RESUMEN
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.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Compuestos Ferrosos/química , Compuestos Ferrosos/farmacología , Células Precursoras de Granulocitos/efectos de los fármacos , Leucemia Promielocítica Aguda/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Alquilación/efectos de los fármacos , Línea Celular Tumoral , Glutatión/metabolismo , Células Precursoras de Granulocitos/metabolismo , Células Precursoras de Granulocitos/patología , Humanos , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Luminiscentes/antagonistas & inhibidores , Oligonucleótidos Fosforotioatos/farmacología , ARN Mensajero/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Estructura Molecular , Oligonucleótidos Fosforotioatos/síntesis química , Oligonucleótidos Fosforotioatos/química , ARN Mensajero/genética , Relación Estructura-Actividad , Proteína Fluorescente RojaRESUMEN
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.
