[6]-Gingerol Induces Cell Cycle Arrest and Cell Death of Mutant p53-expressing Pancreatic Cancer Cells

Yonsei Medical Journal
Vol. 47, No. 5, pp. 688 - 697, 2006
[6]-Gingerol Induces Cell Cycle Arrest and Cell Death of
Mutant p53-expressing Pancreatic Cancer Cells
Yon Jung Park,1 Jing Wen,1 Seungmin Bang,2 Seung Woo Park,2 and Si Young Song2
1Brain Korea 21 Project for Medical Science, 2Internal Medicine and Institute of Gastroenterology, Yonsei University College
of Medicine, Seoul, Korea.
[6]-Gingerol, a major phenolic compound derived from
micals are known to have ability to interfere with
ginger, has anti-bacterial, anti-inflammatory and anti-tumor
carcinogenesis and tumorigenesis. There are many
activities. While several molecular mechanisms have been de-
evidences that many phytochemicals, such as
scribed to underlie its effects on cells in vitro and in vivo, the
underlying mechanisms by which [6]-gingerol exerts anti-tu-
epigallocatechin-3-gallate (EGCG), genestine, tan-
morigenic effects are largely unknown. The purpose of this
geretin, silymarin, silibinin, and quercetin, can
study was to investigate the action of [6]-gingerol on two
inhibit the proliferation or survival of various
human pancreatic cancer cell lines, HPAC expressing wild-
cancer cells and also induce cell cycle arrest.1-5
type (wt) p53 and BxPC-3 expressing mutated p53. We found
Plant of ginger (Zingeiber officinale Roscoe,
that [6]-gingerol inhibited the cell growth through cell cycle
arrest at G1 phase in both cell lines. Western blot analyses
Zingiberaceae) family is one of the most highly
indicated that [6]-gingerol decreased both Cyclin A and
consumed dietary substances in the world. In
Cyclin-dependent kinase (Cdk) expression. These events led to
China and Malaysia, the rhizome of ginger has
reduction in Rb phosphorylation followed by blocking of S
been used in traditional oriental herbal medicine
phase entry. p53 expression was decreased by [6]-gingerol
for the management of common cold, digestive dis-
treatment in both cell lines suggesting that the induction of
Cyclin-dependent kinase inhibitor, p21cip1, was p53-indepen-
orders, rheumatism, neuralgia, colic and motion-
dent. [6]-Gingerol induced mostly apoptotic death in the mutant
sickness.6 The oleoresin from rhizome of ginger
p53-expressing cells, while no signs of early apoptosis were
contains pungent ingredients including gingerol,
detected in wild type p53-expressing cells and this was related
shoagol, and zingerone.7 Recently, these phenolic
to the increased phosphorylation of AKT. These results suggest
substances have been found to possess many in-
that [6]-gingerol can circumvent the resistance of mutant p53-
expressing cells towards chemotherapy by inducing apoptotic
teresting pharmacological and physiological acti-
cell death while it exerts cytostatic effect on wild type p53-
vities. Of these, [6]-gingerol (1-[4'-hydroxy-3'-
expressing cells by inducing temporal growth arrest.
methoxyphenyl]-5-hydroxy-3-decanone), the major
Key Words: [6]-gingerol, pancreatic cancer, G1 phase,
pungent principle of ginger, has anti-oxidant,
apoptosis, AKT
anti-inflammation and anti-tumor promoting acti-
vities.7
Anti-cancer and/or chemopreventive activities
INTRODUCTION
of [6]-gingerol have been reported. For instance,
[6]-gingerol inhibited pulmonary metastasis in
Various plant-derived compounds or phytoche-
mice bearing B16F10 melanoma cells through the
activation of CD8+ T cells.8 It also inhibited tumor
promotion of ICR mice induced skin tumor by
Received February 1, 2006
Accepted May 4, 2006
tumor promoter (TPA),9 and blocked the azoxy-
This work was supported in part by the Brain Korea 21 Project
methane-induced intestinal carcinogenesis in
for Medical Science.
