1-2-dioleoyloxy-3-(trimethylammonium)propane and Carcinoma--Non-Small-Cell-Lung

In the present study, we have used plasmid-based RNA interference (RNAi) strategy to downregulate the expression of epidermal growth factor receptor (EGFR) in EGFR wild-type (H292) and mutant (H1975) lung tumor models. The targeted knockdown of EGFR by small hairpin RNA not only inhibited growth of H292 xenograft but also inhibited H1975 lung cancer cell and xenograft, which bore L858R/T790M EGFR and was resistant to EGFR tyrosine kinase inhibitors. These data demonstrated that small hairpin RNA was an effective therapy against mutant EGFR-expressing cancer cells and thus considered to be a promising strategy in the treatment of lung cancers.

Topics: Animals; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; ErbB Receptors; Fatty Acids, Monounsaturated; Female; Humans; Liposomes; Lung Neoplasms; Mice; Mutation; Neoplasm Invasiveness; Quaternary Ammonium Compounds; RNA, Small Interfering

The antisense oligonucleotide 2'-O-methyl-RNA is a selective telomerase inhibitor targeting the telomerase RNA component and represents a potential candidate for anticancer therapy. The poor cellular uptake of 2'-O-methyl-RNA is a limiting factor that may contribute to the lack of functional efficacy. To improve delivery of 2'-O-methyl-RNA and consequently antitumoral efficiency in human lung cancer cells, we have investigated several transfection reagents. The transfection reagents DOTAP, MegaFectin 60, SuperFect, FuGENE 6 and MATra-A were tested for intracellular delivery. A FAM-labeled 2'-O-methyl-RNA was used to assess the intracellular distribution by confocal laser scanning microscopy in A549 human non-small cell lung cancer cells. Telomerase activity was measured using the telomeric repeat amplification protocol. Cell viability after transfection was quantified by the MTT assay. All transfection reagents enhanced 2'-O-methyl-RNA uptake in A549 cells but the cationic lipid reagents DOTAP and MegaFectin 60 were most efficient in the delivery of 2'-O-methyl-RNA resulting in telomerase inhibition. Among both DOTAP exhibited the lowest cytotoxicity. Our experiments show that DOTAP is the most suitable transfection reagent for the delivery of 2'-O-methyl-RNA in human lung cancer cells according to its relatively low cytotoxicity and its ability to promote efficient uptake leading to the inhibition of telomerase.

Topics: Antineoplastic Agents; Capillary Electrochromatography; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Cholesterol; Drug Delivery Systems; Enzyme Inhibitors; Excipients; Fatty Acids, Monounsaturated; Humans; Indicators and Reagents; Liposomes; Lung Neoplasms; Microscopy, Confocal; Oligonucleotides; Oligonucleotides, Antisense; Quaternary Ammonium Compounds; RNA, Antisense; Telomerase; Tetrazolium Salts; Thiazoles; Transfection

NPRL2 is one of the novel candidate tumor suppressor genes identified in the human chromosome 3p21.3 region. The NPRL2 has shown potent tumor suppression activity in vitro and in vivo and has been suggested to be involved in DNA mismatch repair, cell cycle checkpoint signaling, and regulation of the apoptotic pathway. In this study, we analyzed the endogenous expression of the NPRL2 protein and the cellular response to cisplatin in 40 non-small-cell lung cancer cell lines and found that expression of NPRL2 was significantly and reciprocally correlated to cisplatin sensitivity, with a Spearman correlation coefficient of -0.677 (P < 0.00001). Exogenously introduced expression of NPRL2 by N-[1-(2,3-dioleoyloxyl)propyl]-NNN-trimethylammoniummethyl sulfate:cholesterol nanoparticle-mediated gene transfer significantly resensitized the response to cisplatin, yielding a 40% greater inhibition of tumor cell viability and resulting in a 2- to 3-fold increase in induction of apoptosis by activation of multiple caspases in NPRL2-transfected cells compared with untransfected cells at an equal dose of cisplatin. Furthermore, a systemic treatment with a combination of NPRL2 nanoparticles and cisplatin in a human H322 lung cancer orthotopic mouse model significantly enhanced the therapeutic efficacy of cisplatin and overcame cisplatin-induced resistance (P < 0.005). These findings implicate the potential of NPRL2 as a biomarker for predicting cisplatin response in lung cancer patients and as a molecular therapeutic agent for enhancing response and resensitizing nonresponders to cisplatin treatment.

