lipofectamine and Glioma

High expression of multidrug resistance-associated protein 1 (MRP1) in tumor cells reduces effectiveness of chemotherapy drugs. In this study, we screened MRP1 interfering RNA (MRP1-siRNA) molecules that are able to reverse etoposide (VP16) resistance in multidrug resistance rat glioma cell line C6/VP16, and identified one siRNA molecule that is able to effectively deplete the expression of MRP1 gene and reverse tumor cells resistance to etoposide. Since siRNA instability limits its application in treatment of diseases, we next tested silencing effect of chitosan-MRP1-siRNA nanoparticles and found that the nanoparticles with N:P ratio 175 are able to effectively inhibit MRP1 mRNA and protein expression. Our data demonstrate that chitosan can be used as siRNA carrier for high efficient gene silencing in tumor cells.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Chitosan; Down-Regulation; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Etoposide; Gene Expression Regulation, Neoplastic; Gene Silencing; Glioma; Inhibitory Concentration 50; Lipids; Multidrug Resistance-Associated Proteins; Nanoparticles; Rats; RNA, Small Interfering; Transfection

Polyamidoamine (PAMAM) dendrimer and Tat peptides were conjugated to bacterial magnetic nanoparticles (BMPs) for the construction of an efficient and targeted gene delivery system with transmembrane ability for the gene therapy of brain tumors. Tat-BMPs-PAMAM was complexed with small interfering RNA expression plasmid (psiRNA) corresponding to the open reading frame of the human epidermal growth factor receptor gene (psiRNA-EGFR) to downregulate the EGFR gene by electrostatic interaction. The antitumor effect of psiRNA-EGFR delivered via Tat-BMPs-PAMAM was assessed both in human glioblastoma U251-MG cells and in nude mouse models. Compared with control groups, Tat-BMPs-PAMAM/psiRNA-EGFR resulted in better suppression of EGFR expression and a more obviously arrested effect on the proliferation and invasion ability of U251 cells in vitro. In addition, the growth rate of tumor in the U251 subcutaneous nude mouse model treated with Tat-BMPs-PAMAM/psiRNA-EGFR was slower than in those treated with phosphate-buffered saline or Lipofectamine 2000/psiRNA-Scr. Also, compared with control groups, the expression of oncoproteins (EGFR, p-AKT, MMP2/9, PCNA, VEGF, Bcl-2, and cyclin D1) was obviously downregulated and the number of apoptotic cells was clearly increased in the Tat-BMPs-PAMAM/psiRNA-EGFR treatment groups. In addition, there was no significant difference between the results in vitro and in vivo for the Tat-BMPs-PAMAM/psiRNA-EGFR treatment groups and those of the Lipofectamine 2000/psiRNA-EGFR treatment groups. These results show that Tat-BMPs-PAMAM, with its targeted delivery and transmembrane ability, may be a novel gene delivery system with potential applications in the targeted gene therapy of brain tumors.

Topics: Animals; Brain Neoplasms; Dendrimers; Drug Delivery Systems; ErbB Receptors; Genetic Therapy; Glioma; Humans; Lipids; Metal Nanoparticles; Mice; Mice, Nude; Peptide Fragments; RNA Interference; RNA, Small Interfering; tat Gene Products, Human Immunodeficiency Virus; Treatment Outcome; Xenograft Model Antitumor Assays

Several gene delivery reagents were analyzed for their transfection efficiency. Genes studied belonged to the class of mammalian proteins termed regulators of G-protein signaling (RGS), ranged in size up to 2.2 Kb long and were transfected into the NG108-15, SH-SY5Y and CHO-K1 cell lines. Prior to transfection, genes were cloned into a nonviral vector pcDNA 6.2/EmGFP, so as to express a green fluorescent protein tag at the 3' end. Flow cytometry was used to analyze cell fluorescent activity and thereby transfection efficiency. Gene delivery reagents Lipofectamine 2000 and ExGen 500 produced more effective transfection in NG108-15 cells whereas Lipofectamine 2000, ExGen 500 and TurboFectin 8.0 were more effective in CHO-K1 cells. In both these cell lines, transfection efficiency reached 60-80%. In SH-SY5Y cells, TurboFectin 8.0 produced the best transfection result; however efficiency level was only 5%. Gene size had no effect on transfection efficiency. Unlike Lipofectamine 2000, cells transfected using ExGen 500 showed morphological deformation. Our results suggest that Lipofectamine 2000 is the most suitable transfection medium for gene delivery to NG108-15 and CHO-K1 cells.

