(-)-Epigallocatechin gallate derivatives reduce the expression of both urokinase plasminogen activator and plasminogen activator inhibitor-1 to inhibit migration, adhesion, and invasion of MDA-MB-231 cells

Article abstract of DOI:10.1002/ptr.6154

Urokinase plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor-1 (PAI-1) are established independent biomarkers for high metastasis risk in breast cancer. In this study, we investigated the regulatory activity of (-)-epigallocatechin-3-gallate (EGCG) and its derivatives on uPA and PAI-1 expression and thereby their anti-metastatic potential. EGCG showed only marginal effects on the uPA system and on the metastatic behavior of breast cancer cells (MDA-MB-231). However, the EGCG derivative 3e with a methyl-substituted carbonate substituent at the 4″-position showed potent inhibition of PAI-1 (62%) and uPA (50%) expression. The Ras-extracellular-signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and phosphatidylinositol-3-kinase (PI3K)/Akt/NF-κB pathways, which regulate uPA and PAI-1 expression, were also affected by 3e (25%, 45%, and 25% reduction, respectively). In line with these findings, substantial reduction in metastatic behavior of MDA-MB-231 cells, such as adhesion (40%), invasion (56%), and migration (40%), was observed in the presence of 3e. It is also noteworthy that, in MDA-MB-231 cells, 3e did not exert any beneficial effect on the expression of matric metalloprotein (MMP) 2 and 9, which indicates that the anti-metastatic activity of 3e in MDA-MB-231 cells is not related to its regulation of the expression of MMPs. Taken together, we have shown that the EGCG derivative 3e could suppress the metastatic behavior of MDA-MB-231 cells through regulation of uPA and PAI-1.

Full text of DOI:10.1002/ptr.6154

Received: 27 February 2018
DOI: 10.1002/ptr.6154
Revised: 16 June 2018
Accepted: 18 June 2018
R E S E A R C H A R T I C L E
(−)‐Epigallocatechin gallate derivatives reduce the expression
of both urokinase plasminogen activator and plasminogen
activator inhibitor‐1 to inhibit migration, adhesion, and invasion
of MDA‐MB‐231 cells
Sunhye Shin1 Mi Kyoung Kim1 Woong Jung2 Youhoon Chong1
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1 Department of Integrative Bioscience and
Biotechnology, Bio/Molecular Informatics
Center, Konkuk University, Seoul, Korea
Urokinase plasminogen activator (uPA) and its inhibitor plasminogen activator inhibi-
tor‐1 (PAI‐1) are established independent biomarkers for high metastasis risk in breast
cancer. In this study, we investigated the regulatory activity of (−)‐epigallocatechin‐3‐
gallate (EGCG) and its derivatives on uPA and PAI‐1 expression and thereby their anti‐
metastatic potential. EGCG showed only marginal effects on the uPA system and on
the metastatic behavior of breast cancer cells (MDA‐MB‐231). However, the EGCG
derivative 3e with a methyl‐substituted carbonate substituent at the 4″‐position
showed potent inhibition of PAI‐1 (62%) and uPA (50%) expression. The Ras‐extracel-
lular‐signal‐regulated kinase (ERK), p38 mitogen‐activated protein kinase (MAPK), and
phosphatidylinositol‐3‐kinase (PI3K)/Akt/NF‐κB pathways, which regulate uPA and
PAI‐1 expression, were also affected by 3e (25%, 45%, and 25% reduction, respec-
tively). In line with these findings, substantial reduction in metastatic behavior of
MDA‐MB‐231 cells, such as adhesion (40%), invasion (56%), and migration (40%),
was observed in the presence of 3e. It is also noteworthy that, in MDA‐MB‐231 cells,
3e did not exert any beneficial effect on the expression of matric metalloprotein
(MMP) 2 and 9, which indicates that the anti‐metastatic activity of 3e in MDA‐MB‐
231 cells is not related to its regulation of the expression of MMPs. Taken together,
we have shown that the EGCG derivative 3e could suppress the metastatic behavior
of MDA‐MB‐231 cells through regulation of uPA and PAI‐1.
2 Department of Emergency Medicine, Kyung
Hee University Hospital at Gangdong, Seoul,
Korea
Correspondence
Youhoon Chong, PhD, Department of
Integrative Bioscience and Biotechnology,
Bio/Molecular Informatics Center, Konkuk
University, Hwayang‐dong, Gwangjin‐gu,
Seoul 143‐701, Korea.
Email: chongy@konkuk.ac.kr
Funding information
Ministry of Education, Science and Technol-
ogy, Grant/Award Number: 2009‐0093824;
Ministry of Science, ICT and future Planning,
Grant/Award Number:
2015R1A2A1A10053461
KEYWORDS
breast cancer, EGCG, metastasis, uPA system
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1
INTRODUCTION
Harbeck, 2002; Harbeck, Kates, & Schmitt, 2002; Jänicke et al.,
2001; Konecny et al., 2001; Look et al., 2002).
