Novel mechanism of gastric proton...
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Transcript of Novel mechanism of gastric proton...
의학의학의학의학 석사학위석사학위석사학위석사학위 논문논문논문논문
Novel mechanism of gastric proton
pump inhibitor (PPI) : Suppression of
Helicobacter-pylori induced
angiogenesis and selective induction of
apoptosis in gastric cancer cells.
아아아아 주주주주 대대대대 학학학학 교교교교 대대대대 학학학학 원원원원
의의의의 학학학학 과과과과
김김김김 동동동동 규규규규
Novel mechanism of gastric proton pump inhibitor
(PPI) : Suppression of Helicobacter-pylori induced
angiogenesis and selective induction of apoptosis in
gastric cancer cells.
by
Dong Kyu Kim
A Dissertation Submitted to The Graduate School of Ajou University
in Partial Fulfillment of the Requirements for the Degree of
MASTER OF MEDICAL SCIENCE
Supervised by
Sung Won Cho, M.D., Ph.D.
Department of Medical Sciences
The Graduate School, Ajou University
August, 2008
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김동규의김동규의김동규의김동규의 의학의학의학의학 석사학위를석사학위를석사학위를석사학위를 인준함인준함인준함인준함.
심사위원장심사위원장심사위원장심사위원장 조조조조 성성성성 원원원원 인인인인
심 사 위 원심 사 위 원심 사 위 원심 사 위 원 이이이이 광광광광 재재재재 인인인인
심 사 위 원심 사 위 원심 사 위 원심 사 위 원 정정정정 재재재재 연연연연 인인인인
아아아아 주주주주 대대대대 학학학학 교교교교 대대대대 학학학학 원원원원
2008 2008 2008 2008 년년년년 6 6 6 6 월월월월 23 23 23 23 일일일일
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ABSTRACT
Novel Mechanism of Gastric Proton Pump (PPI) Inhibitor :
Suppression of Helicobacter pylori Induced Angiogenesis and
Selective Induction of Apoptosis in Gastric Cancer Cells.
To survival in an ischemic microenvironment with a lower extracellular pH, ability to
up-regulate proton extrusion is critical for cancer cell survival. Gastric H+/K+-ATPase
exchanges luminal K+ for cytoplasmic H+ and is the enzyme primarily responsible for gastric
acidification. On the basis of the fact that blocking the clearance of acidic metabolites are
known to induce the cell death, we hypothesized that pantoprazole (PPZ), one of gastric
H+/K+-ATPase inhibitors used frequently to treat acid-related diseases, could inhibit growth
of tumor cells. And, although activation of mitogen activated protein kinases (MAPKs) by
Helicobacter pylori infection is associated with induction of host angiogenesis, which may
contribute to H.pylori associated gastric carcinogenesis, the strategy for its prevention has
not been identified. As we previously reported a strong inhibitory action of gastric proton
pump inhibitors (PPIs) on MAPK extracellular signal regulated kinase (ERK) 1/2
phosphorylation, we investigated whether PPIs could suppress the H.pylori induced
angiogenesis via inhibition of MAPK ERK1/2.
To detect PPZ-induced apoptosis, we performed that Genomic DNA fragmentation,
terminal deoxynucleotidyl transferase (Tdt)-mediated nick end labeling assay, and annexin V
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staining. And mitogen-activated protein kinase activation and heat shock proteins expression
were determined by immunoblot with specific antibodies. The antitumor effect of PPZ was
evaluated in vivo by a xenograft model of nude mice. And, to address the relationship
between H.pylori infection and angiogenesis, comparative analysis of density of CD34+
blood vessel was performed in tissues obtained from 20 H.pylori positive gastritis and 18
H.pylori negative gastritis patients. Expression of hypoxia inducible factor 1 (HIF-1α) and
vascular endothelial growth factor (VEGF) was tested by reverse transcription-polymerase
chain reaction and secretion of interleukin 8, and VEGF was measured by ELISA. To
evaluate the direct effect of H.pylori infection on the tubular formation of human umbilical
vein endothelial cells (HUVEC), an in vitro angiogenesis assay was employed. Activation of
MAPK and nuclear factor κB was detected by immunoblotting.
After PPZ treatment, apoptotic cell death was seen selectively in cancer cells and was
accompanied with extracellular signal-regulated kinase deactivation. By contrast, normal
gastric mucosal cells showed the resistance to PPZ-induced apoptosis through the
overexpression of anti-apoptotic regulators including HSP70 and HSP27. In a xenograft
model of nude mice, administration of PPZ significantly inhibited tumorigenesis and induced
large-scale apoptosis of tumor cells. And, H.pylori positive gastritis patients showed a higher
density of CD34+ blood vessels (mean 40.9(SEM 4.4)) than H.pylori negative gastritis
patients (7.2±0.8), which was well correlated with expression of HIF-1α. Conditioned media
from H.pylori infected gastric epithelial cells directly induced tubular formation of HUVEC
and the increase of in vitro angiogenesis was suppressed by PPI treatment. Infection of
H.pylori significantly upregulated expression of HIF-1α and VEGF in gastric epithelial cells
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and expression of proangiogenic factors was mediated by MAPK activation and partially
responsible for NFκB activation. PPIs effectively inhibited the phosphorylation of MAPK
ERK1/2 that is a principal signal for H.pylori induced angiogenesis.
In conclusion, PPZ selectively induced in vivo and in vitro apoptotic cell death in
gastric cancer, suggesting that proton pump inhibitors could be used for selectively
anticancer effects. And, the fact that PPZ could downregulate H.pylori induced angiogenesis
indicates that antiangiogenic treatment using a PPZ could be a promising protective
therapeutic approach for H.pylori associated carcinogenesis.
Key words : Proton pump inhibitor, Apoptosis, Gastric cancer cell, H+/K+-ATPase, anticancer
effect, Helicobacter pylori, Angiogenesis, VEGF, HIF-1α, Carcinogenesis
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TABLE OF CONTENTS
ABSTRACT ........................................................................................................................ 1
TABLE OF CONTENTS ................................................................................................. vii
LIST OF FIGURES ........................................................................................................... ix
I. INTRODUCTION ........................................................................................................... 1
II. MATERIALS AND METHODS .................................................................................. 6
A. Cell culture, bacteria strain, and reagents ............................................................ 6
B. Tissue samples ......................................................................................................... 7
C. Measurements of Intracellular pH ......................................................................... 8
D. Detection of Apoptosis . ............................................................................................ 8
E. Western Blot Analysis .............................................................................................. 9
F. Reverse transcription-polymerase chain reaction (RT-PCR) analysis ............. 10
G. Enzyme linked immunosorbent assay (ELISA) ................................................. 11
H. Immunohistochemistry for counting vessles in the gastric mucosa . ................. 11
I. Immunofluorescence Staining ................................................................................ 12
J. In vitro angiogenesis assay .................................................................................... 12
K. Tumor Xenograft . .................................................................................................. 13
L. Statistics . ................................................................................................................. 13
III. RESULTS .................................................................................................................... 14
IV. DISSCUTION ............................................................................................................. 43
REFERENCES ................................................................................................................. 50
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국문요약국문요약국문요약국문요약 ............................................................................................................................ 66
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LIST OF FIGURES
Fig. 1. Effects of pH of culture medium on cell viability and cellular expression of
H+/K+-ATPase............................................................................................................15
Fig. 2. Inhibition of cancer cell viability by PPZ treatment.................................................18
Fig. 3. Effect of proton pump inhibitors on gastric cancer cell viability. ............................19
Fig. 4. Selective induction of apoptosis with PPZ...............................................................21
Fig. 5. Distinct MAPK signaling in cancer cells (A) and noncancer cells (B) by PPZ. ......23
Fig. 6. Effect of PPZ on expression of HSP70 and HSP27 in gastric cancer and
noncancer cells. .........................................................................................................25
Fig. 7. Antitumorigenic potency of PPZ in xenograft model...............................................27
Fig. 8. Immunohistochemical staining of the CD34 endothelial cell marker. .....................30
Fig. 9. Expression of hypoxia inducible factor 1 (HIF-1α) mRNA in human gastric
mucosa................................................................................................................. 31
x
Fig. 10. In vitro angiogenesis assay. ....................................................................................33
Fig. 11. Release and expression of vascular endothelial growth factor (VEGF) and
interleukin 8 (IL-8) from Helicobacter pylori infected AGS cells. ...........................36
Fig. 12. Effects of proton pump inhibitor (PPI) on expression of angiogenic growth
factors interleukin 8 (IL-8), hypoxia inducible factor 1 (HIF-1α), and vascular
endothelial growth factor (VEGF).............................................................................37
Fig. 13. Involvement of mitogen activated protein kinase and nuclear factor κB
(NFκB) in Helicobacter pylori induced mRNA expression of hypoxia inducible
factor 1 (HIF-1α) and vascular endothelial growth factor (VEGF)...........................39
Fig. 14. Deactivation of mitogen activated protein kinase (MAPK) extracellular signal
regulated kinase (ERK)1/2 signaling with proton pump inhibitor (PPI). ..................41
1
I. INTRODUCTION
The H+/K+-ATPase of gastric parietal cell exchanges luminal K+ for cytoplasmic H+ and
is the enzyme primarily responsible for gastric acidification (Marie et al, 2004; Scott et al,
1993; Besancon et al, 1993; Pouyet et al, 1992). The enzyme consists of two subunits, a 114
kDa α-subunit and a 35 kDa β-subunit. The α-subunit containing ATP and cation binding
sites carries out the catalytic and transfporting function of the proton pump. The heavily
glycosylated β-subunit is required for endocytic retrieval of the H+/K+-ATPase from the
canalicular membranes and is also essential for protecting proton pump from the
environment of acid milieu. Because abnormally controlled gastric acids secreated by
H+/K+-ATPase caused several gastrointestinal acid-related diseases including
gastroesophageal reflux disease, gastric ulcer, duodenal ulcer, and Barrett’s esophagus,
gastric proton pump inhibitors have been developed as the treatment for these acid-related
diseases (Horn et al, 2000; Sachs et al, 1997; Sachs et al,1995; Fitton et al, 1996).
Pantoprazole (PPZ) of these proton pump inhibitors, a substituted 2- pyridylmethyl/sulfinyl
benzimidazole derivative, is a prodrug requiring protonation for functional activation at
acidic conditions, accumulating selectively in acidic gastric luminal space and ultimately
inhibiting acid secretion by the covalent binding with cystein residues in α-subunit of
H+/K+-ATPase.
