Novel therapeutic approach for bladder cancer: Synergistic effects with combined radiation and suicide gene therapy using a chimeric adenovirus vector

Radiation therapy is an established local therapy for muscle invasive transitional cell carcinoma of urinary bladder. Radiation has been shown to improve gene transduction and transgene integration for gene therapy. On the other hand, cancer cells expressing a cytotoxic gene may not repair radiation induced DNA damage. Decrease or loss of CAR (coxsackie-adenoviral receptor) expression is associated with higher grade and stage, metastatic potential and poorer prognosis in bladder cancer. The purpose of this study is to evaluate the efficacy of a novel CAR independent Ad (adenoviral) vector suicide gene therapy in combination with radiation for bladder cancer. Three human bladder cancer cell lines: 5637 (CAR-positive), T24 (CAR deficient) and TCCSUP (CAR deficient) were used. Increasing doses of radiation (2.5, 5, 7.5 and 10 Gy) were delivered to a monolayer of cancer cells. 2.5 Gy was selected for use in combination (radio-gene therapy) treatment. The therapeutic chimeric Ad vector (Ad5/F35) incorporates the Ad35 fiber gene into the original Ad5 backbone and expresses HSV-tk (herpes-simplex virus thymidine kinase gene) driven by the CMV (cytomegalovirus) promoter. Efficacy has been previously established in combination with ganciclovir (GCV) in CAR+ and CAR- bladder cancer cells. The efficacy of combination therapy was evaluated using increasing MOI’s (multiplicity of infections) of Ad5/F35HSV-tk followed by a single dose of radiation (2.5 Gy) that was delivered prior to initiation of GCV (ganciclovir) treatment. The MTT assay was used to assess cell viability. Induction of apoptosis was evaluated by annexin V-FITC. The effect on cell cycle and induction of apoptosis were analyzed using flow cytometry. In all three bladder cancer cell lines, anti-tumor effects were observed in a dose- dependent manner with the Ad5/F35HSV-tk vector. A 10-fold significant increase in anti-tumor effect was demonstrated in combination therapy (radio-gene therapy) when compared to gene therapy alone. Suicide gene therapy as monotherapy as well as in combination with radiation both caused a significant increase in G0/G1 arrest. However, radiation alone demonstrated G2/M arrest. Gene therapy alone demonstrated predominant induction of necrosis. Combination therapy showed higher early induction of apoptosis compared to gene therapy alone. The cell killing effect of this combination strategy, was similar in CAR-positive and -deficient bladder cancer cells. We have demonstrated that the combination radiation and suicide gene therapy elicits significantly greater anti-tumor effect compared to suicide gene therapy alone in both CAR-positive and -deficient bladder cancer cells. Combination radio-gene therapy leads to G0/G1 arrest in cell cycle and predominantly early induction of apoptosis. We are currently evaluating this novel treatment strategy in animal models in order to demonstrate a rationale for enhanced local-regional control in the bladder-sparing therapeutic approach for muscle invasive bladder cancer