Suppression of bladder cancer growth by adeno-associated virus vector-mediated combination of HSV-TK and endostatin in vitro.

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. OBJECTIVES: 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.

Potent antitumor activity of the combination of HSV-TK and endostatin by adeno-associated virus vector for bladder cancer in vivo.

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. METHODS: 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 intratumorally delivered HSV-TK and endostatin adeno-associated viruses (AAV). We constructed plasmid AAV-TK-IRES-Endostatin (pAAV-TIE) and packaged the AAV particles containing gene fragments of HSV-TK and endostatin. The combined anticancer effect of recombinant AAV-TIE (rAAV-TIE) was measured in vivo with rAAV-HSV-TK and rAAV-Endostatin as the control groups. RESULTS: The inverted terminal repeat sequence was 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 vivo, our results showed that the tumors in mice injected with the rAAV-TIE not only took significantly longer to emerge but also that their growth, once established, was significant slower than that of tumors grown with single HSV-TK or endostatin treated animals. 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 compared to the single gene based therapy in BTCC.

Potent antitumour activity of the combination of HSV-TK and endostatin armed oncolytic adeno-associated virus for bladder cancer in vitro and in vivo.

BACKGROUND: Gene therapy may offer a new tool for the treatment of bladder cancer. Previously, we have shown a significant antitumour effect in bladder cancer xenografts in a nude mouse model using intratumoural herpes simple virus thymidine (HSV-TK) and Endostatin gene therapy. OBJECTIVES: Given the high vascularity of human bladder cancer and the ability of HSV-TK or Endostatin monotherapy to eradicate the tumours, we decided to test a novel combination of cytotoxicity and antiangiogenisis gene therapy using intratumourally delivered HSV-TK and Endostatin adeno-associated viruses (AAVs). METHODS: We constructed plasmid AAV-TK-IRES-Endostatin (pAAV-TIE) and packaged the AAV particles contain gene fragments of HSV-TK and Endostatin. The function of HSV-TK and Endostatin was evaluated separately in vitro via T24 bladder tumour cells and human umbilical vein endothelial (HUVEC) cells. The combination anticancer effect of recombinant AAV-TIE (rAAV-TIE) was measured in vivo while rAAV-HSV-TK and rAAV-Endostatin as control groups. RESULTS: In vitro, rAAV-TIE was found to induce a significant increase in apoptosis in HUVEC cells equally as rAAV-Endostatin and confirmed that the inhibition of endothelial cells mediated by rAAV-TIE was associated with the apoptotic process. rAAV-TIE was found to induce a significant increase in apoptosis in T24 cells equally as rAAV-HSV-TK and confirmed that the inhibition of T24 cells mediated by rAAV-TIE was associated with the apoptotic process too. In vivo, our results showed that the tumours in mice injected with the rAAV-TIE not only took significantly longer to emerge but also that their growth, once established, was significant slower than that of tumours grown, compared with single HSV-TK or Endostatin treated animals. CONCLUSIONS: We concluded that the inhibition of angiogenesis using Endostatin gene transfer, together with the cytotoxicity HSV-TK gene therapy, resulted in a significant antitumour effect compared to the single gene based therapy in BTCC. The results warrant further development of the combination gene therapy, and suggest that this approach, directed towards systemic efficiency, could be used as an additional treatment for human BTCC.

Suppression of bladder cancer growth in mice by adeno-associated virus vector-mediated endostatin expression.

Novel treatment strategies such as gene therapy are warranted in view of the failure of current treatment approaches to cure a high percentage of patients with advanced bladder cancers. Testing of the hypothesis that blocking the angiogenic switch may keep tumour growth in check has been facilitated by the discovery of endogenous inhibitors of angiogenesis and has also added another research dimension to the field of cancer gene therapy. Consequently, the concept of targeting the tumour vasculature with anti-angiogenic agents has emerged as an attractive new strategy in the treatment of cancer. Targeted biological therapies that selectively interfere with tumour angiogenesis could improve survival among patients with bladder cancer. Endostatin is a tumour-derived angiogenesis inhibitor and is the first endogenous inhibitor of angiogenesis to be indentified in a matrix protein. Gene therapy represents an attractive approach to treat cancers and other chronic diseases. The development of an effective delivery system is absolutely critical to the usefulness and safety of gene therapy. At present, the adeno-associated virus (AAV) vector has the most promising potential in view of its non-pathogenicity, wide tropisms and long-term transgene expression in vivo. Gene therapy studies using different serotypes of recombinant AAV (rAAV) as delivery vehicles have proved rAAVs to be an effective modality of cancer gene therapy. In the present study, an IgG fragment was inserted at the start of the sequence coding for endostatin with the aim of enabling continuous secretion of endostatin the serum. We also investigated the suppression effect of AAV-mediated endostatin expression on endothelial cells and in mice xenograft models of bladder cancer. Our data demonstrates that rAAV-endostatin controlled tumour cell growth and achieves strong anti-tumour efficacy in vivo.