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1.
Clin Cancer Res ; 20(5): 1249-58, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24423613

RESUMO

PURPOSE: Azanucleoside DNA methyltransferase (DNMT) inhibitors are currently approved by the U.S. Food and Drug Administration for treatment of myelodysplastic syndrome. The relative contributions of DNMT inhibition and other off-target effects to their clinical efficacy remain unclear. Data correlating DNA methylation reversal and clinical response have been conflicting. Consequently, it is necessary to investigate so-called off-target effects and their impact on cell survival and differentiation. EXPERIMENTAL DESIGN: Flow cytometry was used for cell cycle, apoptosis, and reactive oxygen species (ROS) accumulation analysis. Gene expression analysis was performed using real-time PCR. DNA methylation was detected by methylation-specific PCR. Mitochondrial membrane potential was analyzed using JC-1 dye staining. Western blotting was used for quantitative protein expression analysis. RESULTS: 5-Aza-2'-deoxycytidine (DAC) induced cell-cycle arrest and apoptosis in leukemia cells. p53 expression was dispensable for DAC-induced apoptosis. DAC induced delayed ROS accumulation in leukemia cells but not in solid tumor cells and p53 expression was dispensable for ROS increase. ROS increase was deoxycytidine kinase dependent, indicating that incorporation of DAC into nuclear DNA is required for ROS generation. ROS accumulation by DAC was caspase-independent and mediated the dissipation of the mitochondrial membrane potential. Concordantly, ROS scavengers diminished DAC-induced apoptosis. DAC induced the expression of different NADPH oxidase isoforms and upregulated Nox4 protein expression in an ATM-dependent manner, indicating the involvement of DNA damage signaling in Nox4 upregulation. CONCLUSION: These data highlight the importance of mechanisms other than DNA cytosine demethylation in modulating gene expression and suggest investigating the relevance of ROS accumulation to the clinical activity of DAC.


Assuntos
Azacitidina/análogos & derivados , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia/enzimologia , Leucemia/genética , Espécies Reativas de Oxigênio/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Azacitidina/farmacologia , Caspases/metabolismo , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Decitabina , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores Enzimáticos/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Metaloproteinases da Matriz/metabolismo , Oxirredução , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo
2.
Clin Cancer Res ; 15(19): 6241-9, 2009 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-19789320

RESUMO

PURPOSE: This was a phase I trial to determine the minimal effective dose and optimal dose schedule for 5-azacytidine (5-AC) in combination with sodium phenylbutyrate in patients with refractory solid tumors. The pharmacokinetics, pharmacodynamics, and antineoplastic effects were also studied. EXPERIMENTAL DESIGN: Three dosing regimens were studied in 27 patients with advanced solid tumors, and toxicity was recorded. The pharmacokinetics of the combination of drugs was evaluated. Repeat tumor biopsies and peripheral blood mononuclear cells (PBMC) were analyzed to evaluate epigenetic changes in response to therapy. EBV titers were evaluated as a surrogate measure for gene re-expression of epigenetic modulation in PBMC. RESULTS: The three dose regimens of 5-AC and phenylbutyrate were generally well tolerated and safe. A total of 48 cycles was administrated to 27 patients. The most common toxicities were bone marrow suppression-related neutropenia and anemia, which were minor. The clinical response rate was disappointing for the combination of agents. One patient showed stable disease for 5 months whereas 26 patients showed progressive disease as the best tumor response. The administration of phenylbutyrate and 5-AC did not seem to alter the pharmacokinetics of either drug. Although there were individual cases of targeted DNA methyltransferase activity and histone H3/4 acetylation changes from paired biopsy or PBMC, no conclusive statement can be made based on these limited correlative studies. CONCLUSION: The combination of 5-AC and phenylbutyrate across three dose schedules was generally well tolerated and safe, yet lacked any real evidence for clinical benefit.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Azacitidina/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Dose Máxima Tolerável , Neoplasias/tratamento farmacológico , Fenilbutiratos/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Azacitidina/efeitos adversos , Azacitidina/farmacocinética , Metilases de Modificação do DNA/antagonistas & inibidores , Metilases de Modificação do DNA/metabolismo , Relação Dose-Resposta a Droga , Feminino , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/metabolismo , Fenilbutiratos/efeitos adversos , Fenilbutiratos/farmacocinética , Resultado do Tratamento
3.
Blood ; 114(16): 3448-58, 2009 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-19652201

