Objective: To package recombinant adeno-associated virus-endostatin (rAAV-ES) and study its anti-tumor effect in vitro and in vivo.
Methods: rAAV-ES was packaged with co-transfection technique and transfected into the human bladder cancer cells of the line EJ. 24 h later ELISA was used to examine the concentration of ES in the supernatant. The inhibition of human umbilical veins endothelial cells (HUVECs) chemotactic movement were examined by Transwell system. Nude Balb/c mice were divided into 4 groups: (1) 5 mice were inoculated with the EJ cells transfected with rAAV-ES or rAAV-enhanced yellow fluorescence protein (rAAV-EYFP) for 3 days to the subcutaneous tissues of bilateral shoulders so as to observe the growth of tumor. (2) 24 mice were injected with rAAV-ES intramuscularly and then the serum ES was examined every 10 days since the 10 th day after the injection. (3) 36 mice were randomly subdivided into 3 equal subgroups to be injected with rAAV-ES, rAAV-EYFP, or RPMI medium, inoculated with EJ cells 2 weeks later, and then killed 50 days later to observe the size of tumor. (4) 4 healthy mice and 4 mice injected with rAAV-ES for 8 weeks were killed with their hearts and brains taken out to observe the side effects.
Results: rAAV-ES was packaged successfully. The ES concentration in the supernatant of culture fluid of the EJ cell transfected with rAAV-ES was 54.09 ng/ml. The inhibition rate of the HUVECs chemotactic movement was 37.45%. The xenograft formation rate was 2/5 for the EJ cells transfected with rAAV-ES. The serum ES levels of the mice injected with rAAV-ES remained high. The tumor size in the mice injected with rAAV-ES was significantly smaller than those of the other groups (both P < 0.01). No pathological changes was found in the hearts and brains in the mice injected with rAAV-ES.
Conclusion: rAAV-ES inhibits tumor angiogenesis, and tumor formation and progression. Successful packaging of rAAV-ES has laid a foundation for gene therapy of bladder cancer.