In breast cancer patients, the main cause of death is metastases at dis-
tant sites, not the primary tumor. However, it is not possible to accu-
rately predict the risk of metastasis development, which necessitates
new prognostic markers for breast cancer metastasis. The serine pro-
tease urokinase plasminogen activator (uPA) and its inhibitor plasmin-
ogen activator inhibitor‐1 (PAI‐1) are newly established independent
biomarkers for breast cancer, and high protein levels of uPA and
PAI‐1 are associated with high metastasis risk (Foekens et al., 2000;
The metastatic cascade is initiated by the degradation of the
extracellular matrix followed by the invasion of the surrounding host
tissue by cancer cells; matrix metalloproteinases and uPA play key
roles in this process (Andreasen, Kjoller, Christensen, & Duffy, 1997).
uPA converts plasminogen to plasmin, which degrades matrix compo-
nents to activate latent metalloproteinases and latent growth factors.
Interestingly, in addition to uPA, increased levels of its inhibitor PAI‐
1 were paradoxically also reported to be a prognostic marker in breast
Phytotherapy Research. 2018;1–11.
wileyonlinelibrary.com/journal/ptr
© 2018 John Wiley & Sons, Ltd.
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SHIN ET AL.
cancer patients (Jänicke, Schmitt, & Graeff, 1991). However, it should
be noted that, independent of its protease‐inhibitory capacity, PAI‐1
also has a role in cell migration (Andreasen et al., 1997) and promotes
tumor invasion and angiogenesis (Bajou et al., 2004).
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2
MATERIALS AND METHODS
Chemicals and reagents
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2.1
(−)‐EGCG (1) is the product of Xi'an Le Sen Bio‐Technology Co., Ltd.
(China). AcEGCG (2) and its derivatives (3) were prepared according
to previous methods (Seo et al., 2017; Scheme 1). Briefly, AcEGCG
(2), obtained from the reaction of EGCG with excess amount of
Ac2O in pyridine, was directly converted to desired 4″‐O‐alkylated
AcEGCG (3a–3j) upon treatment with appropriate alkyl halide in the
presence of KI and K2CO3 (5–40% yield; spectroscopic data can be
found in Supporting Information).
Of the two markers, PAI‐1 has been the focus of many studies, with
numerous reports regarding PAI‐1 inhibition with small molecules [PAI‐
039 (tiplaxtinin) (Gomes‐Giacoia, Miyake, Goodison, & Rosser, 2013;
Gorlatova et al., 2007), TM5001/TM5007 (Izuhara et al., 2008),
TM5257 (W. T. Huang, Vayalil, Miyata, Hagood, & Liu, 2012; Izuhara
et al., 2010), TM5441 (Boe et al., 2013; Pelisch et al., 2015)], monoclonal
antibodies (Fortenberry, 2013), and aptamers (Blake, Sullenger, Law-
rence, & Fortenberry, 2009; Brandal, Blake, Sullenger, & Fortenberry,
2011; Fortenberry, Brandal, Carpentier, Hemani, & Pathak, 2016;
Madsen et al., 2010). However, less attention has been paid to control
the gene expression of uPA and PAI‐1 and thus reduce their levels,
which would serve to inhibit the metastasis of breast cancer cells.
Green tea polyphenols have been reported to block the invasive
behavior of human breast cancer MDA‐MB‐231 cells by inhibiting
the transcription factors for uPA (activator protein‐1 and NF‐κB
[nuclear factor κB]) and by forming signaling complexes responsible
for cell adhesion and migration (uPA, uPA‐receptor, vitronectin, and
integrin receptor; Slivova et al., 2005). (−)‐Epigallocatechin gallate
[(−)‐EGCG (1), Figure 1], the major polyphenol in green tea, has also
been reported to play an important role in inhibiting the invasive
behavior of breast cancer (Kushima et al., 2009), but its direct relation-
ship with uPA/PAI‐1 levels has not yet been established. Thus, we
aimed to evaluate the inhibitory potential of EGCG against the inva-
sive behavior of MDA‐MB‐231 cells in conjunction with its effects
on uPA and PAI‐1 protein levels. Moreover, peracetylated EGCG
[AcEGCG (2), Figure 1] (Chao et al., 2010; Chiou et al., 2013; Lambert
et al., 2006; Landis‐Piwowar et al., 2007; Wang et al., 2013) and
AcEGCG derivatives with alkyl substituents at the D‐ring 4″‐position
(3, Figure 1; Seo, Kim, Choo, & Chong, 2017), which had been investi-
gated to overcome low stability and high polarity of EGCG, were also
included for biological assay.