Besides of gastic H+/K+-ATPase, several kinds of vacuolar-type H+-ATPase are
ubiquitously found on the membrane of various intracellular compartments of eukaryotic
cells such as lysosomes, endosomes, the Golgi complex, and secretary granules (Nelson et al,
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1995). Essential regulateion of pH in cytoplasmic, intraorganellar, and local extracellular
spaces through vacuolar-type H+-ATPase has been suggested to play an important role in the
mechanism of cell survival. These facts can be credited by several studies showing that
vacuolar-type H+-ATPase inhibitor, bafilomycin A or concanamycin A, strongly induced
apoptotic cell death (Nishihara et al, 1995; Ishisaki et al, 1999; Ohta et al, 1998; Aiko et al,
2002).
Cell survival and cell death are tightly controlled by numerous signal enzymes and
regulators such as mitogen-activated protein kinases (MAPKs) and heat shock proteins
(HSPs). MAPK signal enzymes are divided into three major groups, extracellular signal-
regulated kinases (ERKs), c-Jun NH2-terminal kinases (JNKs)/stress-activated kinases, and
p38 (Franklin et al, 2000; Johnson et al, 2002). The ERKs appear to play a crucial role in the
process of extracellular signals to the nucleus leading to induction of cellular growth,
proliferation, and differentiation (Ballif et al, 2001; Dent et al, 1998). Heat shock proteins,
which function mainly as molecular chaperones, allow cells to adapt to their environmental
changes and to survive in otherwise lethal conditions (Garrido et al, 2001; Mehlen et al,
1996; Garrido et al, 1999; Bruey et al, 2000; Mosser et al, 1997; Buzzard et al, 1998; Beere
et al, 2000). Because HSPs include pro- and antiapoptotic proteins that interact with a variety
of cellular proteins, the type of HSP induced and its level of expression can determine the
fate of a cell in response to a death stimulus. Generally, HSP60, HCS70, and HSP90 are
constitutively expressed in mammalian cells, whereas HSP70 and HSP27 are strongly
induced by different stresses, such as heat, oxidative stress, or anticancer drugs. It is well
known that HSP27 and HSP70 play a cytoprotective role in gastrointestinal damage
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including apoptosis (Tsukiml et al, 2001; Rokutan et al, 2000; Ethridge et al, 1998; Shichijo
et al, 2003).
Maintenance of intra- or extracellular pH is very much important for cell function, and
cancer cells in vivo often exist in an ischemic microenvironment with a lower extracellular
pH than surrounding normal cells (Stubbs et al, 1999; Helmlinger et al, 1997; Stubbs et al,
1992; Suubbs et al, 1994; Frenzel et al, 1994; Gillies et al, 1994). The acidity in tumors is
due to the increased production of acidic metabolites from rapid and large amounts of
glycolysis and is provoked by the limited ability of the tumor vasculature to remove these
acidic products. To overcome this hypoxic microenvironment and to prevent the
accumulation of the increased acidic metabolites, the ability to dispose of intracellular
protons is critical for cancer cell survival (Mccoy et al, 1995; Tannock et al, 1989; Maritinez-
et al, 1993; Lee et al, 1998). These findings support the rationale of the present study that the
inhibition of proton extrusion might be more susceptible or vulnerable to cell death of cancer
cells than normal cells.
In this study, we have demonstrated for the first time that PPZ, the H+/K+-ATPase
inhibitor, induced apoptosis selectively in gastric cancer cells and significantly inhibited
tumorigenesis in a tumor xenograft model. We also documented the mechanism of the
selectivity of this proton pump inhibitor on apoptosis of cancer cells. Our novel finding
suggests that proton pump inhibitors could be considered as the selective anticancer agents in
gastric cancer.
And another PPZ study, we investigated that PPZs could exert angiogenesis actions
through MAPK inhibitions in H.pylori induced angiogenesis. That chronic persistent gastric
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inflammation associated with Helicobacer pylori may play a crucial role in either the
development or progression of gastric cancers has been generally agreed (Peek et al, 2002;
Stolte et al, 2002; Scheiman et al, 1999) but the exact molecular mechanisms of how
longstranding H.pylori infection can induce and make the procancer microenvironment
favourable for the survival of tumor cells have not yet been clearly identified. The
mechanisms fostering the neoplastic process of H.pylori infection have been revealed to
include : (1) induction of neoplastic mutation by a considerable burden of oxidative stress
(Touati et al, 2003; Bagchi et al, 1996); (2) imbalance between cell proliferation and
apoptosis (Maeda et al, 2002; Gupta et al, 2001); (3) production of proteases and growth
factors providing the environment for cell migration (Betten et al, 2001; Allen et al, 2000;
Crawford et al, 2003); and (4) induction of host angiogenesis.
Among the diverse host cellular response related to H.pylori associated inflammation or
carcinogenesis, some investigators reported that angiogenic growth factors induced by
H.pylori might be primarily important (Kitadai et al, 2003; Cox et al, 2001; Strowski et al,
2004; Innocenti et al, 2002; Franceschi et al, 2002). Kitadai and colleagues and Cox and
colleagues (Cox et al, 2001) found that H.pylori infection induced several angiogenic factors
and proteases, such as interleukin 8 (IL-8), vascular endothelial growth factor (VEGF),
angiogenin, urokinase-type plasminogen activator, and metalloprotease 9 using high
throughput technology of cDNA microarray analysis. Strowski and colleagues
(Strowski et al, 2004) also reported that H.pylori stimulated host VEGF gene expression via
a mitogen activated protein kinase (MAPK) pathway. These data imply that H.pylori are
capable of inducing host angiogenesis, which may play a critical role in the development and
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progression of gastric cancer. However, trials documenting the precise mechanism and
preventive therapeutic approaches have not been performed.
The last decade has seen stanardisation of the treatment regimens for H.pylori
eradication, with the use of triple therpy consisting of a PPI and two antibiotics, mainly
clarithromycin and amoxicillin. Blockage of gastric acid secretion by PPIs contributes
towards eradication of H.pylori via the rising pH of the gastric lumen. Appropriately high pH
values increase antimicrobial susceptibility of H.pylori because the minimum inhibitory
concentration of most antibiotics against H.pylori is very dependent on the pH of the
environment (Iwahi et al, 1991; Nakao et al, 1998; Hirai et al, 1995; Tsuchiya et al, 1995;
McGowan et al, 1994; Mauch et al, 1993).
Previously, we found that PPIs could exercise selective induction of apoptosis in gastric
cancer cells, which was due to a significant inhibitory action of PPIs on MAPK activation
(Yeo et al, 2004). As Stowski and colleagues (Hirai et al, 1995) reported that H.pylori
stimulated host VEGF gene expression via the MAPK pathway, we hypothesized that PPIs
could exert antiangiogenesis actions through MAPK inhibition in H.pylori induced
angiogenesis. Here, we have found that infection with H.pylori significantly upregulated
angiogenesis of the gastric mucosa by strong induction of proangiogenic factors, including
IL-8, hypoxia inducible factor 1 (HIF-1α), and VEGF and, remarkably, angiogenesis induced
by H.pylori was attenuated by PPI treatment.
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II. MATERIALS AND METHODS
A. Cell culture, bacteria strain, and reagents
Human gastric cancer cell lines (AGS, Kato III, SNU-1, SNU-601, MKN-28, and
MKN-45) were cultured in RPMI 1640 (Life Technologies, Inc., Grand Island, NY)
containing 10% fetal bovine and 100 units/ml penicillin in a humidified 5% CO2 atmosphere.
As counter cells of these cancer cells, we cultured normal rat gastric mucosal RGM-1 cells
and normal rat intestinal epithelial IEC-6 cells, which were maintained with DMEM-F12 and
DMEM, high glucose (Life Technologies, Inc.) supplemented with 10% bovine insulin,
respectively, and COS-1 cell, normal human fibroblast cells with RPMI 1640. Human
umbilical vein endothelial cells (HUVEC) were obtained by collagenase treatment of
umbilical cord veins, as previously described (Jappe EA et al, 1973). Cells were cultured on
gelatin coated dishes and propagated in RPMI 1640 medium supplemented with 20% bovine
calf serum, 90 ug/ml heparin (Sigma Chemical Co, St Louis, Missouri, USA), and 50 ug/ml
endothelial cell growth factor.
A cagA+ and vacA+ standard strain of H.pylori (ATCC 43504) was obtained from the
American Type Culture Collection (ATCC, Manassas, Virginia, USA). H.pylori were
recovered from frozen stock by seeding on a blood agar plate including 7% sheep blood at
37℃ for five days under microaerophilic conditions (5% O2, 10% CO2) generated with
campy pouch (Becton Dickinson Microbiology Systems, Sparks, Maryland, USA). For
inoculation of the bacteria, H-pylori were resuspended in phosphate buffered saline (PBS) to
an A450 of 1.2 units, which corresponds to a bacterial concentration of 5 x 108 colony forming
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units (CFU)/ml, and cocultured with AGS cells at a concentration of 5 x 107 CFU/ml.
Solutions of pantoprazole (PPZ) and omeprazole were obtained from Altana Pharma
AG (Konstanz, Germany) and AstraZeneca, respectively, and lansoprazole (Takeda, Japan)
was solved in PBS (adjusted pH 2.0) with HCl overnight at room temperature. PD98059
(50µM, extracellular signal regulated kinase (ERK) 1/2 inhibitor; Cell Signaling Technology,
Beverly, Massachusetts, USA), SB203580 (10µM, p38 inhibitor; Cell Signaling Technology),
1-pyrrolidinecarbodithioic acid (PDTC 100µM, ammonium salt, nuclear factor κB (NF-κB)
inhibitor; Calbiochem, La Jolla, Califonia, USA), and BAYII-7082 (5µM, NF-κB inhibitor;
Calbiochem) were used in the cell culture experiments. Briefly, to evaluate the effect of these
inhibitors or PPIs, they were preincubated with AGS cells for eight hours, washed with PBS,
and inoculated with H.pylori.
B. Tissue samples
Biopsied samples were obtained from five patients (mean age 48 years) with functional
dyspepsia without H.pylori infection, 20 patients (means age 55 years) with chronic active
H.pylori positive gastritis, and 18 patients (means age 54 years) with H.pylori negative
gastritis induced mostly by non-steroidal anti-inflammatory drugs or other cause during
gastroscopy. The presence of H.pylori was determined using the following tests :
haematoxylin-eosin staining and Giemsa staining of biopsied tissues, rapid urease test, and
urea breath test. When all of the above were negative, the case was defined as H.pylori
negative and if more than two of these tests were positive, the case were defined as H.pylori
positive. Gastritis was evaluated histologically and scored according to a modified Sydney
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classification (Dixon MF et al, 1996); two different pathologists scored the degree of gastritis
independently. Informed written consent was obtained from patients and the study was
approved by Institutional Review Board.
