRE for 24 (A) and 72 (B) hours. (C) Western blot showing EMT markers in
different breast cancer lines. Triple negative breast cancer cell lines are indicated with an asterisk. (D) (left) Western blot showing
successful vimentin siRNA depletion (right) Cytotoxciity assay using sWRE in isogenic control and vimentin siRNA depleted cells.
doi: 10.1371/journal.pone.0075069.g002
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Figure 3. sWRE inhibits migration and invasion in MDA-MB-231 and 4T1 cell lines. (A and B) Cell wounding assay in (A)
MDA-MB-231 and (B) 4T1 cells treated with increasing concentrations of sWRE for 24 hours. (C and D) Line graph showing the rate
of cellular invasion through Matrigel embedded in a Boyden chamber in (C) MDA-MB-231 and (D) 4T1 cells treated with increasing
concentrations of sWRE. (E) Confocal immunofluorescence imaging of vimentin (green), actin (red), and DAPI (blue) in MDA-
MB-231 cells treated with 9% EtOH control, 0.5µM or 1.0µM sWRE.
doi: 10.1371/journal.pone.0075069.g003
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sWRE prevents TGFβ-induced EMT
Since vimentin plays a key role in EMT, we wanted to test if
sWRE could inhibit EMT and EMT-induced motility in breast
cancer. To test this, we used MCF10A cells, where treatment
with 4ng/ml TGFβ causes these cells to undergo EMT, as
assessed by an increase in the mesenychmal markers vimentin
and fibronectin, and loss of the epithelial marker, E-cadherin
[8,9]. These data show that sWRE inhibits MCF10A motility in a
wound healing assay with TGFβ (Figure 4C) at doses similar to
those used in 4T1 and MDA-MB-231cell lines (Figure 3). To
assess how sWRE impacts EMT, MCF10A cells were treated
with TGFβ in the presence of sWRE or Withaferin A. TGFβ
alone induced vimentin and fibronectin protein expression and
decreased E-cadherin protein levels indicating successful EMT
induction. In contrast, treatment with 500nM sWRE or 500nM
Withaferin A potently inhibited TGFβ-induced EMT by keeping
vimentin and fibronectin at pre-induction levels and increasing
E-cadherin levels (Figure 4A, B). To determine if this occurs at
the transcriptional level, real-time PCR of the vimentin
transcript was performed and these results showed that TGFβ
increased vimentin mRNA as expected, but sWRE did not
impact vimentin mRNA levels (Figure 4D), similar to that
observed using Withaferin A [30]. Therefore the results show
that sWRE does not inhibit vimentin expression at the mRNA
level but rather on the protein level.
These studies were then moved into a spheroid model of
invasion to determine if sWRE inhibits EMT-induced invasion.
In this model, TGFβ induced potent invasion into the Matrigel
extracellular matrix using live cell imaging to visualize invasion
(Figure 4E, Movie S1); however, upon treatment with doses as
low as 100nM sWRE, invasion was potently inhibited (Figure
4E, Movie S2). Taken together, these results show that sWRE
can inhibit EMT and EMT-induced motility and invasion.
sWRE in vivo toxicity
To study the anti-metastatic efficacy of sWRE in vivo, sWRE
toxicity was first assessed in normal female BALB/c mouse.
Mice were given either 4 or 8 mg/kg sWRE in 9% EtOH and the
mean body weight gain after 35 days in treated mice were not
significantly different from the control group given 9% EtOH
alone (Figure 5A). After four weeks of treatment, the histology
of the heart, lung, liver, spleen and kidney were graded for
fibrosis, necrosis and inflammation. Histological data show no
significant difference between sWRE-treated and vehicle
control groups (Figure 5B, C).
sWRE anti-metastatic efficacy
To determine the anti-metastatic efficacy of sWRE and
compare it to Withaferin A in a mouse model, the mouse
mammary carcinoma 4T1 metastatic model was used. This
model develops metastatic lesions in the lung, liver, and spleen
4-6 weeks post-injection of cells into the mammary fat pad.
Mice were divided randomly into 4 groups with 10 mice in each
group, and sWRE was given by oral gavage and Withaferin A
by i.p. injection at 1, 4, and 8 mg/kg 3 times per week for 4
weeks. The dose range was selected based on the previous
sWRE toxicity experiment in the BALB/c mouse (Figure 5). At
all doses, primary tumor volume was decreased after 36 days
of treatment with sWRE or Withaferin A (Figure 6A, B).
Representative gross specimens of primary tumors show that
both sWRE and Withaferin A treated tumors were smaller
(Figure 6C). Importantly, the number of metastatic lung nodules
significantly decreased in both 4 and 8 mg/kg groups in sWRE
and Withaferin A treated mice (Figure 6D, E, and examples in
Figure 6G). H&E staining confirmed the presence of micro-
metastatic lesions in the lung (Figure 6F).
