title: Characterisation of enzyme prodrug gene therapy combinations in coated spheroids and vascular networks in vitro.
abstract: BACKGROUND
  Enzyme prodrug gene therapy is designed as a targeted cancer treatment, destroying gene-modified and bystander cells via exogenous enzyme-generated cytotoxins. Targeting of tumour blood vessels using gene therapy is attractive, although optimal enzyme prodrug combinations have yet to be identified.
  
  
  METHODS
  Seven enzyme prodrug combinations were ranked in two endothelial (HUVEC, HMEC-1) and one tumour cell line (T24) for their ability to reduce proliferation and viability. The ability to destroy bystander cells in two dimensions (2D) and three dimensions (3D), mode of cell kill, and the ability to disrupt vascular networks were measured.
  
  
  RESULTS
  Endothelial cell proliferation (bromodeoxyuridine uptake) was reduced most effectively by Herpes simplex virus thymidine kinase (TK) with ganciclovir (GCV), followed by Escherichia coli nitroreductase NfsB (NTR) with CB1954; viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay] was reduced most efficiently by NTR/CB1954 followed by TK/GCV. Of the seven combinations, only NTR/CB1954 displayed measurable bystander effects in 2D monolayers, and none demonstrated bystander killing in coated spheroids, a 3D spatially distinct model with tissue-like cell density. NTR-expressing endothelial cells displayed increased apoptosis, necrosis and caspase-3 activity after CB1954 treatment. Despite good antiproliferative activity, TK/GCV was ineffective at disrupting vascular network-like structures of endothelial cells, whereas NTR/CB1954 was efficient. NTR/metronidazole and the vascular disrupting agent, combretastatin A-4 phosphate, were the only other effective agents.
  
  
  CONCLUSIONS
  Collectively, these data demonstrate that cytotoxic rather than cytostatic activity is necessary for efficient vascular disruption in vitro, and bystander killing is not essential. We identify NTR/CB1954 and NTR/metronidazole as candidates for in vivo investigation of vascular-targeted gene therapy.
authors.affiliation: Angiogenesis and Cancer Research Group, Department of Pathology, University of Otago, Christchurch, New Zealand.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Hunt
authors.forename: Dan
authors.initials: D
authors.lastname: Li
authors.forename: Michael P
authors.initials: MP
authors.lastname: Hay
authors.forename: Margaret J
authors.initials: MJ
authors.lastname: Currie
authors.forename: Bridget A
authors.initials: BA
authors.lastname: Robinson
authors.forename: Adam V
authors.initials: AV
authors.lastname: Patterson
authors.forename: Gabi U
authors.initials: GU
authors.lastname: Dachs
mesh_terms: D001808:Blood Vessels / Q000166:cytology* / Q000187:drug effects*; D024201:Bystander Effect / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002999:Clone Cells; D003131:Combined Modality Therapy; D004798:Enzymes / Q000494:pharmacology*; D015316:Genetic Therapy / Q000379:methods*; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000187:drug effects; D006801:Humans; D011355:Prodrugs / Q000494:pharmacology*; D018874:Spheroids, Cellular / Q000166:cytology* / Q000187:drug effects*; D014162:Transfection
chemical_list: D004798:Enzymes; D011355:Prodrugs