Dimethyl sulfoxide (DMSO) and MTT [(3‐(4,5‐dimethylthiazol‐2‐
yl)‐2,5‐diphenyltetrazolium bromide)] were purchased from Sigma‐
Aldrich. Roswell Park Memorial Institute (RPMI) 1640 media, penicillin,
streptomycin, and fetal bovine serum (FBS) were purchased from
HyClone Laboratories. A 100‐mm cell culture plate was purchased
from Dishes Biousing™. A Bio‐Rad DC protein assay kit was purchased
from Bio‐Rad.
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2.2
Cell culture
Human breast cancer MDA‐MB‐231 cells were obtained from the
American Type Culture Collection (Manassas, VA, USA) and cultured
in RPMI‐1640 medium, supplemented with 10% FBS, penicillin (100
units/ml), and streptomycin (100 μg/ml), at 37°C with 5% CO2 in a
humidified atmosphere.
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2.3
Cell viability assay
MDA‐MB‐231 cells (8 × 103 cells/well) were seeded in 96‐well plates
and cultured in RPMI with 10% FBS. After 24 hr, various concentra-
tions of EGCG derivatives (0–100 μM) were added, and the cells
were incubated for a further 48 hr. Then, 10 μl of MTT solution
(5 mg/ml) was added, and the cells were incubated for another
4 hr. The MTT formazan was dissolved with DMSO, and absorbance
was measured at 570 nm, using a CYTATION 5 imaging reader
(BioTek).
Herein, we report the inhibition of the invasion, adhesion, and
migration of human breast cancer MDA‐MB‐231 cells by treatment
with EGCG (1), AcEGCG (2), and AcEGCG derivatives (3) through reg-
ulation of the expression of both uPA and PAI‐1.
FIGURE 1 Structures of EGCG (1), AcEGCG (2), and AcEGCG derivatives (3)

SHIN ET AL.
3
SCHEME 1 Synthesis of the title
compounds
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2.4
Immunoblotting
2.4.3
NF‐κB expression
NF‐κB protein in MDA‐MB‐231 cells was isolated using a cytoplasmic
and nuclear proteins lysis buffer, which consisted of nuclear extract
lysis buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 25% glyc-
erol, 1× protease inhibitors) and cytoplasmic extract (CE) lysis buffer
(10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.3% NP‐40, 1×
protease inhibitor). MDA‐MB‐231 cells (3 × 105 cells/well) were
seeded in 6‐well plates and, 24 hr later, incubated with either control
media or media supplemented with EGCG derivatives. After 48 hr,
the cells were washed with cold PBS and scraped. Samples were then
centrifuged (5 min at 1500× g) at 4°C. The pellet was resuspended in
CE buffer, vortexed, and incubated on ice for 5 min. Samples were again
centrifuged (5 min at 3300× g) at 4°C. The supernatant, that is, the CE,
was harvested. The pellet was resuspended in washing buffer (CE
buffer without NP‐40) and then centrifuged (5 min at 3300× g) at
4°C. The resulting pellet was then resuspended in nuclear extract
buffer, vortexed, and incubated on ice for 10 min. The sample was then
centrifuged (20 min at 16000× g) at 4°C. The supernatant, that is, the
nuclear extract, was harvested. NF‐κB protein was detected by immu-
noblotting using NF‐κB rabbit monoclonal antibody (Cell Signaling
Technology). Western blotting protocols were as described above.
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2.4.1
PAI‐1 expression
MDA‐MB‐231 cells (3 × 105 cells/well) were seeded in 6‐well plates
and, 24 hr later, incubated with either control media or media sup-
plemented with EGCG derivatives. After 48 hr, the cells were
washed with cold PBS and scraped. Samples were then centrifuged
(5 min at 1500× g) at 4°C, and the pellet was resuspended in lysis
buffer (1 M HEPES, pH 7.9, 400 mM NaCl, 0.1 mM EDTA, 5% glyc-
erol, 1 mM DTT) containing protease inhibitors (Thermo Fisher) and
phenylmethylsulfonyl fluoride (Thermo Fisher). The samples were
again centrifuged (20 min at 16100× g) at 4°C, and the protein con-
centration in the resulting protein lysate was determined by the
bicinchoninic acid assay. Total cell lysate (30 μg/well) was separated
by SDS‐PAGE (Bio‐Rad) and transferred to nitrocellulose membranes
(Bio‐Rad). The membranes were blocked in 5% nonfat milk in TBST
(Tris‐buffered saline containing 0.1% Tween 20) at room tempera-
ture for 1 hr. The membranes were then incubated with TBST with
mouse anti‐PAI‐1 (Abcam) at 4°C overnight, followed by incubation
with horse anti‐mouse IgG conjugated to horseradish peroxidase
(1:1000 in TBST, Cell Signaling Technology) for 1 hr. Bands were
visualized by enhanced chemiluminescence (Bio‐Rad), followed by
autoradiography, and quantified by densitometry (ChemiDoc™
Imaging Systems, Bio‐Rad).
