DOI:http://dx.doi.org/10.7314/APJCP.2015.16.15.6753
Induction of Apoptosis by Eugenol and Capsaicin - Role of p53
RESEARCH ARTICLE
Induction of Apoptosis by Eugenol and Capsaicin in Human
Gastric Cancer AGS Cells - Elucidating the Role of p53
Arnab Sarkar, Shamee Bhattacharjee*, Deba Prasad Mandal*
Abstract
Background: Loss of function of the p53 gene is implicated in defective apoptotic responses of tumors to
chemotherapy. Although the pro-apoptotic roles of eugenol and capsaicin have been amply reported, their
dependence on p53 for apoptosis induction in gastric cancer cells is not well elucidated. The aim of the study was
to elucidate the role of p53 in the induction of apoptosis by eugenol and capsaicin in a human gastric cancer cell
line, AGS. Materials and Methods: AGS cells were incubated with or without various concentrations of capsaicin
and eugenol for 12 hrs, in the presence and absence of p53 siRNA. Cell cycling, annexin V and expression of
apoptosis related proteins Bax, Bcl-2 ratio, p21, cyt c-caspase-9 association, caspase-3 and caspase-8 were studied.
Results: In the presence of p53, capsaicin was a more potent pro-apoptotic agent than eugenol. However, silencing
of p53 significantly abrogated apoptosis induced by capsaicin but not that by eugenol. Western blot analysis
of pro-apoptotic markers revealed that as opposed to capsaicin, eugenol could induce caspase-8 and caspase-3
even in the absence of p53. Conclusions: Unlike capsaicin, eugenol could induce apoptosis both in presence and
absence of functional p53. Agents which can induce apoptosis irrespective of the cellular p53 status have immense
scope for development as potential anticancer agents.
Keywords: Apoptosis - p53 - eugenol - capsaicin - AGS cell line
Asian Pac J Cancer Prev, 16 (15), 6753-6759
Introduction
Evasion of cell death is one of the important hallmarks
of malignant transformation and hence provides an
attractive opportunity for intervention. To this day,
induction of apoptosis is considered to be a key strategy
in the treatment of cancer. A host of literature suggests
that the success of many anticancer therapies lies in
their ability to generate a potent pro-apoptotic stimulus.
However, due to the severe side-effects of conventional
chemotherapies, phytochemicals from dietary sources are
receiving increasing prominence as potential sources of
more compatible anticancer drugs. A myriad of literature
encompassing both in vitro and in vivo studies indicate
apoptosis induction as a mechanism of action for a wide
range of dietary phytochemicals.
Eugenol (4-allyl (-2-methoxyphenol)), a phenolic
compound, found abundantly in the essential oil of a
commonly consumed spice, clove (Syzgium aromaticum),
is one such phytochemical. It has been exploited for
various medicinal applications such as antiseptic,
analgesic, antioxidant, antiviral and antibacterial agent
(Pramod et al., 2010). Furthermore, many recent studies
have explored the anticancer potential of eugenol and
its ability to induce apoptosis in diverse cancer cell
lines as well as in in vivo tumor models (Jaganathan and
Supriyanto, 2012; Majeed et al., 2014).
Capsaicin is yet another phytochemical found naturally
as the pungent constituent of hot chilli peppers of the genus
Capsicum (family Solanaceae). There are various reports
which highlight the physiological and pharmacological
properties of capsaicin including its anticancer property.
The capacity of capsaicin to suppress the growth of cancer
cells is considered to be primarily mediated through
induction of apoptosis (Lin et al., 2013).
Despite increasing evidences and molecular
unmasking of apoptotic induction by the above mentioned
phytochemicals, the involvement of p53 in the apoptotic
cascade induced by eugenol and capsaicin in gastric cancer
cells is not very clear. Although a fair number of studies
have focused on the anticancer potential of capsaicin
and eugenol, the influence of p53 status on induction
of apoptosis by these two phytochemicals is not well
characterized. Various mechanisms for the induction of
apoptosis have been proposed for capsaicin and eugenol.
While, several studies have reported that capsaicin induces
apoptosis by upregulating p53, there are also few instances
of p53 independent induction of apoptosis by capsaicin
(Mori et al., 2006). Similarly, there are reports of both p53
dependent as well as independent induction of apoptosis
Department of Zoology, West Bengal State University, Berunanpukuria, Malikapur, Kolkata, West Bengal, India *For correspondence:
[email protected], [email protected]
Asian Pacific Journal of Cancer Prevention, Vol 16, 2015
6753
Arnab Sarkar et al
by eugenol in diverse cellular systems (Al-Sharif et al.,
2013).
