Fluorescent microplate assay for cancer cell-associated cathepsin B

Cathepsin B and in particular cell-surface and secreted cathepsin B has bee n implicated in the invasive and metastatic phenotype of numerous types of cancer. We describe here a method to easily survey cancer cell lines for ca thepsin B activity using the highly selective substrate Z-Arg-Arg-AMC. Inta ct human U87 glioma cells hydrolyze Z-Arg-Arg-AMC with a K-m of 460 mu m at pH 7.0 and 37 degrees C. This is nearly the same as the K-m of 430 mu m ob tained with purified cathepsin B assayed under the same conditions. The per icellular (i.e. both cell-surface and released) cathepsin B activity was in hibited by the cysteine protease inhibitors E-64, leupeptin, Mu-Np2-HphVS-2 Np, Mu-Leu-HpHVSPh and the cathepsin B selective inhibitor Mu-Tyr(3,5 I-2)- HphVSPh with IC50 values similar to those observed for the inhibition of pu rified human liver cathepsin B. Other human cancer cell lines with measurab le pericellular cathepsin B activity included HT-1080 fibrosarcoma, MiaPaCa pancreatic, PC-3 prostate and HCT-116 colon. Cathepsin B activity correlat ed with protein levels of cathepsin B as determined by immunoblot analysis. Pericellular cathepsin B activity was also detected in the rat cell lines MatLyLu prostate and Mat B III adenocarcinoma and in the murine lines B16a melanoma and Lewis lung carcinoma. The ability to determine pericellular ca thepsin B activity will be useful in selecting appropriate cell lines for u se in vivo when analyzing the effects of inhibiting cathepsin B activity on tumor growth and metastasis.