In vitro efficacy of Fas ligand gene therapy for the treatment of bladder cancer

Abstract

Previous investigations have revealed that bladder cancer cells are generally resistant to Fas-mediated apoptosis by conventional Fas agonists. However, the ability of these cell lines to undergo Fas-mediated apoptosis may have been underappreciated. As a result, we investigated the in vitro efficacy of Fas ligand gene therapy for bladder cancer. Three human bladder cancer lines (T24, J82, and 5637) were treated with the conventional Fas agonist CH-11, a monoclonal antibody to the Fas receptor. Cells were also treated with a replication-deficient adenovirus containing a modified murine Fas ligand gene fused to green fluorescent protein (GFP), AdGFPFasL. A virus containing the GFP gene alone was used to control for viral toxicity (AdGFP). Cell death was quantified using a tetrazolium-based (MTS) assay. Cells were also evaluated by Western blotting to evaluate poly (ADP-ribose) polymerase, caspase 8, and caspase 9 cleavage and by flow cytometry to determine the presence of coxsackie/adenovirus receptor (CAR). These studies confirmed bladder cancer resistance to cell death by the anti-Fas monoclonal antibody CH-11. This resistance was overcome with AdGFPFasL at a multiplicity of infection (MOI) of 1000 achieving over 80% cell death in all cell lines. Furthermore, greater than 80% cell death was evident in 5637 cells treated with low-dose AdGFPFasL (MOI ¼ 10). 5637 cells expressed significantly higher levels of surface CAR than J82 or T24 cells (Po.05). AdGFPFasL is cytotoxic to bladder cancer cells that would otherwise be considered Fas resistant, supporting its in vivo potential. Enhanced sensitivity to AdGFPFasL may be in part due to increased cell surface CAR levels. Cancer Gene Therapy ( Keywords: apoptosis B ladder cancer is the second most-common cancer of the genitourinary tract. Currently, there are a wide range of therapeutic modalities available based on the extent of the disease including intravesical chemotherapy, surgery, radiation therapy, and systemic chemotherapy. Unfortunately, patients with advanced bladder cancer face a 5-year survival rate of approximately 20-40% despite the wide range of treatment modalities.

Combined therapeutic use of AdGFPFasL and small molecule inhibitors of ceramide metabolism in prostate and head and neck cancers: a status report

“…Our initial approach was to develop tissue specificity of transgene expression, by using tissue or tumor-restricted promoters to regulate a TET-derived transcription factor (tTA) which drives transcription of the selected transgene (FasL, Apoptin, Bax or TRAIL) [1][2][3][4][5][6][7][8][9] (See Figure 1a). We successfully carried out such studies with adenoviral vectors and demonstrated restricted gene expression.…”

FasL gene therapy: a new therapeutic modality for head and neck cancer

“…Induction of apoptosis by AdGFPFasL has also been established in other CH-11 cancer resistant cells including prostate and bladder lines. 12,17 AdGFPFasL leads to significant tumor regression in vivo Although the SCC-14a cell line appeared to be quite sensitive to apoptotic induction by AdGFPFasL, it was not clear if the cells would behave similarly in an in vivo environment. To address this, an experiment was initiated to compare the growth of SCC-14a cells with and without AdGFPFasL treatment over a period of 3 months.…”

Role for Hedgehog signaling in hepatic stellate cell activation and viability

“…Apoptotic activity was assayed in parallel cultures using the Apo-ONE Homogeneous Caspase 3/7 Apoptosis Assay (Promega, Madison, WI, USA) according to the manufacturer's instructions. 38,39 Hh-Responsive Luciferase Reporter Assay…”