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2.5
Cell invasion assay (trans‐well invasion assay)
Cell invasion was analyzed using a Transwell (Corning Incorporated,
Corning, NY, USA). To analyze cell invasion, the upper surface of a filter
membrane in the upper compartment of a Transwell was coated with
5 μg/well vitronectin and incubated at 37°C overnight. MDA‐MB‐231
cells (2.5 × 105) suspended in 200 μl of serum‐free medium were
seeded onto the upper compartment of the Transwell, and the lower
chambers were filled with 750 μl of medium containing 10% FBS and
30 μM of different EGCG derivatives. After 48 hr, the cells remaining
in the upper chamber were removed, and cells on the undersurface of
the filters were fixed with 4% paraformaldehyde (Sigma Aldrich)
followed by staining with 0.5% crystal violet (Sigma Aldrich) for
15 min. The invaded cells were visualized and counted in six randomly
selected fields under a CYTATION 5 imaging reader (BioTek).
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2.4.2
Expression of uPA, p38‐MAPK, and ERK1/2
Cellular proteins in MDA‐MB‐231 cells were isolated as described
above. uPA, p38‐mitogen‐activated protein kinase (MAPK), and
ERK1/2 proteins were detected by immunoblotting using uPA mouse
monoclonal antibody (Santa Cruz), p38‐MAPK rabbit polyclonal anti-
body (Cell Signaling Technology), and ERK1/2 rabbit monoclonal anti-
body (Cell Signaling Technology), respectively. For detection of the
phosphorylated p38‐MAPK and ERK1/2, phospho‐p38 MAPK
(Thr180/Thy182) rabbit polyclonal antibody (Cell Signaling Technol-
ogy) and phospho‐ERK1/2 rabbit monoclonal antibody (Cell Signaling
Technology), respectively, were used. Western blotting protocols were
as described above.

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SHIN ET AL.
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with cold PBS and scraped. Samples were centrifuged for 5 min at
1500× g at 4oC, and the pellet was resuspended by lysis buffer (1 M
HEPES, pH 7.9, 400 mM NaCl, 0.1 mM EDTA, 5% glycerol, 1 mM
DTT) containing protease inhibitors (Thermo Fisher) and
phenylmethylsulfonyl fluoride (Thermo Fisher). Cell lysate was
obtained by centrifugation (20 min at 16100× g) at 4°C. MMP‐2 and
MMP‐9 ELISA assay (Abcam) was performed according to the
manufacturer's instructions.
2.6
Cell migration assay (wound healing assay)
Six‐well tissue culture plates were coated with vitronectin (Sigma
Aldrich) at a concentration of 5 μg/well and incubated at 4°C over-
night. All plates were washed with medium, and cells were seeded
(1 × 106 cells/well) and incubated at 37°C for 24 hr. Confluent cells
were wounded by scraping the cell monolayer with a sterile pipette
tip. After removal of the cellular debris, the MDA‐MB‐231 cells were
exposed to EGCG derivatives at concentrations of 30 μM for 48 hr,
with the medium being replaced every 24 hr. Wound closure was
recorded and photographed using a CYTATION 5 imaging reader
(BioTek). The extent of the wound was expressed as average percent
compared with that at zero time.
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2.9
Statistical analysis
Data are expressed as means
SD of at least three independent
experiments. Tests used for nonparametric data included one‐way
analysis of variance withTukey's post hoc test, using a GraphPad soft-
ware (Prism 5). Unless otherwise indicated, p values <0.05 are consid-
ered statistically significant.
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2.7
Cell adhesion assay
Ninety‐six‐well tissue culture plates were coated with vitronectin
(Sigma Aldrich) at a concentration of 5 μg/well and incubated at 4°C
overnight. All plates were washed twice with washing buffer (0.1%
BSA in RPMI) and blocked with blocking buffer (0.5% BSA in RPMI)
at 37°C for 1 hr. Plates were again washed with washing buffer, and
cells were seeded (4 × 105 cells/well) with each compound and incu-
bated at 37°C for 48 hr. Non‐adherent cells were removed, and the
plates were washed twice with washing buffer. Adherent cells were
fixed with 4% paraformaldehyde and incubated at room temperature
for 15 min. Plates were washed with washing buffer and stained with
0.5% crystal violet for 10 min. Plates were then washed with water
and dried up completely. SDS (2%, 100 μl) was added to each well
and incubated at 37°C for 30 min. Absorbance was measured at
550 nm, using a CYTATION 5 imaging reader (BioTek).
