Hesperidin inhibited acetaldehyde-induced matrix metalloproteinase-9 gene expression in human hepatocellular carcinoma cells
Previous studies have revealed that acetaldehyde-induced cell invasion and matrix metalloproteinase-9 (MMP-9) activation and are directly involved in hepatic tumorigenesis and metastasis. Acetaldehyde is an important substance for tumor regression. We designed this study to aid in the development of powerful anti-cancer drugs with specific tumor regression and anti-metastatic potentials. Optimal drugs should possess both specific MMP-9 enzyme and gene transcriptional activities at the molecular level. Hesperidin, a flavonoid present in fruits and vegetables, possess anti-inflammatory and chemopreventive activities. Hesperidin suppressed acetaldehyde-induced cell invasion and inhibited the secreted and cytosolic MMP-9 forms in HepG2 cells with acetaldehyde. Hesperidin suppressed acetaldehyde-induced MMP-9 expression through the inhibition of nuclear factor-kappaB (NF-kappaB) and AP-1, and suppressed acetaldehyde-stimulated NF-kappaB translocation into the nucleus through IkappaB inhibitory signaling pathways. Hesperidin also inhibited acetaldehyde-induced AP-1 activity by the inhibitory phosphorylation of p38 kinase and c-Jun N-terminal kinase (JNK) signaling pathways. Results from our study revealed that hesperidin suppressed both acetaldehyde-activated NF-kappaB and activator protein 1 (AP-1) activity by IkappaB, JNK, and p38 signaling pathways. This resulted in the reduction of MMP-9 expression, secretion, and hepatocarcinoma cellular invasion. Our result confirmed the therapeutic potential of hesperidin an anti-metastatic and its involvement in the acetaldehyde-induced cell invasiveness of hepatocellular carcinoma in alcoholic patients.
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Toxicology Letters 184 (2009) 204–210
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/toxlet
Hesperidin inhibited acetaldehyde-induced matrix metalloproteinase-9 gene
expression in human hepatocellular carcinoma cells
, Shung-Te Kao
, Che-Ming Hung
, Ching-Ju Liu
, Chia-Chou Yeh
School of Medicine, Department of Medicine, Tzu Chi University, Hualien, Taiwan
Department of Chinese Medicine, Buddhist Dalin Tzu Chi General Hospital, Chia-Yi, Taiwan
School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, Taiwan
Division of Chinese Medicine, China Medical University Hospital, Taichung, Taiwan
Animal Industry Division, Livestock Research Institute, Council of Agriculture, Tainan, Taiwan
Hsin-Chu Branch Station, Council of Agriculture-TLI, Hsin-Chu, Taiwan
Received 26 September 20 08
Received in revised form
16 November 2008
Accepted 17 November 2008
Available online 6 December 2008
Activator protein 1
Previous studies have revealed that acetaldehyde-induced cell invasion and matrix metalloproteinase-9
(MMP-9) activation and are directly involved in hepatic tumorigenesis and metastasis. Acetaldehyde is an
important substance for tumor regression. We designed this study to aid in the development of powerful
anti-cancer drugs with speciﬁc tumor regression and anti-metastatic potentials. Optimal drugs should
possess both speciﬁc MMP-9 enzyme and gene transcriptional activities at the molecular level.Hesperidin,
a ﬂavonoid present in fruits and vegetables, possess anti-inﬂammatory and chemopreventive activi-
ties. Hesperidin suppressed acetaldehyde-induced cell invasion and inhibited the secreted and cytosolic
MMP-9 forms in HepG2 cells with acetaldehyde. Hesperidin suppressed acetaldehyde-induced MMP-9
expression through the inhibition of nuclear factor-B (NF-B) and AP-1, and suppressed acetaldehyde-
stimulated NF-B translocation into the nucleus through IB inhibitory signaling pathways. Hesperidin
also inhibited acetaldehyde-induced AP-1 activity by the inhibitory phosphorylation of p38 kinase and
c-Jun N-terminal kinase (JNK) signaling pathways.