The purpose of this study is to elucidate the mechanism
of apoptotic induction by eugenol and capsaicin in gastric
cancer cells and to investigate the outcome of treatment by
these phytochemicals in the presence and absence of p53.
Materials and Methods
Chemicals
Annexin V-assay Kit was purchased from (Abcam,
USA). Cycle TEST PLUS DNA reagent kit procured
from Becton Dickinson Immunocytometry system (San
Jose, CA). p53 siRNA transfection kit (Dharmacon
ON-TARGET Plus siRNA) was purchased from GE
Healthcare. Anti-mouse anti-bodies against p53, p21,
Bax, Bcl-2, Cyt c, caspase-3, caspase-8, procaspase-9 and
PCNA, were procured from Santa Cruz (USA), bacitracin,
leupeptin, pepstatin A, PMSF, phosphatase inhibitor
cocktails. A-Sepharose beads, RNase, NAC and NBT were
purchased from Sigma (St. Louis, MO). Nitrocellulose
membrane, and filter papers were obtained from Pall
Corporation, USA. Acetonitrile, methanol and ethanol
were HPLC grade and were purchased from Merck. The
remaining chemicals and materials were purchased from
local firms (India) and were of highest grade.
Cell culture
AGS cells were routinely maintained in RPMI 1640
supplemented with 10% fetal bovine serum, insulin
(0.1units/mL), L-glutamine (2 mM), sodium pyruvate
(100μg/mL), non-essential amino acids (100μM),
streptomycin (100μg/mL), penicillin (50unit/mL) and
tetracycline (1μg/mL) (Sigma Chemical Co.) at 37°C
in a humidified incubator containing 5% CO2. Cells
were incubated with or without various concentrations
of capsaicin and eugenol for 12 hrs. The cells were then
processed for the analysis of cell count and cell cycle,
detection of apoptosis and Western blotting of many proand anti-apoptotic proteins as described in the following
sections.
Hemolytic assay
Fresh human blood was centrifuged at 4000Xg for 10
min and the cell pellet was washed thrice and re-suspended
in 10mM PBS at pH 7.4 to obtain a final concentration of
1.6x109 erythrocytes/ml. Equal volumes of erythrocytes
were incubated with varying concentrations of capsaicin
and eugenol and with shaking at 37ºC for 1hr. Samples
were then subjected to centrifugation at 3500Xg for 10
min at 4ºC. RBC lysis was measured at different peptide
concentrations by taking absorbance at an OD of 540 nm.
Complete hemolysis (100%) was determined using 1%
Triton X 100 as a control. Hemolytic activity of the spice
active components was calculated in percentage using the
following Equation:
H=100 X (Op-Ob)/(Om-Ob)
where, Op is the optical density of given peptide
concentration, Ob is the optical density of buffer and Om
is the optical density of Triton X 100.
6754
Asian Pacific Journal of Cancer Prevention, Vol 16, 2015
Cell viability assay
The effect of capsaicin and eugenol on the viability
of AGS cells was determined by Trypan blue exclusion
test. Cells were treated with different doses of these two
phytochemicals and at definite time point (12hrs) the cells
that could exclude the Trypan blue dye were counted in
haemocytometer as viable cells. Vehicle-treated cells were
considered as control and viability of these cells was taken
as 100%. IC50, the dose which induced 50% cell-killing,
was determined for the compounds.
MTT assay
The cytotoxicity of the spice principles was tested on
AGS cells by the MTT-assay. Briefly, cells were seeded
in a 96-well microtitre plate (2 x 104 cells/well in 100 µL
of complete medium) and then incubated with different
concentration of these two phytochemicals. After 12 h of
exposure to the phytochemicals, 50 µl of MTT (5 mg/5
mL) was added to each well, and the cells incubated in
the dark at 37°C for an additional 4 h. Thereafter, the
medium was removed, the formazan crystals dissolved
in 200 µL of dimethyl sulfoxide, and the absorbance
measured at 570 nm.
siRNA transfection
Gene Silencing of p53 was done using siRNA in
accordance with the manufacturer’s instructions. For
siRNA transfection experiments, AGS cells were plated
and transfected after 24 h at ~70% confluency by using
DharmaFECT 1 siRNA transfection reagent, according to
the manufacturer’s instructions. After 60 h of transfection,
cells were exposed to capsaicin and eugenol for 12 h prior
to analysis of experimental parameters. A non-targeting
control siRNA (“scramble” siRNA) was used as negative
control. Siglo incorporation was used to analyze the extent
of transfection, which in this case was approximately 90%.
