Fluorescent microplate assay for cancer cell‐associated cathepsin B Journal Articles uri icon

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abstract

  • 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 µm at pH 7.0 and 37 °C. This is nearly the same as the Km of 430 µm 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.

authors

  • Hulkower, Keren I
  • Butler, Christopher
  • Linebaugh, Bruce E
  • Klaus, Jeffrey L
  • Keppler, Daniel
  • Giranda, Vincent L
  • Sloane, Bonnie F

publication date

  • July 2000