rodents.10 [6]-Gingerol interfered with EGF-in-
Reprint address: requests to Dr. Si Young Song, Department
duced transformation of mouse epidermal JB6 cell
of Internal Medicine, Yonsei University College of Medicine, 134
line, and reduced the activation of Activator Pro-
Shinchon-dong, Seodaemoon-gu, Seoul 120-752, Korea. Tel: 82-2-
2228-1957, Fax: 82-2-2227-7900, E-mail: sysong@yumc.yonsei.ac.kr
tein-1 (AP-1), which plays a critical role in tumor
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Pancreatic Cancer Cell Death Induced by [6]-Gingerol
promotion.11 Moreover, [6]-gingerol exerted in-
Gingerol's antiproliferative effects are associated
hibitory effects on the cell viability and DNA
with changes in the expression or phosphoryla-
synthesis, also induced apoptosis of human
tion of Cyclin A, Cdks, p21 and Rb, and most
promyelocytic leukemia HL-60 cells.12 Recently, it
commonly, causes cell cycle arrest at the G1
has been reported that ginger root extracts and
phase. We found that [6]-gingerol is capable of
gingerol inhibit the growth of Helicobacter pylori
inducing cell death in the mutant p53-expressing
CagA+ strains, which has a specific gene linked
cells, while arresting mutant p53-expressing cells
to the development of gastric premalignant and
at G1 phase. Thus, [6]-gingerol can circumvent
malignant lesions.13 It suggests that ginger and
drug resistance induced by p53 mutations.
gingerol have effects of chemoprevention to the
gastric-intestinal cancers.
Pancreatic ductal adenocarcinoma (pancreatic
MATERIALS AND METHODS
cancer) is the fifth leading cause of cancer death
in Korea. Pancreatic cancer is a fatal disease, with
Chemicals and cell culture
a 5-year survival rate of less than 5%.14 In the
majority of cases (> 80%), at first diagnosis,
A purified preparation of [6]-gingerol (> 98.0%
pancreatic cancer has already become metastatic
pure) was purchased from Wako Pure Chemicals
so that conventional treatment regimens provide
(Osaka, Japan). It was dissolved in sterile DMSO
minimal, if any, clinical benefit in prolonging life
at a stock concentration of 50 mM and stored at
or ameliorating the negative prognosis of this
-20 . Human pancreatic cancer cells, BxPC-3 and
disease.15 Resistance of recurrent disease to
HPAC, were obtained from the American Type
cytotoxic drugs is the principal factor limiting
Culture Collection (Manassas, VA, USA) and main-
long-term treatment success against pancreatic
tained in RPMI1640 and DMEM/F12 medium
cancer. The oncogenesis of pancreatic cancer in
respectively, containing 10% fetal bovine serum.
particular appears to favor the development and
Both cell lines were incubated at 37
in a
subsequent expansion of cell clones that are
humidified atmosphere with 5% CO2.
resistant to apoptotic triggers. The basis for failed
apoptosis and more specifically the cause of
Assessment of cell viability
chemotherapy resistance in pancreatic cancer is
multifactorial. Molecular mechanisms implicated
The viability of the cells was measured by the
to date include expression of P-glycoproteins and
MTT [3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-
other multidrug resistance proeins, mutations of
zolium bromide] (Sigma, St. Louis, MO, USA)
K-ras and p53, and high-level expression of Bcl-2
assay. The cells (4 × 103 cells/well) were seeded
and other inhibitors of apoptosis. 16-18 Therefore, an
into 96-well plates, after 24 hr they were treated
important research objective is the identification
with various concentration of [6]-gingerol or
of lead compounds that circumvent the resistance
vehicle alone (0.1% DMSO) in serum containing
mechanisms that limit the success of conventional
media. After incubation for indicated times, MTT
drugs.
solution was added to the plate at a final con-
Although anticancer activities of ginger extract
centration of 0.5 mg/mL. The cells were incubated
and constituents have been examined, the under-
for 4 hr in dark at 37 . The resulting MTT-pro-
lying mechanism has not been clarified yet. The
ducts were dissolved by DMSO and determined
purpose of this work was to develop an under-
by measuring the absorbance at 570 nm with
standing of [6]-gingerol's effects on pancreatic
ELISA reader (Molecular Devices, Sunnyvale, CA,
cancer cells to begin to determine its therapeutic
USA). Each point represents the mean of tripli-
value in preventing or treating this disease.
cated experiments.
Therefore we examined the anticancer activities of
[6]-gingerol, and investigated its mechanism in
Cell cycle analysis
two different pancreatic cancer cell lines, HPAC
with wt p53 or BxPC-3 with mutant p53. [6]-
Flow cytometry was performed as previously
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Yon Jung Park, et al.
described.19 Briefly, cells were seeded into 100 mm
then centrifuged at 13,200 rpm for 30 min at 4 .
dishes, after 24 hr treatment with the indicated
The protein concentrations were measured by the
concentrations of [6]-gingerol for indicated hours.
