Effect of pertussis toxin and herbimycin A on proteinase-activated receptor 2-mediated cyclooxygenase 2 expression in Helicobacter pylori-infected gastric epithelial AGS cells.
ABSTRACT Helicobacter pylori (H. pylori) is an important risk factor for chronic gastritis, peptic ulcer, and gastric cancer. Proteinase-activated receptor 2 (PAR2), subgroup of G-protein coupled receptor family, is highly expressed in gastric cancer, and chronic expression of cyclooxygenase-2 (COX-2) plays an important role in H. pylori-associated gastric carcinogenesis and inflammation. We previously demonstrated that H. pylori induced the expression of PAR2 and COX-2 in gastric epithelial cells. Present study aims to investigate whether COX-2 expression induced by H. pylori in Korean isolates is mediated by PAR2 via activation of G(i) protein and Src kinase in gastric epithelial AGS cells. Results showed that H. pylori-induced COX-2 expression was inhibited in the cells transfected with antisense oligonucleotide for PAR2 or treated with Gi protein blocker pertussis toxin, Src kinase inhibitor herbimycin A and soybean trypsin inbitor, indicating that COX-2 expression is mediated by PAR2 through activation of Gi protein and Src kinase in gastric epithelial cells infected with H. pylori in Korean isolates. Thus, targeting the activation of PAR2 may be beneficial for prevention or treatment of gastric inflammation and carcinogenesis associated with H. pylori infection.
Article: Localisation of cyclooxygenase 1 and cyclooxygenase 2 in Helicobacter pylori related gastritis and gastric ulcer tissues in humans.[show abstract] [hide abstract]
ABSTRACT: Prostaglandin endoperoxide synthase/cyclooxygenase (COX) is the key enzyme in gastric mucosal protection and repair but its cellular localisation in the human stomach is still unclear. To investigate immunohistochemically the cellular distribution of COX-1 and COX-2 proteins in the human stomach with or without gastritis or ulceration. Tissues were obtained by surgical resection of gastric ulcers associated with perforation (n = 9) or by biopsy from Helicobacter pylori positive patients with gastric ulcers (n = 45) and H pylori negative healthy subjects (n = 15). COX expression was detected by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), western blotting, and light and electron microscopic immunohistochemistry. COX-2 mRNA and protein were detected in gastric ulcer tissues but not in intact gastric mucosa. COX-1 mRNA and protein were detected in the intact mucosa. COX-2 immunostaining was exclusively localised in macrophages and fibroblasts between necrotic and granulation tissues of the ulcer bed. The percentage of COX-2 expressing cells was significantly higher in open than in closed ulcers, and in gastritis than in gastric mucosa without H pylori infection. COX-1 immunoreactivity localised in lamina propria mesenchymal cells was similar in various stages of ulcer disease and in intact gastric mucosa. Electron microscopic immunohistochemistry revealed both COX-1 and COX-2 on the luminal surfaces of the endoplasmic reticulum and nuclear envelope of macrophages and fibroblasts. Our results showed that COX-2 protein was induced in macrophages and fibroblasts in gastric ulcers and H pylori related gastritis, suggesting its involvement in the tissue repair process.Gut 07/2000; 46(6):782-9. · 10.11 Impact Factor
Article: Pharmacological and biochemical demonstration of the role of cyclooxygenase 2 in inflammation and pain.[show abstract] [hide abstract]
ABSTRACT: Nonsteroidal antiinflammatory drugs (NSAIDs) are widely used for the treatment of inflammatory diseases, but significant side effects such as gastrointestinal erosion and renal damage limit their use. NSAIDs inhibit the enzyme cyclooxygenase (COX), which catalyzes the conversion of arachidonic acid to prostaglandins (PGs) and thromboxane. Two forms of COX have been identified--COX-1, which is constitutively expressed in most tissues and organs, and the inducible enzyme, COX-2, which has been localized primarily to inflammatory cells and tissues. In an animal model of acute inflammation (injection of carrageenan into the footpad), edema was produced that was associated with marked accumulation of COX-2 mRNA and thromboxane. A selective inhibitor of COX-2 (SC-58125) inhibited edema at the inflammatory site and was analgesic but had no effect on PG production in the stomach and did not cause gastric toxicity. These data suggest that selective inhibition of COX-2 may produce superior antiinflammatory drugs with substantial safety advantages over existing NSAIDs.Proceedings of the National Academy of Sciences 01/1995; 91(25):12013-7. · 9.68 Impact Factor
