Dataset Information

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.

SUBMITTER: Achilefu S

PROVIDER: S-EPMC1142399 | biostudies-literature | 2005 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Synergistic effects of light-emitting probes and peptides for targeting and monitoring integrin expression.

Achilefu Samuel S Bloch Sharon S Markiewicz Mary A MA Zhong Tuoxiu T Ye Yunpeng Y Dorshow Richard B RB Chance Britton B Liang Kexian K

Proceedings of the National Academy of Sciences of the United States of America 20050523 22

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 molecul ...[more]

PMID: 15911748

Similar Datasets

Project description:This study sought to evaluate FITC-conjugated cyclic RGD peptides (FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2) as fluorescent probes for in vitro assays of integrin αvβ3/αvβ5 expression in tumor tissues. FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were prepared, and their integrin αvβ3/αvβ5 binding affinity was determined using the displacement assay against (125)I-echistatin bound to U87MG glioma cells. IC50 values of FITC-Galacto-RGD2, FITC-3P-RGD2, and FITC-RGD2 were calculated to be 28 ± 8, 32 ± 7, and 89 ± 17 nM, respectively. The integrin αvβ3/αvβ5 binding affinity followed a general trend: FITC-Galacto-RGD2 ∼ FITC-3P-RGD2 > FITC-RGD2. The xenografted tumor-bearing models were established by subcutaneous injection of 5 × 10(6) tumor cells into shoulder flank (U87MG, A549, HT29, and PC-3) or mammary fat pad (MDA-MB-435) of each athymic nude mouse. Three to six weeks after inoculation, the tumor size was 0.1-0.3 g. Tumors were harvested for integrin αvβ3/αvβ5 staining, as well as hematoxylin and eosin (H&E) staining. Six human carcinoma tissues (colon cancer, pancreatic cancer, lung adenocarcinoma, squamous cell lung cancer, gastric cancer, and esophageal cancer) were obtained from recently diagnosed cancer patients. Human carcinoma slides were deparaffinized in xylene, rehydrated with ethanol, and then used for integrin αvβ3/αvβ5 staining, as well as H&E staining. It was found that the tumor staining procedures with FITC-conjugated cyclic RGD peptides were much simpler than those with the fluorescence-labeled integrin αvβ3 antibodies. Since FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were able to co-localize with the fluorescence-labeled integrin β3 antibody, their tumor localization and tumor cell binding are integrin αvβ3-specific. Quantification of the fluorescent intensity in five xenografted tumors (U87MG, MDA-MB-435, A549, HT29, and PC-3) and six human carcinoma tissues revealed an excellent linear relationship between the relative integrin αvβ3/αvβ5 expression levels determined with FITC-Galacto-RGD2 and those obtained with the fluorescence-labeled anti-human integrin β3 antibody. There was also an excellent linear relationship between the tumor uptake (%ID/g) of (99m)Tc-3P-RGD2 (an integrin αvβ3/αvβ5-targeted radiotracer) and the relative integrin αvβ3/αvβ5 expression levels from the quantification of fluorescent intensity in the tumor tissues stained with FITC-Galacto-RGD2. These results suggest that FITC-conjugated cyclic RGD peptides might be useful to correlate the in vitro findings with the in vivo imaging data from an integrin αvβ3/αvβ5-targeted radiotracer. The results from this study clearly showed that the FITC-conjugated cyclic RGD peptides (particularly FITC-3P-RGD2 and FITC-Galacto-RGD2) are useful fluorescent probes for assaying relative integrin αvβ3/αvβ5 expression levels in tumor tissues.

Dataset Information

Synergistic effects of light-emitting probes and peptides for targeting and monitoring integrin expression.

Publications

Synergistic effects of light-emitting probes and peptides for targeting and monitoring integrin expression.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets