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