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RESEARCH ARTICLE
Anti-invasion effects of 6-shogaol and 6-gingerol,
two active components in ginger, on human
hepatocarcinoma cells
Chia-Jui Weng
1,2
, Cheng-Feng Wu
1
, Hsiao-Wen Huang
1
, Chi-Tang Ho
3
and Gow-Chin Yen
1
1
Department of Food Science and Biotechnology, National Chung Hsing University, Taichung, Taiwan
2
Graduate Institute of Applied Science of Living, Tainan University of Technology, Yongkang, Tainan, Taiwan
3
Department of Food Science, Rutgers University, New Brunswick, NJ, USA
Received: March 2, 2010
Revised: March 26, 2010
Accepted: March 29, 2010
Scope: Hepatocellular carcinoma is the most common type of liver cancer and is highly
metastatic. Metastasis is considered to be the major cause of death in cancer patients. Ginger
is a natural dietary rhizome with anti-oxidative, anti-inflammatory, and anti-carcinogenic
activities. The aims of this study were to evaluate the anti-invasion activity of 6-shogaol and
6-gingerol, two compounds found in ginger, on hepatoma cells.
Methods and results: The migratory and invasive abilities of phorbol 12-myristate 13-acetate
(PMA)-treated HepG2 and PMA-untreated Hep3B cells were both reduced in a dose-depen-
dent manner by treatment with 6-shogaol and 6-gingerol. Upon incubation of PMA-treated
HepG2 cells and PMA-untreated Hep3B cells with 6-shogaol and 6-gingerol, matrix metal-
loproteinase (MMP)-9 activity decreased, whereas the expression of tissue inhibitor metallo-
proteinase protein (TIMP)-1 increased in both cell types. Additionally, urokinase-type
plasminogen activator activity was dose-dependently decreased in Hep3B cells after incuba-
tion with 6-shogaol for 24 h. Analysis with semi-quantitative reverse transcription-PCR
showed that the regulation of MMP-9 by 6-shogaol and 6-gingerol and the regulation of
TIMP-1 by 6-shogaol in Hep3B cells may on the transcriptional level.
Conclusions: These results suggest that 6-shogaol and 6-gingerol might both exert anti-
invasive activity against hepatoma cells through regulation of MMP-9 and TIMP-1 and that
6-shogaol could further regulate urokinase-type plasminogen activity.
Keywords:
6-Gingerol / 6-Shogaol / Invasion / Matrix metalloproteinases / Tissue inhibitor
metalloproteinase
1 Introduction
Naturally occurring phytochemicals are present in the
human diet from foodstuffs such as garlic, ginger, soy,
curcumin, onion, tomatoes, chilies, cruciferous vegetables,
and green tea. Many of these compounds are used for the
chemoprevention of cancer and contribute to lowering the
risk of cancer [1]. Ginger (Zingiber officinale) is a natural
dietary rhizome that is widely used as a flavoring agent and
occasionally used as a traditional medicinal herb. The anti-
oxidative, anti-inflammatory, and anti-carcinogenic proper-
ties of ginger have been verified [2–4], and treatment with
ginger is useful in preventing the development of colorectal
cancer [3]. However, the literature concerning the identifi-
cation of the constituent(s) of ginger active in preventing
cancer invasion or metastasis is still limited. Several
pungent compounds, such as gingerols, shogaols, paradols,
Abbreviations: ECM, extracellular matrix; MMP, matrix metallo-
proteinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide; PMA, phorbol 12-myristate 13-acetate; TIMP,
tissue inhibitor of metalloproteinase protein; uPA, urokinase-
type plasminogen activator
These authors have contributed equally to this work.
Correspondence: Dr. Gow-Chin Yen, Department of Food Science
and Biotechnology, National Chung Hsing University, 250
Kuokuang Road, Taichung 40227, Taiwan
E-mail: gcyen@nchu.edu.tw
Fax: 1886-4-2285-4378
&2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com
1618 Mol. Nutr. Food Res. 2010, 54, 1618–1627DOI 10.1002/mnfr.201000108
and gingerdiols, have been identified in ginger. Among
these compounds, shogaols and gingerols are two phenolic
substances contained in a volatile oil extracted from ginger
root and provide ginger its characteristic odor and flavor [5].
