Jian-Yun Dong

Downregulation of c-FLIP Sensitizes DU145 Prostate Cancer Cells to Fas-Mediated Apoptosis

Although DU145 prostate cancer cells are resistant to exogenously applied Fas agonist CH-11 (anti-Fas monoclonal antibody), Fas-resistance can be overcome using a FasL expressing adenovirus (AdGFPFasLTET) (Hyer et al., Mol. Therapy, 2: 348-58, 2000). The purpose of this study was to try to understand why DU145 cells are resistant to CH-11 and determine the signaling pathway utilized by AdGFPFasLTET to induce apoptosis in these Fas-resistant cells. Using immunoblot analysis, we show that AdGFPFasLTET is capable of initiating the classic Fas-mediated apoptotic pathway in DU145 cells, which includes activation of caspases-8, -3, -7, and -9, BID cleavage, cytochrome c release from mitochondria, and PARP cleavage. In contrast, CH-11 binds to Fas, but is unable to transmit the death signal beyond the plasma membrane suggesting a block at the DISC (death inducing signaling complex). The anti-apoptotic protein c-FLIP (cellular Flice-like inhibitory protein), which has been shown to inhibit Fas-mediated apoptosis at the DISC, was down-regulated following AdGFPFasLTET treatment prompting us to investigate its role in inhibiting CH-11-induced cell death. Using c-FLIP anti-sense oligonucleotides to down-regulate c-FLIP we sensitized DU145 cells to CH-11-induced apoptosis. These data suggest that c-FLIP may play a critical role in regulating Fas-mediated apoptosis in prostate cancer cells and that modulation of c-FLIP may enhance Fas signaling based therapies.

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

As of January 2005, there were 1020 gene therapy clinical trials ongoing worldwide with 675 or 66.2% devoted to cancer gene therapy. The majority are occurring in the US and Europe (http://www.wiley.co.uk/genetherapy/clinical/). At the present time, to our knowledge there are no trials that employ gene delivery of Fas Ligand (FasL). As an important note, and in contrast to somatic cell therapy trials, there are no reported deaths due to therapeutic vector administration in any cancer gene therapy trial. That said, from our studies and from the published literature, the issue of gene delivery remains the major obstacle to successfully employing gene therapy for cancer treatment. Numerous laboratories are studying this with many different approaches. My co-workers and I have focused on the delivery issue by using various approaches that address tumor targeting and transgene expression. In addition, we are focusing on enhancing tumor cell killing via the bystander effect and through use of small molecules to enhance bystander activity.

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

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=10). 5637 cells expressed significantly higher levels of surface CAR than J82 or T24 cells (P<.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.

Quantification and characterization of the bystander effect in prostate cancer cells following adenovirus-mediated FasL expression

Inducing Fas-mediated apoptosis in prostate cancer (PCa) is a promising new therapeutic approach with the potential to overcome delivery issues currently problematic in cancer gene therapy. We have previously demonstrated that a Fas Ligand (FasL) expressing adenovirus (AdGFPFasLTET) was able to induce Fas-mediated apoptosis in a panel of PCa cell lines regardless of their Fas-sensitivity as determined by the agonistic Fas antibody CH-11. We now report that AdGFPFasLTET-infected cells produce apoptotic bodies and cellular debris that continues to elicit FasL-mediated bystander killing in uninfected neighboring cells. Using light microscopy, we demonstrate that AdGFPFasLTET-infected cells release apoptotic bodies and cellular debris into the local environment and that this material will induce bystander killing in Jurkat, PPC-1, and PC-3 target cells, but not in DU145 and K-562 cells. The bystander killing mechanism is mediated through Fas/FasL interaction because it is significantly inhibited if target cells are pretreated with the pan spectrum caspase inhibitor Z-VAD-FMK or the Fas neutralizing antibody ZB-4. Coincubation of PPC-1 target cells with apoptotic bodies and cellular debris (effector material) induce nearly complete target cell killing at a ratio of 1:1 target to effector. Collectively, these data indicate that AdGFPFasLTET-infected PCa cells release apoptotic and cellular debris capable of inducing bystander killing in PCa and supports the development of FasL as a gene therapy agent.

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

In this study, we investigated the in vitro and in vivo efficacy of Fas ligand (FasL) gene therapy for the treatment of head and neck cancer. Three head and neck squamous cell carcinoma (HNSCC) cell lines (SCC-1, SCC-12, and SCC-14a) were treated with the Fas agonist CH-11, a monoclonal antibody to the Fas receptor, or with a replication-incompetent adenovirus (AdGFPFasL) expressing a modified murine Fas ligand gene fused to green fluorescent protein (GFP). A replication-incompetent adenovirus containing the GFP gene alone was used as a control for viral transduction toxicity (AdGFP). Cell death was quantified using a tetrazolium-based (MTS) assay. Cells were analyzed by flow cytometry to determine the expression of adenoviral and Fas receptors on the surface of the cells. Our results showed that the head and neck cancer cell lines are resistant to cell death induction when treated with the anti-Fas monoclonal antibody CH-11. This resistance can be overcome with AdGFPFasL, which was able to induce cell death in all three cell lines. Apoptosis induction was demonstrated using Western blotting by evaluating poly(ADP-ribose) polymerase, and caspase 9 cleavages. In addition, intratumoral injections of AdGFPFasL into SCC-14a xenografts induced significant growth suppression of tumors, indicating that FasL gene therapy may provide a new efficient therapeutic modality for HNSCC that is worthy of a clinical trial.

