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
World distribution of H. pylori infection and its gastric consequences from common chronic gastritis
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+
Importance steps during H. pyroli interaction with its host in the gastric mucosa
Med Sci Monit 9:sr 53-66 (2003)
1. Inflammatory responseHost responses induced by H. pylori ：
JOURNAL OF CELLULAR PHYSIOLOGY 200:334–342 (2004)
2. Proliferation (G1/S transition)
Host responses induced by H. pylori ：
3. Induction or prevention gastric epithelial-cell apoptosis
NATURE REVIEWS CANCER 2:28-37(2002)
Host responses induced by H. pylori ：
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
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
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.
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
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
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
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
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.
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.
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
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
