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| Chemical name: | Cy5.5-GGSGRSANAK(Fitc)C-poly-L-lysine-methoxy polyethylene glycol | |
| Abbreviated name: | Cy5.5-GGSGRSANAK(Fitc)C-PL-MPEG, Cy5.5-uPA-PGC | |
| Synonym: | ||
| Agent category: | Peptide | |
| Target: | Urokinase-type plasminogen activator | |
| Target category: | Enzyme | |
| Method of detection: | Optical, near-infrared (NIR) fluorescence | |
| Source of signal: | Cy5.5 | |
| Activation: | Yes | |
| Studies: |
| Click on MMP-2 and MMP-9 for protein information. |
[PubMed]
Extracellular matrix (ECM) adhesion molecules consist of a complex network of fibronectins, collagens, chondroitins, laminins, glycoproteins, heparin sulfate, tenascins, and proteoglycans that surround connective tissue cells, and they are mainly secreted by fibroblasts, chondroblasts, and osteoblasts (1). Cell substrate adhesion molecules are considered essential regulators of cell migration, differentiation, and tissue integrity and remodeling. These molecules play an important role in inflammation and atherogenesis, but they also participate in the process of invasion and metastasis of malignant cells in the host tissue (2). Invasive tumor cells adhere to the ECM, which provides a matrix environment for permeation of tumor cells through the basal lamina and underlying interstitial stroma of the connective tissue. Overexpression of matrix metalloproteinases (MMPs) and other proteases by tumor cells allows intravasation of tumor cells into the circulatory system after degrading the basement membrane and ECM (3). Several families of proteases are involved in atherogenesis, myocardial infarction, angiogenesis, and tumor invasion and metastasis (4-7).
Urokinase-type plasminogen activator (uPA) is a serine protease (8, 9). The uPA and uPA receptor (uPAR) system is responsible for tissue degradation after plasminogen activation to plasmin, which leads to a cascade of proteolysis or thrombolysis depending on the physiological conditions. uPA also directly activates MMPs, vascular endothelial growth factor, and human growth factor (10). Malignant tumors often express high levels of uPA and uPAR (11); therefore, the uPA/uPAR system is linked to vascular diseases and cancer. A synthetic, protected-graft copolymer (PGC), consisting of poly-L-lysine (PL) with multiple methoxy polyethylene glycol (MPEG) side chains, has been used as a drug carrier with efficient accumulation in tumors (12, 13). The peptide GGSGRSANAKC was found to be an uPA substrate that is cleaved between the Arg (R) and Ser (S) residues. Law et al. (14) used this sequence to attach a Cy5.5 near-infrared (NIR) dye molecule to the unmodified PL side chains of PGC to form Cy5.5-GGSGRSANAK(Fitc)C-PL-MPEG (Cy5.5-uPA-PGC), a fluorescence-quenched polymer. 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 M-1cm-1. The Cy5.5 molecules are in close proximity, which causes fluorescence quenching as a result of efficient fluorescence resonance energy transfer. The NIR fluorescence signal will increase when the Arg-Ser bond is cleaved by uPA, releasing fragments containing Cy5.5. Cy5.5-uPA-PGC is being developed for NIR fluorescence imaging of uPA proteolytic activity in tumors, atherosclerosis, myocardial infarction, and other diseases (15).
[PubMed]
PL-MPEG was formed by reaction of the hydroxysuccinimde ester of MPEG succinate to the NH2 group of PL (35.5 kDa) (16). The purified PL-MPEG had a calculated average molecular mass of 480 kDa as determined with elemental analysis. The PL-MPEG was then iodoacetylated by reaction with excess iodoacetic acid. GGPRQITAGK(Fitc)C was coupled to the iodoacetylated PL-MPEG via a thiol-specific reaction at the C-terminal amino acid (C) (17). The resulting PGC was further modified with Cy5.5-hydroxysuccinimide ester via the free N-terminal amino acid (G) of the attached peptide. Cy5.5-MMP-PGC was purified with column chromatography. Each PL backbone contained an average of 92 MPEG molecules, 17–19 copies of GGPRQITAGK(Fitc)C peptide, and 15–19 Cy5.5 molecules.
[PubMed]
Law et al. (17) showed thatCy5.5-uPA-PGC (200 nM), a uPA substrate, was activated by uPA in a time-dependent manner with a six-fold increase in NIR fluorescence signal after 120 min of incubation. Co-incubation with amileride (uPA inhibitor) markedly reduced NIR fluorescence signal in a dose-dependent manner with a 50% inhibition concentration value of 63 µM. Hsiao et al. (15) showed that MMP-2, MMP-7, MMP-9, cathepsin B, and cathepsin D did not activate the uPA probe.
[PubMed]
Hsiao et al. (15) performed reflectance fluorescence imaging studies of Cy5.5-uPA-PGC in mice bearing HT-1080 (n = 16) or HT-28 (n = 10) tumors. Images were obtained after injection of 10 nmol Cy5.5-uPA-PGC. The initial NIR signal in the HT-1080 tumors was 75 ± 1 arbitrary units (AU) at 0 h and increased to 735 ± 52 AU at 6 h and 1,206 ± 107 AU at 24 h. The NIR fluorescence signal in the HT-28 tumors was 935 ± 106 AU at 6 h and 1,217 ± 87 AU at 24 h. The tumor/muscle ratio for both tumors was 3.1–3.2 at 24 h. Histological immunofluorescence colocalized with immunoreactivity for uPA in the tumor sections. Zymography experiments also showed uPA expression in both tumor extracts. No blocking or uPA inhibition experiments were performed.
P50 CA86355, R01 CA99385
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