a-Tomatine inactivates PI3K/Akt and ERK signaling pathways in human

lung adenocarcinoma A549 cells: Effect on metastasis

Yuan-Wei Shih
a
, Jiunn-Min Shieh
b
, Pei-Fen Wu
c
, Yi-Chieh Lee
a
, Yi-Zhi Chen
a
, Tai-An Chiang
d,
*
a
Department of Biological Science and Technology and Graduate Institute of Biomedical Science, Chung Hwa University of Medical Technology, Tainan 717, Taiwan
b
Department of Pulmonary Medicine, Chi Mei Medical Center, No. 901, Chung-Hua Road, Yong-Kang, Tainan 710, Taiwan
c
Department of Occupational Safety and Hygiene, Tajen University, Pingtung 907, Taiwan
d
Department of Medical Technology and Graduate Institute of Biological Science and Technology, Chung Hwa University of Medical Technology, Tainan 717, Taiwan
a r t i c l e i n f o
Article history:
Received 6 January 2009
Accepted 12 May 2009
Keywords:
a-Tomatine
Invasion
Migration
PI3K/Akt
ERK
a b s t r a c t
This study rst investigates the anti-metastastic effect of a-tomatine in the human lung adenocarcinoma
cell line: A549. In this study, we rst noted a-tomatine inhibited A549 cells invasion and migration by
wound-healing assay and Boyden chamber assay. The data also showed a-tomatine could inhibit phos-
phorylation of Akt and extracellular signal-regulated kinase 1 and 2 (ERK1/2), which is involved in the
up-regulating matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) or urokinase-
type plasminogen activator (u-PA), whereas it did not affect phosphorylation of c-Jun N-terminal kinase
(JNK) and p38. Next, a-tomatine signicantly decreased the nuclear levels of nuclear factor kappa B
(NF-jB), c-Fos, and c-Jun. Also, treating A549 cells with a-tomatine also leads to a dose-dependent inhi-
bition on the binding abilities of NF-jB and activator protein-1 (AP-1). Further, the treatment of inhibitors
specic for PI3K (Wortmannin) or ERK (U0126) to A549 cells could cause reduced activities of MMP-2,
MMP-9, and u-PA. These results showed a-tomatine could inhibit the metastatic ability of A549 cells by
reducing MMP-2, MMP-9, and u-PA activities through suppressing phosphoinositide 3-kinase/Akt
(PI3K/Akt) or ERK1/2 signaling pathway and inhibition NF-jB or AP-1 binding activities. These ndings
proved a-tomatine might be an anti-metastastic agent against human lung adenocarcinoma.
Crown Copyright 2009 Published by Elsevier Ltd. All rights reserved.
1. Introduction
Lung cancer is the major cause of malignancy-related deaths
worldwide, and its incidence is rising in many countries (Greenlee
et al., 2001). About 40% of lung cancers are adenocarcinomas. Ade-
nocarcinomas belonging to the subgroup of the non-small cell lung
cancers are the most common type in US and Asia (Shivapurkar
et al., 2003). Studies have shown lung cancer cases are caused by
smoking, air pollution, environmental risk factors (for example,
exposure to radiation, asbestos, heavy mental, and polycyclic aro-
matic hydrocarbon) and oncogene (for example, slug gene) (Lee
et al., 2005; Shih et al., 2005). Most diagnosed patients with lung
adenocacinoma are in an advanced stage because of its highly met-
astatic properties, and such patients are not candidates for surgical
resection.
One of the glycoalkaloids, a-tomatine, occurs naturally in toma-
toes (Lycopersicon esculentum). Immature green tomatoes contain
up to 500 mg a-tomatine/kg fresh fruit weight. The compound is
partly degraded as the tomato ripens until at maturity levels in
red tomatoes are about 5 mg/kg fresh fruit weight (Friedman and
Levin, 1995). Fig. 1A shows a-tomatine is constructed of an aglycon
moiety (tomatidine), and a tetrasaccharide moiety (b-lycotetrose)
that contains two molecules of D-glucose and one each of D-galact-
ose and D-xylose. Previous studies demonstrated a-tomatine has
exhibited anti-proliferative and apoptotic effects on the growth
of cancer cells originating from the human colon and liver (Lee
et al., 2004). Although it was quite clear a-tomatine may inhibit
the growth of various cancers by inducing cancer cells toward
apoptosis and anti-proliferation, whereas the precise impact and
related molecular mechanism of a-tomatine on metastasis of can-
cer cells was still unclear.
Metastasis is a multistep process involving overexpression of
proteolytic enzymes, such as matrix metalloproteinases (MMPs)
and u-PA. MMP-2 and MMP-9 (also known as type IV collagenases
or gelatinases) which can degrade most ECM components that
forming the basal membrane (Bernhard et al., 1994). In addition,
u-PA may initiate the activation of an enzymatics cascade and con-
vert the zymogen plasminogen to plasmin. The activation of these
0278-6915/$ - see front matter Crown Copyright 2009 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2009.05.011
Abbreviations: MMPs, matrix metalloproteinases; u-PA, urokinase-type plas-
minogen activator; ECM, extracellular matrix; MAPK, mitogen-activated protein
kinase; ERK, extracellular signaling-regulating kinase; JNK/SAPK, c-Jun N-terminal
kinase/stress-activated protein kinase; PI3K, phosphoinositide 3-kinase; PTEN,
phosphatase and tensin homolog deleted on chromosome 10; NF-jB, nuclear factor
kappa B; AP-1, activator protein-1, IjB, Inhibitor of NF-jB.
* Corresponding author. Tel.: +886 6 2674567x450; fax: +886 6 2605598.
E-mail address: giantful@mail.hwai.edu.tw (T.-A. Chiang).