Phenolic substances present in natural dietary foods, such
as fruits and vegetables, have been found to protect against
cancer both in vitro and in vivo [6–8]. 6-Shogaol [1-(4-
hydroxy-3-methoxyphenyl)-4-decen-3-one] is a lipid-soluble
organic compound that exhibits significant anti-hepatotoxic
effect against galactosamine-induced cytotoxicity in primary
cultured rat hepatocytes [9] and protects against LPS-
induced inflammation in RAW 264.7 macrophage cells [10].
The administration of 6-shogaol prevents secondary patho-
logical events following traumatic spinal cord injuries and
promotes the recovery of motor function in an animal
model [9]. 6-Gingerol [5-Hydroxy-1-(40-hydroxy-30-methoxy-
phenyl)-3-decanone] is an abundant constituent of ginger
and also possesses anti-oxidative and anti-inflammatory
activities [11, 12]. It acts to prevent the metastasis of B16
melanoma cells [13] and limits the invasion of the rat ascite
hepatoma AH109A cell line [14]. Both 6-shogaol and
6-gingerol can significantly inhibit tumor necrosis factor-a
mediated downregulation of adiponectin in 3T3-L1 adipo-
cytes [15]. Additionally, 6-shogaol is more potent than
6-gingerol in inhibiting the proliferation and inducing the
apoptosis of COLO 205 cells [16]. The studies mentioned
above suggest that 6-shogaol and 6-gingerol are the active
components in ginger and that 6-gingerol possesses anti-
metastatic and anti-invasive pharmacological activities on
cancer cells. However, the effect of 6-shogaol on the inva-
sion of cancer cells has not been verified, and the literature
regarding the effects of 6-gingerol on inhibiting the invasion
of hepatoma cells is still limited.
Hepatocellular carcinoma is a highly metastatic cancer,
representing 83% of all liver cancer cases, and is the third
leading cause of cancer deaths worldwide [17]. Metastasis is
responsible for the majority of failures of cancer treatment
and is the major cause of death in cancer patients. As a
result, treatments that can block cancer invasion and
metastasis, in addition to minimizing the growth of existing
tumors, are being actively pursued to enhance the survival
of cancer patients. The invasion and metastasis of cancer
cells involves the degradation of the environmental extra-
cellular matrix (ECM) and basement membrane by various
proteolytic enzymes and results in the mobility of cancer
cells [18–20]. Among these proteases, matrix metalloprotei-
nase (MMP)-2 and MMP-9 are highly expressed in various
malignant tumors and are closely related to the invasion and
metastasis of cancer cells [21, 22].
MMP-2 and MMP-9 are activated by plasmin, which is
generated from specifically cleaved zymogen plasminogen
through the enzyme urokinase-type plasminogen activator
(uPA) when it associates with its receptor, uPAR. uPA
initiates an enzymatic cascade leading to the activation of
both the MMP-2 and the MMP-9 enzymes and making them
capable of degrading type IV collagen, which is a major
constituent of the basement membrane, making mobility
both possible and easy. Therefore, several inhibitors against
uPA or MMPs have been tested in clinical trials for
prevention of tumor invasion and metastasis [23]. Tissue
inhibitor metalloproteinase proteins (TIMPs) are a
mammalian protein family composed of TIMP-1, -2, -3, and
-4, which together display wide-ranging sequence homology
and structural identity. TIMPs have been reported as natural
MMP inhibitors that prevent the degradation of the ECM by
abolishing the hydrolytic activity of all activated members of
the metalloproteinase family, in particular that of
membrane type 1-MMP (MT1-MMP), MMP-2, and
MMP-9 [24].
Human hepatocarcinoma HepG2 and Hep3B cells are
two common cell models for cancer research, which secrete
both MMP-2 and MMP-9 simultaneously with or without
induction, respectively [25]. The aims of this study were to
evaluate the anti-invasion activity of 6-shogaol and
6-gingerol (structures shown in Fig. 1) on inducer-treated
HepG2 and inducer-untreated Hep3B cells. To explore the
anti-invasive mechanisms involved in human liver cancer
cells by 6-shogaol and 6-gingerol, the impacts of these
compounds on MMP-2, MMP-9, TIMP-1, and uPA were
evaluated.
2 Materials and methods
2.1 Materials and reagents
6-Shogaol and 6-gingerol were isolated and purified through
column chromatography; the purity and identity of the
isolated compounds were confirmed by HPLC and NMR
6-shogaol
[1-(4-hydroxy-3-methoxyphenyl)-4-decen-3-one]
6-gingerol
[5-H
y
drox
y
-1-(4'-h
y
drox
y
-3'-methox
yp
hen
y
l)-3-decanone]
A
B
Figure 1. Chemical structures of (A) 6-shogaol and (B) 6-gingerol.
