RESUMO
In this study, we prepared oligodeoxynucleotides (ODNs) containing the uridine base modified by an alkyl chain at the 5-position (AU) and characterized their aggregate formation, localization, and functions in cells. These experiments revealed that aggregates of these ODNs were readily transported into cells, but their localization was dependent upon the number of hydrophobic units. ODNs with one modified AU were transported in the cytosol, while ODNs with multiple AU modifications resulted in their accumulation at the cell membrane. We also examined the ability of the AU-modified ODNs to capture small molecules at the cell membrane and their cellular uptake. We positioned a thioflavin-T (ThT)-binding aptamer on the cell membrane by means of hybridization with ODNs with three AUs at the strand end. Treatment with ThT resulted in its efficient uptake into cells, due to the capture of the ThT by the aptamers on the cell membrane. Thus, we demonstrated the functionalization of cell membranes with modified ODNs and the efficient delivery of small molecules into the cells.
Assuntos
Membrana Celular/metabolismo , Oligodesoxirribonucleotídeos/metabolismo , Uridina/metabolismo , Células A549 , Membrana Celular/química , Humanos , Estrutura Molecular , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/química , Espectrometria de Fluorescência , Células Tumorais Cultivadas , Uridina/químicaRESUMO
Among the various enzymes, reductases that catalyze one-electron reduction are involved in the selective activation of functional compounds or materials in hypoxia, which is one of the well-known pathophysiological characteristics of solid tumors. Enzymatic one-electron reduction has been recognized as a useful reaction that can be applied in the design of tumor hypoxia-targeting drugs. In this report, we characterized the enzymatic reaction of 5-fluorodeoxyuridine (FdUrd) prodrug bearing an indolequinone unit (IQ-FdUrd), which is a substrate of reductases. IQ-FdUrd was activated to release FdUrd under hypoxic conditions after treatment with cytochrome NADPH P450 reductase. We also confirmed that IQ-FdUrd showed selective cytotoxicity in hypoxic tumor cells.
Assuntos
Hipóxia Celular/efeitos dos fármacos , Floxuridina/farmacologia , Indolquinonas/farmacologia , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Pró-Fármacos/farmacologia , Relação Dose-Resposta a Droga , Ativação Enzimática , Floxuridina/química , Floxuridina/metabolismo , Humanos , Indolquinonas/química , Indolquinonas/metabolismo , Estrutura Molecular , NADP/metabolismo , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Relação Estrutura-AtividadeRESUMO
We previously designed and reported a novel class of drugs, namely hybrid peptides, which are chemically synthesized and composed of a targeted binding peptide and a lytic-type peptide containing cationic amino acid residues that cause cancer cell death. In the present study, we screened for peptides that bind to interleukin-13 receptor alpha 2 (IL-13Rα2) by using a T7 random peptide phage display library system and isolated several positive phage clones. The A2b11 peptide, which was one of the positive clones, was shown to bind to IL-13Rα2 protein by Biacore analysis and a binding assay using glioblastoma (GB) cell lines. This peptide was linked with a lytic peptide containing a linker sequence to form the IL-13Rα2-lytic hybrid peptide. The IL-13Rα2-lytic hybrid peptide showed cytotoxic activity against GB cell lines in vitro. The IL-13Rα2-lytic hybrid peptide also affected Akt and Erk1/2 activation following treatment with interleukin-13 and induced rapid ATP dynamics in GB cells. Anti-tumor activity of the IL-13Rα2-lytic hybrid peptide was observed in vivo after intratumoral injection in a mouse xenograft model of human GB cells. These results suggest that the IL-13Rα2-lytic hybrid peptide might be a potent therapeutic option for patients with GB.
Assuntos
Antineoplásicos/química , Subunidade alfa2 de Receptor de Interleucina-13/antagonistas & inibidores , Peptídeos/química , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Humanos , Subunidade alfa2 de Receptor de Interleucina-13/genética , Subunidade alfa2 de Receptor de Interleucina-13/metabolismo , Camundongos , Camundongos Nus , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Biblioteca de Peptídeos , Peptídeos/metabolismo , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Exogenous nucleic acids showed low efficiency regarding cellular uptake and low stability in biological conditions; therefore, a number of techniques have been developed to improve their basic properties. One of the best solutions is the application of nanosized particles consisting of oligonucleotides that penetrate the cell membrane without any additives and exhibit high stability in cells. In this report, we employed a simple approach to address the basic properties of nanoparticles of oligonucleotides in biological systems. We prepared BODIPY-labeled oligonucleotides that carried an exclusive modification at the strand end. BODIPY shows high hydrophobicity and fluorescent emission; therefore, the oligonucleotides formed nanosized aggregates in aqueous solution and their behaviors in cells or tissues were easily tracked. Detailed experiments revealed that aggregate formation was indispensable for the high cellular uptake of the oligonucleotides via scavenger-receptor-mediated endocytosis. In addition, the aggregates provided an efficient gene regulation in living cells and tumor tissues transplanted into mice.
RESUMO
The use of DNA aggregates could be a promising strategy for the molecular imaging of biological functions. Herein, phosphorescent oligodeoxynucleotides were designed with the aim of visualizing oxygen fluctuation in tumor cells. DNA-ruthenium conjugates (DRCs) that consisted of oligodeoxynucleotides, a phosphorescent ruthenium complex, a pyrene unit for high oxygen responsiveness, and a nitroimidazole unit as a tumor-targeting unit were prepared. In general, oligonucleotides have low cell permeability because of their own negative charges; however, the DRC formed aggregates in aqueous solution due to the hydrophobic pyrene and nitroimidazole groups, and smoothly penetrated the cellular membrane to accumulate in tumor cells in a hypoxia-selective manner. The oxygen-dependent phosphorescence of DRC in cells was also observed. In vivo experiments revealed that aggregates of DRC accumulated in hypoxic tumor tissue that was transplanted into the left leg of mice, and showed that oxygen fluctuations in tumor tissue could be monitored by tracking of the phosphorescence emission of DRC.
