Kalopanaxsaponin A inhibits PMA-induced invasion by reducing matrix metalloproteinase-9 via PI3K/Akt- and PKCdelta-mediated signaling in MCF-7 human breast cancer cells.

Abstract

Induction of matrix metalloproteinase (MMP)-9 is particularly important for the invasiveness of breast cancers. We investigated the inhibitory effect of kalopanaxsaponin A (KPS-A) on cell invasion and MMP-9 activation in phorbol 12-myristate 13-acetate (PMA)-treated MCF-7 human breast cancer cells. KPS-A inhibited PMA-induced cell proliferation and invasion. PMA-induced cell invasion was blocked in the presence of a primary antibody of MMP-9, and KPS-A suppressed the increased expression and/or secretion of MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1. Using specific inhibitors, we confirmed that PMA-induced cell invasion and MMP-9 expression is primarily regulated by nuclear factor-kappa B (NF-kappaB) activation via phosphatidylinositol 3-kinase (PI3K)/Akt and activator protein-1 (AP-1) activation via extracellular signal-regulated kinase (ERK)1/2. KPS-A decreased PMA-induced transcriptional activation of NF-kappaB and AP-1 and inhibited PMA-induced phosphorylation of ERK1/2 and Akt. Treatment with the protein kinase C (PKC)delta inhibitor rottlerin caused a marked decrease in PMA-induced MMP-9 secretion and cell invasion, as well as ERK/AP-1 activation, and KPS-A reduced PMA-induced membrane localization of PKCdelta. Furthermore, oral administration of KPS-A led to a substantial decrease in tumor volume and expression of proliferating cell nuclear antigen, MMP-9, TIMP-1 and PKCdelta in mice with MCF-7 breast cancer xenografts in the presence of 17beta-estradiol. These results suggest that KPS-A inhibits PMA-induced invasion by reducing MMP-9 activation, mainly via the PI3K/Akt/NF-kappaB and PKCdelta/ERK/AP-1 pathways in MCF-7 cells and blocks tumor growth and MMP-9-mediated invasiveness in mice with breast carcinoma. Therefore, KPS-A may be a promising anti-invasive agent with the advantage of oral dosing.

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Carcinogenesis vol.30 no.7 pp.1225–1233, 2009
doi:10.1093/carcin/bgp111
Advance Access publication May 6, 2009
Kalopanaxsaponin A inhibits PMA-induced invasion by reducing matrix
metalloproteinase-9 via PI3K/Akt- and PKCd-mediated signaling in MCF-7
human breast cancer cells
Sun Kyu Park1,2,y, Young Sun Hwang3,y, Kwang-Kyun
Park1,2,3, Hee-Juhn Park4, Jeong Yeon Seo3and
Won-Yoon Chung1,2,3,?
1Department of Applied Life Science, The Graduate School, Yonsei
University,2Department of Oral Biology, Brain Korea 21 project and
3Oral Cancer Research Institute, Yonsei University College of Dentistry, 134
Shinchon-dong, Seodaemun-gu, Seoul 120-752, Korea and4Departments of
Botanical Resources, Sangji University, Wonju 220-702, Korea
?To whom correspondence should be addressed. Tel: þ82 2 2228 3057;
Fax: þ82 2 364 7113;
Email: wychung@yuhs.ac
Induction of matrix metalloproteinase (MMP)-9 is particularly
important for the invasiveness of breast cancers. We investigated
the inhibitory effect of kalopanaxsaponin A (KPS-A) on cell in-
vasion and MMP-9 activation in phorbol 12-myristate 13-acetate
(PMA)-treated MCF-7 human breast cancer cells. KPS-A in-
hibited PMA-induced cell proliferation and invasion. PMA-
induced cell invasion was blocked in the presence of a primary
antibody of MMP-9, and KPS-A suppressed the increased expres-
sion and/or secretion of MMP-9 and tissue inhibitor of metallo-
proteinase (TIMP)-1. Using specific inhibitors, we confirmed that
PMA-induced cell invasion and MMP-9 expression is primarily
regulated by nuclear factor-kappa B (NF-kB) activation via phos-
phatidylinositol 3-kinase (PI3K)/Akt and activator protein-1
(AP-1) activation via extracellular signal-regulated kinase
(ERK)1/2. KPS-A decreased PMA-induced transcriptional acti-
vation of NF-kB and AP-1 and inhibited PMA-induced phosphor-
ylation of ERK1/2 and Akt. Treatment with the protein kinase
C (PKC)d inhibitor rottlerin caused a marked decrease in
PMA-induced MMP-9 secretion and cell invasion, as well as
ERK/AP-1 activation, and KPS-A reduced PMA-induced mem-
brane localization of PKCd. Furthermore, oral administration of
KPS-A led to a substantial decrease in tumor volume and expres-
sion of proliferating cell nuclear antigen, MMP-9, TIMP-1 and
PKCd in mice with MCF-7 breast cancer xenografts in the pres-
ence of 17b-estradiol. These results suggest that KPS-A inhibits
PMA-induced invasion by reducing MMP-9 activation, mainly via
the PI3K/Akt/NF-kB and PKCd/ERK/AP-1 pathways in MCF-7
cells and blocks tumor growth and MMP-9-mediated invasiveness
in mice with breast carcinoma. Therefore, KPS-A may be a prom-
ising anti-invasive agent with the advantage of oral dosing.
Introduction
Breast cancer is the malignancy that is most frequently diagnosed in
women, and its incidence is rapidly increasing in all industrialized
countries. Because of local invasion and metastasis, neither radiation
therapy nor chemotherapy substantially increases the length or quality
of life of patients with advanced breast cancer. Therefore, the control
ofinvasion and metastasis is an important therapeutic strategy, and the
development of effective anti-invasive agents for breast cancer would
be very probably to improve treatment.
