Cyclo(RGDyK)

Cyclo(Arg-Gly-Asp- d-Tyr-Lys)-Cy5.5

Integrins are a family of cell surface heterodimeric glycoproteins that mediate diverse biological events involving cell-cell and cell-matrix interactions (1). They consist of an α and a β subunit. They are important for cell adhesion and signal transduction. The αvβ3 integrin is the most prominent receptor class affecting tumor growth, tumor invasiveness, metastasis, tumor-induced angiogenesis, inflammation, osteoporosis, and rheumatoid arthritis (2-7). The αvβ3 integrin is strongly expressed on tumor cells and activated endothelial cells. In contrast, expression of αvβ3 integrin is weak on resting endothelial cells and most normal tissues. The αvβ3 antagonists are being studied as anti-tumor and anti-angiogenic agents (4, 8, 9) and the agonists as angiogenic agents for coronary angiogenesis (10, 11). A tripeptide sequence consisting of Arg-Gly-Asp (RGD) is identified as a recognition motif used by extracellular matrix proteins (vitronectin, fibrinogen, laminin, and collagen) to bind to a variety of integrins including αvβ3. Various radiolabeled antagonists were introduced for imaging of tumors and tumor angiogenesis (12).

Optical fluorescence imaging is increasingly used to obtain biological functions of specific targets (13, 14). However, the intrinsic fluorescence of biomolecules poses a problem when visible light (350-700 nm) absorbing fluorophores are used. Near-infrared (NIR) fluorescence (700-1000 nm) detection avoids the background fluorescence interference of natural biomolecules, providing a high contrast between target and background tissues. NIR fluorophores have wider dynamic range and minimal background as a result of reduced scattering compared with visible fluorescence detection. They also have high sensitivity, resulting from low infrared background, and high extinction coefficients, which provide high quantum yields. The NIR region is also compatible with solid-state optical components, such as diode lasers and silicon detectors. NIR fluorescence imaging is becoming a non-invasive alternative to radionuclide imaging.

Cyclo(RGDyK) was conjugated with Cy5.5 to study in vivo biodistribution of the tracer in tumor-bearing mice. Cy5.5 is a NIR fluorescent dye with absorbance maximum at 675 nm and emission maximum at 694 nm with a high extinction coefficient of 250,000 (mol/L)-1cm-1. RGD-Cy5.5 or c(RGDyK)-Cy5.5 was found to have a high and long-lasting accumulation in αvβ3-positve U87MG human glioblastoma tumor cells in nude mice (15). The binding of RGD-Cy5.5 to the integrin receptor was found to be specific both in vitro and in vivo.

Cyclo(Cys-Arg-Gly-Asp-Cys)-Gly-Lys-Cy5.5

Optical fluorescence imaging is increasingly being used to monitor biological functions of specific targets (1-3). However, the intrinsic fluorescence of biomolecules poses a problem when fluorophores that absorb visible light (350–700 nm) are used. Near-infrared (NIR) fluorescence (700–1,000 nm) detection avoids the natural background fluorescence interference of biomolecules, providing a high contrast between target and background tissues in small animals. NIR fluorophores have a wider dynamic range and minimal background fluorescence as a result of reduced scattering compared with visible fluorescence detection. NIR fluorophores also have high sensitivity, attributable to low background fluorescence, and high extinction coefficients, which provide high quantum yields. The NIR region is also compatible with solid-state optical components, such as diode lasers and silicon detectors. NIR fluorescence imaging is a non-invasive alternative to radionuclide imaging in small animals or with probes in close proximity of the target in humans (4, 5). Among the various optical imaging agents, only indocyanine green (ICG), with NIR fluorescence absorption at 780 nm and emission at 820 nm, is approved by the United States Food and Drug Administration for clinical applications in angiography, blood flow evaluation, and liver function assessment. It is also under evaluation in several clinical trials for other applications, such as optical imaging and mapping of both the lymphatic vessels and lymph nodes in cancer patients for surgical dissection of tumors and endoscopic imaging of the pancreas and colon.

Integrins are a family of cell-surface heterodimeric glycoproteins that mediate diverse biological events involving cell–cell and cell–matrix interactions (6). They consist of an α and a β subunit, and they are important for cell adhesion and signal transduction. The α_vβ₃ integrin is the most prominent receptor class affecting tumor growth, tumor invasiveness, metastasis, tumor-induced angiogenesis, inflammation, osteoporosis, and rheumatoid arthritis (7-12). The α_vβ₃ integrin is strongly expressed on tumor cells and activated endothelial cells. In contrast, expression of α_vβ₃ integrin is weak on resting endothelial cells and most normal tissues. The α_vβ₃ antagonists are being studied as anti-tumor and anti-angiogenic agents (9, 13, 14), and the agonists are being studied as angiogenic agents for coronary angiogenesis (15, 16). A tripeptide sequence consisting of Arg-Gly-Asp (RGD) has been identified as a recognition motif used by extracellular matrix proteins (vitronectin, fibrinogen, laminin, and collagen) to bind to a variety of integrins, including α_vβ₃. Various radiolabeled antagonists have been introduced for imaging of tumors and tumor angiogenesis (17).