Bradford dye-binding protein assay, using bovine
Following treatment, cells were trypsinized, and
serum albumin (Sigma) as a standard. For Western
washed twice with cold PBS (pH 7.4). The pellet
blot analysis, protein samples (30 g
? ) were solu-
was fixed with cold ethanol (70%) for 12 hr at 4
bilized by boiling in sample buffer and subjected
and washed with cold PBS. Then, they were in-
to SDS-PAGE followed by electrotransfer onto
cubated with RNase (200 ?g/mL final concentra-
nitrocellulose membrane (Amersham Life Science,
tion) and stained with Propidium Iodide (100 g
? /
Buckinghamshire, UK). Blots were blocked for 2
mL final concentration) for 1 hr and analyzed by
hr at rom temperature with 5% nonfat dry milk
flow cytometry. Flow cytometry was performed
and incubated at 4
overnight with the fol-
on a FACSCalibur system equipped with argon-
lowing antibodies: rabbit polyclonal anti-Cyclin A,
ion laser (Becton Dickison Immunocytometry
rabbit polyclonal anti-Cyclin D1, rabbit polyclonal
system, San Jose, CA, USA). Percentages of cells
anti-Cyclin E, rabbit polyclonal anti-Cdk 2 and
in each phase were calculated using Cell Modfit
rabbit polyclonal anti-Cdk 4 (Delta Biolabs, Camp-
software programs (Becton Dickinson).
bell, CA, USA), goat or rabbit polyclonal anti-pRb
(Ser 780) (BioSource, Camarillo, CA, USA), mouse
Assessment of apoptosis
monoclonal anti-Rb, rabbit polyclonal anti-Cdk 6,
rabbit polyclonal anti-p21, mouse monoclonal
To identify cells undergoing apoptosis, Annexin
anti-p53, rabbit polyclonal anti-phosphatidylino-
V-FITC Apoptosis Detection Kit (BD Pharmingen,
sitol-3 kinase (PI3K) p85? subunit, goat polyclo-
Franklin Lakes, NJ, USA) was used. Cells were
nal anti-ERK1, mouse monoclonal anti-pERK,
harvested at different intervals after [6]-gingerol
mouse monoclonal anti-phospho-c-jun-N-terminal
treatment, containing floating and adherent cells.
kinase (pJNK), goat or chicken polyclonal anti-
After washing with cold PBS (pH 7.4), the cells
AKT1, rabbit polyclonal anti-pAKT1/2/3 (Ser
were stained and analyze by the flow cytometry.
473), and mouse monoclonal anti-K-Ras (Santa
For each tube, 20,000 cells were immediately mea-
Cruz Biotechnology Inc., Santa Cruz, CA, USA).
sured on a FACSCalibur flow cytometer. The
The membranes were washed three times and
quantitative analysis was performed with Win
incubated with horseradish peroxidase-conju-
MDI program version 2.8 (provided by the Flow
gated species-appropriate secondary antibodies
Cytometry Core Facility, The Scripps Research
(Santa Cruz). After additional washing, they were
Institute, La Jolla, CA, USA).
developed with enhanced chemiluminescence
reagents (Amersham Life Science), and exposed to
Protein extraction and Western blot analysis
films in a dark room.
Whole cell lysates, used to determine the levels
Statistical analysis
of various proteins, were prepared by following
method. In brief, the pancreatic cancer cells were
When appropriate, statistical significance was
seeded onto culture-dishes, after 24 hr incubation
tested using a two tailed Student's t-test; p
0.05
with [6]-gingerol for the indicated times. The cells
was considered significant. All values shown are
were washed twice with cold PBS (pH 7.4) and
means with the corresponding standard error.