Metastasis is one of the major causes of mortality in cancer patients
and occurs as a complex multistep process involving cancer cell ad-
hesion, invasion and migration. Although a number of proteinases are
involved in tissue lysis, the secretion of matrix metalloproteinases
(MMPs) is crucial in cancer cell metastasis, because MMPs are re-
sponsible for the degradation of environmental barriers, such as the
extracellular matrix and basement membrane (1,2). Among human
MMPs, MMP-2 (also known as gelatinase-A and 72-kDa type IV
collagenase) and MMP-9 (also known as gelatinase-B and 92-kDa
type IV collagenase) have been correlated with the malignancy of
various tumors and with poor survival in patients with breast cancer
(1,2). MMP-2 is commonly constitutively present in tissues, and it is
maximally expressed in malignant neoplasms as part of the host re-
sponse to the presence of neoplastic cells, rather than as part of an
initial response to invasion (3). In contrast, synthesis and secretion of
MMP-9 can be stimulated by a variety of growth factors and inflam-
matory cytokines during pathological processes and by agents such as
phorbol 12-myristate 13-acetate (PMA) (4–7). It has been reported
that induction of MMP-9 is particularly important for the invasiveness
ofhuman cancers, includingbreast cancer (8–12), therebyblockade of
MMP-9-mediated invasion suppresses the metastasis of breast cancer
cells into other organs (13,14).
The activity of MMP-9 in various tumor cells is tightly controlled,
with regulation occurring primarily at the transcription level (15). The
promoter of human MMP-9 contains cis-acting regulatory elements
that bind with transcription factors such as nuclear factor-kappa B
(NF-jB; ?600 bp) or activator protein-1 (AP-1; ?533 bp and ?79 bp)
(16,17). NF-jB and AP-1 are ubiquitous eukaryotic transcription fac-
tors and can be induced by multiple stimuli. NF-jB, a heterodimer of
p50 and p65, is sequestered in the cytoplasm due to its association
with the inhibitory protein IjBa under normal conditions. Stimulation
by inflammatory cytokines or tumor promoters leads to the disso-
ciation of IjBa from NF-jB through the ubiquitin/proteasome-
dependent pathway following its phosphorylation. The released
NF-jB translocates into the nucleus and binds to the promoter region
of MMP-9, leading to gene expression (18). On the other hand, AP-1
is a nuclear transcription factor comprising homodimers and hetero-
dimers of members of the Fos and Jun families (19). NF-jB- and/or
AP-1-dependent MMP-9 expression is regulatedby mitogen-activated
protein kinases (MAPKs) and by the phosphatidylinositol 3-kinase
(PI3K)/Akt pathway, depending on the cell type and on the type of
stimuli (10,16,20–22). Therefore, these upstream molecules that reg-
ulate MMP-9 expression or enzymatic activity can be used as a target
for treating breast cancer metastasis. Recent studies to detect new
anti-metastatic agents have demonstrated that plant-derived com-
pounds with chemopreventive potential inhibit the invasiveness of
several types of cancer by modifying MMP-9 expression (16,23–26).
Kalopanaxsaponin A (KPS-A; 3-O-[L-rhamnopyranosyl-(1/2)-a-
L-arabinopyranosyl]-hederagenin) is an oleanane triterpene saponin
found in Kalopanax pictus Nakai (Araliaceae), a plant that has been
traditionally used for the treatment of rheumatoidal arthritis and di-
abetes mellitus in East Asian countries (27,28). KPS-A displays cyto-
toxicity in J82, T24, Colon 26 and 3LL cancer cells (29), significantly
reduces LL/2 tumor growth in mice and apparently prolongs the life
span of mice with Colon 26 and 3LL Lewis lung carcinomas (27).
In this study, we investigated the inhibitory effect of KPS-A on cell
invasion and MMP-9 activation in PMA-treated MCF-7 human breast
cancer cells. KPS-A reduced MMP-9 expression by blocking the acti-
vation of NF-jB and AP-1 transcription factors via PI3K/Akt- and
Abbreviations: AP-1, activator protein-1; DMEM, Dulbecco’s modified Ea-
gle’s medium; ERK, extracellular signal-regulated kinase; FBS, fetal bovine
serum; JNK, c-jun N-terminal kinase; KPS-A, kalopanaxsaponin A; MAPK,
mitogen-activated protein kinase; MMP, matrix metalloproteinase; NF-jB,
nuclear factor-kappa B; PBS, phosphate-buffered saline; PCNA, proliferating
cell nuclear antigen; PI3K, phosphatidylinositol 3-kinase; PKC, protein
kinase C; PMA, phorbol 12-myristate 13-acetate; TIMP, tissue inhibitor of
metalloproteinase.
yThese authors contributed equally to this work.
? The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org 1225

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proteinkinaseC(PKC)d-mediatedextracellularsignal-regulatedkinase
(ERK)1/2 signaling. In a mouse xenograft model of MCF-7 cells, KPS-
A suppressed tumor growth and the expression of invasive biomarkers.
Materials and methods
Materials
KPS-A was generously provided by Professor Hee-Juhn Park (Sangji Univer-
sity, Wonju, Korea) (Figure 1A) and was dissolved in dimethyl sulfoxide,
followed by dilution with culture medium. Dulbecco’s modified Eagle’s
medium/F12(DMEM/F12,1:1),fetalbovineserum(FBS),antibiotic–antimycotic
(10 000 U/ml penicillin G sodium, 10 000 lg/ml streptomycin sulfate and
25 lg/ml amphotericin B), phosphate-buffered saline (PBS) and 0.25% trypsin–
ethylenediaminetetraacetic acid were purchased from Gibco BRL (Rockville,
MD). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and di-
methyl sulfoxide were purchased from Sigma–Aldrich (St Louis, MO).
