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1.
Mol Pharmacol ; 73(5): 1568-77, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18299310

RESUMO

Reversal of the multidrug-resistant (MDR) phenotype is very important for chemotherapy success. In fact, the expression of the MDR1 gene-encoded P-glycoprotein (P-gp) actively expels antitumor agents such as daunomycin (DNM) out of the cells, resulting in drug resistance. We show that upon conjugation to triplex-forming oligonucleotides, it is possible to address DNM in resistant cells (MCF7-R and NIH-MDR-G185). The oligonucleotide moiety of the conjugate changes the cellular penetration properties of the antitumor agent that is no more the target of P-gp in resistant cells. We observe an accumulation of conjugated DNM in cells up to 72 h. For more efficient delivery in the cells' nuclei, transfectant agents must be used. In addition, the conjugate recognizes a sequence located in exon 3 of MDR1, and it inhibits its gene expression as measured both by Western blot and by reverse transcription-polymerase chain reaction.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , DNA/farmacologia , Daunorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Oligonucleotídeos/síntese química , Oligonucleotídeos/farmacologia , Animais , Sequência de Bases , Linhagem Celular Tumoral , DNA/síntese química , Daunorrubicina/química , Regulação para Baixo/efeitos dos fármacos , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Cinética , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Espectrometria de Fluorescência , Transfecção
2.
Nucleic Acids Res ; 34(20): 5740-51, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17041234

RESUMO

A preferential target of antisense oligonucleotides directed against human PGY/MDR1 mRNA is a hairpin containing a stem with a G*U wobble pair, capped by the purine-rich 5'r(GGGAUG)3' hexaloop. This hairpin is studied by multidimensional NMR and restrained molecular dynamics, with special emphasis on the conformation of south sugars and non-standard phosphate linkages evidenced in both the stem and the loop. The hairpin is found to be highly structured. The G*U wobble pair, a strong counterion binding site, displays structural particularities that are characteristic of this type of mismatch. The upper part of the stem undergoes distortions that optimize its interactions with the beginning of the loop. The loop adopts a new fold in which the single-stranded GGGA purine tract is structured in A-like conformation stacked in continuity of the stem and displays an extensive hydrogen bonding surface for recognition. The remarkable hairpin stability results from classical inter- and intra-strand interactions reinforced by numerous hydrogen bonds involving unusual backbone conformations and ribose 2'-hydroxyl groups. Overall, this work emphasizes numerous features that account for the well-ordered structure of the whole hairpin and highlights the loop properties that facilitate interaction with antisense oligonucleotides.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Modelos Moleculares , Oligonucleotídeos Antissenso/química , RNA Mensageiro/química , Pareamento de Bases , Carboidratos/química , Humanos , Ligação de Hidrogênio , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Fosfatos/química , Purinas/análise , Soluções
3.
Biochem Pharmacol ; 70(10): 1424-30, 2005 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-16214115

RESUMO

MDR1 overexpression is one form of the multidrug resistance (MDR) phenotype, which can be acquired by patients initially responsive to chemotherapy. Because of the high toxicity of the inhibitors of P-glycoprotein (P-gp), the protein encoded by MDR1, attention has been focused on selective modulation of the MDR1 gene. Small interfering RNAs (siRNAs) were shown to be powerful tools for such a purpose, even when used at low concentrations (< or =20 nM) in order to avoid sequence nonspecific effects. Two siRNAs used at 20 nM were shown to lead to efficient down-regulation of MDR1 at the protein level (only ca. 20% total P-gp expression remaining) in the doxorubicin selected MCF7-R human cell line. Cell surface expression of P-gp was inhibited, leading to reversal of the drug efflux phenotype (about 40% reversal with the most efficient siRNA) and enhancement of chemosensitivity (about 35%). At the mRNA level, the down-regulation of MDR1 obtained with the most efficient siRNA increased from about 50% (5 nM siRNA) to 60% (10 or 20 nM). The advantage of using a combination of siRNAs instead of a single one has been suggested.


