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1.
Cancer Cell ; 6(2): 151-8, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15324698

RESUMO

The frequent silencing of tumor suppressor genes by altered cytosine methylation and chromatin structural changes makes this process an attractive target for epigenetic therapy. Here we show that zebularine, a stable DNA cytosine methylation inhibitor, is preferentially incorporated into DNA and exhibits greater cell growth inhibition and gene expression in cancer cell lines compared to normal fibroblasts. In addition, zebularine preferentially depleted DNA methyltransferase 1 (DNMT1) and induced expression of cancer-related antigen genes in cancer cells relative to normal fibroblasts. Our results demonstrate that zebularine can be selective toward cancer cells and may hold clinical promise as an anticancer therapy.


Assuntos
Antineoplásicos/uso terapêutico , DNA de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Nucleosídeos de Pirimidina/uso terapêutico , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Citidina/análogos & derivados , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Genes p16 , Humanos , Neoplasias/genética
2.
Nat Rev Drug Discov ; 5(1): 37-50, 2006 01.
Artigo em Inglês | MEDLINE | ID: mdl-16485345

RESUMO

The initiation and progression of cancer is controlled by both genetic and epigenetic events. Unlike genetic alterations, which are almost impossible to reverse, epigenetic aberrations are potentially reversible, allowing the malignant cell population to revert to a more normal state. With the advent of numerous drugs that target specific enzymes involved in the epigenetic regulation of gene expression, the utilization of epigenetic targets is emerging as an effective and valuable approach to chemotherapy as well as chemoprevention of cancer.


Assuntos
Antineoplásicos , Epigênese Genética , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Metilação de DNA/efeitos dos fármacos , Inibidores de Histona Desacetilases , Humanos , Estrutura Molecular , Neoplasias/genética
3.
Cancer Res ; 67(13): 6400-8, 2007 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-17616700

RESUMO

The major goal of epigenetic therapy is to reverse aberrant promoter hypermethylation and restore normal function of tumor suppressor genes by the use of chromatin-modifying drugs. Decitabine, or 5-aza-2'-deoxycytidine (5-aza-CdR), is a well-characterized drug that is now Food and Drug Administration approved for the treatment of myelodysplastic syndrome. Although 5-aza-CdR is an extremely potent inhibitor of DNA methylation, it is subject to degradation by hydrolytic cleavage and deamination by cytidine deaminase. We show that short oligonucleotides containing a 5-aza-CdR can also inhibit DNA methylation in cancer cells at concentrations comparable with 5-aza-CdR. Detailed studies with S110, a dinucleotide, showed that it works via a mechanism similar to that of 5-aza-CdR after incorporation of its aza-moiety into DNA. Stability of the triazine ring in aqueous solution was not improved in the S110 dinucleotide; however, deamination by cytidine deaminase was dramatically decreased. This is the first demonstration of the use of short oligonucleotides to provide effective delivery and cellular uptake of a nucleotide drug and protection from enzymatic degradation. This approach may pave the way for more stable and potent inhibitors of DNA methylation as well as provide means for improving existing therapeutics.


Assuntos
Antineoplásicos/farmacologia , Azacitidina/análogos & derivados , Sistemas de Liberação de Medicamentos , Terapia Genética/métodos , Oligonucleotídeos/farmacologia , Azacitidina/administração & dosagem , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Metilação de DNA , Decitabina , Relação Dose-Resposta a Droga , Epigênese Genética , Humanos , Modelos Químicos , Síndromes Mielodisplásicas/tratamento farmacológico , Oligonucleotídeos/química , Triazinas/química , Neoplasias da Bexiga Urinária/tratamento farmacológico
4.
Mol Cancer Ther ; 4(10): 1515-20, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16227400

