title: Inhibition of tumor growth by polyarginine-fused mutant cytosine deaminase.
abstract: Gene-directed enzyme-prodrug therapy is a method whereby cancerous tumors are selectively eradicated with minimal impact to healthy tissue. Due to its thermostability, E. coli cytosine deaminase (bCD) is one of the most widely used enzyme-prodrug combinations. However, wild-type bCD (wtbCD) displays a relatively poor turnover of 5-fluorocytosine (5-FC), and also has low permeability as a hexamer macromolecule (∼ 300 kDa), like many other therapeutic proteins. To improve these shortcomings, site-specific mutagenesis was performed by infusing the bCD with R9, a typical and highly effective cell-penetrating peptide. The results obtained by flow cytometry and confocal microscopy showed that the R9 efficiently delivered the enhanced green fluorescent proteins (EGFP) into the human liver hepatocellular carcinoma (HepG2) cells, and gathered at the nucleus, while EGFP alone did not have this ability. The penetrating efficiency of R9-EGPF was time and dose dependent. The results obtained by Western blot showed that R9-bCD, but not bCD proteins alone, could be uptaken into HepG2 cells. In vitro experiments showed that polyarginine enhanced the cytotoxicity of bCD, and R9-bCDmut had a stronger cytotoxicity than R9-bCD proteins. In vivo experiments also showed that R9-bCD and R9-bCDmut could prolong the survival time of tumor mice for 8-10 days. Future therapeutic applications of cell-permeable R9-bCDmut fusion proteins together with a systemic administration of 5-FC prodrug could result in profound anti-tumor activities.
authors.affiliation: College of Life Science, Northeast Agricultural University, Harbin, Heilongjiang, People's Republic of China, wangwenfei@neau.edu.cn.
authors.forename: Wenfei
authors.initials: W
authors.lastname: Wang
authors.forename: Nan
authors.initials: N
authors.lastname: Zhang
authors.forename: Tingting
authors.initials: T
authors.lastname: Zhao
authors.forename: Mingyao
authors.initials: M
authors.lastname: Liu
authors.forename: Tong
authors.initials: T
authors.lastname: Zhang
authors.forename: Deshan
authors.initials: D
authors.lastname: Li
mesh_terms: D000818:Animals; D015153:Blotting, Western; D016923:Cell Death / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D043525:Cytosine Deaminase / Q000302:isolation & purification / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D051379:Mice; D050505:Mutant Proteins / Q000494:pharmacology; D009154:Mutation / Q000235:genetics; D009369:Neoplasms / Q000473:pathology*; D010455:Peptides / Q000494:pharmacology*; D010539:Permeability; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism; D012995:Solubility
chemical_list: D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C015462:polyarginine; D043525:Cytosine Deaminase; C468827:codA protein, E coli