期刊
JOURNAL OF BIOMEDICAL SCIENCE
卷 23, 期 -, 页码 -出版社
BIOMED CENTRAL LTD
DOI: 10.1186/s12929-016-0227-6
关键词
Targeted cancer therapy; Aminopeptidase N; Asparagine-glycine-arginine motif; Cytosine deaminase; 5-fluorocytosine; 5-fluorouracil
资金
- Ministry of Science and Technology of Taiwan [NSC 101 2627 M 010 001, NSC 101 2627 M 010 004, NSC 102-2627-M-010-001, NSC 102-2627-M-010-002, MOST 103-2627-M-010-001, MOST 103-2627-M-010-002, MOST 104-2627-M-010-001, MOST 104-2627-M-010-002]
- Veterans General Hospitals University System of Taiwan Joint Research Program [VGHUST104-G7-4-1, VGHUST104-G7-4-2, VGHUST104-G7-4-3]
Background: 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. Results: 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. Conclusions: 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.
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