Fluorescent microplate assay for cancer cell-associated cathepsin B

Cathepsin B and in particular cell-surface and secreted Cathepsin B has been implicated in the invasive and metastatic phenotype of numerous types of Cancer. We describe here a method to easily survey Cancer cell lines for Cathepsin B activity using the highly selective substrate Z-Arg-Arg-AMC. Intact human U87 glioma cells hydrolyze Z-Arg-Arg-AMC with a Km of 460 microM at pH 7.0 and 37 degrees C. This is nearly the same as the Km of 430 microM obtained with purified Cathepsin B assayed under the same conditions. The pericellular (i.e. both cell-surface and released) Cathepsin B activity was inhibited by the cysteine Protease Inhibitors E-64, leupeptin, Mu-Np2-HphVS-2Np, Mu-Leu-HpHVSPh and the Cathepsin B selective inhibitor Mu-Tyr(3,5 I2)-HphVSPh with IC50 values similar to those observed for the inhibition of purified human liver Cathepsin B. Other human Cancer cell lines with measurable pericellular Cathepsin B activity included HT-1080 fibrosarcoma, MiaPaCa pancreatic, PC-3 prostate and HCT-116 colon. Cathepsin B activity correlated 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 Cathepsin B activity will be useful in selecting appropriate cell lines for use in vivo when analyzing the effects of inhibiting Cathepsin B activity on tumor growth and metastasis.