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Targeted antitumor prodrug therapy using CNGRC-yCD fusion protein in combination with 5-fluorocytosine
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 re...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4724154/ https://www.ncbi.nlm.nih.gov/pubmed/26801910 http://dx.doi.org/10.1186/s12929-016-0227-6 |
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author | Li, Jia-Je Chang, Shun-Fu Liau, I-Iu Chan, Pei-Chia Liu, Ren-Shyan Yen, Sang-Hue Wang, Hsin-Ell Chang, Cheng Allen |
author_facet | Li, Jia-Je Chang, Shun-Fu Liau, I-Iu Chan, Pei-Chia Liu, Ren-Shyan Yen, Sang-Hue Wang, Hsin-Ell Chang, Cheng Allen |
author_sort | Li, Jia-Je |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-4724154 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-47241542016-01-24 Targeted antitumor prodrug therapy using CNGRC-yCD fusion protein in combination with 5-fluorocytosine Li, Jia-Je Chang, Shun-Fu Liau, I-Iu Chan, Pei-Chia Liu, Ren-Shyan Yen, Sang-Hue Wang, Hsin-Ell Chang, Cheng Allen J Biomed Sci Research 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. BioMed Central 2016-01-22 /pmc/articles/PMC4724154/ /pubmed/26801910 http://dx.doi.org/10.1186/s12929-016-0227-6 Text en © Li et al. 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Li, Jia-Je Chang, Shun-Fu Liau, I-Iu Chan, Pei-Chia Liu, Ren-Shyan Yen, Sang-Hue Wang, Hsin-Ell Chang, Cheng Allen Targeted antitumor prodrug therapy using CNGRC-yCD fusion protein in combination with 5-fluorocytosine |
title | Targeted antitumor prodrug therapy using CNGRC-yCD fusion protein in combination with 5-fluorocytosine |
title_full | Targeted antitumor prodrug therapy using CNGRC-yCD fusion protein in combination with 5-fluorocytosine |
title_fullStr | Targeted antitumor prodrug therapy using CNGRC-yCD fusion protein in combination with 5-fluorocytosine |
title_full_unstemmed | Targeted antitumor prodrug therapy using CNGRC-yCD fusion protein in combination with 5-fluorocytosine |
title_short | Targeted antitumor prodrug therapy using CNGRC-yCD fusion protein in combination with 5-fluorocytosine |
title_sort | targeted antitumor prodrug therapy using cngrc-ycd fusion protein in combination with 5-fluorocytosine |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4724154/ https://www.ncbi.nlm.nih.gov/pubmed/26801910 http://dx.doi.org/10.1186/s12929-016-0227-6 |
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