title: Synergistic effects of light-emitting probes and peptides for targeting and monitoring integrin expression.
abstract: Integrins mediate many biological processes, including tumor-induced angiogenesis and metastasis. The arginine-glycine-aspartic acid (RGD) peptide sequence is a common recognition motif by integrins in many proteins and small peptides. While evaluating a small library of RGD peptides for imaging alpha(V)beta(3) integrin (ABI)-positive tumor cell line (A549) by optical methods, we discovered that conjugating a presumably inactive linear hexapeptide GRDSPK with a near-infrared carbocyanine molecular probe (Cypate) yielded a previously undescribed bioactive ligand (Cyp-GRD) that targets ABI-positive tumors. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay with A549 cells showed that Cyp-GRD was not cytotoxic up to 100 muM in cell culture. The compound was internalized by cells, and this internalization was blocked by coincubation with a cyclic RGD peptide (cyclo[RGDfV], f is d-phenylalanine) that binds ABI with high affinity. In vivo, Cyp-GRD selectively accumulated in tumors relative to surrounding normal tissues. Blocking studies with cyclo[RGDfV] inhibited the in vivo uptake of Cyp-GRD, suggesting that both compounds target the same active site of the protein. A strong correlation between the Cyp-GRD peptide and mitochondrial NADH concentration suggests that the new molecule could also report on the metabolic status of cells ex vivo. Interestingly, neither a Cypate-labeled linear RGD peptide nor an (111)In-labeled DOTA-GRD conjugate was selectively retained in the tumor. These results clearly demonstrate the synergistic effects of Cypate and GRD peptide for molecular recognition of integrin expression and suggest the potential of using carbocyanines as optical scaffolds for designing biologically active molecules.
authors.affiliation: Department of Radiology and Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors.forename: Samuel
authors.initials: S
authors.lastname: Achilefu
authors.forename: Sharon
authors.initials: S
authors.lastname: Bloch
authors.forename: Mary A
authors.initials: MA
authors.lastname: Markiewicz
authors.forename: Tuoxiu
authors.initials: T
authors.lastname: Zhong
authors.forename: Yunpeng
authors.initials: Y
authors.lastname: Ye
authors.forename: Richard B
authors.initials: RB
authors.lastname: Dorshow
authors.forename: Britton
authors.initials: B
authors.lastname: Chance
authors.forename: Kexian
authors.initials: K
authors.lastname: Liang
mesh_terms: D000818:Animals; D002232:Carbocyanines / Q000378:metabolism; D005470:Fluorometry; D016023:Integrins / Q000378:metabolism*; D008024:Ligands; D051379:Mice; D008819:Mice, Nude; D008856:Microscopy, Fluorescence; D015336:Molecular Probe Techniques; D009369:Neoplasms / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*; D010456:Peptides, Cyclic; D012910:Snake Venoms; D013053:Spectrophotometry; D013778:Tetrazolium Salts; D013844:Thiazoles; D014407:Tumor Cells, Cultured
chemical_list: D002232:Carbocyanines; D016023:Integrins; D008024:Ligands; D009842:Oligopeptides; D010456:Peptides, Cyclic; D012910:Snake Venoms; D013778:Tetrazolium Salts; D013844:Thiazoles; C422910:Cilengitide; C047981:arginyl-glycyl-aspartic acid; C022616:thiazolyl blue