Impact of bile acids on development of Barrett's esophagus and its progression to esophageal adenocarcinoma [Elektronische Ressource] : the role of apoptosis, COX-2, {NF-_k63B [NF-Kappa-B], reactive oxygen species and CDX-2 / vorgelegt von Grzegorz Burnat

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Impact of bile acids on development of Barrett’s esophagus and its progression to esophageal adenocarcinoma: the role of apoptosis, COX-2, NFκB, reactive oxygen species and CDX-2. Den Naturwissenschaftlichen Fakultäten der Friedrich-Alexander-Universität Erlangen- Nürnberg zur Erlangung des Doktorgrades vorgelegt von Grzegorz Burnat aus Krakau (Polen) 1 Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultäten der Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 30.07.2008 Vorsitzender der der Promotionskommission: Prof.Dr. Eberhard Bänsch Erstberichterstatter: PD Dr. Robert Slany Zweitberichterstatter: Prof. Dr. med. Peter Konturek 2 Dedicated to my parents 3 Contents 1. Introduction .................................................................................................................8 1.1. Barrett’ esophagus definition and epidemiology ..............................................8 1.2. Barrett’s esophagus - current definition.......................................................9 1.3. Epidemiology ...........................................................................................10 1.4.
Publié le : mardi 1 janvier 2008
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Source : WWW.OPUS.UB.UNI-ERLANGEN.DE/OPUS/VOLLTEXTE/2008/1110/PDF/GRZEGORZBURNATIMPACTOFBILEACIDSONDEVELOPMENTOFBARRETTSESOPHAGUSANDITSPROGRESSIONTOESOPHAGEALADENOCARCINOMATHEROLEOFAPOPTOSISCOX2NFKBREACTIVEOXYGENSPECIESANDCDX2.PDF
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Impact of bile acids on development of Barrett’s esophagus and its
progression to esophageal adenocarcinoma: the role of apoptosis, COX-2,
NFκB, reactive oxygen species and CDX-2.








Den Naturwissenschaftlichen Fakultäten
der Friedrich-Alexander-Universität Erlangen-
Nürnberg zur
Erlangung des Doktorgrades






vorgelegt von

Grzegorz Burnat

aus

Krakau (Polen)










1





Als Dissertation genehmigt
von den Naturwissenschaftlichen Fakultäten
der Universität Erlangen-Nürnberg






















Tag der mündlichen Prüfung: 30.07.2008


Vorsitzender der
der Promotionskommission: Prof.Dr. Eberhard Bänsch
Erstberichterstatter: PD Dr. Robert Slany
Zweitberichterstatter: Prof. Dr. med. Peter Konturek




