1-2-dioleoyloxy-3-(trimethylammonium)propane and Ovarian-Neoplasms

The DNA transfer efficiency in liposomal versus viral transfection is very low, mainly due to an insufficient nuclear transport of the delivered DNA after its endocytotic uptake to the cell. Ligand activation of intracellular steroid receptors and their subsequent mobilization to the nucleus could result in a co-transport of DNA to the nucleus. The augmentation of nuclear transport of DNA after steroid addition might cause enhanced transfection efficiency. We used cell lines from gynecologic malignoma expressing steroid receptors, such as T47D and Mcf-7 breast cancer cell lines, as well as receptor-negative cell lines, such as Hec1A from endometrium carcinoma or the breast cancer cell line MDA-MB-231. The cells were transfected by the liposomal transfection agent Dotap with the gene for firefly-luciferase as a reporter gene and transfection efficiencies were determined in the luciferase assay. We compared the effect of the addition of cholesterol and steroids in different cell lines on the transfection efficiency. The addition of cholesterol to transfection agents led to an enhancement of the luciferase activity in all cell lines. Steroids enhanced the transfection efficiency only in receptor-positive cell lines. The transfection efficiencies of HEC-1A or MDA-MB-231 cells were not enhanced by steroids. A progesterone preincubation of receptor-positive T47D cells resulted in a decrease of progesterone receptors and afterwards the progesterone enhanced transfection was dramatically diminished. We presume that the transfection enhancement by steroids is dependent on increased nuclear import of the delivered DNA only in the presence of steroid receptors. Steroid enhancement of transfection is different from the benefit of cholesterol for transfection that acts on general cellular properties or the transfection complex as such. Liposomal transfection in combination with steroids might be useful for a cell-specific enhancement of gene transfer for example in gynecological malignoma where subgroups are expressing high levels of steroid receptors.

Topics: Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cholesterol; Fatty Acids, Monounsaturated; Female; Fluorescent Dyes; HeLa Cells; Humans; Liposomes; Luciferases; Ovarian Neoplasms; Quaternary Ammonium Compounds; Receptors, Steroid; Steroids; Transfection

Previously we have formulated a new cationic liposome, DDC, composed of dioleoyltrimethylamino propane (DOTAP), 1,2-dioeoyl-3-phosphophatidylethanolamine (DOPE), and cholesterol (Chol), and it efficiently delivered plasmid DNA into ovarian cancer cells. Mutations in the p53 tumor suppressor gene are the most common molecular genetic abnormalities to be described in ovarian cancer. However, there has been so far no report of nonviral vector-mediated p53 gene deliveries in ovarian cancer. In this study, wild-type p53 DNA was transfected into the ovarian cancer cells, using the DDC as a nonviral vector and the expression and activity of p53 gene were evaluated both in vitro and in vivo.. DDC liposomes were prepared by mixing DOTAP:DOPE:Chol in a 1:0.7:0.3 molar ratio using the extrusion method. Plasmid DNA (pp53-EGFP) and DDC complexes were transfected into ovarian carcinoma cells (OVCAR-3 cells) and gene expression was determined by reverse transcription-polymerase chain reaction and Western blot analysis. The cellular growth inhibition and apoptosis of DDC-mediated p53 transfection were assessed by trypan blue exclusion assay and annexin-V staining, respectively. The OVCAR-3 cells treated with DDC/pp53-EGFP complexes were inoculated into female balb/c nude mice and tumor growth was observed.. The transfection of liposome-complexed p53 gene resulted in a high level of wild-type p53 mRNA and protein expressions in OVCAR-3 cells. In vitro cell growth assay showed growth inhibition of cancer cells transfected with DDC/pp53-EGFP complexes compared with the control cells. The reestablishment of wild-type p53 function in ovarian cancer cells restored the apoptotic pathway. Following the inoculation of DDC/pp53-EGFP complexes, the volumes of tumors in nude mice were significantly reduced more than 60% compared to the control group.. The DDC-mediated p53 DNA delivery may have the potential for clinical application as nonviral vector-mediated ovarian cancer therapy due to its effective induction of apoptosis and tumor growth inhibition.

Topics: Adenocarcinoma; Apoptosis; Cell Division; Cholesterol; Fatty Acids, Monounsaturated; Female; Genes, p53; Genetic Vectors; Humans; Liposomes; Ovarian Neoplasms; Phosphatidylethanolamines; Plasmids; Quaternary Ammonium Compounds; Transfection; Tumor Cells, Cultured; Tumor Suppressor Protein p53

We investigated, for the first time, by using Energy Dispersive X-ray Diffraction, the structure of a new ternary cationic liposome formulated with dioleoyl trimethylammonium propane (DOTAP), 1,2-dioleoyl-3-phosphatidylethanolamine (DOPE) and cholesterol (Chol) (DDC) which has been recently found to have a selective high gene transfer ability in ovarian cancer cells. Our structural results provide a further experimental support to the widely accepted statement that there is not a simple and direct correlation between structure and transfection efficiency and that the factors controlling cationic lipid/DNA (CL-DNA) complexes-mediated gene transfer depend not only on the formulations of the cationic liposomes and their thermodynamic phase, but also significantly on the cell properties.

Topics: Animals; Coated Materials, Biocompatible; DNA; Drug Carriers; Fatty Acids, Monounsaturated; Female; Genetic Therapy; Humans; Lipid Bilayers; Liposomes; Macromolecular Substances; Materials Testing; Models, Molecular; Molecular Conformation; Ovarian Neoplasms; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Transfection; X-Ray Diffraction

Various strategies have been attempted to design efficient protocols for ovarian cancer gene therapy but there has been little progress in their clinical application. In this study, we formulated and evaluated a new cationic liposome prepared with dioleoyltrimethylaminopropane (DOTAP), 1,2-dioleoyl-3-phosphophatidylethanolamine (DOPE), and cholesterol (Chol) (DDC) for plasmid DNA transfer into ovarian cancer cells.. The DDC liposome was prepared by mixing the DOTAP:DOPE:Cholin a 1:0.7:0.3 molar ratio using the extrusion method. Plasmid DNA (pEGFP-C1) and DDC were complexed at various weight ratios to find the optimum condition and the percentage of transfected cells was determined by selecting a green fluorescence protein (GFP) expressing cells in flow cytometry. The transfection efficiency of the DDC liposome was compared with 3[N-(N,N-dimethylaminoethylene) carbamoyl] cholesterol (DC-Chol)/DOPE liposome and commercially available lifopectin.. The optimal transfection of plasmid DNA was achieved at a 1:4 (w/w) ratio of DDC to DNA. The DDC/DNA complex exhibited higher transfection efficiency in human ovarian cancer cells (OVCAR-3 and SK-OV-3 cells) compared to that in other types of cell lines (NCI-NIH:522 and HepG2 cells). Flow cytometric analysis revealed that the DDC/DNA complex exhibited an over fourfold increase in GFP expression levels compared with DC-Chol/DOPE or lipofectin in OVCAR-3 cells. This result was further confirmed by confocal microscopy and RT-PCR analysis.. These results suggest that our newly formulated cationic liposome (DDC) appears to be a promising nonviral vector for treating ovarian adenocarcinoma because of its selective high gene transfer ability in ovarian cancer cells.

Topics: Adenocarcinoma; Cations; Cholesterol; DNA; Fatty Acids, Monounsaturated; Female; Genetic Therapy; Genetic Vectors; Glycerophospholipids; Green Fluorescent Proteins; Humans; Lipids; Liposomes; Luminescent Proteins; Ovarian Neoplasms; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Tumor Cells, Cultured