added cell lysis buffer (70 mM beta-glycerol-
phosphate (pH 7.2), 0.6 mM sodium vanadate, 2
M MgCl2, 1 mM Ethylene glycol-bis (2-aminoe-
RESULTS
thylether-)N,N,N',N'-tetraacetic acid (EGTA), 1
mM dithiothreitol (DTT), 0.5% Triton X-100, 0.2
[6]-Gingerol inhibits cell growth on both BxPC-3
mM phenylmethylsulfonyl fluoride (PMSF), 1X
and HPAC cells
Protease Inhibitor; Leupeptin, Pepstatin, Aprotinin,
and Antipain each 5 ?g/mL). Cell lysates were
To determine antiproliferative effects of [6]-
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Pancreatic Cancer Cell Death Induced by [6]-Gingerol
gingerol on two pancreatic cancer cells expressing
ated with the induction of apoptosis as repre-
either wt (HPAC) or mutant p53 protein (BxPC-3),
sented by the large sub-G1 peak. On the other
we treated cells with [6]-gingerol ranging from 50
hand, cell cycle arrest of HPAC by [6]-Gingerol
to 800 ?M. As shown in Fig. 1, cells treated with
different concentrations of [6]-gingerol for 0, 24,
72, and 120 h resulted in the growth inhibition in
a dose- and time-dependent manner. [6]-Gingerol
at the highest concentration (800 ?M) had similar
mean cytotoxicity over the 72 h of 89.4% and
91.2% on BxPC-3 and HPAC cells, respectively.
There was no significant difference in IC50 values
between BxPC-3 cells and HPAC cells (387.4 ?M
and 405.3 ?M at day 3, respectively, p < 0.006).
IC25 values were 186.1 and 146.3 ?M for BxPC-3
and HPAC, respectively.
[6]-Gingerol induces cell cycle arrest
Although it was apparent that [6]-gingerol was
growth inhibitory to the pancreatic cancer cell
lines, the sustained effects on culture growth
raised suspicion that it might also affect the
proliferation of surviving cells. To test this pos-
sibility, we examined whether [6]-gingerol affects
cell cycle progression. BxPC-3 and HPAC cells
Fig. 2. [6]-Gingerol induces cell cycle arrest in both
were treated with 400 ?M of [6]-gingerol (approxi-
BxPC-3 and HPAC cell lines. Exponentially growing cells
mate IC
were exposed to either 0.1% DMSO (control) or [6]-
50 at day3). As shown in Fig. 2, [6]-
gingerol (400 ?M) for 24, 48 or 72 hrs. Cells were then
gingerol caused BxPC-3 cells to accumulate in
harvested, washed in PBS, and fixed in 70% ethanol.
G1-phase (59.2% in [6]-gingerol-treated vs. 42.3%
DNA content was evaluated with propidium iodide
in control) within 24 hr at the expense of cells
staining and fluorescence measured and analyzed as
described in Materials and Methods. Data are repre-
mainly in S phase. Furthermore, this was associ-
sentative of three independent experiments.
Fig. 1. Effect of [6]-gingerol on survival of BxPC-3 and HPAC cells. [6]-Gingerol inhibited growth of both BxPC-3 and
HPAC cells in a dose-dependent manner. Cells were treated with different concentrations of [6]-gingerol. Control cells
(empty circle) were treated with vehicle alone (0.1% DMSO). Cells were harvested at day 1, 3 and day 5 and cell viability
was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described in Materials
and methods. Percent viability was calculated by comparison with controls. Values shown as the mean ± SEM obtained
from four independent experiments.
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Yon Jung Park, et al.
was less dramatic in HPAC cells where cellular
accumulation in G0/G1 phase was slightly higher
in [6]-gingerol treated group (64.6%) compared to
the control (54.6%). In contrast to BxPC-3 cells, we
did not detect an increase in HPAC cells with
hypodiploid DNA content, suggesting that
apoptotic cell death was not induced. At 72 hr,
HPAC cells partially recovered from [6]-gingerol-
induced growth arrest suggesting the operation of
Fig. 4. Effects of [6]-gingerol on the expression of p21cip
compensatory mechanism(s).
and p53. Both BxPC-3 and HPAC were treated with 400
M
?
of [6]-ingerol, and harvested every 12 hr. The levels
of p21cip1, a Cyclin dependent kinase inhibitor, and p53
[6]-Gingerol caused changes in various cell cycle
changed by [6]-Gingerol in BxPC-3 and HPAC cells. Three
related protein expressions
replicate experiments were done with similar results.