Mol. Nutr. Food Res. 2010, 54, 1618–1627 1619
&2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com
[10, 16]. All chemicals were dissolved in DMSO. Type IV
gelatin, phorbol 12-myristate 13-acetate (PMA), DMSO, and
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) were purchased from Sigma Chemical (St. Louis, MO).
DMEM, fetal bovine serum, and SuperScript
s
III First-Strand
Synthesis SuperMix for quantitative reverse transcription-PCR
kit were purchased from Invitrogen (Carlsbad, CA). PCR
Master Mix 2X Kit was purchased from Fermentas (Glen
Burnie,MD).Transwell
s
Permeable Support was purchased
from Corning (Lowell, MA).
2.2 Cell culture
Human hepatoma HepG2 and Hep3B cells were
obtained from the Bioresource Collection and Research
Center (BCRC, Food Industry Research and Develop-
ment Institute, Hsin Chu, Taiwan). Cells were grown as
described in our previous study [25]. Cells were cultured
in serum-free medium for the invasive and metastatic
experiments.
2.3 Cell viability assay
Cell viability was determined with an MTT assay as descri-
bed in our previous study [25], and the percent viability of
the treated cells was calculated as follows:
½ðA570nm A630nmÞsample =ðA570nm A630nmÞcontrol 100
2.4 Gelatin and casein zymography
HepG2 and Hep3B cells were incubated in the presence and
absence, respectively, of the PMA concentrations indicated,
and serum-free DMEM with or without compounds (in
DMSO) for a given time. The conditioned media were then
collected as samples. The zymography was performed
according to the method described in our previous study [25].
2.5 Cell migration and invasion assay
The cell invasion assay was performed according to the
method described by Repesh [26]. The detailed procedure
has been described in our previous study [25].
2.6 Western blotting
Ten micrograms of each of the total cell lysates were sepa-
rated by SDS-PAGE on 10% polyacrylamide gels and
transferred onto a polyvinylidene fluoride membrane using
a Bio-Rad Mini Protean electrotransfer system. The probe
detection and signal development were performed according
to the methods described in our previous study [25].
2.7 Reverse transcription-PCR
Total RNA preparation and reverse transcription-PCR (RT-
PCR) were performed according to the methods described in
our previous study [25]. Then, the resulting cDNA was
amplified by PCR with the following primers: MMP-9 (94 bp),
50-GGGCTTAGATCATTCCTCAGTG-30(sense) and 50-
GCCATTCACGTCGTCCTTAT-30(antisense); TIMP-1 (95 bp )
50-ACTTCCACAGGTCCCACAAC-30(sense) and 50-AGCC-
ACGAAACTGCAGGTAG-30(antisense); glyceraldehyde-3-
phosphate dehydrogenase (110 bp), 50-ATCGACCACTACC-
TGGGCAA-30(sense), and 50-AGGATAACGCAGGCGAT-
GT-30(antisense). PCR amplification was performed under
the following conditions: 35 cycles of 941Cfor1min,591Cfor
1min, 721C for 2 min (for MMP-9); 28 cycles of 941Cfor
1min, 591C (for TIMP-1) or 581C (for glyceraldehyde-3-
phosphate dehydrogenase) for 1 min, 721C for 2 min; then,
followed by a final incubation at 721Cfor10min.
2.8 Protein concentration determination
The protein concentration was determined according to the
method described by Bradford [27] using bovine serum
albumin as a standard.
2.9 Statistical analysis
Data are indicated as the mean7SD for three different
determinations. Statistical comparisons were made by
means of one-way analysis of variance followed by a
Duncan’s multiple-comparison test. Values of po0.05 were
considered statistically significant.
3 Results
3.1 Effects of 6-shogaol and 6-gingerol on the
viability of hepatoma cells
The MTT assay was used to evaluate the cytotoxicity of
6-shogaol and 6-gingerol on hepatoma cells. A range of
0–100 mM of these two compounds was used to treat HepG2
and Hep3B cells for 24 and 48 h. The results showed that the
viability of both hepatoma cells was over 80% when treated
with 6-shogaol and 6-gingerol at a range of concentrations
from 0 to 10 and 0 to 50 mM, respectively, for 24 h (Fig. 2).