C. Measurements of Intracellular pH.
Intracellular pH was measured in the monolayers using the pH-sensitive fluorescent
probe 2',7'-bis-(2-carboxyethyl)-5-carboxyfluorescein (BCECF). Cells were loaded with
BCECF for 10 minutes at room temperature in solution A containing 2.5µM/L BCECF-AM
and mounted in the miniature Ussing chamber described for [Ca2+] ί measurements. BCFCF
fluorescence was recorded and calibrated using a protocol described previously (Namkung
W et al, 2003). Briefly, the fluorescence at excitation wavelengths of 490 and 440nm was
recorded using a recording setup (Delta Ram; PTI Inc., St Louis, MO), and the 490;440
ratios were calibrated intracellularly by perfusing the cells with solutions containing
145mmol/L KCl, 10mmol/L HEPES, and 5µmol/L nigericin with the pH adjusted to 6.2 to
7.6.
D. Detection of Apoptosis.
Induction of apoptosis was determined by assaying a genomic DNA fragmentation.
Briefly, cells were lysed for 15minutes in 10 mmol/L Tris.Cl (pH 7.4), 5 mmol/L EDTA, and
1% Triton X-100 and centrifuged at 12,000 rpm for 15 minutes. The supernatant was
incubated with 0.1mg/ml proteinase K at 37℃ for 1 hour and extracted with an equal
volume of phenol-chloroform, and the cellular DNA was precipitated with 1:10 volumes of
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0.3 mol/L sodium acetate and 2 volumes of absolute EtOH overnight at -70℃. The
precipitate was dissolved in 20 µL TE buffer containing 200 µg/ml RNase and incubated for
1 hour at 37℃. The extracted DNA was resolved on 1.8% agarose gel and stained with
ethidium bromide.
Caspase-3 and poly(ADP-ribose) polymerase (PARP) proteolysis were assessed by
immunoblotting with specific antibodies (Cell Signaling Technology, Beverly, MA).
Terminal deoxynucleotidyl transferase (Tdt)-mediated nick end labeling (TUNEL) and
annexin V/propidium iodide staining were detected by Fluorescent FragEL DNA fragment
detection kit (Oncogene, Boston, MA) and annexin V-FITC apoptosis detection kit (BD
Bioscience, San Diego, CA), according to the manufacture’s instructions, respectively.
E. Western Blot Analysis.
Human gastric cancer cell line, MKN-45 and normal gastric mucosal cell line, RGM-1
cells were incubated with PPZ(0.5 mmol/L PPZ for 0.5, 2, 4 or 8 hours and 0.3 or 0.6
mmol/L PPZ for 24 hours). And Human gastric cancer cell line, AGS cells were incubated
with 0, 100, 200, 400 µmmol/L PPZ for eight hours, washed with PBS three times, and then
inoculated with H.pylori (5 x 107 CFU/ml) for 15 minutes (western blotting for ERK1/2) or
two hours (western blotting for NFκB). Cells were resuspended in lysis buffer (20mM Tris,
pH 7.5, 150mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, and protease inhibitor
cocktail; Roche, Mannheim, Germany). The suspension was sonicated for approximately 30
seconds and centrifuged at 15,000g for 30 minutes. For documenting NFκB activation,
nuclear/cytosolic fractionation was performed using NE-PER Nuclear and Cytoplasmic
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Extraction Kit (Pierce, Rockford, Illinois, USA) following the manufacturer’s protocol.
Isolated protein was subjected to western blotting. Proteins were extracted from the cells,
electrophoresed on 12% sodium dodecyl sulphate-polyacrlyamide gels, and transferred to
PVDF membranes using a semidry transfer system (Hoeffer Phamacia Biotech, San
Francisco, California, USA). Membranes were blocked in 5% non-dry milk and probed with
1:1000 dilution of specific antibodies corresponding to phospho-p38 (Cell Signaling
Technology, Beverly, MA), phosphor-ERK, phosphor-JNK, HSP70, HSP60, total ERK,
NFκB p65, HSP27 or α-tubulin; all antibodies were purchased from Santa Cruz
Biotechnology (Santa Cruz, California, USA).
F. Reverse transcription-polymerase chain reaction (RT-PCR) analysis
Total RNA was isolated from cells with appropriate treatment using TRIzol reagent
(Life Technologies, Milan, Italy), and 2 µg total RNA were reverse transcribed according to
the manufacturer’s instructions (M-MLV reverse transcriptase; Promega, Madison,
Wisconsin, USA). PCR was performed using the Premix Ex Tag kit (Takara, Chiba, Japan)
with specific primers as follows : 5'-CTC AAA GTC GGA CAG CCT CA-3' and 5'-CCC
CGC AGT TTT CTG CT-3' for HIF-1α; 5'-TCG GGC CTC CGA AAC CAT G-3' and 5'-
GGT TCC CGA AAC CCT GAG G-3' for VEGF; 5'-TTG TTG CCA TCA ATG ACC CC-3'
and 5'-TGA CAA AGT GGT CGT TGA GG-3' for GAPDH. The PCR reaction was carried
out for 28 thermal cycles of 94℃ for one minute at 55℃ (for HIF-1α and GAPDH) or
60℃ (for VEGF) for one minute, and 72℃ for one minute. The product was resolved on
1% agarose gel and stained with ethidium bromide.
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G. Enzyme linked immunosorbent assay (ELISA)
Immunoreactive human IL-8 and VEGF were measured in culture supernatants of AGS
cells using enzyme linked immunosorbent assay (ELISA) kits according to the manufacture’s
instructions (HyCult Biotechnology, Uden, the Netherlands). AGS cells were grown in six
well cell culture dishes, incubated in the presence or absence of PPZ for eight hours, and
after washing with PBS, cocultrued with H.pylori for various times. Culture supernatant
(200µl) was used for analysis of IL-8 and VEGF production.
H. Immunohistochemistry for counting vessles in the gastric mucosa.
Immunohistochemistry staining of CD34+ endothelial cells was performed to analyse
the degree of angiogenesis in the gastric mucosa of gastric patients. For
immunohistochemical detection, 10% buffered formalin fixed paraffin embedded sections
were deparaffinised, rehydrated, and then boiled in 100 mM Tris buffered saline (pH 7.6)
with 5% urea in an 850 W microwave oven for five minutes, followed by two more
treatments of five minutes each. Then sections were stained with Histostain-Plus kit (Zymed
Laboratories Inc., San Francisco, California, USA) according to the manufacture’s
instructions. Primary antibodies against the CD34 endothelial cell marker were purchased
from Novocastra Laboratories (clone QBEnd/10; UK). Sections were counterstained with
haematoxylin. CD34 positive blood vessles were counted on three separate sites (x100
magnified field) and presented as mean (SEM) of 20 H.pylori positive and 18 H.pylori
negative cases.
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I. Immunofluorescence Staining.
Dispersed single cells (2 x 105 cells per well) were grown on 22 x 22 x 1 ㎣ glass
coverslips in 6-well cultrure plates. After 24-hour culture, cells were fixed in ice-cold
methanol for 5 minutes in a -20℃ freezer and permeabilized with 1% Triton X-100/PBS for
10 minutes at room temperature. The cells were blocked with 5％ bovine serum albumin for
30 minutes and probed with anti-α subunit of H+/K+-ATPase antibodies (1:100 diluted in
5％ bovine serum albumin, Santa Cruz Biotechnology) for 2 hours. Cy3-conjugated
secondary antibodies were used to visualize under a confocal microscope (BX50F, Olympus,
Japan).
J. In vitro angiogenesis assay
In vitro angiogenesis assay was slightly modified from Kitadal and colleagues.
Briefly, AGS cells (1 x 107 cells/100 ㎟ culture dish) were incubated with 0,100,200, or 400
µM PPZ for eight hours, washed with PBS three times, and then inoculated with H.pylori (5
x 107 CFU/ml) for 24 hours. The cell culture supernatant was harvested and centrifuged at
5000 g for 30 minutes. Conditioned media was prepared by 1:1 dilution of the culture
supernatant with HUVEC endothelial cell medium. The conditioned media were filtered
through 0.4µM pore filters (Millipore, Boston, Massachusetts, USA) to remove H.pylori and
then the media were added to HUVEC culture and changed every three days. After nine days,
the HUVEC were observed for tubular formation under microscopy and confirmed
expression of the endothelial cell marker, CD31, by immunocytofluorescence staining.
HUVEC were fixed in ice cold methanol for five minutes, frozen at -20℃, and treated with
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1％ Triton X-100/PBS for 10 minutes at room temperature. Cells were blocked with 5％
bovine serum albumin for 30 minutes and probed with anti-CD31 antibodies (1:100 dilution
in 5％ bovine serum albumin; Santa Cruz Biotechnology) for two hours. Cy3-conjugated
secondary antibodies were used to visualize under a inverted fluorescence microscope
(Olympus, IX71, Tokyo, Japan).
K. Tumor Xenograft.
Subconfluent MKN-45 cells were dissociated with 0.25％ trypsin and 1 mmol/L EDTA
(Life Technology, Inc.) and suspended in PBS at density of 5 x 107 cells/ml. Each mouse was
s.c. inoculated with MKN-45 cells (5 x 106 per site) on the left and right side of the back on
day 0. The animals were randomly divided into two groups (9 per group). Group A received
an intratumoral injection of PBS daily from day 14; Group B daily received an intratumoral
injection of 0.4 ㎎/㎏ PPZ daily from day 14. The shortest and longest intervals, and the
volume of each tumor (㎣) was calculated. Mice were sacrified at day 22, and isolated tumor
tissues were analyzed for microscopic gross finding and TUNEL stain. These studies were
approved by the Institutional Animal Care and Use Committee and complied with the highest
international criteria for human use of animals in research.
L. Statistics.
All values are expressed as mean (SEM) and the Mann-Whitney U test and Friedman
ANOVA test were used for statistical calculations.
14
III. RESULTS
Human gastric cancer cells were more tolerant of acidity in the culture media than
normal cells.