Enhanced apoptosis of glioma cell lines is achieved by co-delivering FasL-GFP and TRAIL with a complex Ad5 vector

Brain tumors (BTs) are among the most malignant forms of human cancer. Unfortunately, current treatments are often ineffective and produce severe side effects. Cytotoxic gene therapy is an alternative treatment strategy, with the potential advantages of reduced toxicity to normal brain tissue. Apoptosis-inducing “death ligands” Fas ligand and TNF-related apoptosis-inducing ligand (TRAIL) are genes with substantial cytotoxic activity in susceptible tumor cells. Here, we compared the effectiveness of Ad vector-mediated delivery of Fas ligand-green fluorescent protein (FasL-GFP) fusion protein, human TRAIL, and both genes simultaneously. We examined a panel of 13 cell lines (eight derived from primary isolates) for susceptibility to Ad5-based vector infection and for sensitivity to FasL- and TRAIL-mediated apoptosis. All cell lines were efficiently transduced, but, as expected, varied in their sensitivity to ligand-induced apoptosis. Generally, sensitivity to FasL-GFP correlated with cell surface FasR levels, but no such correlation was seen for TRAIL and its functional receptors, DR4 and DR5. The vector expressing both FasL-GFP and TRAIL was more effective than either of the single-gene vectors at comparable transduction levels, and it was effective against a broader range of cell lines. In five cell lines, coexpression resulted in apoptosis levels greater than those predicted for strictly additive activity of the two death ligands. We believe that Ad vector-mediated delivery of multiple death ligands may be developed as a potential BT therapy, either alone or in conjunction with surgical resection of the primary tumor.

The present and future for gene and viral therapy of directly accessible prostate and squamous cell cancers of the head and neck

Gene therapy has been in a continuous evolutionary process since the first approved trial occurred in 1990 at the National Institute of Health. In the USA, as of March 2004, there were 619 approved gene therapy/transfer protocols and 405 of these were for cancer treatment. Another 294 trials are in progress worldwide, with most concentrated in Europe. However, cancer gene therapy is in its relative infancy when compared with the well-established use of chemo-radiotherapy for treating cancer. As the field develops it is becoming clear that using gene therapy in conjunction with established chemo-radiotherapy approaches is yielding the best results. This concept shall be reviewed in the context of the status of the field, and a future direction based on a combination of gene therapy with small molecule modification of sphingolipid metabolism shall be discussed.

Ceramide, Ceramidase, and FasL Gene Therapy in Prostate Cancer

Glycerolipid-derived second messengers such as diacylglycerol, phosphatidylinositides, and eicosanoids are now well-established mediators of signal transduction. Sphingolipids, which are even more structurally complex than glycerophospholipids, are also appreciated to serve as potential reservoirs for bioactive lipids (1, 2, 3, 4, 5, 6, 7, 8, 9). Thus, regulation of sphingolipid metabolism appears involved in regulation of cell growth, differentiation, senescence, and programmed cell death, and possibly, as proposed herein, favoring growth of a subset of prostate cancers.

676. A Role for Ceramide in AdGFPFasLTET Gene Therapy|[ast]|

Top of pageAbstract The application of gene therapy to cancer has many options in the choice of therapeutic genes but delivery of a corrective signal to every cell in the cancer is impossible. Thus, studies on improving the delivery of therapeutic genes or how to amplify the therapeutic response to gene therapy are urgently needed. In the DU145 model of prostate cancer, resistance to induction of apoptosis via Fas receptor signaling is due to overexpression of apoptotic resistance genes including cFLIPs [Hyer et al., Can Biol. and Ther. 1(4): 405–410, 2002]. We have also determined that expression of a FasL-GFP fusion gene overcomes resistance in infected cells [Hyer et al., Mol. Ther. 2(4):348–358, 2000] and kills the cell apoptotically. During this process apoptotic vesicles that signal through Fas to induce apoptosis are also produced and can be demonstrated to exert bystander activity [Hyer et al., Can. Gene Ther. 10(4):330–339, 2003]. DU145 cells overexpress apoptotic resistance genes and acid ceramidase (i.e. lowers ceramide levels), which we have shown (unpublished) produces a FasL resistant phenotype and reduces the bystander effects. This led us to examine the role of ceramide elevation in the bystander process by using small molecule ceramidase inhibitors. We have now demonstrated that acid ceramidase inhibitors, are highly efficient at activating cell death in DU145 cells in vitro using nontoxic doses in combination with AdGFPFasL virus at MOIs achievable in vivo. Administration of inhibitors activates proteasome function and transiently elevates ceramide. Mitochondrial depolarization also is observed. In vivo experiments using 75mg/kg of acid ceramidase inhibitor and AdGFPFasL provides real support for this combined gene therapy/small molecule therapy approach. We will present selected data at the meeting that explain, in part, the mechanisms of this combined therapy.