2





























Dedicated to
my parents









3 Contents


1. Introduction .................................................................................................................8
1.1. Barrett’ esophagus definition and epidemiology ..............................................8
1.2. Barrett’s esophagus - current definition.......................................................9
1.3. Epidemiology ...........................................................................................10
1.4. Risk factors for Barrett’s esophagus..........................................................10
1.4.1. GERD.....................................................................................................11
1.4.1.1. Symptoms of GERD .............................................................................11
1.4.1.2. Pathogenesis of GERD..........................................................................12
1.4.2 Obesity ...................................................................................................13
1.4.3. Smoking .................................................................................................13
1.5. Duodendogastroesophageal reflux-DGER.................................................14
1.5.1. Bile acids- synthesis and circulation........................................................15
1.5.2. Bile salts as potent factors in development of intestinal metaplasia of the
esophagus.............................................................................................................16
1.5.3. Barrett’s metaplasia an intermediate stage in development of the
esophagealcancer………………………………………………………………...18
1.6. Diagnosis of Barrett’s esophagus ..............................................................19
1.6.1. Endoscopy ..............................................................................................19
1.6.2. Histology................................................................................................21
1.6.3. Other methods useful in diagnosis of Barrett’s esophagus .......................23
1.7. Treatment options of Barrett's esophagus ..................................................24
1.7.1. Antacids..................................................................................................24
1.7.2. Histamine receptor antagonists (H RA) ..................................................25 2
1.7.3. Proton pomp inhibitors............................................................................25
1.7.4. Photodynamic therapy (PDT)..................................................................25
1.7.5. Endoscopic mucosal resection (EMR).....................................................26
1.7.6. Thermal ablation (APC)..........................................................................26
1.7.7. Esophagectomy.......................................................................................26
1.8. Molecular events during development of Barrett's esophagus and Barrett'
adenocarcinoma ...................................................................................................27
1.9. Inflammation as an important initiating factor in the Barrett’s esophagus
carcinogenesis ......................................................................................................28
1.9.1. Cytokines..............................................................................................29
1.9.2. Reactive oxygen species (ROS) ..............................................................30
4 Contents
1.9.3. Nuclear factor κ B (NFκB) .....................................................................31
1.9.4. Cyclooxygenase (COX) and prostaglandins ............................................33
1.9.5. Peroxisome proliferator-activated receptor (PPAR) ............................36
1.10. Angiogenesis ..............................................................................................37
1.11. Proliferation and cell cycle..........................................................................38
1.12. Apoptosis ...................................................................................................40
1.12.1. The Extrinsic Pathway ............................................................................42
1.12.2. The Intrinsic Pathway .............................................................................42
1.13. The molecular changes of DNA.................................................................43
1.14. Other factors...............................................................................................44
1.14.1. Ghrelin...................................................................................................44
1.14.2. Adiponectin ...........................................................................................45
1.14.3. Caudal type homebox transcription factor (CDX)...................................46
2. Aims ....................................................................................................................47
3. Materials and methods.......................................................................................48
3.1. Materials...................................................................................................48
3.1.1. Cell culture .............................................................................................48
3.1.2. Chemicals for RNA isolation: .................................................................49
3.1.3. Complementary DNA synthesis: .............................................................49
3.1.4. Polymerase chain reaction.......................................................................49
3.1.5. Chemicals for DNA electrophoresis ........................................................51
3.1.6. Chemicals and reagents for Western Blot................................................52
3.1.7. Immunohistochemical staining................................................................53
3.1.8. Laboratory equipments ...........................................................................54
3.1.9. Software .................................................................................................54
3.2. Methods....................................................................................................55
3.2.1. 24 hour pH and bile monitoring ..............................................................55
3.2.2. Human biopsies collection ......................................................................56
3.2.3. Isolation of total RNA from biopsies and cell culture. .............................56
3.2.4. Quantification of total RNA....................................................................57
3.2.5. Reverse transcription-polymerase chain reaction (RT-PCR), generation of
complementary DNA ...........................................................................................57
3.2.6. Polymerase chain reaction PCR ..............................................................58
3.2.7. Gel electrophoresis .................................................................................59
5 Contents
3.2.8. Real-Time PCR.......................................................................................59
3.2.9. Western blotting .....................................................................................60
3.2.9.1. Protein isolation. ...................................................................................60
3.2.9.2. Preparation nuclear extract of proteins ..................................................61
3.2.9.3. Bicinchoninic Acid (BCA) Protein Assay .............................................62
3.2.9.4. Gels preparation and proteins electrophoresis........................................62
3.2.9.5. Electrophoretic transfer.........................................................................64
3.2.9.6. Blotting.................................................................................................64
3.2.10. Immunohistochemical staining................................................................65
3.2.11. Fluorescence-activated cell sorting – FACS ............................................66
3.2.12. MTT-assay..............................................................................................67
3.2.13. ELISA (Enzyme-Linked ImmunoSorbent Assay)....................................68
3.2.13.1. NFκB p65 – transcription factor activation assay (Active Motif) ..........68
3.2.13.2. Quantitative determination of human VEFG in cell culture medium
(BIOSURCE) .......................................................................................................69
3.2.14. RNA Interference Experiments ...............................................................70
3.2.15. Statistics .................................................................................................70
4. Results.................................................................................................................71
4.1. In vivo results............................................................................................71
4.1.1. 24 h monitoring of bile and acid reflux....................................................71
4.1.2. Measurement of NFκB activity in esophageal biopsies specimens...........71
4.1.3. Expression of mRNA and proteins for COX-2, IL-8, catalase, PPAR γ and
p21…………………………………………………………………………………73
4.1.4. Expression of apoptotic related genes (bax and bcl-2) in biopsies
specimens from Barrett’s mucosa .........................................................................76
4.1.5. Estimation of CDX-2 expression.............................................................78
4.1.6. Analysis of mRNA expression for PTEN, IL-4, VEGF, HGF, gastrin,
CCK-2, MUTYH, OGG-1 and cyclin D1..............................................................81
4.1.7. Expression of ghrelin and its receptor in human esophageal biopsies from
Barrett’s esophagus patients .................................................................................84
4.2. In vitro Results .........................................................................................87
4.2.1. Effects of DCA and UDCA on cell viability (MTT assay).......................87
4.2.2. Influence of bile acids on apoptosis and cell cycle...................................88
6 Contents
4.2.3. Modulatory effect of bile acids on COX-2, MUTYH, OGG-1 and CDX-2
mRNA expression in OE19 cells ..........................................................................92
4.2.4. Effect of bile acids on transactivation of NFκB.......................................96
4.2.5. Effects of NFκB inactivation by siRNA ..................................................97
4.2.6. Effect of bile acids on VEGF expression...............................................101
4.2.7. Comparison of effect of DCA and UDCA on the esophageal cell line
incubated in neutral medium pH and after short incubation in acid medium........103
4.2.8. Effect of ciglitazone (PPARγ ligand) alone or in combination with gastrin
on cell proliferation and caspas-3 mRNA expression..........................................107
4.2.9. Effect of adiponectin on esophageal adenocarcinoma cell line
apoptosis…………………………………………………………………………109
4.2.10. Assessment of anti-inflammatory properties of ghrelin..........................113
5. Discussion .........................................................................................................117
6. Summary ..........................................................................................................134
7. Zusammenfassung............................................................................................136
8. Shortcuts...........................................................................................................138
9. References.........................................................................................................141
10. Acknowledgements...........................................................................................161













