title: 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.
authors.affiliation: Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, No. 155, Sec. 2, Linong St., Beitou District, Taipei, 112, Taiwan, ROC. uvpxyz@gmail.com.|Program in Molecular Medicine, National Yang-Ming University and Academia Sinica, Taipei, 112, Taiwan, ROC. uvpxyz@gmail.com.
authors.forename: Jia-Je
authors.initials: JJ
authors.lastname: Li
authors.affiliation: Department of Medical Research and Development, Chang Gung Memorial Hospital-Chiayi Branch, Chiayi, 613, Taiwan, ROC. sfchang@cgmh.org.tw.
authors.forename: Shun-Fu
authors.initials: SF
authors.lastname: Chang
authors.affiliation: Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, No. 155, Sec. 2, Linong St., Beitou District, Taipei, 112, Taiwan, ROC. everleycat@hotmail.com.
authors.forename: I-Iu
authors.initials: II
authors.lastname: Liau
authors.affiliation: Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, No. 155, Sec. 2, Linong St., Beitou District, Taipei, 112, Taiwan, ROC. rene1122@gmail.com.
authors.forename: Pei-Chia
authors.initials: PC
authors.lastname: Chan
authors.affiliation: Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, No. 155, Sec. 2, Linong St., Beitou District, Taipei, 112, Taiwan, ROC. rsliu@vghtpe.gov.tw.|Department of Nuclear Medicine and National PET/Cyclotron Center, Taipei Veterans General Hospital, Taipei, 112, Taiwan, ROC. rsliu@vghtpe.gov.tw.|Biophotonics & Molecular Imaging Research Center, National Yang-Ming University, Taipei, 112, Taiwan, ROC. rsliu@vghtpe.gov.tw.
authors.forename: Ren-Shyan
authors.initials: RS
authors.lastname: Liu
authors.affiliation: Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, No. 155, Sec. 2, Linong St., Beitou District, Taipei, 112, Taiwan, ROC. shyen@vghtpe.gov.tw.|Department of Oncology Medicine, Taipei Veterans General Hospital, Taipei, 112, Taiwan, ROC. shyen@vghtpe.gov.tw.
authors.forename: Sang-Hue
authors.initials: SH
authors.lastname: Yen
authors.affiliation: Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, No. 155, Sec. 2, Linong St., Beitou District, Taipei, 112, Taiwan, ROC. hewang@ym.edu.tw.|Program in Molecular Medicine, National Yang-Ming University and Academia Sinica, Taipei, 112, Taiwan, ROC. hewang@ym.edu.tw.|Biophotonics & Molecular Imaging Research Center, National Yang-Ming University, Taipei, 112, Taiwan, ROC. hewang@ym.edu.tw.
authors.forename: Hsin-Ell
authors.initials: HE
authors.lastname: Wang
authors.affiliation: Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, No. 155, Sec. 2, Linong St., Beitou District, Taipei, 112, Taiwan, ROC. cachang@ym.edu.tw.|Biophotonics & Molecular Imaging Research Center, National Yang-Ming University, Taipei, 112, Taiwan, ROC. cachang@ym.edu.tw.|Biomedical Engineering Research and Development Center, National Yang-Ming University, Taipei, 112, Taiwan, ROC. cachang@ym.edu.tw.
authors.forename: Cheng Allen
authors.initials: CA
authors.lastname: Chang
mesh_terms: D000970:Antineoplastic Agents / Q000493:pharmacokinetics / Q000494:pharmacology; D045744:Cell Line, Tumor; D043525:Cytosine Deaminase / Q000235:genetics / Q000494:pharmacology*; D016503:Drug Delivery Systems / Q000379:methods*; D005437:Flucytosine / Q000493:pharmacokinetics / Q000494:pharmacology; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D009369:Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D009842:Oligopeptides / Q000235:genetics / Q000494:pharmacology*; D011355:Prodrugs / Q000493:pharmacokinetics / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; C526428:NGR peptide; D009842:Oligopeptides; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D005437:Flucytosine; D043525:Cytosine Deaminase