Ga-1,4,7-Triazacyclononane-1,4,7-triacetic acid-c(RGDfK)-human serum albumin-tissue inhibitor of matrix metalloproteinase 2 fusion protein

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 a role in inflammation and atherogenesis, but they also participate in the process of invasion and metastasis of malignant cells in the host tissue (2). 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 MMPs are involved in atherogenesis, myocardial infarction, angiogenesis, and tumor invasion and metastases (4-7). MMP expression is highly regulated in normal cells, such as trophoblasts, osteoclasts, neutrophils, and macrophages. Elevated levels of MMPs have been found in tumors associated with a poor prognosis for cancer patients (8). There are four members of endogenous tissue inhibitors of metalloproteinases (TIMPs), which regulate the activity of MMPs leading to inhibition of tumor growth and metastasis (9, 10). TIMP-2 (TIMP2) is a bifunctional inhibitor of angiogenesis by inhibition of proteinase activity of MMPs and endothelial cell proliferation binding to αβ (the N-terminal domain) and by MMP-independent anti-angiogenic activity (the C-terminal domain) (11, 12). Kang et al. (13) fused the N-terminal domain of TIMP2 to the C-terminus of human serum albumin (HSA) to form HSA/TIMP2 fusion protein (HSA-TIMP2), which is readily secreted by the transfected yeast . HSA-TIMP2 retains its anti-angiogenic activity at the C-terminal domain with little MMP inhibitory activity at the N-terminal domain. Lee et al. (14) have evaluated Cy5.5-HSA/TIMP2 (Cy5.5-HSA-TIMP2) for near-infrared (NIR) fluorescence imaging of rat prostate MLL tumors in nude mice showing maximum tumor accumulation at 2 d after injection. Integrins are a family of heterodimeric glycoproteins on cell surfaces that mediate diverse biological events involving cell–cell and cell–matrix interactions (15). Integrins consist of an α and a β subunit and are important for cell adhesion and signal transduction. The αβ integrin is the most prominent receptor affecting tumor growth, tumor invasiveness, metastasis, tumor-induced angiogenesis, inflammation, osteoporosis, and rheumatoid arthritis (16-21). Expression of the αβ integrin is strong on tumor cells and activated endothelial cells, whereas expression is weak on resting endothelial cells and most normal tissues. 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 αβ. The αβ antagonists are being studied as anti-tumor and anti-angiogenic agents (18, 22, 23). Various radiolabeled RGD peptides (antagonists) have been introduced for imaging of tumors and tumor angiogenesis (24). Choi et al. (25) conjugated multiple c(RGDfK) peptides to HSA-TIMP2 to enhance the binding capacity of the protein (RGD-HSA-TIMP2) to tumors and their vasculatures. Ga-NOTA-RGD-HSA-TIMP2 and Ga-NOTA-HSA-TIMP2 have been studied as potential positron emission tomography (PET) probes for imaging αβ integrin receptors in nude mice bearing human glioblastoma U87MG tumors.