Capsaicin-induced cell death in a human gastric adenocarcinoma cell line

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World Journal of Gastroenterology ISSN 1007-9327
wjg@wjgnet.com © 2005 The WJG Press and Elsevier Inc. All rights reserved.
E L S E V I E R
• GASTRIC CANCER •
Capsaicin-induced cell death in a human gastric adenocarcinoma
cell line
Yi-Ching Lo, Yuan-Chieh Yang, I-Chen Wu, Fu-Chen Kuo, Chi-Ming Liu, Hao-Wei Wang, Chao-Hung Kuo, Jeng-Yi Wu,
Deng-Chyang Wu
Yi-Ching Lo, Chi-Ming Liu, Hao-Wei Wang, Department and
Apoptosis; Bcl-2
Graduate Institute of Pharmacology, Kaohsiung Medical University,
Kaohsiung City, Taiwan, China
Lo YC, Yang YC, Wu IC, Kuo FC, Liu CM, Wang HW, Kuo CH,
Yuan-Chieh Yang, Department of Clinical Pathology, Kaohsiung
Wu JY, Wu DC. Capsaicin-induced cell death in a human
Medical University Hospital, Kaohsiung City, Taiwan, China
gastric adenocarcinoma cell line. World J Gastroenterol
I-Chen Wu, Chao-Hung Kuo, Jeng-Yi Wu, Deng-Chyang Wu,
2005; 11(40): 6254-6257
Division of Gastroenterology, Department of Internal Medicine,
http://www.wjgnet.com/1007-9327/11/6254.asp
Kaohsiung Medical University Hospital. Kaohsiung City, Taiwan,
China
Fu-Chen Kuo, Department of Gynecology and Obstetrics, E-DA
Hospital, I-Shou University, Kaohsiung, Taiwan, China
Supported by Grants from the National Science Council of the
INTRODUCTION
ROC, No. NSC 89-2314-B-037-073 and NSC-89-2315-B-037-004
Correspondence to:
Carcinoma of the stomach is currently the leading cause
Dr. Deng-Chyang Wu, Chief of Gastroenterology,
of cancer-related death among the Chinese. The two most
Kaohsiung Medical University Hospital, 100 Zih-You 1st Road
Kaohsiung City, 807 Taiwan, China. dechwu@yahoo.com
likely factors for such a high incidence of stomach cancer
Telephone: +886-7-3121101-7451 Fax: +886-7-3135612
has been thought to be either Helicobacter pylori[1] or diet.
Received: 2004-12-23 Accepted: 2005-02-18
Capsaicin (8-methyl-N-vanillyl-6-nonenamide), the main
ingredient of hot chili peppers, has long been used in spices,
food additives, and drugs[2]. Capsaicin has been shown to
protect the gastric mucosa of animals and humans against
Abstract
various kinds of damage[3-6]. It is generally considered that
AIM: Capsaicin, a pungent ingredient found in red pepper,
capsaicin’s effect results from the activation of sensory
has long been used in spices, food additives, and drugs.
afferent neurons in the stomach, and is mediated by various
Cell death induced by the binding of capsaicin was examined
physiological functions, such as mucosal blood flow[7-10],
in a human gastric adenocarcinoma cell line (AGS cells).
mucus secretion[11], and bicarbonate secretions[12]. Vanilloid
receptor subtype 1 (VR1), a receptor responsible for
METHODS: By using XTT-based cytotoxicity assay, flow
capsaicin action, has been cloned[13] and demonstrated to
cytometry using the TUNEL method, and quantitation of
be located in both neural and non-neural cells[14]. VR1 has
DNA fragmentation, both cell death and DNA fragmentation
also been found to be expressed peripherally in gastric
were detected in AGS cells treated with capsaicin. By using
mucosal epithelial cells, playing a role in cell protection[15].
Western blotting methods, capsaicin reduced the
Recently, a series of studies have demonstrated that
expression of Bcl-2, the antiapoptotic protein, in AGS cells
capsaicin inhibits mutagenicity and DNA binding of some
in a concentration-dependent manner.
chemical carcinogens, possibly by suppressing their metabolic
activation[16-18]. With cells in culture, capsaicin-inhibited
RESULTS: After incubation of AGS cells with capsaicin for
24 h, cell viability decreased significantly in a dose-dependent
proliferation of HeLa, ovarian carcinoma, and mammary
manner. After incubation of AGS cells with capsaicin for
adenocarcinoma by decreasing NADH oxidase activity[19].
24 h, apoptotic bodies also significantly increased, and
Capsaicin can also alter the expression of tumor forming-
were again correlated with the dose of capsaicin. When
related genes by mediating the overexpression of p53 and/
the concentration of capsaicin was 1 mmol/L, the amount
or c-myc genes in a Korean stomach cancer cell line[20].
of DNA fragments also increased. Similar results also were
Capsaicin was found to induce apoptosis in T cells by
in the lower traces.
increasing the reactive oxygen species and by a subsequent
mitochondrial transmembrane potential[21]. In this report,
CONCLUSION: These results suggest that capsaicin-
we examined the underlying mechanism by which
induced cell death might be via a Bcl-2 sensitive apoptotic
capsaicin induces apoptotic cell death in a human gastric
pathway. Therefore, capsaicin might induce protection
adenocarcinoma cell line (AGS).
from gastric cancer.
© 2005 The WJG Press and Elsevier Inc. All rights reserved.
MATERIALS AND METHODS
Cell line
Key words: Capsaicin; Human gastric adenocarcinoma;
A human gastric adenocarcinoma cell line (AGS) was