PD98059, SB203580, SP600125, LY294002, GF109203X, Go ¨6976, rottlerin
and PMA were purchased from Calbiochem (La Jolla, CA). The following
antibodies were purchased from their respective sources: p38 and phospho-
p38 MAPKs (New England Biolabs, Beverly, MA); total/phospho form of Akt,
ERK1/2, c-jun N-terminal kinase (JNK) and IjBa (Cell Signaling Technology,
Denver, MA); MMP-9, c-Fos, c-Jun, p50, p65, PKCa, PKCb1, PKCd, tissue
inhibitor of metalloproteinase (TIMP)-1 and Lamin A/C (Santa Cruz Biotech-
nology, CA); b-actin (Sigma–Aldrich); proliferating cell nuclear antigen
(PCNA) (DAKO Diagnostics, Ontario, Canada); horseradish peroxidase-
conjugated secondary antibodies (Amersham Life Science, Little Chalfont, UK)
and biotinylated anti-mouse/anti-rabbit immunoglobulin G (H þ L) and horserad-
ish peroxidase-conjugated streptavidin (Vector Laboratories, Burlingame, CA).
Cell culture
The estrogen receptor (ER)-positive human breast cancer cell line MCF-7 was
obtained from the American Type Culture Collection (Rockville, MD). Cells
were maintained in DMEM:F12 (1:1) supplemented with 10% FBS and 1%
antibiotic–antimycotic at 37?C in a humidified atmosphere of 5% CO2.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay
MCF-7 cells (5 ? 103cells/well) were plated into a 96-well culture plate with
10% FBS–DMEM and left overnight to adhere. The cells were cultured in
serum-free medium containing 0–10 lg/ml KPS-A in the absence or presence
of 0.5 lM PMA for 24 and 48 h, respectively. The cells were then incubated
with a 5 mg/ml 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-
mide solution for an additional 4 h at 37?C. The formazan product was dis-
solved with200 l1 dimethylsulfoxide. Absorbancewasmeasured at570 nm in
a microplate reader (Bio-Rad, Hercules, CA).
5-Bromo-2#-deoxyuridine incorporation assay
MCF-7 cells (5 ? 103cells/well) were seeded onto a 96-well culture plate and
incubated overnight at 37?C. The cells were treated with serum-free medium
containing 0–10 lg/ml KPS-A and 0.5 lM PMA for 24 h, followed by labeling
with 10 lM 5-Bromo-2#-deoxyuridine for an additional 4 h. The cells were
fixed with 200 l1 FixDenat solution (Roche Diagnostics, Mannheim,
Germany) for 30 min at ?20?C. DNA synthesis in proliferating cells was
measured using the 5-Bromo-2#-deoxyuridine labeling and detection Kit III
(Roche Diagnostics) according to the manufacturer’s protocol. Absorbance
was measured at 450 nm using a microplate reader.
Invasion assay
MCF-7 cells were cultured in 10% FBS–DMEM:F12 (1:1) with 10 lCi/ml
[3H]-thymidine for 24 h. The 8 lm pore size polycarbonate nucleopore filter
inserts in a 24-well transwell chamber (Corning Costar, Cambridge, MA) were
coated with 30 lg/well Matrigel (Becton Dickinson, Lincoln Park, NJ). The
[3H]thymidine-labeled cells (5 ? 104cells) were seeded into the upper part of
the Matrigel-coated filter, and serum-free DMEM/F12 with 0.5 lM PMAwas
added to the lower part with 0–10 lg/ml KPS-A, 1 lg/ml anti-MMP-9 anti-
body, 50 lM PD98059, 20 lM SB203580, 50 lM LY294002, 40 lM
SP600125, 2 lM GF109203X, 2 lM Go ¨6976 or 0.5 lM rottlerin for 48 h.
Fig. 1. KPS-AsuppressesPMA-induced proliferationandinvasionofMCF-7 cells.(A) Chemicalstructureof KPS-A.(B) MCF-7cells were treated with 0–10lg/
ml KPS-A in the absence or presence of 0.5 lM PMA for 24 and 48 h, and cell viability was measured using an 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide assay;#P , 0.05,?P , 0.05,??P , 0.01 versus vehicle alone-treated cells,‡P , 0.05,‡‡P , 0.01 versus PMA alone-treated cells. (C)
Cells were treated with KPS-A and/or PMA for 24 h, and the amount of the newly synthesized DNA in proliferating cells was measured by a 5-Bromo-2#-
deoxyuridine (BrdU) incorporation assay;#P , 0.01 versus vehicle alone-treated cells,?P , 0.01,??P , 0.001 versus PMA alone-treated cells. (D)
[3H]thymidine-labeled MCF-7 cells were treated with or without PMA and KPS-A for 48 h. Invasion activities were determined by a Matrigel-coated in vitro
invasionassay and expressedas changesin invasionrelativeto control;#P , 0.001versus vehicle alone-treatedcells,?P , 0.01,??P , 0.001versus PMAalone-
treated cells. Data represent the mean ± SE of three independent experiments.
S.K.Park et al.
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The radioactivity of the cells that invaded through the Matrigel into the lower
chamber was counted using a LS6500 liquid scintillation counter with liquid
scintillation cocktail (Beckman Coulter, Fullerton, CA). Results are expressed
as the changes in invasion relative to control.