To determine whether cancer cells tolerate acidic conditions better than noncancer cells,
we cultured several gastric cancer cell lines (AGS, KATO III, MKN-28, MKN-45, SNU-1,
and SNU-601) and noncancer cell lines (RGM-1, IEC-6, and COS-1) in media maintained at
different pHs, including 7.4, 6.9, 6.4, 5.9, and 5.4. The cancer cells adapted well to lower pH,
whereas the normal cells were much less tolerant of acidity in the culture media (Fig. 1A). At
pH 5.4, most of cancer cell lines tested showed >80％ viability, but normal cell lines RGM-
1 significantly decreased viability by 21.6％.
To investigate whether this difference in cell survival at lower pH was due to ability to
dispose or disperse H+, we immunocytochemically stained two cancer cells (AGS and MKN-
45) and two noncancer cells (RGM-1 and IEC-6) with antibodies against the gastric proton
pump, α subunit of H+/K+-ATPase. We found that there was a much larger amount of proton
pump in cancer than noncancer cells (Fig. 1B). Immunofluorescence images showed a
predominant expression of the α subunit of H+/K+-ATPase in the membrane and cytoplasm
of cancer cells. These results indicated the enhanced ability to dispose of H+ due to
overexpression of H+/K+-ATPase, which might contribute to cancer cell survival in an acidic
microenvironment.
15
Fig. 1. Effects of pH of culture medium on cell viability and cellular expression of
H+/K +-ATPase.
Human gastric cancer cell lines (MKN-45, MKN-28, AGS, Kato III, SNU-601, and SNU-1)
and noncancer cell lines (RGM-1, Cos-1, and IEC-6) were cultured in media at different pH
for 24 hours, and cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide assay (A). Expression of H+/K+-ATPase, α-subunit was
detected with specific antibodies and visualized with Cy3-conjugated secondary antibodies.
Note that the α-subunit of H+/K+-ATPase was more highly expressed in the plasma
membrane and cytoplasm of cancer than noncancer cells (B).
A.
B.
16
Treatment of PPZ significantly inhibits cancer cell viability in a pH-dependent manner.
Because PPZ is a protonatable weak base, which can convert to active form in an acidic
environment with a low pH, we tested the effects of PPZ on cell growth in culture media
maintained at various pHs (Fig. 2). Nevertheless, cancer cells showed tolerance of acidity in
culture media as shown in Fig. 1A, and administration of the proton pump inhibitor PPZ led
to significant reduction of cancer cell survival (Fig. 2A). At pH 5.0, PPZ treatment reduced
cell viability by 6.9％ on average in MKN-45 cancer cells (Fig. 2A). On the contrary,
normal cell line RGM-1 did not showed a significant difference in cell viability between the
PPZ-treated and nontreated groups (data not shown). Despite resistance of cancer cells to the
acidic environment, cancer cells were much more susceptible to growth inhibition of PPZ at
a lower pH.
We evaluated whether this attenuation of cancer cell viability is induced by a decrease
of intracellular pH due to the blocking of disposal of intracellular H+ by specific protonation
of this drug. As shown in Fig. 2B, PPZ treatment significantly increased acidity of
intracellular pH in AGS, MKN-28, and MKN-45, but SNU-601 did not show a remarkable
change (Fig. 2B). Of note, AGS decreased intracellular pH from 7.6 to 7.2. These findings
showed that PPZ-induced cell death was caused by the specific protonation of this proton
pump inhibitor.
We also investigated whether other proton pump inhibitors such as omeprazole and
lansoprazole has a similar effect to pantoprazole on cancer cell viability. Human gastric
MKN-45 cells were cultured with each proton pump inhibitor in pH 6.0 of media for 24
hours, and cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-
17
diphenyltetrazolium bromide assay. The results showed that whereas the PPZ or omeprazole-
treated cells remarkably reduced cell viability in a dose-dependent manner, lansoprazole did
not show any changes in cancer cell viability (Fig. 3). This difference suggested that the
decreased cell viability comes from the specific effect of proton pump inhibition.
Omeprazole and PPZ are available for parenteral injection, but lansoprazole is only available
for oral administration, suggesting that lansoprazole lost its proton pump inhibiting ability in
aqueous form.
18
Fig. 2. Inhibition of cancer cell viability by PPZ treatment.
In different pH media (7.4, 6.9, 6.4, 5.9, 5.0), gastric MKN-45 cancer cells were incubated in
the presence or absence of 0.5 mmol/L PPZ, and cell viability was measured by 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (A). Although MKN-45
showed resistance to acidity of culture media, PPZ significantly decreased this cancer cell
viability in a pH-dependent manner. To evaluate effect of PPZ on intracellular pH (pHi) of
cancer cell, gastric cancer cells were plated in a 6-well culture dish, treated with 0.5 mmol/L
PPZ for 16 hours, and measured according to Materials and Methods as described previously
(B).
A. B.
19
Fig. 3. Effect of proton pump inhibitors on gastric cancer cell viability.
To compare effect of other proton pump inhibitors (omeprazole and lansoprazole) with PPZ
on cancer cell viability, MKN-45 cells were cultured in the presence of each proton pump
inhibitor indicated at Ph 6.0 for 24 hours and were measured cell viability by 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell viability (％) was
calculated by formula as follows ; Cell viability (％) = (sample absorbance － total
absorbance)/(spontaneous absorbance － total absorbance) × 100. Total absorbance was
obtained from absorbance of 0.1％ Triton X-100-treated cells as negative control, and
spontaneous absorbance indicated absorbance of PPZ-untreated control cells.
20
PPZ selectively induced apoptosis cell death in human gastric cancer cells.
To determine whether PPZ preferentially induces apoptosis in cancer cells, we
administered this agent to two cell lines, a normal gastric mucosal cell line, RGM-1, and a
gastric cancer cell line, MKN-45 (Fig. 4). Although normal gastric mucosal RGM-1 cells
significantly reduced cell viability in a low pH media as shown in Fig. 1A, an apoptotic
genomic DNA fragmentation was detected by neither pH change nor PPZ treatment (Fig. 4A).
However, human gastric cancer cell MKN-45 showed a significant apoptotic genomic DNA
fragmentation, caspase-3 and its substrate, PARP, were cleaved by PPZ treatment in a dose-
dependent manner in gastric cancer cells but not detected in normal gastric mucosal cell (Fig.
4B). PPZ also induced alteration of phosphatidylserine distribution and permeability of
plasma membrane only in cancer that which were detected by annexin V/propidium iodide
staining (Fig. 4C). These results indicated much higher vulnerability and selective sensitivity
to apoptosis in gastric cancer MKN-45 cells than normal mucosal RGM-1 cells.
21
Fig. 4. Selective induction of apoptosis with PPZ.
Cells were cultured in the presence or absence of PPZ for 24 hours, and induction of
apoptosis was detected by genomic DNA fragmentation (A), caspase-3 activation and PARP
cleavage (B), and localization of annexin V and propidium iodide (C). Annexin V-positive
cells were stained green, and red represented propidium iodide-positive cells.
A.
B.
C.
22
PPZ suppressed ERK phosphorylation in human gastric cancer cells.
To assay the involvement of MAPKs in PPZ-induced apoptosis, phosphorylation of
ERKs, JNKs/stress-activated kinases, and p38 after PPZ treatment was detected (Fig. 5A and
B). In MKN-45 cells, PPZ significantly and selectively inhibited the phosphorylation of ERK
and increased the phosphorylation of p38 but had no apparent effects on the phosphorylation
of JNK. After 8-hours treatment of 0.5 mmolL PPZ, ERK phosphorylation was completely
inhibited in MKN-45 cells despite an equal amount of total proteins controlled by α-tubulin
(Fig. 5A). In adition, inhibition of p38 by SB203580 blocked PPZ-induced apoptosis in
MKN-45 cells (data not shown). As shown is Fig. 5B, RGM-1 cells did not change the
degree of phosphorylation of ERK and p38, but phosphorylation of JNK was significantly
decreased in a time-dependent manner. Taken together, these findings suggest that inhibition
of ERK phosphorlyation may be responsible for the attenuation of cancer cell survival,
whereas activation of p38 contributes to proton pump inhibitor-induced apoptosis.
23
Fig. 5. Distinct MAPK signaling in cancer cells (A) and noncancer cells (B) by PPZ.
Cells were treated in the presence or absence of 0.5 mmol/L PPZ for various times, and
total proteins were extracted from the cells, electrophoresed on 12％ SDS-PAGE gels, and
transferred to polyvinylidene difluoride membranes. The membranes were probed with
specific antibodies for p-ERK, p-P38, p-JNK, and α-tubulin, respectively.
A. B.
24
Overexpression of HSP27 and HSP70 played a cytoprotective role in PPZ-induced
apoptosis of normal gastric mucosal cells.
Interestingly, we found that HSP70 and HSP27 were overexpression in RGM-1 after
PPZ treatment but not in MKN-45 cells (Fig. 6A and B). Notably, treatment of 0.6 mmol/L
PPZ in RGM-1 cells showed a 3.3- and 36.3-fold increased of HSP27 and HSP70 compared
with that of nontreated cells, respectively (Fig. 6C). However, HSP27 and HSP70, as well as
HSP60, were not grossly changed in PPZ-treated gastric cancer MKN-45 cells (Fig. 6A). To
compare the HSP induction, we also treated geranylgeranylacetone (GGA), which is well
known as a HSP inducer in gastric mucosal cells. GGA slightly increased HSP60 and HSP27
in MKN-45 cell, but deceased HSP60 and HSP27 in RGM-1. In comparisom with GGA, the
ablilty of PPZ to induce HSP70 is much higher at 5.6-fold than GGA. These data suggest
that overexpression of HSP27 and HSP70 might play a cytoprotective role in PPZ-induced
apoptosis of normal gastric mucosal cells.
25
Fig. 6. Effect of PPZ on expression of HSP70 and HSP27 in gastric cancer and
noncancer cells.
RGM-1 (A) and MKN-45 cells (B) were treated with PPZ or generylgenerylacetone (GGA)
for 24 hours, and expression of HSP70, HSP60, and HSP27 was assayed by immunoblotting
with specific antibodies. Note that PPZ induced expression of HSP70 and HSP27 in RGM-1
normal gastric mucosal cells but not in MKN-45 gastric cancer cells. Relative intensities of
HSP70 and HSP27 compared with nontreated control cells were represented in C.
A. B.
C.
26
In a xenograft model of nude mice, administration of PPZ significantly inhibited
tumorigenesis and induced large-scale apoptosis of tumor cells.
