lipofectamine and Breast-Neoplasms

The present study compared the effects of ultrasonic irradiation and SonoVue microbubbles (US) or Lipofectamine 3000 on the transfection of small interfering RNA for PRR11 (siPRR11) and Proline-rich protein 11 (PRR11) overexpression plasmid into breast cancer cells. SiPRR11 and PRR11 overexpression plasmid were transfected into breast cancer MCF7 cells mediated by US and Lipofectamine 3000. PRR11 expressions in breast cancer and normal tissues were determined using Gene Expression Profiling Interactive Analysis (GEPIA). The viability, proliferation, migration, invasion and apoptosis of breast cancer cells were respectively measured by MTT assay, clone formation assay, scratch wound-healing assay, Transwell assay and flow cytometry. PRR11 and epithelial-to-mesenchymal transition (EMT)-related and apoptosis-related (B-cell lymphoma 2, Bcl-2; Bcl-2-associated protein X, Bax) proteins' expressions were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot as appropriate. As ultrasonic intensity increased, the viability of MCF7 cells was decreased. Results from GEPIA suggested that PRR11 was up-regulated in breast cancer. Silencing PRR11 mediated by US showed a higher efficiency than by Lipofectamine 3000. SiPRR11 transfected by Lipofectamine 3000 suppressed cells growth and metastasis, while promoted cell apoptosis. Moreover, E-cadherin (E-cad) and Bax expressions were high but N-cadherin (N-cad), Snail and Bcl-2 expressions were low. However, overexpressed PRR11 caused the opposite effects. More importantly, transfection of siPRR11 and PRR11 overexpression plasmid using US had a higher efficacy than using Lipofectamine 3000. US transfection of PRR11 siRNA showed better effects on inhibiting breast cancer progression. The current findings contribute to a novel treatment for breast cancer.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Breast Neoplasms; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Lipids; MCF-7 Cells; Microbubbles; Neoplasm Invasiveness; Phospholipids; Proteins; RNA Interference; RNA, Small Interfering; RNAi Therapeutics; Signal Transduction; Sulfur Hexafluoride; Transfection; Ultrasonic Waves

Hyperbranched poly(amidoamine) (HPAMAM), structurally analogous to polyamidoamine dendrimer (PAMAM) dendrimers, has been suggested to be an effective carrier for gene delivery. In the present study, glutamic acid-modified hPAMAM was developed as a novel non-viral gene carrier for the first time. The hPAMAM was synthesized by using a modified one-pot method. DNA was found to be bound to hPAMAM at different weight ratios (WhPAMAM/WDNA). The resulting HPAMAM-Glu20 was able to efficiently protect the encapsulated-DNA against degradation for over 2 h. In addition to low cytotoxicity, the transfection efficiency of hPAMAM-Glu20 represented much higher (p < 0.05) than that of Lipofectamine 2000 in both MCF7 and MDA-MB231 cells. Cellular uptake of the hPAMAM-Glu20 in MDA-MB231 cells, 173.56 ± 1.37%, was significantly higher than that of MCF7 cells, 65.00 ± 1.73% (p < 0.05). The results indicated that hPAMAM-Glu20-mediated gene delivery to breast cancer cells is a feasible and effective strategy that may provide a new therapeutic avenue as a non-viral gene delivery carrier. In addition, it was found that hPAMAM-glutamic amino acid (Glu)-based gene delivery is an economical, effective and biocompatible method.

Topics: Breast Neoplasms; Cell Line, Tumor; Dendrimers; DNA; Female; Gene Transfer Techniques; Genetic Therapy; Glutamic Acid; Humans; Lipids; MCF-7 Cells; Polyamines; Transfection