We examined the effect of [6]-gingerol on the
levels of G1 phase-regulatory proteins by Western
Next, we studied the difference in basal and
blot analyses (Fig. 3). Both BxPC-3 and HPAC
[6]-gingerol-induced levels of p53 and p21cip1,
cells were treated with 400 ?M of [6]-gingerol and
between wt and mutant p53-expressing cells. In
harvested in every 12 hr. In BxPC-3 and HPAC
line with previous report,20 the basal levels of p53
cells, expression levels of various proteins were
were elevated in mutant p53-expressing cells.
changed by [6]-gingerol. In BxPC-3, [6]-gingerol
[6]-Gingerol reduced the p53 levels of wt, but less
induced decrease in Cyclin A, Cdk 2, Cdk 4 and
significantly of mutant, p53-expressing cells (Fig.
Cdk 6 expression and increase in Cyclin D1 expres-
4). Expression of p21cip1 was increased by [6]-
sion. [6]-Gingerol decreased the Cyclin A and Cdk
gingerol in both BxPC-3 and HPAC cells. It is well
6 expression, but not Cdk 2 and Cdk 4, in HPAC
known that induction of p21cip1 is associated with
cells. Phosphorylated Rb protein was decreased in
the expression of p53.21 The treatment of [6]-
both BxPC-3 and HPAC cells by [6]-gingerol.
gingerol reduced the p53 level in the both cells
implying that the overexpression of p21cip1 by
[6]-gingerol might be caused by p53-independent
events in both cell lines.
[6]-Gingerol induces apoptosis in BxPC-3 cell
Next, we assessed potential differences between
the [6]-gingerol-induced cell death in wt and
mutant p53-expressing cells. Since mutations in
p53 often compromise the ability of cells to un-
dergo apoptosis, we analyzed the mode of cell
death involved. As can be seen in Fig. 5, early
apoptosis (Annexin-V+/propidium iodide-) could
be detected in the mutant p53-expressing BxPC-3
cells 24 hr after treatment with [6]-gingerol. The
Fig. 3. Western blot analyses of the cell cycle regulatory
percentage of early-apoptotic cells and late
proteins. Both BxPC-3 and HPAC were treated with 400
apoptotic/necrotic cells (Annexin-V+/propidium
?M of [6]-gingerol, and harvested every 12 hr, and levels
iodide+) continued to rise, reaching a peak at 72
of Cyclins, Cdks, Rb and pRb were determined by Western
blotting. The upper band of Rb indicates phosphorylated
hr. On the other hand, [6]-gingerol did not induce
form. pRb antibody detects specifically on Ser-780
death in wild type p53 expressing HPAC cells.
phophorylated Rb. Data are representative of three inde-
HPAC cells displayed few early apoptosis and late
pendent experiments. Actin was used as a standard for
each sample.
apoptotic/necrotic fractions until 72 hr suggesting
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Pancreatic Cancer Cell Death Induced by [6]-Gingerol
Fig. 5. Differential mode of death induced by [6]-Gingerol in pancreatic cancer cells expressing wt versus mutant p53.
Cells were treated with 400 M
?
of [6]-gingerol for 24, 28 and 72 hrs. Annexin-V and PI staining revealed increasing
percentage of Annexin-positive and PI-positive cells with increasing time of [6]-gingerol in BxPC-3 cells. On the other
hand, [6]-gingerol treatment on HPAC cells showed no changes in cell viability under the identical culture condition.
The X and Y axis represents annexin V-FITC and Propidium Iodide (PI) fluorescence respectively. Population in lower-
left part (Annexin V-FITC and PI negative) is viable, and lower-right part (Annexin V-FITC positive and PI negative)
is undergoing apoptosis. Cells observed in Annexin V-FITC and PI positive (upper-right and upper-left parts) indicates
either late stage of apoptosis or dead cells. The percentage of each part is calculated in the bottom table. The figure
is representative of three independent experiments.
that HPAC cells are highly resistance to [6]-
AP-1, cyclooxygenase-2 (COX-2) and p38 mitogen
gingerol induced apoptosis. This observation cor-
activated protein kinase (MAPK).23 Thus, we ex-
responds to the data of cell cycle analysis where
amined the levels of pERK, pJNK and PI3K/AKT
HPAC cells had minimal sub-G1 population upon
pathway proteins by Western blot analysis (Fig.
[6]-gingerol treatment even after 72 hr.
