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[6]-Gingerol Suppresses Colon Cancer Growth by Targeting Leukotriene A4 Hydrolase

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  • College of Pharmacy, Keimyung University

Abstract and Figures

[6]-Gingerol, a natural component of ginger, exhibits anti-inflammatory and antitumorigenic activities. Despite its potential efficacy in cancer, the mechanism by which [6]-gingerol exerts its chemopreventive effects remains elusive. The leukotriene A(4) hydrolase (LTA(4)H) protein is regarded as a relevant target for cancer therapy. Our in silico prediction using a reverse-docking approach revealed that LTA(4)H might be a potential target of [6]-gingerol. We supported our prediction by showing that [6]-gingerol suppresses anchorage-independent cancer cell growth by inhibiting LTA(4)H activity in HCT116 colorectal cancer cells. We showed that [6]-gingerol effectively suppressed tumor growth in vivo in nude mice, an effect that was mediated by inhibition of LTA(4)H activity. Collectively, these findings indicate a crucial role of LTA(4)H in cancer and also support the anticancer efficacy of [6]-gingerol targeting of LTA(4)H for the prevention of colorectal cancer.
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[6]-Gingerol Suppresses Colon Cancer Growth by Targeting
Leukotriene A
4
Hydrolase
Chul-Ho Jeong,1Ann M. Bode,1Angelo Pugliese,1Yong-Yeon Cho,1Hong-Gyum Kim,1
Jung-Hyun Shim,1Young-Jin Jeon,1,2 Honglin Li,3,4 Hualiang Jiang,3,4 and Zigang Dong1
1The Hormel Institute, University of Minnesota, Austin, Minnesota; 2Department of Pharmacology, College of Medicine, Chosun University,
Gwanju, Republic of Korea; 3Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia
Medica, Chinese Academy of Science; and 4School of Pharmacy, East China University of Science and Technology, Shanghai, China
Abstract
[6]-Gingerol, a natural component of ginger, exhibits anti-
inflammatory and antitumorigenic activities. Despite its
potential efficacy in cancer, the mechanism by which [6]-
gingerol exerts its chemopreventive effects remains elusive.
The leukotriene A
4
hydrolase (LTA
4
H) protein is regarded as a
relevant target for cancer therapy. Our in silico prediction
using a reverse-docking approach revealed that LTA
4
H
might be a potential target of [6]-gingerol. We supported
our prediction by showing that [6]-gingerol suppresses
anchorage-independent cancer cell growth by inhibiting
LTA
4
H activity in HCT116 colorectal cancer cells. We showed
that [6]-gingerol effectively suppressed tumor growth in vivo
in nude mice, an effect that was mediated by inhibition of
LTA
4
H activity. Collectively, these findings indicate a crucial
role of LTA
4
H in cancer and also support the anticancer
efficacy of [6]-gingerol targeting of LTA
4
H for the prevention
of colorectal cancer. [Cancer Res 2009;69(13):5584–91]
Introduction
Chemoprevention by plant-derived compounds or dietary
phytochemicals has emerged as an accessible and promising
approach to cancer control and management (1). Of the many
phytochemicals displaying a wide array of biochemical and
pharmacologic activities, [6]-gingerol, the major pharmacologically
active component of ginger, was reported to exhibit antioxidant
and anti-inflammatory properties and exert substantial anticarci-
nogenic and antimutagenic activities (2). Several lines of evidence
suggest that [6]-gingerol is effective in the suppression of the
transformation, hyperproliferation, and inflammatory processes
that initiate and promote carcinogenesis, as well as the later steps
of carcinogenesis, namely, angiogenesis and metastasis (3–7).
Despite its anticancer activity against several human cancers, the
exact molecular mechanism by which [6]-gingerol exerts its
chemopreventive effects is not fully understood. Identification of
molecular and cellular targets, which are associated with the
suppression of cell malignancy, is important in the prevention of
cancer and will provide a better understanding of anticancer
mechanisms. Therefore, the delineation of the molecular mecha-
nism of action exerted by [6]-gingerol merits further investigation.