7 Introduction

1. Introduction

1.1. Barrett’ esophagus definition and epidemiology

Barrett’s esophagus (BE) is a condition in which squamous epithelium that occurs
normally in the esophagus has been replaced by an abnormally columnar epithelium
completed with goblet cells. This condition is termed specialized intestinal metaplasia
(SIM) [1, 2]. The morphological changes that replace the normal squamous epithelium by
columnar epithelium are called metaplasia. Metaplastic tissue differs from normal tissues
occurring in the body. The normal esophageal squamous epithelium consists of multilayer
cells that are similar to those of the skin in pinkish-white colour. They protect the
esophagus against mechanical damage and allow for easy passage of swallowed food. The
metaplastic columnar epithelium is red and looks like a stomach tissue but have a many
abnormal features. For example, the Barrett’s metaplastic lining include few groups of
cells. First one is similar to cells from small intestinal. Others cells resemble these from a
stomach region lining.
The Barrett’s alterations appear in the distal part of the esophagus which is exposed
to the acid and bile. The metaplastic changes differ in their length. They could be up to 3
cm (short segment Barrett’s esophagus SSBE) or longer (long-segment Barrett’s esophagus
LSBE).
















Fig. 1. Normal esophageal epithelium (A) versus different length variants of
Barrett’s esophagus (B-D).
8 Introduction
A. Normal squamous esophageal mucosa B. Metaplastic Barrett‘s esophagus
Luminal surface
Goblet
cell
Columnar
epithelium Stratified
squamous
epithelim Basemant
membrane
Mucosal
layer
Lamina
propia
Lamina
propia Inflammatory
cell Mucous
secreting
gland
Muscularis
mucosae

Fig. 2. Key morphological differences between normal squamous and Barrett’s
epithelial phenotypes.

1.2. Barrett’s esophagus - current definition

The definition of Barrett’s esophagus has evolved during last century. In the 1950
the Barrett’s esophagus was described by the British surgeon Norman Barrett (1903-1979).
He proposed that the red-color part of the esophagus, which appears in some patients, was
actually the part of stomach. He supposed that those patients were born with the shorter
length of squamous esophageal lining [3].
In current time according to the guidelines of American Collage of
Gastroenterology, Barrett’s esophagus is defined as an alteration in the esophageal
epithelium of any length that can be recognized during upper endoscopy and is confirmed
by pathologist to have intestinal metaplasia by biopsy [4]. According to this definition
metaplasia must be present in the esophageal side. In some cases patients have intestinal
metaplasia localized in the cardia on a border between the esophagus and the stomach.
This kind of changes is called intestinal metaplasia of the gastric cardia and should not be
classified as Barrett’ esophagus.
9 Introduction
Both types of metaplasia look very similar to the normal stomach tissue in upper
endoscopy but they have different origin and prognosis. To distinguish which kind of
metaplasia is present or if it is an abnormal epithelium, quadrant biopsies must by taken for
histological analysis under microscope.

1.3. Epidemiology

Barrett's esophagus is mostly a disease of Caucasian males. The mean age of
development of BE is estimated to be 40 years but mean age of diagnosis is 55-63 years [5,
6]. Barrett's esophagus is found in approximately 6%-12% of patients in the United States
undergoing endoscopy for symptoms of GERD and in 1-3% or less of unselected patients
undergoing endoscopy [7-9]. In Europe, the prevalence of Barrett's esophagus is
approximately 1-4% in adults undergoing upper endoscopy (Germany 4,6%; Spain 0,46%)
[10]. Barrett's esophagus is uncommon in blacks and Asians and this phenomenon may be
due to their style of life and diet.
The frequency of adenocarcinoma of the esophagus increased 30 to 50 fold in the
western world during the last 4 decades [11]. Esophageal cancer is the sixth leading cancer
type leading to death in the world and represents about 1% of the cancers diagnosed in the
United States [12]. In 2006, approximately 16000 new cases (more than the previous
years) of esophageal cancers in US were noted and 60% of them were classified as
adenocarcinoma with poor survival (11% of survival in 5-year for this cancer) [13].
Similarly to Barrett's esophagus, esophageal adenocarcinoma is uncommon in Africa and
the Asia (with exception of Japan, Hong Kong, and China) what emphasize association
between prevalence of BE, BA and ethnical factors [14, 15].

1.4. Risk factors for Barrett’s esophagus

For many years the reason of metaplastic changes in the esophagus was unknown.
Initially, this fact was explained by a natural inappropriate development of distal part of
the esophagus, during formation of the gastrointestinal system. In last two decades a
significant progress in techniques used in medicine and biology shed a new light on the
pathogenesis of Barrett’s esophagus.

10

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