RESUMO
Class II histone deacetylases (HDACs) are important in regulation of gene transcription during T cell development. However, our understanding of their cell-specific functions is limited. In this study, we reveal that class IIa Hdac4 and Hdac7 (Hdac4/7) are selectively induced in transcription, guiding the lineage-specific differentiation of mouse T-helper 17 (Th17) cells from naive CD4+ T cells. Importantly, Hdac4/7 are functionally dispensable in other Th subtypes. Mechanistically, Hdac4 interacts with the transcription factor (TF) JunB, facilitating the transcriptional activation of Th17 signature genes such as Il17a/f. Conversely, Hdac7 collaborates with the TF Aiolos and Smrt/Ncor1-Hdac3 corepressors to repress transcription of Th17 negative regulators, including Il2, in Th17 cell differentiation. Inhibiting Hdac4/7 through pharmacological or genetic methods effectively mitigates Th17 cell-mediated intestinal inflammation in a colitis mouse model. Our study uncovers molecular mechanisms where HDAC4 and HDAC7 function distinctively yet cooperatively in regulating ordered gene transcription during Th17 cell differentiation. These findings suggest a potential therapeutic strategy of targeting HDAC4/7 for treating Th17-related inflammatory diseases, such as ulcerative colitis.
Assuntos
Diferenciação Celular , Colite , Histona Desacetilases , Correpressor 1 de Receptor Nuclear , Células Th17 , Animais , Células Th17/citologia , Células Th17/metabolismo , Células Th17/imunologia , Histona Desacetilases/metabolismo , Histona Desacetilases/genética , Camundongos , Colite/genética , Colite/metabolismo , Colite/imunologia , Transcrição Gênica , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Correpressor 2 de Receptor Nuclear/metabolismo , Correpressor 2 de Receptor Nuclear/genética , Interleucina-17/metabolismo , Regulação da Expressão Gênica , Camundongos Endogâmicos C57BL , Humanos , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Interleucina-2/metabolismoRESUMO
BRD4-NUT, a driver fusion mutant in rare and highly aggressive NUT carcinoma, acts in aberrant transcription of anti-differentiation genes by recruiting histone acetyltransferase (HAT) p300 and promoting p300-driven histone hyperacetylation and nuclear condensation in chromatin. However, the molecular basis of how BRD4-NUT recruits and activates p300 remains elusive. Here, we report that BRD4-NUT contains two transactivation domains (TADs) in NUT that bind to the TAZ2 domain in p300. Our NMR structures reveal that NUT TADs adopt amphipathic helices when bound to the four-helical bundle TAZ2 domain. The NUT protein forms liquid-like droplets in-vitro that are enhanced by TAZ2 binding in 1:2 stoichiometry. The TAD/TAZ2 bipartite binding in BRD4-NUT/p300 triggers allosteric activation of p300 and acetylation-driven liquid-like condensation on chromatin that comprise histone H3 lysine 27 and 18 acetylation and transcription proteins BRD4L/S, CDK9, MED1, and RNA polymerase II. The BRD4-NUT/p300 chromatin condensation is key for activating transcription of pro-proliferation genes such as ALX1, resulting ALX1/Snail signaling and epithelial-to-mesenchymal transition. Our study provides a previously underappreciated structural mechanism illuminating BRD4-NUT's bipartite p300 recruitment and activation in NUT carcinoma that nucleates a feed-forward loop for propagating histone hyperacetylation and chromatin condensation to sustain aberrant anti-differentiation gene transcription and perpetual tumor cell growth.
Assuntos
Carcinoma , Proteínas de Ciclo Celular , Cromatina , Proteínas de Neoplasias , Proteínas Nucleares , Humanos , Acetilação , Carcinoma/metabolismo , Carcinoma/patologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas de Neoplasias/metabolismoRESUMO
BRD4 is a well-recognized transcriptional activator, but how it regulates gene transcriptional repression in a cell type-specific manner has remained elusive. In this study, we report that BRD4 works with Polycomb repressive complex 2 (PRC2) to repress transcriptional expression of the T-helper 2 (Th2)-negative regulators Foxp3 and E3-ubiqutin ligase Fbxw7 during lineage-specific differentiation of Th2 cells from mouse primary naïve CD4+ T cells. Brd4 binds to the lysine-acetylated-EED subunit of the PRC2 complex via its second bromodomain (BD2) to facilitate histone H3 lysine 27 trimethylation (H3K27me3) at target gene loci and thereby transcriptional repression. We found that Foxp3 represses transcription of Th2-specific transcription factor Gata3, while Fbxw7 promotes its ubiquitination-directed protein degradation. BRD4-mediated repression of Foxp3 and Fbxw7 in turn promotes BRD4- and Gata3-mediated transcriptional activation of Th2 cytokines including Il4, Il5, and Il13. Chemical inhibition of the BRD4 BD2 induces transcriptional de-repression of Foxp3 and Fbxw7, and thus transcriptional downregulation of Il4, Il5, and Il13, resulting in inhibition of Th2 cell lineage differentiation. Our study presents a previously unappreciated mechanism of BRD4's role in orchestrating a Th2-specific transcriptional program that coordinates gene repression and activation, and safeguards cell lineage differentiation.