Food and Chemical Toxicology 47 (2009) 19851995
Contents lists available at ScienceDirect
Food and Chemical Toxicology
j our nal homepage: www. el sevi er . com/ l ocat e/ f oodchemt ox
enzymes enable the degradation of extracellular matrix (ECM) by
tumor cells, allowing their access to the vasculature, migration
and invasion into the target organ and development of tumor
metastasis (Duffy and Duggan, 2004; Itoh and Nagase, 2002).
As well as MMPs and u-PA, the mitogen-activated protein ki-
nases family members (MAPK) are also known to mediate metasta-
sis. The MAPK serine/threonine kinase superfamily is activated by
numerous extracellular stimuli and is involved in signal transduc-
tion cascades playing an important regulatory role in cell growth,
differentiation, apoptosis, and metastasis (Chan-Hui and Weaver,
1998). Three major mammalian MAP kinases have been described:
ERK1/2 or p44/42 MAPK, c-Jun N-terminal kinase/stress-activated
protein kinase (JNK/SAPK), and p38 MAPK. The diverse MAP kinase
members are activated in response to different extracellular stimuli
and have distinct downstream targets, thus serving different roles
in cellular responses. ERK1/2, p38 MAPK, and JNK/SAPK play a cen-
tral role in regulating the expression of MMPs and u-PA (Chen et al.,
2005; Kwon et al., 2008; Lee et al., 2008). In addition, PI3K/Akt sig-
nal transduction pathway regulates the cell metastasis of non-small
cell lung cancer (NSCLC) and is closely associated with the develop-
ment and progression of various tumors. Overexpression of PI3K
and low expression of phosphatase and tensin homolog deleted
on chromosome 10 (PTEN) are closely correlated with the develop-
ment, invasion and metastasis of NSCLC (Liao et al., 2006).
NF-jB is a multisubunit transcription factor, which is involved
in immune response, inammation and malignant transformation.
The active NF-jB consists of p50, p52, p65 (RelA), Rel B, and c-Rel.
under normal condition, NF-jB is maintained in the cytoplasm
through interactions with an inhibitor of NF-jB (IjB), but upon
dissociation, moves into the nucleus and promotes cancer cells
proliferation, angiogenesis and metastasis. AP-1 is a nuclear tran-
scription, which is involved in cell proliferation, differentiation,
apoptosis and neoplastic transformation. AP-1 consists of homodi-
mers and heterodimers of members from Fos (c-Fos, Fos B, Fra-1,
and Fra-2) and Jun (c-Jun, Jun B, and Jun D) families (Karin and
Ben-Neriah 2000; Lee et al., 2007). Previous papers have showed
the MMP-2, MMP-9, and u-PA promoters are coordinately regu-
lated by both NF-jB and AP-1 (Aguirre Ghiso et al., 1999; Ma
et al., 2004; Westermarck and Kahari, 1999). As with NF-jB and
AP-1 regulates the expression of matrix metalloproteinase and u-
PA, consistent with a role for this protein in regulating metastasis.
Further, to establish anti-metastastic mechanism of a-tomatine,
the objective of this work was to examine the inhibitory effects
and the related signaling pathways of a-tomatine on the inva-
sion/migration of human lung adenocarcinoma A549 cells in vitro.
2. Materials and methods
2.1. Chemicals and reagents
a-tomatine (purity >97%), DMSO, TrisHCl, EDTA, SDS, phenylmethylsulfonyl
uoride, bovine serum albumin (BSA), gelatin, casein, plasminogen, leupeptin, Non-
idet P-40, deoxycholic acid, sodium orthovanadate, wortmannin, and U0126 were
purchased from SigmaAldrich (St. Louis, MO); the protein assay kit was obtained
from Bio-Rad Labs. (Hercules, CA). Dulbeccos phosphate buffer solution (PBS), tryp-
sin-EDTA, and powdered Dulbeccos modied Eagles medium (DMEM) were pur-
chased from Gibco/BRL (Gaithersburg, MD). Matrigel was from BD Biosciences
(Bedford, MA). Antibody against Akt, MAPK/ERK1/2, JNK/SAPK and p38 MAPK, pro-
teins and phosphorylated proteins were purchased from Cell Signaling Tech. (Bev-
erly, MA). PI3K (p85), NF-jB (p65), c-Fos, c-Jun, b-actin, and C23 antibodies were
from BD Transduction Laboratories (San Diego, CA). The enhanced chemilumines-
cence (ECL) kit was purchased from Amersham Life Science (Amersham, UK).
2.2. Cell culture and a-tomatine treatment
A549, a human lung adenocarcinoma cell line, was obtained from BCRC (Food
Industry Research and Development Institute in Hsin-Chu, Taiwan). Cells were cul-
tured in DMEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/
ml of penicillin, 100 mg/ml streptomycin mixed antibiotics and 1 mM sodium pyru-
vate. All cell cultures were maintained at 37 C in a humidied atmosphere of 5%
CO
2
95% air. The culture medium was renewed every 23 days. Adherent cells were
detached by incubation with trypsin. For a-tomatine treatment, the stock solution
of a-tomatine was dissolved in dimethyl sulfoxide (DMSO) and sterilized by ltra-
tion through 0.2 lm disc lters. Suitable amounts of stock solution (1 mg/ml in
DMSO) of a-tomatine were added into the cultured medium to achieve the indi-
cated concentrations (Final DMSO concentration was less than 0.2%) and then incu-
bated with cells for the indicated time periods.
2.3. Analysis of cell viability (MTT assay)
To evaluate the cytotoxicity of a-tomatine, an MTT [3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl-tetrazolium bromide] assay was performed to determine cell via-
bility (Mosmann, 1983). Briey, cells were seeded at a density of 4 10
4
cells/ml in
a 24-well plate for 24 h. Then, the cells were treated with a-tomatine at various
concentrations (0, 1, 1.5, 2, 2.5, 3, and 3.5 lM) for various periods of time (24 and
48 h). Each concentration was repeated three times. After the exposure period,
the medium was removed that was followed by washing the cells with PBS. Then,
the medium was changed and incubated with MTT solution (5 mg/ml)/well for 4 h.