Topics: Animals; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; Glioma; Humans; Lipids; Mice; Neuroblastoma; Rats; Transfection

Gene transfer into cells of CNS origin is an important tool to counteract neurodegeneration by introducing, e.g., neuroprotective molecules. Although viral gene transfer reveals the highest gene transfer efficiency, liposome-mediated gene transfer seems to become an attractive alternative. In this study we investigated the lipid-mediated gene transfer into primary neurons in vitro by using the novel nonliposomal lipid FuGene and compared it to primary glia and malignant C6 glioma cells. FuGene-mediated gene transfer was useful to transfer the reporter gene beta-galactosidase into C6 glioma cells, primary glia, and primary neurons. Lipofection was highly dependent on the surface bottom and did not result in good efficiencies when using glass compared to plastic dishes. Comparing to a standard lipofection (1 x 8 h), lipofection on 3 consecutive days for 6 h each ("boosting") markedly increased the gene transfer efficiency in primary glia (up to sevenfold) and in primary neurons (up to sixfold). The use of endotoxin-free DNA only slightly increased the transfection efficiency. Immunohistochemistry demonstrated MAP-2-positive neurons (up to 1614 neurons/16-mm well; 2.4% of total neurons) as well as TH-positive neurons (up to 48 neurons/16-mm well; 12.7% of TH neurons) expressing beta-galactosidase. We conclude that FuGene-mediated gene transfer is an attractive alternative to introduce genes of interest into cells of glial and neuronal origin; however, this technique lacks sufficient gene transfer efficiency.

Topics: Animals; beta-Galactosidase; Cation Exchange Resins; Cell Survival; Cells, Cultured; Culture Media; DNA; Evaluation Studies as Topic; Gene Transfer Techniques; Genes, Reporter; Glial Fibrillary Acidic Protein; Glioma; Immunohistochemistry; Lipid Metabolism; Lipids; Liposomes; Microtubule-Associated Proteins; Neuroglia; Neurons; Rats; Transfection; Tyrosine 3-Monooxygenase

Malignant gliomas are the most common intracranial tumors and are considered incurable. Therefore, exploration of novel therapeutic modalities is essential. Telomerase is a ribonucleoprotein enzyme that is detected in the vast majority of malignant gliomas but not in normal brain tissues. We, therefore, hypothesized that telomerase inhibition could be a very promising approach for the targeted therapy of malignant gliomas. Thus, 2-5A (5'-phosphorylated 2'-5'-linked oligoadenylate)-linked antisense against human telomerase RNA component (2-5A-anti-hTER) was investigated for its antitumor effect on an intracranial malignant glioma model. 2-5A is a mediator of one pathway of IFN actions by activating RNase L, resulting in RNA degradation. By linking 2-5A to antisense, RNase L degrades the targeted RNA specifically and effectively. Prior to the experiments using intracranial tumor models in nude mice, we modified the in vitro and in vivo treatment modality of 2-5A-anti-hTER using a cationic liposome to enhance the effect of 2-5A-anti-hTER. Here we demonstrate that 2-5A-anti-hTER complexed with a cationic liposome reduced the viability of five malignant glioma cell lines to 20-43% within 4 days but did not influence the viability of cultured astrocytes lacking telomerase. Furthermore, treatment of intracranial malignant gliomas in nude mice with 2-5A-anti-hTER was therapeutically effective compared with the control (P < 0.01). These findings clearly suggest the therapeutic potentiality of 2-5A-anti-hTER as a novel approach for the treatment of intracranial malignant gliomas.

Topics: Adenine Nucleotides; Animals; Brain Neoplasms; Cation Exchange Resins; Cations; Female; Glioma; Humans; Lipids; Liposomes; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Oligoribonucleotides; Oligoribonucleotides, Antisense; RNA, Neoplasm; Telomerase; Tumor Cells, Cultured

Growth factors play an important role in proliferation and differentiation of malignant brain gliomas in humans. Glial cell line-derived neurotrophic factor (GDNF) has been shown recently to be highly expressed in human glioblastomas and in rat glial cell lines B49 and C6. The aim of the present study was to knockdown GDNF, its receptor GFR-alpha1, and the related family member persephin by using antisense oligonucleotides and to observe the effects on cell proliferation. To enhance cellular uptake into C6 glioma cells, 15-mer phosphorothioate oligonucleotides were complexed with the cationic lipid Lipofectamine. The complex was applied for 3 x 12 hours to C6 glioma cells, and cells were allowed to recover for 24 hours after each transfection and then analyzed. This protocol markedly reduced GDNF and GFR-alpha1 protein levels in C6 glioma cells compared with control oligonucleotides. Knockdown of C6 cells with GDNF and GFR-alpha1 but not with persephin antisense oligonucleotides significantly decreased the number of C6 glioma cells and also inhibited the incorporation of bromodeoxyuridine as a sign of reduced DNA synthesis. In conclusion, it is shown that GDNF but not persephin is a potent proliferation factor for rat glioma cells. Knockdown of GDNF using antisense oligonucleotides complexed with lipids as carriers may be useful in gene therapeutic approaches in vitro and possibly also in vivo.