Assuntos
Proteínas Nucleares , Complexo Repressor Polycomb 2 , Camundongos , Animais , Complexo Repressor Polycomb 2/metabolismo , Proteínas Nucleares/metabolismo , Proteína 7 com Repetições F-Box-WD/metabolismo , Interleucina-13/metabolismo , Interleucina-4/genética , Interleucina-5/metabolismo , Lisina , Diferenciação Celular/genética , Fatores de Transcrição Forkhead/genéticaRESUMO
SignificanceSTAT3 (signal transducer and activator of transcription 3) is a master transcription factor that organizes cellular responses to cytokines and growth factors and is implicated in inflammatory disorders. STAT3 is a well-recognized therapeutic target for human cancer and inflammatory disorders, but how its function is regulated in a cell type-specific manner has been a major outstanding question. We discovered that Stat3 imposes self-directed regulation through controlling transcription of its own regulator homeodomain-interacting protein kinase 2 (Hipk2) in a T helper 17 (Th17) cell-specific manner. Our validation of the functional importance of the Stat3-Hipk2 axis in Th17 cell development in the pathogenesis of T cell-induced colitis in mice suggests an approach to therapeutically treat inflammatory bowel diseases that currently lack a safe and effective therapy.
Assuntos
Colite , Fator de Transcrição STAT3 , Animais , Diferenciação Celular/genética , Colite/genética , Colite/metabolismo , Ativação Linfocitária , Camundongos , Proteínas Serina-Treonina Quinases/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Células Th17RESUMO
The BET (bromodomain and extra-terminal domain) family proteins, consisting of BRD2, BRD3, BRD4, and testis-specific BRDT, are widely acknowledged as major transcriptional regulators in biology. They are characterized by two tandem bromodomains (BDs) that bind to lysine-acetylated histones and transcription factors, recruit transcription factors and coactivators to target gene sites, and activate RNA polymerase II machinery for transcriptional elongation. Pharmacological inhibition of BET proteins with BD inhibitors has been shown as a promising therapeutic strategy for the treatment of many human diseases including cancer and inflammatory disorders. The recent advances in bromodomain protein biology have further uncovered the complex and versatile functions of BET proteins in the regulation of gene expression in chromatin. In this review article, we highlight our current understanding of BET proteins' functions in mediating protein-protein interactions required for chromatin-templated gene transcription and splicing, chromatin remodeling, DNA replication, and DNA damage repair. We further discuss context-dependent activator vs. repressor functions of individual BET proteins, isoforms, and bromodomains that may be harnessed for future development of BET bromodomain inhibitors as emerging epigenetic therapies for cancer and inflammatory disorders.