This study aimed to investigate the relationship between miR-506 and proliferation and migration of breast cancer cells.. MiR-506 mimics, inhibitor, and negative control (NC) were transfected into MDA-MB-231 breast cancer cells. Cell proliferation, cell counting, colony formation assay, and Transwell assay were applied to evaluate the proliferation and migration of breast cancer cells. Data are shown as mean ± standard deviation and the experiment was performed 3 times. Statistical analyses were performed with SPSS version 10.0.. At 1 day after transfection, cell proliferation detected by CCK-8 assay was significantly promoted in miR-506 inhibitor when compared with the miR-506 mimics group and the NC group (P<0.05). At 3 days or 5 days after transfection, cell proliferation was markedly inhibited in the miR-506 mimics group, and miR-506 inhibitor was still significantly promoted. Cell counting with a hemocytometer showed similar results to cell proliferation. Colony formation assay showed that the number of colonies in the miR-506 mimics group was significantly smaller than that in the miR-506 inhibitor group and NC group. Transwell assay revealed that the number of migrated cells in miR-506 mimics was markedly smaller than that in the miR-506 inhibitor group and NC group.. MiR-506 over-expression significantly inhibits the proliferation, colony formation, and migration of breast cancer cells. miR-506 over-expression may thus be able to improve the malignant phenotype of breast cancer cells.

Topics: Breast Neoplasms; Cell Count; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; In Vitro Techniques; Lipids; MicroRNAs; Molecular Mimicry; Neoplasm Metastasis; Transfection; Tumor Stem Cell Assay

The aim of this study was to investigate the effect of a Lipofectamine2000 (Life2000) Transfection Reagent transfected psiRNA-STAT3 plasmid on 4T1 breast cancer cells.. MTT was used to detect the cell proliferation of breast cancer 4T1 cells at different periods (0h, 6h, 8h, 10h); the cell cycle was assessed by flow cytometry; variation of apoptosis and mitochondrial membrane potential was observed under a fluorescence microscope; immunohistochemical staining was used to determine the expression of caspase-3 and cyclin-D1 protein.. An obvious effect of inhibition to 4T1 cancer cells could be observed at 8h after the psiRNA-STAT3 was transfected. Typical alterations of apoptotic morphological features were visible in the psiRNA-STAT3 treatment group. Mitochondrial membrane potential decreased significantly, the number of cells was increased in G0/G1 phase, and the number of cells was decreased in S phase, and the data were statistically significant (p<0.05), compared with the Scramble and Mock groups. Expression of caspase-3 protein was increased significantly, while that of cyclin D1 was significantly decreased.. Life2000 transfected psiRNA-STAT3 plasmid can inhibit 4T1 tumor cell proliferation and promote apoptosis of 4T1 tumor cells, which process depends on the regulation of expression of cyclin D1 and caspase-3 protein.

Topics: Animals; Apoptosis; Breast Neoplasms; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; G1 Phase Cell Cycle Checkpoints; Lipids; Membrane Potential, Mitochondrial; Mice; RNA, Small Interfering; S Phase Cell Cycle Checkpoints; STAT3 Transcription Factor; Transfection

To investigate cellular uptake pathways of novel anionic siRNA-lipoplexes as a function of formulation composition.. Anionic formulations with anionic lipid/Ca(2+)/siRNA ratio of 1.3/2.5/1 (AF1) and 1.3/0.3/1 (AF2) were utilized. Uptake mechanisms were investigated using uptake inhibition and co-localization approaches in breast cancer cells. Actin-mediated uptake was investigated using actin polymerization and rearrangement assays. Silencing efficiency and endosomal escaping capability of lipoplexes were evaluated. The cationic formulation Lipofectamine-2000 was used as a control.. Anionic lipoplexes entered the breast cancer cells via endocytosis specifically via macropinocytosis or via both macropinocytosis and HSPG (heparin sulfate proteoglycans) pathways, depending on the Ca(2+)/siRNA ratio. Additionally, uptake of these lipoplexes was both microtubule and actin dependent. The control cationic lipid-siRNA complexes (Lipofectamine-2000) were internalized via both endocytic (phagocytosis, HSPG) and non-endocytic (membrane fusion) pathways. Their uptake was microtubule independent but actin dependent. Silencing efficiency of the AF2 formulation was negligible mainly due to poor endosomal release (rate-limiting step).. Formulation composition significantly influences the internalization mechanism of anionic lipoplexes. Uptake mechanism together with formulation bioactivity helped in identification of the rate-limiting steps to efficient siRNA delivery. Such studies are extremely useful for formulation optimization to achieve enhanced intracellular delivery of nucleic acids.