Western blot analysis
To examine the expression and/or activation of MMP-9, TIMP-1, MAPKs and
Akt, cells were pretreated with KPS-A for 2 h or PKC inhibitors for 1 h and then
stimulated with 0.5lM PMAfor 24 h(MMP-9and TIMP-1) or 20min(MAPKs
and Akt). Total lysates were prepared as described previously (6). Cytoplasmic
and nuclear fractions to examine the activation of transcription factors were
obtained from cells treated with PMA for 1 h (NF-jB) or 2 h (AP-1) after 2 h
pretreatment with KPS-A (30). In addition, cytosolic and membrane fractions to
identify the localization of PKC isotypes were also prepared with cells treated
with PMA for the indicated time, orcells pretreatedwith KPS-Afor 2 h and then
stimulated with PMA for 15 min (20). Equal amounts of protein (50 lg) were
separated on 10% sodium dodecyl sulfate–polyacrylamide gels. The proteins
were transferred onto a polyvinylidene difluoride membrane (Millipore, Bill-
erica, MA). The membrane was blocked with 5% skim milk in PBS-containing
0.1% Tween-20 and then incubated with each primary antibody (1:1000) against
specific proteins in 5% skim milk overnight at 4?C. The blots were incubated
with a 1:3000 dilution of the respective horseradish peroxidase-conjugated
secondary antibodies for 2 h at room temperature and were again washed with
PBS-containing 0.1% Tween-20. The targeted proteins were visualized with an
enhanced chemiluminescence detection kit (Amersham Life Science) according
to the protocol of the manufacturer.
Gelatin zymography
MCF-7 cells (1 ? 105cells) were incubated in serum-free medium including
0.5 lM PMA for 24 h, following pretreatment with 0–10 lg/ml KPS-A for 2 h,
or specific inhibitors of MAPKs (PD98059, SB203580 and SP600125), PI3K
(LY294002) and PKC isotypes (GF109203X, Go ¨6976 and rottlerin) for 1 h,
respectively. The conditioned medium was collected and protein concentration
was determined by the Bradford method (Bio-Rad). Equal amounts of protein
(10 lg) were separated on 8% sodium dodecyl sulfate–polyacrylamide gels-
containing 0.1% (wt/vol) gelatin. The gel was washed with 2.5% Triton X-100
for 30 min at room temperature and then incubated in a buffer containing
10 mM CaCl2, 0.01% NaN3and 50 mM Tris–HCl (pH 7.5) for 16 h at
37?C. The gel was stained with 0.2% Coomassie Brilliant Blue and photo-
graphed on a light box. MMP-9 gelatinolytic activity was detected as clear
bands in a dark blue background.
Electrophoretic mobility shift assays
Electrophoretic mobility shift assay was performed using a DNA–protein bind-
ing detection kit (Promega, Madison, WI) according to the manufacturer’s
protocol. MCF-7 cells were pretreated with 5 lg/ml KPS-A for 2 h, or specific
inhibitors of MAPKs (PD98059,SB203580 and SP600125),PI3K (LY294002)
and PKC (GF109203X, Go ¨6976 and rottlerin) for 1 h, followed by treatment
with 0.5 lM PMA for 1 h or 2 h to determine DNA-binding activity of NF-jB
and AP-1, respectively. Nuclear extract was prepared as described previously
(30). Double-stranded oligonucleotides containing the NF-jB (5#-AGTT-
GAGGGGACTTTCCCAGGC-3#) or AP-1 (5#-CGCTTGATGAGTCAGC-
CGGAA-3#)consensus sequences
[c-32P]adenosine triphosphate (3000 Ci/mmol) using T4 polynucleotide kinase
and purified with a NICK column (Amersham Pharmacia Biotechnology, Pis-
cataway, NJ). The eluted solution was used as probes for electrophoretic mo-
bility shift assay. Nuclear extracts (10 lg) were incubated with the binding
buffer [10 mM Tris–HCl (pH 7.5), 100 mM NaCl, 1 mM dithiothreitol, 1 mM
ethylenediaminetetraacetic acid, 4% (vol/vol) glycerol and 1 lg/ll poly dI-dC]
for 10 min at room temperature, then treated with 0.5 pmol-labeled probe for
20 min. The DNA–protein complex was separated on a 6% polyacrylamide gel
in 0.5? Tris–Borate–ethylenediaminetetraacetic acid buffer at 10 V for 3 h.
Gels were dried and exposed to Kodak-XAR film.
were endlabeled withKlenow
Transient transfection and luciferase reporter assays
TranscriptionalactivitiesofNF-jBandAP-1weremeasuredbyluciferasereporter
assayusingthepNF-jB-LucandpAP-1-Lucreporterplasmid(Clontech,PaloAlto,
CA).MCF-7cells(1 ? 105cells/well) were seeded into6-well plates.The cellsat
70–80% confluence were cotransfected with 1 lg of NF-jB or AP-1 reporter
constructs and 0.5 lg pSV-b-galactosidase for 8 h in serum- and antibiotics-free
Opti-MEM (Gibco BRL) with Lipofectamine 2000 reagent (Invitrogen, Carlsbad,
CA). The transfected cells were pretreated with KPS-A at the indicated concen-
trations for 2 h and then incubated with 0.5 lM PMA for 8 h. Luciferase and
b-galactosidase activities were assayed according to the manufacturer’s protocol
(Promega), using a microplate spectrofluorometer (Molecular Devices, Palo Alto,
CA). Luciferase activity was normalized by b-galactosidase activity in cell lysate
and expressed as an average of three independent experiments.
Xenograft assays in nude mice
Female Balb/C athymic nude mice (5-week-old mice; Central Lab Animal,
Seoul, Korea) were maintained at 20–22?C on a 12 h light–dark cycle. Animal
studies were performed in accordance with experimental protocols that were
approved by the animal ethics committee of Yonsei University College of
Dentistry (Seoul, Korea). MCF-7 cells (1 ? 107cells/0.2 ml PBS) cultured
in 10% FBS–DMEM/F12 were subcutaneously injected into the right flanks
of the mice. To stimulate the growth of MCF-7 cells, a 17b-estradiol pellet
(Innovative Research of America, Sarasota, FL) was implanted into the intra-
capular region before the injection of MCF-7 cells. Two days later, the mice
were administered three times/week with KPS-A in 0.1 ml PBS (5 and 10
mg/kg body wt) by oral gavage for 23 days, and the control group received
PBS alone. Tumor volume was measured with a digital electric caliper and
calculated by the following formula: (width in mm)2? (length in mm)/2. The
mice were killed under anesthesia. The tumors were collected for immunohis-
tochemical studies.