RESUMO
The importance of BET protein BRD4 in gene transcription is well recognized through the study of chemical modulation of its characteristic tandem bromodomain (BrD) binding to lysine-acetylated histones and transcription factors. However, while monovalent inhibition of BRD4 by BET BrD inhibitors such as JQ1 blocks growth of hematopoietic cancers, it is much less effective generally in solid tumors. Here, we report a thienodiazepine-based bivalent BrD inhibitor, MS645, that affords spatially constrained tandem BrD inhibition and consequently sustained repression of BRD4 transcriptional activity in blocking proliferation of solid-tumor cells including a panel of triple-negative breast cancer (TNBC) cells. MS645 blocks BRD4 binding to transcription enhancer/mediator proteins MED1 and YY1 with potency superior to monovalent BET inhibitors, resulting in down-regulation of proinflammatory cytokines and genes for cell-cycle control and DNA damage repair that are largely unaffected by monovalent BrD inhibition. Our study suggests a therapeutic strategy to maximally control BRD4 activity for rapid growth of solid-tumor TNBC cells.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Transcrição Gênica/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Feminino , Humanos , Subunidade 1 do Complexo Mediador/genética , Subunidade 1 do Complexo Mediador/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismoRESUMO
The BET proteins are major transcriptional regulators and have emerged as new drug targets, but their functional distinction has remained elusive. In this study, we report that the BET family members Brd2 and Brd4 exert distinct genomic functions at genes whose transcription they co-regulate during mouse T helper 17 (Th17) cell differentiation. Brd2 is associated with the chromatin insulator CTCF and the cohesin complex to support cis-regulatory enhancer assembly for gene transcriptional activation. In this context, Brd2 binds the transcription factor Stat3 in an acetylation-sensitive manner and facilitates Stat3 recruitment to active enhancers occupied with transcription factors Irf4 and Batf. In parallel, Brd4 temporally controls RNA polymerase II (Pol II) processivity during transcription elongation through cyclin T1 and Cdk9 recruitment and Pol II Ser2 phosphorylation. Collectively, our study uncovers both separate and interdependent Brd2 and Brd4 functions in potentiating the genetic program required for Th17 cell development and adaptive immunity.
Assuntos
Imunidade Adaptativa , Diferenciação Celular , Cromatina/enzimologia , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Nucleares/metabolismo , Células Th17/enzimologia , Fatores de Transcrição/metabolismo , Transcrição Gênica , Acetilação , Animais , Fator de Ligação a CCCTC , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Cromatina/genética , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Ciclina T/genética , Ciclina T/metabolismo , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Regulação da Expressão Gênica , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Camundongos Endogâmicos C57BL , Modelos Moleculares , Proteínas Nucleares/genética , Fenótipo , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , RNA Polimerase II/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Células Th17/imunologia , Fatores de Transcrição/genética , Transfecção , CoesinasRESUMO
While it is well established that treatment of cancer patients with 5-Fluorouracil (5-FU) can result in immune suppression, the exact function of 5-FU in the modulation of immune cells has not been fully established. We found that low dose 5-FU selectively suppresses TH17 and TH1 cell differentiation without apparent effect on Treg, TH2, and significantly suppresses thymidylate synthase (TS) expression in TH17 and TH1 cells but has a lesser effect in tumor cells and macrophages. Interestingly, the basal expression of TS varies significantly between T helper phenotypes and knockdown of TS significantly impairs TH17 and TH1 cell differentiation without affecting the differentiation of either Treg or TH2 cells. Finally, low dose 5-FU is effective in ameliorating colitis development by suppressing TH17 and TH1 cell development in a T cell transfer colitis model. Taken together, the results highlight the importance of the anti-inflammatory functions of low dose 5-FU by selectively suppressing TH17 and TH1 immune responses.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Fluoruracila/farmacologia , Células Th17/efeitos dos fármacos , Timidilato Sintase/antagonistas & inibidores , Animais , Antimetabólitos Antineoplásicos/farmacologia , Western Blotting , Linhagem Celular Tumoral , Colite/metabolismo , Colite/prevenção & controle , Citocinas/genética , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Camundongos Endogâmicos C57BL , Camundongos Knockout , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Th1/efeitos dos fármacos , Células Th1/metabolismo , Células Th17/metabolismo , Timidilato Sintase/genética , Timidilato Sintase/metabolismoRESUMO
Mutations in adenomatous polyposis coli (APC) gene are found in more than 80% of colorectal cancer (CRC) patients. The nuclear transcription factor Nrf2 plays a central role in the regulation of oxidative stress and inflammation. Previously, we have shown that chronic inflammation in Nrf2(-/-) (Nrf2 knockout; KO) mice resulted in higher expression of inflammatory markers and cytokines, coupled with higher inflammatory damage to the colonic crypt cells, as compared to the Nrf2(+/+) (wild type; WT) mice. Induction of mutation in the colon by administration of carcinogen, AOM prior to DSS-induced inflammation resulted in higher tumor incidence and numbers in Nrf2KO mice. These results indicate that Nrf2-dependent inhibition of inflammation appears to be critical in inhibiting mutation-initiated colorectal carcinogenesis. In this study, we aim to investigate if loss of Nrf2 would dose-dependently promote intestinal tumorigenesis in Apc(min/+) mice. To demonstrate the in vivo mechanisms, we constructed both Apc mutated and Nrf2 deficient strain Apc(min/+) mice with C57BL/6 Nrf2KO mice to obtain F1, Apc(min/+) ;Nrf2(+/-) and F2, Apc(min/+) ;Nrf2(-/-) mice. Nrf2KO decreased the protein expression of antioxidant enzyme NQO1 in Apc(min/+) . In contrast, Nrf2KO enhanced the expression of inflammatory markers such as COX-2, cPLA, LTB4 in Apc(min/+) . Finally, Nrf2KO resulted in higher level of PCNA and c-Myc expression in intestinal tissue, indicating the deficiency of Nrf2 promotes proliferation of intestinal crypt cells in Apc(min/+) . Taken together, our results suggest that Nrf2KO attenuates anti-oxidative stress pathway, induces inflammation, and increases proliferative potential in the intestinal crypts leading to enhanced intestinal carcinogenesis and adenomas in Apc(min/+) .