West blot of protein expression was tallied with the result.
Cell cycle distribution analysis
For the determination of cell cycle phase distribution
of nuclear DNA, In vitro AGS cells (1x106 cells) were
harvested. Then, cells were fixed with 3% p-formaldehyde,
permeabilized with 0.5% Triton X-100, and nuclear DNA
was labeled with propidium iodide (PI, 125 μg/mL) after
RNase treatment using Cycle TEST PLUS DNA reagent
kit. Cell cycle phase distribution of nuclear DNA was
determined on FACS Calibur using Cell Quest Software
(Becton-Dickinson Histogram display of DNA content
(x-axis, PI fluorescence) versus counts (y-axis) has been
displayed. Cell Quest statistics was employed to quantitate
the data at different phases of the cell cycle.
Annexin V assay
Apoptosis assays were carried out based on the
instruction from the Annexin V Apoptosis Kit (Biovision).
Briefly, PI and Annexin V were added directly to AGS
cells. The mixture was incubated for 15 min at 37oC. Cells
were fixed and then analyzed on FACS Calibur (equipped
with 488 nm Argon laser light source; 515 nm band pass
filter, FL1-H, and 623 nm band pass filter, FL2-H) (Becton
Dickinson). Electronic compensation of the instrument
DOI:http://dx.doi.org/10.7314/APJCP.2015.16.15.6753
Induction of Apoptosis by Eugenol and Capsaicin - Role of p53
was done to exclude overlapping of the emission spectra.
Total 10,000 events were acquired, the cells were properly
gated and dual parameter dot plot of FL1-H (x-axis; Fluosfluorescence) versus FL2-H (y-axis; PI-fluorescence)
shown in logarithmic fluorescence intensity.
β−glycerophosphate, p-nitrophenylphos-phate and
sodium vanadate) cocktails. The lysate (200 μg protein)
was incubated for 4 h in rocking condition at 4°C with
2 μg antibody of a cyt c and the immunecomplex was
then incubated with protein A-Sepharose beads. The
immunoprecipitate was centrifuged at 12,000 rpm for 2
min at 4°C and the pellet was washed with ice-cold PBS
containing phosphatase inhibitor. The immunopurified
protein was then used to detect the presence of associated
protein, procaspase-9, by Western blot analysis using
specific antibody of protein of interest as described above.
Western blot analysis
AGS cell lysates were obtained and equal amounts
of protein from each sample were diluted with loading
buffer, denatured, and separated by 10% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
followed by protein transfer to polyvinylidene fluoride
membranes (PVDF). The effect of the two spice principles
on the expression of certain cell cycle proteins such as
p53, p21 and PCNA and on apoptotic proteins such as
Bax/Bcl-2 ratio, caspase-3 and caspase-8, was determined.
Proteins were detected by incubation with corresponding
primary antibodies (anti p53, anti-p21, anti-PCNA, antiBax, anti-Bcl-2 and anti-caspase-3, and anti-caspase-8)
antibodies followed by blotting with HRP-conjugated
secondary antibody. The blots were then detected by using
a chemiluminescent kit from Thermofisher. This analysis
was performed three times.
Statistical analysis
The experiments were repeated three times and the
data were analyzed statically. Values have been shown as
standard error of mean, except where otherwise indicated.
Data were analyzed and one-way ANOVA were used to
evaluate the statistical differences. Statistical significance
was considered when P<0.05 or P<0.01.
Results
Selection of a sub-lethal concentration of capsaicin and
eugenol
Trypan blue exclusion test and MTT assay revealed
that more than 40% cell killing was observed for capsaicin
and eugenol at and above a concentration of 250μM and
1mM respectively. Hemolytic data also suggests that
capsaicin above 250 μM and eugenol above 1mM is very
toxic. Hence, we selected a concentration of 200 μM for
capsaicin and 0.7mM for eugenol for our experiments
(Figure 1).
20
10
Eugenol (mM)
0
20
0
25
0
30
0
35
0
0
10
15
ro
0.4
0.3
0.2
0.1
1.