Assuntos
Substâncias Luminescentes/química , Oligodesoxirribonucleotídeos/química , Oxigênio/análise , Células A549 , Animais , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Complexos de Coordenação/efeitos da radiação , Corantes Fluorescentes/química , Humanos , Concentração de Íons de Hidrogênio , Luz , Substâncias Luminescentes/síntese química , Substâncias Luminescentes/efeitos da radiação , Camundongos Endogâmicos BALB C , Imagem Molecular , Nitroimidazóis/síntese química , Nitroimidazóis/química , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/efeitos da radiação , Oxazinas/química , Oxigênio/química , Pirenos/síntese química , Pirenos/química , Rutênio , Hipóxia TumoralRESUMO
Hypoxia is a characteristic feature of solid tumors. Herein, we have developed novel hypoxia-sensitive probes (IM-ACs) for Raman spectroscopic analysis, consisting of nitroimidazole as a hypoxia-targeting unit and acetylene group as the signal-emitting unit. Among IM-ACs synthesized in this study, IM-AC possessing a diacetylene group (IM-AC 3), showed suitable properties as a hypoxia indicator. When administered to A549 cells, we observed a strong signal of IM-AC 3 around 2200cm-1 in the Raman spectra from hypoxic cells. Ex vivo experiments suggest that IM-AC 3 remained in hypoxic tumor tissue and emitted a strong signal.
Assuntos
Alcinos/química , Hipóxia Celular , Neoplasias Pulmonares/patologia , Nitroimidazóis/química , Análise Espectral Raman/métodos , Células A549 , Humanos , Sondas MolecularesRESUMO
Chemical conversion of specific bioactive molecules by external stimuli in living cells is a powerful noninvasive tool for clarification of biomolecular interactions and to control cellular functions. However, in chaotic biological environments, it has been difficult to induce arbitrary photochemical reactions on specific molecules because of their poor molecular selectivity. Here we report a selective and nontoxic photochemical reaction system utilizing photoactivated mesoporous silica nanoparticles to control biological functions. Methylene blue modification within nanoparticle pores for photosensitization produced singlet oxygen confined to the pore that could mediate selective oxidation of small molecules without any damage to living cells. This intracellular photochemical system produced bioactive molecules in situ and remotely controlled the cell cycle phase. We also confirmed that this photoreaction could be applied to control cell cycle phase in tumor tissue transplanted in mice. The cell cycle phase in the cells in mice, to which our system was administered, was arrested at the G2/M phase upon photoirradiation. We demonstrate a simple and promising method for the exogenous conversion of an intracellular biomolecule to another functional compound.
Assuntos
Nanopartículas , Processos Fotoquímicos , Dióxido de Silício , Oxigênio Singlete/química , Animais , Camundongos , Oxirredução , Espectrometria de FluorescênciaRESUMO
A Cu(i)-catalyzed azide-alkyne cycloaddition reaction (CuAAC) has been utilized to prepare novel triazole-linked cationic porphyrin-psoralen conjugates that exhibited significant photocytotoxicity against A549 cancer cells (IC50 = 84 nM).
Assuntos
DNA/química , Ficusina/síntese química , Porfirinas/síntese química , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Catálise , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cobre/química , Ficusina/química , Ficusina/farmacologia , Fluorescência , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Porfirinas/química , Porfirinas/farmacologiaRESUMO
A novel cationic porphyrin-quinoxaline conjugate 8 was prepared in good yield by the coupling of activated quinoxaline carboxylic acid 5 with an appropriate aminoporphyrin. The UV-vis spectra of conjugate 8 with the addition of ctDNA shows substantial hypochromicity (39%) and a red shift (12 nm) in the Soret band indicating intercalation and self stacking along the surface. The binding constant of conjugate 8 with ctDNA was determined to be 1.26×10(6) M(-1). The porphyrin-quinoxaline conjugate 8 displayed enhanced photocytotoxicity (IC50=0.06 µM) when compared to TMPyP against A549 cancer cells.
Assuntos
Antineoplásicos/química , Porfirinas/química , Quinoxalinas/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/toxicidade , Cátions/química , Bovinos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , DNA/química , DNA/metabolismo , Clivagem do DNA/efeitos dos fármacos , Humanos , Porfirinas/síntese química , Porfirinas/toxicidade , Quinoxalinas/síntese química , Quinoxalinas/toxicidade , Espectrometria de Fluorescência , Raios UltravioletaRESUMO
A poly(ethylene glycol) (PEG)-modified dendritic poly(L-lysine) (PEG-WeKG6) containing tryptophan residues in its core was synthesized as an oligonucleotide carrier to tumors after systemic injection. PEG- WeKG6 formed a stable complex with double-stranded deoxyoligonucleotide (ODN). The size and the zeta-potential of the complex were smaller than those of a dendritic poly(L-lysine) without PEG (WeKG6). To study the biodistribution of the complexes in tumor-bearing mice after intravenous injection, the den- drimers and the oligonucleotide were labeled with gadolinium and Cy5, respectively. Our results show that PEG modification of the dendrimer improved the stability of ODN in blood circulation. Effective accumulation of the PEG-WeKG6/ODN complex in the tumor tissue was found 24 h after the injection. These results indicate that PEG-WeKG6 is suitable for forming a complex with any genetic or therapeutic material for efficient delivery to tumors.