Topics: Actins; Anions; Breast; Breast Neoplasms; Calcium; Cell Line, Tumor; Female; Humans; Lipids; Pinocytosis; RNA Interference; RNA, Small Interfering

Small interfering RNA (siRNA) has been widely investigated as a potential therapeutic approach for diseases with genetic defects. However, its application was greatly hampered by the rapid degradation and poor cellular uptake. Recently, chitosan (CS) and its derivant have been considered as a promising siRNA transporter with the advantages of low toxicity, good biodegradability and biocompatibility. Chitosan of different molecular weight (Mw) and degrees of deacetylation (DD) showed significantly varied target gene silencing efficacy, and it is still not well clarified how these characteristics influence CS mediated siRNA transfection. To compare the aspects of cell permeability and intracellular unpacking of CS/siRNA complex on the effect of CS/siRNA transfection. A radiolabeled siRNA, targeting firefly luciferase gene, was loaded by chitosan of different molecular weight (varying from 2000 to 800,000 Da) and subjected to the transfection against MDA-MB-231/Luc human breast cancer cell line which stably expressed knocked in firefly Luciferase reporter gene. Following transfection intracellular radioactivity was measured to represent cell entrance ability of the CS/siRNA, while, luciferase activity in the cell lysate was also determined to reflect target gene silencing effect. The results revealed that although low molecular weight chitosan (LMWC) condensed siRNA has the highest cell permeability of almost two folds of medium molecular weight chitosan and lipofactamine, its target gene silencing effect is really low of almost eight times less than lipofectamine. This conspicuous contradiction gave us the hypothesis that LMWC generated more condensed CS/siRNA complex to facilitate cell entrance but the tight electrostatic interaction probably limited intracellular siRNA unpacking as well and unfavorably hindered target gene silencing as the final consequence. To approve this hypothesis a phosphorylatable short peptide conjugated LMWC was adopt to promote intracellular siRNA unpacking. Which was demonstrated of perfect target gene knock down ability to the extent of being even superior to lipofactamine 2000. In a conclusion, low molecular weight chitosan has the great potential to be an ideal siRNA vehicle if the issue of siRNA unpacking could be properly resolved.

Topics: Acetylation; Breast Neoplasms; Cell Line, Tumor; Chitosan; Female; Humans; Lipids; Luciferases; Molecular Weight; Nanoparticles; Nucleic Acid Conformation; Particle Size; Peptides; Permeability; Phosphorylation; RNA Interference; RNA, Small Interfering; Transfection

The therapeutic humanized monoclonal antibody IgG1 known as Herceptin® has shown remarkable antitumor effects. Although this type of therapy has increased the cancer-free survival of patients, not all tumors respond to this treatment and cancers often develop resistance to the antibody. Despite the fact that Herceptin function has been extensively studied, the precise mechanism underlying its antitumor activity still remains incompletely defined. We previously demonstrated on human breast MCF-7 carcinoma and T-lymphoblastoid CEM cells that monoclonal antibody in combination with Lipoplex consisting of Lipofectamine mixed with plasmid DNA showed a more profound effect on cancer cell viability than antibody alone. The analyses of N-glycans isolated from cancer cells showed dramatic differences in profiles when cells were exposed to Herceptin. Moreover, the investigation of glycosylated peptides from the same cancer cell models after treatment revealed further alterations in the post-translational modifications. Tandem mass spectra obtained from the samples treated confirmed the presence of a series of glycopeptides bearing characteristic oligosaccharides as described in IgG1. However some of them differed by mass differences that corresponded to peptide backbones not described previously and more of them were detected from Herceptin treated samples than from cells transfected with Heceptin/Lipoplex. The results indicate that the presence of Lipoplex prevents antibody transformation and elongates its proper function. The better understanding of the multipart changes described in the glycoconjugates could provide new insights into the mechanism by which antibody induces regression in cancers.

Topics: Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; Carbohydrate Sequence; Cell Line, Tumor; Cell Survival; Chromatography, High Pressure Liquid; Drug Resistance, Neoplasm; Female; Glycomics; Glycopeptides; Humans; Lipids; Molecular Sequence Data; Plasmids; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Protein Processing, Post-Translational; Proteomics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transfection; Trastuzumab; Trypsin