The leukotrienes compose a class of structurally related para-
crine hormones derived from the oxidative metabolism of
arachidonic acid and are implicated in human cancer and chronic
inflammation (8, 9). Leukotrienes are found at high levels in most
inflammatory lesions and are involved in the physiologic changes
that are characteristic of the inflammatory process (10). Previous
studies showed that leukotrienes, such as leukotriene B
4
(LTB
4
), a
potent chemoattractant that induces a vigorous inflammatory
response, are implicated in cancer development (11–14). Because
LTB
4
was shown to play a role in carcinogenesis, recent studies
focused on leukotriene A
4
hydrolase (LTA
4
H) as an attractive target
for chemoprevention and cancer therapy (15). LTA
4
H is a
bifunctional zinc enzyme that catalyzes the final rate-limiting step
in the biosynthesis of LTB
4
. Besides catalyzing the production of
LTB
4
, LTA
4
H also possesses aminopeptidase activity (16). Although
few physiologic substrates have been identified, the suggestion was
made that LTA
4
H might participate in the processing of peptides
related to inflammation and carcinogenesis. LTA
4
H was shown to
exhibit high levels of protein expression in certain types of cancers,
and its inhibition leads to reduced cancer incidence in animal
models (17, 18). The analysis of the cocrystal structure of LTA
4
H
with its inhibitor has provided excellent opportunities for
structure-based drug development (19).
Here we found that LTA
4
H is overexpressed in several human
cancer cell lines, including colorectal cancers. Knockdown of
LTA
4
H provided new direct evidence showing that LTA
4
H is
implicated in the anchorage-independent growth of HCT116 colon
cancer cells. Moreover, our findings showed that [6]-gingerol
suppresses tumor growth of HCT116 cells implanted in nude mice
by inhibiting the enzymatic activity of LTA
4
H. These data indicate
that LTA
4
H might be a highly desirable target for the prevention of
colorectal cancers.
Materials and Methods
Reagents. [6]-Gingerol (98% purity verified by TLC) was from Dalton
Chemical Laboratories. Basal medium Eagle (BME), gentamicin, and
L-glutamine were purchased from Life Technologies, Inc. CNBr-Sepharose
4B was purchased from Amersham Pharmacia Biotech. The LTA
4
H human
recombinant protein and its antibody for Western blot analysis were
purchased from Cayman Chemical. The 29-mer small hairpin RNA
(shRNA) construct against LTA
4
H used in this study was from OriGene
Technologies, Inc.
Cell culture and transfection. H520, H1299, HCT15, and LNCaP cells
were cultivated in RPMI supplemented with 10% fetal bovine serum (FBS)
and antibiotics in a 5% CO
2
incubator. HCT116, HT29, and SKBR3 cells were
maintained in McCoy’s 5A medium. For transfection experiments, jetPEI
(Qbiogen, Inc.) transfection reagent was used following the manufacturer’s
instructions.
In silico target identification. To find the potential binding proteins of
[6]-gingerol, the potential drug target database (PDTD; ref. 20; v. 2007) was
Note: Supplementary data for this article are available at Cancer Research Online
(http://cancerres.aacrjournals.org/).
Requests for reprints: Zigang Dong, University of Minnesota, 801 16th Avenue
Northeast, Austin, MN 55912-3679. Phone: 507-437-9600; Fax: 507-437-9606; E-mail:
zgdong@hi.umn.edu.
I2009 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-09-0491
Cancer Res 2009; 69: (13). July 1, 2009 5584 www.aacrjournals.org
Research Article
used. The PDTD contains structural information (e.g., active site) of more
than 830 known or potential protein drug targets. [6]-Gingerol was docked
to each target in PDTD with the reverse docking tool TarFisDock (21). More
details on the reverse docking procedure are given elsewhere (21–25). The
protein ‘‘hits’’ identified through the reverse docking method (i.e., the top
2% of ranked list) are considered as potential target candidates for further
validation studies.
Molecular modeling. Considering the structural similarity between [6]-
gingerol and bestatin, the LTA
4
H crystal structure (PDB code 1HS6) was
chosen for further docking studies, which were carried out using the Maestro
suite of software (Maestro, version 7.5, Schro¨dinger). [6]-Gingerol was docked
within the LTA
4
H binding site using the QM-Polarized ligand docking (26).
Soft agar formation assay. Cells (8 !10
3
per well) were suspended in
BME (1 mL with 10% FBS and 0.33% agar) and plated over a layer of
solidified BME/10% FBS/0.5% agar (3.5 mL) with various concentrations of
[6]-gingerol. The cultures were maintained at 37jC in a 5% CO
2
incubator
for 6 to 7 d, and the colonies were counted under a microscope using the
Image-Pro Plus software (v. 4) program (Media Cybernetics).
Western blot analysis. Proteins were resolved by SDS-PAGE and
transferred onto polyvinylidene difluoride membranes (Amersham Phar-
macia Biotech), which were blocked and hybridized with specific primary
antibodies. The protein bands were visualized using an enhanced
chemiluminescence reagent (Amersham Biosciences Corp.) after hybridiza-
tion with a horseradish peroxidase–conjugated secondary antibody.
