RESUMO
We observed that electrophilic iron(II)-clathrochelates exhibit significant cytotoxicity in human promyelocytic leukemia cells (IC50=6.5±4.6µM), which correlates with the enhancement of intracellular oxidative stress (17-fold increase with respect to the cells treated with the solvent only). Based on in vitro studies we suggested that this effect is caused by alkylation of glutathione leading to inhibition of the cellular antioxidative system and by catalytic generation of reactive oxygen species by products of the alkylation reaction.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Compostos Ferrosos/química , Compostos Ferrosos/farmacologia , Células Precursoras de Granulócitos/efeitos dos fármacos , Leucemia Promielocítica Aguda/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Alquilação/efeitos dos fármacos , Linhagem Celular Tumoral , Glutationa/metabolismo , Células Precursoras de Granulócitos/metabolismo , Células Precursoras de Granulócitos/patologia , Humanos , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Phosphorothioates are excellent antisense inhibitors, which are active both in cells and in vivo. Since their affinity to complementary ribonucleic acids is rather low, long strands (⩾20-mers) are typically required to achieve the desired biological activity. However, mismatch discrimination of long inhibitors is reduced. In contrast, shorter phosphorothioates exhibit better sequence specificity, but have in most cases too low affinity for practical applications in cells. We screened a range of terminal modifiers of a 14-mer phosphorothioate sequence, which is complementary to mRNA of a representative gene, whose protein product is fluorescent (DsRed2) and easy to monitor in cells. We found that optimal combinations of 5'- and 3'-modifications include 5'-trimethoxystilbene with 3'-uracil(anthraquinone)-cap, 5'-chloic acid derivative with 3'-uracyl(anthraquinone)-cap and 5'-cholic acid derivative with three 3'-LNA moieties. In contrast to the LNA, stabilizing and activity-enhancing effects of other mentioned modifiers for PTO/RNA duplexes have not been previously reported. We observed that the 14-mer inhibitor carrying 5'-cholic acid derivative with three 3'-LNA moieties inhibits expression of DsRed2 in cells stronger than the unmodified 21-mer. Mismatch discrimination of this inhibitor was found to be comparable to that of the unmodified 14-mer.
Assuntos
Proteínas Luminescentes/antagonistas & inibidores , Oligonucleotídeos Fosforotioatos/farmacologia , RNA Mensageiro/antagonistas & inibidores , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Células HeLa , Humanos , Proteínas Luminescentes/genética , Estrutura Molecular , Oligonucleotídeos Fosforotioatos/síntese química , Oligonucleotídeos Fosforotioatos/química , RNA Mensageiro/genética , Relação Estrutura-Atividade , Proteína Vermelha FluorescenteRESUMO
We applied 14-mer 2'-OMe RNAs as inhibitors of selected micro RNAs. To improve their properties, we introduced a trimethoxystilbene residue at the 5'-terminus and three 2'-fluoro-2'-deoxynucleotides at the 3'-terminus to obtain potent inhibitors, whose mismatch discrimination is substantially better than that of typically applied >18-mers.
