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1.
Blood ; 119(22): 5229-38, 2012 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-22517893

RESUMO

5-Azacytidine (5-azaC) is an azanucleoside approved for myelodysplastic syndrome. Approximately 80%-90% of 5-azaC is believed to be incorporated into RNA, which disrupts nucleic acid and protein metabolism leading to apoptosis. A smaller fraction (10%-20%) of 5-azaC inhibits DNA methylation and synthesis through conversion to decitabine triphosphate and subsequent DNA incorporation. However, its precise mechanism of action remains unclear. Ribonucleotide reductase (RR) is a highly regulated enzyme comprising 2 subunits, RRM1 and RRM2, that provides the deoxyribonucleotides required for DNA synthesis/repair. In the present study, we found for the first time that 5-azaC is a potent inhibitor of RRM2 in leukemia cell lines, in a mouse model, and in BM mononuclear cells from acute myeloid leukemia (AML) patients. 5-azaC-induced RRM2 gene expression inhibition involves its direct RNA incorporation and an attenuated RRM2 mRNA stability. Therefore, 5-azaC causes a major perturbation of deoxyribonucleotide pools. We also demonstrate herein that the initial RR-mediated 5-azaC conversion to decitabine is terminated through its own inhibition. In conclusion, we identify RRM2 as a novel molecular target of 5-azaC in AML. Our findings provide a basis for its more widespread clinical use either alone or in combination.


Assuntos
Azacitidina/farmacologia , Inibidores Enzimáticos/farmacologia , Leucemia Mieloide Aguda/enzimologia , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismo , Ribonucleosídeo Difosfato Redutase/administração & dosagem , Proteínas Supressoras de Tumor/antagonistas & inibidores , Azacitidina/uso terapêutico , DNA de Neoplasias/biossíntese , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Células K562 , Leucemia Mieloide Aguda/tratamento farmacológico , Masculino , Ribonucleosídeo Difosfato Redutase/metabolismo , Proteínas Supressoras de Tumor/metabolismo
2.
J Pharmacol Exp Ther ; 329(2): 505-14, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19201992

RESUMO

Hypermethylation of 5'-cytosine-guanosine islands of tumor suppressor genes resulting in their silencing has been proposed to be a hallmark of various tumors. Modulation of DNA methylation with DNA methylation inhibitors has been shown to result in cancer cell differentiation or apoptosis and represents a novel strategy for chemotherapy. Currently, effective DNA methylation inhibitors are mainly limited to decitabine and 5-azacytidine, which still show unfavorable toxicity profiles in the clinical setting. Thus, discovery and development of novel hypomethylating agents, with a more favorable toxicity profile, is essential to broaden the spectrum of epigenetic therapy. Parthenolide, the principal bioactive sesquiterpene lactone of feverfew, has been shown to alkylate Cys(38) of p65 to inhibit nuclear factor-kappaB activation and exhibit anti-tumor activity in human malignancies. In this article, we report that parthenolide 1) inhibits DNA methyltransferase 1 (DNMT1) with an IC(50) of 3.5 microM, possibly through alkylation of the proximal thiolate of Cys(1226) of the catalytic domain by its gamma-methylene lactone, and 2) down-regulates DNMT1 expression possibly associated with its SubG(1) cell-cycle arrest or the interruption of transcriptional factor Sp1 binding to the promoter of DNMT1. These dual functions of parthenolide result in the observed in vitro and in vivo global DNA hypomethylation. Furthermore, parthenolide has been shown to reactivate tumor suppressor HIN-1 gene in vitro possibly associated with its promoter hypomethylation. Hence, our study established parthenolide as an effective DNA methylation inhibitor, representing a novel prototype for DNMT1 inhibitor discovery and development from natural structural-diversified sesquiterpene lactones.


