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.
Hulkower
K I
KI
Department of Cancer Research, Abbott Laboratories, Abbott Park, IL, USA. keren.i.hulkower@abbott.com
Butler
C C
CC
Linebaugh
B E
BE
Klaus
J L
JL
Keppler
D
D
Giranda
V L
VL
Sloane
B F
BF
D000818:Animals; D002401:Cathepsin B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005453:Fluorescence; D006801:Humans; D051379:Mice; D009369:Neoplasms / Q000201:enzymology*; D017830:Octoxynol / Q000494:pharmacology; D051381:Rats; D014407:Tumor Cells, Cultured
D017830:Octoxynol; D002401:Cathepsin B