Assuntos
Linhagem Celular Tumoral , Genes MDR/efeitos dos fármacos , Genes MDR/genética , RNA Interferente Pequeno/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/biossíntese , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Contagem de Células/métodos , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Daunorrubicina/análise , Daunorrubicina/farmacologia , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Citometria de Fluxo/métodos , Fluorescência , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Humanos , RNA Mensageiro/análise , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Interferente Pequeno/síntese química , RNA Interferente Pequeno/classificação , Fatores de Tempo , Azul Tripano , Verapamil/farmacologia
4.
Org Biomol Chem ; 2(6): 915-21, 2004 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-15007422

RESUMO

The binding affinity for a 12-bp dsDNA of Antennapedia helix 3 analogues, major groove binders, has been measured by displacement of prebound ethidium bromide, a fluorescent displacement assay proposed for minor groove binders by Boger et al.(J. Am. Chem. Soc., 2000, 122, 6382-6394). Relative binding affinities determined by this method were compared to those obtained by gel mobility shift and footprinting assays for the 12-bp dsDNA and a 178-bp DNA fragment. The present work demonstrates that the fluorescence displacement assay is suitable for rapid screening of major groove binders, even though about 60 to 70% of the prebound ethidium bromide is displaced by these peptides. Total (100%) displacement of ethidium bromide was serendipitously achieved by addition in the peptide sequence, at the N-terminus, of a S-3-nitro-2-pyridinesulfenyl-N-acetyl-cysteine residue. S-3-nitro-2-pyridinesulfenylcysteine was shown to (i) bind to dsDNA with a micromolar affinity and (ii) direct within DNA grooves a peptide with no affinity for dsDNA.


Assuntos
Proteínas de Ligação a DNA/química , DNA/química , Etídio/química , Proteínas de Homeodomínio/química , Proteínas Nucleares/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Proteína do Homeodomínio de Antennapedia , Sequência de Bases , Sítios de Ligação , Cisteína/química , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Estrutura Secundária de Proteína , Fatores de Transcrição/metabolismo
5.
Oligonucleotides ; 14(3): 191-8, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15625914

RESUMO

Small interfering RNAs (siRNAs) are powerful tools in specifically silencing gene expression. Nevertheless, their efficiency can be limited when targeting proteins with an unusually long half-life, such as P-glycoprotein (P-gp), which is involved in the multidrug resistance phenomenon. P-gp is characterized by a long half-life, which may vary depending on the cell line and, for some of them, on serum deprivation or high cell density. In the present paper, involvement of an exponential cell growth phase in the improvement of siRNA efficiency has been suggested. The doxorubicin-selected human line MCF7-R was shown to be a more adapted model than NIH-MDR-G185 cells stably transfected with human mdr1. Nonspecific effects occurring at moderate (100 nM) siRNA concentration have been shown. Two efficient siRNAs led to a very satisfactory P-gp extinction (only 20% P-gp expression remaining) with siRNA concentration as low as 20 nM.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Animais , Resistência a Múltiplos Medicamentos , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Genes MDR , Humanos , Camundongos , RNA Interferente Pequeno/genética , Células Tumorais Cultivadas
6.
Biochem Pharmacol ; 65(5): 747-54, 2003 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-12628488

RESUMO

In the perspective of reversing multidrug resistance through antisense strategy while avoiding non-antisense effects of all-phosphorothioate oligonucleotides which non-specifically bind to proteins, a minimally modified antisense phosphodiester oligodeoxyribonucleotide has been designed against mdr1, one of the multidrug resistance genes. Its stability in lysates prepared from NIH/3T3 cells transfected with the human mdr1 gene has already been demonstrated. Confocal microspectrofluorometry using a fluorescence resonance energy transfer technique allowed its stability inside living cells to be proven. Its internalization into the cells was achieved with different delivery agents (addition of a cholesteryl group, Superfect or an amphotericin B cationic derivative) and has been followed by fluorescence imaging. For each of the delivery systems, Western blotting allowed its antisense efficiency to be compared to that of an all-phosphorothioate antisense oligonucleotide. No antisense efficiency was demonstrated for the minimally modified ODN when internalized with Superfect. In both other cases, the best extinction of the P-glycoprotein expression has always been achieved with the all-phosphorothioate antisense. While the difference was significant in the case the amphotericin B derivative was used as delivery agent (20% remaining protein expression with the all-phosphorothioate vs. 40% with the minimally modified antisense), it was negligible for the cholesterol conjugates (2% vs. 6%). It is of great interest to prove that an almost all-phosphodiester oligonucleotide can be an efficient antisense against an overexpressed gene. The reduction of non-antisense effects as non-specific binding to proteins are of importance in the case relatively high ODN concentrations are used, which can prove to be necessary in the case of overexpressed genes.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Genes MDR/efeitos dos fármacos , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Células 3T3 , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Animais , Transporte Biológico , Estabilidade de Medicamentos , Camundongos , Oligodesoxirribonucleotídeos Antissenso/química , Fosfatos/química
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