Lu-Ping Chow Graduate Institute of Biochemistry and Molecular Biology National Taiwan University
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The proteomics approach to study the role of Helicobacter pyroli in the development of gastric cancer
Lu-Ping ChowGraduate Institute of Biochemistry and Molecular
BiologyNational Taiwan University
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World distribution of H. pylori infection and its gastric consequences from common chronic gastritis
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GC vs. H. pylori
The prevalence of H. pylori in GC patients is much higher than in age- and gender-matched controls.
The association between H. pylori positivity on serology and overall gastric cancer risk is higher than 60% .
Class I carcinogen of GC [WHO, 1994]
Scand J Gastroenterol 37:891–898.(2002)
GC
H. p+
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Importance steps during H. pyroli interaction with its host in the gastric mucosa
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Med Sci Monit 9:sr 53-66 (2003)
1. Inflammatory responseHost responses induced by H. pylori :
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JOURNAL OF CELLULAR PHYSIOLOGY 200:334–342 (2004)
2. Proliferation (G1/S transition)
Host responses induced by H. pylori :
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3. Induction or prevention gastric epithelial-cell apoptosis
NATURE REVIEWS CANCER 2:28-37(2002)
Host responses induced by H. pylori :
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Excision of spotsIn-gel digestionElution of fragments
AGS cells
AGS cells co-cultured
with H. pylori
Cell lysis and fractionationCyDye labeling & 2D gel seperation
Cy5-labeled non-infected cells
Cy2-labeled pooled standard
Cy3-labeled infected cells
Quantification of Decyder
Identification targets by peptide mass fingerprinting (PMF)
1000 1500 2000
Mass (m/z)
Databasesearch
Sample #1 Protein Name Index # Acession # ScoreProtein 1 Probable DNA-Directed RNA Polym 32443 P05472 0.854Protein 2 Mitochondrial Respiratory Chain 30371 P40341 0.731Protein 3 Tyrosine Protein Kinase SRC64B 34968 P00528 0.921
Strategy
3D view
Image View Histogram View
Table View
Sub-cellular and functional proteomic analysis of the cellular responses induced by Helicobacter pylori
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Functional analyses of effectiveness of H. pylori infection of AGS cells using a MOI of 100.
A. Induction of the scattering (" hummingbird" ) phenotype of AGS cells after infection with H. pylori for 4 h.
B. IL-8 release of AGS cells after infection with H. pylori for 24 h by ELISA.
C. Induction of COX-2 protein expression in AGS cells after infection with H. pylori for 24 h by immunoblot analysis.
Scale bar =10μm.
Fig.1
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Cell fractionaion of non-infected and H. pylori-infected AGS cells
HGFR : indicator of membrane fraction
Urease A : indicator of H. pylori contaminant
Sample loading : 50ug per lane
Fig.2UreaseA was present at high amounts in the bacteria plus cell pellet fraction of H. pylori-infected cells, present at low amounts in the membrane fraction, and absent in the host cytosol fraction.
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A
C
Dye swapping strategy was adopted to avoid dye labeling-bias, therefore, Cy3 and Cy5 dyes were interchangeable.
