title: Suppression of bladder cancer growth by adeno-associated virus vector-mediated combination of HSV-TK and endostatin in vitro.
abstract: BACKGROUND
  Gene therapy may offer a new tool for the treatment of bladder cancer. Previously, we have shown a significant antitumor effect in bladder cancer xenografts in a nude mouse model using intratumoral herpes simplex virus thymidine (HSV-TK) and endostatin gene monotherapy.
  
  
  OBJECTIVE
  Given the high vascularity of human bladder cancer and the ability of HSV-TK or endostatin monotherapy to eradicate the tumors, we decided to test a novel combination of cytotoxic and antiangiogenic gene therapy using HSV-TK and endostatin adeno-associated viruses (AAV) in vitro.
  
  
  METHODS
  We constructed the plasmid AAV-TK-IRES-Endostatin (pAAV-TIE) and packaged the AAV particles containing gene fragments of HSV-TK and endostatin. The combination anticancer effect of recombinant AAV-TIE (rAAV-TIE) was measured in vitro while rAAV-HSV-TK and rAAV-Endostatin were used as control groups.
  
  
  RESULTS
  The inverted terminal repeat sequences were amplified using only one primer and the fragment between two ITRs of pAAV-TIE measuring about 4 kb, which indicated a stable sequence of pAAV-TIE. Three clear bands representing the AAV capsid proteins VP1, VP2, and VP3 could be seen on both lanes against a very low background, which demonstrated that chloroform extraction could effectively extract contaminants from rAAV stock without significant loss of the rAAV. In vitro, our results found that the transduction efficiency, measured from GFP-transduced tumors, was about 62%. The combination therapy led to an obvious apoptosis of bladder tumor cells compared with single HSV-TK or endostatin treatment.
  
  
  CONCLUSIONS
  We concluded that the inhibition of angiogenesis using endostatin gene transfer, together with the cytotoxic HSV-TK gene therapy, resulted in a significant antitumor effect in vitro compared to the single gene based therapy in BTCC.
authors.affiliation: The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, China.
authors.forename: Jian Gang
authors.initials: JG
authors.lastname: Pan
authors.forename: Run Qi
authors.initials: RQ
authors.lastname: Luo
authors.forename: Xing
authors.initials: X
authors.lastname: Zhou
authors.forename: Rui Fa
authors.initials: RF
authors.lastname: Han
authors.forename: Ge Wa
authors.initials: GW
authors.lastname: Zeng
mesh_terms: D000818:Animals; D002460:Cell Line; D000229:Dependovirus / Q000235:genetics*; D043169:Endostatins / Q000008:administration & dosage / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D015552:Injections, Intralesional; D051379:Mice; D008819:Mice, Nude; D018139:Simplexvirus / Q000201:enzymology*; D013937:Thymidine Kinase / Q000008:administration & dosage / Q000235:genetics*; D001749:Urinary Bladder Neoplasms / Q000473:pathology / Q000628:therapy*; D023041:Xenograft Model Antitumor Assays
chemical_list: D043169:Endostatins; D013937:Thymidine Kinase