Location of tumour cells in colon tissue by Texas red labelled pentosan polysulphate, an inhibitor of a cell surface protease.
Pentosan polysulphate (PPS), a highly negatively charged polysaccharide, is a significant inhibitor of an isoenzymic form of a cell surface protease referred to as guanidinobenzoatase GB, associated with colonic carcinoma tissues in frozen sections and free GB in solution, in a concentration-dependent manner. However PPS failed to recognise and bind to the isoenzymic form of GB associated with normal colon epithelial cell surfaces. Texas red labelled PPS (TR-PPS) binds to the tumour cell surfaces of colonic carcinoma and colonic polyps and these cells fluoresce red, whilst the normal colon cell surfaces failed to bind the TR-PPS, and hence lacked red fluorescence. Polysulphonated suramin also selectively recognised and inhibited the colonic carcinoma GB isoenzyme. The kinetic data indicated that this inhibition was not caused by a mere polyanionic effect, since highly sulphated heparin failed to show a significant inhibition of colonic carcinoma GB, however trypan blue did show 50% inhibition. Kinetic studies have also shown that PPS is a non-competitive, reversible inhibitor of colonic carcinoma GB, with an apparent Km 6.8 x 10(-7) M. Gel analysis has shown that PPS binds to another site, distinct from the active centre, and after binding PPS changed the conformation of GB. These studies suggest that TR-PPS is a potent inhibitor of colonic carcinoma GB, and can be used as a novel fluorescent probe for the location of tumour cells in frozen sections of human colon tissues. PSS could also have potential as a vehicle for the transport of cytotoxic compounds to carcinoma cells of the colon.
Anees
M
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Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Manchester, UK.
D002265:Carboxylic Ester Hydrolases / Q000494:pharmacology*; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D003110:Colonic Neoplasms / Q000378:metabolism; D003619:Dansyl Compounds / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D007700:Kinetics; D008856:Microscopy, Fluorescence; D010426:Pentosan Sulfuric Polyester / Q000031:analogs & derivatives / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology; D011487:Protein Conformation; D013498:Suramin / Q000494:pharmacology; D014343:Trypan Blue / Q000494:pharmacology; D014966:Xanthenes / Q000378:metabolism*
C017532:4-methylumbelliferylguanidinobenzoate; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D007527:Isoenzymes; D011480:Protease Inhibitors; D014966:Xanthenes; C016338:dansyl fluoride; D010426:Pentosan Sulfuric Polyester; D006923:Hymecromone; D013498:Suramin; C034657:Texas red; D006493:Heparin; D002265:Carboxylic Ester Hydrolases; C037288:guanidinobenzoate esterase; D010450:Endopeptidases; D014343:Trypan Blue