6). The down regulation of pJNK and pERK
MAPK was time-dependently induced by [6]-
[6]-Gingerol affects AKT activation
gingerol in both cell types. In both cell lines,
there was no change of PI3K regulatory subunit,
Based on these findings, we sought to examine
p85?, levels by [6]-gingerol. While [6]-gingerol
whether [6]-gingerol-induced apoptotic cell death
did not change phosphorylation of AKT protein
was accompanied by the activation of common
in BxPC-3 cells, active phosphorylated AKT
pro-apoptotic signaling pathways. The difference
(pAKT) started to appear 3 hr after incubation
in [6]-gingerol response of these two cell lines
and increased further up to 24 hr in [6]-gingerol-
may be caused by the altered PI3K/AKT path-
treated HPAC cells. These results imply that
ways which play important roles in cell cycle
[6]-gingerol-induced apoptosis in HPAC cells
progression and cell survival.22 [6]-Gingerol was
(p53 wild type) is suppressed via the PI3K/AKT
found to inhibit nuclear factor kappa B (NF-kB),
pathway.
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Yon Jung Park, et al.
be the great advantage of [6]-gingerol for the
therapeutic or preventative use. While there were
some reports that various phenolic substances
induce cell cycle arrest in some phases,1-5 this is
the first report that reveals on [6]-gingerol effect
upon cell cycle in cancer cell lines. Western blot
analyses indicated that [6]-gingerol decreased the
expression of Cyclin A and Cdks including Cdk2,
Cdk4, and Cdk6 in BxPC-3. Also, Cyclin A and
Cdk 6 expression levels were decreased in HPAC.
Thus, the reduction of Cyclin or Cdk expressions
Fig. 6. Western bolt analysis for MPAK and PI3K/AKT
results the blocking of Cyclin-Cdk complexes
pathways in [6]-gingerol-treated pancreatic cancer cells.
The BxPC-3 (A) and HPAC (B) were incubated with 400
formation and that lowers the level of phospho-
?M of gingerol for indicated times. Cells were lysed and
Rb. Since Rb proteins remain in unphosphorylated
the cell lysates (30 ?g) were resolved by SDS-PAGE. AKT,
form, E2F cannot be activated and the cells fail to
ERK and JNK activation was analyzed by Western blotting
enter the S phase. Cyclin D1 might be the cause
with anti-phospho-AKT, anti-phosph-ERK, anti-phosph-
JNK or anti-p85-specific antibodies as indicated. Equal
of apoptotic cell death when overexpressed.25,26
protein loading was confirmed by probing the membranes
Alternatively, increase of Cyclin D1 in BxPC-3
with antibodies detecting the respective unphosphorylated
cells may be a feedback response to drug-induced
proteins (ERK and AKT). Three replicate experiments
were done with similar results.
cell cycle arrest.
p53 is a tumor suppressor gene encoding a
transcription factor. Its tumor-suppressive activity
DISCUSSION
involves inhibition of cell proliferation through
cell cycle arrest and/or apoptosis. Mutation in p53
Phenolic compounds comprise one of the largest
occurs in more than half of human cancers.27 Cells
and most ubiquitous groups of plant metabolites.
harboring mutated p53 lose the ability to elicit the
They are formed to protect the plant from photo-
enzymatic DNA repair cascade, to inhibit cell
synthetic stress, reactive oxygen species (ROS),
proliferation and to induce programmed cell
wounds, and herbivores.24 The most commonly
death, resulting in induction of uncontrolled pro-
contained ones in foods are flavonoids and
liferation and malignancy.28,29 Tumors consisting
phenolic substances. Hence, phenolic compounds
of mutant p53-expressing cells exhibit high resis-
take important parts of the human diet. In
tance to radiation and chemotherapeutic drugs.
addition, current interest is raised up by many
Circumventing this abnormal resistance is a major
observations that dietary phenolic compounds
challenge in cancer therapy.20,30 In the present
have various activities such as antioxidant, anti-
study, we showed that [6]-gingerol exerted its
inflammation and anti-carcinogenesis.
cytotoxic activy toward cancer cells harboring
In this present study, we first investigated the
mutant p53. Based on in vitro and in vivo studies,
effects of [6]-gingerol, a phenolic substance de-
the Cip/Kip family including p21Cip1 were ini-
rived from ginger roots, on two pancreatic cancer
tially thought to interfere with the activation of
cell lines. We found that [6]-gingerol inhibited the
G1/S phase related Cyclin/Cdk complexes. The
cell growth, disrupted the cell cycle progression in
Cyclin-dependent kinase inhibitor p21cip1 is a
both HPac cells (wild type p53) and BXPC-3 cells
major transcriptional target of the tumor-suppres-
(mutant p53 protein), and also induced apoptosis
sor p53.21 BxPC-3 cells have point mutated p53
in BxPC-3 cells. Interestingly, it is noticeable that
proteins and the HPAC cells have the wild type
normal cell showed highly resistance to the
p53 proteins. While the basal levels of p53 were
cytotoxic effects of [6]-gingerol. RIE (rat intestinal
elevated in mutant p53-expressing cells as
epithelial cell) showed 50 percents growth inhibi-
previously reported,20 the level of p53 is decreased
tion at over 900 M
?