Results from our study revealed that hesperidin suppressed both acetaldehyde-activated NF-B and
activator protein 1 (AP-1) activity by IB, JNK, and p38 signaling pathways. This resulted in the reduction
of MMP-9 expression, secretion, and hepatocarcinoma cellular invasion. Our result conﬁrmed the ther-
apeutic potential of hesperidin an anti-metastatic and its involvement in the acetaldehyde-induced cell
invasiveness of hepatocellular carcinoma in alcoholic patients.
Crown Copyright © 2008 Published by Elsevier Ireland Ltd. All rights reserved.
Hepatocellular carcinoma is the ﬁfth most common cancer in
the world-wide (Bosch et al., 2004; Donato et al., 2006). Hepatocar-
cinogenesis is a multi-step process with a multifactorial etiology.
Epidemiological studies suggests that alcohol consumption is an
causative factor for HCC in several countries (Morgan et al., 2004;
Pöoschl and Seitz, 2004; Boffetta and Hashibe, 2006). Consumption
of alcohol increases the prevalence of HCC, extracapsular inva-
sion and intrahepatic metastasis (Kubo et al., 1997). Ethanol and
its metabolites are directly injurious to the liver (Lieber, 1990).
Corresponding author at: Department of Chinese Medicine, Buddhist Dalin Tzu
Chi General Hospital, Chia-Yi, Taiwan 2 Min-Sheng Road, Dalin Town, Chia-Yi 62247,
Taiwan. Tel.: +886 5 2648000 8713; fax: +886 5 2648006.
E-mail address: firstname.lastname@example.org (C.-C. Yeh).
These authors contributed equally to the study.
Acetaldehyde, a product of the oxidative metabolism of ethanol,
is a very reactive intermediate that has been suggested to have
a pathogenic role in ALD and its generation within the liver cor-
relates with cell injury (Lieber, 1994). Acetaldehyde is a primary
ethanol metabolite and may be directly involved in the carcinogenic
effects of liver (IARC, 1999). Acetaldehyde treatment produced of
inﬂammatory cytokines and the expression of NF-B and AP-1 at
10–175 M(Gutierrez-Ruiz et al., 2001; Gómez-Quiroz et al., 2005;
Hsiang et al., 2007). Both in vivo and in vitro studies have revealed
that acetaldehyde forms covalent adducts with DNA and proteins,
it leads to the alteration of liver structure and function (Vaca et
al., 1998; Rintala et al., 2002). Acetaldehyde may also inﬂuence
metastatic toxicity in HepG2 cells (Hsiang et al., 2007).
Tumor invasion and metastasis requires increased matrix met-
alloproteinase (MMP) expression (Stamenkovic, 2000). The MMP
family is involved in the degradation of extracellular membrane
and MMPs are also associated with both malignancy and metasta-
sis. The MMP-9 gene is strongly expressed in invasive HCC (Arii et al.,
0378-4274/$ – see front matter. Crown Copyright © 2008 Published by Elsevier Ireland Ltd. All rights reserved.
Author's personal copy
M.-H. Yeh et al. / Toxicology Letters 184 (2009) 204–210 205
1996), and MMP-9 content in HCC is higher than that of surrounding
liver parenchyma. MMP-9 may be used as an important marker of
the invasiveness and metastatic potential of HCC (Arii et al., 1996).
The expression of MMP-9 may also serve as novel HCC markers
since these levels may be reﬂective of vascular invasion (Nelson et
al., 2000). MMP-9 activity is tightly controlled and regulation pri-
marily occurs at the transcription level (Stamenkovic, 2000). The
MMP-9 promoter is highly conserved and contains multiple func-
tional elements including nuclear factor-B (NF-B) and activator
protein 1 (AP-1) elements (Sato and Seiki, 1993). We have demon-
strated that acetaldehyde activated NF-B activity and increased
MMP-9 expression by NF-B or AP-1 activity induction and was
previously associated with tumor metastasis (Hsiang et al., 2007).
Acetaldehyde activated both NF-B and AP-1 activity through IB
and p38 signaling pathways, which induced MMP-9 expression and
resulting cell invasion.
