Article révisé par les pairs
Résumé : Anaplastic thyroid cancer (ATC) represents the deadliest thyroid tumor in humans. Current treatments have not really demonstrated a long-term benefit. Accordingly, targeted drug delivery should be considered to improve the prognosis of patients, but also the early-stage diagnosis. Epidermal Growth Factor Receptor (EGFR) is commonly studied in oncology as it is overexpressed in cancer cells and is actively investigated in the framework of receptor-mediated drug delivery due to its intracellular trafficking. Therefore, an EGFR-targeted peptide was developed in the present work by taking advantage of the versatility of phage display technique. The selected EGFR-targeted P20 peptide was investigated by a wide range of in silico, in vitro, in vivo and ex vivo methods, allowing us to formulate the following main conclusions: (1) P20 has a theoretical half-life of 100 h and binds to EGFR domains that harbor the EGF binding site; (1) the peptide binds in a higher level to cancer cells and tissues compared to healthy ones; (2) it induces EGFR endocytosis and follows the non-degradative EGFR intracellular pathway; (3) P20 does not interfere with EGF binding and acts as a non-competitive inhibitor of EGFR; (4) it could contribute to the therapeutic effect of anti-cancer drugs by decreasing the expression and activation of EGFR, as well as of AKT phosphorylation in ATC cells; (5) P20 does not induce in vivo toxic effects in the main tissues and organs; (6) it is concentrated in tumors, where the peptide is retained for longer time than in other tissues due to its binding to EGFR. Abbreviations: 7-AAD, 7-Aminoactinomycin D; AA%, amino acid composition expressed as a percentage; ABC, Avidin/Biotinylated enzyme Complex; ABTS, 2,2′-azino-bis-3-ethylbenzothioazoline-6-sulfonic acid; A.I., aliphatic index; AKT, protein kinase B; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ATC, anaplastic thyroid carcinoma; Bad, the Bcl2 associated agonist of cell death protein; BCA, Pierce BiCinchoninic Acid; BUN, Blood Urea Nitrogen; CMMI, Center for Microscopy and Molecular Imaging; DAB, 3,3′-diaminobenzidine tetrahydrochloride solution; DAPI, 4′,6-diamidino-2-phenylindole; DTT, dithiothreitol; ECACC, European Collection of Authenticated Cell Cultures; ECL, Enhanced chemiluminescence; ED, extracellular domain; ED-EGFR, Extracellular domain of EGFR; EGF, epidermal growth factor; EGFR, the epidermal growth factor receptor; ER, endoplasmic reticulum; FLI, Fluorescence Lifetime Imaging; GAPDH, the glyceraldehyde-3-phosphate dehydrogenase; IC50, the half-maximal inhibitory concentration; IF, immunofluorescence, IHC, immunohistochemistry; IL7R, interleukin 7 receptor; K*d, the apparent dissociation constant; LDS, lithium dodecyl sulfate sample buffer; MAPK, Mitogen-Activated Protein Kinase; mTOR, mammalian target of rapamycin; NA: not available; NMRI, The Naval Medical Research Institute (mouse model); NSP: non-stimulated cells incubated with P20-rhodamine; NSPE: non-stimulated cells incubated with P20-rhodamine and soluble EGFR; PAM, the PI3K/AKT/mTOR signaling pathway; PEG, polyethylene glycol; peptide-TR: peptide-Texas Red; PFBB, Protein-Free Blocking Buffer; PI3K, Phosphoinositide 3-kinase; PLCγ/PKC, phospholipase Cγ/protein kinase C; PMSF, phenylmethylsulphonyl fluoride; PS, phosphatidylserine; SP: cells stimulated with EGF and incubated with P20-rhodamine; SPE: cells stimulated with EGF and incubated with P20-rhodamine and soluble EGFR; RET, Rearranged during transfection; RRFL, Relative Ratio of Fluorescent Labeling; RRIL, Relative Ratio of IHC Labeling; RTK, receptor tyrosine kinases; SD: standard deviation; SDS: Sodium Dodecyl Sulfate; SI: signal intensity; T-Bil: total bilirubin; TC, thyroid carcinoma; T-Chol, Total cholesterol; TGF-α, Transforming growth factor alpha; TK, tyrosine kinase; TMB, 3,3′,5,5′-Tetramethylbenzidine; VEGFR, Vascular Endothelial Growth Factor Receptor.

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