2D DIGE analysis of alterations in the cytosolic fraction of AGS cells induced by H. pylori infection
H. pylori-infected : green non-infected : red
Fig.3
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Spot no. Accession no. Protein ID Theoretical Sequence coverage (%)pI / Mr (Da)
Cellular organization / cytoskeleton
3 gi | 24657579 VCL protein (porcine vinculin protein) 5.8 / 116,737 35
5 gi | 2804273 Alpha actinin 4 5.3 / 102,269 58
8 gi | 46249758 Villin 2 5.9 / 69,242 24
16 gi | 18088719 Beta tubulin 4.7 / 49,672 40
17 gi | 57013276 Alpha tubulin 4.9 / 50,152 37
20 gi | 2506774 Cytokeratin 8 5.5 / 53,675 22
25 gi | 1070608 Cytokeratin 19 5.0 / 44,092 51
Protein synthesis and folding
7 gi | 15010550 Heat shock protein gp96 precursor 4.7 / 90,159 31
9 gi | 5729877 Heat shock 70kDa protein 8 isoform 1 5.4 / 70,899 32
10 gi | 12653415 Heat shock 70 KDa protein 9B, precursor 6.0 / 73,728 22
11 gi | 4885431 Heat shock 70kDa protein 1B 5.5 / 70,026 44
12 gi | 135538 T-complex protein 1, alpha subunit 5.8 / 60,344 17
13 gi | 51702252 Mitochondrial matrix protein P1 5.7 / 61,055 29
Metabolic enzymes
4 gi | 35830 Ubiquitin-activating enzyme E1 5.6 / 117,791 31
6 gi | 6005942 Valosin-containing protein 5.1 / 89,323 42
TABLE IProteins in the cytosolic fraction of AGS cells showing up- or down-regulation after 24 h of H. pylori infection identified by MS
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Spot no. Accession no. Protein ID Theoretical Sequence coverage (%)pI / Mr (Da)
15 gi | 45767857 Preteosome 26S ATPase subunit 1 6.0 / 49,127 28
21 gi | 4503571 Enolaseα 7.0 / 47,169 44
23 gi | 4506209 Proteasome 26S ATPase subunit 2 5.7 / 48,634 35
26 gi | 13489087 Protease inhibitor 2 (anti-elastase) 5.9 / 42,742 37
Angiogenesis / metastasis
1 gi | 126369 Laminin gamma-1 chain precursor 5.0 / 177,609 24
22 gi | 34234 Laminin-binding protein 4.8 / 31,794 34
Oxygen-regulated protein
2 gi | 5453832 Oxygen regulated protein (150kD) 5.2 / 111,336 49
14 gi | 14250470 ERO1-like 5.6 / 54,392 31
Transcription and translation
18 gi | 4503729 FKBP4 5.4 / 51,805 44
19 gi | 135191 Tryptophanyl-tRNA synthetase(TrpRS) 5.8 / 53,116 24
Cell communication and signal transduction
24 gi | 6598323 GDP dissociation inhibitor 2 6.1 / 50,664 33
28 gi | 1345590 14-3-3 β / α 4.8 / 28,082 26
Others
27 gi | 10441386 TPM4-ALK fusion oncoprotein type 2 4.8 / 27,530 39
TABLE I-continued
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metabolic enzymes
cytoskeleton proteins
protein synthesis and folding-related proteins
angiogenesis/metastasis-related proteins
oxygen-regulated proteins
transcription and translation-related proteins
others
cell communication and signal transduction proteins
25%
21.4%17.9% 7.1%
7.1%
7.1%7.1%3.6%
Bioinformatics ontology of the identified proteins
1. 2-fold up-regulation after H. pylori-infection
2. Potential cancer-associated proteins
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2-DE immunoblot analysis and three-dimensional fluorescence intensity profiles of non-infected AGS cells and AGS cells infected with H. pylori
Fig.4
The greatest changes were seen for laminin γ-1, VCP, HSP70, and 14-3-3 β, while moderate changes were seen for FKBP4, MMP-P1, TCP1α, and enolase α.
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Immunoblot analysis of expression profiles of lamininγ-1, VCP, HSP70, TCP 1, MMP-P1, FKBP4, Enolaseα, and 14-3-3βin paired cancerous (T) a
nd noncancerous (N) gastric tissues
Fig.5 Increased spots were seen in 9 of the 10 paired samples for laminin γ-1, 6 for VC
P, 7 for HSP70, 7 for MMP-P1, 10 for FKBP4, 6 for TCP1, 10 for enolase α, and 10 for 14-3-3 β.
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Immunohistochemical study of VCP, TCP 1, MMP-P1, Enolase, and 14-3-3βin gastric cancer tissue
Expression of VCP, MMP-P1, TCP1, enolaseα, and 14-3-3βwas more abundance in gastric canceorus cells than in paired normal cells whereas most cases had similar expression amount of lamininγ-1, HSP70 and FKBP4 proteins.
Fig.6
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1. An in vitro model was established using a MOI 100 and evaluating the effectiveness of H. pylori infection by functional analyses.
2. Twenty-seven differential expressed proteins in H. pylori- infected AGS cells were identified by proteomic approach.
3. The identified protein were classified as cytoskeleton proteins, protein synthesis and folding-related proteins, metabolic enzymes, angiogenesis/metastasis-related proteins, oxygen- regulated proteins, transcription and translation-related proteins, or cell communication / signal transduction-related proteins by bioinformatics ontology.
4. Valosin-containing protein, mitochondrial matrix protein P1, T-complex protein 1, enolaseα and 14-3-3βwere found to be overexpressed in cancerous tissues by immunoblot assay and immunohistochemical staining.
Summary