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Mucosa Intestinal/metabolismo , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/genética , Transdução de Sinais , Animais , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Técnicas de Inativação de Genes , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Pólipos Intestinais/genética , Pólipos Intestinais/patologia , Intestinos/patologia , Leucotrieno B4/metabolismo , Masculino , Camundongos , Camundongos Knockout , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismoRESUMO
Cancer chemopreventive activities of various phytochemicals have been attributed to the modulation of xenobiotic disposition, which includes absorption, distribution, metabolism, and excretion. The interaction between xenobiotics and xenobiotic-metabolizing enzymes (XMEs) is bidirectional. XMEs are responsible for the biotransformation of xenobiotics such as bioactivation and detoxification. Conversely, xenobiotics affect XMEs through transcriptional regulation (induction or suppression) and post-translational interactions (inhibition or activation). Similar relationships also exist between xenobiotics and their transporters. Studies conducted over the past decade have demonstrated that the transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), plays a critical role in the regulation of detoxifying enzymes and transporters through a signaling system that senses and responds to redox imbalance. The role of Nrf2 in the interaction between chemopreventive phytochemicals and detoxifying enzymes/transporters has become an important topic in cancer chemoprevention. In this review, the genetic and epigenetic factors that contribute to Nrf2-mediated regulation of detoxifying XMEs and transporters are discussed in the context of cancer chemoprevention. Phytochemicals may modulate the genome as well as epigenome, altering the regulation of XMEs and transporters, which may be critical for both cancer chemoprevention and the prevention of other oxidative stress- and inflammatory-related diseases, including cardiovascular, metabolic and neurological pathologies. The pharmacogenomic expression of XMEs and transporters, with an emphasis on both genomics and epigenetics, will also be discussed.
Assuntos
Anticarcinógenos/farmacologia , Proteínas de Membrana Transportadoras/genética , Fator 2 Relacionado a NF-E2/metabolismo , Oxirredutases/genética , Compostos Fitoquímicos/farmacologia , Transferases/genética , Animais , Epigenômica , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/prevenção & controle , Oxirredutases/metabolismo , Polimorfismo Genético , Transferases/metabolismo , Xenobióticos/metabolismoRESUMO
Nrf2 plays a critical role in the regulation of cellular oxidative stress. MEK-ERK activation has been shown to be one of the major pathways resulting in the activation of Nrf2 and induction of Nrf2 downstream targets, including phase II detoxifying/antioxidant genes in response to oxidative stress and xenobiotics. In this study, IQGAP1 (IQ motif-containing GTPase-activating protein 1), a new Nrf2 interaction partner that we have published previously, was found to modulate MEK-ERK-mediated Nrf2 activation and induction of phase II detoxifying/antioxidant genes. Nrf2 binds directly to the IQ domain (amino acids 699-905) of IQGAP1. Knockdown of IQGAP1 significantly attenuated phenethyl isothiocyanate- or MEK-mediated activation of the MEK-ERK-Nrf2 pathway. Knockdown of IQGAP1 also attenuated MEK-mediated increased stability of Nrf2, which in turn was associated with a decrease in the nuclear translocation of Nrf2 and a decrease in the expression of phase II detoxifying/antioxidant genes. In the aggregate, these results suggest that IQGAP1 may play an important role in the MEK-ERK-Nrf2 signaling pathway.
