CATHEPSIN B-LIKE CYSTEINE PROTEINASES AND TUMOR INVASION

蛋白水解酶参与了肿瘤的侵袭过程

Proteolytic enzymes have been implicated in invasive processes of the metastatic cascade. Our long term objective is to determine the role(s) of the cysteine proteinase cathepsin B and endogenous cysteine proteinase inhibitors (CPI) in tumor cell metastasis. We have purified two forms of cathepsin B (35 and 25 kDa) from human tumors and shown their immunological relatedness to human liver cathepsin B (25 kDa) by Western blots. We have shown that tumor cathepsin B's have enhanced activity against the extracellular matrix substrate laminin at a physiologically relevant pH (6.5). The hypothesis that cathepsin B participates in tumor cell extravasation during the metastatic cascade will be tested in vitro in a series of assays which encompass the invasive process: 1) tumor cell adhesion the basement membrane, 2) tumor cell chemotaxis during extravasation, 3) tumor cell degradation of the basement membrane or isolated components and/or 4) tumor cell invasion through the amnion basement membrane. Two classes of inhibitors of cathepsin B will be tested in these assays: endogenous low Mr protein CPI and a series of synthetic inhibitors screened for selectivity against the purified tumor cathepsin B's. CPI will be purified to homogeneity from human liver and tumors. Studies to date indicate that tumor cell chemotaxis, adhesion and invasion are all blocked by CPI as well as by specific synthetic inhibitors. In murine tumors (melanoma and hepatoma) and in human breast tumors, the subcellular distribution of cathepsin distribution of cathepsin B is bimodal with as much as 37% of cathepsin B activity cosedimenting with plasma membrane fractions in the highly metastatic B16 amelanotic melanoma (B16a) rather that with the lysosmal fraction as in normal cells. The 35 kDa tumor cathepsin B may well be a precursor of mature lysosmal cathepsin B as a 35 kDa precursor predicted from the recently isolated cDNA clones. One hypothesis which will be tested is that the processing of cathepsin B in tumors may be defective. The proposed studies should further our understanding of the role of cathepsin B in a proteolytic cascade resulting in metastasis. It might be possible to interrupt this cascade by designing therapeutic interventions to inhibit cathepsin B and/or to correct the proposed defect in processing, i.e., direct tumor cathepsin B to lysosmes.