Article: Cyclooxygenase-2 overexpression and tumor formation are blocked by sulindac in a murine model of familial adenomatous polyposis.[show abstract] [hide abstract]
ABSTRACT: Inducible cyclooxygenase (Cox-2), also known as prostaglandin H synthase 2 (PGH-2) is a key enzyme in the formation of prostaglandins and thromboxanes. Cox-2 is the product of an immediate-early gene that is expressed in response to growth factors, tumor promoters, or cytokines. Overexpression of Cox-2 is associated with both human colon cancers and suppression of apoptosis in cultured epithelia] cells, an activity that is reversed by the nonsteroidal anti-inflammatory drug, sulindac sulfide. To address the relationship between Cox-2, apoptosis, and tumor development in vivo, we studied C57BL/6J-Min/+(Min) mice, a strain containing a fully penetrant dominant mutation in the Apc gene, leading to the development of gastrointestinal adenomas by 110 days of age. Min mice were fed AIN-76A chow diet and given sulindac (0.5 +/- 0.1 mg/day) in drinking water. Control Min mice and homozygous C57BL/6J-+/+ normal littermates lacking the Apc mutation (+/+) were fed AIN-76A diet and given tap water to drink. At 110 days of age, all mice were sacrificed, and their intestinal tracts were examined. Control Min mice had 11.9 +/- 7.8 tumors per mouse compared to 0.1 +/- 0.1 tumors for sulindac-treated Min mice. As expected, +/+ littermates had no macroscopic tumors. Examination of histologically normal-appearing small bowel from Min animals revealed increased amounts of Cox-2 and prostaglandin E(2) compared to +/+ littermates. Using two different in situ techniques, terminal transferase-mediated dUTP nick end labeling and a direct immunoperoxidase method, Min animals also demonstrated a 27-47% decrease in enterocyte apoptosis compared to +/+ animals. Treatment with sulindac not only inhibited tumor formation but decreased small bowel Cox-2 and prostaglandin E(2) to baseline and restored normal levels of apoptosis. These data suggest that overexpression of Cox-2 is associated with tumorigenesis in the gastrointestinal epithelium, and that both are inhibited by sulindac administration.Cancer Research 07/1996; 56(11):2556-60. · 7.86 Impact Factor
Yonsei Med J http://www.eymj.org Volume 52 Number 3 May 2011
pISSN: 0513-5796, eISSN: 1976-2437
Yonsei Med J 52(3):522-526, 2011
Effect of Pertussis Toxin and Herbimycin A on Proteinase-
Activated Receptor 2-Mediated Cyclooxygenase 2 Expression
in Helicobacter pylori-Infected Gastric Epithelial AGS Cells
Ji Hye Seo,1 Jeong Yeon Seo,2 Hae-Yun Chung,3 and Hyeyoung Kim4
1Department of Pharmacology, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul;
2Department of Physiology, Tissue Injury Defense Research Center, College of Medicine, Ewha Womans University, Seoul;
3Institute of Food and Nutritional Science, College of Human Ecology, Yonsei University, Seoul;
4Department of Food and Nutrition, Brain Korea 21 Project, College of Human Ecology, Yonsei University, Seoul, Korea.
Received: August 17, 2009
Revised: October 9, 2009
Accepted: October 15, 2009
Corresponding author: Dr. Hyeyoung Kim,
Department of Food and Nutrition,
College of Human Ecology, Yonsei University,
50 Yeonse-ro, Seodaemun-gu,
Seoul 120-749, Korea.
Tel: 82-2-2123-3125, Fax: 82-2-364-5781
∙ The authors have no financial conflicts of
Yonsei University College of Medicine 2011
This is an Open Access article distributed under the
terms of the Creative Commons Attribution Non-
Commercial License (http://creativecommons.org/
licenses/by-nc/3.0) which permits unrestricted non-
commercial use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Helicobacter pylori (H. pylori) is an important risk factor for chronic gastritis, pep-
tic ulcer, and gastric cancer. Proteinase-activated receptor 2 (PAR2), subgroup of
G-protein coupled receptor family, is highly expressed in gastric cancer, and
chronic expression of cyclooxygenase-2 (COX-2) plays an important role in H.