Hesperidin (30,5,9-dihydroxy-40-methoxy-7-orutinosyl ﬂa-
vanone, HES) is a naturally occurring ﬂavonoid present in fruits
and vegetables (Justesen et al., 1998; Nielsen et al., 2002). Dietary
hesperidin exerts anti-carcinogenic actions in the tongue, colon,
esophagus, and urinary bladder in rat carcinogenesis models
(Tanaka et al., 1997b, 2000; Yang et al., 1997). These effects occur
both alone and in combination with diosmin. Conversely, other
studies have suggested that ﬂavonoids such as hesperidin possess
anti-inﬂammatory activities (Meloni et al., 1995; Yeh et al., 2007),
and chemopreventive activities of hesperidin are correlated.
Hesperidin may down-regulate MMP expression in response to
nicotine in rats ( Balakrishnan and Menon, 2007), although there is
minimal information on hesperidin’s inﬂuence on acetaldehyde-
inducing MMP-9 expression. We therefore investigated the
inhibitory effects of hesperidine on acetaldehyde-induced MMP-9
expression in HepG2 cells.
2. Materials and methods
2.1. Cell culture, transfection, and acetaldehyde treatment
HepG2 cells were maintained in Dulbecco’s modiﬁed Eagle medium (DMEM)
(Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum
(FBS) (HyClone, Logan, UT). HepG2 cells were transiently transfected with 5 gof
plasmid DNA using the SuperFect
transfection reagent (Qiagen, Valencia, CA), and
weretreated with acetaldehyde. Acetaldehyde (Sigma, St. Louis, MO) was prepared in
phosphate-buffered saline (137 mM NaCl, 1.4 mM KH
, 4.3 mM Na
, 2.7 mM
KCl, pH 7.2). HepG2 cells were cultured in 25-cm
ﬂasks at 37
C. Cells were treated
with 100 M acetaldehyde in DMEM after 24 h incubation. Flasks were immediately
capped and sealed with paraﬁlm to control evaporation.
2.2. Invasion assay
Cell invasion was measured with Matrigel-coated ﬁlm inserts (8 m pore
size) which were ﬁt into 24-well invasion chambers (Becton-Dickinson Bioscience,
Franklin Lakes, NJ). HepG2 cells (5 × 10
) were resuspended in 200 l DMEM and
were added to the upper compartment of the invasion chamber in the presence or
absence of 100 M acetaldehyde. DMEM (500 l) was added to the lower invasion
chamber compartment. Matrigel invasion chambers were incubated at 37
. The ﬁlter inserts were removed from the wells after 24 h incubation, and cells
on the upper sides of ﬁlters were removed with cotton swabs. Cells in the lower
surfaces of the ﬁlter were stained and cell numbers were counted microscopically.
Final values were calculated as the average of total cell numbers from three ﬁlters.
2.3. Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA (1 g) was reverse transcribed using an oligo(dT)15 primer and
SuperScript TMIII (Invitrogen, Carlsbad, CA) in a total volume of 20 l with
2 l of reverse transcription mixture to measure MMP-9 and glyceraldehyde-
3-phosphate dehydrogenase (GAPDH) mRNAs. PCR ampliﬁcation was performed
with Taq polymerase (Promega, Madison, WI) for 36 cycles at 92
45 s, 55
C for 45 s, and 72
C for 2 min. PCR primers for MMP-9 included
) and antisense (5
). GAPDH ampliﬁcation also used sense (5
) and antisense (5
) primers. The
intensity of band on the gel was calculated by Gel-Pro
Analyzer (Media Cybernetics,
Inc., Silver Spring, MD).
2.4. Construction of NF-B and AP-1 promoter/reporter plasmids
Wild-type sequences were present for NF-B (GGAATTCCCC) and AP-1
(TGAGTCA) sites. Reporter plasmids pNF-B-Luc and pAP-1-Luc were purchased
from Stratagene (La Jolla, CA). Plasmid DNAs were prepared with the Qiagen plasmid
midi kit (Qiagen, Valencia, CA).
2.5. Luciferase assay
HepG2 cells were treated with 100 M acetaldehyde for 8 h, and luciferase activ-
ity was determined as previously described (Hsiang et al., 2005). Relative luciferase
activity was calculated by dividing the relative luciferase unit (RLU) of NF-BorAP-1
reporter plasmid-transfected cells by the RLU of pGL3-basic-transfected cells.
