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Systems-Biology Study of RGD Mimetic Peptide in Breast Cancer

Systems-Biology Study of RGD Mimetic Peptide in Breast Cancer. 阮雪芬 Nov 30, 2003. So Many Choices, So Little Money. SCIENCE, 2001, 294, 82-85. Outline. Introduction cDNA microarray Proteomics Bioinformatics. Introduction. Systems Biology. Hiroaki Kitano Science, 2002 Mar.

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Systems-Biology Study of RGD Mimetic Peptide in Breast Cancer

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  1. Systems-Biology Study of RGD Mimetic Peptide in Breast Cancer 阮雪芬 Nov 30, 2003

  2. So Many Choices, So Little Money SCIENCE, 2001, 294, 82-85

  3. Outline • Introduction • cDNA microarray • Proteomics • Bioinformatics

  4. Introduction

  5. Systems Biology • Hiroaki Kitano • Science, 2002 Mar. • Nature, 2002 Nov.

  6. 牛頓雜誌2003年11月號

  7. Traditional Biology & Systems Biology • Traditional biology : • Single genes or proteins • Systems biology: • Simultaneously study the complex interaction of many levels of biological information to understand how they work together Genomic DNA mRNA Proteins Functional proteins Informational pathways Informational networks

  8. Systems Biology and Omics Data Genenomic Transcriptomic Systems Biology Metabonomic Proteomic Drug discovery Development process Understanding drug toxicology

  9. The Structure of an Integrin • Hynes in 1987 to emphasize the role of these RGD receptors in integrating the extracellular matrix outside the cell with the actin-containing cytoskeleton inside the cell.

  10. The Interactions of Integrins with Other Proteins

  11. Schematic Model of the Protein-protein Interactions Signal are presumably transmitted into the nucleus, where they stimulate the transcription of gene involved in cell growth and proliferation

  12. Integrin-activated Survival Signals Ras Raf MEK MAPK Shc Grb2/Sos Cell survival Integrins PI 3-kinase FAK Trends in Cell Biology, 1997, 7, 146-150

  13. How RGD Trigger Apoptosis? • By integrin-mediated signal? • Directly interact with the proteins in cytosol?

  14. How RGD Trigger Apoptosis? a. Cell survive b. RGD trigger apoptosis via integrin c. Cell apoptosis by activating procaspase-3 Nature, 1999, 397, 534-539

  15. RGD and Cell Death RGD(Arg-Gly-Asp) is the smallest motif that bind with the integrin receptor on the cell surface and play important role in cell cycle. Control Aggregation Cell Death

  16. Our Study

  17. Our Study Human breast cancer cell MCF-7 Genomic Study RGD mimetic peptides Proteomics Bioinformatics Cell Apoptosis

  18. Gly Asp Arg Trp Asp Tpa Pro Arg Gly Cys Cyclic-RGD The Structures of RGD Mimetic Peptides RGD

  19. RGD cRGD control control 1mM 0.5mM 5mM 1mM

  20. Crystal Structure RGD and Integrin • Crystal Structure of the extracellular segment of integrin alpha Vbeta3 in complex with an RGD ligand Science 296 pp. 151 (2002)

  21. The 3D Structure of Cyclic-RGD Cis form Trans form

  22. Apoptosis Mechanism in MCF7 C-RGD Cis or Trans? C-RGD Cis or Trans?

  23. cDNA Microarray

  24. Introduction to Microarray • A powerful approach to accurately measure changes in global mRNA expression levels. • Used to • Discover novel genes • Determine gene functions • Evaluate drugs • Dissect pathways • Classify clinical samples.