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3
RESULTS
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3.1
Treatment of MDA‐MB‐231 cells with AcEGCG
derivative 3e results in diminished protein levels
associated with the uPA system (1): Inhibition of PAI‐1
and uPA expression
The screening assay for an inhibitor of PAI‐1 expression was initiated
by treating MDA‐MB‐231 cells with 10 μM of EGCG (1), AcEGCG (2),
or AcEGCG derivatives (3a–3h), and the PAI‐1 levels were estimated
by western blot analysis (Figure 2a). Among the series, compounds
3c, 3d, and 3e, which have an ester or a carbonate functionality at
4″‐O position showed specific PAI‐1 reducing activity. Effects of 3c,
3d, and 3e on viability of the MDA‐MB‐231 cells were then evaluated
by MTT assay, which showed concentration‐dependent cytotoxic
effects (CC50 = 36.5–53.9 μM; Figure 2b). However, compounds 3c,
3d, and 3e did not demonstrate cytotoxicity at concentrations below
30 μM (Figure 2b). At these concentrations, the viability of MDA‐
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2.8
Matrix metalloprotein (MMP) 2 and 9 ELISA
assay
MDA‐MB‐231 cells (3 × 105 cells/well) were seeded in 6‐well plates
and, 24 hr later, cells were incubated with either control or media sup-
plemented with the EGCG derivatives. After 48 hr, cells were washed
FIGURE 2 (a) Effect of 10 μM each of EGCG (1), AcEGCG (2), and EGCG derivatives (3a–3j) on the expression of PAI‐1 in MDA‐MB‐231 cells.
(b) Cytotoxicity profiles of 3c, 3d, and 3e against MDA‐MB‐231 cells. Data are the mean SD of triplicate determinations. Asterisks denote
significant difference from control condition: **p < 0.01; ***p < 0.001. EGCG: epigallocatechin gallate

SHIN ET AL.
5
MB‐231 cells was not notably affected by the EGCG‐4″‐carbonates
(3c, 3d, and 3e): Compound 3e was least toxic, and more than 90%
of the cells were found to be viable in the presence of 30 μM of this
compound (Figure 2b).
significant attenuation of the levels of phospho‐ERK, phospho‐p38
MAPK, and NF‐κB, with 3e being more effective than 2 (Figure 4).
Thus, MDA‐MB‐231 cells treated with 3e exhibited reduced
phospho‐ERK and phospho‐p38 MAPK levels by 25% and 45%,
respectively (Figure 4a,b). A similar trend was observed for NF‐κB
level in the breast cancer cells treated with 3e, which resulted in
25% reduction (Figure 4c).
Taken together, 3e was found to be the most promising com-
pound with PAI‐1 modulating activity. The EGCG derivative 3e
showed concentration‐dependent PAI‐1‐reducing activity (Figure 3a)
and, at optimal concentration of 30 μM, it reduced PAI‐1 expression
by 62% compared with the control (Figure 3b). Compound 3e was also
evaluated for its inhibitory activity against PAI‐1, but it did not inhibit
PAI‐1 activity at 30 μM (data not shown). The selected AcEGCG deriv-
ative 3e was then investigated for its effect on reducing uPA levels in
MDA‐MB‐231 cells (Figure 3c). At 30 μM, compound 3e showed 50%
reduction in uPA level, but other compounds including EGCG (1) and
AcEGCG (2) were not effective.
The effect of 3e on the expression of NF‐κB was further demon-
strated by addition of betulinic acid (BetA; 10 μM), an activator of
NF‐κB (Kasperczyk et al., 2005), to the incubation mixture (Figure 5).
Upon addition of BetA, the level of NF‐κB was shown to increase by
130% in the DMSO control (Figure 5). In the case of 3e, the reduced
NF‐κB expression was rescued to 100% of the control by BetA
(Figure 5).
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3.3
Inhibition of uPA/PAI‐1‐promoted metastatic
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3.2
Treatment of MDA‐MB‐231 cells with AcEGCG
behavior of MDA‐MB‐231 cells by 3e
derivative 3e results in diminished protein levels
associated with the uPA system (2): Reduction of
phospho‐ERK, phospho‐p38 AMPK, and NF‐κB levels
Metastasis is the cumulative result of multiple changes in tumor cells
and their microenvironment that enables cellular migration and inva-
sion into healthy host tissue. As proliferating neoplastic cells attempt
to escape the primary tumor site, local cell adhesion and invasion of
the surrounding tissue must occur (Friedl & Alexander, 2011). Effects
of the EGCG derivatives on the metastatic behavior of MDA‐MB‐
231 cells were evaluated.