7
4
0
1.
1.
7
0.
4
7
1.
4
1.
0
1.
7
0.
4
1
0.
0.
l
ro
7
4
1.
0
1.
1.
7
0.
4
1
0.
0.
l
ro
nt
Co
Eugenol (mM)
0.5
0
0
0
nt
Co
10
30
0.6
1
5
40
MTT Assay
0.7
0.
10
50
Capsaicin(µM)
0.8
0.
15
Trypan blue
F
l
20
0
ro
25
Capsaicin(µM)
60
0.2
Co
nt
30
0.4
Cell number ₍x 105₎
E
35
0.6
0
30
0
35
0
l
50
ro
Co
Capsaicin(µM)
MTT Assay
0.8
25
0
20
0
25
0
30
0
35
0
0
20
0
10
1.0
l
50
20
C
Cell number ₍x 105₎
30
Co
nt
Haemolysis ₍%₎
D
40
nt
Co
nt
10
ro
l
50
0
0
2
50
0
4
Trypan blue
0
6
60
15
Haemolysis ₍%₎
8
Modulation of Cell cycle phase distribution of AGS cells
15
B
10
Cell number ₍x 104₎
A
Cell number ₍x 104₎
Co-immunoprecipitation
For the determination of direct interaction between
cyt-c and procaspase-9, co-immunoprecipitation technique
was applied. For this purpose AGS cells were harvested
and lysed in IP buffer (50 mM Hepes, pH 7.6, 200 mM
NaCl, 1 mM EDTA, 0.5% Noniodet P-40) containing
protease (10 μg/mL each of benzamidine, trypsin
inhibitor, bacitracin, 5g/mL each of leupeptin, pepstatin
A and PMSF) and phosphatase inhibitor (5μM each of
o-phosphoserine, o-phosphotyrosine, o-phosphothreonine,
Eugenol (mM)
Figure 1. Effect of Treatment with Capsaicin and Eugenol on AGS Cell Viability. Percentage hemolytic activity of
capsaicin (A) and eugenol (D) on human red blood cells. B) Cell count at various concentration of capsaicin (B) and eugenol (E)
using Trypan Blue exclusion method. Percentage cell viability at various concentration of capsaicin (C) and eugenol (F) measured
by MTT assay
Asian Pacific Journal of Cancer Prevention, Vol 16, 2015
6755
Arnab Sarkar et al
tagged annexin V and PI and measuring the fluorescence
intensity in a flowcytometer. Results suggest a significant
increase in the percentage of annexin positive cells from
5.18%±0.04% in the AGS control group to 8.48%±
0.19% and 23.9%±0.2% respectively in eugenol and
capsaicin treated groups. Percentage of PI positive cells
also increased significantly by both the phytochemicals
(9.03% by eugenol and 17.15% by capsaicin) (Figure 3).
by capsaicin and eugenol
In order to analyze the effect of the spice principles on
AGS cells flowcytometric analysis of the cell cycle phase
distribution was performed (Figure 2).
Treatment with capsaicin resulted in a significant
increase in the sub-G0 region (hypolpoidy population);
Figure 2 shows representative data of the various
experiments. As compared to 5.33%±0.058% cells in the
hypoploidy region of the AGS control group, capsaicin
treatment recorded 19.86%±0.78% cells. Eugenol
treatment at 0.7 mM, also caused an increase in the
hypolpoidy peak (7.92%±0.06%) although to a much
lesser extent than capsaicin. However, the synthetic phase
or S-phase, showed distinctive depression in the eugenol
treated group as compared to the untreated counterparts.
