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

Exp Cell Res. 2003 Feb 15;283(2):206-14. doi: 10.1016/s0014-4827(02)00055-1.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal / biosynthesis
  • Antibodies, Monoclonal / immunology
  • Antibodies, Monoclonal / pharmacology
  • Antibody Affinity
  • Antibody Specificity
  • Breast Neoplasms / pathology*
  • Cathepsin B / antagonists & inhibitors
  • Cathepsin B / metabolism
  • Cathepsin B / physiology*
  • Cell Culture Techniques / methods
  • Collagen Type IV / metabolism
  • Cysteine Proteinase Inhibitors / pharmacology
  • Extracellular Matrix / metabolism*
  • Female
  • Humans
  • Hybridomas / immunology
  • Immunohistochemistry
  • Neoplasm Invasiveness*
  • Tumor Cells, Cultured

Substances

  • Antibodies, Monoclonal
  • Collagen Type IV
  • Cysteine Proteinase Inhibitors
  • Cathepsin B