Targeted antitumor prodrug therapy using CNGRC-yCD fusion protein in combination with 5-fluorocytosine

• Published in: Journal of biomedical science Vol. 23; no. 14; p. 15
• Main Authors: Li, Jia-Je, Chang, Shun-Fu, Liau, I-Iu, Chan, Pei-Chia, Liu, Ren-Shyan, Yen, Sang-Hue, Wang, Hsin-Ell, Chang, Cheng Allen
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 22.01.2016; BioMed Central
• Subjects: Angiogenesis; Antineoplastic Agents - pharmacokinetics; Antineoplastic Agents - pharmacology; Cell Line, Tumor; Chemotherapy; Cytosine Deaminase - genetics; Cytosine Deaminase - pharmacology; Drug Delivery Systems - methods; Endothelium; Enzymes; Flucytosine - pharmacokinetics; Flucytosine - pharmacology; Gene therapy; Glycine; Human Umbilical Vein Endothelial Cells; Humans; Neoplasms - drug therapy; Neoplasms - metabolism; Neoplasms - pathology; Oligopeptides - genetics; Oligopeptides - pharmacology; Prodrugs - pharmacokinetics; Prodrugs - pharmacology; Protein binding; Proteins; Pyrimidines; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - pharmacology; Recombinant proteins; Registered nurses; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - pharmacology; Tumors
• ISSN: 1423-0127; 1021-7770; 1423-0127
• DOI: 10.1186/s12929-016-0227-6

The enzyme-prodrug system is considered a promising tool for tumor treatment when conjugated with a targeting molecule. The asparagine-glycine-arginine (NGR) motif is a developing and interesting targeting peptide that could specifically bind to aminopeptidase N (APN), which is an NGR receptor expressed on the cell membrane of angiogenic endothelial cells and a number of tumor cells within the tumor tissues. The objective of this study was to develop a novel targeted enzyme-prodrug system using 5-fluorocytosine (5-FC) and an NGR-containing peptide fused with yeast cytosine deaminase (yCD), i.e. CNGRC-yCD fusion protein, to target APN-expressing cells within the tumor tissues and to convert 5-FC into 5-fluorouracil (5-FU) to kill tumors. Both yCD and CNGRC-yCD proteins were cloned into the pET28a vector and expressed by an Escherichia coli host. Both yCD and CNGRC-yCD proteins were purified and the yields were approximately 20 mg/L with over 95 % purity. The binding assay demonstrated that the CNGRC-yCD fusion protein had specific binding affinity toward purified APN recombinant protein and high-APN-expressing cells, including human endothelial cells (HUVECs) and various types of human tumor cell lines, but not low-APN-expressing tumor cell lines. Moreover, the enzyme activity and cell viability assay showed that the CNGRC-yCD fusion protein could effectively convert 5-FC into 5-FU and resulted in significant cell death in both high-APN-expressing tumor cells and HUVECs. This study successfully constructs a new targeting enzyme-prodrug system, CNGRC-yCD fusion protein/5-FC. Systematic experiments demonstrated that the CNGRC-yCD protein retained both the APN-binding affinity of NGR and the enzyme activity of yCD to convert 5-FC into 5-FU. The combined treatment of the CNGRC-yCD protein with 5-FC resulted in the significantly increased cell death of high-APN-expressing cells as compared to that of low-APN-expressing cells.