To further test the anti-metastatic efficacy of sWRE, a similar
experiment using a xenograft model with human metastatic
breast cancer MDA-MB-231 cells was performed. Cells were
injected subcutaneously into the mammary fat pad of female
athymic nude mice. Mice were administrated sWRE by oral
gavage and Withaferin A by i.p. injection at concentration of 1,
4, and 8mg/kg 3 times a week for 4 weeks. Primary tumor
Figure 4. sWRE inhibits TGFβ1-induced EMT in MCF10A
cells. (A) Western blot of vimentin in TGFβ1-stimulated
MCF10A cells treated with increasing concentrations of sWRE.
(B) Western blot of vimentin, fibronectin, and E-cadherin in
TGFβ1 stimulated MCF10A cells treated with 500nM sWRE or
Withaferin A. (C) Cell migration in wound-healing assay with
increasing concentrations of WRE. (D) Relative vimentin
mRNA levels detected by real-time PCR in TGFβ1-stimulated
MCF10A cells treated with 500nM sWRE or WFA. (E) Live cell
time lapse images of MCF10A 3D spheroids embedded in
Matrigel and stimulated with TGFβ1. Cell were treated with 9%
EtOH control, or 100nM or 500nM sWRE.
doi: 10.1371/journal.pone.0075069.g004
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PLOS ONE | www.plosone.org 7 September 2013 | Volume 8 | Issue 9 | e75069

volume was inhibited by sWRE at 4 and 8mg/kg doses.
Similarly Withaferin A treatment also resulted in a reduction in
tumor volume at 4 and 8mg/kg dose (Figure 7A, B).
Importantly, both sWRE and Withaferin A showed similar
efficacy and decreased the number of metastatic nodules in the
lung at 8 mg/kg (Figure 7C, D).
Discussion
Our data show that sWRE has similar anti-metastatic efficacy
to pure Withaferin A in two in vivo mouse models (Figures 6
and 7). Specifically, sWRE significantly decreased metastatic
lung nodule formation in the 4T1 model when given orally at 4
or 8 mg/kg and at 8 mg/kg in the MDA-MB-231 xenograft
model, where in general sWRE had a more graded, dose-
dependent effect on metastatic lung nodule formation. These
results are similar to those observed with pure Withaferin A,
which inhibited metastatic lung nodules at 4 and 8 mg/kg in
both models (Figures 6 and 7). Furthermore, sWRE had nearly
no toxicity based upon pathological analysis and monitoring of
mouse weight at 8 mg/kg (Figure 5), which is similar to the
previous observation that pure Withaferin A given i.p. also had
minimal toxicity at a similar concentration [35]. Therefore,
based upon these in vivo mouse studies we conclude that oral
Figure 5. Toxicity of sWRE in female BALB/c mice. (A) Relative body weight in mice treated with vehicle, or sWRE at 4mg/kg
and 8mg/kg. (B) Histological grading of inflammation, fibrosis, and necrosis in organs from mice treated with vehicle (9% EtOH) or
sWRE at 8mg/kg. (C) Representative images of H&E stained histological sections.
doi: 10.1371/journal.pone.0075069.g005
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Figure 6. Anti-metastatic activity of sWRE and Withaferin A in an allograft breast cancer mouse model. (A and B) Mean
tumor volume in mice treated with 1, 4, 8mg/kg of (A) sWRE or (B) Withaferin A (WFA) (* p<0.05 compared to control). (C)
Representative images of the primary tumor in sWRE or WFA treated mice. (D and E) Bar graph showing the mean number of
metastatic lung nodules in (D) sWRE or (E) WFA-treated mice (* p<0.05 compared to control). (F) Representative H&E staining
images showing the histology of metastatic nodules in mouse lung; two examples shown. (G) Representative images of lung
metastatic nodules (black arrows) in mice treated with vehicle control (9% EtOH) or sWRE or WFA at 8mg/kg.
doi: 10.1371/journal.pone.0075069.g006
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PLOS ONE | www.plosone.org 9 September 2013 | Volume 8 | Issue 9 | e75069

administration of sWRE has similar anti-metastatic efficacy as
pure Withaferin A. Furthermore, these data would suggest that
oral administration of WRE capsules that contain active
Withaferin A could retain anti-metastatic efficacy in a clinical
setting.
sWRE also inhibits cell motility and invasion in vitro at 0.5µM
(Figure 3), which is well below its cytotoxic 24hr IC50 of 8µM or
higher depending on the cell line (Figure 2). These data
suggest that its ability to inhibit cell motility is distinct from its
anti-proliferative activity, and thus may not be due to general
cytotoxicity. We propose that inhibition of motility, could occur
via vimentin inhibition, since we observed a prominent
disruption of vimentin morphology in cells treated at 0.5µM and
1µM sWRE. It was previously shown that Withaferin A can bind
directly to vimentin [28] and also disrupt vimentin morphology
[29,30,35]; therefore, these results with sWRE are consistent
with these previous studies showing vimentin inhibitory activity.