  25. Introduction to Microarray Control or treatment mRNA Reverse transcriptase to generate Cy3/Cy5 cDNA probes Hybridization to the gene chip Data analysis Eur J Nucl Med 2002, 29, 115-32

  26. cDNA Microarray C-RGD, 24hr C-RGD, 6hr C-RGD, 48hr C-RGD, 72hr

  27. Apoptosis • Total 34 genes, but after filtering there are only 19 genes • Total 11genes have expression fold >2 (up or down changes)

  28. Apoptosis Regulator

  29. Apoptosis Regulator

  30. Using Linear Model to Construct Gene Network • Linear Model D’haeseleer, 2000

  31. Weights Matrix of Apoptosis Regulator

  32. Gene Network of Apoptosis Regulator

  33. Signal Transducer 1 2

  34. Weights Matrix of Signal Transducer

  35. Gene Network of Signal Transducer

  36. Proteomics

  37. Two-dimensional Gel Approach Nature 2000, 405, 837-846

  38. 97000 1 66000 45000 30000 20100 3.2 4.0 5.0 5.5 6.0 7.0 8.0 9.0 10.0 14400 3.2 4.0 5.0 5.5 6.0 7.0 8.0 9.0 10.0 Control vs c-RGD (6hr) Control cRGD

  39. 97000 17 66000 2 3 18 45000 4 30000 7 5 6 8 9 20100 10 12 11 15 13 14400 16 14 3.2 4.0 5.0 5.5 6.0 7.0 8.0 9.0 10.0 3.2 5.5 8.0 9.0 4.0 5.0 6.0 7.0 10.0 Control vs c-RGD (24hr) c-RGD Control

  40. 97000 66000 20 45000 19 21 22 30000 20100 14400 3.2 4.0 4.0 5.0 5.0 5.5 5.5 6.0 6.0 7.0 7.0 8.0 9.0 10.0 10.0 8.0 9.0 Control vs c-RGD (48hr) Control c-RGD

  41. 97000 66000 23 45000 24 25 26 30000 20100 14400 3.2 4.0 4.0 5.0 5.0 6.0 6.0 7.0 8.0 9.0 10.0 3.2 5.5 5.5 7.0 8.0 9.0 10.0 Control vs c-RGD (72hr) Control c-RGD

  42. 1. Semenogelin I protein precursor (SGI) 2. Cell division protein kinase 6 3. Zinc finger protein 74 isoform 4 4. Keratin 5. Similar to presomitic mesoderm specific gene 6. Unnamed protein product 7. RNA-binding protein regulatory subunit 8. Similar to Claudin-6 (Skullin) 9. Unnamed protein product 10. Similar to Per-hexamer repeat protein 5 11. Similar to L1 repetitive element ORF 12. Hypothetical protein 13. Zinc finger protein 189 ISOFORM 2 14. Hypothetical protein 15. Similar to stretch response protein 553 16. Zinc finger protein 83 17. Immunoglobulin heavy chain variable region 18. Hypothetical protein 19. Cytokeratin 8 20. Cytokeratin 8 21. Zinc-alpha-2-glycoprotein precursor 22. Keratin 18 23. Platelet-activating factor acetylhydrolase precursor 24. Topoisomerase II alpha 25. 13kD differentiation-associated protein 26. Purified protein derivative-specific T-cell receptor beta chain Proteomics Results Red color: up-regulated Black color: down-regulated

  43. Bioinformatics

  44. RGD Peptides Can Be Used in Many Diseases • Thrombosis • Osteoporosis • Cancer • Any one else ??

  45. Blood Clots Form • Blood clots form when platelets adhere to one another through fibrinogen bridges that bind to the platelet integrin

  46. Clustering Analysis of Proteins http://uranus.csie.ntu.edu.tw:9000/index.jsp

  47. RGD-containing Proteins in Swiss-Prot Database • In Swiss-Prot database, there are738 human RGD-containing proteins which containing 5 caspase proteins . • Caspase 1, caspase 2, caspase 3 and caspase7, caspase 8.

  48. RGD-containing Proteins in Swiss-Prot Database • Heat shock protein Dna • Chaperone DnaJ Alzheimer's disease • SM22 Leiomyoma

  49. Conclusion and Discussion

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