Validating the intrinsic pathway of apoptosis
The increase in the percentage of apoptotic cells in
the capsaicin treated group was further confirmed by coimmunoprecipitating caspase-9 with cyt c, two proteins
which participate in apoptosome formation. Capsaicin
as well as eugenol treatment increased the interaction of
the two proteins as compared to the AGS control group
(Figure 4)
200
400
800
600
200
400
100
800
600
400
AGS + Eugenol
200
400
600
800
S
Hypo G0/G1
G2/M
p53 siRNA
p53 siRNA
0
1000
6.4% 34.4% 12.7% 37.8%
80
G2/M
800
600
100
200
Counts
40
2
0
S
Hypo G0/G1
10
1000
0
1000
12.8% 38.7% 25.7% 21.1%
80
G2/M
Counts
40
2
0
S
800
600
60
Cell population ﴾%﴿
80
Counts
40
2
0
G2/M
p53 wild type
AGS+ Capsaicin
10
10
0
400
p53 siRNA
Control
0
S
10
100
80
1000
Hypo G0/G1
19.9% 36.6% 25.8% 17.7%
Hypo G0/G1
Hypo
B
S+G2/M
G0/G1
p53 wild type
p53siRNA
50
40
30
20
10
AGS+ Capsaicin
0
0
10
Scrambled
control
0
200
5.4% 36.2% 13.4% 42.6%
Counts
40
2
0
G2/M
0
1000
10
S
800
0
100
Counts
40
2
0
80
4.4% 39.5% 14.3% 39.4%
Hypo G0/G1
600
AGS + Eugenol
Western Blot analysis of protein expressions
Capsaicin and eugenol treatment was found to
0
Counts
40
2
0
10
0
Counts
400
G2/M
p53 wild type
Control
200
S
Hypo G0/G1
100
G2/M
p53 wild type
0
7.5% 59.2% 15.1% 14.1%
80
S
Counts
40
2
0
80
4.0% 52.3% 20.7% 21.7%
Hypo G0/G1
0
100
A
100
Effect of capsaicin and eugenol on AGS apoptosis
In order to confirm the increase in apoptotic induction
by the phytochemicals, we performed annexin V/PI assay
in all the groups by staining the AGS cells with FITC-
1000
0
200
400
600
800
1000
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0 ic
20 .7m le c
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20 m c tro
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0
Propidium Iodide
Figure 2. Cell Cycle Analysis of Treated and Untreated AGS Cells by Flowcytometer using Propidium Iodide
(PI) as DNA-binding Fluorochrome. A) Histogram display of DNA content (x-axis, PI-fluorescence) versus counts (y-axis)
0.08%
10 1
10 2
10 3
10 4
10 0
10 3
10 4
6.41%
0.27%
4
10
3
10
10 0
10 1
2
10
9.97%
p53siRNA
0.7mM Eugenol
0.32%
10 1
10 2
10 3
p53siRNA
10 4
10 0
10 2
10 3
10 4
35
p53 wild type
30
0.25%
10 1
Annexin V- FITC
10 2
10 3
10 4
20
15
10
5
0
e
cl
hi
Ve
p53siRNA
25
200mM Capsaicin
0
10
Vehicle control
10 2
p53 wild type
Scramble control
Annexin positive cells ﴾%﴿
10 1
18.48%
1
10
1
10
p53siRNA
B
7.28%
2
10
1
10
10 0
4
10
4.54%
2.01%
0
10
10 4
3
10
3
10
10 3
p53 wild type
1
10
10 2
5.31%
1
10
2
10
10 1
3.91%
0
10
10 0
23.73%
200mM Capsaicin
2
10
4.18%
0.33%
10 0
4
10
10 4
2
10
3
10
10 3
0
10
10 2
4
10
10 1
p53 wild type
0.7mM Eugenol
0
10
0.41%
10 0
1.54%
0
10
3
10
14.61%
2
10
2.21%
1
10
1
10
p53 wild type
Vehicle control
0
10
PI
4
10
4
10
4.77%
3
10
3
10
1.41%
2
10
A
4
10
has been shown. The upper and lower panels display cell cycle phase distribution of treated and untreated AGS cells with wild
type p53 and silenced p53 respectively. B) Bar diagram representation of cell cycle phase distribution of AGS (with wild type and
p53 knock out cells) from different experimental groups. The data represented as mean+S.D. for the three different experiments
performed in triplicate
l
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Figure 3. Annexin V Assay in AGS cells A) In a double Label System, Unfixed AGS Cells were Labeled with PI
and Annexin V and then fixed and Analyzed on a Flowcytometer. Dual parameter dot plot of FITC-fluorescence (x-axis)
versus PI-fluorescence (y-axis) has been shown in logarithmic fluorescence intensity. Quadrants: lower left, live cells; lower right,
apoptotic cells; upper right, necrotic cells. The upper and lower panels demonstrated apoptosis induction in treated and untreated
AGS cells with wild type p53 and silenced p53 respectively. B) Bar diagram representation of percent Annexin V positive AGS
cells from different experimental groups are also given. The data represented as mean+S.D. for the three different experiments
performed in triplicate
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Asian Pacific Journal of Cancer Prevention, Vol 16, 2015
DOI:http://dx.doi.org/10.7314/APJCP.2015.16.15.6753
Induction of Apoptosis by Eugenol and Capsaicin - Role of p53
substantially increase the expressions of pro-apoptotic
proteins such as caspase-3, Bax and caspase-8 with a
simultaneous decrease in Bcl-2 and PCNA expression
as compared to AGS control cells (Figure 4). The extent
of decrease in PCNA expression was much more in the
eugenol treated group than capsaicin treatment which
corroborates the decrease in the S-phase cell population
as mentioned above. Interestingly, the phytochemicals
differed remarkably in their ability to induce p53 and
p21 proteins. Whereas, there was a significant induction
of these proteins by capsaicin, eugenol treatment didn’t
cause any significant change in the expressions of p53 and
p21 as compared to AGS control.
