[PubMed]

The ephrin (Eph) receptors constitute the largest member of the receptor tyrosine kinase family (1, 2). The Eph receptors and their ligands (ephrins) mediate numerous biological processes in normal development, particularly in the nervous and cardiovascular systems (3-5). Based on their structures and sequence relationships, ephrins are divided into the ephrin-A class, which are anchored to the cell membrane by a glycosylphosphatidylinositol linkage, and the ephrin-B class, which are transmembrane proteins. The Eph family of receptors is divided into two groups, EphA and EphB, on the basis of the similarity of their extracellular domain sequences and their affinities for binding ephrin-A and ephrin-B ligands. The Eph receptors transmit forward signals via their kinase domain and reverse signals via their transmembrane ephrin ligands (6). EphB–ephrin-B interactions are capable of mediating bi-directional signaling events upon cell–cell contact, either into the receptor-expressing cell as "forward signaling" or into the ligand-expressing cell as "reverse signaling" (7).

Ephrin-2 is expressed on arterial and activated endothelial cells, whereas EphB4 is normally expressed on venous endothelial cells and various blood cells (8). EphB4 selectively binds to ephrin-2 to promote cell signaling and angiogenesis. EphB4 has been implicated in cancer progression and in pathological forms of angiogenesis. Overexpression of EphB4 has been observed in cancer cells and is associated with tumorigenesis via forward signaling and angiogenesis via reverse signaling through ephrin-2 interaction (9). EphB4 forward signaling stimulates cellular proliferation. Koolpe et al. (10) identified a 15-mer peptide, Tyr-Asn-Tyr-Leu-Phe-Ser-Pro-Asn-Gly-Pro-Ile-Ala-Arg-Ala-Trp (TNYL-RAW), to be a selective antagonist of EphB4 using phage display screening. Xiong et al. (11) reported the development of ⁶⁴Cu-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-TNYL-RAW (⁶⁴Cu-DOTA-TNYL-RAW) for positron emission tomography (PET) imaging of EphB4 in nude mice bearing tumor xenografts. Zhang et al. (12) conjugated TNYL-RAW to polyethylene glycol–coated core-cross-linked polymeric micelles (CCPMs) labeled with Cy7 and ¹¹¹In (¹¹¹In-TNYL-RAW-CCPM) for multimodality detection of EphB4 in tumors.

[PubMed]

CCPM nanoparticles were obtained from Carestream Health (Rochester, NY) (12). There were ~21 Cy7 dye molecules, 180 amino groups, and 19 DTPA moieties per CCPM nanoparticle. Ac-Cys-TNYL-RAW was conjugated to the maleimide-CCPM by sulfhydryl-maleimide bonding. TNYL-RAW-CCPM nanoparticles were purified with column chromatography. The average hydrodynamic diameter of TNYL-RAW-CCPMs was determined to be ~23 nm. There were 60 TNYL-RAW peptides per nanoparticle. The maximum excitation and emission intensities were 755 nm and 781 nm, respectively. ¹¹¹In-TNYL-RAW-CCPM nanoparticles were prepared by incubation of TNYL-RAW-CCPM with ¹¹¹InCl₃ in sodium acetate buffer (pH 5.2) for 30 min at room temperature with a labeling efficiency of >98%. The specific activity was 180 MBq/nmol (4.9 mCi/nmol). Control ¹¹¹In-CCPM nanoparticles without TNYL-RAW were prepared similarly.

[PubMed]

Zhang et al. (12) performed binding experiments with TNYL-RAW-CCPM and CCPM using a Biacore sensor chip immobilized with EphB4-Fc. There were increasing amounts of TNYL-RAW-CCPM bound in a dose-dependent manner (0.16–20 nM). The binding kinetics of TNYL-RAW-CCPM to EphB4 is complex due to multi-valency interactions. No binding to EphB4 was detected with CCPM. In vitro binding of TNYL-RAW-CCPM to PC-3M human prostate cancer cells (EphB4-expressing) and A549 human lung adenocarcinoma cells (EphB4-negative) was determined with fluorescence microscopy. PC-3M cells, but not A549 cells, were readily stained with TNYL-RAW-CCPM. The binding of TNYL-RAW-CCPM to PC-3M cells was almost completely blocked by co-incubation with 100-fold excess TNYL-RAW. ¹¹¹In-TNYL-RAW-CCPM exhibited high cellular uptake (cell/medium ratio = 170) in PC-3M cells but not in A549 cells after 15 min of incubation. Incubation with 100-fold excess of TNYL-RAW almost completely inhibited the radioactivity in PC-3M.

[PubMed]

No publication is currently available.

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