Abstract
In this paper we describe the establishment of an efficient visual method for screening heparanase inhibitors, and we present the results of screening 10,000 microbial culture broths. Heparanase-overexpressing stable clones of the human hepatocellular carcinoma HepG2 cells were established and used as an enzyme source. Digestion of heparan sulfate (HS) was detected using novel HS-containing tablets or SDS-polyacrylamide gel electrophoresis. This method was able to find suramin, a known heparanase inhibitor, from a library of typical enzyme inhibitors. By screening 10,000 culture broths of microorganisms (actinomycetes, fungi, and bacteria) an actinomycete strain, RK99-A234, was found to have heparanase inhibitory activity. RK-682 was identified in the fermentation broth as a heparanase inhibitor, IC50 = 17 microM.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Actinobacteria / metabolism
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Animals
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Blotting, Western
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Carcinoma, Hepatocellular / drug therapy
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Carcinoma, Hepatocellular / pathology
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Cell Line, Tumor
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Clone Cells
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Culture Media
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DNA, Complementary / biosynthesis
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DNA, Complementary / genetics
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Drug Evaluation, Preclinical
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Electrophoresis, Polyacrylamide Gel
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Enzyme Inhibitors / chemistry
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Enzyme Inhibitors / isolation & purification
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Enzyme Inhibitors / pharmacology*
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Glucuronidase / antagonists & inhibitors*
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Glucuronidase / biosynthesis
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Heparitin Sulfate / metabolism
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Humans
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Liver Neoplasms / drug therapy
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Liver Neoplasms / pathology
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Phosphoprotein Phosphatases / antagonists & inhibitors*
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Phosphoprotein Phosphatases / chemistry
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Phosphoprotein Phosphatases / isolation & purification
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Phosphoprotein Phosphatases / pharmacology*
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Transfection
Substances
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Culture Media
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DNA, Complementary
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Enzyme Inhibitors
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RK 682
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Heparitin Sulfate
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Phosphoprotein Phosphatases
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heparanase
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Glucuronidase