Guanylyl cyclase C as a reliable immunohistochemical marker and its ligand Escherichia coli heat-stable enterotoxin as a potential protein-delivering vehicle for colorectal cancer cells.
mRNA-based technologies and preclinical research in a variety of animal models have shown that guanylyl cyclase C (GCC) is a highly sensitive and specific molecular marker for the diagnosis of colorectal cancer (CRC). GCC is also a receptor for Escherichia coli (E. coli) heat-stable enterotoxin (STa) and can be used for STa-directed delivery of small-sized imaging agents to human CRC tumours. In this study, we have evaluated GCC as a new immunohistochemical (IHC) marker for CRC tissues and STa as a suitable vector for delivering high-sized protein molecules to CRC cells. Firstly, we have developed a highly sensitive EnVision(+)-based IHC staining method for detecting GCC in serial paraffin-embedded sections of primary and metastatic CRC (38 cases) or non-CRC (14 cases) adenocarcinomas. Carcinoembryonic antigen (CEA) and cytokeratin 20 (CK20) were chosen as controls. Our results indicate that GCC staining was positive in 100% of CRC tumours and was comparable to CEA (95%) or CK20 (92%). In contrast to CEA and CK20, GCC was negative in all of the extra-intestinal non-CRC tumours examined. GCC appears to display higher specificity than either CEA or CK20 while retaining high sensitivity, suggesting that it is a better CRC marker than CEA or CK20. Secondly, STa was genetically coupled to green fluorescent protein (GFP) and the resulting GFP-tagged STa was characterized for expression in E. coli and enterotoxicity in mouse. The binding characteristics of GFP-STa in CRC Caco-2 cells were followed by immunofluorescence microscopy. In this work we show that GFP-tagged STa is biologically active and has retained its ability to internalise into Caco-2 cells making it a potential vehicle for the delivery of anticancer therapeutic protein agents.
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Service de Chirurgie Générale et Digestive, Hôtel-Dieu, Clermont-Ferrand, France.
Vartanian
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Darcha
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Déchelotte
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Pezet
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D000328:Adult; D000368:Aged; D001427:Bacterial Toxins / Q000378:metabolism*; D014408:Biomarkers, Tumor; D018938:Caco-2 Cells; D015179:Colorectal Neoplasms / Q000201:enzymology*; D004768:Enterotoxins / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005260:Female; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000008:administration & dosage / Q000378:metabolism; D006162:Guanylate Cyclase / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D008875:Middle Aged; D014677:Pharmaceutical Vehicles; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide / Q000378:metabolism*
D001427:Bacterial Toxins; D014408:Biomarkers, Tumor; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D014677:Pharmaceutical Vehicles; D018000:Receptors, Peptide; C034151:heat stable toxin (E coli); D049452:Green Fluorescent Proteins; D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled