Intracellular and extracellular cathepsin B facilitate invasion of MCF-10A neoT cells through reconstituted extracellular matrix in vitro.
Lysosomal cysteine proteinase cathepsin B is implicated in remodeling the extracellular matrix, a crucial step in the process of tumor cell invasion. In this study the contributions of intracellular and extracellular cathepsin B activities in the invasion of ras-transformed human breast epithelial cells, MCF-10A neoT, were assessed using specific cathepsin B neutralizing monoclonal antibody (Mab) 2A2, together with other general and specific cysteine proteinase inhibitors. We showed that the degradation of extracellular matrix by living MCF-10A neoT cells was predominantly intracellular, as imaged by confocal assays using quenched fluorescent substrate DQ-collagen IV. CA-074, a membrane-impermeable cathepsin B-selective inhibitor and its membrane-permeable analogue CA-074Me showed similar inhibition of invasion at 10 microM, i.e., 24.9 and 27.0%, respectively. Neutralizing monoclonal antibody exhibited a significantly higher inhibitory effect, decreasing invasion at 0.5 microM by 42.7%. Tumor cells may internalize monoclonal antibody; therefore, 2A2 Mab could impair both the intracellular and the extracellular fractions of cathepsin B activity. However, both 2A2 Mab and cathepsin B-selective inhibitors were less potent than the general cysteine proteinase inhibitors chicken cystatin and E-64, indicating that other cysteine proteinases, presumably cathepsin L, are involved in invasion. Our results show that intracellular and extracellular cathepsin B activity contribute to in vitro invasion of MCF-10A neoT cells and suggest that inhibitors capable of impairing both fractions have a potential as new anticancer drugs.
Premzl
Ales
A
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia. ales.premzl@ijs.si
Zavasnik-Bergant
Valentina
V
Turk
Vito
V
Kos
Janko
J
D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology / Q000494:pharmacology; D000915:Antibody Affinity; D000918:Antibody Specificity; D001943:Breast Neoplasms / Q000473:pathology*; D002401:Cathepsin B / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D018929:Cell Culture Techniques / Q000379:methods; D024141:Collagen Type IV / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D005109:Extracellular Matrix / Q000378:metabolism*; D005260:Female; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007150:Immunohistochemistry; D009361:Neoplasm Invasiveness; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D024141:Collagen Type IV; D015853:Cysteine Proteinase Inhibitors; D002401:Cathepsin B