Hence, the indicated doses and treatment times of 6-shogaol
and 6-gingerol for maintaining at least 80% cell viability
were used for the subsequent experiments on hepatoma
cells.
1620 C.-J. Weng et al.Mol. Nutr. Food Res. 2010, 54, 1618–1627
&2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com
3.2 Effects of 6-shogaol and 6-gingerol on migration
and invasion of HepG2 and Hep3B cells
Cell–matrix interaction and cell motility are important for
cancer cell invasion. To examine the potential anti-invasive
effects of 6-shogaol and 6-gingerol, migration and invasion
assays were performed on HepG2 and Hep3B cells. The
results indicated that the migratory and invasive activities of
HepG2 cells without induction were not affected by treatment
with these two compounds (data not shown). We have
previously shown [25] that the invasive activity and MMP-9
secretion of HepG2 cells can be induced by 200 nM PMA, so
HepG2 cells were treated with PMA first for the subsequent
tests. As expected, the migratory and invasive abilities of the
HepG2 cells were induced by PMA, and the PMA-induced
migration and invasion were further reduced in a dose-
dependent manner by a 24-h treatment with 6-shogaol and
6-gingerol at concentrations of 42.5 and 45mM, respectively
(Figs. 3A and 4A). After treatment of PMA-treated HepG2
cells with either 2.5 mM 6-shogaol or 5 mM 6-gingerol for 24 h,
the migratory abilities were significantly (po0.05) reduced by
25 and 22% (Fig. 3A), respectively, and the invasive abilities
were significantly (po0.05) reduced by 53 and 52% (Fig. 4A),
respectively, relative to PMA treatment alone. When 6-shogaol
and 6-gingerol were applied to Hep3B cells, dose-dependent
inhibitory effects on migration and invasion were also
observed (Figs. 3B and 4B). The migratory abilities were
significantly (po0.05) reduced to 83 and 69% (Fig. 3B), and
the invasive activities were significantly (po0.05) reduced to
53 and 71% (Fig. 4B) by treat ment with 1 mM6-shogaoland
5mM 6-gingerol on Hep3B cells, respectively. The results
suggest that 6-shogaol and 6-gingerol are inhibitors of hepa-
toma cell migration and invasion and that the effective inhi-
bitory concentration of 6-shogaol on cell invasion is markedly
lower than that of 6-gingerol.
3.3 6-Shogaol and 6-gingerol inhibit the MMP-9
activities of HepG2 and Hep3B cells
To clarify whether the activity of MMP-2 and MMP-9 is
involved in the invasion of hepatoma cells, the effective anti-
invasive dosages of 6-shogaol and 6-gingerol were used to
analyze the effects of these two compounds on MMP-2 and
MMP-9 in hepatoma cells. The PMA-treated HepG2 and PMA-
untreated Hep3B cells were incubated in serum-free medium
with or without a given compound for 24 h. The conditioned
mediawerethenusedtoanalyzeMMP-9andMMP-2activity
by gelatin zymography. The activity of MMP-9 was suppressed
in a dose-dependent manner, but the MMP-2 activity was not
significantly changed when HepG2 and Hep3B cells were
treated with 6-shogaol (1, 2.5, 5, and 10 mM) or 6-gingerol
(5, 10, 25, and 50 mM), respectively (Fig. 5). The dose-inde-
pendent and insignificant decrease of MMP-2 activity might be
concentration (µ
µ
M)
0 1 5 102550
concentration (
µ
M)
015102550
concentration (
µ
M)
015102550
concentration (
µ
M)
015102550
cell viability (% of control)
0
20
40
60
80
100
120
24 h
48 h
*
*
*
*
*
*
*
cell viability (% of control)
0
20
40
60
80
100
120
24 h
48 h
*
*
*
*
*
*
*
0
20
40
60
80
100
120
24 h
48 h
cell viability (% of control)
*
*
*
**
*
*
*
*
0
20
40
60
80
100
120
140
24 h
48 h
cell viability (% of control)
*
*
**
*
*
*
(a)
(b)
(a)
(b)
AB
Figure 2. Effects of 6-shogaol
(a) and 6-gingerol (b) on the
viability of HepG2 (A) and Hep3B
(B) cells. Cells were incubated in
a serum-free medium with
various concentrations of 6-
shogaol and 6-gingerol for 24
and 48 h. Cells in a serum-free
medium without 6-shogaol and
6-gingerol were used as the
control. Data represent the
mean7SD of three indepen-
dent experiments. (
po0.05
compared with the control).