2.6. Biotinylated electrophoretic mobility shift assay (EMSA)
HepG2 cells were treated with 100 M acetaldehyde and hesperidin at 5 and
50 M concentrations. Nuclear extracts were prepared as previously described (Yeh
et al., 2007). The biotin-labele d complementary oligonucleotides corresponding to
NF-B and AP-1-binding sites were annealed. Biotinylated EMSAs were performed
as previously described (Sato and Seiki, 1993), and gels were transferred to nylon
membranes after electrophoresis. Membranes were blocked in solution and detected
with alkaline phosphatase-conjugated streptavidin (Chemicon, Australia) followed
by chemiluminescence (Roche, Germany).
2.7. Western blot analysis
HepG2 cells were treated with 100 M acetaldehyde and hesperidin at 5 and
50 M concentrations and lysed with 250 l sample buffer (62.5 mM Tris–HCl, 2%
SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% bromophenol blue, pH 6.8). We also
collected the supernatant of treated with acetaldehyde and hesperidin. The super-
natant was concentrated 40-fold using a Minicon ﬁlter (Millipore, Billerica, MA) with
a 15-kDa cutoff pore diameter. The protein concentration was determined with a
BCA protein assay kit (Pierce, Rockford, IL). Proteins (10 g for cell lyses or 40 gfor
supernatant) were separated by 10% SDS-polyacrylamide gel electrophoresis and
protein bands were electrophoretically transferred to nitrocellulose membranes.
Membranes were probed with polyclonal antibodies against IKK, IB-␣, JNK, p38,
and ERKs (Cell Signaling Technology, Beverly, MA). Bound antibodies were detected
with peroxidase-conjugated anti-rabbit antibodies followed by chemiluminescence
(ECL system, Amersham, Buckinghamshire, UK) and autoradiographic exposure. The
intensity of band on the gel was calculated by Gel-Pro
Analyzer (Media Cybernetics,
Inc., Silver Spring, MD).
2.8. Statistical analysis
A one-way ANOVA was used to determine if the means were signiﬁcantly dif-
ferent (p<0.05). If means were signiﬁcantly different, a Tukey–Kramer post hoc test
multiple group comparison test was used to compare individual groups. Error bars
in ﬁgures represent ± S.E.M.
3.1. Ef fects of hesperidin on acetaldehyde promoted cell invasion
and induced MMP-9 activity in HepG2 cells
HepG2 cells were treated with acetaldehyde and hesperidin
in the invasion chamber to assess the effects of hesperidin on
acetaldehyde-induced cell invasion. We calculated the resulting
number of invasive cells. Acetaldehyde-induced a 6-fold increase
of HepG2 cells migrated through Matrigel-coated ﬁlters (Fig. 1).
Increased number of HepG2 cells was signiﬁcantly inhibited by
hesperidin at 50 M. Tumor invasion requires increased expres-
sion of MMP-9, and we performed zymographic analysis to assess
whether MMP-9 activity was induced by acetaldehyde with or
without hesperidin treatment in HepG2 cells. Acetaldehyde sig-
niﬁcantly stimulated MMP-9 activation, and this activation was
suppressed by hesperidine in HepG2 cells (Fig. 2). We also eval-
uated the effects of hesperidin on acetaldehyde-induced MMP-9
expression by measuring MMP-9 activity in quantiﬁcation of MMP-
9(Fig. 2A), and quantiﬁcation of MMP-9 mRNA levels (Fig. 2C).
MMP-9 expression was increased after a 16 h acetaldehyde treat-
ment and suppressed by hesperidin in a dose-dependent manner
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206 M.-H. Yeh et al. / Toxicology Letters 184 (2009) 204–210
Fig. 1. Effects of hesperidin (HES) on acetaldehyde-induced cell invasion in HepG2 cells. (A) Cell invasion assay. HepG2 cells (5 × 10
) were re-suspended in 200 lDMEM
and added to the upper compartments of Matrigel invasion chambers supplemented with medium (A), 100 M acetaldehyde (B), acetaldehyde + 5 M hesperidin (C) and
acetaldehyde + 50 M hesperidin (D). After a 24 h incubation, the total number of cells on the lower surface of the insert chamber was stained and microscopically counted
with 200 × magniﬁcation (E). Values were the mean ± S.D. of three independent experiments. *p < 0.01 from untreated cells.