p53 is essential for induction of apoptosis by capsaicin
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0.
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ro
nt
1.0
1.5
1.0
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ic
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1.5
0.5
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Caspase 3
l
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in
1.5
2.0
1.5
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ci
ai
ps
Ca
Bax
Bcl2
p21
p53
co
but not for eugenol
Silencing p53 caused a significant decrease in the
ability of capsaicin to induce apoptosis in AGS. As shown
in Figure 2 lower panel. the percentage of hypoploid cells
by capsaicin treatment was brought down to 8.62%±0.32%
in p53-/- AGS as compared to 19.86%±0.78% in AGS
with wild type p53 expression. Annexin V/PI assay also
reflected a similar same trend with a significant reduction
in the percentage of annexin positive cells by capsaicin
treatment in p53-/- AGS cells.
However, p53 knock down did not influence the
apoptotic activity of eugenol in AGS as evident from
both the hypoploidy peak and percentage of annexin
positive cells induced by eugenol treatment (Figure 3).
le
co
M
Eu
n
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ol
hi
7m
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l
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2.5
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2.5
2.0
2.0
2.0
1.5
1.5
1.5
1.5
1.0
1.0
1.0
1.0
0.5
0.5
0.5
0.5
0
0
0
0
Figure 4. Western Blot detection of Pro-apoptotic and Proliferative Proteins in Treated and Untreated AGS
with wild type p53. Detection of p53, p21, Bcl-2, Bax (upper panel) caspase-3, caspase-8 and PCNA (lower panel) in vehicle
control, capsaicin and eugenol treated AGS. Co-IP of pro-caspase 9 with cyt c suggesting formation of apoptosome. Equal loading
of protein in the lanes was confirmed by GAPDH. Indicated proteins are represented as bar diagrams of mean + SD of their relative
densities as measured from three independent experiments. Each test was performed 3 times and images presented were typical of
3 independent experiments. The data were presented as mean ± SD
Bcl-2
caspase3
Bax
2.0
p53siRNA
2.0
1.5
p53siRNA
1.5
p53siRNA
1.5
0
l
l
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Caspase9-cyt c
p53
1.5
wild type
p53siRNA
1.0
0.5
n
n
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Eu
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p53siRNA
GAPDH
3.0
2.5
p53siRNA
1.2
1.0
2.0
2.0
0.8
1.5
1.5
0.6
1.0
1.0
0.4
0.5
0.5
0.2
0
p53siRNA
0.5
PCNA
3.0
wild type
0
caspase8
2.0
p53
1.0
0.5
0.5
2.0
Bax/Bcl2
1.5
1.0
1.0
0.5
2.5
Relative Density
1.0
0
2.0
2.5
l
l
n
ro
ro
ol
ci
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ai
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p53siRNA
0
l
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0.
Sc
Sc
20
20
0
Figure 5. Western Blot detection of Pro-apoptotic and Proliferative Proteins in Treated and Untreated p53 Knock
Down AGS. Detection of Bcl-2, Bax caspase-3 (upper panel), caspase-8 and PCNA (lower panel) in vehicle control, capsaicin
and eugenol treated AGS. Equal loading of protein in the lanes was confirmed by GAPDH. Indicated proteins are represented as
bar diagrams of mean + SD of their relative densities as measured from three independent experiments. Comparison of Bax/Bcl-2
ratio (upper panel) and pro-caspase 9-cyt c association (lower panel) in AGS cells with wild type and silenced p53. Each test was
performed 3 times and images presented were typical of 3 independent experiments. The data were presented as mean ± SD
Asian Pacific Journal of Cancer Prevention, Vol 16, 2015
6757
Arnab Sarkar et al
Quite interestingly, silencing p53 significantly affected the
capacity of eugenol to reduce the percentage of S-phase
cell population.