We also evaluated the antitumorigenic effects of PPZ in a gastric cancer xenograft
model (Fig. 7). The animals were randomly divided into two groups: an intratumoral
injection of PBS and an intratumoral injection of 0.4 ㎎/㎏ PPZ. Intratumor administration
of PPZ significantly suppressed tumor growth in athymic nude mice, with decreased in
tumor volume at day 22 of 44.69％ compared with mice injected with PBS (Fig. 7A and B).
The isolated tumor from mice with intratumor administration of PPZ was remarkably smaller
than that of mice intramorally injected with PBS (Fig. 7C). Histopathological examination
revealed that PPZ treatment provoked considerable apoptosis. Only a few tumor cells
survived, and most tumor cells were replaced by apoptosis cells (Fig. 7D). TUNEL staining
also showed considerably higher apoptotic cell death in the PPZ-treated group compared
with the control group (Fig. 7E).
27
Fig. 7. Antitumorigenic potency of PPZ in xenograft model.
MKN-45 human gastric cancer cells were inoculated s.c. into the backs of athymic nude
mice. PPZ (40mg/kg) was injected intrautmorally (IT) 14 days after xenograft. Mice were
sacrificed 22 days after tumor inoculation. Tumor volume was measured and represented (A
and B). The isolated tumors (C) were sectioned and processed for H&E staining (D, ×100
and ×400 magnification) and TUNEL assay (E, ×100 and ×200 magnification); bars, ±SD.
A
B
C
D
E
28
Distinct expression of CD34+ blood vessels between H.pylori positive and negative
gastric patients
Stomach tissue samples were obtained from gastritis patients during endoscopy
examination and were evaluated histologically by haematoxylin-eosin staining. Finally, we
choose 20 H.pylori positive gastritis and 18 H.pylori negative gastritis cases with a similar
degree of gastritis, scored according to the modified Sydney classification, as the degree of
gastric inflammations itself can affect angiogenesis. Immunohistochemical staining using
antibodies against the CD34 endothelial cell marker was performed to evaluate the difference
in angiogenesis according to H.pylori infection. The results showed that patients with
H.pylori positive gastritis (Fig. 8A (b, d)) showed significantly higher expression of CD34
positive blood vessels in the gastric mucosa layer than that of patients with H.pylori negative
gastritis (Fig. 8A (a, c)). The number of blood vessels was counted in three sites, for each
specimen, with equal dimensions, and mean levels are shown in Fig. 8B. While H.pylori
negative gastritis samples had a mean of 7.2 (SEM 0.8) blood vessels per x100 magnified
field, H.pylori positive gastritis samples had a significant increased number (mean 40.9
(SEM 4.4)) of blood vessels (p<0.01). Moreover, blood vessels observed in cases with
H.pylori positive gastritis (Fig. 8A (b, d), arrow) were thicker and larger than those of
H.pylori negative cases (Fig. 8A (a, c), arrow). Interestingly, blood vessels were found more
abundantly in the mesenchymal stromal layer below the mocosa layer but the number of
blood vessels in the stromal layer was not different between H.pylori positive gastritis and
H.pylori negative gastritis cases, suggesting that H.pylori infection may be associated with
induction of angiogenesis in H.pylori infected gastric mucosa.
29
We then evaluated if there were any differences in expression of HIF-1α, the potent
angiogenic transcriptional factor (Fig. 9). As a control, we used gastric biopsies obtained
from five cases diagnosed with functional dyspepsia with no significant abnormal
gastroscopic findings and no H.pylori infection. Compared with HIF-1α mRNA from five
normal stomachs, expression was not altered in five H.pylori negative gastritis but was
significantly increased in H.pylori positive gastritis (p<0.01), suggesting that HIF-1α is
responsible for H.pylori induced angiogenesis (Fig. 9B).
30
05
101520253035404550
HP(-) HP(+) Gastritis Gastritis
No
of B
lood
ves
sela b
A.
B.p<0.01
c d
Fig. 8. Immunohistochemical staining of the CD34 endothelial cell marker.
((((AAAA) Immunohistological analysis of CD34 positive blood vessels in gastric biopsy ) Immunohistological analysis of CD34 positive blood vessels in gastric biopsy ) Immunohistological analysis of CD34 positive blood vessels in gastric biopsy ) Immunohistological analysis of CD34 positive blood vessels in gastric biopsy
specimens of specimens of specimens of specimens of Helicobacter Helicobacter Helicobacter Helicobacter pyloripyloripyloripylori negative (a, negative (a, negative (a, negative (a, ×100; c, 100; c, 100; c, 100; c, ×200) and 200) and 200) and 200) and H.pyloriH.pyloriH.pyloriH.pylori positive (b, positive (b, positive (b, positive (b,
×100; d, 100; d, 100; d, 100; d, ×200) gastritis. Specimens obtained from 200) gastritis. Specimens obtained from 200) gastritis. Specimens obtained from 200) gastritis. Specimens obtained from H.pyloriH.pyloriH.pyloriH.pylori negative gastritis or negative gastritis or negative gastritis or negative gastritis or
H.pyloriH.pyloriH.pyloriH.pylori positive gastritis during endoscopic examination were used for positive gastritis during endoscopic examination were used for positive gastritis during endoscopic examination were used for positive gastritis during endoscopic examination were used for
immunohistological analysis with antiimmunohistological analysis with antiimmunohistological analysis with antiimmunohistological analysis with anti----CD34CD34CD34CD34 antibodies. CD34 antibodies. CD34 antibodies. CD34 antibodies. CD34++++ blood vessels were blood vessels were blood vessels were blood vessels were
stained by a dark red color (arrow), counterstained with haematoxylin showing a blue stained by a dark red color (arrow), counterstained with haematoxylin showing a blue stained by a dark red color (arrow), counterstained with haematoxylin showing a blue stained by a dark red color (arrow), counterstained with haematoxylin showing a blue
color. (color. (color. (color. (BBBB) Mean number of blood vessels stained with CD34. Number of blood vessels ) Mean number of blood vessels stained with CD34. Number of blood vessels ) Mean number of blood vessels stained with CD34. Number of blood vessels ) Mean number of blood vessels stained with CD34. Number of blood vessels
with equal dimensions were counted in triplicate fwith equal dimensions were counted in triplicate fwith equal dimensions were counted in triplicate fwith equal dimensions were counted in triplicate for each sample and are represented or each sample and are represented or each sample and are represented or each sample and are represented
as means (SD). There was a statistically significant difference between the two groups as means (SD). There was a statistically significant difference between the two groups as means (SD). There was a statistically significant difference between the two groups as means (SD). There was a statistically significant difference between the two groups
31
(p<0.01). (p<0.01). (p<0.01). (p<0.01). Hp, Helicobacter pyloriHp, Helicobacter pyloriHp, Helicobacter pyloriHp, Helicobacter pylori....
Normal HP+- gastritis HP--gastritis
HIF-1αααα
GAPDH
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
A.
0
500
1000
1500
2000
2500
3000
Normal HP¯
gastritis HP+
gastritis
Mea
n in
ten
sity
/ m
m2
B.p<0.01
p<0.01
Fig. 9. Expression of hypoxia inducible factor 1 (HIF-1α) mRNA in human gastric
mucosa.
((((AAAA) Reverse transcription) Reverse transcription) Reverse transcription) Reverse transcription----polymerase chain reaction of HIFpolymerase chain reaction of HIFpolymerase chain reaction of HIFpolymerase chain reaction of HIF----1111α was done with total was done with total was done with total was done with total
RNA isolated from RNA isolated from RNA isolated from RNA isolated from Helicobacter pyloriHelicobacter pyloriHelicobacter pyloriHelicobacter pylori ( ( ( (HpHpHpHp) negative gastritis (n=5), ) negative gastritis (n=5), ) negative gastritis (n=5), ) negative gastritis (n=5), H.pyloriH.pyloriH.pyloriH.pylori positive positive positive positive
gastritis (n=5), and normal stomachs gastritis (n=5), and normal stomachs gastritis (n=5), and normal stomachs gastritis (n=5), and normal stomachs (n=5). ((n=5). ((n=5). ((n=5). (BBBB) Relative expression of HIF) Relative expression of HIF) Relative expression of HIF) Relative expression of HIF----1111α mRNA mRNA mRNA mRNA
is represented as mean intensity/mmis represented as mean intensity/mmis represented as mean intensity/mmis represented as mean intensity/mm2222. HIF. HIF. HIF. HIF----1111α expression was significantly higher in expression was significantly higher in expression was significantly higher in expression was significantly higher in
gastric mucosa of gastric mucosa of gastric mucosa of gastric mucosa of H. pyloriH. pyloriH. pyloriH. pylori positive gastritis than in positive gastritis than in positive gastritis than in positive gastritis than in H.pyloriH.pyloriH.pyloriH.pylori negative gastritis in spite negative gastritis in spite negative gastritis in spite negative gastritis in spite
of similar expression of GAPDH (p<of similar expression of GAPDH (p<of similar expression of GAPDH (p<of similar expression of GAPDH (p<0.01). 0.01). 0.01). 0.01).
32
Suppression of H.pylori induced in vitro angiogenesis by gastric PPZ
To prove a direct effect of H.pylori infection on angiogenesis, we performed an in vitro
angiogenesis assay. Conditioned media obtained from H.pylori infected AGS cells were
added to HUVEC culture flasks and morphological changes in the endothelial cells were
observed. After nine days, HUVEC became long in shape and formed a tubular structure (Fig.
10B) compared with conditioned media of non-H.pylori infected AGS (Fig. 10A). CD31
immunofluorescence staining showed a dense intensity of CD31 molecules in HUVEC cells
incubated with the culture supernatants of H.pylori infected AGS (Fig. 10C) while control
media obtained from non-H.pylori infected AGS stimulated neither tubular formation of
HUVEC nor expression of the CD31 endothelial cell marker (Fig. 10A, and F).
Results of in vitro angiogenesis assay strongly suggested that H.pylori infection
stimulated infected gastric epithelial cells to secrete proangiogenic factors which induce
growth and differentiation of endothelial HUVEC. Interestingly, pretreatment with PPZ
(200µM or 400µM, for eight hours) on AGS cells prior to H.pylori inoculation significantly
inhibited tubular formation (Fig. 10D, and E) and CD31 expression of endothelial HUVEC
(Fig. 10I, and J). However, no significant changes were noted in HUVEC incubated with
400µM PPZ alone (Fig. 10C, and H), suggesting that PPZ itself did not influence tubular
formation of HUVEC. The data clearly indicate that PPZ suppressed H.pylori induced in
vitro angiogenesis, suggesting that antiangiogenic treatment with PPZ could be a promising
therapeutic approach for H.pylori associated carcinogenesis.