3.2. Effects of hesperidin on acetaldehyde-activated transcription
of NF-B and AP-1 promoters
We have evaluated promoter activity of the NF-B and AP-1
genes by the luciferase assay to investigate whether transcrip-
tional NF-B and AP-1 was regulated by acetaldehyde. A genomic
fragments containing either NF-B or AP-1 promoter region was
sub-cloned into the pGL3-basic vector and transfected into HepG2
cells. Cells were treated with acetaldehyde alone or with hes-
peridin treatment for 24 h, and luciferase activity was measured
with the luciferase assay. The NF-B promoter was activated by
acetaldehyde and expression was approximately 6-fold over the
pGL3-basic-transfected cells, and was inhibited by hesperidin in
a dose-dependent manner (Fig. 3A). Acetaldehyde activated the
AP-1 promoter levels approximately 4.5-fold over the pGL3-basic-
transfected cell levels, and was also inhibited by hesperidin in a
dose-dependent manner (Fig. 3B).
The effects of hesperidin on LPS-stimulated NF-B–speciﬁc-
and AP-1-speciﬁc-DNA–protein binding activities were examined
to evaluate whether hesperidin inhibits NF-B and AP-1 inhibi-
tion in LPS-treated animals. A biotinylated EMSA revealed that
acetaldehyde increased DNA binding abilities of NF-B and AP-
1 at 1 h. Hesperidin at a 50 M concentrated inhibit LPS-induced
NF-B-speciﬁc DNA–protein binding (Fig. 4A) and also inhibit AP-
1-speciﬁc DNA–protein binding (Fig. 4B) in hesperidin-treated cells
were compared to acetaldehyde-induced cells.
3.3. Effects of hesperidin on acetaldehyde-activated IB and
mitogen-activated protein kinases
Previous reports have suggested that transcriptional activity
of both NF-B and AP-1 may be regulated by IB and MAPKs.
Therefore, we examined the roles of ERK1/2, p38, and JNK on
acetaldehyde-induced NF-B and AP-1-DNA–protein binding activ-
NF-B activation is preceded by NF-B translocation to the
nucleus following IB phosphorylation and degradation. We deter-
mined the levels of IB and IKK-␣/␤ levels in acetaldehyde-treated
HepG2 cells to investigate the involvement of the NF-B signaling
pathway in the activation of MMP-9 expression. IB phospho-
rylation and degradation is a predominant pathway for NF-B
activation (Fig. 5), and we therefore determined IB levels in
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M.-H. Yeh et al. / Toxicology Letters 184 (2009) 204–210 207
Fig. 2. Effect of hesperidin on acetaldehyde-induced MMP-9 expression in HepG2
cells. HepG2 cells were cultured in 25-cm
ﬂasks, treated with 100 M acetalde-
hyde for 16 h, and pretreated 5 and 50 M hesperidin concentrations for 1 h. MMP-9
secretion forms in supernatants and cytosol were demonstrated by Western blot
analysis (A). Resulting cDNAs were ampliﬁed by PCR with human MMP-9 or GAPDH
primers for RT-PCR analysis (B). PCR products were resolved on 1% agarose gels and
visualized with ethidium bromide.
the cellular extracts of acetaldehyde-exposed HepG2 cells. IB
phosphorylation and degradation was stimulated by acetalde-
hyde. Reduced IB levels were correlated with a constant increase
in phosphorylated IB levels. Hesperidin (50 M) blocked the
activation of acetaldehyde-induced IB phosphoryation in a dose-
dependent manner (Fig. 5).
We determined acetaldehyde-induced MAP kinase levels in
HepG2 cells treated with varying concentration of hesperidin.