In vitro pull-down assay. Recombinant human LTA
4
H (0.5 Ag) or
endogenous cell lysates (500 Ag) were incubated with [6]-gingerol-
Sepharose 4B (or Sepharose 4B only as a control) beads (50 AL, 50% slurry)
in reaction buffer [50 mmol/L Tris (pH 7.5), 5 mmol/L EDTA, 150 mmol/L
NaCl, 1 mmol/L DTT, 0.01% NP40, 2 Ag/mL bovine serum albumin,
0.02 mmol/L phenylmethylsulfonyl fluoride (PMSF), 1!protease inhibitor
mixture]. After incubation with gentle rocking overnight at 4jC, the beads
were washed five times with buffer [50 mmol/L Tris (pH 7.5), 5 mmol/L
EDTA, 150 mmol/L NaCl, 1 mmol/L DTT, 0.01% NP40, 0.02 mmol/L PMSF],
and proteins bound to the beads were analyzed by Western blotting.
Cell proliferation assay. Cells were seeded (2 !10
3
per well) in 96-well
plates. After incubating for various periods of time, 20 AL of CellTiter96
Aqueous One Solution (Promega) were added and then cells were further
incubated for 1 h at 37jC in a 5% CO
2
incubator. Absorbance was measured
at 492 nm.
LTA
4
H enzymatic assay. Aminopeptidase activity was determined by a
modification of a published procedure (27). Recombinant human LTA
4
H
(0.5 Ag) was incubated for 15 min at room temperature in assay buffer
Figure 1. [6]-Gingerol specifically binds with Glu271 of LTA
4
H. A, proposed molecular model of [6]-gingerol binding with LTA
4
H. The catalytic, NH
2
-terminal, and
COOH-terminal LTA
4
H domains are in cartoon representation in yellow, orange, and green colors, respectively. [6]-Gingerol is depicted in stick and transparent surface
area, and the zinc ion is represented as a purple sphere. B, close-up view of the interactions of [6]-gingerol within the LTA
4
H catalytic site. The hydrogen bond between
the ligand and Glu271 (both in stick representation) is shown as a dotted black line. The amino acids coordinating the zinc ion are in stick representation and the
rest of the catalytic domain is shown as a transparent cartoon. C, [6]-gingerol specifically binds with LTA
4
Hin vitro and ex vivo . The in vitro (top ) and ex vivo (bottom )
binding of [6]-gingerol with LTA
4
H was confirmed by pull-down assay using [6]-gingerol-Sepharose 4B beads and subsequent Western blot analysis. D, LTA
4
H from cell
lysates was pulled down using [6]-gingerol-Sepharose 4B beads, and the binding affinity with [6]-gingerol was determined by Western blot analysis.
[6]-Gingerol Suppresses Colon Cancer
www.aacrjournals.org 5585 Cancer Res 2009; 69: (13). July 1, 2009
[50 mmol/L Tris-Cl (pH 8.0), 100 mmol/L KCl] in the presence of various
concentrations of [6]-gingerol. Then the substrate (L-alanine-4-nitro-anilide
hydrochloride, Sigma Chemical Co.) was added to a final concentration of
5 mmol/L. To measure the LTB
4
levels, HCT116 or HT29 colon cancer cells
were preincubated with [6]-gingerol for 24 h and then incubated
with serum-free medium containing 5 Amol/L calcium ionophore A23187,
1.6 mmol/L CaCl
2
, and 10 Amol/L arachidonic acid at 37jC for 30 min.
Immunoreactive LTB
4
was quantified by ELISA (Cayman Chemical)
following the supplier’s instructions.
Mice. Athymic mice [Cr:NIH(S), NIH Swiss nude, 6–9 wk old] were
purchased from the National Cancer Institute (NIH) and were maintained
under ‘‘specific pathogen-free’’ conditions according to guidelines estab-
lished by Research Animal Resources, University of Minnesota.
In vivo tumor growth. Mice were divided into three groups: untreated
control group (n= 5; 3 males, 2 females), [6]-gingerol group (n= 21; 10
males, 11 females), and vehicle group (n= 20; 10 males, 10 females). A
separate group of 5 untreated control mice was maintained as a negative
control for comparison of body weights and spontaneous tumor
development. For the [6]-gingerol group, 500 Ag of [6]-gingerol in ethanol
(0.001 AL) suspended in 50 AL autoclaved water were fed to each mouse by
gavage in this group three times a week. The dose of [6]-gingerol was based
on preliminary pilot studies and also extrapolated from cell culture
experiments. For the vehicle-treated group, 0.001 AL of 100% ethanol
suspended in 50 AL autoclaved water was fed to each mouse by gavage in
this group three times a week. Before tumor cell injection, mice were fed
either 500 Ag of [6]-gingerol or vehicle (ethanol) three times a week for 2 wk.