The Ras‐extracellular‐signal‐regulated kinase (ERK), p38 MAPK, and
phosphatidylinositol‐3‐kinase (PI3K)/Akt/NF‐κB pathways, in part,
have been suggested to participate in invasive breast cell migration
by regulating the expression of uPA (S. Huang, New, Pan, Han, &
Nemerow, 2000; Nguyen et al., 2000; Sliva, Rizzo, & English, 2002)
and PAI‐1 (Hamaguchi, Takamura, Shimizu, & Nagai, 2003; Norata
et al., 2004; Takeda et al., 2001). Thus, the effects of the EGCG deriv-
atives on activation of ERK and p38 MAPK and expression of NF‐κB
were investigated by western blot analysis (Figure 4). EGCG (1) was
not effective in reducing phosphorylation of ERK/p38 MAPK and
expression of NF‐κB, but AcEGCG (2) and its derivative 3e showed
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3.3.1
Inhibition of cell adhesion
First, to investigate whether the EGCG derivatives affect the adhesion
of breast cancer cells, MDA‐MB‐231 cells were pretreated with EGCG
(1), AcEGCG (2), and 3e for 48 hr, and cell adhesion to the ECM
protein vitronectin was determined. The breast cancer cells treated
with EGCG (1) did not show any change in adhesion, but AcEGCG
FIGURE 3 (a) Concentration‐dependent effect of 3e on the expression of PAI‐1. (b and c) Effect of 30 μM each of EGCG (1), AcEGCG (2), and 3e
on the expression of PAI‐1 (b) and uPA (c) in MDA‐MB‐231 cells. Data are the mean SD of triplicate determinations. Asterisks denote significant
*
difference from control condition: p < 0.05; **p < 0.01; ***p < 0.001. DMSO: dimethyl sulfoxide; EGCG: epigallocatechin gallate; PAI‐1:
plasminogen activator inhibitor‐1

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SHIN ET AL.
FIGURE 4 Effects of 30 μM each of EGCG (1), AcEGCG (2), and 3e on the levels of (a) phospho‐ERK, (b) phospho‐p38 MAPK, and (c) NF‐κB in
MDA‐MB‐231 cells. Data are the mean SD of triplicate determinations. Asterisks denote significant difference from control condition: *p < 0.05;
**p < 0.01; ***p < 0.001. DMSO: dimethyl sulfoxide; EGCG: epigallocatechin gallate; ERK: extracellular‐signal‐regulated kinase; MAPK: mitogen‐
activated protein kinase; NF‐κB: nuclear factor κB
FIGURE 5 Effects of 10 μM of BetA on the
expression of NF‐κB in MDA‐MB‐231 cells in
the absence or presence of 3e. Data are the
mean SD of triplicate determinations.
Asterisks denote significant difference from
control condition: ***p < 0.001. BetA: betulinic
acid; DMSO: dimethyl sulfoxide; NF‐κB:
nuclear factor κB
(2) reduced the attachment of cells by 20% (Figure 6). The AcEGCG
derivative 3e was even more effective than AcEGCG (2), showing inhi-
bition of MDA‐MB‐231 cells' adhesion to vitronectin by 40%
(Figure 6).
effectively suppressed cell invasion (56% reduction compared with
the control; Figure 7b).
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3.3.3
Inhibition of cell migration
Cell migration is one of the characteristics of highly invasive MDA‐
MB‐231 breast cancer cells, and the effects of the EGCG derivatives
on cell migration was investigated by scratch wounding assay
(Figure 8). The migration of the breast cancer cells into the scratch
area was observed in control (DMSO), EGCG‐treated, and AcEGCG‐
treated cells after 48 hr; the migration was significantly reduced in
MDA‐MB‐231 cells treated with 3e (Figure 8a). The ratio of the area
occupied by cells in the scratch region to the total scratch area was
estimated and showed 40% reduction in cancer cell migration upon
treatment with 3e (Figure 8b).
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3.3.2
Inhibition of cell invasion
The inhibition of the proteolytic activity of MDA‐MB‐231 cells by the
EGCG derivatives was also determined by cell invasion assay. The cells
were seeded on the vitronectin‐coated filters in the presence of the
EGCG derivatives, and the number of invaded cells was counted. Nei-
ther EGCG (1) nor AcEGCG (2) was effective in inhibiting cancer cell
invasion to the vitronectin filter, but 3e showed visible reduction in
the number of MDA‐MB‐231 cells to penetrate the filter (Figure 7a).
The number of cells traversed was then counted to show that 3e

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FIGURE 6 Effects of 30 μM each of EGCG (1), AcEGCG (2), and 3e
on the adhesion of MDA‐MB‐231 cells to vitronectin. Data are the
mean SD of triplicate determinations. Asterisks denote significant
difference from control condition: ***p < 0.001. DMSO: dimethyl
sulfoxide; EGCG: epigallocatechin gallate
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3.4
Regulation of expression of matrix
metalloproteinase (MMP) 2 and 9 in MDA‐MB‐231
cells by 3e
Overexpression of MMPs has also been known to correlate closely
with tumor cell invasion and spread through degradation of basement
membranes and extracellular matrix (Parsons, Watson, Brown, Collins,
& Steele, 1997). EGCG is known to block expression of MMP‐2 and
MMP‐9 in various cancer cell lines including human breast cancer
(MCF‐7), human fibrosarcoma (HT‐1080), hepatocellular carcinoma
(SK‐Hep‐1), glioblastoma (T‐98G), and uterine leiomyosarcoma (SK‐
UT‐1) cell lines (Kim et al., 2004; Roomi, Monterrey, Kalinovsky, Rath,
& Niedzwiecki, 2010). In MDA‐MB‐231 cells, EGCG is also known to
reduce the expression levels of MMP‐9 (Sen, Dutta, & Chatterjee,
2010) and MMP‐2 (Farabegoli, Govoni, Spisni, & Apai, 2017).