Mol. Nutr. Food Res. 2010, 54, 1618–1627 1621
&2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com
a result of the reduction in cell numbers but not the inhibitory
effects of 6-shogaol and 6-gingerol treatment.
3.4 Both 6-shogaol and 6-gingerol increase protein
levels of TIMP-1 but only 6-shogaol suppreses
uPA activity
The physiological activity of MMP-9 is significantly related to the
activity of uPA and TIMPs (especially TIMP-1). The Western
blot method and casein zymography were used to determine the
effects of these two compounds on the protein levels of TIMP-1
and the activity of uPA, respectively, in both PMA-treated
HepG2 and PMA-untreated Hep3B cells. After quantifying the
expression of TIMP-1 protein in each condition and correcting
by the corresponding b-actin expression, the data revealed that
the protein level of TIMP-1 in HepG2 and Hep3B cells was
increased in a dose-dependent manner along with a gradual
increase in the concentrations of 6-shogaol and 6-gingerol used
for 24 h treatment (Fig. 6A). uPA activity was not detectable in
HepG2 cells with or without PMA induction, but it dose-
dependently decreased in Hep3B cells after incubation with
cell motility (% of PMAtreatm
ent only)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
6-Shogaol
PMA (200 nM) -+++++
6-Shogaol (µM) 0012.5 510
#
*
*
*
concentration (µM)
0 1 2.5 5 10
cell motility (%of control)
0
20
40
60
80
100
120 6-Shogaol
*
*
**
cell motility(
%of PMA treatment only)
0.0
0.2
0.4
0.6
0.8
1.0
1.2 6-Gingerol
PMA (200 nM) -+++++
6-Gingerol (µM) 0 0510 25 50
#
*
*
*
*
concentration (µM)
0 5 10 25 50
cell motility (% of control)
0
20
40
60
80
100
120
6-Gingerol
**
**
(b) (b)
(a) (a)
AB
Figure 3. Effects of 6-shogaol (a)
and 6-gingerol (b) on the motility
of PMA-treated HepG2 (A) and
PMA-untreated Hep3B (B) cells.
The photographic image (100 )
represents the cells migrating
through PCF membrane. The bar
graphs represent the migratory
cell numbers from cells treated
with various concentrations
of 6-shogaol or 6-gingerol for
24 h. Values are reported as
mean7SD, n=3.(Inpanel(A),#
indicates po0.05 compared with
the control and
indicates
po0.05 compared with the PMA
treatment only. In panel (B),
indicates po0.05 compared
with the control).
1622 C.-J. Weng et al.Mol. Nutr. Food Res. 2010, 54, 1618–1627
&2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com
6-shogaol for 24 h (Fig. 6B). Taken together, we conclude that
the inhibitory effect on MMP-9 in hepatoma cells by 6-shogaol
may be through the regulation of both uPA and TIMP-1 but
that the effect of 6-gingerol may be through TIMP-1 only.
3.5 6-Shogaol transcriptionally regulates MMP-9
and TIMP-1, but 6-gingerol regulates only
MMP-9
According to the results shown in Figs. 5 and 6, the
expression of MMP-9 and TIMP-1 in PMA-treated HepG2
and PMA-untreated Hep3B cells was significantly influ-
enced by 6-shogaol and 6-gingerol treatment. Semi-quanti-
tative RT-PCR was further employed to analyze the effects of
6-shogaol and 6-gingerol on the mRNA expression of MMP-
9 and TIMP-1 in each hepatoma cell line. After treatment of
PMA-treated HepG2 cells with 0–10 mM 6-shogaol or
0–50 mM 6-gingerol for 24 h, the mRNA expression of MMP-
9 and TIMP-1 was nearly unchanged, except in the case of
1mM 6-shogaol treatment, which led to dramatically
decreased levels of MMP-9 mRNA (Fig. 7A). The dose-
independent reduction of MMP-9 mRNA by treatment of
PMA-treated HEPG2 cells with a single dosage of 6-shogaol
cell invasion (% of PMA tre atment only)
0
20
40
60
80
100
120 6-Shogaol
PMA (200 nM) -++
+++
6-Shogaol ( M) 0 012.5 510
#
**
*
*
concentration ( M)
012.5510
cell invasion (% of control)
0
20
40
60
80
100
120 6-Shogaol
*
*
**
cell invasion (% of PMA treatment only)
0
20
40
60
80
100
120 6-Gingerol
PMA (200 nM) -+++++
6-Gingerol (
μ
M)
00510 25 50
#
**
*
*
concentration (
μ
M)
0 5 1 0 25 50
cell invasion (% of control)
0
20
40
60
80
100
120
6-Gingerol
**
**
(b b
)()
(a) (a)
μ
μ
AB
Figure 4. Concentration-depen-
dent inhibitory effects of 6-
shogaol (a) and 6-gingerol (b)
on the invasion of PMA-treated
HepG2 (A) and PMA-untreated
Hep3B (B) cells. Photographic
images (100 ) represent the
cells invading through Matri-
gel-coated membrane. The bar
graphs represent the invasive
cell numbers from 24 h treat-
ments various concentrations
of 6-shogaol or 6-gingerol.