At the beginning of the 3rd week, HCT116 colon cells (3 !10
6
) were
injected into the right flank of each mouse. Following injection, mice
continued to be fed 500 Ag [6]-gingerol or vehicle three times a week. Mice
were weighed and tumors measured by caliper twice a week. Tumor volume
was calculated from measurements of two diameters of the individual
tumor according to the formula: tumor volume (mm
3
) = [longer diameter !
shorter diameter
2
]/2. Mice were monitored until tumors reached 1 cm
3
total
volume at which time mice were euthanized and tumors extracted. All
studies were done according to guidelines approved by the University of
Minnesota Institutional Animal Care and Use Committee.
Statistical analysis. All quantitative data are presented as mean value F
SD unless indicated otherwise. The statistical significance of compared
measurements was measured using the Student’s ttest or one-way ANOVA,
and P< 0.05 was considered significant.
Results
[6]-Gingerol specifically binds with LTA
4
Hin vitro and
ex vivo. We conducted in silico screening using a reverse-docking
approach to elucidate potential targets of [6]-gingerol. [6]-Gingerol
was reversely screened against the Potential Drug Target Database
(PDTD; ref. 20) of f1,200 protein entries. The top 2% of the ranked
list of molecules identified by reverse docking for [6]-gingerol with
all potential targets is shown in Supplementary Table S1. Among
others, LTA
4
H was identified as a possible molecular target for [6]-
gingerol (Supplementary Table S1 and Supplementary Discussion).
Accumulating evidence supports a functional role for LTA
4
H in
cancer development, and therefore targeting LTA
4
H is regarded as
a useful strategy in chemoprevention and cancer therapy (15).
Interestingly, our docking model (Fig. 1A) showed that [6]-gingerol
might bind to LTA
4
H in a manner similar to bestatin (19), which is
a well-known inhibitor of LTA
4
H and other aminopeptidases. In
fact, bestatin and [6]-gingerol may share the same localization
within the LTA
4
H catalytic pocket (Supplementary Fig. S1). Similar
to bestatin, [6]-gingerol, with its carbonyl and hydroxyl oxygens,
seems to be able to participate in the coordination of the zinc
ion with its hydroxyl group to form a hydrogen bond with Glu271
(Fig. 1B).
Figure 2. LTA
4
H is highly expressed and
required for the growth of HCT116 cells. A,
Western blot analysis of LTA
4
H expression
in several cancer cell lines. Top, H520 and
H1299: lung adenocarcinoma; HCT15,
HT29, and HCT116: colorectal carcinoma;
LNCaP: prostate carcinoma; SKBR3:
breast carcinoma. Bottom, densitometric
analysis of expression level of LTA
4
H
normalized against h-actin. B, HCT116
cells were transfected with shRNA pRS
noneffective GFP plasmid (CONT) or
shRNA pRS LTA
4
H plasmid (KD1, KD2),
and stable colonies were selected by
puromycin. Knockdown of LTA
4
H was
analyzed by Western blot. The
percent knockdown was assessed by
densitometry (top). C, using control
(CONT), KD1-LTA
4
H, and KD2-LTA
4
H
stable cells, cell proliferation was
determined at 24-h intervals up to 72 h.
Points, mean from three independent
experiments; bars, SD. *, P< 0.01,
significant decrease in proliferation rate
compared with control group. D, colony
formation in soft agar using control,
KD1-LTA
4
H, or KD2-LTA
4
H cells. Cells
were grown in soft agar for 6 d and then
colonies were counted using a microscope
and the Image-Pro PLUS software
program (v.4). Columns, mean from three
independent experiments; bars, SD.
*, P< 0.01, significant decrease in colony
formation versus control cells.
Cancer Research
Cancer Res 2009; 69: (13). July 1, 2009 5586 www.aacrjournals.org
To confirm this prediction, we performed an in vitro pull-down
assay using [6]-gingerol–conjugated to Sepharose 4B beads. Results
revealed that recombinant LTA
4
H binds with [6]-gingerol-
Sepharose 4B beads, but not with Sepharose 4B beads alone
(Fig. 1C, top)in vitro . We also confirmed the ex vivo binding of
[6]-gingerol with endogenous LTA
4
H in HCT116 cells (Fig. 1C,
bottom). These results clearly support our hypothesis that LTA
4
H
is a target for [6]-gingerol in vitro and ex vivo.