FIGURE 7 Effects of 30 μM each of EGCG (1), AcEGCG (2), and 3e
on cell invasion. (a) After treatment with the EGCG derivatives,
invasion of MDA‐MB‐231 cells through vitronectin was assessed after
48 hr of incubation. (b) MDA‐MB‐231 cells inside the vitronectin filter
was counted. Data are the mean SD of triplicate determinations.
Asterisks denote significant difference from control condition:
***p < 0.001. DMSO: dimethyl sulfoxide; EGCG: epigallocatechin
gallate [Colour figure can be viewed at wileyonlinelibrary.com]
MDA‐MB‐231 cells is also related albeit partially to its regulation
of the expression of MMPs.
However, presumably, due to the differences in assay conditions,
we were not able to reproduce the previous results and, at 30 μM of
concentration, neither EGCG (1) nor AcEGCG (2) was found to mod-
ulate the expression levels of MMP‐2 (Figure 9a) and MMP‐9
(Figure 9b). On the contrary, a slight but subtle decrease (~20%) in
the amount of MMP‐2 and MMP‐9 was observed in the presence
of 3e (Figure 9a,b). Considering the fact that the uPA system acti-
vates pericellular proteolysis (Breuss & Uhrin, 2012), this result indi-
cates that the anti‐metastatic activity of 3e in the metastasis‐probe
|
4
DISCUSSIONS
Metastasis, a process whereby cancer cells spread from a primary site
to distant organs, is responsible for most cancer‐related deaths. Thera-
peutic strategies against cancer metastasis have thus been intensively
explored, and EGCG has been highlighted due to its possible cancer‐
preventive activity and inhibition of multiple signaling pathways

8
SHIN ET AL.
FIGURE 8 Effects of EGCG derivatives on
the migration of MDA‐MB‐231 cells. (a)
MDA‐MB‐231 cells were pretreated with
30 μM each of EGCG (1), AcEGCG (2), and 3e
for 48 hr, and cell motility was determined
after 24 and 48 hr of incubation. (b) Ratio of
area of the scratched region occupied by cells
to the total scratch area. Data are the
mean SD of triplicate determinations.
Asterisks denote significant difference from
control condition: ***p < 0.001. DMSO:
dimethyl sulfoxide; EGCG: epigallocatechin
gallate [Colour figure can be viewed at
wileyonlinelibrary.com]
FIGURE 9 Effects of EGCG derivatives on
the expression of (a) MMP‐2 and (b) MMP‐9
in MDA‐MB‐231 cells. Data are the
mean SD of triplicate determinations.
Asterisks denote significant difference from
control condition: ***p < 0.001. DMSO:
dimethyl sulfoxide; EGCG: epigallocatechin
gallate
involved in metastasis (Khan, Afaq, Saleem, Ahmad, & Mukhtar, 2006;
Khan & Mukhtar, 2010; Takahashi et al., 2014; Xiang et al., 2016).
EGCG has been reported to inhibit multiple proteins involved in the
invasiveness of cancer cells including vascular endothelial growth
factor (Neuhaus et al., 2004) and MMPs (Fassina et al., 2004). High pro-
tein levels of uPA and PAI‐1 are also associated with high metastasis
risk, and EGCG is known to inhibit uPA in vitro (Jankun, Selman,
Swiercz,
& Skrzypczak‐Jankun, 1997) and TNF‐α‐induced PAI‐1

SHIN ET AL.
9
production in vascular endothelial cells (Cao et al., 2013). Nevertheless,
in metastasis‐prone breast cancer cells, the anti‐metastatic activity of
EGCG in relation to inhibition or downregulation of uPA and PAI‐1
has only rarely been scrutinized (Slivova et al., 2005). Also, uPA and
PAI‐1 are gaining more and more attention as biomarkers for metasta-
sis‐prone breast cancer. Thus, uPA/PAI‐1‐targeted anti‐metastasis
activity of EGCG in breast cancer cells deserves intensive investigation.