(data not shown) of [6]-
by [6]-gingerol in both cell lines. Thus, p21cip1 in-
gingerol for 3 days treatment. This selectivity may
duction by [6]-gingerol was not necessarily depen-
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Pancreatic Cancer Cell Death Induced by [6]-Gingerol
dent on p53 wild-type status since we detected
to the pathogenesis of malignancy.36 More re-
p21 induction in mutated p53 cells.
cently evidences have suggested that this pathway
It has been reported Ras signaling through the
is also associated with the regulation of cell cycle
Raf/MAPK pathway also elevates levels of p21cip1
progression.22 The anti-cancer activities of [6]
in some cell types.31-33 However it is still unclear
gingerol could be associated with a control of
whether the over-expression of p21cip1 by [6]-
signal transduction of PI3K/AKT pathway and
gingerol is related with Ras signaling activations.
this led us to investigate the change of the
A number of phytochemicals, including EGCG,34
survival pathway-associated proteins. In the both
tangeretin,3 genestein and silymarin4 have been
cells [6]-gingerol could not affect the expression of
shown to induce cell cycle arrest accompanied by
the regulatory subunit of PI3K, p85?. However,
increased p21cip1 expression, an important Cdk
[6]-gingerol increased phosphorylation of AKT,
inhibitor in G1 and S phase. [6]-Gingerol also
which is regulated by PI3K, in only the HPAC
increased the level of p21cip1 in both cell lines. The
cells. Activated AKT is known to promote the cell
overexpression of p21cip1 facilitated cell cycle
survival by anti-apoptoic mechanism.37 Additio-
arrest effects of [6]-gingerol. Unlike other Cyclins,
nally, activated AKT also phosphorylates and
Cyclin D1 level was increased in BxPC-3. It is pro-
inactivates the proapoptotic protein, BAD.22,36,38 In
bably as a feedback response to G1 arrest. More
HPAC cells, the increase of phospholated AKT
recent study, however, has altered this view and
might protect apoptosis, despite of cell cycle arrest
revealed that even p21cip1 proteins are specific
by [6]-gingerol. On the other hand, there was no
inhibitors of Cyclin E- and A-dependent Cdk2,
change in phosphorylation of AKT by [6]-gingerol-
they act as positive regulators of Cyclin D-depen-
treated BxPC-3 cells and thus failed to counteract
dent kinases.31 Thus, the increase of Cyclin D1
the gingerol-induced apoptotic cell death.
level in BxPC-3 cells may be the consequence of
In conclusion, we describe experiments that
the overexpression of p21cip1. However, it is un-
show [6]-gingerol induces apoptotic cell death in
clear why there was no change of Cyclin D1 level
p53-mutant cancer cells. The death mechanism
in spite of G1 phase arrest and overexpression of
was characterized, revealing that [6]-gingerol not
p21cip1 in HPAC cells. The fact that [6]-gingerol
only initiated cell cycle arrest but ultimately
has different effects on the cell cycle in different
caused cell death through apoptosis. Thus, [6]-
cell lines is intriguing and suggests that the ability
gingerol, is capable of killing cancer cells ex-
of [6]-gingerol to affect the cell cycle may be
pressing mutant p53, overcoming the phenotypic
dependent on other genetic alterations that the
resistance to chemotherapy- and irradiation-in-
tumor harbors. For example, the levels of Cyclins,
duced cell death. These findings support the
Cyclin-dependent kinases, their inhibitors, or the
importance of studying [6]-gingerol and gingerol-
status of tumor suppressor genes such as p53 and
related compounds as anticancer agents that can
Rb, that are all involved in cell cycle regulation,
potentially eradicate tumors resistant to radiation
may determine whether a chemical inhibitor
and to currently available chemotherapy.
(drug) results in cell cycle arrest or not.
Exposure of mammalian cells to growth factors
or genotoxic stress elicits a variety of cellular
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