RESUMO

Increasing evidence shows aberrant hypermethylation of genes occurring in and potentially contributing to pathogenesis of myeloid malignancies. Several of these diseases, such as myelodysplastic syndromes (MDSs), are responsive to DNA methyltransferase inhibitors. To determine the extent of promoter hypermethylation in such tumors, we compared the distribution of DNA methylation of 14 000 promoters in MDS and secondary acute myeloid leukemia (AML) patients enrolled in a phase 1 trial of 5-azacytidine and the histone deacetylase inhibitor entinostat against de novo AML patients and normal CD34(+) bone marrow cells. The MDS and secondary AML patients displayed more extensive aberrant DNA methylation involving thousands of genes than did the normal CD34(+) bone marrow cells or de novo AML blasts. Aberrant methylation in MDS and secondary AML tended to affect particular chromosomal regions, occurred more frequently in Alu-poor genes, and included prominent involvement of genes involved in the WNT and MAPK signaling pathways. DNA methylation was also measured at days 15 and 29 after the first treatment cycle. DNA methylation was reversed at day 15 in a uniform manner throughout the genome, and this effect persisted through day 29, even without continuous administration of the study drugs. This trial was registered at www.clinicaltrials.gov as J0443.


Assuntos
Azacitidina/administração & dosagem , Metilação de DNA/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Inibidores Enzimáticos/administração & dosagem , Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Segunda Neoplasia Primária/tratamento farmacológico , Segunda Neoplasia Primária/metabolismo , Antígenos CD34 , Células da Medula Óssea/metabolismo , Feminino , Inibidores de Histona Desacetilases , Histona Desacetilases/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/metabolismo , Regiões Promotoras Genéticas , Fatores de Tempo , Proteínas Wnt/metabolismo
4.
Blood ; 114(13): 2764-73, 2009 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-19546476

RESUMO

Sequential administration of DNA methyltransferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors has demonstrated clinical efficacy in patients with hematologic malignancies. However, the mechanism behind their clinical efficacy remains controversial. In this study, the methylation dynamics of 4 TSGs (p15(INK4B), CDH-1, DAPK-1, and SOCS-1) were studied in sequential bone marrow samples from 30 patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) who completed a minimum of 4 cycles of therapy with 5-azacytidine and entinostat. Reversal of promoter methylation after therapy was observed in both clinical responders and nonresponders across all genes. There was no association between clinical response and either baseline methylation or methylation reversal in the bone marrow or purified CD34(+) population, nor was there an association with change in gene expression. Transient global hypomethylation was observed in samples after treatment but was not associated with clinical response. Induction of histone H3/H4 acetylation and the DNA damage-associated variant histone gamma-H2AX was observed in peripheral blood samples across all dose cohorts. In conclusion, methylation reversal of candidate TSGs during cycle 1 of therapy was not predictive of clinical response to combination "epigenetic" therapy. This trial is registered with http://www.clinicaltrials.gov under NCT00101179.


Assuntos
Azacitidina/administração & dosagem , Benzamidas/administração & dosagem , Dano ao DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/tratamento farmacológico , Piridinas/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Análise Citogenética , Dano ao DNA/fisiologia , Esquema de Medicação , Epigênese Genética/fisiologia , Feminino , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Fatores de Tempo
5.
Blood ; 109(7): 2781-90, 2007 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-17179232

RESUMO

MS-275 is a benzamide derivative with potent histone deacetylase (HDAC) inhibitory and antitumor activity in preclinical models. We conducted a phase 1 trial of orally administered MS-275 in 38 adults with advanced acute leukemias. Cohorts of patients were treated with MS-275 initially once weekly x 2, repeated every 4 weeks from 4 to 8 mg/m2, and after 13 patients were treated, once weekly x 4, repeated every 6 weeks from 8 to 10 mg/m2. The maximum-tolerated dose was 8 mg/m2 weekly for 4 weeks every 6 weeks. Dose-limiting toxicities (DLTs) included infections and neurologic toxicity manifesting as unsteady gait and somnolence. Other frequent non-DLTs were fatigue, anorexia, nausea, vomiting, hypoalbuminemia, and hypocalcemia. Treatment with MS-275 induced increase in protein and histone H3/H4 acetylation, p21 expression, and caspase-3 activation in bone marrow mononuclear cells. No responses by classical criteria were seen. Our results show that MS-275 effectively inhibits HDAC in vivo in patients with advanced myeloid leukemias and should be further tested, preferably in patients with less-advanced disease.


