title: [Effects of double targeting gene therapy by suicide gene combined with endostatin gene in treatment of bladder cancer: an experimental study].
abstract: OBJECTIVE
  To investigate the effect of double targeting gene therapy by using recombinant adeno-associated virus-thymidine kinase (TK)-internal ribosome entry site (IRES)-endostatin (ES) (rAAV-TIE).
  
  
  METHODS
  Bladder cancer cells of the line T24 were cultured and transfected with rAAV-ES, rAAV-MCS (blank virus), and rAAV-TIE respectively. 72 hours later the levels of ES in the supernatants were measured by ELISA and annexin V apoptosis test kit was used to examine the apoptosis. Human umbilical vein endothelial cells (HUVECs) were transfected with rAAV-ES, rAAV-TIE, and rAAV-MCS respectively. MTT method and flow cytometry were used to detect the apoptosis of the HUVECs. Balb/c nude rats were inoculated subcutaneously with T24 cells. Twenty rats with tumor were randomly divided into 4 equal groups to be treated by rAAV-MCS, rAAV-TK, rAAV-ES, or rAAV-TIE, and 5 rats were used as control group. Four weeks later, blood samples were collected to detect the ES level by ELISA. The tumors were taken out to undergo microscopy to calculate the microvessel density(MVD).
  
  
  RESULTS
  72 h after transfection, ES could be detected in the supernatants of the T24 cells transfected with rAAV-ES, and rAAV-TIE. The apoptotic rates of the T24 cells transfected with rAAV-TK and rAAV-TIE were 34.12% and 36.91% respectively, significantly higher than those of the T24 cells transfected with rAAV-MCS and of the control group (3.08% and 0.84%, all P < 0.05). Transfection of rAAV-ES and rAAV-TIE increased the apoptotic rate of the HUVECs time-dependently. Nine days after the transfection rAAV-ES, rAAV-TK, rAAV-TIE, the tumor volumes of the rAAV-ES, rAAV-TK, and rAAV-TIE groups were (0.75 +/- 0.08), (0.71 +/- 0.11), and (0.52 +/- 0.09) cm(3) respectively, all significantly lower than those of the rAAV-MCS group and control group [(1.27 +/- 0.13) and (1.24 +/- 0.17) cm(3) respectively, all P < 0.05].
  
  
  CONCLUSIONS
  rAAV-TIE effectively inhibits the tumorigenesis and angiogenesis in bladder cancer. Double targeting gene therapy against bladder cancer can be achieved by using rAAV.
authors.affiliation: Department of Urology, the Second Affiliated Hospital of Guang Zhou Medical College, Guangzhou 510260, China.
authors.forename: Jian-gang
authors.initials: JG
authors.lastname: Pan
authors.forename: Xing
authors.initials: X
authors.lastname: Zhou
authors.forename: Rui-fa
authors.initials: RF
authors.lastname: Han
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D000229:Dependovirus / Q000235:genetics*; D043169:Endostatins / Q000235:genetics*; D018014:Gene Transfer Techniques; D041722:Genes, Transgenic, Suicide / Q000235:genetics*; D015316:Genetic Therapy; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D009389:Neovascularization, Pathologic; D014162:Transfection; D001749:Urinary Bladder Neoplasms / Q000473:pathology / Q000628:therapy*
chemical_list: D043169:Endostatins