Since AP-1 activity is controlled by signaling through MAP
kinases. JNK, p38, and ERK protein levels were similar in cells
treated with acetaldehyde at 1 h (Fig. 5). Acetaldehyde stimu-
Fig. 3. Effects of hesperidin on acetaldehyde-induced NF-B and AP-1-dependent
luciferase reporter gene expression. HepG2 cells were treated with varying hes-
peridin concentrations and were transiently transfected with NF-B-containing
plasmids linked to the luciferase gene. Cell supernatants were collected and assayed
for luciferase activity as described in Section 2 after 16 h in culture with 100 M
acetaldehyde. Results are expressed as fold activity over the untreated transfected
Fig. 4. Effects of hesperidin on acetaldehyde-induced NF-B and AP-1 activation in HepG2 cells. EMSAs of nuclear extracts of lung tissue were performed after 24 h, and
hesperidin inhibited acetaldehyde-induced NF-B and AP-1 activity.
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208 M.-H. Yeh et al. / Toxicology Letters 184 (2009) 204–210
Fig. 5. Effects of hesperidin on acetaldehyde-induced IB, ERK1/2, p38, or JNK acti-
vation in HepG2 cells. In A, Cells were pretreated with 5 or 50 M hesperidin for
30 min prior to incubation with acetaldehyde for 60 min, and whole cell lysates were
prepared and subjected to Western blotting using antibodies speciﬁc for the phos-
phorylated form of IB, ERK1/2, p38, JNK, or for IB, ERK2, p38, or JNK as described
under Section 2 (A).
lated JNK and p38 phosphorylation, and exhibited no effects on
ERK phosphorylation. Hesperidin (50 M) blocked the activation of
acetaldehyde-induced phosphorylation of p38 and JNK in a dose-
dependent manner, with no effects on ERK1/2 (Fig. 5).
HCC is the most common cancer in the world-wide (Morgan et
al., 2004). Alcohol consumption enhances liver metastasis in col-
orectal carcinoma patients and affects the HCC malignancy grade
(Kubo et al., 1997; Maeda et al., 1998), and these studies suggested
that acetaldehyde might contribute to the carcinogenic effects of
alcoholism. Therefore, suppression of acetaldehyde-induced HCC
metastasis is an important area of study. The MMP family is strongly
associated with proteolysis of various extracellular matrix com-
ponents. MMP-2 and MMP-9 degrade type IV collagen (a major
basement membrane constituent membrane in cancer invasion
and metastasis) and are expressed in various tumor cells (Nelson
et al., 2000; Chung et al., 2002). Such observations suggested the
utility of MMP inhibitors in the prevention of tumor metastasis.
Previous studies have suggested that MMP-9 expression may be
associated with cancer progression and invasion (Rao et al., 1993,
1996; Scorilas et al., 2001), Several novel MMP inhibitors are being
investigated in clinical trials (Sugita, 1999, Wojtowicz-Praga, 1999).
In addition, MMP-9 over-expression has been associated with HCC
capsular inﬁltration and growth (Arii et al., 1996; Sakamoto et al.,
2000). Elevated MMP-9 plasma levels have been observed in HCC
patients, particularly in patients with macroscopic portal vein inva-
sion (Hayasaka et al., 1996). Further, acetaldehyde has been shown
to increase MMP-9 activity and increase invasion potential (Hsiang
et al., 2007). In our present study we have shown that hesperidin
reduced acetaldehyde-induced effects of HCC tumor invasion and
metastasis by suppression of MMP-9 activity.
