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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 xeno-grafts in a nude mouse model using intratumoral herpes simplex virus thymidine (HSV-TK) and endostatin gene monotherapy.
Objectives: Given the high vascularity of human bladder cancer and the ability of HSV-TK or endostatin mono-therapy to eradicate the tumors, we decided to test a no-vel combination of cytotoxic and antiangiogenic gene therapy using HSV-TK and endo-statin 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 combina-tion 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 pri-mer and the fragment between two ITRs of pAAV-TIE measuring about 4 kb, which indi-cated 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 back-ground, which demonstrated that chloroform extraction could effectively extract con-taminants from rAAV stock without significant loss of the rAAV. In vitro, our re-sults 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 signifi-cant antitumor effect in vitro compared to the single gene based therapy in BTCC.
DOI: 10.7754/Clin.Lab.2012.121021
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