Invasion of ras-Transformed Breast Epithelial Cells Depends on the Proteolytic Activity of Cysteine and Aspartic Proteinases

Abstract

It has been suggested that the lysosomal proteinases cathepsin B, L and D participate in tumour invasion and metastasis. Whereas for cathepsins B and L the role of active enzyme in invasion processes has been confirmed, cathepsin D was suggested to support tumour progression via its pro-peptide, rather than by its proteolytic activity. In this study we have compared the presence of active cathepsins B, L and D in ras-transformed human breast epithelial cells (MCF-10A neoT) with their ability to invade matrigel. In this cell line high expression of all three cathepsins was detected by immunofluorescence microscopy. The effect of proteolytic activity on cell invasion was studied by adding various natural and synthetic cysteine and aspartic proteinase inhibitors. The most effective compound was chicken cystatin, a general 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 of cathepsin L, showed a lower effect than chicken cystatin and E-64. Pepstatin A weakly inhibited invasion, whereas the same molar concentrations of squash aspartic proteinase (SQAPI)-like inhibitor, isolated from squash Cucurbita pepo, showed significant inhibition (65.7+/-1.8%). We conclude that both cysteine and aspartic proteinase activities are needed for invasion by MCF-10A neoT cells in vitro.

Poly(lactide-co-glycolide) nanoparticles as a carrier system for delivering cysteine protease inhibitor cystatin into tumor cells

“…MCF-10A neoT cells, as used in our previous in vitro studies of tumor invasiveness [4,15,16], were provided by Prof. Bonnie F. Sloane (Wayne State University, Detroit, MI). Their origin was a human breast epithelial cell line (MCF-10), transformed with a neomycin resistance gene and c-Ha-ras oncogene.…”

Regulation of Cell Transformation by Rb-Controlled Redox Homeostasis

“…We used a lentiviral construct to knockdown Rb (shRb) [12] or over-expressed Ras V12 to transform human MCF-10A mammary epithelial cells [36] (Fig. 9A).…”

The association between RASSF1 gene polymorphisms and lung cancer susceptibility among people in Hubei Province of China

“…RASSF1 can adjust the microtubules stability by binding to microtubules and modulating the function of centrosomes and spindles, and may also inhibit the phosphorylation activity of anaphase-promoting complex and block the cell cycle at G 2 /M phase [13,14] ; (3) Suppressing cancer metastasis. Exogenous expression of RASSF1A may lead to down-regulation of CTSL (a metastasis promoter) and up-regulation of SPARC (a metastasis inhibitor) [15,16] .…”