Topics: Animals; Antineoplastic Agents, Alkylating; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cholesterol; Cisplatin; Drug Resistance, Neoplasm; Fatty Acids, Monounsaturated; Female; Gene Transfer Techniques; Genetic Vectors; Humans; Lung Neoplasms; Mice; Mice, Nude; Nanoparticles; Neoplasm Proteins; Quaternary Ammonium Compounds; Random Allocation; Recombinant Fusion Proteins; Tumor Stem Cell Assay; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays

We have investigated the effects of an improved liposomal formulation (extruded DOTAP:cholesterol (DOTAP:Chol)-DNA complex) on transgene expression in tumor cells and normal cells of murine and human origin both in vitro and in vivo. In vitro, transgene expression was significantly increased (P = 0.01) in human tumor cells compared to normal human cells. The increased transgene expression was due to increased uptake of the liposome-DNA complex by tumor cell phagocytosis. Furthermore, immunohistochemical analysis demonstrated a greater transgene expression in lung tumors than in surrounding normal tissues. Increased transgene expression due to enhanced uptake of the liposome-DNA complexes by tumor cells in vivo was also demonstrated using fluorescently labeled DOTAP:Chol liposomes. Finally, evaluation of lung tissue explants obtained from patients undergoing pulmonary resection demonstrated significantly higher (P = 0.001) transgene expression in tumor cells than in normal cells. Thus, we demonstrated that intravenous injection of DOTAP:Chol-DNA complex results in increased transgene expression in tumor and is due to increased phagocytosis of the complexes by tumor cells.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Survival; Cholesterol; DNA; Fatty Acids, Monounsaturated; Genetic Therapy; Humans; Immunoenzyme Techniques; Injections, Intravenous; Liposomes; Luciferases; Lung Neoplasms; Mice; Mice, Inbred C3H; Phagocytosis; Pinocytosis; Quaternary Ammonium Compounds; Transgenes; Tumor Cells, Cultured

ras mutations represent one of the most common oncogenetic lesions in human non-small cell lung cancer (NSCLC) and adversely affect the survival of patients afflicted with this disease. ras-directed gene therapy in the past employed primarily antisense oligonucleotides (AS-ODN) or expression vectors (such as a viral vector construct) that deliver the antisense sequence to inactivate the mutant oncogene message. These approaches produced minimal toxicity, and yet were limited in efficacy. Ribozymes present a viable alternative in antisense therapy by virtue of their renewable catalytic capability for site-specific RNA cleavage. We recently produced an adenoviral vector with a hammerhead ribozyme transgene (KRbz) that is specific for the K-ras codon 12 mutant sequence GUU, given the considerations that (a) in the United States, approx 30% of human NSCLCs express K-ras oncogene mutations, nearly all of which reside in codon 12; (b) anti-K-ras, anti-H, as well as anti-N-ras hammerhead ribozymes are potent growth inhibitors in various human cancers tested; and (c) in vitro and animal model studies suggest that ribozymes directed at oncogene (K- and H-ras C-fos, BCR-ABL) or human immunodeficiency viral gene messages are more effective than their antisense counterpart. This article describes the techniques involved in the production of the KRbz-adenoviral vector that is specific for the K-ras mutation GTT, and summarizes its in vivo antitumor effect against NSCLC xenografts expressing the relevant K-ras mutation in athymic mice.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line; DNA Mutational Analysis; Fatty Acids, Monounsaturated; Genes, ras; Genetic Techniques; Genetic Vectors; Humans; Liposomes; Lung Neoplasms; Mutation; Plasmids; Quaternary Ammonium Compounds; Recombination, Genetic; RNA, Catalytic; Sequence Analysis, DNA; Transfection