Assuntos
Antineoplásicos/farmacologia , Metilação de DNA/efeitos dos fármacos , Lactonas/farmacologia , Sesquiterpenos/farmacologia , Alquilação , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Domínio Catalítico , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Imunoprecipitação da Cromatina , Cisteína/metabolismo , Citocinas/genética , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/genética , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Humanos , Immunoblotting , Lactonas/química , Lactonas/uso terapêutico , Camundongos , Camundongos Nus , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Regiões Promotoras Genéticas , Sesquiterpenos/química , Sesquiterpenos/uso terapêutico , Fator de Transcrição Sp1/metabolismo , Proteínas Supressoras de Tumor/genética , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Pharm Res ; 26(6): 1504-15, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19291372

RESUMO

PURPOSE: To simultaneously quantify intracellular nucleoside triphosphate (NTP) and deoxynucleoside triphosphate (dNTP) pools and to assess their changes produced by interfering with ribonucleotide reductase (RNR) expression in leukemia cells. METHODS: A HPLC-MS/MS system was used to quantify intracellular NTP and dNTP pools. RESULTS: The assay was linear between 50 nM, the lower limit of quantification (LLOQ), and 10 muM in cell lysate. The within-day coefficients of variation (CVs, n = 5) were found to be 12.0-18.0% at the LLOQ and 3.0-9.0% between 500 and 5,000 nM for dNTPs and 8.0-15.0% and 2.0-6.0% for NTPs. The between-day CVs (n = 5) were 9.0-13.0% and 3.0-11.0% for dNTPs and 9.0-13.0% and 3.0-6.0% for NTPs. The within-day accuracy values were 93.0-119.0% for both NTPs and dNTPs. ATP overlapped with dGTP and they were analyzed as a composite. This method was applied to measure basal intracellular dNTPs/NTPs in five leukemia cell lines exposed to the RNR antisense GTI-2040. Following drug treatment, dCTP and dATP levels were found to decrease significantly in MV4-11 and K562 cells. Additionally, perturbation of dNTP/NTP levels in bone marrow sample of a patient treated with GTI-2040 was detected. CONCLUSIONS: This method provides a practical tool to measure intracellular dNTP/NTP levels in cells and clinical samples.


Assuntos
Cromatografia Líquida/métodos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/terapia , Leucemia/metabolismo , Leucemia/terapia , Nucleotídeos/análise , Espectrometria de Massas em Tandem/métodos , Antimetabólitos Antineoplásicos/uso terapêutico , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Citarabina/uso terapêutico , Humanos , Modelos Lineares , Nucleotídeos/isolamento & purificação , Oligodesoxirribonucleotídeos/uso terapêutico , Sensibilidade e Especificidade
4.
J Med Chem ; 48(16): 5269-78, 2005 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-16078845

RESUMO

Carbohydrate moiety is found in many anticancer nature products. To explore the carbohydrate moiety of daunorubicin in enhancing anticancer efficacy, several daunorubicin derivatives bearing disaccharide (1-8) have been synthesized. Their cytotoxicities were tested in leukemia K562 and colon cancer SW620 cells. Topoisomerase II (topo II) poisoning was performed with the in vivo complex of topoisomerase bioassay. In both cell lines, compounds with various terminal 2,6-dideoxy sugars (compounds 1, 3, 5, and 8) showed 30- to 60-fold higher anticancer activity than compounds with 2-deoxy- or 6-deoxy sugar (compounds 6 and 7). Compounds with an alpha-linkage between two sugar units (compound 3) showed 35-fold higher anticancer activity than compounds with a beta-linkage (compound 4). In addition, the anticancer activities of these compounds correlated with their ability to target topo II mediated genomic DNA damage in vivo. Compounds 1 and 3 with 2,6-dideoxy sugars produced more covalent topo-DNA complex than compounds with 2-deoxy sugar (6) and 6-deoxy sugar (7). Compounds with an alpha-configuration of terminal 2,6-dideoxy sugar (compounds 1 and 3) showed higher topo II poisoning than their counterparts with the beta-configuration (compounds 2 and 4). These results indicate that sugar moieties in daunorubicin play a significant role in its anticancer activity and topo II inhibition. The second sugar of disaccharide daunorubicin should possess 2,6-dideoxy with alpha-linkage to the first sugar to exhibit better anticancer activity.