Immunohistochemical analysis
The collected MCF-7 xenograft tumors were fixed with 4% paraformaldehyde
solution for 24 h and then embedded in paraffin. Serial tissue sections (4 lm
thick) were prepared and mounted onto slides. The sections were deparaffinized
inxylene,rehydratedinalcoholandtreatedwith3%(vol/vol)H2O2for10minat
room temperature to suppress endogenous peroxidase activity. The antigen re-
trieval was performed by microwave treatment of the sections for 10 min in 10
mMsodium citrate buffer (pH6.0).After rinsing three timeswithPBS for 5min,
the sections were incubated with 10% normal goat serum for 20 min at room
temperature toblocknon-specificbindingsitesandreactedovernightat4?Cwith
a primary antibody against PCNA, MMP-9, TIMP-1 and PKCd at a dilution of
1:100 in PBS. The sections were rinsed with PBS and incubated with biotiny-
lated anti-mouse/anti-rabbit immunoglobulin G (H þ L) (1:100 dilution in 1%
bovine serum albumin) at room temperature for 30 min, followed by exposure to
horseradishperoxidase-conjugatedstreptavidin(1:200dilutionin1%bovineserum
albumin) at room temperature for 30 min. The sections were reacted with 0.02%
3,3#-diaminobenzidine as chromogen and then counterstained with hematoxylin.
Statistical analysis
Statistical analysis was performed using InStat statistical software (GraphPad
Software, San Diego, CA). The results are presented as mean ± SE. The
statistical significance of differences between groups was analyzed via re-
peated measures of one-way analysis of variance followed by Student’s t-test.
A P value ,0.05 was considered to be significant.
Results
KPS-A inhibits PMA-induced proliferation and invasion of MCF-7
cells
We first determined the effect of KPS-A on PMA-induced prolifera-
tion of MCF-7 cells. PMA treatment for 24 and 48 h significantly
increased the viability of MCF-7 cells, but KPS-A at .6 lg/ml re-
duced cell viability in the absence or presence of PMA (Figure 1B).
When the amount of the newly synthesized DNA was quantified by
measuring the incorporated 5-bromo-2#-deoxyuridine in MCF-7 cells
treated with PMA and KPS-A for 24 h, KPS-A dose dependently
inhibited PMA-induced DNA synthesis (Figure 1C). Next, we exam-
ined whether KPS-A could inhibit PMA-induced invasion in MCF-7
cells. PMA caused a 3.6-fold increase in the invasion of MCF-7 cells,
but PMA-induced cell invasion was inhibited by KPS-A in a dose-
dependent manner (Figure 1D). Moreover, KPS-A significantly in-
hibited PMA-induced cell invasion at non-cytotoxic doses of 6 lg/
ml or less.
KPS-A inhibits PMA-induced invasion of MCF-7 cells by reducing
MMP-9 activation and TIMP-1 expression
To confirm whether MMP-9 activity is involved in increased PMA-
induced invasion of MCF-7 cells, the cells were treated with PMA
and/or a primary antibody of MMP-9. Addition of an MMP-9 primary
antibody significantly blocked PMA-induced cell invasion (Figure
2A). We next studied the effect of KPS-A on MMP-9 expression in
cells and its activity in the conditioned media. Western blot analysis
and gelatin zymography revealed that the basal level of MMP-9 in
MCF-7 cells was low but that its protein expression and secretion
were markedly induced by PMA treatment. KPS-A inhibited
KPS-A inhibits PMA-induced invasion in MCF-7 cells
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increased MMP-9 expression (Figure 2B) and secretion (Figure 2C) in
PMA-treated MCF-7 cells. In addition, PMA-induced TIMP-1 ex-
pression was also blocked by KPS-A treatment (Figure 2B). These
results indicate that KPS-A alleviates cell invasion by reducing MMP-
9 expression and secretion as well as TIMP-1 expression in PMA-
treated MCF-7 cells.
PMA increases cell invasion and MMP-9 activity through PI3K/Akt-
and ERK-mediated activation of transcription factors in MCF-7 cells
To explore signaling pathways regulating the invasiveness of PMA-
treated MCF-7 cells, cells were treated with PD98059, SB203580,
SP600125 and LY294002, which are specific inhibitors of ERK1/2,
p38 MAPK, JNK and PI3K, respectively. PMA-stimulated cell inva-
sion was suppressed by all the inhibitors but was significantly in-
hibited by the ERK1/2 and PI3K inhibitors (Figure 3A). PMA-
induced MMP-9 secretion (Figure 3B) and DNA binding of NF-jB
(Figure 3C) were also inhibited by MAPK inhibitors and the PI3K
inhibitor, but DNA binding of AP-1 was abolished only by the ERK1/
2 inhibitor. These results indicate that PMA stimulates cell invasion
and MMP-9 secretion by modulating PI3K/Akt-mediated NF-jB ac-
tivation and ERK-mediated AP-1 activation.