Values are reported as
mean7SD, n=3. (In panel (A), #
indicates po0.05 compared
with the control and
indicates
po0.05 compared with the
PMA treatment only. In panel
(B),
indicates po0.05 compa-
red with the control).
Mol. Nutr. Food Res. 2010, 54, 1618–1627 1623
&2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com
is unexpected, and the cause needs to be verified in further
specifically designed studies. Therefore, we ignored this
phenomenon in HepG2 cells and paid more attention to the
effects of these two compounds on Hep3B cells. Upon
treatment of the Hep3B cells with 6-shogaol or 6-gingerol
for 24 h, the mRNA expression of MMP-9 decreased but the
expression of TIMP-1 increased along with an increased
concentration of 6-shogaol, whereas that of the internal
control (G6PD) remained unchanged. However, a decrease
in the levels of MMP-9 mRNA was only seen after treatment
with 6-gingerol (Fig. 7B). Given these results, both 6-shogaol
and 6-gingerol affected MMP-9 and TIMP-1 in PMA-treated
HepG2 cells via a method other than transcriptional regu-
lation; however, transcriptional regulation was at least partly
responsible for the regulation of MMP-9 and TIMP-1 in
Hep3B cells by 6-shogaol and MMP-9 by 6-gingerol.
4 Discussion
Metastasis is the major cause of death in cancer patients.
Thus, active compounds demonstrating anti-invasive and
anti-metastatic properties are defined as a new catalog of
chemopreventive agents. 6-Shogaol is the most effective
individual component of ginger for the inhibition of growth
of ovarian cancer cells [28]. 6-Gingerol, the most abundant
component in ginger, can inhibit the oxidant-induced
invasion of AH109A, a hepatoma cell line that does not
produce MMPs, in a dose-dependent manner (50–200 mM)
by means of its anti-oxidative activity [14]. Metastasis occurs
through a complex multistep process consisting of invasion
of cells from a primary tumor into the circulation, immi-
gration of these cells to distant organs, adhesion to endo-
thelial cells and infiltration into tissue. The compounds
which are effective to suppress cell migration could be
contributed to the inhibition of cell invasion. In this study,
6-shogaol and 6-gingerol at concentrations of o10 and
o50 mM, respectively, could effectively inhibit both the
migratory and the invasive activities of PMA-treated HepG2
and PMA-untreated Hep3B cells (Figs. 3 and 4). Although
the anti-invasive activity of 6-gingerol on hepatoma cells has
been tested and verified in the AH109A cell model, the
underlying mechanism of 6-gingerol action on invasion of
HepG2 and Hep3B cells was supposed to be different from
that of AH109A cells due to the different MMP-producing
properties of these cells. 6-Gingerol can inhibit cell adhe-
sion, invasion, motility, and MMP-2 and -9 activities in
MDA-MB-231 human breast cancer cells [29]. It also inhibits
angiogenesis both in vitro and in vivo, and suppresses the
formation of lung metastases of B16F10 melanoma in an
experimental tumor-bearing mouse model [30]. Therefore,
the anti-MMP-2 and -9 and anti-angiogenesis effects could
be the anti-invasive strategies of 6-gingerol.