Cyclo(RGDyK) was conjugated with Cy5.5 to study in vivo biodistribution of the tracer in tumor-bearing mice. Cy5.5 is a NIR fluorescent dye with an absorbance maximum at 675 nm and an emission maximum at 694 nm, with a high extinction coefficient of 250,000 (mol/L)^–1cm^–1. c(RGDyK)-Cy5.5 was found to have high and long-lasting accumulation in α_vβ₃-positve U87MG human glioblastoma tumor cells in nude mice (18). The binding of c(RGDyK)-Cy5.5 to the integrin receptor was found to be specific both in vitro and in vivo. von Wallbrunn et al. (19) performed optical NIR fluorescence imaging studies using a two-dimensional planar fluorescence reflectance imaging system and three-dimensional fluorescence-mediated tomography (FMT) of cyclo(Cys-Arg-Gly-Asp-Cys)-Gly-Lys-Cy5.5 (c(CRGDC)-GK-Cy5.5) in tumor-bearing nude mice.

ZW800-1, a zwitterionic near-infrared fluorophore, and its cyclic RGD peptide derivative cyclo-(RGDyK)-ZW800-1

Indocyanine green (ICG) is a noninvasive near-infrared (NIR) fluorescence (NIRF) imaging dye that is approved by the United States Food and Drug Administration (FDA) for ophthalmic angiography to determine cardiac output and liver blood flow and function. This dye is also used in cancer patients to map sentinel lymph nodes (SLN), for the detection of solid tumors, and for angiography during reconstructive surgery (2). Methylene blue (MB) is another NIRF dye that has been approved by the FDA for the treatment of drug-induced methemoglobinemia, but MB is also used to visualize SLNs with ultrasound imaging in the clinic (3). Two other commercially available NIRF dyes, Cy5.5 and IRDye800-CW (CW800), have been coupled with peptides or antibodies and successfully used for the targeted visualization of neoplastic tumors in rodents (4). Although the NIRF dyes exhibit low autofluorescence, tissue absorbance, and scatter at NIR wavelengths (700–900 nm), a major limitation of using these dyes in the clinic is that they often show high nonspecific binding and have high tissue uptake and retention (5). In addition, these dyes are cleared from circulation primarily through the hepatobiliary pathway and generate high background signals in the gastrointestinal tract, which results in the masking of signals from cancerous lesions that may be present in tissues located around the digestive system (5). The high background signals interfere with distinguishing the tumors from normal tissues during NIRF-guided resection of the lesions (6). Physiochemical properties such as positive/negative charge density, charge distribution, and hydrophilicity/hydrophobicity (or lipophilicity) are known to affect the protein binding, serum stability, and in vivo biodistribution characteristics of compounds and imaging agents, including probes of the NIRF variety (7). In this regard, MB carries a small net positive charge (+1), whereas ICG, Cy5.5, and CW800 carry a net negative charge (?1, ?4, and ?4, respectively) and are hydrophobic (Table 1) (6). The physiochemical properties of these NIRF dyes are discussed in detail by Gioux et al. (6).

Results obtained from a biodistribution study of NIRF nanoparticles (NPs) with different chemical compositions, hydrodynamic diameters, shapes, and surface charges administered to rats through the pulmonary route showed that zwitterionic NPs (i.e., NPs with a net zero charge or charge-neutral), compared with cationic or anionic charged NPs, were rapidly absorbed from the lungs and were moved to the SLN and into blood circulation for clearance through the kidneys (8). Similar results were reported with zwitterionic quantum dots (QDs) conjugated to CW800 and decorated with a limited number of small molecule (2-(3-amino-3-carboxypropyl)) or peptide (cyclo-RGD-yK) ligands that targeted the prostate-specific membrane antigen and the α_vβ₃ integrin receptor, respectively (9). On the basis of these studies, it was hypothesized that NIRF agents with a net zwitterionic charge would probably behave in a manner similar to the zwitterionic NPs and QDs under in vivo conditions (7). In addition, it was expected that the charge-neutral NIRF agents would generate superior images compared with NIRF probes that carried a net anionic or cationic charge. To test this hypothesis, the biodistribution of a series of heptamethine indocyanine NIRF probes with varying net charges, e.g., ICG (net charge ?1), CW800 (net charge ?4), RS800 (a derivative of CW800, net charge ?2), ZW800-1 (a derivative of CW800, net charge 0), and ZW800-3a (a derivative of CW800, net charge +2) was investigated in mice (7). In another study, cyclo-(RGDyK) conjugated to ZW800-1 (cRGD-ZW800-1) was compared with cyclo-(RGDyK) conjugated to CW800 (cRGD-CW800) and cyclo-(RGDyK) conjugated to Cy5.5 (cRGD-Cy5.5) for the visualization of xenograft tumors that overexpressed integrin α_vβ₃ receptors in mice (5). In addition, fibrinogen (FBG) labeled with the NIRF dyes (FBG-ZW800, FBG-CW800, and FBG-Cy5.5, respectively) was evaluated for the detection of fibrinogen-positive thrombi (blood clots) in mice (5).