The medium was removed, and formazan was solubilized in isopropanol and mea-
sured spectrophotometrically at 563 nm. The percentage of viable cells was esti-
mated by comparing with untreated control cells.
2.4. Analysis of MMP-2, MMP-9 and u-PA activity (zymography assay)
The activities of MMP-2 and MMP-9 were assayed by gelatin zymography as de-
scribed previously (Chu et al., 2004). Briey, conditioned media from cells cultured
in the absence of serum for 24 h were collected. Samples were mixed with loading
buffer and electrophoresed on 8% SDSpolyacrylamide gel containing 0.1% gelatin.
Electrophoresis was performed at 140 V and 110 V for 3 h. Gels were then washed
twice in zymography washing buffer (2.5% Triton X-100 in double-distilled H
2
O) at
room temperature to remove SDS, followed by incubation at 37 C for 1216 h in
zymography reaction buffer (40 mM TrisHCl (pH 8.0), 10 mM CaCl
2
, 0.02%
NaN
3
), stained with Coomassie blue R-250 (0.125% Comassie blue R-250, 0.1% ami-
no black, 50% methanol, 10% acetic acid) for 1 h and destained with destaining solu-
tion (20% methanol, 10% acetic acid, 70% double-distilled H
2
O). Non-staining bands
representing the levels of the latent form of MMP-2 and MMP-9 were quantied by
densitometer measurement using a digital imaging analysis system.
Fig. 1. Effect of a-tomatine on the viability in A549 cells. (A) Chemical structure of
glycoalkaloid a-tomatine isolated from the leaves and fruits of tomato (Lycopersicon
esculentum). (B) Cells (4 10
4
cells/ml) were treated with various concentrations
(0, 1, 1.5, 2, 2.5, 3, and 3.5 lM) of a-tomatine for 24 and 48 h. Cell viability was
determined by MTT assay. The cell viability was directly proportional to the
production of formazan, which was measured spectrophotometrically at 563 nm.
Values are expressed as mean SD of three independent experiments. **p < 0.01,
***p < 0.001 compared with the untreated control (dose 0).
1986 Y.-W. Shih et al. / Food and Chemical Toxicology 47 (2009) 19851995
Visualization of u-PA activity was performed by casein-plasminogen zymogra-
phy. Briey, 2% casein and 20 lg/ml plasminogen were added to 8% SDSPAGE gel.
Samples with a total protein of about 20 lg were then loaded onto the gels. The
u-PA activity of cells treated or untreated with a-tomatine was measured as de-
scribed in the gelatin zymography section.
2.5. Wound-healing assay
For cell motility determination, A549 cells (1 10
5
cells/ml) were plated in six-
well tissue culture plate and grown to 8090% conuence. After aspirating the med-
ium, the centers of the cell monolayers were scraped with a sterile micropipette tip
to create a denuded zone (gap) of constant width. Subsequently, cellular debris was
washed with PBS, and A549 cells were exposed to various concentrations of a-tom-
atine (0, 1, 1.5, and 2 lM). The wound closure was monitored and photographed at
0, 12, 24, 36, and 48 h with an Olympus CKX-41 inverted microscope and an Olym-
pus E-410 camera. To quantify migrated cells, pictures of the initial wounded mon-
olayers were compared with the corresponding pictures of cells at the end of the
incubation. Articial lines tting the cutting edges were drawn on pictures of the
original wounds and overlaid on the pictures of cultures following incubation. Mi-
grated cells across the white lines were counted in six random elds from each trip-
licate treatment, and the data were presented as mean SD.
2.6. Boyden chamber invasion and migration assay
The ability of A549 cells to pass through Matrigel-coated lters was measured
by the Boyden chamber invasion assay (Ochi et al., 1993). Matrigel was diluted to
200 lg/ml with cold ltered distilled water and applied to the top side of the 8-
lm pore polycarbonate lter. Briey, A549 cells were treated with various con-
centrations of a-tomatine. After 48 h, cells were detached by trypsin and resus-
pended in serum-free medium. Medium containing 10% FBS-medium was
applied to the lower chamber as chemoattractant, and then the cells were seeded
on the upper chamber at a density of 1 10
5
cells/well in 50 ll of serum-free
medium. The chamber was incubated for 8 h at 37 C. At the end of incubation,
the cells in the upper surface of the membrane were carefully removed with a
cotton swab and cells invading across the Matrigel to the lower surface of the
membrane were xed with methanol and stained with 5% Giemsa solution. The
invasive cells on the lower surface of the membrane lter were counted with a
light microscope. The data are presented as the average number of cells attached
to the bottom surface from randomly chosen elds. Each experiment was carried
out in triplicate.
To measure the ability of A549 cells on migration, cells were seeded into a Boy-
den chamber with 8 lm pore polycarbonate lters which were not coated with
Matrigel. The migration of cells was treated with various concentrations of a-tom-
atine. The migration assay was measured as described in the invasion assay.
