938. Ceramide May Have a Role in Sensitizing ACHN Renal Cancer Cells (RCC) to Fas Induced Apoptosis
Year: 2006
Type: article
Abstract: 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. METHODS: ACHN treated with CH-11, AdGFPFasLTETTET, or AdGFP. AdGFPFasLTETTET is replication-deficient adenovirus containing a modified murine Fas ligand gene fused to green fluorescent protein (GFP), with a promoter can down-regulate gene expression in presence of doxycycline. AdGFP that contain the GFP gene alone was used as a control. Viability of the cells was assessed using MTS assay. Cells were also evaluated for apoptosis by alteration of mitochondrial membrane potential (MMP) by FACS and caspase 3/7 assay. Ceramide level with AdGFPFasLTET treatment was measured by HPLC-Ms in Lipidomics core . C6-De erythro-ceramide and lysosomotropic ceramide analogue LCL-385 were combined with CH-11 and viability was assessed by MTS. Apoptosis was confirmed using casapase 3/7 assay. RESULTS: ACHN cells were resistant to apoptosis when treated with CH-11. However, ACHN were extremely sensitive to AdGFPFasLTET while minimal cytotoxicity was seen with control virus AdGFP at the same MOI. Caspase 3/7 was increased by treatment with AdGFPFasLTET and not with AdGFP or CH-11. Increased ceramide level was observed with treatment with AdGFPFasLTET. C6-De-Erythro ceramide or LCL-385 were able to sensitize ACHN cells to CH-11 as assessed by MTS and casapse 3/7 assay. CONCLUSIONS: ACHN cells are extremely sensitive to AdGFPFasLTET treatment and resistant to CH-11. AdGFPFasLTET treatment results in Ceramide generation. Treating ACHN with Ceramide or ceramide analogue lead to sensitizing these cells to CH-11. These findings support the significance to investigate further role of ceramide in sensitizing resistant cancer cells to gene therapy or other chemotherapy.
Source: Molecular Therapy
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $4,500
OpenAlex ID: W1987977793