pylori-associated gastric carcinogenesis and inflammation. We previously demon-
strated that H. pylori induced the expression of PAR2 and COX-2 in gastric epi-
thelial cells. Present study aims to investigate whether COX-2 expression induced
by H. pylori in Korean isolates is mediated by PAR2 via activation of Gi protein
and Src kinase in gastric epithelial AGS cells. Results showed that H. pylori-in-
duced COX-2 expression was inhibited in the cells transfected with antisense oli-
gonucleotide for PAR2 or treated with Gi protein blocker pertussis toxin, Src ki-
nase inhibitor herbimycin A and soybean trypsin inbitor, indicating that COX-2
expression is mediated by PAR2 through activation of Gi protein and Src kinase in
gastric epithelial cells infected with H. pylori in Korean isolates. Thus, targeting
the activation of PAR2 may be beneficial for prevention or treatment of gastric in-
flammation and carcinogenesis associated with H. pylori infection.
Key Words: Helicobacter pylori, cyclooxygenase-2, proteinase-activated receptor
2, AGS cells
H. pylori-associated acute and chronic antral inflammation has been associated
with the expression of cyclooxygenase-2 (COX-2) in gastric epithelial cells,1
which is strongly correlated with the extent of chronic inflammatory cell infiltrate.2
Chronic expression of COX-2 is related to H. pylori-associated gastric carcinogen-
esis in addition to propagation of gastric inflammation since prostaglandins pro-
duced via COX-2 are reported to contribute to inflammation3 and carcinogenesis.4
Our previous study demonstrated that oxidant-sensitive transcription factor NF-κB
mediates COX-2 expression, which may be related to cell proliferation in gastric
epithelial cells.5 H. pylori-induced activation of NF-κB mediates the expression of
PAR2-Mediated COX-2 Expression
Yonsei Med J http://www.eymj.org Volume 52 Number 3 May 2011
In the present study, we investigated whether H. pylori-
induced COX-2 expression is mediated by PAR2 via activa-
tion of Gi protein and Src kinase in gastric epithelial AGS
cells. Thus, the cells were transfected with sense oligonu-
cleotide (S ODN) and antisense oligonucleotide (AS ODN)
for PAR2 and cultured in the presence of H. pylori. In other
sets of experiments, the cells were treated with Gi protein
blocker pertussis toxin, Src kinase inhibitor herbimycin A
or soybean trypsin inhibitor for 1 hour and cultured in the
presence of H. pylori. Subsequently, mRNA and protein ex-
pression of COX-2 were determined by RT-PCR analysis
and Western blot analysis, respectively. Pertussis toxin in-
hibits Gi protein by coupling the inhibitory receptors to the
adenylate cyclase system.21 Herbimycin A is known to be an
irreversible inhibitor of Src kinase.22
We used HP99 which was isolated from gastric antral mu-
cosa of Korean patients with gastric and duodenal ulcer, and
identified it as cagA+, vacA s1b, m2, iceA1 H. pylori strain.23
H. pylori was added to human gastric epithelial AGS cells
(ATCC CRL 1739, American Type Culture Collection,
Manassas, Virginia, USA) at a bacterium/cell ratio of 100 : 1
in a 3 mL volume. The cells were transfected with S ODN
and AS ODN for PAR2 for 16 hours and cultured in the pres-
ence of H. pylori. In other sets of experiments, the cells were
treated with Gi protein blocker pertussis toxin (400 ng/mL),
Src kinase inhibitor herbimycin A (10 µM), or soybean tryp-
sin inhibitor (1, 2, 5 nM) for 1 hours and cultured in the pres-
ence of H. pylori. mRNA and protein expression of COX-2
were determined by reverse transcription-polymerase chain
reaction (RT-PCR) analysis (at 12 hours) and Western blot
analysis (at 24 hours), respectively. For ODN preparation,
single-stranded ODNs were prepared commercially (GIB-
CO-BRL, New York, USA). ODNs were phosphorothioate-
modified to reduce intracellular nuclease digestion. AS ODN
and S ODN target the ATG start codon of the PAR2 mRNA.
