High-throughput methods for measuring heparanase activity and screening potential antimetastatic and anti-inflammatory agents

• Published in: Analytical biochemistry Vol. 333; no. 2; pp. 389 - 398
• Main Authors: Huang, Kuo-Sen, Holmgren, Janna, Reik, Linda, Lucas-McGady, Debra, Roberts, John, Liu, Chao-Min, Levin, Wayne
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15-10-2004
• Subjects: Animals; Anti-Inflammatory Agents - isolation & purification; Anti-Inflammatory Agents - pharmacology; Antineoplastic Agents - isolation & purification; Antineoplastic Agents - pharmacology; Biotin - metabolism; Cattle; Cells, Cultured; CHO Cells; Cloning, Molecular; Cricetinae; Drug Evaluation, Preclinical - methods; Escherichia coli - genetics; Fibroblast Growth Factors - metabolism; Fluorescein-5-isothiocyanate; Gene Expression; Glucuronidase - analysis; Glucuronidase - genetics; Glucuronidase - metabolism; Heparitin Sulfate - analysis; Heparitin Sulfate - metabolism; Humans; Kidney - enzymology; Staining and Labeling; Tritium
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2004.06.023

Heparanase plays an important role in the degradation of the extracellular matrix. It is implicated in inflammation, tumor angiogenesis and metastasis. We have developed two high-throughput methods for measuring heparanase activity and screening potential inhibitors. The first method involves coating fibroblast growth factor (FGF) on microtiter plates and capturing fluorescein isothiocyanate (FITC)-labeled heparin sulfate (HS), which is used as a substrate for heparanase digestion. Labeled HS fragments are released into the medium and quantitated by fluorescence intensity measurement. We have implemented this assay method into a Zeiss uHTS system and screened compound libraries for heparanase inhibitors. The second method involves labeling HS with biotin followed by FITC to generate a dual-labeled HS. The labeled material is bound to streptavidin-coated plates and used as a substrate for heparanase digestion. Both methods are sensitive and easily applicable to robotic systems. In addition, we have labeled both HS and biotin-HS with Eu-chelate, a fluorophore that exhibits long decay fluorescence. Assays using Eu-labeled HS and Eu-labeled biotin-HS have been developed and show higher sensitivity than those using FITC-labeled material. Furthermore, assays using Eu-chelate HS (or biotin-HS) should eliminate the interference of fluorescence compounds.