2.7. Preparation of whole-cell lysates and nuclear extracts
The cells were lysed with iced-cold RIPA buffer (1% NP-40, 50 mM Tris
base, 0.1% SDS, 0.5% deoxycholic acid, 150 mM NaCl, pH 7.5) and then the fol-
lowing were added phenylmethylsulfonyl uoride (10 mg/ml), leupeptin (17 mg/
ml), and sodium orthovanadate (10 mg/ml). After vortexing for 30 min on ice,
the samples were centrifuged at 12,000 g for 10 min, and then the superna-
tants were collected, denatured, and subjected to SDSPAGE and Western blot-
ting. Nuclear extracts were prepared as previously described (Hoppe-Seyler
et al., 1991) and then used for NF-jB, c-Fos, c-Jun, and AP-1 detection. Each nu-
clear pellet was resuspended in nuclear extract buffer (1.5 mM MgCl
2
, 10 mM
HEPES, pH 7.9, 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsul-
fonyl uoride, 25% glycerol, and 420 mM NaCl). The nuclear suspension was
incubated on ice for 20 min and then centrifuged at 14,000 g for 5 min. The
supernatant (corresponding to the soluble nuclear fraction) was saved, and
the remaining pellet was solubilized by sonication in PBS. The protein content
was determined with Bio-Rad protein assay reagent using bovine serum albu-
min as a standard.
2.8. Western blotting assay
To analyze the migration-related proteins, Western blotting was performed as
follows. The denatured samples (50 lg puried protein) were resolved on 1012%
SDSPAGE gels. Proteins were then transferred onto nitrocellulose membranes.
Non-specic binding of the membranes was blocked with Tris-buffered saline
(TBS) containing 1% (w/v) non-fat dry milk and 0.1% (v/v) Tween-20 (TBST) for more
than 2 h. Membranes were washed with TBST three times for 10 min and incubated
with a suitable dilution of specic primary antibodies in TBST overnight at 4 C.
Subsequently, the membranes were washed with TBST and incubated with an
appropriate secondary antibody (horseradish peroxidase-conjugated goat anti-
mouse or antirabbit IgG) for 1 h. After washing the membrane three times for
10 min in TBST, the band detection was revealed by enhanced chemiluminescence
using ECL Western blotting detection reagents and exposed ECL hyperlm in a UVP
Luminescent image analyzer.
2.9. Analysis of NF-jB and AP-1 binding assay (electrophoretic mobility shift assay)
Cell nuclear proteins were extracted with a nuclear extract buffer and measured
by an electrophoretic mobility shift assay (EMSA) (Ma et al., 2001). Cells (1 10
5
/
ml) were collected in PBS buffer (pH 7.4) and centrifuged at 2000 g for 5 min at
4 C. Cells were lysed with buffer A (10 mM HEPES, 1.5 mM MgCl
2
, 10 mM KCl,
0.5 mM DTT, and 0.5 mM PMSF (pH 7.9) containing 5% NP-40) for 10 min on ice,
and this was followed by vortexing to shear the cytoplasmic membranes. The ly-
sates were centrifuged at 2000 g for 10 min at 4 C. The pellet containing the nu-
clei was extracted with high salt buffer B (20 mM HEPES, 420 mM NaCl, 1.5 mM
MgCl
2
, 0.5 mM DTT, 0.5 mM PMSF, 0.2 mM EDTA, and 25% glycerol) for 15 min on
ice. The lysates were claried by centrifuge at 13,000 g for 10 min at 4 C. The
supernatant containing the nuclear proteins was collected and frozen at 80 C un-
til use. The protein content of nuclear fractions was determined with Bio-Rad pro-
tein assay. Five microgram aliquot of nuclear proteins were mixed with either
biotin-labeled NF-jB or AP-1 oligonucleotide probes for 15 min at room tempera-
ture or with oligonucleotides containing (sense of NF-jB, 5
0
-AGTTGAGGGGACTTTCC
CAGGC-3
0
, antisense of NF-jB, 3
0
-TCAACTCCCCTGAAAGGGTCCG-5
0
; sense of
AP-1, 5
0
-CG CTTGATGACTCAGCCGGAA-3
0
, antisense of AP-1, 3
0
-GCGAACTACT-
GAGTCGGCCTT). DNA probes were added to 10 ll binding reactions containing dou-
ble-distilled H
2
O, 5 lg nuclear protein, 1 ll poly (dI-dC), 1 ll biotin-labeled double-
stranded NF-jB or AP-1 oliginucleotides and 2 ll of 10-fold binding buffer into a
microcentrifuge tube and were incubated for 15 min at room temperature. Specic
competition binding assays were performed by adding 200-fold excess of unlabeled
probe as a specic competitor. Following protein-DNA complexes formation, sam-
ples were loaded on a 6% non-denaturing polyacrylamide gel in 0.5 TBE buffer
and were then transferred to positively charged nitrocellulose membranes (Mili-
pore, Bedford, MA) by a transfer blotting apparatus and cross-linked in a Stratagene
cross-linker. Gel shifts were visualized with streptavidin-horseradish peroxidase
followed by chemiluminescent detection.
2.10. Statistical analysis
Data were expressed as mean SD of three independent experiments and ana-
lyzed by Students t-test (Sigmaplot 2001). Signicant differences were established
at p 6 0.05.
3. Results
3.1. Cytotoxicity of a-tomatine in A549 cells
We rst assayed the cytotoxicity of a-tomatine by treating
A549 cells with a-tomatine at various concentrations (0, 1, 1.5, 2,
2.5, 3, and 3.5 lM) for 24 and 48 h followed by MTT assay. As
shown in Fig. 1B, a-tomatine showed a dose- and time-dependent
inhibitory effect on the growth of A549 cells. Compared to 0 lM
(DMSO was treated alone, data not shown), after 24 h and 48 h
treatment with a-tomatine at a concentration between 0 to 2 lM
was not signicantly altered, indicating that a-tomatine was not
toxic to A549 cells at these dosages. When cells were treated with
2.53.5 lM a-tomatine for 24 and 48 h, cell viability was signi-
cantly decreased. These results demonstrated the treatment of a-
tomatine with doses higher than 2 lM for 24 and 48 h resulted
in dose- and time-dependent loss of cell viability in A549 cells,
but doses lower than 2 lM for 24 and 48 h did not cause cytotox-
icity. In the following experiments, these doses below 2 lM of a-
tomatine were applied in all subsequent experiments.