6758
Asian Pacific Journal of Cancer Prevention, Vol 16, 2015
6.3
12.8
56.3
51.1
33.1
31.3
Newly diagnosed without treatment
Chemotherapy
None
Remission
Persistence or recurrence
Newly diagnosed with treatment
Newly diagnosed without treatment
activation of caspase-8 and capsase-3 (Kim et al., 2007),
it can be hypothesized that while the intrinsic pathway
of apoptotic induction by eugenol is dependent on p53,
the extrinsic pathway is not. In case of capsaicin, on the
Study of protein expressions in p53-/- AGS cells treated
other hands, both the intrinsic and extrinsic pathways
with spice principles
were dependent on cellular p53 status. In line with our
The significant elevation in the expression of prostudy, Al-Sharif et al. (2013) had previously demonstrated
apoptotic proteins such as caspase-3, caspase-8 and
the p53-independent mechanism of apoptosis induction
bax, decrease in that of anti-apoptotic protein bcl-2 and
by eugenol in breast cancer cells. The dependence of
proliferative marker PCNA by capsaicin in AGS with
capsaicin on p53 for the induction of apoptosis has also
100.0been reported in an earlier study (Jin et al., 2014).
100.0
wild type p53 was completely offset by silencing p53
in AGS (Figure 5) suggesting a decreased ability of
This study
showed eugenol to be a potent inhibitor
6.3 also 10.1
20.3
capsaicin to induce apoptosis in the absence of p53. In
of cell proliferation and the antiproliferative activity of
eugenol treated cells, silencing p53, didn’t cause much
eugenol was compromised in the absence
of p53. P53 is 30.0
25.0
75.0
75.0
alteration in the expression of either caspase 3 and 8 as
known to directly control DNA replication and repair by
compared to eugenol treated cells with wild type p53.
modulating the levels
46.8 of PCNA and some studies have
56.3
Eugenol, however, was unable to modulate the Bax/bclfound that higher levels of p53 represses PCNA promoter
54.2
2 ratio and cyt c-capsapse 9 association in p53-/- cells.50.0(Xu and Morris, 1999). The decrease
in31.3
the percentage of50.0
30.0
Moreover, extent of reduction in PCNA expression was
S-phase cells in eugenol treated AGS was matched by a
much lesser in eugenol treated p53-/- cells suggesting
similar reduction in PCNA expression. However, in the
that eugenol modifies the expression of PCNA in a p5325.0absence of p53, the repression on the PCNA promoter25.0
dependent manner.
couldn’t be affected
by eugenol leading to obliteration of
38.0
31.3
31.3
its antiproliferative effect. Capsaicin,
on the other hand, 30.0
23.7
was not found to exert any anti-proliferative on AGS cells
Discussion
0either in the presence or absence of p53.
0
p53 mutation is a common event in human cancers
In conclusion, we demonstrated that both eugenol
which causes defects in apoptosis and makes cancer
and capsaicin induced apoptosis in AGS by the intrinsic
cells resistant to chemotherapeutic agents (Do et al.,
as well as extrinsic apoptotic pathways. Between the
2012). Alterations in p53 occurs in gastric carcinoma
two phytochemicals, capsaicin caused a more potent
and it increases in frequency during the course of gastric
apoptotic induction than eugenol in AGS cells in the
carcinoma developments (Fenoglio-Preiser et al., 2003;
presence of p53. In the absence of p53, however, eugenol
Busuttil et al., 2014). Chemosensitivity of gastric cancer
was a better apoptotic agent than capsaicin because of its
towards chemotherapy has been shown to be abrogated
ability to induce the extrinsic pathway of apoptosis in a
in the absence of p53 (Osaki et al., 1997). Therefore,
p53 independent manner. Loss of function of p53 tumor
identification of agents that can kill cancer cells with
suppressor gene is implicated in defective apoptotic
mutated/deleted p53 is a promising anticancer strategy.
response of tumors to chemotherapy. Thus agents which
In the present study we showed that capsaicin induces
can induce apoptosis irrespective of the cellular p53
apoptosis in AGS cells through upregulation of p53 and
status have immense scope for development as potential
that the apoptotic activity of capsaicin is p53-dependent.
anticancer agents. Therefore, eugenol warrants further
In contrast, eugenol was found to induce apoptosis
investigation for its potential use as anticancer agent
independent of p53. Moreover, eugenol was also found
against p53 defective or null tumors with poor prognosis.
to be a potent inhibitor of cellular proliferation as evident
from a significant decrease in the population of S-phase
Acknowledgements
cells and a corresponding decrease in PCNA expression
by eugenol treatment. This antiproliferative activity of
This work was supported by the Department of
eugenol was, however, dependent on p53.