Hesperidin is a bioﬂavonoid and is abundantly present in cit-
rus fruits. It has antioxidant, anti-inﬂammatory, and anti-cancer
properties. Flavonoids act as powerful antioxidants, provide signif-
icant protection against oxidative stress and free radical scavenging
action and it may act as a vasodilator with therapeutic efﬁcacy
in hypertension. Recently, hesperidin was protective against CCl4-
induced oxidative stress in rat livers and kidneys, and this was
attributed to its antioxidant effects (Tirkey et al., 2005). Hesperidin
was also protective against gamma-irradiation induced hepatocel-
lular damage and oxidative stress in rats, likely due to its protective
effects against hepatocellular necrosis secondary to free radi-
cal scavenging and membrane-stabilizing abilities (Pradeep et al.,
2008). Hesperidin displayed preventive anti-inﬂammatory effects
in mouse skin which were the result of a tumor promoter (Koyuncu
et al., 1999; Berkarda et al., 1998), and also acts as a chemopre-
ventive agent against colon, esophageal, oral, and urinary bladder
carcinogenesis (Tanaka et al., 1997a,b,c; Yang et al., 1997). Dietary
hesperidin is an important chemopreventive agent and inﬂuences
tumor yields in mice (Corpet and Pierre, 2003). We identiﬁed the
potential for hesperidin-mediated suppression of acetaldehyde-
induced cancer invasion potential in HepG2 cells. In this study we
suggest that hesperidin inhibit the MMP activity and its leads to
prevention of HCC metastasis.
Several stimulators induce MMP-9 expression by various sig-
naling pathways and results in the invasiveness of cell lines.
Transforming growth factor-␤ activated the p38 signaling pathway
which induce d MMP-2 and MMP-9 expression (Kim et al., 2004).
Phorbol ester-induced MMP-9 secretion primarily through protein
kinase C-dependent activation of the Ras/ERK signaling pathway
(Liu et al., 2002). Radiation enhanced HCC cell invasiveness through
MMP-9 expression through the PI3K/Akt/NF-B signal transduction
pathway (Cheng et al., 2006). We performed an MMP-9 promoter
luciferase assay and an EMSA in a previous study to determine
the inhibitory effects of hesperidin on MMP-9 gene transcrip-
tion through the suppression of transcription factor. The luciferase
activity signiﬁcantly increased in HepG2 cells that was transiently
transfected with the wild-type MMP-9 promoter through acetalde-
hyde treatment as seen with luciferase promoter assay. Conversely,
acetaldehyde-induced luciferase activity was signiﬁcantly reduced
in NF-B and AP-1 mutant MMP-9 promoters. This suggested that
NF-B and AP-1 have important roles as MMP-9 promoters, and
hesperidin inhibited acetaldehyde-induced NF-B and AP-1 pro-
moter activity in HepG2 cells by the luciferase assay and EMSA.
Acetaldehyde-induced MMP-9 activation was associated with both
increased NF-B and AP-1 activity by the IB, JNK, and p38 signaling
pathways in a previous study (Hsiang et al., 2007). Several stud-
ies also suggested that hesperidin inhibited both NF-B and Ap-1
activity through the IB, p38, and JNK signal transduction pathways
(Yeh et al., 2007; Kim et al., 2006). Our study demonstrated that hes-
peridin suppressed MMP-9 expression at both the transcription and
secretion levels, and inhibited acetaldehyde-induced NF-B activa-
tion mediated by IKK and IB phosphorylation and degradation.
Hesperidin also suppressed acetaldehyde-induced AP-1 activation
through inhibitory effects on JNK and p38 phosphor ylation. These
results suggest that hesperidin inhibited acetaldehyde-induced
MMP-9 expression and activity in HepG2 cells (Fig. 6). NF-B and
Author's personal copy
M.-H. Yeh et al. / Toxicology Letters 184 (2009) 204–210 209
Fig. 6. Schematic diagram illustrates the molecular mechanism of hesperidin inhib-
ited acetaldehyde-induced MMP-9 expression.
AP-1 activation could partially regulate inhibitory effects of hes-
peridin. The inhibitory effect of hesperidin could be associated with
the anti-metastatic toxicity by acetaldehyde in HCC, and may have
therapeutic beneﬁts for HCC patients who engage in alcohol con-
sumption. We will further evaluate the anti-metastatic effects of
dietary hesperidin on acetaldehyde-induced HCC in experimental
animal model in future studies.
Conﬂict of interest statement
None of the authors has a ﬁnancial relationship with a commer-
cial entity that has an interest in the subject of this manuscript.
We thank Tin-Yun Ho for her technical assistant. This work was
supported by grants from National Science Council and Buddhist
Dalin Tzu Chi General Hospital (DTCRD97(2)-03), Taiwan, ROC.
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