KPS-A inhibits PMA-induced transcriptional activity of MMP-9 and
phosphorylation of ERK1/2 and Akt in MCF-7 cells
To determinewhether MMP-9 expressionwas regulated at a transcrip-
tional level by KPS-A, we performed a promoter assay using
transiently transfected MCF-7 cells with a luciferase reporter gene
linked to the MMP-9 promoter sequence, including NF-jB and
Fig. 3. PMA enhances cell invasion and MMP-9 secretion via PI3K/Akt/NF-jB and ERK1/2/AP-1 pathways in MCF-7 cells. (A) [3H]thymidine-labeled MCF-7
cells were seeded into the upper part of a Matrigel-coated filter, and MAPK [PD98059 (PD, 50 lM), SB203580 (SB, 20 lM) and SP600125 (SP, 40 lM)] or PI3K
[LY294002 (LY, 50 lM)] inhibitors were added to the lower chamber of the Transwell apparatus in the presence of PMA for 48 h. Invasion activities were
determined by radioactivity of the invaded cells and expressed as changes in invasion relative to control conditions. Data represent the mean ± SE of three
independent experiments.?P , 0.01 versus PMA-treated cells. (B) Cells were treated with specific inhibitors of MAPKs or PI3K for 1 h and then stimulated with
PMA for 24 h. Gelatine zymography was performed with the conditioned medium to detect MMP-9 activity. (C) Cells were pretreated with specific inhibitors of
MAPKs or PI3K for 1 h and then stimulated with PMA for 1 and 2 h. Electrophoretic mobility shift assay was performed as described in Materials and Methods;
NS, non-specific.
Fig. 2. KPS-A inhibits PMA-induced MMP-9 activation and TIMP-1 expression in MCF-7 cells. (A) [3H]thymidine-labeled MCF-7 cells were incubated in
Matrigel-coated transwellswith or withoutPMA and anti-MMP-9polyclonalantibody for48 h. Invasionactivities were determinedby radioactivity of the invaded
cells and expressed as changes in invasion relative to control conditions. Data represent the mean ± SE of three independent experiments;?P , 0.01 versus PMA
alone-treated cells. (B) Cells were pretreated with KPS-A for 2 h and then stimulated with PMA for 24 h. Western blot analysis was performed with MMP-9 and
TIMP-1-specific antibodies. (C) Cells were pretreated with KPS-A for 2h followed by PMA stimulation for 24 h. MMP-9 enzyme activity in the conditioned
medium was analyzed by gelatin zymography.
S.K.Park et al.
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AP-1-binding site motifs. NF-jB-dependent luciferase activity was
increased 13.5-fold and AP-1-dependent luciferase activity was
increased 34-fold in PMA-stimulated MCF-7 cells, compared with
unstimulated cells. KPS-A treatment decreased the NF-jB- and AP-
1-dependent luciferase activities induced by PMA in a dose-related
manner (Figure 4A). To further evidence the inhibitory effect of KPS-
A on the transcriptional activity of MMP-9, we examined the effect of
KPS-A on the DNA-binding activity of NF-jB and AP-1 by electro-
phoretic mobility shift assay. The DNA-binding activity of NF-jB
was maximally induced at 1 h and returned to control levels 4 h after
PMA stimulation, whereas AP-1 DNA-binding activity was signifi-
cantly enhanced at 2 h and remained so for 8 h (data not shown).
Building upon these results, when MCF-7 cells were treated with
PMA for 1 h or 2 h after pretreatment with KPS-A for 2 h, PMA-
induced increases in the DNA-binding activity of NF-jB and AP-1
were substantially inhibited by KPS-A (Figure 4B). In western blot
analysis, PMA stimulated the phosphorylation of IjBa in cytoplasm
and, thereby, the nuclear translocation of NF-jB subunits p50
and p65. In the case of AP-1, c-Jun expression was considerably
augmented, but c-Fos expression was only negligibly induced in
PMA-treated cells. However, KPS-A reduced the cytoplasmic level
of phospho-IjBa and the nuclear levels of NF-jB subunits and c-Jun
(Figure 4C). Moreover, KPS-A inhibited the phosphorylation of
ERK1/2 and Akt, but not p38 MAPK or JNK, in PMA-treated
MCF-7 cells (Figure 4D). Our data suggest that KPS-A inhibits the
activation of NF-jB and AP-1 by blocking Akt and ERK1/2 activation
in PMA-treated MCF-7 cells.
KPS-A inhibits memebrane localization of PMA-induced PKCd
To determinewhether PMA causes the activation of any PKC isotypes
in MCF-7 cells, we analyzed the levels of PKCa, PKCb1 and PKCd in
cytosol and membrane fractions. PMA stimulated translocation from
the cytosol to the membrane of PKCd only, after 10 min of stimula-
tion, although PKCa and PKCb1 isotypes were also expressed in
MCF-7 cells (Figure 5A). Furthermore, to confirm whether PKCd,
but not PKCa and PKCb1, is involved in PMA-induced MMP-9 ac-
tivation and cell invasion, MCF-7 cells were exposed to PMA together
with PKC inhibitors. PMA-induced MMP-9 secretion and cell inva-
sionwereblockedbyaspecificPKCdinhibitorrottlerinandbyabroad
inhibitor of PKC GF109203X, but not by a specific PKCa and PKCb1
inhibitor Go ¨6976 (Figure 5B). Interestingly, rottlerin and GF109203X
inhibited PMA-induced phosphorylation of ERK1/2 (Figure 5C) and
DNA-binding activity of AP-1, not NK-jB (Figure 5D). PMA-
induced membrane localization of PKCd was blocked by pretreatment
with KPS-A for 2 h (Figure 5E). These results indicate that PMA
mainly stimulates MMP-9-mediated cell invasion through PKCd-
triggered ERK/AP-1 activation in MCF-7 cells, and KPS-A sup-
presses PMA-induced PKCd activation.
