title: Effects of enzymatic activation on the distribution of fluorescently tagged MMP-2 cleavable peptides in cancer cells and spheroids.
abstract: A peptide tagged at the N-terminus with FITC, at the C-terminus with coumarin-343, and incorporating a sequence selectively cleaved by the matrix metalloproteinase, MMP-2, was synthesized to investigate the effect of peptide cleavage on both cellular accumulation and distribution in cancer cell spheroids. The peptide was shown by HPLC and mass spectroscopy to be cleaved in the presence of MMP-2 at the expected site. The cellular and spheroid distribution of each of the fragments was monitored using confocal fluorescence microscopy. The intact peptide had minimal accumulation in 2D-cultured DLD-1 cells that do not express MMP-2 in these conditions. Following addition of serum containing MMP-2 to the cell media, the cleaved C-terminal fragment was seen to enter the cells, while the N-terminal fragment remained extracellular, evidently blocked by the presence of the FITC group. 3D culture of DLD-1 cells as spheroids resulted in measurable MMP-2 activity. Different distribution patterns of the two fluorophores were seen in spheroids treated with the intact peptide, consistent with cleavage occurring. Different rates of accumulation of each of the fragments were observed within the spheroid over time, which is attributed to the extent of accumulation and sequestration of the fragments by cells residing in the periphery of the spheroids. The outcomes suggest that tumor-associated enzymes have the potential to modify the distribution of peptides and peptide fragments in solid tumors by modifying the cellular uptake of those peptides.
authors.affiliation: School of Chemistry, The University of Sydney , NSW 2006, Australia.
authors.forename: Natsuho
authors.initials: N
authors.lastname: Yamamoto
authors.forename: Nicole S
authors.initials: NS
authors.lastname: Bryce
authors.forename: Nils
authors.initials: N
authors.lastname: Metzler-Nolte
authors.forename: Trevor W
authors.initials: TW
authors.lastname: Hambley
mesh_terms: D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D003374:Coumarins / Q000032:analysis; D004789:Enzyme Activation; D016650:Fluorescein-5-isothiocyanate / Q000032:analysis; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis* / Q000378:metabolism; D010455:Peptides / Q000032:analysis* / Q000138:chemical synthesis / Q000378:metabolism*; D018874:Spheroids, Cellular; D014407:Tumor Cells, Cultured
chemical_list: D003374:Coumarins; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D010455:Peptides; C552279:coumarin 343; D020778:Matrix Metalloproteinase 2; D016650:Fluorescein-5-isothiocyanate