Shogaol is a dehydrated product of the structurally
similar gingerols. As a large quantity of gingerols is found
in fresh ginger, shogaols are abundant in dried and ther-
mally treated ginger. The only structural difference between
6-shogaol and 6-gingerol is the double bond on the carbon
side chain forming a,b-unsaturated ketone moiety in
6-shogaol or the hydroxyl moiety in 6-gingerol. Previous
studies have suggested that the a,b-unsaturated ketone
moiety is very susceptible to nucleophilic addition reactions
with thiols [31] and is essential for exerting cytotoxic activity
via this susceptibility [32]. The a,b-unsaturated carbonyl
group in 6-shogaol might influence the conformation of the
compound and modulate its inhibitory effect. Additionally,
the hydroxyl group might reduce the lipophilic property and
cell membrane permeability of 6-gingerol and impede its
bioavailability to cells. In other words, the difference in
chemical structural of 6-shogaol and 6-gingerol might be an
influential factor of their bioactivity. Several studies have
illustrated a consistent difference in the bioactivity of
6-shogaol and 6-gingerol. For example, 6-shogaol is far more
potent than 6-gingerol in inhibiting the proliferation of and
(a)
(b)
(a)
(b)
0.3 1.0 0.8 0.7 0.7 0.6
0.9 1.0 1.0 0.9 0.9 0.8
1.0 0.9 0.7 0.7 0.6 0.6
1.0 0.9 0.8 0.8 0.8 0.8
1.0 0.7 0.8 0.6 0.6 0.6
1.0 0.9 0.9 0.8 0.8 0.8
0.3 1.0 0.7 0.6 0.6 0.5
1.0 1.0 0.9 0.9 0.9 0.8
AB
Figure 5. Effects of 6-shogaol (a)
and 6-gingerol (b) on activity of
both MMP-9 and MMP-2 in
PMA-treated HepG2 (A) and
PMA-untreated Hep3B cells.
Cells were incubated in a serum-
free medium with various
concentrations of 6-shogaol and
6-gingerol for 24 h. Activities of
these proteins were subse-
quently quantified by densito-
metric analyses with that of
PMA treatment only or control
set to 100%.
1624 C.-J. Weng et al.Mol. Nutr. Food Res. 2010, 54, 1618–1627
&2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com
inducing apoptosis in COLO 205 cells [16] and in inhibiting
iNOS and COX-2 expressions in LPS-activated macrophages
[10]. In this study, we also found that 6-shogaol is more
effective than 6-gingerol in inhibiting the migration and
invasion of hepatoma cells. Taking into account the
compounds’ dietary availability, the ordinary daily human
consumption of ginger has approximately 250 mg–1 g and
1.0–3.0% 6-gingerol and its derivatives [5]. The concentra-
tions of 6-gingerol (5–50 mM(1.4–14 mg/mL)) and
6-shogaol (1–10 mM(0.28–2.8 mg/mL)) used in this study
should be similar to what is present in vivo.
The MMP-mediated degradation of the ECM is a well-
known factor in tumor invasion and metastasis. TIMPs are
endogenous inhibitors that can block the hydrolytic activities
of MMPs. The balance between the levels of activated MMPs
and free TIMPs determines overall MMP activity and
contributes to tumor invasion and metastasis. The over-
expression of TIMPs has been demonstrated to reduce
experimental metastasis. TIMP-1 has been shown to have a
statistically significant association with the response to
chemotherapy in metastatic breast cancer [33]. Davidsen
et al. [34] showed that TIMP-1-positive or TIMP-1-deficient
cancer cell lines displayed significant differences in their
sensitivity toward chemotherapeutics. TIMP-1 is a general
prototype inhibitor for most MMP family members and is
28.5 kda
TIMP-1
ββ
β
-actin
(fold)
(M)
control 1.0 2.5 5.0 5 10 25
6-shogaol 6-gingerol
TIMP-1/
β
β
-actin
1.0 1.3 1.8 1.5 1.5 1.7 2.0
TIMP-1
β
β
-actin
(fold)
(μ
μ
M)
control 1.0 2.5 5.0 5 10 25
6-shogaol
PMA
1.0 1.5 1. 7 2.1 1.2 2.4 2.3
0.9
28.5 kda
6-gingerol
TIMP-1
β
β
-actin
(a)
(b)
A
B
Figure 6. Effects of 6-shogaol and 6-gingerol on the protein level
of TIMP-1 in PMA-treated HepG2 [A(a)] and PMA-untreated
Hep3B [A(b)] cells, and on the activity of uPA in PMA-untreated
Hep3B cells (B). Cells were treated with 6-shogaol and 6-gingerol
at the indicated concentrations for 24 h; the conditioned medium
was subjected to casein zymography for uPA assay, whereas
cytosolic extracts were subjected to SDS-PAGE followed by
Western blotting with TIMP-1 antibody, as described in Section
2. Determined activities or protein levels were subsequently
quantified by densitometric analyses and the relative density
was compared with that of PMA treatment alone or control,
which was set as 100%.