Fig. 4. KPS-A inhibits PMA-induced transcriptional activation of MMP-9 and phosphorylation of ERK1/2 and Akt in MCF-7 cells. (A) Cells were transfected
with pTAL-Luc, pNF-jB-Luc or pAP-1-Luc. The transfected cells were treated with KPS-A for 2 h and then PMA for 8 h. Data for the NF-jB- and AP-1-
dependent luciferase activity were normalized to b-galactosidase activity from cotransfection of pRSV b-galactosidase. The data represent the mean ± SE of three
independent experiments;#P , 0.001 versus unstimulated cells,?P , 0.01 versus PMA alone-treated cells. (B) Cells were pretreated with KPS-A for 2 h and
then stimulated with PMA for 1 h for NF-jB or 2 h for AP-1. Prepared nuclear extract was reacted with radioactive oligonucleotides containing the NF-jB or the
AP-1 motif of the MMP-9 promoter. Reacted bound complexes were separated by 6% non-denaturating polyacrylamide electrophoresis; NS, non-specific. (C)
Cells were pretreated withKPS-Afor 2 h followedby PMA stimulation for 1 or 2 h. Western blottingwas performedto determine the nuclear levelsof NF-jB (p50
and p65) and AP-1 (c-Fos and c-Jun) subunits as well as the cytoplasmic levels of NF-jB subunits, IjBa and pIjBa. (D) Cells were incubated with KPS-A for 2 h
followed by PMA stimulation for 20 min, and the levels of total/phospho MAPKs and Akt were determined by western blotting.
KPS-A inhibits PMA-induced invasion in MCF-7 cells
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KPS-A inhibits breast cancer growth and MMP-9 expression in
athymic nude mice
To verify in vitro anti-invasive activity of KPS-A, we investigated its
inhibitory effect on tumorgrowth and MMP-9 expression in micewith
MCF-7 breast cancer xenografts. Tumor volume increased markedly
for 23 days in mice inoculated with MCF-7 cells in the presence of
17b-estradiol pellets, but orally administered KPS-A resulted in a sig-
nificant dose-related inhibition of tumor growth (Figure 6A). Further-
more, immunohistochemical analysis indicated that while the
expression of PCNA, MMP-9, TIMP-1 and PKCd in the tumors
was noticeably higher in MCF-7 cell-injected mice, the expression
of these proteins was inhibited by orally administered KPS-A (Figure
6B). Therefore, KPS-A can suppress MMP-9-mediated invasion of
breast cancer in mice.
Discussion
The current study was designed to estimate the anti-invasive potential
of KPS-A and to explore the molecular mechanisms underlying its
activity. We first evaluated the inhibitory effect of KPS-A on PMA-
induced invasion in MCF-7 cells. PMA is a well-known inflammatory
stimulus and tumor promoter that activates almost all PKC isozymes
by direct binding. It causes dramatic PKC-mediated induction of in-
vasiveness in ER-positive MCF-7 human breast cancer cells, which
are usually weakly invasive (31). Our data show that KPS-A inhibited
PMA-induced cell proliferation and invasion. As described in previ-
ous studies (16,26,32), treatment with PMA ranging from 10 nM to 1
lM stimulated MMP-9 secretion in a dose-related manner (data not
shown). We confirmed that PMA-induced cell invasion was blocked
in the presence of a primary antibody of MMP-9, evidence that MMP-
9 plays a central role in the PMA-induced invasion of MCF-7 cells. In
PMA-treated MCF-7 cells, KPS-A suppressed the increased expres-
sion and secretion of MMP-9. In addition, PMA-induced TIMP-1
expression was inhibited by KPS-A. Although TIMPs were thought
to inhibit tumor growth and MMP-induced proteolysis of the sur-
rounding matrix, a growing body of evidence suggests that TIMP-1
is a multifunctional protein that inhibits apoptosis in breast epithelial
cells and breast carcinoma cells and therefore may promote tumor
growth and development (33,34).
PMA increases the invasiveness of various types of cancer cells by
activating MMP-9 via transcription factors and the PKC, MAPKs and
PI3K/Akt pathways (20,25,31). However, the distinct mechanisms
regulating PMA-induced MMP-9 expression in different cell types
are not defined clearly. To gain a comprehensive understanding of
the PMA-induced signaling cascade underlying MMP-9 expression
in MCF-7 human breast cancer cells, we assessed the effects of spe-
cific inhibitors of three MAPKs and PI3K on PMA-induced invasion,
MMP-9 activity in the conditioned medium and DNA binding of
Fig. 5. KPS-A inhibits PMA-induced PKCd activation. (A) MCF-7 cells were treated with PMA, and the levels of PKC isotypes were analyzed in cytosol and
membrane fractions by western blotting. (B, top) Cells were pretreated for 1 h with GF109203X (GF, 2 lM), Go ¨6976 (Go ¨, 2 lM) or rottlerin (Rot, 0.5 lM)
followed by PMA stimulation for 24 h. The MMP-9 activity in the conditioned media was analyzed by gelatin zymography; bottom, [3H]thymidine-labeled MCF-
7 cells were incubated in serum-free media with PKC inhibitors and PMA for 48 h. Invasion activities were measured using the Matrigel-coated transwell assay.
Data represent the mean ± SE of three independent experiments;?P , 0.01 versus PMA alone-treated cells. (C) Cells were stimulated with PMA for 20 min after
pretreatment with PKC inhibitors for 1 h, and the levels of total/phospho MAPKs and Akt were determined by western blotting. (D) Cells were pretreated with
specific PKC inhibitors for 1 h and then stimulated with PMA for 1 and 2 h. Electrophoretic mobility shift assay was performed as described in Materials and
Methods; NS, non-specific. (E) Cells were pretreated with KPS-A for 2 h and then stimulated with PMA for 15 min. Western blot analysis was performed to detect
the PKCd level in cytosol and membrane fractions.
S.K.Park et al.
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transcription factors. PMA-induced invasion was more significantly
inhibited by treatment with an ERK1/2 inhibitor (PD98059) or
a PI3K/Akt inhibitor (LY294002), than it was with a p38 MAPK
inhibitor (SB203580) or a JNK inhibitor (SP600125). PMA-induced
MMP-9 secretion and DNA binding of NF-jB was inhibited to some
extent by all of the MAPK inhibitors and was completely inhibited by
the PI3K/Akt inhibitors, whereas DNAbinding ofAP-1 was abolished
only by an ERK1/2 inhibitor. These results indicate that cell invasion
and MMP-9 expression is mainly regulated by NF-jB activation via
PI3K/Akt and by AP-1 activation via ERK1/2, although p38 MAPK
and JNK may also partially contribute to PMA-induced cell invasion
and MMP-9 expression by activating NF-jB.
