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

Abstract

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.

Fig. 1

Schematic diagram of gene construction and identification of fusion proteins. a The genes encoding CNGRC-yCD and yCD were cloned into the pET28a expression vector using a PCR cloning strategy, and digested with the restriction enzymes, Nco I and Xho I. b The respective purified yCD and CNGRC-yCD fusion proteins were identified by Coomassie brilliant blue stained gels (left panel) and Western blot using His₆-tag-specific antibody (right panel). The experiments were completed at least three times with similar results

Fig. 2

Enzyme activities of yCD and CNGRC-yCD proteins to transform 5-FC to 5-FU and the binding affinity of both proteins to the recombinant APN. a The recombinant APN protein was coated on a 96-well plate and then an increasing dose of protein solutions (0.0625, 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0 μM) was added into the wells. The binding affinities of both yCD and CNGRC-yCD proteins to APN were determined by ELISA assay using horseradish peroxidase (HRP)–tagged anti-His₆ antibody. b Increasing concentrations of 5-FC solutions (0.181, 0.363, 0.725, 1.5 and 3.0 mM) were added into a solution containing 50 nM of yCD or CNGRC-yCD fusion protein. The concentrations of 5-FC and 5-FU were determined by measuring the UV absorbance (λ₂₅₅ and λ₂₉₀) at 0.5, 1.0, 2.0 and 3.0 min after mixing 5-FC with the proteins. At least three repeats were performed with similar results

Fig. 3

APN expression levels in various types of cell lines. Tumor cells were grown to 90 % confluent and HUVECs was grown to 50 % and 90 % confluent. Cells were probed with anti-APN antibody (WM15) and then with secondary antibody conjugated with Alexa488. The levels of APN expression for all cell lines were analyzed by flow cytometry. At least three repeats were performed with similar results

Fig. 4

CNGRC-yCD fusion protein has high binding affinity to APN-expressing cells. a-b. HT-1080 a, and HT-29 b. cells were cultured on a 96-well plate. CNGRC-yCD or yCD protein (0.0625, 0.125, 0.25, 0.5, 1.0 and 2.0 μM) was added into the wells to determine its specific binding affinity by ELISA assay using horseradish peroxidase (HRP)–tagged anti-His₆ antibody. c-d. High- c. and low- d. APN-expressing cells were cultured on a 96-well plate. 2.0 μM of yCD or CNGRC-yCD protein was added into the wells and the binding capacities of both proteins were determined by ELISA assay using horseradish peroxidase (HRP)–tagged anti-His₆ antibody. At least three repeats were performed with similar results (*, t test, P < 0.05 relative to the yCD group)

Fig. 5

CNGRC-yCD/5-FC combination treatment significantly reduces cell viability of high-APN-expressing cells. a-b. HT-1080 a. and HT-29 cells b. were treated with 2.0 μM of proteins and with an increasing dose of 5-FC (0.1, 1.0, 10, 100 and 1000 μM). Then, the cell viability was determined by MTT assay. c-d. High-c and low-d APN-expressing cells were treated with 2.0 μM of protein and 60 μM of 5-FC and then the cell viability was determined by MTT assay. Treatments with 5-FC and 5-FU alone were used as negative and positive controls, respectively. At least three repeats were performed with similar results (*, t test, P < 0.05 relative to the 5-FC treatment group; #, t test, P < 0.05 relative to the yCD/5-FC treatment group)