3.2. a-Tomatine inhibits the activation of MMP-2, MMP-9, and u-PA in
A549 cells
For the cell migration and invasion processes, pointing to the
inevitable involvement of matrix-degrading proteinases, the ef-
fects of a-tomatine on MMP-2, MMP-9, and u-PA activities were
investigated by gelatin and casein zymography. The conditioned
media were collected, concentrated, and the inhibition of metasta-
sis was measured after A549 cells were treated for 24 h by a-tom-
atine. As shown in Fig. 2A, dose-dependent and markedly reduced
MMP-2 and MMP-9 activities were noted in the serum-free med-
ium treated with 2 lMa-tomatine for 24 h. Similarly, u-PA activity
was also inhibited in a dose-dependent manner by a-tomatine
treatment (Fig. 2B). These results suggested the anti-metastatic
Y.-W. Shih et al. / Food and Chemical Toxicology 47 (2009) 19851995 1987
effect of a-tomatine was related to the inhibition of the enzymat-
ically degradative processes of tumor metastasis. This study gives a
rst glimpse to demonstrate a-tomatine reduced the metastasis in
human lung adenocarcinoma cells. The activities of MMP-2, MMP-
9, and u-PA have been shown to play a critical role in degrading the
basement membrane in cancer invasion and migration.
3.3. a-Tomatine inhibits the migration and invasion in A549 cells
To investigate the inhibitory effect of a-tomatine on A549 cells
migration and invasion process, a wound-healing assay and a Boy-
den chamber assay were used. In wound-healing assay, the conu-
ent monolayer was scraped with a sterile micropipette tip to create
a scratch wound. After incubation with 1.5 and 2 lM of a-tomatine
for 24 and 48 h, the cells migrated to the denuded zone, and they
were counted. The results demonstrated a-tomatine dose-depen-
dently suppressed A549 cell migration to the denuded zone.
According to a quantitative assessment, treatment with 1.5 and
2 lM of a-tomatine inhibited 60% and 69% of cell migration after
24 h, respectively; and such doses of a-tomatine inhibited 45%
and 55% of cell migration at 48 h, respectively. Also, the cells were
treated with various concentrations of a-tomatine for 0, 12, 24, 36,
and 48 h. The results showed 2 lM of a-tomatine exhibited the
most inhibiting effect on cell motility after 48 h incubation. Espe-
cially, compared with the untreated cells, the level of A549 cells
number decreased almost 2.2-fold with the treatment of 2 lM a-
tomatine for 48 h (Fig. 3A). Although the cells were not treated
by a-tomatine, the number of cells in migration property was
increased with increasing time (Because A549 cells is under non-
cytotoxic concentrations). Also, the cells were treated with non-
a-tomatine for 24, 36 and 48 h compared with the untreated
control (dose 0), the results was presented signicantly different
and dened as constituting statistical signicance. These results
revealed that a-tomatine signicantly inhibited the motility of
A549 cells.
One important characteristic of metastasis is the migratory and
invasive ability of tumor cells. We used Boyden chamber assay to
quantify the migratory and invasive potential of A549 cells. The re-
sults showed a-tomatine induced a dose-dependent decrease in
migration with increasing concentrations of a-tomatine (Fig. 3B).
At 1.5 lM, the migration was reduced to 54.9% and at 2 lM the
migration was reduced to less than 43%. Subsequently, a-tomatine
also induced a dose-dependent decrease in invasion with increas-
ing concentrations of a-tomatine (Fig. 3C). At 1.5 lM the invasion
was reduced to 68.1% and at 2 lM the invasion was reduced to less
than 51%. The results demonstrated a-tomatine signicantly inhib-
ited the migration and invasion of A549 cells.
3.4. a-Tomatine inhibits phosphorylation of ERK and Akt
Since we have shown treatment of A549 cells with a-tomatine
inhibited the cell metastasis and activities of MMP-2, MMP-9, and
u-PA, the underlying mechanisms were further investigated. Sev-
eral studies have indicated the transcription factors (for example,
NF-kB, c-Fos, and c-Jun), JNK1/2, ERK1/2, p38 MAPK, and Akt that
are involved in activity of MMP-2, MMP-9, and u-PA on different
cell types (Chen et al., 2005; Turner et al., 2007). To assess whether
a-tomatine mediates and/or inhibits phosphorylation of JNK1/2,
ERK1/2, p38 MAPK, Akt, and the protein level of PI3K, we investi-
gated the effect of a-tomatine on the phosphorylated status of
MAPK family members (JNK1/2, ERK1/2, and p38 MAPK) and Akt
in A549 cells which were treated with various concentrations of
a-tomatine for 3 h and 2 lM of a-tomatine for various periods of
time (0, 1, 2, 3, and 6 h). Fig. 4A and B showed a-tomatine signi-
cantly inhibited the activation of ERK1 and ERK2 as shown by
decreasing the phosphorylation of ERK1 and ERK2. In contrast,
Fig. 2. Effect of a-tomatine on MMP-2/MMP-9 and u-PA activities in A549 cells. Cells were treated with various concentrations (0, 1, 1.5, and 2 lM) of a-tomatine for 24 h.