The effective targeting of malignant cell surface antigens is essential in cancer therapy. Resistance to treatment and rapid invasion of cancer cells are the main causes of cancer mortality. Despite intense research efforts, treatments often have demonstrated insufficient outcomes in clinical applications. The aim of the present study was to determine whether combined administration of monoclonal antibody (Herceptin, trastuzumab) and anti-HER-2 (clone CB11) with hyaluronic acid (HA) and lipoplex (containing lipofectamine (LipA) and plasmid DNA) can produce a synergistic reaction to increase the therapeutic effect of monoclonal antibodies. To assess the treatment response, we cultured a 3-D MCF-7 cell line overexpressing HER-2 and CD44 receptors. The high density 3-D cell aggregation in the hollow fiber bioreactor (HFB) used for the cell culture was monitored with the use of proton magnetic resonance imaging ((1)H MRI). In addition, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used in combination with HPLC (high performance liquid chromatography) to evaluate structural changes in the proteins contained in treated cells. The study showed that incorporation of antibodies into targeted lipoplex results in more efficient delivery of the complex to tumor cells. The viability of cells decreased mostly due to cellular uptake of lipoplex and binding of the antibodies to the cellular surface receptor. The data also demonstrate that HA could be used to enhance treatment efficacy of trastuzumab and anti-HER-2 (clone CB11) in breast cancer cell cultures.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Chromatography, High Pressure Liquid; DNA; Drug Delivery Systems; Drug Resistance, Neoplasm; Drug Synergism; Female; Humans; Hyaluronan Receptors; Hyaluronic Acid; Lipids; Magnetic Resonance Imaging; Plasmids; Receptor, ErbB-2; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trastuzumab

The aim of this study was to assess the herceptin efficacy in ex vivo cultures of MCF-7 breast carcinoma cells. Herceptin was used with perfluorooctyl bromide (PFOB) and conjugated with lipoplex, containing plasmid DNA and lipofectamine (LipA), to allow fluorine-19 magnetic resonance imaging ((19)F MRI) study. Treatments such as herceptin, herceptin/PFOB and herceptin/PFOB/lipoplex were used for ex vivo targeting of MCF-7 cells cultured in three-dimensional (3D) geometry using hollow fiber bioreactor (HFB) device. The viability of MCF-7 cells after 72h treatments decreased to 54+/-2%, 50+/-3% and 45+/-1% for herceptin, herceptin/PFOB and herceptin/PFOB/Lipoplex, respectively. The EC(50) values were 1000microg/ml, 930microg/ml and 730microg/ml, respectively. The significant correlation between the treatment concentration and efficacy was observed in MCF-7 cell cultures.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Bioreactors; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Chemistry, Pharmaceutical; Contrast Media; Dose-Response Relationship, Drug; Emulsions; Female; Fluorocarbons; Humans; Hydrocarbons, Brominated; Inhibitory Concentration 50; Lipids; Magnetic Resonance Imaging; Time Factors; Trastuzumab

The humanized monoclonal antibody IgG1 in combination with chemotherapy has been demonstrated to enhance survival benefit in cancer treatment. Despite positive outcomes, some cancer cells develop multidrug resistance. Numerous mechanisms in cancers can be involved in the process of treatment therapy and most of them are not still well understood. To address how the carbohydrate moieties of cells are affected during treatment, the glycan profiles from the two most common cancer cell lines - human breast MCF-7 carcinoma and T-lymphoblastoid CEM cells - were studied here and compared with profiles after treatment with Herceptin alone or in combination with Lipofectamine mixed with plasmid DNA to form Lipoplex. N-Glycans were released from total cells by digestion with PNGaseF and analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). In summary, both original cell lines showed a dominant occurrence of high-mannose glycans. After treatment, these structures were suppressed and biantennary core-fucosylated glycans originating from IgG1 were the major carbohydrate products identified in cells. The high incidence of additional fucosylated or nonfucosylated galactosylated oligosaccharides, which were not detected in original cells or Herceptin, varied with conditions and time of exposure of cells to the antibody. The results presented in this study provide strong evidence for a role of glycosylation during antibody treatment.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Breast Neoplasms; Cell Line, Tumor; DNA; Female; Glycomics; Glycosylation; Humans; Lipids; Mass Spectrometry; Plasmids; Polysaccharides; Reproducibility of Results; T-Lymphocytes; Trastuzumab