Topics: Animals; Blotting, Western; Bromodeoxyuridine; Cation Exchange Resins; Cell Count; Cell Division; Cell Survival; Central Nervous System; Drosophila Proteins; Enzyme-Linked Immunosorbent Assay; Genetic Therapy; Glial Cell Line-Derived Neurotrophic Factor; Glial Cell Line-Derived Neurotrophic Factor Receptors; Glioma; Lipid Metabolism; Lipids; Liposomes; Nerve Growth Factors; Nerve Tissue Proteins; Oligonucleotides, Antisense; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ret; Rats; Receptor Protein-Tyrosine Kinases; Transfection; Trypan Blue; Tumor Cells, Cultured

Vascular endothelial growth factor (VEGF) is most promising in therapeutic angiogenesis for ischemic vascular disease. This paper aimed to study VEGF gene therapy for the treatment of cerebral ischemia. The glial cell was chosen as the target cell for gene transfer, and the expression of VEGF was studied in vitro. VEGF plasmid/liposome complexes were constructed by mixing VEGF plasmid with liposome, and then cultured C6 glioma cells were transfected with these complexes by lipofectamine method. As control, the same kind of cells were exposed to liposome only. Immunohistochemistry was performed to both groups at 24, 48 and 72 hours after transfection. The transfected cells expressed VEGF significantly higher than the control. The present result demonstrated the feasibility of choosing the glial cell as the target cell for VEGF gene transfer, and found the rationale for the cerebral VEGF gene therapy.

Topics: Animals; Brain Ischemia; Brain Neoplasms; Cation Exchange Resins; DNA, Complementary; Endothelial Growth Factors; Feasibility Studies; Genetic Therapy; Genetic Vectors; Glioma; Immunoenzyme Techniques; Lipids; Liposomes; Lymphokines; Neuroglia; Rats; Recombinant Fusion Proteins; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

A synthetic polyamino polymer with a glucose backbone was used for gene transfer in vitro and in vivo. Gene transfer in vitro to various human carcinoma cell lines was achieved with an efficiency superior to a commercially available cationic liposome preparation. The polymer was resistant to inhibition by serum, which allowed for efficient gene transfer in vivo. Direct Intracranial tumor injection using this reagent resulted in reporter gene expression levels comparable to those achieved by a recombinant adenoviral vector. Thus, this compound represents a new class of agent that may have broad utility for gene transfer and gene therapy applications.

Topics: Animals; beta-Galactosidase; Biocompatible Materials; Brain Neoplasms; Cation Exchange Resins; Cell Survival; Gene Transfer Techniques; Genes, Reporter; Glioma; Green Fluorescent Proteins; Humans; Lipids; Liposomes; Luciferases; Luminescent Proteins; Mice; Mice, SCID; Plasmids; Polyamines; Sarcoma; Transfection; Transplantation, Heterologous; Tumor Cells, Cultured

We recently have developed a unique cytoplasmic transient gene expression system based on cotransfection of target cells with bacteriophage T7 RNA polymerase (RNAP) and plasmid DNA vectors containing a T7 autogene. Because this T7 system is self-initiating, self-maintaining, and requires no cellular factors for transcription, it is therefore likely to function in any mammalian cell with any gene both in vitro and, more importantly, in vivo. In this study we demonstrate that the T7 DNA vector and T7 RNAP could be efficiently codelivered to cultured cells by lipofection. Different target genes were expressed by the T7 system in a wide variety of mammalian cells including several tumor cell lines. Gene expression could be detected in more than 30% of the cells of some tumor cell lines transiently transfected by the T7 vector. Average activity of the reporter enzyme (luciferase) expressed by a transfected cell was relatively constant regardless of the cell line used. When a T7-luciferase vector was directly injected into various tissues of mice without the use of liposomes, luciferase activity could be found in the injected liver, muscle, brain and tail connective tissues. The luciferase levels expressed by the T7 system were found to be up to 200-fold higher, depending upon the injected tissues, than levels achieved with a traditional nuclear gene expression vector. Direct tumor injection with a T7-beta-galactosidase (beta-gal) construct resulted in beta-gal gene expression in tumor cells near the injection sites. In addition, direct injection of the T7 system in mice did not generate detectable quantities of antibodies against the T7 RNAP. These results suggest that this gene expression system may be useful in many different medical applications such as cancer gene therapies and DNA vaccination, where transient but rapid and efficient gene expression is required.

Topics: Animals; Antibodies, Viral; Bacteriophage T7; beta-Galactosidase; Cation Exchange Resins; CHO Cells; Cricetinae; Cytoplasm; DNA-Directed RNA Polymerases; Fibrosarcoma; Gene Expression; Genes, Reporter; Genes, Viral; Genetic Therapy; Genetic Vectors; Glioma; Humans; L Cells; Lipids; Liver; Luciferases; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Sequence Data; Osteosarcoma; Phosphatidylethanolamines; Plasmids; Promoter Regions, Genetic; Rats; Transfection; Tumor Cells, Cultured; Viral Proteins