Invasion of ras-transformed breast epithelial cells depends on the proteolytic activity of cysteine and aspartic proteinases

It has been suggested that the lysosomal proteinases cathepsin B, L and D p articipate in tumour invasion and metastasis. Whereas for cathepsins B and L the role of active enzyme in invasion processes has been confirmed, cathe psin D was suggested to support tumour progression via its pro-peptide, rat her than by its proteolytic activity. In this study we have compared the pr esence of active cathepsins B, L and D in ras-transformed human breast epit helial cells (MCF-10A neoT) with their ability to invade matrigel. In this cell line high expression of all three cathepsins was detected by immunoflu orescence microscopy. The effect of proteolytic activity on cell invasion w as studied by adding various natural and synthetic cysteine and aspartic pr oteinase inhibitors. The most effective compound was chicken cystatin, a ge neral 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 o f cathepsin L, showed a lower effect than chicken cystatin and E-64. Pepsta tin A weakly inhibited invasion, whereas the same molar concentrations of s quash aspartic proteinase (SQAPI)-like inhibitor, isolated from squash Cucu rbita pepo, showed significant inhibition (65.7 +/- 1.8%). We conclude that both cysteine and aspartic proteinase activities are needed for invasion b y MCF-10A neoT cells in vitro.