It is important to note that the role of vimentin in cell motility
has remained controversial. Numerous reports show that
vimentin is a classic EMT biomarker that is expressed in
aggressive cell lines and tumors [46-53], and correlates with
high grade cancer and metastatic disease [54-58];
Nevertheless, the precise molecular role of vimentin in cell
motility remains largely undefined and there are several reports
that induced vimentin expression in vimentin null cell lines does
not impact motility [59,60]. Thus, it is still debatable as to why
vimentin expression in certain contexts correlates with invasion
(e.g., metastatic disease) while in other systems re-expression
does not. Though Withaferin A and now sWRE are both shown
to disrupt vimentin, we cannot directly rule out the possibility
Figure 7. Anti-metastatic activity of sWRE and Withaferin
A in an MDA-MB-231 xenograft breast cancer model. (A
and B) Mean tumor volume in mice treated with 1, 4, 8mg/kg of
(A) sWRE or (B) WFA (*, p<0.05 compared to vehicle control).
Bar graph showing the mean number of metastatic lung
nodules in (C) sWRE or (D) WFA-treated mice compared to
vehicle control (9% EtOH; * indicated p<0.05 compared to
control).
doi: 10.1371/journal.pone.0075069.g007
that both treatments inhibit metastasis through a vimentin-
independent pathway.
sWRE can prevent EMT induction in the MCF10A EMT
model (Figure 4) at 0.5µM, whereby sWRE treatment reverses
vimentin and fibronectin induction and promotes E-cadherin
expression. Furthermore, sWRE also potently inhibits TGFβ
induced 3-D MCF10A spheroid invasion at both 0.1µM and
0.5µM (supplemental Movies). It remains unclear if the anti-
EMT efficacy of sWRE is tied to its ability to inhibit vimentin, but
one possibility is that vimentin inhibition by sWRE leads to its
degradation and consequently a reversal of the EMT program.
We did not observe changes in the vimentin transcript after
sWRE treatment; therefore, we do not suspect that sWRE
affects transcription of the EMT markers.
Though the major focus of these studies was on metastasis,
it is interesting to note that higher concentrations of sWRE
inhibited cell proliferation (Figure 2). Interestingly, the greatest
anti-proliferative activity was observed in cell lines that were
vimentin-positive suggesting a potential correlation between
vimentin expression and cytotoxicity. Though vimentin is
primarily linked to cell motility, there are reports that it functions
in proliferation [61-64] and perhaps that is responsible for the
observed cytotoxicity.
In triple negative breast cancers (estrogen, progesterone,
and HER-2 negative), vimentin expression is correlated with
poor prognosis as well as an aggressive and metastatic
phenotype [64-68]. We observed that two of the three triple
negative cell lines (MDA-MB-231 and Hs578-T) express
vimentin. These cell lines were also the most sensitive to
sWRE in cytotoxicity assays and sWRE inhibited metastasis in
the MDA-MB-231 xenograft model. Though this data set is
correlative and we cannot directly attribute the sensitivity to
vimentin expression, we believe that these efficacy data
suggest that sWRE has the potential to be used as an anti-
metastatic in vimentin-positive tumors. Additional
pharmacokinetic and pharmcodynamic data with sWRE will
likely prove to be useful and will be the focus of future work.
Supporting Information
Figure S1. (A) Wounding assay in MDA-MB 231 cells using
low concentrations of sWRE (B) Western blotting of vimentin in
MDA-MB 231 cells and BEAS-2B lung epithelial cells (C) MTT
anti-proliferative assay in BEAS-2B cells with sWRE(D)
Wounding assay in BEAS-2B lung epithelial cells using a range
of sWRE concentrations.
(PDF)
Figure S2. (A) BCA assay quantifying protein concentration
per cell in MDA-MB 231 cells after treatment at different
timepoints with sWRE (*p<0.05).To do this, the total protein
was divided by the number of cells in the well after treatment.
(B) BCA assay quantifying protein levels per well after
treatment at different timepoints with sWRE (*p<0.05).
(PDF)
Movie S1. Live cell imaging of MCF10A spheroids embedded
in Matrigel and treated with TGF-beta.Time is in hours.
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PLOS ONE | www.plosone.org 10 September 2013 | Volume 8 | Issue 9 | e75069

(AVI)
Movie S2. Live cell imaging of MCF10A spheroids embedded
in Matrigel and treated with TGF-beta and sWRE at 100
nM.Time is in hours.
(AVI)
Acknowledgements
We would like to thank the Winship Pathology Core, Emory
Integrated Cellular Imaging Core, and the Winship Cancer
Animal Models Core for their assistance. We would also like to
thank Anthea Hammond for editorial assistance on this
manuscript.
Author Contributions
Conceived and designed the experiments: ZY AG DRP PMV
AIM. Performed the experiments: ZY AG SX DRP AIM.
Analyzed the data: ZY AG SX DRP PMV SS AIM. Contributed
reagents/materials/analysis tools: PMV SS AIM. Wrote the
manuscript: ZY SS AIM.
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Root Extract Inhibits Metastasis
PLOS ONE | www.plosone.org 12 September 2013 | Volume 8 | Issue 9 | e75069