The conditioned media were collected, and then (A) MMP-2/MMP-9 and (B) u-PA activities were determined by gelatin zymography or casein zymography. MMP-2/MMP-9
and u-PA activities were quantied by densitometric analysis. The densitometric data were expressed as mean SD of three independent experiments. *p < 0.05, **p < 0.01,
***p < 0.001 compared with the untreated control (dose 0).
1988 Y.-W. Shih et al. / Food and Chemical Toxicology 47 (2009) 19851995
Fig. 3. Effect of a-tomatine on the migration and invasion in A549 cells. (A) In wound-healing assay, A549 cell monolayers were scraped by a sterile micropipette tip and the
cells were treated with various concentrations (0, 1, 1.5, and 2 lM) of a-tomatine for 0, 12, 24, 36, and 48 h. The number of cells in the denuded zone was quantitated after
indicated times (0, 12, 24, 36, and 48 h) by inverted microscopy. White lines indicate the wound edge. Pictures only were presented 24 and 48 h. Migrated cells across the
white lines were counted in six random elds from each treatment. (B) In Boyden chamber migration assay, cells were treated with various concentrations of setin for 48 h,
then cell migration were measured by Boyden chamber for 6 h with polycarbonate lters (pore size, 8 lm); (C) In Boyden chamber invasion assay, cells were treated with
various concentrations of setin for 48 h, then cell invasion were measured by Boyden chamber for 8 h; polycarbonate lters (pore size, 8 lm) were precoated with Matrigel.
Migration and invasion ability of A549 cells were quantied by counting the number of cells that invaded to the underside of the porous polycarbonate membrane under
microscopy. Values are expressed as mean SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the untreated control (dose 0);
##
p < 0.01,
###
p < 0.001 compared with the 0-h treated time.
Y.-W. Shih et al. / Food and Chemical Toxicology 47 (2009) 19851995 1989
a-tomatine did not signicantly affect phospho-JNK1/2 and phos-
pho-p38 activity (Fig. 4CF). In addition, a-tomatine inhibited
the protein level of PI3K and phosphorylation of Akt in a dose-
and time-dependent manner (Fig. 4G and H).
To further investigate whether the inhibition of a-tomatine was
mainly occurred through the inhibition of ERK1/2 or PI3K/Akt sig-
naling pathway, A549 cells were pretreated with a PI3K inhibitor
(Wortmannin; 5 or 10 lM) or ERK inhibitor (U0126; 10 or
20 lM) for 1 h and then incubated in the present or absence of
a-tomatine (1 lM) for 24 h. Results of gelatin zymography assay
has shown that a sole treatment of Wortmannin (5 or 10 lM)
and U0126 (10 or 20 lM) or a-tomatine separately reduced the
expressions of MMP-9 or MMP-2 or by 8.7%, 28.3%, 14%, 34.3%
and 12.3% or 4.7%, 14.2%, 7.3%, 30% and 3.3%, respectively, and
the combination treatment could even dramatically reduced the
secretions of MMP-9 or MMP-2 by 50.7% or 25% (5 lM Wortman-
nin + 1 lM a-tomatine), 63.3% or 40% (10 lM U0126 + 1 lM a-
tomatine) and 79.2% or 62.5% (5 lM Wortmannin + 10 lM
U0126 + 1 lM a-tomatine) (Fig. 5A). Similarly, in a casein zymog-
raphy assay, a sole treatment of Wortmannin (5 or 10 lM) and
U0126 (10 or 20 lM) or a-tomatine reduced the expression of u-
PA by 12%, 27.5%, 14.5%, 32% and 15%, respectively, and the com-
bination treatment could further reduce the secretion of u-PA by
67% (5 lM Wortmannin + 10 lM U0126 + 1 lM a-tomatine)
(Fig. 5B). The data nding revealed the inhibition of the expres-
sions of MMP-2 and MMP-9 by a-tomatine on A549 cells could
partly occur through ERK1/2 and Akt inactivation, while the inhi-
bition of the expression of u-PA could partly occur through
ERK1/2 inactivation.
3.5. a-Tomatine inhibits the DNA binding activities of NF-jB, c-Fos,
and c-Jun
NF-jB and AP-1 family of transcriptional factors have been
known to translocate to the nucleus and regulate the expression
of multiple genes involved in MMPs or u-PA secretion. To clarify
Fig. 4. Dose- and time-dependent effect of a-tomatine on the phosphorylation of ERK, JNK, p38, Akt, and the protein expression level of PI3K. In dose-dependent assay (A, C, E,
and G), A549 cells were treated with various concentrations (0, 1, 1.5, and 2 lM) of a-tomatine for 3 h. In time-dependent assay (B, D, F, and H), A549 cells were treated with
2 lM of a-tomatine for 0, 1, 2, 3, and 6 h. Activities of ERK phosphorylation, ERK, JNK phosphorylation, JNK, p38 phosphorylation, p38, Akt phosphorylation, Akt, and the
expression of PI3K were analyzed by Western blotting. b-Actin was used as a loading control. Values are expressed as mean SD of three independent experiments. *p < 0.05,
**p < 0.01, ***p < 0.001 compared with the untreated control (dose 0).
1990 Y.-W. Shih et al. / Food and Chemical Toxicology 47 (2009) 19851995
the involvement of NF-jB and AP-1 proteins in the mechanism of
a-tomatines action, the effect of a-tomatine on the DNA binding
activities of NF-jB and AP-1 in A549 cells was explored by EMSA.
As shown in Fig. 6A, A549 cells were treated with 02 lM of a-
tomatine for 24 h, and a-tomatine inhibited NF-jB and AP-1 tran-
scriptional activities in a dose-dependent manner. Especially, the
binding activities of NF-jB and AP-1 were strongly inhibited by
treating with 2 lM a-tomatine. Further, the expressions of NF-
jB, c-Fos, and c-Jun in nuclear extracts were analyzed by Western
blotting to assess the possible inhibitory effect of a-tomatine on
NF-jB, c-Fos, and c-Jun. As explained in Fig. 6B, the nuclear levels
of NF-jB, c-Fos, and c-Jun were gradually diminished by a-toma-
tine in a dose-dependent manner. Especially, data was shown to
be strongly inhibited by treating with 2 lM a-tomatine.