Assuntos
Sistema de Sinalização das MAP Quinases , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Ativadoras de ras GTPase/fisiologia , Sequência de Bases , Núcleo Celular/metabolismo , Primers do DNA , Ativação Enzimática , Células HEK293 , Humanos , Reação em Cadeia da PolimeraseRESUMO
3,3'-diindolylmethane (DIM) is currently being investigated in many clinical trials including prostate, breast, and cervical cancers and has been shown to possess anticancer effects in several in vivo and in vitro models. Previously, DIM has been reported to possess cancer chemopreventive effects in prostate carcinogenesis in TRAMP mice; however, the in vivo mechanism is unclear. The present study aims to investigate the in vitro and in vivo epigenetics modulation of DIM in TRAMP-C1 cells and in TRAMP mouse model. In vitro study utilizing TRAMP-C1 cells showed that DIM suppressed DNMT expression and reversed CpG methylation status of Nrf2 resulting in enhanced expression of Nrf2 and Nrf2-target gene NQO1. In vivo study, TRAMP mice fed with DIM-supplemented diet showed much lower incidence of tumorigenesis and metastasis than the untreated control group similar to what was reported previously. DIM increased apoptosis, decreased cell proliferation and enhanced Nrf2 and Nrf2-target gene NQO1 expression in prostate tissues. Importantly, immunohistochemical analysis showed that DIM reduced the global CpG 5-methylcytosine methylation. Focusing on one of the early cancer chemopreventive target gene Nrf2, bisulfite genomic sequencing showed that DIM decreased the methylation status of the first five CpGs of the Nrf2 promoter region, corroborating with the results of in vitro TRAMP-C1 cells. In summary, our current study shows that DIM is a potent cancer chemopreventive agent for prostate cancer and epigenetic modifications of the CpG including Nrf2 could be a potential mechanism by which DIM exerts its chemopreventive effects.
Assuntos
Anticarcinógenos/uso terapêutico , Epigênese Genética/fisiologia , Indóis/uso terapêutico , Fator 2 Relacionado a NF-E2/fisiologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/terapia , Membro 25 de Receptores de Fatores de Necrose Tumoral/genética , Animais , Anticarcinógenos/farmacologia , Linhagem Celular Tumoral , Feminino , Indóis/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
PALB2/FANCN is mutated in breast and pancreatic cancers and Fanconi anemia (FA). It controls the intranuclear localization, stability, and DNA repair function of BRCA2 and links BRCA1 and BRCA2 in DNA homologous recombination repair and breast cancer suppression. Here, we show that PALB2 directly interacts with KEAP1, an oxidative stress sensor that binds and represses the master antioxidant transcription factor NRF2. PALB2 shares with NRF2 a highly conserved ETGE-type KEAP1 binding motif and can effectively compete with NRF2 for KEAP1 binding. PALB2 promotes NRF2 accumulation and function in the nucleus and lowers the cellular reactive oxygen species (ROS) level. In addition, PALB2 also regulates the rate of NRF2 export from the nucleus following induction. Our findings identify PALB2 as a regulator of cellular redox homeostasis and provide a new link between oxidative stress and the development of cancer and FA.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Transformação Celular Neoplásica , Reparo do DNA , Proteína do Grupo de Complementação N da Anemia de Fanconi , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch , Neoplasias/patologia , Oxirredução , Estresse Oxidativo , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismoRESUMO
Curcumin (CUR), a major bioactive polyphenolic component from turmeric curry, Curcuma longa, has been shown to be a potent anti-cancer phytochemical with well-established anti-inflammatory and anti-oxidative stress effects. Chromatin remodeling-related epigenetic regulation has emerged as an important mechanism of carcinogenesis, chemoprevention, and chemotherapy. CUR has been found to inhibit histone acetyltransferase activity, and it was also postulated to be a potential DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitor. In this study, we show that when human prostate LNCaP cells were treated with CUR, it led to demethylation of the first 14 CpG sites of the CpG island of the Neurog1 gene and restored the expression of this cancer-related CpG-methylation epigenome marker gene. At the protein level, CUR treatment had limited effects on the expression of epigenetic modifying proteins MBD2, MeCP2, DNMT1, and DNMT3a. Using ChIP assay, CUR decreased MeCP2 binding to the promoter of Neurog1 dramatically. CUR treatment showed different effects on the protein expression of HDACs, increasing the expression of HDAC1, 4, 5, and 8 but decreasing HDAC3. However, the total HDAC activity was decreased upon CUR treatment. Further analysis of the tri-methylation of histone 3 at lysine 27 (H3K27me3) showed that CUR decreased the enrichment of H3K27me3 at the Neurog1 promoter region as well as at the global level. Taken together, our present study provides evidence on the CpG demethylation ability of CUR on Neurog1 while activating its expression, suggesting a potential epigenetic modifying role for this phytochemical compound in human prostate cancer cells.