02 BOOK OF ABSTRACTS .indd

Assessment of cathepsin B activity in tumour invasion
and angiogenesis
Ale{ Premzl 1 and Janko Kos 2
1
Department of Biochemistry and Molecular Biology, Jo`ef Stefan Institute, Jamova 39,
SI-1000 Ljubljana, Slovenia; e-mail: ales.premzl@ijs.si; 2 Faculty of Pharmacy, University
of Ljubljana, A{ker~eva 7, SI-1000 Ljubljana, Slovenia
Lysosomal cysteine protease cathepsin B is implicated in degradation and remodelling
of extracellular matrix (ECM), creating a local environment that facilitates progression
of solid tumours and formation of new blood vessels. Besides pericellular proteolyitic
activity of this enzyme the evidence about the contribution of intracellular cathepsin
B to these processes has emerged in recent years.
The aim of our study was to investigate the role of both extracellular and intracellular
cathepsin B activity in the invasion of ras-transformed human breast epithelial
MCF-10A neoT cells and in the formation of capillary-like tubes by HUVEC
endothelial cells in angiogenesis in vitro. We used general and specific cysteine
protease inhibitors and cathepsin B neutralising monoclonal antibody to assess the
role of both fractions of cathepsin B in selected cell models. Additionally, fluorescent
and confocal microscopy was used to (co-)localise total proteolytic activity detected
by degradation of quenched fluorescent protein substrate DQ-collagen IV and
intracellular cathepsin B activity detected by degradation of Z-Arg-Arg-cresyl violet
substrate in living tumour and endothelial cells. We have shown that in MCF-10A
neoT cells activity of both extracellular and intracellular cathepsin B contributes to
their invasive potential, and that inhibitors acting on both fractions of the enzyme
are more effective in preventing tumour cell invasion. However, contrary to our
expectations, in HUVECs only intracellular cathepsin B activity showed significant
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effect on the formation of capillary-like tubes by cells grown on Matrigel, despite the
fact that the total extracellular proteolysis was more intense when compared to the
total intracellular proteolysis, as observed by degradation of DQ-collagen IV.
Our results clearly demonstrate that intracellular and extracellular cathepsin B activity
differs in its contribution to the degradation of ECM in tumour cell invasion and
angiogenesis in vitro. Further, they also indicate that localisation of target protease
activity should be taken into consideration when planning therapeutic strategies for
treatment of cancer with protease inhibitors.
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