4. Discussion
Lung cancer is the most common neoplasm in humans in both
developed and developing countries (Erridge et al., 2007; Gajra
et al., 2003; McCracken et al., 2007). This research has conrmed
a-tomatine can inhibit the invasion and migration of A549 human
adenocarcinoma cells in vitro model. We found a-tomatine can
suppress cancer cell invasion and migration possibly occurs
through inactivation of PI3K/Akt or ERK signaling pathways, exert-
ing inhibitory effects on NF-jB, c-Fos, and c-Jun transcriptional fac-
tors, inhibiting NF-kB and AP-1 DNA binding activities, decreasing
MMP-2, MMP-9, and u-PA activities, and then having an anti-met-
astatic effect. Our results strengthen the potential of a-tomatine as
a new strategy for anti-cancer therapy.
Fig. 4. (continued)
Y.-W. Shih et al. / Food and Chemical Toxicology 47 (2009) 19851995 1991
a-Tomatine, a tomato glycoalkaloid, may also have benecial ef-
fects. Glycoalkaloids are reported to inactivate the Herpes simplex
and Herpes zoster viruses in humans (Chataing et al., 1997), to en-
hance the duration of action of anesthetics, which act by inhibiting
acetylcholinesterase (McGehee et al., 2000), and to potentiate the
immune response of vaccines in mice (Rajananthanan et al.,
2000). a-Tomatine may benet cancer chemotherapy by inhibiting
multidrug resistance in human cancer cells (Lavie et al., 2001). As
part of an effort designed to improve food safety through identica-
tion and reduction of the content of the most toxic alkaloids in plant
foods using safety evaluation. In previous study has demonstrated
a-tomatine dose not appear to be toxic when consumed orally in
moderate amount, and observation that the absence of a 5, 6-dou-
ble bond in the B-ring of tomatidine results in a much less toxic
molecule in mice (Friedman et al., 2000). Wilson et al. studied the
pharmacology and toxicology of a-tomatine. In mice, a-tomatine
appears to be non-toxic following oral consumption, presumably
because of poor absorption from the digestive tract into the blood-
stream due to formation of an insoluble complex with dietary and
endogenous cholesterol which is then eliminated in the faeces
(Cayen, 1971; Roddick, 1979). Nevertheless, further studies need
to be done in order to investigate the anti-metastatic effect of a-
tomatine in humans.
Malignant tumors invade the tissue, involving three indepen-
dent processes: the degradation of the extracellular matrix
(ECM), cell metastasis and proliferation. Metastasis has been found
to be accompanied by various physiological alterations involved in
degrading ECM, such as the overexpression of proteolytic enzyme
activity as in MMPs or u-PA, as well as the migration and invasion
of tumor cells into the bloodstream or lymphatic system to spread
to other tissues or organs (Kleiner and Stetler-Stevenson, 1999).
More specically, the ability to penetrate the basement membrane
(BM) is related to an increased potential for metastasis. Basement
membranes are thin extracellular matrices underlying cells in vivo.
Matrigel Basement Membrane Matrix is a solubilized basement
membrane preparation extracted from the Englebreth-Holm
Swarm (EHS) mouse tumor. It major component is lamin, collagen
I, entactin, heparin sulfate proteoglycan (perlecan), growth factors,
and so on. A number of methods have been developed using Matri-
gel Matrix to investigate the invasion of the basement membrane
matrix by tumor in vitro. The invasive ability of A549 cells to pass
through Matrigel-coated lters was measured by the Boyden
chamber invasion assay. So, our study demonstrated treatment
with a-tomatine at a non-cytotoxic concentration below 2 lM
for 24 h exerted an inhibitory effect in a dose- and time-dependent
manner on the migration and invasion of the highly metastatic
A549 cells. In recent years, attention has been drawn to the phys-
iological relevance of MMPs and u-PA markers related to the met-
astatic ability and malignancy of tumor cells (Chen et al., 2005;
Shih et al., 2007). Thus, many studies have shown proteinases re-
lated to degradation of matrix are required for tumor cell metasta-
sis and heightened production of MMPs and u-PA correlates with
the invasion, migration and angiogenesis of the tumors (Mackay
et al., 1990). To further explore the exact expression of a-toma-
tine-induced inhibition on migration and invasion, we performed
gelatin or casein-plasminogen zymographic assays, to detect activ-
ities of MMP-2, MMP-9, and u-PA. The result showed a-tomatine
noticeably downregulated the activities of MMP-2, MMP-9, and
u-PA. These results suggested the anti-metastastic effect of
a-tomatine was associated with the inhibition of the enzymatically
degradative processes of tumor metastasis. One important charac-
teristic of metastasis is the migratory and invasive ability of tumor
cells. Further, we used wound-healing assay and Boyden chamber
assay to quantify the migratory potential of A549 cells. The results
demonstated a-tomatine signicantly inhibited the migration and
invasion of A549 cells.
A major mechanism through which signals from extracellular
stimuli are transmitted to the nucleus involves the activation of ki-
nases. These kinases, serine/threonine kinases related to the mito-
gen-activated proteins kinase (MAPK) superfamily, mediate signals
from cell membrane receptors triggered by growth factors, cyto-
kines, and cell-matrix interactions. MAPKs are intricately involved
in the expression of the components involved in MMPs or u-PA
promoters induction by NF-kB, AP-1, and its association with c-
Fos and c-Jun. At least three subgroups of MAPK family members
have been implicated: extracellular signal-regulated kinases
(ERKs), c-Jun N-terminal kinase/stress-activated protein kinase
(JNK/SAPK), and p38MAPK (Robinson and Cobb, 1997). Also, the
PI3K and Akt signal pathways also play a critical role in MMP-9
gene regulation (Yao et al., 2001).