To further identify the amino acid residues of LTA
4
H that are
required for its binding with [6]-gingerol, we constructed full-
length wild-type (WT) LTA
4
H, mock, and three LTA
4
H mutants,
including [E271A]LTA
4
H, [H295A]LTA
4
H, and [D375A]LTA
4
H. The
mutants were based on the molecular modeling results, which
suggested that Glu271 of LTA
4
H might be involved in the binding of
[6]-gingerol. The WT, mock, and mutant plasmids of LTA
4
H were
transfected into HEK293 cells to determine whether the substitu-
tion of alanine (Ala) for Glu271, His295, or Asp375 would affect the
binding affinity of LTA
4
H with [6]-gingerol. The ectopically
expressed WT LTA
4
H interacted strongly with [6]-gingerol
(Fig. 1D). In contrast, the [E271A]LTA
4
H mutant displayed a
markedly reduced binding affinity with [6]-gingerol compared with
the WT, H295A, or D375A mutant (Fig. 1D). This result indicated
that the Glu271 residue is required for LTA
4
H binding with [6]-
gingerol confirming the docking results (Fig. 1Aand B). The docking
model showed no direct interaction between [6]-gingerol and
His295, and indeed, as expected, [H295A]LTA
4
H did not disrupt the
protein-ligand binding (Fig. 1Band D). The [D375A]LTA
4
H mutant
showed a small reduction in binding affinity with [6]-gingerol,
which might be due to a structural change in the binding pocket
that could only partially affect the binding.
Knockdown of LTA
4
H inhibits anchorage-independent
growth of HCT116 colon cancer cells. Previous immunohisto-
chemical analysis suggested that LTA
4
H is highly expressed in
several human cancers including esophageal adenocarcinomas (15).
To determine whether LTA
4
H activity is directly associated with the
tumorigenic properties of cancer cells, we first evaluated the
expression of LTA
4
H in several human cancer cell lines. Compared
with other cancer cell lines, LTA
4
H expression was relatively higher
in colorectal cancer cell lines, especially in HCT116 cells (Fig. 2A).
These data suggested that LTA
4
H might be associated with the
tumorigenic potential of colorectal cancer cells. Based on the finding
that LTA
4
H is highly expressed in HCT116 cells, we investigated the
function of LTA
4
H in the growth of this cell line. To assess the effects
of LTA
4
H inhibition on HCT116 colorectal cancer cell growth, we
established two stable HCT116 clones (KD1-LTA
4
H and KD2-LTA
4
H)
that express shRNAs targeting different sequences of LTA
4
H. The
specificity of shRNA targeting of LTA
4
H was confirmed by Western
blot analysis. Notably, a substantially reduced expression level of
LTA
4
H was observed in clone KD2-LTA
4
H (KD2) compared with the
control cells that express GFP-shRNA (Fig. 2B). Additional results
indicated that the rate of proliferation of KD2-LTA
4
H cells was
delayed compared with control cells (Fig. 2C).
Based on the finding that knockdown of LTA
4
H is associated with
a reduced proliferation rate, we examined whether knockdown of
Figure 3. [6]-Gingerol inhibits LTA
4
H
activity and suppresses the growth of colon
cancer cells. A, secretion of LTB
4
in HT29
or HCT116 cells was quantified by ELISA.
Columns, mean from three independent
experiments; bars, SD. *, P< 0.01,
significant decrease in LTB
4
secretion
in [6]-gingerol–treated cells compared
with the DMSO-treated group.
B, aminopeptidase activity was determined
in a spectrophotometric assay at 405 nm.
Columns, mean from three independent
experiments; bars, SD. *, P< 0.01,
significant decrease in activity in cells
treated with [6]-gingerol compared with the
DMSO-treated group. C, [6]-Gingerol
suppresses anchorage-dependent growth
of HCT116 or HT29 cells. Cell proliferation
was estimated by MTS assay. Absorbance
(A
492
) was read at 24-h intervals up to
72 h. Points, mean from three independent
experiments; bars, SD. *, P< 0.01,
significant decrease in proliferative
rate compared with control group. D,
[6]-gingerol inhibits anchorage-independent
growth of HCT116 or HT29 cells.
Columns, mean from three independent
experiments; bars, SD. *, P< 0.01,
significant decrease in colony formation in
cells treated with [6]-gingerol compared
with the DMSO-treated group.
[6]-Gingerol Suppresses Colon Cancer
www.aacrjournals.org 5587 Cancer Res 2009; 69: (13). July 1, 2009
LTA
4
H would affect cell growth under anchorage-independent
conditions. Anchorage-independent growth ability is an in vitro
indicator and a key characteristic of the transformed cell phenotype
(28). Our results revealed that the knockdown of LTA
4
H in HCT116
cells by shRNA (KD1 or KD2) resulted in fewer colonies being
formed in soft agar compared with control cells (Fig. 2D). These
results suggest that blocking LTA
4
H in HCT116 colon cancer cells
reduces the malignant potential of these cells.