MDA‐MB‐231 breast cancer cell lines are known to frequently and
reliably metastasize to distant organs (Iorns et al., 2012). In this study,
we evaluated inhibitory activity of EGCG and AcEGCG against uPA/
PAI‐1 and metastatic behavior of MDA‐MB‐231 cells, but they showed
only marginal effects. We reasoned that the low bioactivity of EGCG
and AcEGCG might be attributable to their low stability, and novel
EGCG derivatives (3a–3h) with various functionalities at 4″‐O position
were prepared (Figure 1). The EGCG derivatives (3a–3h) were then
evaluated for their effects on downregulation of PAI‐1 and, among
those, compounds 3c, 3d, and 3e that have an ester or a carbonate
functionality at 4″‐O position showed specific PAI‐1‐reducing activity
(Figure 3). Considering the hydrolysable nature of an ester or a carbon-
ate linkage, it might be assumed that the PAI‐1‐reducing 3c, 3d, and 3e
might have behaved as EGCG prodrugs. Particularly, the methyl‐
substituted carbonate 3e showed the most promising properties in
terms of toxicity and PAI‐1‐reducing activity, and 3e was further inves-
tigated for its potency as anti‐metastasis agent in breast cancer cells.
The EGCG derivative 3e showed concentration‐dependent PAI‐1‐
reducing activity (Figure 3a), and at optimal concentration of 30 μM,
it reduced PAI‐1 expression by 62% compared with the control
(Figure 3b). Also, at 30 μM, 3e showed 50% reduction in uPA level
(Figure 3c). To our knowledge, 3e is the first example that potently
downregulated PAI‐1 and uPA protein levels at the same time in the
metastasis‐prone MDA‐MB‐231 cells. The inhibitory activity of 3e
against expression of uPA/PAI‐1 proteins was in line with its downreg-
ulation of uPA/PAI‐1 regulators such as Ras‐ERK (25%), p38 MAPK
(45%), and PI3K/Akt/NF–κB (25%) pathways (Figure 4). The blockage
of uPA/PAI‐1 signaling pathway resulted in substantial reduction in
metastatic behavior of MDA‐MB‐231 cells, such as adhesion (40%,
Figure 6), invasion (56%, Figure 7), and migration (40%, Figure 8).
Overexpression of MMPs has also been known to correlate closely
with tumor cell invasion and spread, and EGCG is known to block
expression of MMP‐2 and MMP‐9 in various cancer cell lines. How-
ever, in MDA‐MB‐231 cells, EGCG (1), AcEGCG (2), and 3e did not
exert any beneficial effect on the expression of MMP‐2/9 (Figure 9),
which indicates that the anti‐metastatic activity of 3e in MDA‐MB‐
231 cells is not related to its regulation of the expression of MMPs.
metastatic activity in MDA‐MB‐231 cells. In addition, to tackle the
well‐known physicochemical problems of EGCG such as instability
and high polarity, several derivatives including AcEGCG and its D‐ring
4″‐substituted congeners were included in bioassay. Interestingly,
EGCG and AcEGCG showed only the marginal effects on the uPA sys-
tem and the metastatic behavior of MDA‐MB‐231 cells, but the
AcEGCG derivative 3e with a methyl‐substituted carbonate at the
4″‐position showed potent inhibition of PAI‐1 (62%) and uPA (50%)
expression. The Ras‐ERK, p38 MAPK, and PI3K/Akt/NF‐κB pathways,
which are suggested to participate in invasive breast cell migration by
regulating uPA and PAI‐1 expression, were also affected by 3e (25%,
45%, and 25% reduction, respectively). In line with these findings, sub-
stantial reduction in metastatic behavior of MDA‐MB‐231 cells, such
as adhesion (40%), invasion (56%), and migration (40%), was observed
in the presence of 3e. It is also noteworthy that, in MDA‐MB‐231
cells, EGCG (1), AcEGCG (2), and 3e did not exert any beneficial effect
on the expression of MMP‐2/9, which indicates that the anti‐metasta-
tic activity of 3e in MDA‐MB‐231 cells is not related to its regulation
of the expression of MMPs. Taken together, we have shown that the
EGCG derivative 3e could suppress the metastatic behavior of MDA‐
MB‐231 cells through regulation of uPA and PAI‐1.
ACKNOWLEDGMENTS
This research was supported by the Basic Science Research Pro-
gram through the National Research Foundation of Korea (NRF)
funded by the Ministry of Science, ICT and future Planning
(2015R1A2A1A10053461), as well as by a grant from the Priority
Research Centers Program through the National Research Founda-
tion of Korea (NRF) funded by the Ministry of Education, Science
and Technology (2009‐0093824).
CONFLICT OF INTEREST
The authors declare that they have no conflicts of interest.
ETHICAL STATEMENT
This article does not contain any studies with human participants or
animals performed by any of the authors.
ORCID
Youhoon Chong http://orcid.org/0000-0002-9683-5402
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SUPPORTING INFORMATION
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How to cite this article: Shin S, Kim MK, Jung W, Chong Y.
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