Steroid hormones such as 17beta-estradiol (E2) are critical to diverse cellular processes including tumorigenesis. A number of cofactors such as nuclear receptor corepressor (NCoR), CREB-binding protein (CBP), and steroid receptor coactivator 1 (SRC-1) interact with estrogen receptors (ERs) to regulate transcriptional repression or activation of target genes. Estrogen signaling in non-reproductive tract tissues such as skin is less well characterized and the effectiveness of anti-estrogen therapy for cancer arising from these tissues is unknown. We show that tamoxifen (TAM) treatment inhibited cell cycle progression and proliferation of human cancer lines derived from stratified squamous epithelium squamous cell carcinoma (SCC). E2 had no effect on proliferation of these lines despite low levels of ERalpha expression. The E2 treatment promoted displacement of the NCoR from ERalpha and recruitment of CBP to the receptor. SRC-1 expression was not detected in these SCC lines; however, transient transfection of SRC-1, CBP, or both coactivators enhanced transactivation of an estrogen responsive promoter in cancer cells treated with E2 or TAM. In stable clones expressing SRC-1, the coactivator was recruited to ERalpha along with CBP in E2 but not in TAM-treated cells. SRC-1 expression restored the E2-mediated proliferative response to human SCC lines. This increased proliferation correlated with increased extracellular signal regulated kinase 1 (ERK1) expression. SRC-1 and CBP were recruited to the proximal ERK1 promoter region in E2 but not in TAM-treated cells. We concluded that SRC-1 was a key molecular determinant of estrogen-mediated proliferation in human SCC lines.

Topics: Blotting, Western; Breast Neoplasms; Carcinoma, Squamous Cell; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chromatin Immunoprecipitation; CREB-Binding Protein; Estrogen Antagonists; Estrogen Receptor alpha; Estrogens; Extracellular Signal-Regulated MAP Kinases; Histone Acetyltransferases; Humans; Lipids; Nuclear Receptor Coactivator 1; Reverse Transcriptase Polymerase Chain Reaction; Tamoxifen; Transcription Factors; Transfection

To investigate the feasibility of transfer antisense oligodeoxynucleotides (AS-ODNs) by the phospholipids-based gas-filled microbubbles (PGM) under ultrasound activation.. An antisense oligodeoxynucleotides sequence ZL combined with luciferase reporter plasmid was used. A breast cancer cell line SK-BR-3 was exposed to different conditions to investigate the effects of such factors as ZL concentration, PGM concentration, mechanical index (MI) and ultrasound exposure duration on transfection efficiency and cell viability. The transfection efficiency and cell viability by other lipid vectors such as lipofectamine and liposome were also tested, whose results were comparied with that of PGM. Transfection efficiency was detected by fluorescence microscopy. Cell viability was verified by PI (propidium iodide) assay.. Among the factors tested, ultrasound exposure duration, MI and PGM concentration had obvious impacts on transfection efficiency and cell viability. The results showed that the optimal ultrasound condition was the exposure to ultrasound at MI 1.0 for 30 s with 2% PGM concentration, which gave an overall transfection efficiency of 78% +/- 10%, increased nearly 18 folds over the transfection by PGM (4.0%) or lipofectamine (4.3%) without ultrasound. Under same ultrasound conditions, different vectors showed significant difference in transfection efficiency while there are similar results in cell viability.. Under proper ultrasound conditions, PGM can markedly enhance AS-ODNs transfection efficiency.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Survival; Drug Carriers; Female; Humans; Lipids; Liposomes; Luciferases; Microbubbles; Microscopy, Fluorescence; Oligodeoxyribonucleotides, Antisense; Phospholipids; Transfection; Ultrasonics

The influence of estradiol on the delivery of plasmid DNA to estrogen receptor positive MCF-7 human breast cancer cells was studied by the use of a reporter assay and by histochemical staining. Continuous exposure to estradiol enhanced the lipofectamine-mediated delivery of both pSV40-luciferase and pCMV beta-galactosidase in a concentration-dependent manner. Estradiol increased both the amount of pCMV beta-galactosidase per cell and the total fraction of cells competent to receive the transgene. The efficiency of transgene delivery to MCF-7 cells was further improved by repeating the transfection procedure in the presence of estradiol. Although overall gene uptake was reduced in control cells when studies were performed at room temperature (as opposed to 37 degrees C), potentiation of gene uptake by estradiol was maintained. At a concentration of 100 microM, estradiol also enhanced delivery of the transgene to estrogen receptor negative MDA-MB-231 breast tumor cells, indicating that the potentiating effects of estradiol are not mediated through the estrogen receptor. These studies are the first to raise the possibility that gene delivery to breast tumor cells can be improved by estradiol in single- or repeated-treatment regimens.