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Lo YC et al. Cell death induced by capsaicin
6255
obtained from American Type Culture Collection. The cells
ice. Lysates were centrifuged at 2 500 g for 5 min. Protein
were maintained in RPMI 1640 medium (Life Technologies,
concentration was determined by means of the Bradford
Inc., Melbourne, Australia) supplemented with 10% heat-
protein assay (BioRad Lab., Richmond, CA) using bovine
inactivated fetal bovine serum with penicillin (100 U/mL)
serum albumin as the standard. Thirty micrograms of protein
and streptomycin (100 ?g/mL). Cultures in 75- or 25-mL
were resolved by electrophoresis on 10% polyacrylamide
culture flasks were incubated at 37 in a humidified gas
gels, electrotransferred to a polyvinylidene difluoride filter
mixture containing 50 mL/L CO2 balanced with air.
(Millipore, Bedford, MA), and then blotted with mouse
monoclonal antibody for Bcl-2 (1:500 dilution). Blots were
XTT-based cytotoxicity assay
developed with peroxidase-labeled anti-mouse IgG (1:400
Unlabeled AGS cells (1×104/well) were distributed on to a
dilution) using a Lumi-Light Plus Western Blotting Kit
96-well flat-bottomed plate. Appropriate numbers of cells
(Boehringer Mannheim).
were added, resulting in triplicate wells, and a final volume
of 100 ?L of RPMI medium with 10% bovine serum was
Statistical analysis
obtained. After incubation of AGS cells for 24 h, the
All values in the text and figures are expressed as mean±SE.
medium was then replaced with serum-free RPMI medium
Statistical differences were evaluated by Student’s t-test in
containing capsaicin (0.05 ?mol/L-10 mmol/L) at 37
unpaired samples, by paired t-test in paired samples, or by
for 24 h. The quantity of viable cell was determined using
Wilcoxon’s sum of ranks test. When evaluating multiple
a cell proliferation ELISA kit from Boehringer Mannheim
values, the Bonferroni correction was used. Probability
(No. 1465015), according to the recommendations of the
values less than 0.05 were considered to be significant in
supplier. Each sample was tested in triplicate.
all experiments. Analysis of the data and plotting of the
figures were done with the aid of software (SigmaStat and
Detection of apoptotic cells by flow cytometry using TUNEL
SigmaPlot, Version 5.0, Jandel, USA; PHARM/PCS, Version
method
4.2, MCS, USA) run on an IBM PC-AT computer. Protein
For demonstration of apoptosis, TUNEL assay was performed
blot images were captured by an Imaging Densitometer with
with an in situ cell death detection kit (POD, Boehringer
the aid of software (Bio-ID, V.97 software for Windows
Mannheim) according to the manufacturer’s recommendations.
95, Vilber Laurmat, France). Comparisons were made only
After incubation of AGS cells for 24 h, cells were analyzed
between averaged values of bands within the same gel.
by Epics Elite ESP flow cytometer. Apoptosis was analyzed
using the Wincycle Software (Coulter). Adherent cells were
RESULTS
harvested and stained in hypotonic fluorochrome solution
(propidium iodide 50 ?g/mL in sodium citrate plus 0.1%
Based on the XTT assay, DNA fragmentation and flow
Triton X-100, Sigma). Apoptotic nuclei were identified as a
cytometric analysis, induction of apoptosis by capsaicin was
subgenomic DNA peak and were distinguished from cell
reconfirmed in stomach tumor cell line AGS. In this study,
debris on the basis of both forward light scatter and
after incubation of AGS cells with capsaicin for 24 h, cell
fluorescence of propidium iodide.
viability decreased significantly in a dose-dependent manner
(Figure 1A). This reduction in cell viability induced by
Quantitation of DNA fragmentation
treatment with capsaicin was ascribed to apoptotic DNA
Apoptosis was also evaluated with a cell death ELISA kit
fragmentation, and the amounts of fragmented DNA were
(Boehringer Mannheim, Indianapolis, IN) which utilizes a
also dependent on the dose of capsaicin (Figure 1B). After
monoclonal antibody against histone to detect DNA
incubation of AGS cells with capsaicin for 24 h, apoptotic
fragments in the cytosolic fraction of lysed cells. Cells treated
bodies also significantly increased, and were again correlated
with or without capsaicin were harvested and lysed according
with the dose of capsaicin. The upper traces of Figure 2
to the manufacturer’s instructions. The samples were
show capsaicin-induced, concentration-dependent positive
transferred into 96-well dishes coated with a mouse
TUNEL staining. When the concentration of capsaicin was
monoclonal antibody against histone. After incubation and
1 mmol/L, the amount of DNA fragments also increased.
washing, anti-DNA-peroxidase was added to the wells. The
Similar results also were in the lower traces of Figure 2,
reaction was developed with the substrate supplied by the
where apoptotic bodies increased due to the application of
manufacturer and the absorbance of the wells was read at
capsaicin. Figure 3 shows that the expression of Bcl-2, the
410 nm. The ratio of the absorbance of the treated cells to
antiapoptotic protein, was significantly reduced in AGS cells
the untreated cells was calculated as an enrichment factor,
by capsaicin in a concentration-dependent manner.
which provides a qualitative assessment of apoptosis. Each
sample was tested in triplicate.
DISCUSSION
Apoptosis, or programmed cell death, is a natural form of
Western blotting
cell death controlled by a constitutively expressed machinery
After incubation of AGS cells for 24 h, about 107 cells
that induces condensation of the nucleoplasm and
were washed in PBS and solubilized buffer (50 mmol/L
cytoplasm, blebbing of cytoplasmic membranes, and
Tris-HCl pH 7.4, 125 mmol/L NaCl, 0.1% NP-40, 5 mmol/L
fragmentation of the cell into apoptotic bodies that are
NaF, 1 mmol/L PMSF, 1 ng/mL leupeptin, 10 ng/mL
rapidly recognized and eliminated by adjacent cells[22-24].
soybean trypsin inhibitor, 1 ng/mL aprotinin, 10 ng/mL
Induction of apoptosis by the vanilloid compound capsaicin
N-tosyl-L-phenylalanyl chloromethyl ketone) for 60 min on
has been reported in several studies. Vanilloid compounds

