1-2-dioleoyloxy-3-(trimethylammonium)propane and Carcinoma--Squamous-Cell

Suicide gene therapy has been used for the treatment of a variety of cancers. We reported previously the in vitro efficacy of the Herpes Simplex Virus Thymidine kinase (HSV-tk)/ganciclovir (GCV) system to mediate cytotoxicity in oral squamous cancer cells, using transferrin (Tf)-lipoplexes, prepared from cationic liposomes composed of 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and cholesterol. In the present study, we evaluated the antitumoral efficacy mediated by this lipoplex formulation in two suicide gene therapy strategies, HSV-tk/GCV and cytosine deaminase (CD)/5-fluorocytosine (5-FC), using a syngeneic, orthotopic murine model for head and neck squamous cell carcinoma. The cellular and molecular events associated with the antitumoral response elicited by both the therapeutic approaches were investigated by analyzing tumor cell death, tumor-infiltrating immune cells and tumor cytokine microenvironment. Significant tumor reduction was achieved upon intratumoral delivery of HSV-tk or CD genes mediated by Tf-lipoplexes, followed by intraperitoneal injection of GCV or 5-FC, respectively. Enhanced apoptosis, the recruitment of NK cells, CD4 and CD8 T-lymphocytes and an increase in the levels of several cytokines/chemokines were observed within the tumors. These observations suggest that suicide gene therapy with lipoplexes modifies the tumor microenvironment, and leads to the recruitment of immune effector cells that can act as adjuvants in reducing the tumor size.

Topics: Animals; Antimetabolites; Antiviral Agents; Apoptosis; Carcinoma, Squamous Cell; Cholesterol; Cytokines; Fatty Acids, Monounsaturated; Female; Flucytosine; Ganciclovir; Gene Transfer Techniques; Genes, Transgenic, Suicide; Genetic Therapy; Liposomes; Lymphocytes; Mice; Mouth Neoplasms; Quaternary Ammonium Compounds; Simplexvirus; Thymidine Kinase; Transferrin; Viral Proteins

Delivery of genes to airway mucosa would be a very valuable method for gene therapy and vaccination. However, there have been few reports on suitable gene delivery systems for administration. In this study, we use a cationic emulsion system, which is physically stable and facilitates the transfer of genes in the presence of up to 90% serum, as a mucosal gene carrier.. Cationic lipid emulsion was formulated with squalene and 1,2-dioleoyl-sn-glycero-3-trimethylammoniumpropane (DOTAP) as major components. Emulsions formed stable complexes with DNA and protected and transferred DNA to target cells against DNase I digestion in the presence of mucosal destabilizers such as heparin sulfate (a polysaccharide of the glycosaminoglycan family in mucosa) and Newfectan (a natural lung extract of bovine) in an in vitro system. In contrast, commercial liposomes and counter liposomes, made with an identical lipid composition of emulsions, failed. After in vivo intranasal instillation, the cationic emulsion showed at least 200 times better transfection activity than the liposomal carriers in both nasal tissue and lung.. These findings show that cationic emulsions can mediate gene transfection into airway epithelium, making it a good choice for transferring therapeutic genes and for genetic vaccination against an pathogenic infection via an airway route.

Topics: Carcinoma, Large Cell; Carcinoma, Squamous Cell; Cations; Cell Culture Techniques; DNA; Emulsions; Epithelial Cells; Fatty Acids, Monounsaturated; Gene Expression; Gene Transfer Techniques; Genes, Reporter; Humans; Lipids; Liposomes; Luciferases; Lung Neoplasms; Nasal Mucosa; Phosphatidylethanolamines; Plasmids; Polysorbates; Quaternary Ammonium Compounds; Squalene; Transfection; Tumor Cells, Cultured; X-Ray Diffraction

The 5-year survival rate of patients with squamous cell carcinoma of the head and neck (HNSCC) has remained poor despite innovative surgery and new radiation and chemotherapeutic strategies. In such patients, gene therapy relying on the modification of tumor cells by gene transfer may have great potential as a new treatment modality in the therapy of HNSCC. In the present study we developed an in vitro model to show the efficacy and technical feasibility of cationic liposome-mediated gene transfer into HNSCC. Five adherent squamous cell carcinoma cell lines were transfected with SV40- or CMV-promoter-driven CAT (chloramphenicol-acetyl-transferase)-expression plasmids using DOTAP as the liposome carrier. The level of CAT expression was shown to correlate directly with the amount of transfected DNA and could be measured by a CAT-enzyme-linked immunosorbent assay. The results of gene transfer by liposome-DNA complexes obtained for all cell lines showed a dose-dependent efficacy correlating to the amount of DOTAP employed. The data demonstrate the successful in vitro transfection of epithelial cell lines with DNA, suggesting its usefulness as a new tool for head and neck cancer therapy in vivo.

Topics: Carcinoma; Carcinoma, Squamous Cell; Cell Line; Chloramphenicol O-Acetyltransferase; Cytomegalovirus; DNA, Viral; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Epithelium; Fatty Acids, Monounsaturated; Feasibility Studies; Gene Transfer Techniques; Genetic Therapy; Head and Neck Neoplasms; HeLa Cells; Humans; Liposomes; Plasmids; Promoter Regions, Genetic; Quaternary Ammonium Compounds; Simian virus 40; Survival Rate; Transfection; Tumor Cells, Cultured