RESUMO

DNA cytosine methylation plays a considerable role in normal development, gene regulation, and carcinogenesis. Hypermethylation of the promoters of some tumor suppressor genes and the associated silencing of these genes often occur in certain cancer types. The reversal of this process by DNA methylation inhibitors is a promising new strategy for cancer therapy. In addition to the four well-characterized nucleoside analogue methylation inhibitors, 5-azacytidine, 5-aza-2'-deoxycytidine (5-Aza-CdR), 5-fluoro-2'-deoxycytidine, and zebularine, there is a growing list of non-nucleoside inhibitors. However, a systemic study comparing these potential demethylating agents has not been done. In this study, we examined three non-nucleoside demethylating agents, (-)-epigallocatechin-3-gallate, hydralazine, and procainamide, and compared their effects and potencies with 5-Aza-CdR, the most potent DNA methylation inhibitor. We found that 5-Aza-CdR is far more effective in DNA methylation inhibition as well as in reactivating genes, compared with non-nucleoside inhibitors.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/análogos & derivados , Catequina/análogos & derivados , Metilação de DNA/efeitos dos fármacos , Hidralazina/farmacologia , Procainamida/farmacologia , Azacitidina/farmacologia , Carcinoma de Células de Transição/tratamento farmacológico , Carcinoma de Células de Transição/genética , Catequina/farmacologia , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Citidina/análogos & derivados , Citidina/farmacologia , Metilases de Modificação do DNA/antagonistas & inibidores , Decitabina , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Inativação Gênica/efeitos dos fármacos , Células HT29 , Humanos , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
5.
Ann N Y Acad Sci ; 1058: 246-54, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16394141

RESUMO

1-(Beta-d-ribofuranosyl)-1,2-dihydropyrimidin-2-one (zebularine) corresponds structurally to cytidine minus the exocyclic 4-amino group. The increased electrophilic character of its simple aglycon endows the molecule with unique biologic properties as a potent inhibitor of both cytidine deaminase and DNA cytosine methyltransferase. The latter activity makes zebularine a promising antitumor agent that is hydrolytically stable, preferentially targets cancer cells, and shows activity both in vitro and in experimental animals, even after oral administration.


Assuntos
Antineoplásicos/farmacologia , Citidina/análogos & derivados , Epigênese Genética , Neoplasias/tratamento farmacológico , Administração Oral , Animais , Citidina/farmacologia , Citidina Desaminase/metabolismo , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Humanos , Cinética , Modelos Químicos , Neoplasias/genética
6.
Artigo em Inglês | MEDLINE | ID: mdl-16247946

RESUMO

1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one (zebularine) is structurally 4-deamino cytidine. The increased electrophilic character of this simple aglycon endows the molecule with unique chemical and biological properties, making zebularine a versatile starting material for the synthesis of complex nucleosides and an effective inhibitor of cytidine deaminase and DNA cytosine methyltransferase. Zebularine is a stable, antitumor agent that preferentially targets cancer cells and shows activity both in vitro and in experimental animals, even after oral administration.


Assuntos
Antineoplásicos/farmacologia , Citidina/análogos & derivados , Neoplasias/tratamento farmacológico , Administração Oral , Animais , Citidina/farmacologia , Citidina Desaminase/antagonistas & inibidores , DNA/química , DNA/efeitos dos fármacos , Epigênese Genética , Humanos , Modelos Químicos , Neoplasias/genética
7.
Mol Cancer Ther ; 9(5): 1443-50, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20442312

RESUMO

Methylation of CpG islands in promoter regions is often associated with gene silencing and aberrant DNA methylation occurs in most cancers, leading to the silencing of some tumor suppressor genes. Reversal of this abnormal hypermethylation by DNA methylation inhibitors is effective in reactivating methylation-silenced tumor suppressor genes both in vitro and in vivo. Several DNA methylation inhibitors have been well studied; the most potent among them is 5-aza-2'-deoxycytidine (5-Aza-CdR), which can induce myelosuppression in patients. S110 is a dinucleotide consisting of 5-Aza-CdR followed by a deoxyguanosine, which we previously showed to be effective in vitro as a DNA methylation inhibitor while being less prone to deamination by cytidine deaminase, making it a promising alternative to 5-Aza-CdR. Here, we show that S110 is better tolerated than 5-Aza-CdR in mice and is as effective in vivo in inducing p16 expression, reducing DNA methylation at the p16 promoter region, and retarding tumor growth in human xenograft. We also show that S110 is effective by both i.p. and s.c. deliveries. S110 therefore is a promising new agent that acts similarly to 5-Aza-CdR and has better stability and less toxicity.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/análogos & derivados , Proliferação de Células/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Neoplasias/patologia , Oligonucleotídeos/farmacologia , Carga Tumoral/efeitos dos fármacos , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Azacitidina/química , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Decitabina , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Biológicos , Neoplasias/tratamento farmacológico , Oligonucleotídeos/uso terapêutico , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Mol Cell Biol ; 29(19): 5366-76, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19620278