MMP-9/
G6PD
(a)
(b)
0.3 1.0 0.6 0.9 0.9 1.0
TIMP-1/
G6PD
(a)
1.0 0.7 0.7 0.5
1.0 69.1 65.3 146.0
MMP-9/
G6PD
TIMP-1/
G6PD
MMP-9/
G6PD
TIMP-1/
G6PD
(b)
MMP-9/
G6PD
TIMP-1/
G6PD
A
B
Figure 7. Effects of 6-shogaol and 6-gingerol on MMP-9 and
TIMP-1 mRNA expression in PMA-treated HepG2 (A) and PMA-
untreated Hep3B (B) cells. HepG2 and Hep3B cells in the
presence or absence of 200 nM PMA, respectively, were incu-
bated with the indicated concentrations of 6-shogaol (a) or
6-gingerol (b) for 24 h. The RNA was extracted from cells and
subjected to a semi-quantitative RT-PCR. G6PD was used as an
internal control. The final PCR products were quantified by
densitometric analyses with that of PMA treatment alone or
control, which was set as 100%.
Mol. Nutr. Food Res. 2010, 54, 1618–1627 1625
&2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com
present in various cell types [35]; however, TIMP-1 has a
particularly high affinity for MMP-9 [36]. This study indi-
cated that 6-shogaol and 6-gingerol reduced MMP-9 activity
(Fig. 5), partially through the increase of TIMP-1 protein
level (Fig. 6A), and further led to the inhibition of migratory
(Fig. 3) and invasive (Fig. 4) activities of PMA-treated
HepG2 and PMA-untreated Hep3B cells. Moreover, the
suppression of uPA activity by 6-shogaol was also seen in
Hep3B cells. Therefore, the increase of TIMP protein levels
and the reduction of uPA activity may be a possible alter-
native strategy for the inhibition of MMP activity, with the
added benefit of anti-invasion activity. We further demon-
strated that the regulation of MMP-9 and TIMP-1 expression
by 6-shogaol and 6-gingerol in PMA-treated HepG2 cells
and the regulation of TIMP-1 expression by 6-gingerol in
PMA-untreated Hep3B cells was not done through tran-
scriptional regulation (Fig. 7), but rather on the translational
or post-translational level. Owing to this complexity, further
study of the detailed underlying mechanism is needed.
HepG2 and Hep3B are hepatoma cells with and without
the tumor suppressor p53, respectively, while PMA is a well-
known selective tumor activator that is often used to induce
MMP-9 activity and enhance the invasion of HepG2 cells [37].
Wang et al. [38] indicated that p53 suppresses cancer cell
invasion by inducing the MDM2-mediated degradation of an
invasion promoter, Slug. This mechanism may provide a
reasonable explanation for the migratory and invasive activ-
ities conferred by PMA treatment in HepG2 but not Hep3B
cells. The different responses (e.g. mRNA expression of MMP-
9 and TIMP-1) of HepG2 and Hep3B cells when treated with
6-shogaol and 6-gingerol are to be expected.
5 Concluding remarks
The results in this study suggest that 6-shogaol and 6-
gingerol could possess potential anti-invasive activity against
hepatoma cells, with 6-shogaol being more effective than 6-
gingerol. The proposed anti-invasion mechanisms for these
two compounds on hepatoma cells might be mediated
through the inhibition of MMP-9 and the induction of
TIMP-1, but only 6-shogaol decreased uPA activity in Hep3B
cells. Further analysis with semi-quantitative RT-PCR
showed that the regulation of MMP-9 by 6-shogaol and 6-
gingerol and the regulation of TIMP-1 by 6-shogaol in
Hep3B cells may on the transcriptional level. From the
results presented here, 6-shogaol and 6-gingerol could be
used to further test the effect of their signal transduction
pathways on MMP-9 suppression and TIMP-1 induction for
the prevention of hepatoma invasion or metastasis.
This research was partially supported by National Science
Council (NSC98-2622-B005-010-CC2), Republic of China.
The authors have declared no conflict of interest.
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