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1.
Cancer Lett ; 250(2): 311-22, 2007 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-17184909

RESUMO

2-Methoxyestradiol (2ME) is an estradiol metabolite with anti-tumor and anti-angiogenic properties. We studied the effect of 2ME on apoptosis of MCF-7 breast cancer cells and explored a combination therapy using 2ME and a polyamine analogue, bis(ethyl)norspermine (BE-3-3-3). Determination of viable cells on day 4 of treatment with 2ME/BE-3-3-3 combinations showed synergistic effects by Chou-Talalay analysis. APO-BRDU analysis showed that there was only 1.5+/-0.5% apoptosis at 200 nM 2ME and 3.7+/-1.7% in the presence of 2.5 microM BE-3-3-3. Combination of 200 nM 2ME and 2.5 microM BE-3-3-3 resulted in 52.2+/-2.6% apoptosis. Up to 90% of the cells underwent apoptosis in the presence of 1000 nM 2ME and 2.5 microM BE-3-3-3. Combination treatments resulted in total disruption of microtubules and depletion of putrescine, spermidine and spermine. In addition, phosphorylation of Akt and nuclear localization of cyclin D1 were altered by 2ME/BE-3-3-3 combination. Our results suggest an important strategy to induce apoptosis of breast cancer cells, with potential applications in therapy.


Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Estradiol/análogos & derivados , Espermina/análogos & derivados , 2-Metoxiestradiol , Western Blotting , Neoplasias da Mama/enzimologia , Neoplasias da Mama/metabolismo , Bromodesoxiuridina , Ciclo Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Ciclina D1/metabolismo , Sinergismo Farmacológico , Estradiol/farmacologia , Humanos , Microscopia Confocal , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espermina/farmacologia
2.
Clin Cancer Res ; 12(7 Pt 1): 2038-48, 2006 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-16609013

RESUMO

PURPOSE: The purpose of this investigation is to determine the effects of physiologic levels (10-50 nmol/L) of 2-methoxyestradiol (2ME) on the growth of estrogen receptor (ER)-positive breast cancer cells and provide insights into its mechanism(s) of action. EXPERIMENTAL DESIGN: Using the ERalpha-positive breast cancer cells, we studied the effects of 2ME on cell proliferation and cell signaling. Our hypothesis is that 17beta-estradiol (E(2)) and 2ME can affect shared cell signaling pathways, leading to different outcomes in cell proliferation, depending on the absence/presence of E(2). RESULTS: E(2) stimulated the growth of MCF-7 and T-47 D cells and induced Akt phosphorylation, a nongenomic signaling pathway. In the absence of E(2), 10 to 50 nmol/L of 2ME enhanced cell growth and Akt phosphorylation. However, in the presence of E(2), 2ME inhibited E(2)-induced cell growth and prevented E(2)-induced Akt phosphorylation. Confocal microscopic studies showed that 2ME inhibited subcellular distribution of ERalpha in response to E(2) in MCF-7 and T-47D cells. 2ME also down-regulated E(2)-induced increases in cyclic AMP and ornithine decarboxylase activity. In addition, treatment of MCF-7 cells with 2ME in the presence of E(2) resulted in a decrease in ERalpha level by 72 hours. Accelerated down-regulation of ERalpha may contribute to growth inhibition in the presence of E(2)/2ME combinations. In contrast, a concentration of up to 2.5 mumol/L 2ME had no effect on the growth of ER-negative SK-BR-3 cells, either in the presence or absence of E(2). CONCLUSIONS: Our results provide evidence for the nongenomic action of 2ME in ER-positive cells. In the presence of E(2), 2ME suppressed E(2)-induced cell growth, Akt signaling, and generation of cyclic AMP, whereas it acted as an estrogen in the absence of E(2). The intriguing growth-stimulatory and growth-inhibitory effects of 2ME on breast cancer cells suggests the need for its selective use in patients.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Estradiol/análogos & derivados , Estradiol/farmacologia , Transdução de Sinais/efeitos dos fármacos , 2-Metoxiestradiol , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , AMP Cíclico/biossíntese , Relação Dose-Resposta a Droga , Receptor alfa de Estrogênio/efeitos dos fármacos , Feminino , Humanos , Fosforilação , Poliaminas/metabolismo , Relação Estrutura-Atividade , Fatores de Tempo , Células Tumorais Cultivadas
3.
Int J Biochem Cell Biol ; 38(7): 1191-1206, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16767802

RESUMO

Estrogenic regulation of gene expression is mediated by the binding of the hormone to its receptors (ERalpha and ERbeta) followed by their binding to estrogen response element (ERE). Previous studies showed that natural polyamines -- putrescine, spermidine, and spermine -- facilitated ERalpha.ERE recognition. We determined the effects of natural and synthetic polyamines on the bending of a 27-mer oligonucleotide (ODN) harboring the ERE (ERE-ODN), using fluorescence resonance energy transfer (FRET) technique. Complementary strands of the ERE-ODN were labeled with fluorescein and tetramethylrhodamine, as donor and acceptor, respectively. The ERE-ODN was intrinsically bent with an end-to-end distance of 76 +/- 2 Angstrom, compared to a theoretical value of 98 Angstrom. The end-to-end distance of the ERE-ODN was reduced to 64 Angstrom in the presence of 250 microM spermine. A control ODN with scrambled sequence did not show intrinsic bending or spermine-induced bending. Alkyl substitution at the pendant amino groups reduced the ability of spermine to bend the ERE-ODN. Both ERalpha and ERbeta decreased the end-to-end distance of the ERE-ODN, although ERalpha was more efficient than ERbeta in inducing ERE bending. Spermine-induced bending of the ERE-ODN was significantly increased by ERalpha. Fluorescence anisotropy measurement showed that the equilibrium association constant of ERalpha-ERE binding increased by 12-fold in the presence of 250 microM spermine compared to control. The free energy change (Delta G) of ERalpha.ERE complex formation was -13.1 kcal/mol at 22 degrees C in the presence of spermine. Our results suggest that polyamine-induced bending of the ERE might be a mechanism for enhancing ERalpha-ERE binding affinity and thereby fine-tuning the transcriptional response of estrogen-responsive genes.


Assuntos
DNA/química , Receptor alfa de Estrogênio/química , Receptor beta de Estrogênio/química , Oligonucleotídeos/química , Poliaminas/química , Elementos de Resposta/genética , Regulação Alostérica/fisiologia , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Estrogênios/química , Estrogênios/metabolismo , Transferência Ressonante de Energia de Fluorescência , Cinética , Conformação de Ácido Nucleico , Oligonucleotídeos/síntese química , Poliaminas/farmacologia , Putrescina/química , Proteínas Recombinantes/biossíntese , Espermidina/química , Espermina/química , Relação Estrutura-Atividade
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