33
Control H. pylori (24 h)H. pylori (24 h)
+PPI (200 µµµµM, 8 h)H. pylori (24 h)
+PPI (400 µµµµM, 8 h)
Pha
se-c
ontr
ast
αα αα-C
D31
Ab
A B C D E
F G H I J
PPI (400 µµµµM, 8 h)
Fig. 10. In vitro angiogenesis assay.
AGS cells (1×107 cells/100mm2 culture dish) were incubated with 0, 200, or 400 µM proton
pump inhibitor (PPZ) for eight hours, washed with phosphate buffered saline three time, and
inoculated with Helicobacter pylori (5×107 CFU/ml) for 24 hours. Conditioned media were
prepared from 1:1 dilution of the cell culture supernatant and the human umbilical vein
endothelial cell (HUVEC) medium. Conditioned media were filtered through a 0.4 µM pore
filter to remove H.pylori and then added to the HUVEC culture which was change every
three days. After nine days, HUVEC were observed in a tubular formation under microscopy
(A-E) and expression of the endothelial cell marker, CD31, was confirmed by
immunocytofluorescence staining (F-J). (A, F) Control HUVEC cells; (B, G) HUVEC cells
34
incubated with conditioned media of H.pylori infected AGS; (C, H) HUVEC cells incubated
with conditioned media of 400 µM PPZ treated AGS; (D, I) HUVEC cells incubated with
conditioned media of 200 µM PPZ/H.pylori infected AGS; (E, J) HUVEC cells incubated
with conditioned media of 400 µM PPZ/H.pylori infected AGS.
35
Production of proangiogenic factors from H.pylori infected gastric epithelium and its
inhibition by PPZ
Following H.pylori infection, AGS cells significantly secreted VEGF and IL-8, well
characterized as proangiogenic factors, in a time dependent manner (Fig. 11A). Maximal
induction of IL-8 (mean 1019 (SEM 278) pg/ml) and VEGF (1597 (94) pg/ml) was observed
after 24 hours of inoculation (Fig. 11A). We also examined mRNA expression of these
angiogenic factors using RT-PCR analysis (Fig. 11B). Expression of VEGF mRNA, one of
the HIF-1α target genes, was induced after 16 hours of H.pylori infection, showing the
correlation with HIF-1α expression (Fig. 11B). IL-8 mRNA was also significantly induced
after H.pylori infection (Fig. 11B). All of these results suggest that synthesis of angiogenic
epithelial cells, which could induce proliferation and differentiation of endothelial cells.
Because PPZ showed a strong antiangiogenic action in the in vitro angiogenesis assay
(Fig. 10), we measured the effect of PPZ on expression of these angiogenic factors (Fig. 12).
Secretion of IL-8 in the supernatants of H.pylori infected AGS cells was found to be
remarkably suppressed after PPZ treatment in a dose dependent manner (Fig. 12A).
Following eight hours of infection with H.pylori, IL-8 production increased up to 870 pg/ml
but this increment in IL-8 production was significantly attenuated by PPZ pretreatment.
Pretreatment with PPZ showed a considerable regulatory effect on H.pylori mediated VEGF
synthesis (Fig. 12A). Suppression of thses angiogenic factors by PPZ was evidenced by
transcriptional inhibition of the genes (Fig. 12B). At 400 µM of PPZ, expression of VEGF
and HIF-1α seemed to decline relevant to that of control AGS cells.
36
A.
0
200
400
600
800
1000
1200
1400
1600
1800
2000IL
-8 (
pg
/ml)
Times after H. pylori
0 0.5 1 2 4 8 16 24 h
VE
GF
(p
g/m
l)
0
200
400
600
800
1000
1200
Times after H. pylori
HIF-1αααα
VEGF
IL-8
GAPDH
0 1 2 4 8 16 24 h
B.
0.5
1
1.5
0.5
1
1.5
0.5
1
1.5
2
2.5
3
Times after H. pylori (h)
0 1 2 4 8 16 24
HIF-1αααα
Times after H. pylori (h)
0 1 2 4 8 16 24
VEGF
Times after H. pylori (h)
0 1 2 4 8 16 24
IL-8
Rel
ativ
ein
tens
ity (
-fol
ds)
Rel
ativ
ein
tens
ity (
-fol
ds)
Rel
ativ
e in
tens
ity (
-fol
ds)
Times after H. pylori
0 0.25 0.5 1 2 4 8 24h
C.
Fig. 11. Release and expression of vascular endothelial growth factor (VEGF) and
interleukin 8 (IL-8) from Helicobacter pylori infected AGS cells.
(A) Production of VEGF (top) and IL-8 (bottom) was measured by ELISA with the culture
supernatant of AGS cells infected with H.pylori for the indicated times. (B) Induction of
hypoxia inducible factor 1 (HIF-1α), VEGF, and IL-8 mRNA by H.pylori infection was
tested by reverse transcription-polymerase chain reaction analysis in AGS cells incubated
with the bacterium for the indicated times. (C) Relative band intensity is presented as fold
ratio.
37
IL-8
(p
g/m
l)
00100200300400500600700800900
1000
GAPDH
HIF-1αααα
VEGF
H. pylori (16 h) - + + + +PPI (µM, 8h) - - 100 200 400
B.
A.
0
100
200300
400
500
600
700800
900
1000
VE
GF
(p
g/m
l)
- + + + + + H. pylori (16 h)- - 50 100 200 400 PPI (µM, 8 h)
- + + + + + H. pylori (16 h)- - 50 100 200 400 PPI (µM, 8 h)
Fig. 12. Effects of proton pump inhibitor (PPI) on expression of angiogenic growth
factors interleukin 8 (IL-8), hypoxia inducible factor 1 (HIF-1α), and vascular
endothelial growth factor (VEGF).
(A) To examine the inhibitory effect of PPI on (A) To examine the inhibitory effect of PPI on (A) To examine the inhibitory effect of PPI on (A) To examine the inhibitory effect of PPI on Helicobacter pyloriHelicobacter pyloriHelicobacter pyloriHelicobacter pylori induced angiogenic induced angiogenic induced angiogenic induced angiogenic
growth factor expression, AGS cells (1growth factor expression, AGS cells (1growth factor expression, AGS cells (1growth factor expression, AGS cells (1×101010107777 cells/100 mm cells/100 mm cells/100 mm cells/100 mm
2222 culture dish) were culture dish) were culture dish) were culture dish) were
incubated with 0, 50, 100, 200, or 400 incubated with 0, 50, 100, 200, or 400 incubated with 0, 50, 100, 200, or 400 incubated with 0, 50, 100, 200, or 400 µM PPI for eight hours, washed with phosphateM PPI for eight hours, washed with phosphateM PPI for eight hours, washed with phosphateM PPI for eight hours, washed with phosphate
buffered saline three times, and inoculated with buffered saline three times, and inoculated with buffered saline three times, and inoculated with buffered saline three times, and inoculated with H.pyloriH.pyloriH.pyloriH.pylori (5 (5 (5 (5×101010107 7 7 7 CFU/ml) for 16 hours. CFU/ml) for 16 hours. CFU/ml) for 16 hours. CFU/ml) for 16 hours.
Production of ILProduction of ILProduction of ILProduction of IL----8 (top) and VEGF (bottom) was measured in culture supernatant of 8 (top) and VEGF (bottom) was measured in culture supernatant of 8 (top) and VEGF (bottom) was measured in culture supernatant of 8 (top) and VEGF (bottom) was measured in culture supernatant of
the cells by ELISA. (B) Total RNA extractethe cells by ELISA. (B) Total RNA extractethe cells by ELISA. (B) Total RNA extractethe cells by ELISA. (B) Total RNA extracted from cells was used in the reverse d from cells was used in the reverse d from cells was used in the reverse d from cells was used in the reverse
transcriptiontranscriptiontranscriptiontranscription----polymerase chain reaction analysis of HIFpolymerase chain reaction analysis of HIFpolymerase chain reaction analysis of HIFpolymerase chain reaction analysis of HIF----1111α and VEGF. and VEGF. and VEGF. and VEGF.
38
Expression of H.pylori induced angiogenic factors is mediated by activation of ERK1/2
As VEGF and IL-8 expression was found to be regulated by MAPK and NKκB on
H.pylori induced angiogenesis using specific inhibitors (Fig. 13). PD098059 (50µM), one of
the ERK inhibitors, strongly inhibited H.pylori induced HIF-1α and VEGF expression, and
SB203580 (10µM), a p38 inhibitor, was also able to inhibit expression of these angiogenic
factors. PDTC (ammonium salt, 100µM) a NFκB inhibitor, potently suppressed HIF-1α and
VEGF expression induced by H.pylori. BAY11-7082 (5µM) reversibly increased expression
of the genes. These data suggest that H.pylori induced VEGF induction was mediated via the
MAPK pathway, and partially by the NFκB pathway.
39
Fig. 13. Involvement of mitogen activated protein kinase and nuclear factor κB
(NFκB) in Helicobacter pylori induced mRNA expression of hypoxia inducible factor 1
(HIF-1α) and vascular endothelial growth factor (VEGF).
Prior to inoculation with H.pylori, AGS cells were treated with each inhibitor (50 µM
PD08059, 10 µM SB203580, 100 µM PDTC, or 5 µM BAYII-7082 (BAY)) for eight hours,
and their effects on H.pylori induced HIF-1α and VEGF expression were evaluated by
reverse transcription-polymerase chain reaction. PD, PD098059, extracellular signal
regulated kinase (ERK)1/2 inhibitor; SB, SB203580, p38 inhibitor; PDTC, 1-
pyrrolidinecarbodithioic acid, ammonium salt, NFκB inhibitor; BAY, BAYII-7082, NFκB
inhibitor.