RESUMO

Proper DNA methylation patterns are essential for mammalian development and differentiation. DNA methyltransferases (DNMTs) primarily establish and maintain global DNA methylation patterns; however, the molecular mechanisms for the generation and inheritance of methylation patterns are still poorly understood. We used sucrose density gradients of nucleosomes prepared by partial and maximum micrococcal nuclease digestion, coupled with Western blot analysis to probe for the interactions between DNMTs and native nucleosomes. This method allows for analysis of the in vivo interactions between the chromatin modification enzymes and their actual nucleosomal substrates in the native state. We show that little free DNA methyltransferase 3A and 3B (DNMT3A/3B) exist in the nucleus and that almost all of the cellular contents of DNMT3A/3B, but not DNMT1, are strongly anchored to a subset of nucleosomes. This binding of DNMT3A/3B does not require the presence of other well-known chromatin-modifying enzymes or proteins, such as proliferating cell nuclear antigen, heterochromatin protein 1, methyl-CpG binding protein 2, Enhancer of Zeste homolog 2, histone deacetylase 1, and UHRF1, but it does require an intact nucleosomal structure. We also show that nucleosomes containing methylated SINE and LINE elements and CpG islands are the main sites of DNMT3A/3B binding. These data suggest that inheritance of DNA methylation requires cues from the chromatin component in addition to hemimethylation.


Assuntos
Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Nucleossomos/enzimologia , Linhagem Celular , Cromatina , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Ligação Proteica , Sequências Repetitivas de Ácido Nucleico , DNA Metiltransferase 3B
9.
Mol Cancer Ther ; 8(6): 1579-88, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19509260

RESUMO

DNA methylation, histone modifications, and nucleosomal occupancy collaborate to cause silencing of tumor-related genes in cancer. The development of drugs that target these processes is therefore important for cancer therapy. Inhibitors of DNA methylation and histone deacetylation have been approved by the Food and Drug Administration for treatment of hematologic malignancies. However, drugs that target other mechanisms still need to be developed. Recently, 3-deazaneplanocin A (DZNep) was reported to selectively inhibit trimethylation of lysine 27 on histone H3 (H3K27me3) and lysine 20 on histone H4 (H4K20me3) as well as reactivate silenced genes in cancer cells. This finding opens the door to the pharmacologic inhibition of histone methylation. We therefore wanted to further study the mechanism of action of DZNep in cancer cells. Western blot analysis shows that DZNep globally inhibits histone methylation and is not selective. Two other drugs, sinefungin and adenosine dialdehyde, have similar effects as DZNep on H3K27me3. Intriguingly, chromatin immunoprecipitation of various histone modifications and microarray analysis show that DZNep acts through a different pathway than 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor. These observations give us interesting insight into how chromatin structure affects gene expression. We also determined the kinetics of gene activation to understand if the induced changes were somatically heritable. We found that upon removal of DZNep, gene expression is reduced to its original state. This suggests that there is a homeostatic mechanism that returns the histone modifications to their "ground state" after DZNep treatment. Our data show the strong need for further development of histone methylation inhibitors.


Assuntos
Adenosina/análogos & derivados , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Adenosina/química , Adenosina/farmacologia , Azacitidina/análogos & derivados , Azacitidina/química , Azacitidina/farmacologia , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Decitabina , Proteína Potenciadora do Homólogo 2 de Zeste , Inibidores Enzimáticos/farmacologia , Perfilação da Expressão Gênica , Humanos , Queratina-7/genética , Queratina-7/metabolismo , Metilação/efeitos dos fármacos , Estrutura Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Complexo Repressor Polycomb 2 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
J Med Chem ; 51(23): 7593-601, 2008 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-19006382

RESUMO

We report herein the application of the phosphoramidate ProTide technology to improve the metabolism of the DNA methytransferase inhibitor, zebularine (Z). Zebularine is a riboside that must undergo a complex metabolic transformation before reaching the critical 2'-deoxyzebularine 5'-triphosphate (dZTP). Because 2'-deoxyzebularine (dZ) is not phosphorylated and therefore inactive, the ProTide strategy was employed to bypass the lack of phosphorylation of dZ and the inefficient reduction of zebularine 5'-diphosphate by ribonucleotide-diphosphate reductase required for zebularine. Several compounds were identified as more potent inhibitors of DNA methylation and stronger inducers of p16 tumor suppressor gene than zebularine. However, their activity was dependent on the administration of thymidine to overcome the potent inhibition of thymidylate synthase (TS) and deoxycytidine monophosphate (dCMP) deaminase by dZMP, which deprives cells of essential levels of thymidine. Intriguingly, the activity of the ProTides was cell line-dependent, and activation of p16 was manifest only in Cf-Pac-1 pancreatic ductal adenocarcinoma cells.


