Abstract
The sequence asparagine-glycine arginine (NGR), flanked by Cysteine (Cys) residues so as to form a disulfide-bridge (CNGRC), has previously been found to target and bind specifically to aminopeptidase N (APN), which is highly expressed on the surface of tumor cells. The goal of this study was to develop and evaluate the potential of fusion proteins carrying the CNGRC sequence linked to the enzyme carboxypeptidase G2 (CPG2) for targeted cancer therapy. We refer to this strategy as ligand-directed enzyme prodrug therapy (LDEPT).We constructed two forms of the CNGRC-CPG2 fusions, containing one or two copies of the cyclic NGR motif and designated CNGRC-CPG2 (X-CPG2) and CNGRC-CPG2-CNGRC (X-CPG2-X), respectively. In vitro binding assays of the purified constructs showed that both X-CPG2 and X-CPG2-X bound with high affinity to cancer cells expressing high levels of APN, compared to their binding to cells expressing low levels of APN.Further in vitro studies of the constructs to assess the therapeutic potential of LDEPT were carried out using cells expressing high and low levels of APN. Using methotrexate, it was demonstrated that cancer cell survival was significantly higher in the presence of the fusion proteins, due to the hydrolysis of this cytotoxic drug by CPG2. Conversely, when the prodrug ZD2767P was used, cancer cell killing was higher in the presence of the fused CPG2 constructs than in their absence, which is consistent with CPG2-mediated release of the cytotoxic drug from the prodrug. Furthermore, the doubly-fused CPG2 construct (X-CPG2-X) was significantly more effective than the singly-fused construct (X-CPG2).
Highlights
Metastasis is a key problem preventing cancer treatment, and often results in patient death
We investigated the specific binding of the fusion proteins using cancer cell lines with different levels of aminopeptidase N (APN) expression
The present study investigated the use of peptides, fused with carboxypeptidase G2 (CPG2), that bind to aminopeptidase N (APN), which is often highly expressed on the surfaces of many types of www.oncotarget.com cancer cell
Summary
Metastasis is a key problem preventing cancer treatment, and often results in patient death. In the advanced stages of cancer, tumor cells break the extracellular matrix (ECM) barrier and invade additional tissues in a multistep process [1]. Two classes of enzymes known to be involved in the ECM degradation are aminopeptidase N (APN) and the matrix metalloproteases (MMPs). APN, known as CD13, is a transmembrane receptor with an exopeptidase activity, and is expressed in many tissues and cell types [3]. It plays a crucial role in metastasis by degrading the ECM allowing the www.oncotarget.com escape of tumor cells into the bloodstream [4]. APN promotes angiogenesis and the invasion of neo-endothelial cells through the ECM [5]
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