592. Transfection of Human Oral Cancer Cells Mediated by Transferrin-Associated Lipoplexes: Mechanisms of Cell Death Induced by Herpes Simplex Virus Thimidine Kinase/Ganciclovir Therapy
Authors: Silvia S. Neves, Sergio M. Simoes, Maria C. Pedroso de Lima
Journal: Molecular Therapy (2004)

The Herpes Simplex Virus thymidine kinase (HSV-tk) suicide gene/ganciclovir approach has been used for the treatment of a variety of cancers. Cells that express HSV-tk are rendered sensitive to the pro-drug ganciclovir which is phosphorylated into a toxic agent. Furthermore, there may be another advantage of HSV-tk/GCV therapy. Both HSV-tk-expressing and non-expressing cells can be killed by the bystander effect which allows the possibility of complete tumor eradication even at relatively low levels of cell transfection. Different mechanisms have been proposed to explain this effect: the formation of apoptotic vesicles containing the toxic metabolite, which are internalized by neighboring cells, the activation of immune system in vivo, and diffusion of the toxic metabolite into non-transfected cells trough gap junctions.The purpose of this study was to evaluate the cytotoxic effect of ganciclovir in human tongue cancer cells (HSC-3) previously transfected with transferrin-associated lipoplexes containing DOTAP/cholesterol as well as to investigate the mechanisms involved in the process of cell death and the bystander effect. To optimize the transfection conditions, a plasmid coding for the reporter gene luciferase was used, and the levels of gene expression assessed by luminescence measurements. Our results show that the highest efficiency of transfection was obtained upon association of transferrin (Tf) to lipoplexes prepared at 3/2 (+/-) charge ratio. The delivery of HSV-tk gene to HSC-3 cells mediated by this Tf-lipoplexes followed by ganciclovir treatment for 7 days resulted in 90% cytotoxicity, as assessed by Alamar Blue assay. Cell death was analysed by flow cytometry, by staining the cells with FITC-Annexin V, and propidium iodide. We observed that cell death occur mainly by an apoptotic process. Since only a low number of cells were efficiently transfected, the high cytotoxicity obtained can be attributed to a bystander effect. The absence of any toxic metabolite in the cell culture medium collected from transfected cells followed by incubation with ganciclovir, and the significant decrease in gene expression observed 2 days after transfection, suggest that the observed cytotoxicity was due to diffusion of the toxic agent into neighbouring cells via gap junctions. In order to study the role of gap junctions in the bystander effect, we used dibutyryl-cAMP as a gap junction inducer and α-glycyrrhetinic acid as a gap junction blocker. At five days incubation, cell survival was only reduced to 60 % (50 μM AGA) as compared to 25% in the absence of AGA when transfected cells were treated with 50 μM GCV. On other hand db-cAMP promoted the bystander effect. Cytotoxicity of 25 μM ganciclovir was increased from 30% to 70% when cells were pre-treated with 500 mM db-cAMP. Our results indicate that the observed toxic effect is GCV dose-dependent. Diffusion of the toxic agent into neighbouring cells via gap junctions is most likely involved in the process of cell death which occurs mainly by an apoptotic process. Overall, we can conclude that Tf-associated lipoplexes constitute a promising tool for suicide gene therapy of oral carcinomas