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6256 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol October 28, 2005 Volume 11 Number 40
100
A
B
a
100
80
80
60
a
60
a
a
40
a
40
Cell survival (%) 20
a
20
DNA fragmentation (%)
0 0 200 400 600 800 1000
0
Control 200 300 1000
Capsaicin ( mol/L)
Capsaicin ( mol/L)
Figure 1 A: Effect of capsaicin on cell survival in the cultured gastric cancer cell line, AGS. Survival was analyzed by a cell proliferation ELISA kit from Boehringer
Mannheim. The results are the mean±SE of three experiments. aP<0.05 vs solvent control; B: Effect of capsaicin on the amount of DNA fragmentation in the cultured
gastric cancer cell line, AGS. The amount of fragmented DNA produced was determined with an ELISA kit from Boehringer Mannheim. The results are the mean±SEM
of three experiments. aP<0.05 vs solvent control.
Control
Capsaicin 200 mol/L
Capsaicin 1 mmol/L
A
M=16.35
M=27.31
M=156.69
4
4
Cell number
0
6
0
6
100 101 102 103 104
100 101 102 103 104
100 101 102 103 104
Relative fluorescence intensity
B
FS(1) vs PMT2 LOG(6)
4
FS(1) vs PMT2 LOG(6)
4
FS(1) vs PMT2 LOG(6)
4




1
0





1
0





1
0



3
3
3


1
0


1
0


1
0



2
2
2


1
0


1
0


1
0
Fluorescence



1
1
1

1
0

1
0

1
0



0
0
0
10
10
10
0 1023
0 1023
0 1023
Cell size
Figure 2 Flow cytometry analysis for capsaicin-induced apoptosis in AGS gastric carcinoma cells(A,B).
CTL CAP CAP CAP
are quinone analogs that inhibit the NADH-plasma
A
10 100 200 ( mol/L)
membrane electron transport system and induce apoptosis
Bcl-2
in transformed cells[21]. Capsaicin (3.5-10 ?mol/L) induces
apoptotic cell death in an in vitro Korean stomach tumor cell (SNU-
1), which may possibly be mediated by overexpression of p53
B
and/or c-myc genes, but not by overexpression of those of c-
100
a
erbB-2, c-jun and bcl-2 genes[20], based on XTT assay, DNA
80
fragmentation and flow cytometric analysis. In our study,
a
60
induction of apoptosis by capsaicin was reconfirmed in
another stomach tumor cell line, AGS. In this study, cell
40
viability significantly decreased in a dose-dependent manner
20
after incubation of AGS cells with capsaicin for 24 h. This
b
0
reduction in cell viability induced by treatment with capsaicin
Bcl-2 expression (% of control)
CTL CAP CAP CAP
was ascribed to apoptotic DNA fragmentation, and the
10 100 200 ( mol/L)
amounts of fragmented DNA were also dependent on the
dose of capsaicin. After incubation of AGS cells with
Figure 3 Effect of capsaicin (CAP) on the expression of Bcl-2 in AGS cells.
capsaicin for 24 h, apoptotic bodies increased significantly
Each bar represents means±SE of three experiments(A,B). aP<0.05, bP<0.01
vs solvent control (CTL).
in a dose-dependent manner.

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Lo YC et al. Cell death induced by capsaicin
6257
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