Assuntos
Adenocarcinoma/metabolismo , Amidas/química , Citidina/análogos & derivados , Metilação de DNA/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Genes p16/efeitos dos fármacos , Neoplasias Pancreáticas/metabolismo , Ácidos Fosfóricos/química , Adenocarcinoma/genética , Adenocarcinoma/patologia , Citidina/síntese química , Citidina/química , Citidina/farmacologia , DCMP Desaminase/antagonistas & inibidores , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estereoisomerismo , Timidilato Sintase/antagonistas & inibidores , Células Tumorais Cultivadas
11.
Cancer Prev Res (Phila) ; 1(4): 233-40, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19138966

RESUMO

Recent successes in the application of epigenetic drugs for the treatment of myelodysplastic syndrome have raised questions on the safety of long-term administration of DNA methylation inhibitors. We treated preweaned cancer prone Apc(Min/+) (Min) mice continuously with the DNA methylation inhibitor zebularine in their drinking water to determine the effects of the drug on normal mouse development as well as cancer prevention. Zebularine caused a tissue-specific reduction in DNA methylation at B1 short interspersed nucleotide elements in the small and large intestines of female Min mice but not in other organs examined after chronic oral treatment. No significant difference in the average weights of mice was observed during the treatment. In addition, analysis of global gene expression of colonic epithelial cells from the females indicated that only 3% to 6% of the genes were affected in their expression. We did not detect toxicity and abnormalities from the histopathologic analysis of liver and intestinal tissues. Lastly, we tested whether prevention of tumorigenesis can be achieved with chronic oral administration of zebularine in Min mice. The average number of polyps in Min females decreased from 58 to 1, whereas the average polyp number remained unaffected in Min males possibly due to differential activity of aldehyde oxidase. Taken together, our results show for the first time that long-term oral administration of zebularine causes a gender-specific abrogation of intestinal tumors while causing a tissue-specific DNA demethylation. Importantly, prolonged treatment of mice with epigenetic drugs resulted in only minor developmental and histologic changes.


Assuntos
Carcinoma/prevenção & controle , Citidina/análogos & derivados , Epigênese Genética/efeitos dos fármacos , Neoplasias Intestinais/prevenção & controle , Administração Oral , Animais , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/efeitos adversos , Antimetabólitos Antineoplásicos/farmacologia , Carcinoma/genética , Citidina/administração & dosagem , Citidina/efeitos adversos , Citidina/farmacologia , Metilação de DNA/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Genes APC , Mucosa Intestinal/metabolismo , Neoplasias Intestinais/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos/efeitos dos fármacos , Caracteres Sexuais , Fatores de Tempo
12.
Proc Natl Acad Sci U S A ; 103(38): 14080-5, 2006 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-16963560

RESUMO

Previous studies have shown that DNA methyltransferase (Dnmt) 1 is required for maintenance of bulk DNA methylation and is essential for mouse development. However, somatic disruption of DNMT1 in the human cancer cell line HCT116 was not lethal and caused only minor decreases in methylation. Here, we report the identification of a truncated DNMT1 protein, which was generated by the disruption of DNMT1 in HCT116 cells. The truncated protein, which had parts of the regulatory N-terminal domain deleted but preserved the catalytic C-terminal domain, was present at different levels in all DNMT1 single-knockout and DNMT1/DNMT3b double-knockout cell lines tested and retained hemimethylase activity. DNMT1 RNAi resulted in decreased cell viability in WT and knockout cells and further loss of DNA methylation in DNMT1 knockout cells. Furthermore, we observed a delay in methylation after replication and an increase in hemimethylation of specific CpG sites in cells expressing the truncated protein. Remethylation studies after drug-induced hypomethylation suggest a putative role of DNMT1 in the de novo methylation of a subtelomeric repeat, D4Z4, which is lost in cells lacking full-length DNMT1. Our data suggest that DNMT1 might be essential for maintenance of DNA methylation, proliferation, and survival of cancer cells.


Assuntos
Sobrevivência Celular , DNA (Citosina-5-)-Metiltransferases/metabolismo , Animais , Azacitidina/análogos & derivados , Azacitidina/metabolismo , Linhagem Celular , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Metilases de Modificação do DNA/antagonistas & inibidores , Decitabina , Inibidores Enzimáticos/metabolismo , Engenharia Genética , Humanos , Camundongos , Interferência de RNA
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