40
PPZ disturbs H.pylori induced signaling for angiogenesis via inactivation of MAPK
ERK1/2
Based on the previous findings that H.pylori infection stimulated the synthesis of
angiogenic factors via MAPK ERK activation (Fig. 13) and that the anticancer action of PPZ
is fundamentally attributable to inhibition of phosphorylation of MAPK ERK1/2,
we evaluated whether the antiangiogenic activity of PPZ is caused by block of ERK
activation. Western blot analysis with phosphor-ERK antibodies was performed to determine
the influence of PPZ on MAPK ERK1/2 activation related to H.pylori induced angiogenesis
(Fig. 14A, and B). The ERK inhibitor, PD098059, and the p38 inhibitor, SB203580,
decreased phosphorylation of ERK1/2. Interestingly, PPZ completely attenuated
phosphorylation of ERK1/2 inhibitor PD098059 (Fig. 14A). These inhibitory actions of PPZ
against ERK phosphorylation were dose dependent (Fig. 14B) and maximal inhibitory
activity was seen after four hours (data not shown). However, PPZ did not have any effect on
nuclear translocation of NFκB, suggesting that the antiangiogenic effect of PPZ seems to be
independent of suppression of NFκB (Fig. 14C). In summary, angiogenesis induced by
H.pylori was attenuated by PPZ treatment, which was caused by inactivation of the MAPK
pathway, one of principal signals for H.pylori induced angiogenesis.
41
-ERK1/2
total ERK
P
H. pylori
PPIPPIPPIPPISBSBSBSBPDPDPDPD--------InhibitorInhibitorInhibitorInhibitor
H. pylori - + + + +
- + + + +
NucleicNF-κκκκB p65
total NF-κκκκB p65
-ERK1/2
total ERK
P
A.
B.
C.
400400400400200200200200100100100100--------PPI (PPI (PPI (PPI (µµµµMMMM) ) ) )
H. pylori - + + + +400400400400200200200200100100100100--------PPI (PPI (PPI (PPI (µµµµMMMM) ) ) )
Fig. 14. Deactivation of mitogen activated protein kinase (MAPK) extracellular
signal regulated kinase (ERK)1/2 signaling with proton pump inhibitor (PPI).
(A) To compare the inhibitory effect of MAPK inhibitors and PPI on phosphorylation of
MAPK ERK1/2, AGS cells were treated with 50 µM of the ERK inhibitor (PD098059), 10
µM of the p38 inhibitor (SB203580), or 200 µM PPI for eight hours, and then infected with
Helicobacter pylori for 15 minutes. Proteins isolated from cells were subjected to
immunoblotting with phosphor-ERK antibodies. (B) Effect on phosphorylation of ERK at
different concentration of PPI. (C) Effect of PPI on H.pylori induced nuclear factor κB
(NFκB) translation was evaluated at different concentrations of PPI. AGS cells were treated
with the indicated concentrations of PPI for eight hours and inoculated with H.pylori for two
42
hours. Nucleic proteins were isolated from cells and subjected to western blotting using
specific NFκB in nuclear fractions compared with total NFκB. PD, PD098059 (ERK1/2
inhibitor), SB, SB203580 (p38 inhibitor).
43
IV. DISSCUTION
Because the ultimate aim of anticancer treatment is to kill only the cancer cells, the
therapeutic efficiency of anticancer agents could be increased by specificity and selectivity to
cancer cells. Current understanding of anticancer strategy has led to attempts to screen for
agents that selectively increase in cancer cells or to use apoptosis pathways specific for
tumor cells. Therefore, our current findings suggested that the gastric proton pump inhibitor
PPZ has very considerable advantages as anticancer treatment based on the following novel
findings. First, PPZ selectively induced apoptosis in cancer cells, and this PPZ-induced
apoptosis may be caused by suppressing ERK phosphorylation. This H+/K+-ATPase inhibitor
sitmultaneously stimulated phosphorylation of p38 as well as deactivation of ERK in a dose-
and time-dependent manner only in cancer cells. The different regulation of the individual
MAPK subfamily by the proton pump inhibitor seems to be better considered as anticancer
agents with high selectivity to cancer, because generally p38 is involved on apoptosis and
stress signaling pathway, whereas ERK is activated by stimuli of cell growth and
differentiation. Second, noncancer cells have mechanisms to counteract the PPZ-induced
apoptosis by the induction of antiapoptotic molecules HSP70 and HSP27. We observed that
PPZ-induced significant amounts of HSP70 much more than GGA, an agent generally
known to induce HSP70 as a key action. Thus, the H+/K+-ATPase inhibitor may act to
selectively induce apoptotic cell death in cancer cells without having any significantly
adverse effects on the surrounding noncancer cells, and the feature of this agent contributes
to safety in an aspect of clinical application. Third, PPZ would be selectively conversion of
44
the active form under the hypoxic and acidic condition, resulting in induction of significant
apoptosis. Just like in the canaliculi of parietal cell, pH of inner space of the tumor was
reported to be below 6.8 (Frenzel et al, 1994; Gillies et al, 1994). The finding shown in Fig.
2 showed that with lower pH of media, higher activities of apoptosis were observed. In all,
these results suggest that PPZ can be used clinically as an anticancer agent.
One of the well-known properties of cancer cells is aerobic glycolysis, which may cause
tumor acidification (Warburg et al, 1930; Holm et al, 1995; Vaupel et al, 1989). The cancer
cell will take in glucose and form lactic acid, which will largely dissociate into the lactate ion
and proton (H+). The lactate ion and H+ produced from the glycolysis are effluxed into the
extracellular fluid leading to low extracellular pH and high intracellular pH of cancer. There
are some mechanisms involved in the regulation of tumor pH; the main mechanism by which
proton (H+) is exported is by the sodium-hydrogen antiport, using the energy of the Na+
gradient (Karamazyn et al, 1999; Wakabayashi et al, 1997). Tumor cells may possess an
additional mechanism for H+ export via a vacuolar H+-ATPase (V-ATPase) of plasma
membrane, the bicarbonate transporter, and the proton-lactate synporter (Maritinez-Z et al,
1993; Finbow et al, 1997). In the present study, we used the H+/K+-ATPase inhibitor for
blocking the H+ export of tumor cells. The H+/K+-ATPase inhibitor successfully suppressed
tumor cell viability by inducing apoptotic cell death. Thus, these findings implicate that
blockage of another kind of proton pump predominantly expressed in tumor cells could be
used as a promising anticancer drug.
Several inhibitors have been found to interact with vacuolar-type H+-ATPase,
interfering with both ATP hydrolysis and proton translocation activities (Bowman et al,
45
1988; Zhang et al, 1992; Dross et al, 1993). Theses inhibitors can divided largely into two
classes: inhibitors acting at a soluble cytoplasmic domain (N-ethylmaleimide and 4-
nitrobenzo-2-oxa-1,3-diazole chloride) and inhibitors acting at transmembrane sites
(dicyclohexyl-carbodimide, Bafilomycina A1, and Concanamycin A). Several research
groups have already reported that these inhibitors of vacuolar-type H+-ATPase can induce
apoptotic cell death in several human cancer cell lines including pancreatic cancer,
hepatocellular carcinoma, and B-cell hybridoma cells (Nishihara et al, 1995; Ishisaki et al,
1999; Ohta et al, 1998; Aiko et al, 2002; Hasimoto et al, 2002). They also proved the
involvement of cytochrome C release and caspase activation in vascuolar-type H+-ATPase
inhibitor-induced apoptosis. The specific inhibitors of mammalian vacuolar-type H+-ATPase
belonging to the benzolactone enamide class, such as salicylihalamide, lobatamides, and
oximidines, were developed and appear promising as anticancer agents (Erickson et al, 1997;
Boyd et al, 2001). However, some reports are contradicting the proapoptotic effect of the
proton pump inhibitor, suggesting that the inhibitor of mitochondria F0F1-ATPase proton
pump, oligomycin, prevented apoptotic cell death (Schwerdt ea al, 2003; Matsuyama et al,
1998; Shchepina et al, 2002). .
However, there remain two limitations for anticancer application of a vascuolar-type
H+-ATPase inhibitor like concanamycin A or bafilomycin A. The first one is nonselectivity of
apoptosis of vascuolar-type H+-ATPase inhibitor, and the second problem is that the
vacuolar-type H+-ATPase gene is considered a “housekeeping gene” expressed
indiscriminately on every cell (Torigoe et al, 2002). Hence, there might be similar
cytotoxicity provoked by current cytotoxic anticancer drugs. The feasibility of clinical
46
application of these factors is difficult despite strong apoptotic-inducing capability. On the
other hand, PPZ showed selective induction of apoptosis in cancer cells. It rendered
noncancerous cells escaping from apoptotic activities through the induction of antiapoptotic
signaling molecules. Moreover, the fact the proton pump inhibitor is a prodrug requiring
protonation after administration brings more hope that protonation is more easily performed
within tumor tissues.
In conclusion, our findings provide novel mechanistic insight into the anticancer target
of gastric proton pump, H+/K+-ATPase, and expand the repertoire of clinical use of gastric
proton pump inhibitor as an anticancer drug by implicating the selective induction of
apoptosis in cancer cells.
After H.pylori infection, the signal transduction enzymes, MAPK ERK1/2 and NFκB,
are activation and these molecules are responsible for transcriptional activation of angiogenic
growth factors, including IL-8, HIF-1α, and VEGF. Increase in H.pylori induced angiogenic
factors stimulates the recruitment and activation of endothelial cells in the gastric mucosa,
resulting in significant neovascularisation of the gastric mucosal layer which can provide a
vulnerable and fertile environment for carcinogenesis. Chronic gastric inflammation
triggered by H.pylori infection may predispose to the development and progression of gastric
cancer. H.pylori induced angiogenesis might contribute to this H.pylori associated gastric
carcinogenesis along with other carcinogenic events. In this study, for the first time, we have
documented the mechanistic link between H.pylori infection and carcinogenesis, and the
inhibitory effects of PPIs on H.pylori induced angiogenesis. PPI treatment efficiently
inhibited IL-8, VEGF, and HIF-1α expression in H.pylori infected gastric epithelial cells,
47
which was due to inactivation of MAPK signaling induced by H.pylori infection. Thus these
findings have helped shed light on antiangiogenic treatment with PPIs, drugs popularly
prescribed for gastro-oesophageal acid related diseases and H.pylori eradiation. PPI therapy
could be a promising protective therapeutic approach for H.pylori associated carcinogenesis.