Fig. 6. KPS-A inhibits the growth and invasiveness of MCF-7 breast cancer in nude mice. (A) MCF-7 cells were single injected into the right flank of each female
Balb/C nude mouse with a 17b-estradiol (E2) pellet (n 5 5). Oral administration of KPS-Awas carried out three times a week for 23 days and tumor volume was
calculated. (B) Immunohistochemical analysis for PCNA, MMP-9, TIMP-1 and PKCd was performed on tumor tissue sections from cancer cell alone-injected
mice and cancer cell-injected mice with oral dosing of KPS-A [5 and 10 mg/kg body wt (BW)], using specific antibodies. Original magnification was ?200.
(C) Molecular mechanisms underlying anti-invasive activity of KPS-A in PMA-treated MCF-7 cells.
KPS-A inhibits PMA-induced invasion in MCF-7 cells
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We found that KPS-A decreased PMA-induced transcriptional and
DNA-binding activity of NF-jB and AP-1. KPS-A inhibited the level
of phosphorylated IjBa in the cytoplasm and the translocation of NF-
jB subunits p50 and p65 to the nucleus in PMA-treated MCF-7 cells.
KPS-A also suppressed PMA-induced expression of c-Jun, rather than
c-Fos, both of which are members of the AP-1. In our further study to
test the effect of KPS-A on MAPKs and Akt activation in PMA-
treated MCF-7 cells, KPS-A suppressed PMA-induced phosphoryla-
tion of ERK and Akt, key pathways in PMA-induced cell invasion via
MMP-9 expression. These results demonstrate that KPS-A reduces
MMP-9 expression by blocking NF-jB activation via PI3K/Akt as
well as AP-1 activation via ERK1/2 and consequently inhibits
MMP-9-mediated cell invasion in MCF-7 human breast cancer cells.
Activation of PKC by PMA involves the translocation of PKC
isoforms to the plasma membrane, causing proliferation, differentia-
tion, malignant transformation and cell death in cancer cells. PMA
activates classical (a, bI, bII and c) and novel (d, e, g and h) PKCs by
binding to the C1domain of these isoforms (35). PKCa leads toPMA-
stimulated growth of MCF-7 cells through ERK and JNK signaling
(36), and impairment of PKCa potentiates heregulin-induced apopto-
sis in SKBr3 breast cancer cells (37). However, recent studies
demonstrated that PKCd, not PKCa, plays a critical role in MMP-9
induction in MCF-7 cells. PKCd mediates PMA-induced MMP-9
secretion through the Ras/Raf/MEK/ERK pathway (6) and platelet-
induced invasion and MMP-9 secretion (38). PKCd protects against
tumor necrosis factor-related apoptosis-inducing ligand-mediated ap-
optosis in breast cancer cells (39). Therefore, PKC isoforms are prom-
ising targets for the prevention and treatment of breast cancer. In this
study, PMA stimulation resulted in the translocation of PKCd from
the cytosol to the cell membrane, whereas translocation of PKCa and
PKCb1 was not observed. Treatment with a non-cytotoxic dose of
a PKCd inhibitor (rottlerin) and a broad PKC inhibitor (GF109203X),
but not a PKCa and PKCb1 inhibitor (Go ¨6976), caused marked in-
hibition in PMA-induced activation of ERK1/2 and AP-1, as well as
PMA-induced MMP-9 secretion and cell invasion. These data indicate
that PMA-activated PKCd mediates MMP-9 expression and cell
invasion via ERK1/2 and AP-1. As expected, KPS-A reduced PMA-
induced membrane localization of PKCd.
To confirm invitro anti-invasive activity of KPS-A, we estimated its
inhibitory effect on tumor growth and MMP-9 expression in MCF-7
breast cancer xenografts of mice. 17b-Estradiol pellets were
implanted into mice because tumor growth was not observed in the
absence of 17b-estradiol pellets (data not shown). Like PMA, 17b-
estradiol activates PKCd by direct binding in ER-positive breast can-
cer cells (40–43). Oral administration of KPS-A led to a substantial
inhibition of tumor growth in a dose-related manner. PCNA, MMP-9,
TIMP-1 and PKCd expression was inhibited in the tumor tissues of
mice orally administered with KPS-A. These results indicate that
KPS-A suppresses the breast cancer growth and MMP-9-mediated
invasiveness in mice.
In conclusion, KPS-A inhibited PMA-induced invasion by reducing
MMP-9 activation mainly through the PI3K/Akt/NF-jB and PKCd/
ERK/AP-1 pathways in MCF-7 human breast cancer cells (Figure
6C). Oral administration of KPS-A suppressed tumor growth and
MMP-9-mediated invasiveness in mice implanted with MCF-7 cells
in the presence of 17b-estradiol. It is possible that targeting the sig-
naling molecules that regulate MMPs expression might be a more
effective means of therapeutically inhibiting MMPs. Therefore,
KPS-A is a promising anti-invasive agent; in addition to reducing
MMP-9, it primarily targets the signaling molecules PKCd and
PI3K/Akt that regulate MMP-9 and has the added advantage of oral
dosing.
Funding
Korea Research Foundation, Korean Government (KRF-2005-005-
J05902); BioGreen 21 Program, Rural Development Administration,
Republic of Korea (20050401-034-695-183-02-00).
Acknowledgements
Conflict of Interest Statement: None declared.
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Received November 19, 2008; revised April 4, 2009; accepted May 2, 2009
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