[6]-Gingerol inhibits LTA
4
H activity and suppresses colon
cancer cell growth. Based on our results showing that [6]-gingerol
directly binds with LTA
4
H, we then investigated whether [6]-
gingerol inhibits LTA
4
H enzyme activity. We first measured the
secreted LTB
4
levels in HCT116 and HT29 cells. Results showed
that [6]-gingerol suppresses LTB
4
production in both cell lines
(Fig. 3A). Moreover, the inhibitory effect of [6]-gingerol against
aminopeptidase activity was further evaluated in vitro by using a
p-nitroanilide derivative of alanine (Ala-p-NA) as substrate. The
aminopeptidase activity of LTA
4
H was also potently suppressed by
[6]-gingerol (Fig. 3B).
Next, we evaluated the effect of [6]-gingerol treatment on
proliferation of the colorectal cancer cell lines HCT116 and HT29.
Data indicate that [6]-gingerol treatment significantly inhibits
HCT116 cell growth at 100 Amol/L (Fig. 3C, left) or HT29 growth in
a dose-dependent manner (Fig. 3C, right). In addition, we examined
the effect of [6]-gingerol on anchorage-independent growth of
HCT116 or HT29 cells, which highly express LTA
4
H. Cells were
cultured for 6 days in medium containing various concentrations
(0–100 Amol/L) of [6]-gingerol. Control (DMSO-treated) cells grew
readily and formed many colonies in soft agar (Fig. 3D) in both
HCT116 (Fig. 3D, left) and HT29 cells (Fig. 3D, right). On the other
hand, [6]-gingerol–treated cells showed an impaired anchorage-
independent growth capability, leading to a significant dose-
dependent reduction in colony formation (Fig. 3D).
LTA
4
H activity enhances anchorage-independent growth of
HCT116 cells. Our data indicated that the Glu271 residue of LTA
4
H
was required for binding with [6]-gingerol. Previous data presented
by others (29) suggested that Glu271 is the recognition site for the
NH
2
-terminal amino group of the peptidase substrate. We therefore
determined whether blocking the aminopeptidase activity of
LTA
4
H would have an effect on its ability to induce anchorage-
independent cell growth. WT or [E271A]LTA
4
H was transiently
transfected into HCT116 cells and aminopeptidase activity was
measured. As expected, the expression of WT-LTA
4
H, but not of
[E271A]LTA
4
H, strongly increased the aminopeptidase activity,
indicating the importance of the Glu271 residue of LTA
4
H in its
aminopeptidase activity (Fig. 4A). To determine whether the
aminopeptidase activity of LTA
4
H is involved in anchorage-
independent cell growth, we transfected WT or [E271A]LTA
4
H
Figure 4. LTA
4
H activity is required for
HCT116 cell growth in soft agar.
A, HCT116 cells were transfected with a
pcDNA4A-Mock, pcDNA4A-LTA
4
H,
or mutant [E271A]LTA
4
H plasmid and
LTA
4
H expression level was confirmed by
Western blot analysis using an antibody
against Xpress or LTA
4
H. h-Actin was used
as a loading control (left ). Cell lysates
(50 Ag) were incubated with 5 mmol/L
L-alanine-4-nitro-anilide hydrochloride and
aminopeptidase activity was determined
with a spectrophotometer at 405 nm (right).
*, P< 0.01, versus MOCK. B, stable
KD-LTA
4
H cells were transfected with a
pcDNA4A-LTA
4
H or [E271A]LTA
4
H
plasmid, and expression level was
confirmed by Western blot analysis (left ).
Right, colony formation of KD2-LTA
4
H cells
transfected with WT or [E271A]LTA
4
H.
Columns, mean from three independent
experiments; bars, SD. *, P< 0.01,
WT-transfected cells compared with
MOCK-transfected cells. C, soft agar
formation assay of KD2-LTA
4
H cells
treated with the indicated dose of LTB
4
.
Columns, mean from three independent
experiments; bars, SD. *, P< 0.05, versus
LTB
4
nontreated control.
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Cancer Res 2009; 69: (13). July 1, 2009 5588 www.aacrjournals.org
into KD2-LTA
4
H cells and assessed colony formation in soft agar.