Topics: beta-Galactosidase; Breast Neoplasms; Cation Exchange Resins; Cytomegalovirus; Estradiol; Female; Gene Dosage; Genetic Therapy; Genetic Vectors; Humans; Lipids; Liposomes; Luciferases; Simian virus 40; Temperature; Transfection; Tumor Cells, Cultured

To examine the potential use of adenovirus vectors in cancer gene therapy as a mechanism for purging bone marrow cells of possible breast cancer contaminants, we compared the infection efficiency of adenovirus and the transfection efficiency of plasmid DNA in the presence of adenovirus in human breast cancer and bone marrow cells. Following infection of breast cancer cells with an adenovirus expressing beta-galactosidase gene, high levels of beta-galactoside activity were observed. No beta-galactosidase activity was observed in low-density human bone marrow cells. A replication-deficient adenovirus mutant dl312 enhanced the transfection efficiency of a plasmid DNA-expressing beta-galactosidase gene into breast cancer cells, and addition of a liposome, lipofectamine, further enhanced the transfection efficiency. In contrast, human bone marrow cells treated under the same conditions expressed very low levels of transfected beta-galactosidase DNA. Transfection of cells with plasmid DNA expressing a truncated but fully active Pseudomonas exotoxin gene in the presence of dl312 and lipofectamine resulted in marked breast cancer cell killing, whereas colony-forming unit granulocyte-macrophage (CFU-GM) were relatively resistant to these treatments. A recombinant adenovirus expressing human wild-type p53 protein (AdWTp53) was also highly cytotoxic to breast tumor cells. Infection of breast cancer cells with AdWTp53 (100 plaque-forming units/cell) resulted in 100% loss of the clonogenicity of breast tumor cells. However, colony formation from CFU-GM was relatively resistant to the cytotoxic effects of AdWTp53 alone or in the presence of pULI100 plasmid and lipofectamine. On the basis of these results, it is proposed that human adenoviruses are potentially useful for cancer gene therapy and bone marrow purging.

Topics: Adenoviridae; ADP Ribose Transferases; Bacterial Toxins; beta-Galactosidase; Bone Marrow; Bone Marrow Purging; Breast Neoplasms; Cation Exchange Resins; Colony-Forming Units Assay; Defective Viruses; Exotoxins; Female; Gene Expression Regulation, Enzymologic; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Granulocytes; Humans; Indicators and Reagents; Lipids; Neoplasm Proteins; Pseudomonas aeruginosa Exotoxin A; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Virulence Factors

Direct in vivo transfection of tumor nodules in situ via liposome-DNA complexes has been employed as a strategy to accomplish antitumor immunization. To circumvent the potential safety hazards associated with systemic localization of delivered DNA, the utility of mRNA transcript-mediated gene delivery was explored. Capped, polyadenylated mRNA transcripts encoding the firefly luciferase and Escherichia coli lacZ reporter genes were derived by in vitro transcription. Transfection of the human breast cancer cell line MDA-MB-435 in vitro was accomplished employing cationic liposome-mRNA complexes. Evaluation of a panel of cationic liposome preparations demonstrated significant differences in the capacity of the various preparations to accomplish mRNA-mediated transfection. Quantitative evaluation of in vitro transfection demonstrated that target cells could be transfected at a high level of efficiency. The mRNA liposome-complexes were evaluated for in vivo transfection of tumor nodules in human xenografts in athymic nude mice. It could be demonstrated the liposome-mRNA complexes were comparable in efficacy to liposome-DNA complexes in accomplishing in situ tumor transfection. Thus, mRNA may be considered as an alternative to plasmid DNA as a gene transfer vector for genetic immunopotentiation applications.

Topics: beta-Galactosidase; Breast Neoplasms; Cation Exchange Resins; Cations; Drug Carriers; Fatty Acids, Monounsaturated; Genes, Reporter; Genetic Therapy; Glycine; Humans; Lipids; Liposomes; Luciferases; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Recombinant Fusion Proteins; RNA, Messenger; Safety; Spermine; Transfection; Tumor Cells, Cultured