Neovascularisation, the development of new blood vessels from existing endothelial
precursors, is a general physiological mechanism critically involved in the normal repair
process and in the pathogenesis of inflammatory and ulcerative epithelial lesions as well as
malignant tumour growth, and even tumour metastasis (Jordan et al, 2004; Blagosklonny et
al, 2004). Among the proangiogenic factors known, VEGF response one of the most potent
stimuli of neoangiogenesis. Although previous studies found that enhanced VEGF gene
expression contributed to the healing of peptic lesions in the stomach (Baatar et al, 2002;
Jones et al, 2001), here we demonstrated that H.pylori infection stimulation of blood vessels
in the mucosa layer, which might be positive associated with propagation of gastric
inflammantion as well as gastric carcinogenesis after H.pylori infection. Several
investigations suggested that H.pylori infection stimulates host VEGF-A gene expression and
H.pylori induced angiogenesis may play a critical role in the development of gastric cancer
(Takahashi et al, 1996; Maeda et al, 1999; Kanai et al, 1998). Gastric adenocarcinomas
frequently showed high levels of VEGF expression (Takahashi et al, 1996; Maeda et al,
1999), and neutralization of circulating VEGF with specific VEGF antibodies potently
reduced the growth of gastric cancer (Kanai et al, 1998). Potent stimulators of angiogenesis
related to H.pylori infection, H.pylori VacA toxin (Caputo et al, 2003), reactive oxygen
species synthesized from neutrophils or macrophages (Park et al, 2003), CagA pathogenicity
48
islands (Cox et al, 2001; Innocenti et al, 2002), and lipopolysaccharide have been reported,
and several pathways linking the bacterium to host angiogenesis have been revealed. A wider
spectrum of genes induced by H.pylori in gastric epithelium was identified by high
throughput analysis of cDNA microarray analysis, and most of these genes are responsible
for angiogenesis and tumour invasion stimuli, such as ADAM series, IL-8, VEGF, integrins,
VCAM-1, ICAM-1, E-selectin, GRO-α, and IL-6 (Cox et al, 2001; Innocenti et al, 2002).
The PPI PPZ, a substituted 2-pyridyl methyl/sulfinyl benzimidazole derivative, is a
prodrug requiring protonation under acidic conditions for functions activation, accumulates
selectively in the acidic gastric luminal space, and ultimately inhibits acid secretion by
covalent binding with cysteins residues on the α-subunit of H+/K+-ATPase. These PPIs have
been universally used with antibodies for the eradication of H.pylori and several types of
acid related diseases, including gastro-oesophageal reflux diseases, peptic ulcer diseases, and
Zollinger-Ellison syndrome (Sachs et al, 1997; Fitton et al, 1996). Increased gastric pH by
PPIs stimulates resting H.pylori to activate metabolically and thus H.pylori are more
susceptible to antibiotics (Iwahi et al, 1991; Nakao et al, 1998; Hirai et al, 1995; Tsuchiya et
al, 1995; McGowan et al, 1994; Mauch et al, 1993). Apart from enhancing the susceptibility
of H.pylori to antibiotics, we found, for the first time, that PPIs can directly influence host
angiogenesis induced by H.pylori. Previously, we reported that PPIs had a strong inhibitory
effect on phosphorylation of MAPK ERK1/2 and its administration showed anticancer
activity in the xenograft nude mice model (Yeo et al, 2004). These inhibitory actions of the
drug against ERK phosphorylation also involved suppression of H.pylori induced host
angiogenesis. Blocking of H+ by PPIs caused an increase in extracellular pH (gastric lumen)
49
and a decrease in intracellular pH. The increased pH of the extracellular space may improve
the hypoxic microenvironment surrounding gastric epithelial cells and interrupt H.pylori
induced intracellular signaling via inhibition of MAPK activation. Compared with inhibitors
of ERK1/2 or p38, PPZ showed stronger inhibitory activities than these (fig. 14), in a dose
dependent manner. However, the influence of this drug on NFκB transcriptional activation
was minor (fig. 14).
In conclusion, we have shown that considerable angiogenic activities were stimulated in
the gastric mucosa after H.pylori infection, for which increasing proangiogenic growth
factors from gastric epithelial cells were responsible. PPIs could have significant inhibitory
activities against H.pylori associated angiogenesis. Therefore, the current data indicate that
as H.pylori infection causally promoted host angiogenesis, which has been attributed to
either augmented inflammation or enhanced carcinogenesis, PPIs could be potentially used
for inhibition of H.pylori provoked angiogenesis.
50
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-국문요약-
위산억제제의위산억제제의위산억제제의위산억제제의 새로운새로운새로운새로운 작용기전작용기전작용기전작용기전 : 헬리코박터헬리코박터헬리코박터헬리코박터 파이로리균에파이로리균에파이로리균에파이로리균에
의해의해의해의해 유도되는유도되는유도되는유도되는 신생혈관생성을신생혈관생성을신생혈관생성을신생혈관생성을 억제하고억제하고억제하고억제하고 위암세포에만위암세포에만위암세포에만위암세포에만
선택적으로선택적으로선택적으로선택적으로 세포자사멸을세포자사멸을세포자사멸을세포자사멸을 유도한다유도한다유도한다유도한다.
아주대학교 대학원의학과
김 동 규
( 지도교수 : 조 성 원 )
암세포는 급격한 세포분열을 위한 대사작용으로 인해 암세포내에 많은 양의
수소이온이 축적이 되고 세포밖 환경도 배출된 수소이온들로 인해 낮은 pH가 형
성되어 있다. 따라서, 대사작용 결과 생성된 수소이온을 세포밖으로 충분히 퍼낼
수 있는 능력이 요구된다. H+/K+-ATPase 는 세포내의 H+와 세포밖의 K+를 교환
하는 역할을 하여 위의 산성화에 기여하는 효소이다. 이러한 사실들에 기인하여,
우리는 Gastric H+/K+-ATPase inhibitor (위산억제제) 의 하나인, Pantoprazole (PPZ) 을
사용하여 암세포가 수소이온을 세포밖으로 퍼내는 능력을 차단하면, 암세포의 성
장을 억제하거나 암세포를 죽일 수 있을 것이란 가정을 세웠다. 그리고, 헬리코
박터 파이로리균의 감염으로 인한 mitogen activated protein kinases (MAPKs) 의 활
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성화가 숙주의 신생혈관생성을 유도하고, 더 나아가 위암화에 기여한다는 것은
많이 알려진 사실이나, 그 과정을 억제하는 방법에 관하여는 밝혀진 것이 많지
않다. 그러므로, 우리는 위산억제제가 MAPK ERK1/2 의 인산화를 강하게 억제하
는 능력이 있다는 것을 보고하였기에, 헬리코박터 파이로리균 감염에 의한
MAPK ERK1/2의 활성화를 위산억제제로 억제할 수 있고 그 결과 신생혈관생성
도 억제할 수 있는가를 조사해 보았다.
PPZ에 의해, 암세포가 세포자사멸에 이르는가를 알아보기 위하여 genomic
DNA fragmentation, terminal deoxynucleotidyl transferase (Tdt)-mediated nick end labeling
assay, 그리고 annexin V 염색법을 수행하였다. 그리고, Mitogen-activated protein
kinase 와 Heat shock protein 의 발현과 암세포의 세포자사멸과의 상관관계를 알아
보기 위하여 특정항체를 이용한 immunoblot 을 이용해 검증하였다. 그리고, PPZ가
생체내에서 항암효과가 있는지 검증을 위하여 nude mice 를 이용한 Xenograft
model 실험을 수행하였다. 그리고, 헬리코박터 파이로리균의 감염과 신생혈관생
성과의 관계를 알아보기 위해, 20명의 헬리코박터 파이로리 양성인 환자와 18명
의 헬리코박터 파이로리 음성인 환자의 조직절편에서 CD34+ 혈관의 상대적 발현
량을 확인해 보았다. 그리고, reverse transcription-polymerase chain reaction(RT-PCR)
을 이용해 hypoxia inducible factor 1 (HIF-1α) 와 vascular endothelial growth factor
(VEGF) 의 발현량을 비교해 보았고, ELISA 를 이용해 interleukin 8 과 VEGF의 분
비도 확인해 보았다. 그리고, in vitro angiogenesis assay 를 이용하여, 헬리코박터 파
이로리균의 감염이 human umbilical vein endothelial cells (HUVEC) 의 혈관모양생성
을 직접적으로 유도하는지도 확인해 보았다. 그리고, MAPK 와 nuclear factor κB
(NFκB) 의 활성화도 immunoblotting 으로 확인해 보았다.
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그 결과로, 암세포에 PPZ를 처리해 주면 세포자사멸에 이르는 것을 확인하
였고 이때, extracellular signal-regulated kinase(ERK)의 활성화가 저해되는 것을 확인
하였다. 이와 대조적으로, 정상세포에서는 HSP70 과 HSP27 같은 항세포자사멸인
자들의 과발현을 통하여 PPZ에 의해 유도되는 세포자사멸에 저항성을 보이는 것
을 확인하였다. 그리고, nude mice를 이용한 xenograft model에서도 PPZ의 접종이
암화의 진행을 억제하고, 암세포의 세포자사멸을 유도한다는 것을 확인하였다.
그리고, 헬리코박터 파이로리균 양성인 위염 환자 (40.9±4.4)에서 헬리코박터 파
이로리균 음성인 위염 환자 ( 7.2±0.8) 보다 CD34+ 혈관의 발현량이 현저히 증가
되어 있는 것을 관찰하였고, HIF-1α 의 발현상관관계도 같은 결과치를 보여주었
다. 위상피세포에 헬리코박터 파이로리균을 감염시켜 얻은 배양액을 가지고
HUVEC 세포의 배양액에 혼합하여 배양했을 때, HUVEC 세포가 혈관모양을 생
성하는 것을 확인할 수 있었다. 그러나, 여기에 위산억제제를 처리해주면 현저하
게 HUVEC 세포가 혈관모양을 생성하는 것을 억제해 주었다. 그리고, 헬리코박
터 파이로리균의 감염이 MAPK 의 활성화와 전사인자인 NFκB의 활성화를 통해
혈관생성인자인 HIF-1α 와 VEGF 의 발현을 증가시킨다는 것을 확인할 수 있었
다. 이러한 과정에, 위산억제제가 MAPK ERK1/2 의 인산화를 효과적으로 억제해
헬리코박터 파이로리균의 감염에 의한 신생혈관생성 과정을 억제하는 효과를 보
이는 것을 확인할 수 있었다.
결론적으로, 위산억제제가 생체내와 세포수준에서 위암세포의 세포자사멸을
유도하고, 헬리코박터 파이로리균의 감염으로 인한 신생혈관생성을 억제하는 능
력이 있다면, 이 위산억제제가 더 나아가 항암치료에 이용될 수도 있고, 또한, 헬
리코박터 파이로리균과 관계된 위암화의 진행을 억제할 수 있는 치료제로의 역
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할도 기대해 볼 수 있겠다.
핵심어 : 위산억제제, 세포자사멸, 위암, H+/K+-ATPase, 항암효과, 헬리코박터 파이
로리균, 신생혈관생성, HIF-1α, VEGF, 암화과정
