title: Invasion of ras-transformed breast epithelial cells depends on the proteolytic activity of cysteine and aspartic proteinases.
abstract: It has been suggested that the lysosomal proteinases cathepsin B, L and D participate in tumour invasion and metastasis. Whereas for cathepsins B and L the role of active enzyme in invasion processes has been confirmed, cathepsin D was suggested to support tumour progression via its pro-peptide, rather than by its proteolytic activity. In this study we have compared the presence of active cathepsins B, L and D in ras-transformed human breast epithelial cells (MCF-10A neoT) with their ability to invade matrigel. In this cell line high expression of all three cathepsins was detected by immunofluorescence microscopy. The effect of proteolytic activity on cell invasion was studied by adding various natural and synthetic cysteine and aspartic proteinase inhibitors. The most effective compound was chicken cystatin, a general natural inhibitor of cysteine proteinases, (82.8+/-1.6% inhibition of cell invasion), followed by the synthetic inhibitor trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64). CLIK-148, a specific inhibitor of cathepsin L, showed a lower effect than chicken cystatin and E-64. Pepstatin A weakly inhibited invasion, whereas the same molar concentrations of squash aspartic proteinase (SQAPI)-like inhibitor, isolated from squash Cucurbita pepo, showed significant inhibition (65.7+/-1.8%). We conclude that both cysteine and aspartic proteinase activities are needed for invasion by MCF-10A neoT cells in vitro.
authors.affiliation: Jozef Stefan Institute, Department of Biochemistry and Molecular Biology, Jamova, Ljubljana, Slovenia.
authors.forename: A
authors.initials: A
authors.lastname: Premzl
authors.forename: V
authors.initials: V
authors.lastname: Puizdar
authors.forename: V
authors.initials: V
authors.lastname: Zavasnik-Bergant
authors.forename: N
authors.initials: N
authors.lastname: Kopitar-Jerala
authors.forename: T T
authors.initials: TT
authors.lastname: Lah
authors.forename: N
authors.initials: N
authors.lastname: Katunuma
authors.forename: B F
authors.initials: BF
authors.lastname: Sloane
authors.forename: V
authors.initials: V
authors.lastname: Turk
authors.forename: J
authors.initials: J
authors.lastname: Kos
mesh_terms: D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D001940:Breast / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D001943:Breast Neoplasms / Q000201:enzymology* / Q000209:etiology / Q000473:pathology; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology; D002645:Chickens; D003094:Collagen; D015891:Cystatins / Q000037:antagonists & inhibitors / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism / Q000494:pharmacology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004338:Drug Combinations; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D005260:Female; D006801:Humans; D007797:Laminin; D008856:Microscopy, Fluorescence; D009361:Neoplasm Invasiveness; D011509:Proteoglycans; D018631:ras Proteins / Q000494:pharmacology
chemical_list: D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D003094:Collagen; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D016282:Aspartic Acid Endopeptidases; D018631:ras Proteins