68Ga-1,4,7-Triazacyclononane-1,4-7-triacetic acid-Glu-[c(Arg-Gly-Asp- D-Tyr-Lys)]2

Integrins are a family of heterodimeric glycoproteins on cell surfaces that mediate diverse biological events involving cell–cell and cell–matrix interactions (1). Integrins consist of an α and a β subunit and are important for cell adhesion and signal transduction. The α_vβ₃ integrin is the most prominent receptor affecting tumor growth, tumor invasiveness, metastasis, tumor-induced angiogenesis, inflammation, osteoporosis, and rheumatoid arthritis (2-7). Expression of the α_vβ₃ integrin is strong on tumor cells and activated endothelial cells, whereas expression is weak on resting endothelial cells and most normal tissues. The α_vβ₃ antagonists are being studied as antitumor and antiangiogenic agents, and the agonists are being studied as angiogenic agents for coronary angiogenesis (6, 8, 9). The peptide sequence Arg-Gly-Asp (RGD) has been identified as a recognition motif used by extracellular matrix proteins (vitronectin, fibrinogen, laminin, and collagen) to bind to a variety of integrins, including α_vβ₃. Various radiolabeled antagonists have been introduced for imaging of tumors and tumor angiogenesis (10).

Most of the cyclic RGD peptides are composed of five amino acids. Haubner et al. (11) reported that various cyclic RGD peptides exhibit selective inhibition of binding to α_vβ₃ (50% inhibition concentration (IC₅₀), 7–40 nM) but not to α_vβ₅ (IC₅₀, 600–4,000 nM) or α_IIbβ₃ (IC₅₀, 700–5,000 nM) integrins. Various radiolabeled cyclic RGD peptides and peptidominetics have been found to have high accumulation in tumors in mice (12, 13). Out of these developments [¹⁸F]Galacto-c(RGDfK) has been evaluated in a number of clinical studies for imaging of α_vβ₃ in cancer patients (14-19). Li et al. (20) used 1,4,7-triazacyclononane-1,4-7-triacetic acid (NOTA) as a bifunctional chelator for labeling Glu-[cyclo(RGDyK)]₂ (RGD2) to form (⁶⁸Ga-NOTA-RGD2) for positron emission tomography (PET) imaging of α_vβ₃ receptors in nude mice bearing human glioblastoma U87MG tumors.

Potential Antimetastatic Effect of Timosaponin AIII against Human Osteosarcoma Cells through Regulating the Integrin/FAK/Cofilin Axis

Timosaponin AIII (TSAIII) is a steroidal saponin which demonstrates anti-tumour activities. However, the effect of TSAIII on human osteosarcoma cells remains largely unknown. In this study, we demonstrated that TSAIII exerted a significant inhibitory effect on the distribution of cytoskeletal F-actin and cytoskeletal-related proteins, which contributed to the suppression of cell migration and invasion, without inhibiting cell growth or apoptosis. In the synergistic inhibitory analysis, cotreatment of TSAIII with αVβ3 integrin inhibitor [Cyclo(RGDyK)] or focal adhesion kinase (FAK) inhibitor (PF-573228) exerted greater synergistic inhibitory effects on the expression of Intergin αVβ3/FAK/cofilin axis, thus inhibiting the migration and invasion capacities of human osteosarcoma cells. TSAIII was demonstrated to significantly inhibit the pulmonary metastasis formation of human osteosarcoma cells in vivo in metastasis animal models. These findings reveal the inhibitory effects of TSAIII on the metastasis progression of human osteosarcoma cells and the regulation of integrin-αVβ3-FAK-Src and TESK1/p-cofilin mediated cytoskeletal F-actin pathway. Therefore, TSAIII might represent a novel strategy for the auxiliary treatment of human osteosarcoma cells.