Antiangiogenic and Proteolytic Activities of Prostate-specific Antigen

.............................................................................................................................. 9 INTRODUCTION ................................................................................................................... 10 REVIEW OF THE LITERATURE ......................................................................................... 11 1. Prostate cancer ................................................................................................................ 11 2. Proteases ......................................................................................................................... 12 2.1 Classification ............................................................................................................. 13 2.2 Specificity and catalytic mechanism ......................................................................... 13 2.3 Regulation of proteolytic activity .............................................................................. 15 2.4 Proteases in cancer ..................................................................................................... 16 3. Kallikrein-related peptidases .......................................................................................... 17 4. Prostate-specific antigen ................................................................................................. 20 4.1 Gene structure ............................................................................................................ 20 4.2 Protein structure ......................................................................................................... 21 4.3 Expression .................................................................................................................. 21 4.4 Complexed and nicked forms of PSA ....................................................................... 22 4.5 Proteolytic activity and specificity ............................................................................ 22 4.6 Biological function of PSA ........................................................................................ 23 4.7 Clinical use of PSA .................................................................................................... 27 5. Angiogenesis .................................................................................................................. 30 5.1 Tumor angiogenesis ................................................................................................... 30 5.2 Proteases in angiogenesis .......................................................................................... 32 5.3 Angiogenesis inhibitors ............................................................................................. 33 5.4 Angiogenesis models ................................................................................................. 35 AIMS OF THE STUDY .......................................................................................................... 37 MATERIALS AND METHODS ............................................................................................. 38 1. Proteins, peptides and other compounds ........................................................................ 38 1.1 Purification of PSA (I-V) ........................................................................................... 38 2. Assays for the enzymatic activity of PSA (I-V) ............................................................. 38 3. Peptide binding assay (V) ............................................................................................... 40 4. Proteolytic activity of PSA toward protein substrates .................................................... 40 4.1 Degradation of Matrigel (V) ...................................................................................... 40 4.2 Degradation of protein substrates (V) ........................................................................ 40 4.3 Assay for the degradation of IGFBP-3 (V) ................................................................ 41 5. SDS-PAGE, silver staining and Western blotting (V) ................................................... 41 6. Mass spectrometry .......................................................................................................... 41 6.1 Characterization of PSA isoforms (I) ........................................................................ 41 6.2 Identification of Matrigel-derived PSA substrates (V) .............................................. 41 7. Edman degradation (V) .................................................................................................. 42 8. Cell culture ..................................................................................................................... 42 8.1 LNCaP cells (I) .......................................................................................................... 42 8.2 Human umbilical vein endothelial cells (I-V) ........................................................... 42 8.3 Proliferation and viability assays (I) .......................................................................... 43 9. Immunoassays for endostatin and angiostatin ................................................................ 43 10. Gene expression analyses (IV) ....................................................................................... 43 10.1 RNA isolation, DNA microarray and data analysis ................................................ 43 10.2 Quantitative RT-PCR .............................................................................................. 44 11. Statistical analyses .......................................................................................................... 45 12. Ethics .............................................................................................................................. 45 RESULTS ................................................................................................................................ 46 1. Characterization of PSA isoforms (I) ............................................................................. 46 2. Proteolytic activity of PSA ............................................................................................. 47 2.1 Identification of nidogen-1 as a PSA substrate (V) ................................................... 48 2.2 Degradation of protein substrates by PSA (V) .......................................................... 48 3. Stimulators and inhibitors of PSA activity ..................................................................... 48 3.1 PSA-stimulating peptides (V) .................................................................................... 48 3.2 Small molecule inhibitors of PSA (III) ...................................................................... 51 4. Antiangiogenic activity of PSA (I-III, V) ....................................................................... 51 5. PSA-induced changes in HUVEC gene expression (IV) ............................................... 54 DISCUSSION .......................................................................................................................... 56 1. Characterization of internal cleavage sites and glycosylation of PSA isoforms (I) ....... 56 2. Proteolytic activity of PSA toward protein substrates (V) ............................................. 57 2.1 Stimulators and inhibitors of the enzymatic activity of PSA (III, V) ........................ 60 3. Antiangiogenic activity of PSA (I-V) ............................................................................. 60 3.1 PSA-induced changes in HUVEC gene expression (IV) ........................................... 63 3.2 Significance of the antiangiogenic activity of PSA ................................................... 64 SUMMARY AND CONCLUSIONS ...................................................................................... 66 ACKNOWLEDGEMENTS ..................................................................................................... 67 REFERENCES ........................................................................................................................ 69