Intracellular and extracellular cathepsin B facilitate invasion of MCF-10A neoT cells through reconstituted extracellular matrix in vitro

被引:127
作者
Premzl, A
Zavasnik-Bergant, V
Turk, V
Kos, J
机构
[1] Jozef Stefan Inst, Dept Biochem & Mol Biol, SI-1000 Ljubljana, Slovenia
[2] KRKA, R&D Div, Dept Biochem Res & Drug Design, SI-1000 Ljubljana, Slovenia
关键词
cathepsin B; extracellular matrix; invasion; monoclonal antibody; quenched fluorescent substrate;
D O I
10.1016/S0014-4827(02)00055-1
中图分类号
R73 [肿瘤学];
学科分类号
100214 ;
摘要
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 muM, i.e., 24.9 and 27.0%, respectively. Neutralizing monoclonal antibody exhibited a significantly higher inhibitory effect, decreasing invasion at 0.5 muM 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. (C) 2003 Elsevier Science (USA). All rights reserved.
引用
收藏
页码:206 / 214
页数:9
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