Although an enhanced number of colonies was observed in WT and
[E271A]LTA
4
H–transfected cells compared with mock-transfected
cells, a higher level of recovery in colony formation was detected in
WT compared with [E271A]LTA
4
H–transfected cells, which lack
aminopeptidase activity (Fig. 4B). These results imply that the
aminopeptidase activity of LTA
4
H might be necessary for
anchorage-independent growth of HCT116 cells. In addition,
treatment of KD2-LTA
4
H cells with LTB
4
also enhanced soft agar
colony formation, indicating that the level of LTB
4
that is produced
by the epoxide hydrolase activity of LTA
4
H contributes to
anchorage-independent growth of HCT116 cells (Fig. 4C).
[6]-Gingerol suppresses tumor growth by inhibiting LTA
4
H
activity in vivo.Based on the results of our ex vivo and in vitro
data, we evaluated whether [6]-gingerol could suppress tumor
growth in vivo. The body weights of [6]-gingerol– or vehicle-treated
groups were similar throughout the study (data not shown). The
first measurable tumors (minimum of 13.5 mm
3
) were observed in
both experimental groups on day 15 after injection (data not
shown). However, the vehicle-treated group had 13 measurable
tumors, whereas only 4 tumors were large enough to be measured
in the [6]-gingerol–treated group (data not shown). Furthermore,
all mice in the vehicle-treated group had developed measurable
tumors by day 28 after injection, whereas all mice (except one) in
the [6]-gingerol group did not develop measurable tumors until day
38. Furthermore, results showed that mice fed [6]-gingerol survived
significantly longer than those receiving vehicle, implying that
tumors grew much slower. Specifically, as of day 49 after injection,
all vehicle-treated mice had been euthanized due to tumor size
equal to 1 cm
3
. On the other hand, at day 49, 11 of the [6]-gingerol–
treated mice still had not developed tumors equal to 1 cm
3
(Fig. 5A). Collectively, the results presented in Fig. 5Bshow that
mean tumor volume in the vehicle-treated group increased
significantly faster than that in the [6]-gingerol–treated group
(P< 0.001). To further determine whether the antitumor effect of
[6]-gingerol was associated with inhibition of LTA
4
H, tumor
extracts from each of the three vehicle-treated and three [6]-
gingerol–treated mice (i.e., euthanized on the same day of the
experiment) were prepared and analyzed for LTA
4
H expression and
the production of LTB
4
. Western blot analyses revealed that the [6]-
gingerol–treated tumor extracts exhibited substantially decreased
LTA
4
H expression level compared with vehicle-treated tumors
(Fig. 5C). Consistent with this result, ELISA data showed that [6]-
gingerol–treated tumors exhibited a much lower level of LTB
4
production, suggesting that [6]-gingerol inhibits colon tumor
formation by suppressing LTA
4
H activity in vivo (Fig. 5D).
Discussion
Colorectal carcinoma, the third leading cause of cancer-related
deaths in the United States, is a highly preventable cancer with a
transition from precursor to malignant lesion of 10 to 15 years (25).
Chemoprevention by consumption of edible phytochemicals has
gained considerable attention as a promising strategy for reducing
the incidence of colorectal cancer as well as other cancers. Our
results herein clearly show a role of [6]-gingerol as a chemo-
preventive and/or chemotherapeutic agent for colorectal carcino-
mas and strongly suggest that LTA
4
H is a potential therapeutic
target of [6]-gingerol. Notably, LTA
4
H has long been recognized as
an anti-inflammatory target. Its enzymatic product, LTB
4
, is widely
Figure 5. [6]-Gingerol suppresses tumor growth through inhibition of LTB
4
production. A, mice fed [6]-gingerol survived significantly longer than did mice fed vehicle.
According to the guidelines of the University of Minnesota Institutional Animal Care and Use Committee, mice were to be euthanized when tumor size reached 1 cm
3
.
Thus, based on this stipulation, mice fed [6]-gingerol survived significantly longer than those fed vehicle (ethanol). All vehicle-treated mice were euthanized by
day 50 (after injection) because of attaining maximal allowable tumor size. The study was terminated at day 75 after injection when all but one [6]-gingerol–fed mouse
had reached the maximal allowable tumor size of 1 cm
3
(*, P< 0.05). B, total mean (average) tumor volume in the [6]-gingerol–treated group increased significantly
less than that of the vehicle-treated group. Tumor volume was measured and recorded twice a week for the duration of the study (*, P< 0.001). Points, mean;
bars, SE (Aand B). Significant differences were determined by one-way ANOVA. C, expression of LTA
4
H in vehicle- or [6]-gingerol–treated tumor tissues (n= 3).
D, analysis of vehicle- or [6]-gingerol–treated LTB
4
tissue level by ELISA. The amount of LTB
4
is expressed as picograms per milligram of protein (n= 3).