Assuntos
Antineoplásicos/síntese química , Daunorrubicina/análogos & derivados , Daunorrubicina/síntese química , Dissacarídeos/síntese química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Daunorrubicina/farmacologia , Dissacarídeos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Conformação Molecular , Relação Estrutura-Atividade , Inibidores da Topoisomerase II
5.
AAPS J ; 15(1): 242-9, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23180159

RESUMO

Decitabine (DAC) is used for treatment of patients with myelodysplastic syndromes and acute myeloid leukemia (AML). Following cellular uptake, DAC is activated to DAC-triphosphate (TP) and incorporated into DNA. Once incorporated into the DNA, DAC-TP binds and inactivates DNA methyltransferases (DNMTs), thereby leading to hypomethylation and re-expression of epigenetically silenced tumor suppressor genes and ultimately antileukemia activity. However, direct evidence of in vivo DAC-TP occurrence in DAC-treated patients has been difficult to demonstrate due to a lack of suitable validated analytical methodology. Thus, we developed and validated a nonradioactive sensitive and specific LC-MS/MS assay for quantification of DAC-TP. The assay is linear from 50 to 1,000 nM and from 1 to 10 µM and has a lower limit of quantitation of 50 nM and a coefficient of variation for both within- and between-day precision <20%. Following DAC treatment, we detected DAC-TP in parental and DAC-resistant AML cells (in vitro) and bone marrow (BM) and spleen of normal and leukemic mice (in vivo). Downregulation of DNMTs and correlation of DAC-TP concentration with proteins involved in mechanisms of DAC resistance were also demonstrated. The clinical applicability of this method was proven by measuring DAC-TP level in BM and blood mononuclear cells from DAC-treated AML patients. Higher levels are seemingly associated with clinical response. Monitoring the DAC-TP intracellular level may serve as a novel pharmacological endpoint for designing more effective DAC-based regimens.


Assuntos
Antimetabólitos Antineoplásicos/metabolismo , Azacitidina/análogos & derivados , Metilases de Modificação do DNA/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Polifosfatos/metabolismo , Animais , Azacitidina/metabolismo , Decitabina , Humanos , Camundongos , Camundongos Endogâmicos C57BL
6.
AAPS J ; 13(1): 131-40, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21191677

RESUMO

GTI-2040 is a potent antisense to the M2 subunit of the ribonucleotide reductase (RNR), an enzyme involved in the de novo synthesis of nucleoside triphosphates. We hypothesized that combination of GTI-2040 with the cytarabine (Ara-C) could result in an enhanced cytotoxic effect with perturbed intracellular deoxynucleotide/nucleotide (dNTP/NTP) pools including Ara-C triphosphate (Ara-CTP). This study aims to provide a direct experimental support of this hypothesis by monitoring the biochemical modulation effects, intracellular levels of Ara-CTP, dNTPs/NTPs following the combination treatment of Ara-C, and GTI-2040 in K562 human leukemia cells. GTI-2040 was introduced into cells via electroporation. A hybridization-ligation ELISA was used to quantify intracellular GTI-2040 concentrations. Real-time PCR and Western blot methods were used to measure the RNR M2 mRNA and protein levels, respectively. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay was used to measure the cytotoxicity following various drug treatments. A non-radioactive HPLC-UV method was used for measuring the intracellular Ara-CTP, while a LC-MS/MS method was used to quantify intracellular dNTP/NTP pools. GTI-2040 was found to downregulate M2 mRNA and protein levels in a dose-dependent manner and showed significant decrease in dNTP but not NTP pool. When combining GTI-2040 with Ara-C, a synergistic cytotoxicity was observed with no further change in dNTP/NTP pools. Importantly, pretreatment of K562 cells with GTI-2040 was found to increase Ara-CTP level for the first time, and this effect may be due to inhibition of RNR by GTI-2040. This finding provides a laboratory justification for the current phase I/II evaluation of GTI-2040 in combination with Ara-C in patients with acute myeloid leukemia.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Citarabina/farmacologia , Oligodesoxirribonucleotídeos/farmacologia , Ribonucleotídeo Redutases/antagonistas & inibidores , Antimetabólitos Antineoplásicos/análise , Western Blotting , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Citarabina/análise , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Humanos , Indicadores e Reagentes , Células K562 , Espectrometria de Massas , Nucleotídeos/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrofotometria Ultravioleta
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