Assuntos
Benzamidas/uso terapêutico , Inibidores de Histona Desacetilases , Leucemia/tratamento farmacológico , Piridinas/uso terapêutico , Acetilação , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Benzamidas/administração & dosagem , Benzamidas/farmacocinética , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/uso terapêutico , Feminino , Histonas/metabolismo , Humanos , Leucemia/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Piridinas/administração & dosagem , Piridinas/farmacocinética
6.
Cancer Res ; 66(12): 6361-9, 2006 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-16778214

RESUMO

Optimal reexpression of most genes silenced through promoter methylation requires the sequential application of DNA methyltransferase inhibitors followed by histone deacetylase inhibitors in tumor cell cultures. Patients with myelodysplastic syndrome or acute myeloid leukemia (AML) were treated with the methyltransferase inhibitor 5-azacitidine (aza-CR) followed by the histone deacetylase inhibitor sodium phenylbutyrate. Major responses associated with cytogenetic complete response developed in patients receiving prolonged dosing schedules of aza-CR. Bisulfite sequencing of the p15 promoter in marrow DNA during the first cycle of treatment showed heterogeneous allelic demethylation in three responding patients, suggesting ongoing demethylation within the tumor clone, but no demethylation in two nonresponders. Six of six responding patients with pretreatment methylation of p15 or CDH-1 promoters reversed methylation during the first cycle of therapy (methylation-specific PCR), whereas none of six nonresponders showed any demethylation. Gene demethylation correlated with the area under the aza-CR plasma concentration-time curve. Administration of both drugs was associated with induction of acetylation of histones H3 and H4. This study provides the first demonstration that molecular mechanisms responsible for responses to DNA methyltransferase/histone deacetylase inhibitor combinations may include reversal of aberrant epigenetic gene silencing. The promising percentage of major hematologic responses justifies the testing of such combinations in prospective randomized trials.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Inibidores de Histona Desacetilases , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/enzimologia , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/enzimologia , Acetilação/efeitos dos fármacos , Doença Aguda , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Azacitidina/administração & dosagem , Azacitidina/efeitos adversos , Azacitidina/farmacocinética , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Estudos de Viabilidade , Feminino , Histona Desacetilases/genética , Histonas/metabolismo , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Fenilbutiratos/administração & dosagem , Fenilbutiratos/efeitos adversos , Fenilbutiratos/farmacocinética , Regiões Promotoras Genéticas , Resultado do Tratamento
7.
J Med Chem ; 48(20): 6350-65, 2005 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-16190761

RESUMO

The reversible acetylation of histones is critical for regulation of eukaryotic gene expression. The histone deacetylase inhibitors trichostatin (TSA, 1), MS-275 (2) and suberoylanilide hydroxamic acid (SAHA, 3) arrest growth in transformed cells and in human tumor xenografts. However, 1-3 suffer from lack of specificity among the various HDAC isoforms, prompting us to design and synthesize polyaminohydroxamic acid (PAHA) derivatives 6-21. We felt that PAHAs would be selectively directed to chromatin and associated histones by the positively charged polyamine side chain. At 1 microM, compounds 12, 15 and 20 inhibited HDAC by 74.86, 59.99 and 73.85%, respectively. Although 20 was a less potent HDAC inhibitor than 1, it was more potent than 2, more effective as an initiator of histone hyperacetylation, and significantly more effective than 2 at re-expressing p21Waf1 in ML-1 leukemia cells. On the basis of these results, PAHAs 6-21 represent an important new chemical class of HDAC inhibitors.


Assuntos
Antineoplásicos/síntese química , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/síntese química , Acetilação , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Histona Desacetilases/química , Humanos , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Isoenzimas/antagonistas & inibidores , Relação Estrutura-Atividade , Transplante Heterólogo
8.
Environ Microbiol ; 6(2): 145-58, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14756879

RESUMO

Photobacterium leiognathi forms a bioluminescent symbiosis with leiognathid fishes, colonizing the internal light organ of the fish and providing its host with light used in bioluminescence displays. Strains symbiotic with different species of the fish exhibit substantial phenotypic differences in symbiosis and in culture, including differences in 2-D PAGE protein patterns and profiles of indigenous plasmids. To determine if such differences might reflect a genetically based symbiont-strain/host-species specificity, we profiled the genomes of P. leiognathi strains from leiognathid fishes using PFGE. Individual strains from 10 species of leiognathid fishes exhibited substantial genomic polymorphism, with no obvious similarity among strains; these strains were nonetheless identified as P. leiognathi by 16S rDNA sequence analysis. Profiling of multiple strains from individual host specimens revealed an oligoclonal structure to the symbiont populations; typically one or two genomotypes dominated each population. However, analysis of multiple strains from multiple specimens of the same host species, to determine if the same strain types consistently colonize a host species, demonstrated substantial heterogeneity, with the same genomotype only rarely observed among the symbiont populations of different specimens of the same host species. Colonization of the leiognathid light organ to initiate the symbiosis therefore is likely to be oliogoclonal, and specificity of the P. leiognathi/leiognathid fish symbiosis apparently is maintained at the bacterial species level rather than at the level of individual, genomotypically defined strain types.


Assuntos
Peixes/microbiologia , Photobacterium/genética , Polimorfismo Genético , Simbiose/fisiologia , Animais , DNA Bacteriano/análise , Genoma , Photobacterium/classificação , Photobacterium/fisiologia , Filogenia , Plasmídeos/genética , Plasmídeos/metabolismo , Proteoma
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