1. The proteomics approach to study the role of Helicobacter pyroli in the development of gastric cancer Lu-Ping Chow Graduate Institute of Biochemistry and Molecular Biology National Taiwan University

2. World distribution of H. pylori infection and its gastric consequences from common chronic gastritis

3. 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 +

4. Importance steps during H. pyroli interaction with its host in the gastric mucosa

5. Med Sci Monit 9:sr 53-66 (2003) 1. Inflammatory response Host responses induced by H. pylori ：

6. JOURNAL OF CELLULAR PHYSIOLOGY 200:334–342 (2004) 2. Proliferation (G1/S transition) Host responses induced by H. pylori ：

7. 3. Induction or prevention gastric epithelial-cell apoptosis NATURE REVIEWS CANCER 2:28-37(2002) Host responses induced by H. pylori ：

8. Excision of spots In-gel digestion Elution of fragments AGS cells AGS cells co-cultured with H. pylori Cell lysis and fractionation CyDye 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) Database search Sample #1Protein NameIndex #Acession #Score Protein 1Probable DNA-Directed RNA Polym32443P054720.854 Protein 2Mitochondrial Respiratory Chain30371P403410.731 Protein 3Tyrosine Protein Kinase SRC64B34968P005280.921 Strategy 3D view Image ViewHistogram View Table View Sub-cellular and functional proteomic analysis of the cellular responses induced by Helicobacter pylori

9. 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

10. 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.2 UreaseA 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.

11. 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

12. Spot no.Accession no. Protein IDTheoretical Sequence coverage (%) pI / Mr (Da) Cellular organization / cytoskeleton 3gi | 24657579VCL protein (porcine vinculin protein)5.8 / 116,73735 5gi | 2804273Alpha actinin 45.3 / 102,26958 8gi | 46249758Villin 25.9 / 69,24224 16gi | 18088719Beta tubulin4.7 / 49,67240 17gi | 57013276Alpha tubulin4.9 / 50,15237 20gi | 2506774Cytokeratin 85.5 / 53,67522 25gi | 1070608Cytokeratin 195.0 / 44,09251 Protein synthesis and folding 7gi | 15010550Heat shock protein gp96 precursor4.7 / 90,15931 9gi | 5729877Heat shock 70kDa protein 8 isoform 15.4 / 70,89932 10gi | 12653415Heat shock 70 KDa protein 9B, precursor6.0 / 73,72822 11gi | 4885431Heat shock 70kDa protein 1B5.5 / 70,02644 12gi | 135538T-complex protein 1, alpha subunit5.8 / 60,34417 13gi | 51702252Mitochondrial matrix protein P15.7 / 61,05529 Metabolic enzymes 4gi | 35830Ubiquitin-activating enzyme E15.6 / 117,79131 6gi | 6005942Valosin-containing protein5.1 / 89,32342 TABLE I Proteins in the cytosolic fraction of AGS cells showing up- or down-regulation after 24 h of H. pylori infection identified by MS

13. Spot no.Accession no. Protein IDTheoreticalSequence coverage (%) pI / Mr (Da) 15gi | 45767857Preteosome 26S ATPase subunit 16.0 / 49,12728 21gi | 4503571Enolaseα7.0 / 47,16944 23gi | 4506209Proteasome 26S ATPase subunit 25.7 / 48,63435 26gi | 13489087Protease inhibitor 2 (anti-elastase)5.9 / 42,74237 Angiogenesis / metastasis 1gi | 126369Laminin gamma-1 chain precursor5.0 / 177,60924 22gi | 34234Laminin-binding protein4.8 / 31,79434 Oxygen-regulated protein 2gi | 5453832Oxygen regulated protein (150kD)5.2 / 111,33649 14gi | 14250470ERO1-like5.6 / 54,39231 Transcription and translation 18gi | 4503729FKBP45.4 / 51,80544 19gi | 135191Tryptophanyl-tRNA synthetase(TrpRS)5.8 / 53,11624 Cell communication and signal transduction 24gi | 6598323GDP dissociation inhibitor 26.1 / 50,66433 28gi | 134559014-3-3 β / α4.8 / 28,08226 Others 27gi | 10441386TPM4-ALK fusion oncoprotein type 24.8 / 27,53039 TABLE I-continued

14. 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% 3.6% Bioinformatics ontology of the identified proteins 1.2-fold up-regulation after H. pylori-infection 2.Potential cancer-associated proteins

15. 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.

16. Immunoblot analysis of expression profiles of lamininγ-1, VCP, HSP70, TCP 1, MMP-P1, FKBP4, Enolaseα, and 14-3-3βin paired cancerous (T) and noncancerous (N) gastric tissues Fig.5 Increased spots were seen in 9 of the 10 paired samples for laminin γ-1, 6 for VCP, 7 for HSP70, 7 for MMP-P1, 10 for FKBP4, 6 for TCP1, 10 for enolase α, and 10 for 14-3-3 β.

17. 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

18. 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