[6]-Gingerol Suppresses Colon Cancer
www.aacrjournals.org 5589 Cancer Res 2009; 69: (13). July 1, 2009
implicated in the pathogenesis of several inflammatory diseases,
including asthma, psoriasis, rheumatoid arthritis, and bowel
disease (30). In addition, previous reports provide evidence
supporting a possible role for LTA
4
H and LTB
4
in cancer cell
progression. Notably, higher expression of LTA
4
H (17) and an
elevated production level of LTB
4
(31) in colon cancer tissue have
been reported. In addition, LTB
4
was reported to stimulate the
proliferation of colorectal cancer cells (32). Consistent with these
findings, our observations also showed that LTA
4
H was highly
expressed in most of the human colorectal cancer cell lines tested
and knockdown of LTA
4
H impaired the growth of HCT116 colon
cancer cells, suggesting that LTA
4
H might play an important role in
the promotion and progression of colorectal carcinomas.
Carcinogenesis is a multistep process accompanying molecular
alterations that drive the progressive transformation of normal
cells into highly malignant derivatives. One of the noticeable
characteristics of malignant cancer cells is the ability to survive
and grow in the absence of anchorage to an extracellular matrix
(28, 33). Our new evidence showing that HCT116 cells with
knockdown of LTA
4
H (i.e., KD-LTA
4
H cells) were less capable of
surviving under anchorage-independent growth conditions sug-
gests a crucial role for LTA
4
H in colorectal cancer cell malignancy.
We also showed that LTA
4
H enhanced HCT116 cell growth in soft
agar through its aminopeptidase and epoxide hydrolase activity.
Overall, this evidence strongly indicates that inhibition of LTA
4
H
activity might be a potential target to prevent colorectal carcinoma
promotion and progression.
Bestatin, a classic aminopeptidase inhibitor, is known to bind
the Glu296 residue of LTA
4
H to inhibit both enzyme activities (34).
Notably, our results indicate that [6]-gingerol binds to Glu271 and
also inhibits both the aminopeptidase and epoxide hydrolase
activities of LTA
4
H. Because Glu271 is the recognition site for the
NH
2
-terminal amino group of the peptidase substrate (29), [6]-
gingerol could inhibit the binding of known and unknown
peptidase substrates to LTA
4
H. Although the mechanism is not
entirely clear, [6]-gingerol seems to inhibit the epoxide hydrolase
activity of LTA
4
H in a manner similar to bestatin and results in a
reduced anchorage-independent growth of HCT116 cells in soft
agar. Recovery experiments using knockdown-LTA
4
H cells trans-
fected with wild-type LTA
4
H or treated with LTB
4
confirmed that
these activities of LTA
4
H are required for colony formation in
soft agar.
We and others have reported that [6]-gingerol inhibits cell
transformation and mouse skin carcinogenesis. Indeed, [6]-gingerol
was reported to suppress epidermal growth factor–induced
neoplastic transformation in mouse epidermal JB6 cells (3), 7,12-
dimethylbenz(a)anthracene–induced skin cancer promotion in ICR
mice (35), and 12-O-tetradecanoylphorbol-13-acetate–induced
cyclooxygenase-2 (COX-2) expression in a mouse skin cancer
model (4). In addition, [6]-gingerol inhibits angiogenesis and
metastasis (5, 6), which suggests a broad anticancer activity of [6]-
gingerol mediated by multiple mechanisms in various cancers. Our
results herein are noteworthy in that promotion of colorectal
carcinoma can be delayed and suppressed by [6]-gingerol in vivo.
Moreover, the low in vivo toxicity and potent tumor inhibitory
activity of [6]-gingerol observed in nude mice suggest that [6]-
gingerol is an effective chemopreventive agent for colorectal
carcinoma. In conclusion, we showed here that LTA
4
H is closely
associated with colorectal cancer cell growth, promotion, and
progression. Moreover, we provided clear evidence showing that
[6]-gingerol effectively suppresses anchorage-independent cell
growth and in vivo tumor growth in HCT116 cancer cell–bearing
nude mice by inhibiting LTA
4
H activity. Collectively, these findings
support the anticancer efficacy of [6]-gingerol through its targeting
of LTA
4
H for the prevention of colorectal cancer progression.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
Received 2/25/09; revised 4/16/09; accepted 4/16/09; published OnlineFirst 6/16/09.
Grant support: The Hormel Foundation (Austin, MN), Pediatric Pharmaceuticals
(Iselin, NJ), Korea Research Foundation Grant (Korean Government; KRF-2007-357-
C00084), the 863 Hi-Tech Program of China (grant 2007AA02Z304), and the Shanghai
Committee of Science and Technology (Grant 07dz22004).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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