title: The NFL-TBS.40-63 anti-glioblastoma peptide enters selectively in glioma cells by endocytosis.
abstract: Glioblastoma are the most frequent and aggressive tumour of the nervous system despite surgical resection associated with chemotherapy and radiotherapy. Recently, we showed that the NFL-TBS.40-63 peptide corresponding to the sequence of a tubulin-binding site of neurofilaments, enters selectively in glioblastoma cells where it blocks microtubule polymerization, inhibits their proliferation, and reduces tumour development in rats bearing glioblastoma (Bocquet et al., 2009; Berges et al., 2012a). Here, we characterized the molecular mechanism responsible for the uptake of NFL-TBS.40-63 peptide by glioblastoma cells. Unlike other cell penetrating peptides (CPPs), which use a balance between endocytosis and direct translocation, the NFL-TBS.40-63 peptide is unable to translocate directly through the membrane when incubated with giant plasma membrane vesicles. Then, using a panel of markers and inhibitors, flow cytometry and confocal microscopy investigations showed that the uptake occurs mainly through endocytosis. Moreover, glycosaminoglycans and αVβ3 integrins are not involved in the NFL-TBS.40-63 peptide recognition and internalization by glioblastoma cells. Finally, the signalling of tyrosine kinase receptors is involved in the peptide uptake, especially via EGFR overexpressed in tumour cells, indicating that the uptake of NFL-TBS.40-63 peptide by glioblastoma cells is related to their abnormally high proliferative activity.
authors.affiliation: Laboratoire Neurobiologie & Transgenese, LUNAM, UPRES EA-3143, Université d'Angers, Centre Hospitalier Universitaire, Bâtiment IBS-IRIS, 49033 Angers, France.
authors.forename: Claire
authors.initials: C
authors.lastname: Lépinoux-Chambaud
authors.forename: Joël
authors.initials: J
authors.lastname: Eyer
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D004705:Endocytosis; D005910:Glioma / Q000378:metabolism*; D006801:Humans; D016900:Neurofilament Proteins / Q000494:pharmacology*; D010446:Peptide Fragments / Q000494:pharmacology*; D051381:Rats
chemical_list: C576937:NFL-TBS.40-63 peptide; D016900:Neurofilament Proteins; D010446:Peptide Fragments
keywords: 4′,6′-diamidino-2-phenylindole; 5-(N,N-dimethyl) amiloride hydrochloride; ATP; Antp; BSA; CPP; DAPI; DMA; DMEM; DTT; Dubelcco's modified Eagles medium; EGFR; Endocytosis; FACS; FITC; GPMV; HSPGs; MAPK; MβCD; NFL; NaClO(3); PBS; PDGFR; PFA; PI; PI3K; PMA; PTEN; Peptide; RGD; RNase; RTK; TBS; TP10; Tat; Tyrosine kinase receptor; Uptake; VEGFR; adenosine triphosphate; antennapedia-homeodomain-derived antennapedia; arginine–glycine–aspartic acid; bovine serum albumin; cell penetrating peptide; chlorpr.; chlorpromazine hydrochloride; cytoch. D; cytochalasin D; dithiothreitol; epidermal growth factor receptor; fluorescein isothiocyanate; fluorescence activated cell sorting; giant plasma membrane vesicles; glioblastoma; heparan sulfate proteoglycans; methyl-β-cyclodextrin; mitogen-activated protein kinase; neurofilament light subunit; paraformaldehyde; phorbol 12-myristate 13-acetate; phosphatase and TENsin homolog; phosphate buffered saline; phosphoinositide 3-kinase; platelet-derived growth factor receptor; propidium iodide; ribonuclease; sodium chlorate; trans-activator of transcription; transportan 10; tubulin-binding site; tyrosine kinase receptor; vascular endothelial growth factor receptor