David H. Holman

Lysosomotropic acid ceramidase inhibitor induces apoptosis in prostate cancer cells

PurposeAlterations in ceramide metabolism have been reported in prostate cancer (PCa), resulting in escape of cancer cells from ceramide-induced apoptosis. Specifically, increased expression of lysosomal acid ceramidase (AC) has been shown in some primary PCa tissues and in several PCa cell lines. To determine if this represents a novel therapeutic target, we designed and synthesized LCL204, a lysosomotropic analog of B13, a previously reported inhibitor of ACMethodsProstate cancer cell lines were treated with LCL204 for varying times and concentrations. Effects of treatment on cytotoxicity, sphingolipid content, and apoptotic markers were assessed.ResultsTreatment of DU145 PCa cells resulted in increased ceramide and decreased sphingosine levels. Interestingly, LCL204 caused degradation of AC in a cathepsin-dependent manner. We also observed rapid destabilization of lysosomes and the release of lysosomal proteases into the cytosol following treatment with LCL204. Combined, these events resulted in mitochondria depolarization and executioner caspase activation, ultimately ending in apoptosisConclusionsThese results provide evidence that treatment with molecules such as LCL204, which restore ceramide levels in PCa cells may serve as a new viable treatment option for PCa.

New insights on the use of desipramine as an inhibitor for acid ceramidase

Treatment of different cancer cell lines with desipramine induced a time‐ and dose‐dependent downregulation of acid ceramidase. Desipramines effect on acid ceramidase appeared specific for amphiphilic agents (desipramine, chlorpromazine, and chloroquine) but not other lysomotropic agents such as ammonium chloride and bafilomycin A1, and was not transcriptionally regulated. The cathepsin B/L inhibitor, CA074ME, but not the cathepsin D inhibitor, pepstatin A, blocked desipramines effect on acid ceramidase. Desipramine led to a more pronounced downregulation of sphingosine compared to ceramide suggesting acid ceramidase inhibition is important to desipramines mechanism of action. This study reveals a new mechanism of action for desipramine.

Acid ceramidase inhibition: a novel target for cancer therapy

During the last decade, sphingolipid deregulation, namely the balance between the pro-apoptotic molecule ceramide and the anti-apoptotic sphingolipid sphingosine-1-phosphate, has emerged as an important factor in cancer pathology and resistance to therapy. Thus, our research has been focused on developing drugs that are able to restore normal sphingolipid balance, precisely through increasing the levels of ceramide and decreasing sphingosine-1-phosphate. Particularly, inhibition of the ceramide metabolizing enzyme acid ceramidase, whose over-expression in cancer cells has been implicated in resistance to treatment, is proving to be an efficient and promising strategy. In this review, we consider our recent work with acid ceramidase inhibitors, in combination with radiation or gene therapy as a sensitizer that enhance cancer therapy.

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.

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.

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.

938. Ceramide May Have a Role in Sensitizing ACHN Renal Cancer Cells (RCC) to Fas Induced Apoptosis

INTRODUCTION AND OBJECTIVE: RCC is the sixth leading cause of cancer death in USA. One third of patients present with metastatic disease. Current systemic therapies have limited results. Gene therapy including pro-apoptotic gene therapy is a promising tool under investigation for treatment of RCC. However, inability to deliver lethal genes to all tumor cells is a problem. FasL is a pro-apoptotic gene that is a member of the Tumor Necrosis Factor family. Although ACHN cells were resistant to convention Fas monoclonal Ab (CH-11), we were able to induce apoptosis by AdGFPFasLTET gene therapy. Ceramide generation was observed after treatment. Ceramide, the basic unit of sphingolipid, is involved in cell cycle arrest and/ or apoptosis. On Using Ceramide or ceramide analogue, we were able to sensitize ACHN cells to CH-11. This indicates that ceramide plays a significant role in sensitizing ACHN to gene therapy.

Proapoptotic Strategy in Cancer Gene Therapy

With the recent availability of the human genome sequence and development of bioinformatic tools to analyze these results, the promise of gene therapy is broad. Further, our understanding of the causes of cancer are rapidly expanding with the developing tools of genomics, proteomics, and metabolomics, which when analyzed with continually developing bioinformatics tools will provide insight into the myriad cellular signaling pathways that become disturbed in cancer. Thus, there is now an abundance of genes available for introduction into cancer to correct better-understood growth aberrations.