Fig. 5. Effect of PI3K inhibitor (Wortmannin), ERK inhibitor (U0126), and a-
tomatine on the activities of MMP-2, MMP-9, and u-PA. Cells were plated in six-well
and pre-treated with Wortmannin (5 or 10 lM) or U0126 (10 or 20 lM) for 1 h and
then incubated in the presence or absence of a-tomatine (1 lM) for 24 h.
Afterwards, the culture medium was subjected to gelatin and casein zymography
to analyze the activities of (A) MMP-2/MMP-9 and (B) u-PA. Determined activities
of these proteins were subsequently quantied by densitometric analysis with that
of control being 100% as shown just below the gel data. Data represented the
mean SD of three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001).
1992 Y.-W. Shih et al. / Food and Chemical Toxicology 47 (2009) 19851995
Indeed, we have demonstrated treatment of a-tomatine inhib-
ited phosphorylation of ERK1/2 and Akt. In contrast, a-tomatine
did not signicantly affect phospho-ERK1/2 and phospho-p38
activities. The involvement of the ERK and PI3K/Akt pathways
was further supported by using the ERK and PI3K inhibitors in
our experimental model. Treatment with an inhibitors specic
for ERK could inhibit the MMP-2, MMP-9, and u-PA secretion. Also,
the PI3K/Akt signaling pathway also played a crucial role in MMP-
2, MMP-9, and u-PA gene regulation, cell survival and tumor cell
metastasis (Kim et al., 2001; Rao, 2003; Westermarck and Kahari,
Fig. 6. Effects of a-tomatine on the DNA binding activities of NF-jB and AP-1 in A549 cells. Cells were treated with various concentrations (0, 1, 1.5, and 2 lM) of a-tomatine
for 24 h. Cell nuclear extracts were prepared and analyzed for (A) NF-jB and AP-1 DNA binding activities using biotin-labeled consensus NF-jB and AP-1 specic
oligonucleotide, then EMSA assay were performed as described in materials and methods. Lane 1: nuclear extracts incubated with 100-fold excess unlabeled consensus
oligonucleotide (comp.) to conrm the specicity of binding. Excess free probe is indicated at the bottom. (B) Nuclear extracts were also analyzed by Western blotting with
anti-NF-jB (p65), c-Fos, and c-Jun antibodies. C23 was a nucleus protein loading control. Determined the protein expressions of NF-jB, c-Fos, and c-Jun were subsequently
quantied by densitometric analysis with that of control being onefold. The densitometric results are expressed as mean SD of three independent experiments. *p < 0.05,
**p < 0.01, ***p < 0.001 compared with the untreated control.
Y.-W. Shih et al. / Food and Chemical Toxicology 47 (2009) 19851995 1993
1999). Our ndings suggested a-tomatine-caused decreased MMP-
2 and MMP-9 activities in the culture media could possibly occur
through suppression of phospho-ERK1/2 or phospho-Akt, while
the reduction in u-PA activity may only be associated with a sup-
pression of phospho-ERK1/2. Here, it was demonstrated a-toma-
tine clearly decreased MMP-2, MMP-9, and u-PA activities
through inactivating the PI3K/Akt or ERK signaling pathways, and
such inhibitory effect on proteinases expression may contribute
to the capability of a-tomatine for the inhibition of cell metastasis.
In this study, the activity of u-PA, an upstream activating en-
zyme of MMP-2 and MMP-9 involved in invasion and migration,
was also shown to be inhibited in a dose-dependent manner by
a-tomatine treatment. The transcription of MMPs and u-PA gene
is regulated by upstream regulatory sequences, including NF-jB,
AP-1, and Ets-1 binding sites (Nagase and Woessner, 1999; Roth-
hammer et al., 2004; Sliva, 2004; Westermarck and Kahari,
1999). Therefore, this study provides insight into how a-tomatine
suppresses the ERK1/2 and Akt signaling pathways and reduces
NF-kB and AP-1 transcriptional activities in A549 lung adenocarci-
noma cells. Indeed, one or more of these binding sites have been
implicated in mediating the effects of a diverse set of agents. Here,
we have also found the treatment of a-tomatine to A549 cells re-
sults in an inhibition of NF-kB and AP-1 DNA binding activities,
which was accompanied by the inhibition of nuclear translocation
of these factors. Thus, the inhibitory effect of a-tomatine on the
migration and invasion of non-small lung cancer cells probably oc-
curs by abating the expressions of NF-kB, c-Fos, and c-Jun, then
reducing the activities of MMP-2, MMP-9, and u-PA.
In addition, Chishma et al. (1997) indicated the tumor host-or-
gan chimeric histoculture system developed in the present study
with GFP uorescing tumor cells has signicantly advanced the
ability to understand and treat human metastatic cancer. The re-
sults provide an invaluable new tool for understanding the most
important steps in tumor host-organ interaction, tumor progres-
sion, and metastasis. Because the metastatic colony expansion
takes place in vitro, it offers a unique opportunity for developing
agents for intervention. We will carry out this histoculture exper-
iment in the further.
Finally, the involvement of ERK and PI3K/Akt signaling path-
ways in cell metastasis were further supported by experiments
with ERK and PI3K/Akt inhibitors, showing treatment with inhibi-
tors of ERK and PI3K/Akt to A549 cells inhibited the cell metastasis.
In conclusion, these results imply the therapeutic potential of a-
tomatine for controlling tumor metastasis based on the observa-
tion of its inhibitory effect on migration and invasion of adenocar-
cinoma cancer cell line A549 cells. This study suggested a-
tomatine may serve as an efcient anti-metastastic drug in cancer
treatment.
Conict of interest statement
The authors declare that there are no conicts of interest.
Acknowledgment
This work was supported by the grant from the Center for Re-
gional Industry Academia Collaboration M.O.E, Ministry of Educa-
tion (96-B-17-042, 97-B-17-018).
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