Biotechnology, Govt. of India under Rapid Grant for
Mechanistically, we found that the ability of capsaicin
Young Investigators Scheme [Grant number: BT/
to induce the expression of proapoptotic proteins such
PR15116/GBD/27/327/2011 dated 09/03/2011].
as Bax, caspase-3 and caspase-8 was almost completely
obliterated by knocking down p53. Moreover, the
References
cyt-c-caspase-9 association, which is considered to
Al-Sharif I, Remmal A, Aboussekhra A (2013). Eugenol triggers
be important in caspase-9 mediated apoptosis, didn’t
apoptosis in breast cancer cells through E2F1/survivin downchange significantly in capsaicin treated p53-/- AGS as
regulation. BMC Cancer, 13, 600.
compared to untreated controls. Similarly, eugenol too
Busuttil
RA, Zapparoli GV, Haupt S, et al (2014). Role of p53 in
couldn’t modulate the levels of either Bax/Bcl-2 ratio or
the progression of gastric cancer. Oncotarget, 5, 12016-26.
cyt c-caspase-9 interaction in p53-/- AGS cells suggesting
Do PM, Varanasi L, Fan S, et al (2012). Mutant p53 cooperates
inhibition of the intrinsic pathway of apoptosis in the
with ETS2 to promote etoposide resistance. Genes Dev,
absence of p53. Interestingly, however, eugenol was able
26, 830-45.
to enhance the expression of both caspase 8 and caspase 3
Fenoglio-Preiser CM, Wang NJ, Stemmermann GN, Noffsinger
even in the absence of p53. As it is known that apoptosis
A (2003). TP53 and Gastric Carcinoma: A Review. Human
Mutation, 21, 258-70.
can be induced by the extrinsic pathway via sequential
Jaganathan SK, Supriyanto E (2012). Antiproliferative and
molecular mechanism of eugenol-induced apoptosis in
cancer cells. Molecules, 17, 6290-304.
Jin J, Lin G, Huang H, et al (2014). Capsaicin mediates cell
cycle arrest and apoptosis in human colon cancer cells via
stabilizing and activating p53. Int J Biol Sci, 10, 285-95.
Kim EJ, Park SY, Shin HK, et al (2007). Activation of caspase-8
contributes to 3,3’-Diindolylmethane-induced apoptosis in
colon cancer cells. J Nutr, 137, 31-6.
Lin CH, Lu WC, Wang CW, Chan YC, Chen MK (2013).
Capsaicin induces cell cycle arrest and apoptosis in human
KB cancer cells. BMC Complement Altern Med, 13, 46.
Majeed H, Antoniou J, Fang Z (2014). Apoptotic effects of
eugenol-loaded nanoemulsions in human colon and liver
cancer cell lines. Asian Pac J Cancer Prev, 15, 9159-64.
Mori A, Lehmann S, O’Kelly J, et al (2006). Capsaicin, a
component of red peppers, inhibits the growth of androgenindependent, p53 mutant prostate cancer cells. Cancer Res,
66, 3222-29.
Osaki M, Tatebe S, Goto A, et al (1997). 5-Fluorouracil (5-FU)
induced apoptosis in gastric cancer cell lines: role of the p53
gene. Apoptosis, 2, 221-6.
Pramod K, Ansari SH, Ali J (2010). Eugenol: a natural compound
with versatile pharmacological actions. Nat Prod Commun,
13, 1999-2006.
Xu J, Morris GF (1999). p53-Mediated regulation of proliferating
cell nuclear antigen expression in cells exposed to ionizing
radiation. Molecular and Cellular Biology, 19, 12-20.
DOI:http://dx.doi.org/10.7314/APJCP.2015.16.15.6753
Induction of Apoptosis by Eugenol and Capsaicin - Role of p53
Asian Pacific Journal of Cancer Prevention, Vol 16, 2015
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