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

• Published in: European journal of cancer (1990) 2005-07, Vol.41 (11), p.1618-1627
• Main Authors: Buc, E., Vartanian, M. Der, Darcha, C., Déchelotte, P., Pezet, D.
• Format: Article
• Language: English
• Subjects: Adult; Aged; Bacterial Toxins - metabolism; Biological and medical sciences; Biomarkers, Tumor; Caco-2 Cells; Colorectal cancer; Colorectal Neoplasms - enzymology; Enterotoxins - metabolism; Escherichia coli Proteins; Female; Fluorescent Antibody Technique; Fusion protein; Green Fluorescent Proteins - administration & dosage; Green Fluorescent Proteins - metabolism; Guanylate Cyclase - metabolism; Guanylyl cyclase C; Heat-stable enterotoxin; Humans; Immunofluorescence; Immunohistochemistry; Immunohistochemistry - methods; Life Sciences; Male; Medical sciences; Middle Aged; Pharmaceutical Vehicles; Pharmacology. Drug treatments; Protein delivery; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide - metabolism; Tumors
• ISSN: 0959-8049; 1879-0852
• DOI: 10.1016/j.ejca.2005.02.031

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.