The sequence of PAR-2 AS ODN was 5’-TCCG-
CATCCTCCTGGAA-3’, and that of PAR2 S ODN was
5’-TTCCAGGAGGATGC GGA-3’. For RT-PCR analysis,
the primers used were: PAR2, forward 5’-GATGGCA-
CATCCCACGTC-3’, reverse 5’-GGCATGTATGTGATAG-
GC-3’, giving a 288 bp PCR product; COX-2, forward
5’-AGATCATCTCTGCCTGAGTATCTT- 3’, giving a 296
bp PCR produc; β-actin, forward 5’-ACCAACTGGGAC-
GACATGGAG-3’, reverse 5’-GTGAGGATCTTCATGAG-
GTAGTC-3’, giving a 354 bp PCR product. The PCR prod-
ucts were amplified and visualized by UV transilumination.23
several genes involved in inflammation, such as IL-86 and
adhesion molecules intergrin α5, in gastric epithelial AGS
cells.7 Proteinase-activated receptor 2 (PAR2) regulates cell
proliferation and enhances COX-2 expression in human
pancreatic cancer cells8 and integrin expression in H. pylo-
ri-infected gastric epithelial cells.9
Proteinase-activated receptors (PARs) are G protein-cou-
pled receptors that are activated by the cleavage of their N-
terminal domains by proteases.10,11 Proteinase-activated re-
ceptor 2 (PAR2) activation induces G protein-mediated
signal transduction, generation of inositol triphosphate, mo-
bilization of intracellular calcium, the activation of mito-
gen-activated protein kinase, cell growth, and the release of
cytokines (IL-6, IL-8) and prostaglandins.12,13 PAR2 is acti-
vated by multiple trypsin-like serine proteases including
trypsin and tryptase.13 Inflammatory cytokines (interleukin-
1α, tumor necrosis factor-α) increase mRNA expression of
PAR2.14 Invasive breast and pancreatic cancer cells express
high levels of PAR2 compared with normal cells.15 These
studies show the positive relationship between PAR2 ex-
pression and carcinogenesis. Previously, we demonstrated
that H. pylori induced the expression and activation of PAR2
by stimulating the expression of trypsinogens and trypsin in
gastric epithelial AGS cells.9 In addition, inhibition of PAR2
activation by a soyben trypsin inhibitor (SBTI) suppressed H.
pylori-induced expression of integrins in gastric epithelial
cells.9 Therefore, PAR2 seems to play an important role in H.
pylori-associated gastric inflammation and/or carcinogenesis
by mediating the induction of inflammatory and carcinogenic
genes, including COX-2, in gastric epithelial cells.
In addition, the genetic differences of H. pylori isolates
play a role in the clinical outcome of the infection, particu-
larly H. pylori-virulence associated genes such as vacA,
cagA, and iceA genes.16 Infection by cagA strain is more
likely to result in peptic ulceration, atrophic gastritis, and
gastric carcinoma.17,18 Presence of cagA or vacA in H. pylo-
ri strain showed different expression of genes as compared
to cagA negative or vacA negative H. pylori in gastric epi-
thelial AGS cells.19 The studies suggest that the presence of
virulence factors (vacA, cagA, and iceA) and isotypes of each
virulence factor (vacA s1b m2, vacA s1c m1, iceA1, iceA2
etc.) in H. pylori strain are important to determine disease
incidence related to H. pylori infection. Since the predomi-
nant genotype of H. pylori in Korea has been reported to be
cagA positive and vacA positive genotype,20 H. pylori in
Korean isolates may be used to determine the pathogenic
mechanism of H. pylori-induced gastric diseases in Korea.
Ji Hye Seo, et al.
Yonsei Med J http://www.eymj.org Volume 52 Number 3 May 2011
fection efficiency of ODN was determined by observing
mRNA and protein levels of PAR2 in the cells transfected
(Fig. 1A). H. pylori-induced PAR2 expression was inhibit-
ed in the cells transfected with AS ODN, but not changed
in those transfected with S ODN. H. pylori-induced expres-
sion of COX-2 was similarly inhibited in the cells transfect-
ed with AS ODN compared to those in the cells transfected
with S ODN (Fig. 1B). Since protein level and the activity
of trypsin increased in H. pylori-infected AGS cells,9 trypsin
may activate PAR2 in AGS cells. Previously, we showed the
increase of intracellular Ca mobilization by PAR2 activa-
tion in H. pylori-infected AGS cells,25 which was inhibited
by SBTI (data not shown), demonstrating that SBTI in AGS
cells suppresses PAR activation induced by H. pylori. Using
SBTI, we determined whether SBTI suppresses H. pylori-
induced expression of COX-2 in AGS cells. As seen in Fig.
2A, H. pylori-induced expression of COX-2 was inhibited
dose-dependently by SBTI. G protein coupled receptor
(GPRC) signalings have been shown to be inhibited by per-
tussis toxin, which inactivates ADP-ribosylation of α sub-
For Western blot analysis, the proteins were detected with
polyclonal antibodies for PAR2 (Catalog # SC-13504) and
COX-2 (Catalog # SC-19999), perchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA), at 1 : 1000 dilation
and the immunoreactive proteins were visualized by en-
Since mRNA and protein expressions of COX-2 were evi-
dent at 12 hours and 24 hours in our previous study,24 in the
following experiments using transfection with S ODN and
AS ODN for PAR2 or treatment with Gi protein blocker per-
tussis toxin, Src kinase inhibitor herbimycin A and SBTI, re-
spectively, the time points of 12 hours and 24 hours for
mRNA and protein expressions of COX-2, respectively,
were used. β-Actin (for mRNA) and actin (for protein) were
constitutively expressed in the cells and not changed with
To determine direct involvement of PAR2 in the expres-
sion of COX-2, the cells were transfected with PAR2 AS
ODN or S ODN and cultured in the presence of H. pylori
for 12 hours (mRNA) or 24 hours (protein) (Fig. 1). Trans-
Fig. 2. H. pylori-induced expression of COX-2 is inhibited in AGS cells treat-
ed with soybean trypsin inhibitor (SBTI), pertussis toxin and herbimycin A.
AGS cells in 6-well culture plates were treated with SBTI (1, 2, 5 nM) (A), or
pertussis toxin (400 ng/mL) or herbimycin A (10 µM) (B) for 1 h and cultured
in the presence of H. pylori at a bacterium/cell ratio of 100 : 1 for 12 h (for
mRNA) or 24 h (for protein). COX-2 mRNA expression levels were deter-
mined by RT-PCR and at protein level by Western blotting. COX-2, cycloox-
Fig. 1. H. pylori-induced expressions of PAR2 and COX-2 are inhibited in
AGS cells transfected with PAR2 AS ODN. AGS cells were seeded in 6-well
culture plates at 5×105 cells per well transfected with S or AS ODNs for
PAR-2 for 16 h. The bacterial cells were added to the cultured cells at a
bacterium/cell ratio of 100 : 1 for 12 h (for mRNA) or 24 h (for protein). The
expressions of mRNA and protein for PAR2 (A) and COX-2 (B) were deter-
mined by RT-PCR and Western blotting, respectively. PAR2, proteinase-ac-
tivated receptor 2; COX-2, cyclooxygenase-2.
PAR2-Mediated COX-2 Expression
Yonsei Med J http://www.eymj.org Volume 52 Number 3 May 2011
gram through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education, Science and
Technology (2010-0001669) (to H Kim) and the Korea Re-
search Foundation Grant funded by Korea Government
(MOEHRD) (KRF-2006-353-F00019) (to H-Y Chung). H
Kim is grateful to Brain Korea 21 Project, College of Hu-
man Ecology, Yonsei University.
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Fig. 2B shows that pertussis toxin and herbimycin A inhib-
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These results demonstrated that Gi protein and Src kinase
are involved in PAR2-mediated COX-2 expression in H.
pylori-infected gastric epithelial cells. Gi protein is known
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flammation at molecular and cellular levels. Recent study
showed that advanced oxidation protein products were in-
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activities of antioxidant enzyme catalase in serums were not
different between H. pylori-positive and negative patients.
Since oxygen radicals are important in transcription of COX-
2, the relation between oxidative stress and PAR2-mediated
signaling should be investigated. The novel finding of the
present study is that H. pylori in Korean isolates induced
COX-2 expression, which is mediated by PAR2 through acti-
vation of Gi protein and Src kinase in gastric epithelial cells.
Specific targeting of the activation of PAR2 may be benefi-
cial for prevention or treatment of H. pylori-associated gas-
tric inflammation and carcinogenesis in Korea.
This study was supported by Basic Science Research Pro-
Ji Hye Seo, et al.
Yonsei Med J http://www.eymj.org Volume 52 Number 3 May 2011
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