RESUMO
The catalytic core of an 8-17 DNAzyme directed against STAT 3 was modified using (2'R) and (2'S) 2'-deoxy-2'-C-methyluridine and cytidine. While 2'-deoxy-2'-C-methyluridine significantly diminished the catalytic activity, 2'-deoxy-2'-C-methylcytidine replacement was better accepted, being the kact of modified DNAzymes at 8- and 11-positions comparable to the non-modified one. When 2'-O-methyl and phosphorothioate nucleotides were tested in the binding arms together with core modified DNAzymes the kcat was affected in a non predictable way, emphasizing the fact that both chemical substitutions should be considered globally. Finally, 2'-deoxy-2'-C-methyl modified DNAzymes stability was assayed finding that the double 2'-C-methyl modification in the catalytic core enhanced 70% the stability against a T47D cell lysate compared to a non-modified control.
Assuntos
DNA Catalítico/metabolismo , Biocatálise , DNA Catalítico/química , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Desoxicitidina/metabolismo , Estrutura Molecular , Uridina/análogos & derivados , Uridina/química , Uridina/metabolismoRESUMO
The combination of magnetic hyperthermia therapy with the controlled release of chemotherapeutic agents in tumors may be an efficient therapeutic with few side effects because the bioavailability, tolerance and amount of the drug can be optimized. Here, we prepared magnetoliposomes consisting of magnetite nanoparticle cores and the anticancer drug gemcitabine encapsulated by a phospholipid bilayer. The potential of these magnetoliposomes for controlled drug release and cancer treatment via hyperthermic behavior was investigated. The magnetic nanoparticle encapsulation efficiency was dependent on the initial amount of magnetite nanoparticles present at the encapsulation stage; the best formulation was 66%. We chose this formulation to characterize the physicochemical properties of the magnetoliposomes and to encapsulate gemcitabine. The mean particle size and distribution were determined by dynamic light scattering (DLS), and the zeta potential was measured. The magnetoliposome formulations all had acceptable characteristics for systemic administration, with a mean size of approximately 150 nm and a polydispersity index <0.2. The magnetoliposomes were stable in aqueous suspension for at least one week, as determined by DLS. Temperature increases due to the dissipation energy of magnetoliposome suspensions subjected to an applied alternating magnetic field (AMF) were measured at different magnetic field intensities, and the values were appropriated for cancer treatments. The drug release profile at 37 °C showed that 17% of the gemcitabine was released after 72 h. Drug release from magnetoliposomes exposed to an AMF for 5 min reached 70%.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Preparações de Ação Retardada/química , Desoxicitidina/análogos & derivados , Lipossomos/química , Nanopartículas de Magnetita/química , Animais , Antimetabólitos Antineoplásicos/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Preparações de Ação Retardada/farmacologia , Desoxicitidina/metabolismo , Desoxicitidina/farmacologia , Composição de Medicamentos , Liberação Controlada de Fármacos , Estabilidade de Medicamentos , Óxido Ferroso-Férrico/química , Hipertermia Induzida/métodos , Luz , Lipossomos/farmacologia , Campos Magnéticos , Nanopartículas de Magnetita/ultraestrutura , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Tamanho da Partícula , Fotoquimioterapia/métodos , Cultura Primária de Células , Ratos , Ratos Endogâmicos Lew , GencitabinaRESUMO
Gemcitabine is the first-line treatment for pancreatic adenocarcinoma, but is increasingly used to treat breast, bladder, and non-small cell lung cancers. Despite such broad use, intrinsic and acquired chemoresistance is common. In general, the underlying mechanisms of chemoresistance are poorly understood. Here, current knowledge of gemcitabine metabolism, mechanisms of action, sensitivity and chemoresistance reported over the past two decades are reviewed; and we also offer new perspectives to improve gemcitabine efficacy with particular reference to the treatment of pancreatic cancer.
Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Antimetabólitos Antineoplásicos/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Desoxicitidina/metabolismo , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/fisiologia , Humanos , Mediadores da Inflamação/metabolismo , Neoplasias Pancreáticas/metabolismo , GencitabinaRESUMO
BACKGROUND: Down regulation of genes coding for nucleoside transporters and drug metabolism responsible for uptake and metabolic activation of the nucleoside gemcitabine is related with acquired tumor resistance against this agent. Hydralazine has been shown to reverse doxorubicin resistance in a model of breast cancer. Here we wanted to investigate whether epigenetic mechanisms are responsible for acquiring resistance to gemcitabine and if hydralazine could restore gemcitabine sensitivity in cervical cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: The cervical cancer cell line CaLo cell line was cultured in the presence of increasing concentrations of gemcitabine. Down-regulation of hENT1 & dCK genes was observed in the resistant cells (CaLoGR) which was not associated with promoter methylation. Treatment with hydralazine reversed gemcitabine resistance and led to hENT1 and dCK gene reactivation in a DNA promoter methylation-independent manner. No changes in HDAC total activity nor in H3 and H4 acetylation at these promoters were observed. ChIP analysis showed H3K9m2 at hENT1 and dCK gene promoters which correlated with hyper-expression of G9A histone methyltransferase at RNA and protein level in the resistant cells. Hydralazine inhibited G9A methyltransferase activity in vitro and depletion of the G9A gene by iRNA restored gemcitabine sensitivity. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that acquired gemcitabine resistance is associated with DNA promoter methylation-independent hENT1 and dCK gene down-regulation and hyper-expression of G9A methyltransferase. Hydralazine reverts gemcitabine resistance in cervical cancer cells via inhibition of G9A histone methyltransferase.
Assuntos
Antimetabólitos Antineoplásicos/metabolismo , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Epigênese Genética/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Hidralazina/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico , Antimetabólitos Antineoplásicos/uso terapêutico , Western Blotting , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Primers do DNA/genética , Desoxicitidina/metabolismo , Desoxicitidina/uso terapêutico , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Feminino , Antígenos de Histocompatibilidade , Histona Desacetilases/metabolismo , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , GencitabinaRESUMO
This work has undertaken liquid chromatographic separation of nucleosides and deoxynucleosides. Two different columns with three mobile phases (A, deionized water; B, 50 mM phosphate buffer (pH 4.0); C, methanol) and slightly different gradient programs were used. The elution order was as follows: cytidine (C), 2'-deoxycytidine (dC), uridine (U), 5-methyl-2'-cytidine (5mC), 5-methyl-2'-deoxycytidine (5mdC), guanosine (G), deoxyguanosine (dG), 2'-deoxythymidine (dT), adenosine (A), and 2'-deoxyadenine (dA). Using a Luna C18 Phenomenex column (150 x 4.6 mm, 5 microm), the separation was performed at 40 degrees C with a total flow rate of 1 ml/min and a run time of 10 min. The second column was an Agilent C18 (50 x 3 mm, 1.8 microm), for which the run time was 4.5 min with a flow rate of 0.6 ml/min (25 degrees C). In application to the DNA digests from human THP-1 cells, the quantification of C, dC, U, 5mC, 5mdC, G, dG, and A was performed. The percentages of global methylation were evaluated based on the 5mdC and dC concentrations (c(5mdC) / [c(5mdC)+c(dC)], where c is concentration in microg/ml) and compared with those calculated from the respective peak areas (A(5mdC) / [A(5mdC)+A(dC)], where A is peak area at 254 nm). For peak area measurements, excellent agreement was obtained with the results reported previously in the same cell line. In the quantitative approach, the results of DNA methylation were higher but consistent with the previous data obtained using mass spectrometric detection. Comparing the analytical features of the two procedures, the use of a smaller column could be recommended because it provides efficient separation (capacity factors in the range of 1.29-10.66), a short run time, and feasibility of nucleoside and deoxynucleoside quantification in real-world samples and because it also minimizes the use of reagents.
Assuntos
DNA/química , DNA/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/análise , Animais , Cromatografia Líquida de Alta Pressão , Desoxicitidina/metabolismo , Humanos , Macrófagos/metabolismo , Monócitos/metabolismo , Espectrofotometria , Fatores de TempoRESUMO
We report the synthesis of the triphosphate of 5-methyl 4-N-[6-(p-bromobenzamido)hex-1-yl]-2'-O-deoxycytidine 3A. We also analyzed the formation of intramolecular H-bonds of 5-methyl 4-N-[n-[6-(p-bromobenzamido) caproyl amino]alk-1-yl]-2'-deoxycytidine compounds, and confirmed their presence by 1H-NMR studies. In vitro DNA labeling with modified nucleotides is preliminarily evaluated.