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Ilhas de CpG , Curcumina/farmacologia , Metilação de DNA , Epigênese Genética , Expressão Gênica/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Metilases de Modificação do DNA/antagonistas & inibidores , Primers do DNA , Inibidores de Histona Desacetilases/farmacologia , Humanos , Masculino , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Induction of Nrf2-mediated detoxifying/antioxidant enzymes is an effective strategy for cancer chemoprevention. The goal of this study was to examine the role of calcium [Ca(2+)] in regulating a well-known phenolic chemopreventive compound tertiary-butylhydroquinone (tBHQ) activation of Nrf2 and induction of Nrf2 downstream target gene heme-oxygenase (HO-1). tBHQ alone caused Nrf2 nuclear localization and induced HO-1 mRNA and protein expression in a dose-dependent manner. Using RT-PCR and Western blotting, we showed that tBHQ-induced transcription of HO-1 is Ca(2+)-dependent. Chelation of [Ca(2+)](ext) or [Ca(2+)](intra) by EGTA or BAPTA attenuated tBHQ-induced HO-1. Cotreatment of tBHQ with inhibitors of [Ca(2+)]-sensitive protein kinase C and camodulin kinase did not attenuate HO-1 induction. Nuclear translocation of Nrf2 induced by tBHQ was also not affected by treatment of EGTA or BAPTA. Additionally, EGTA and BAPTA treatments decreased basal nuclear phosphorylation of CREB and decreased tBHQ-induced Nrf2-CBP binding and Nrf2 binding to enhancer as well as polymerase II binding to the promoter of HO-1 gene. Furthermore, tBHQ in combination with higher [Ca(2+)](ext) augmented HO-1 induction both in vitro and in vivo, indicating that the modulation of [Ca(2+)](int) could be used as an adjuvant to increase the efficacy of chemopreventive agents. Taken together, our results indicated that in addition to tBHQ-induced oxidative stress-mediated Nrf2 translocation, HO-1 induction by tBHQ also appears to be dependent on a series of Ca(2+)-regulated mechanisms.
Assuntos
Anticarcinógenos/farmacologia , Antioxidantes/farmacologia , Cálcio/metabolismo , Heme Oxigenase-1/genética , Hidroquinonas/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias/prevenção & controle , Linhagem Celular Tumoral , DNA Polimerase II/metabolismo , Elementos Facilitadores Genéticos , Ativação Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glutationa Transferase/genética , Células Hep G2 , Humanos , Fator 2 Relacionado a NF-E2/genética , Regiões Promotoras Genéticas , Transporte Proteico/efeitos dos fármacosRESUMO
Carcinogenesis is a multi-step process which could be prevented by phytochemicals. Phytochemicals from dietary plants and other plant sources such as herbs are becoming increasingly important sources of anticancer drugs or compounds for cancer chemoprevention or adjuvant chemotherapy. Phytochemicals can prevent cancer initiation, promotion, and progression by exerting anti-inflammatory and anti-oxidative stress effects which are mediated by integrated Nrf2, NF-kappaB, and AP-1 signaling pathways. In addition, phytochemicals from herbal medicinal plants and/or some dietary plants developed in recent years have been shown to induce apoptosis in cancer cells and inhibition of tumor growth in vivo. In advanced tumors, a series of changes involving critical signaling molecules that would drive tumor cells undergoing epithelial-mesenchymal transition and becoming invasive. In this review, we will discuss the potential molecular targets and signaling pathways that mediate tumor onset and metastasis. In addition, we will shed light on some of the phytochemicals that are capable of targeting these signaling pathways which would make them potentially applicable to cancer chemoprevention, treatment and control of cancer progression.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Metástase Neoplásica/prevenção & controle , Neoplasias/prevenção & controle , Fitoterapia , Animais , Transformação Celular Neoplásica/efeitos dos fármacos , Transição Epitelial-Mesenquimal , Humanos , Neoplasias/patologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is a transcription factor which regulates the expression of many cytoprotective genes. In the present study, we found that the expression of Nrf2 was suppressed in prostate tumor of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice. Similarly, the expression of Nrf2 and the induction of NQO1 were also substantially suppressed in tumorigenic TRAMP C1 cells but not in non-tumorigenic TRAMP C3 cells. Examination of the promoter region of the mouse Nrf2 gene identified a CpG island, which was methylated at specific CpG sites in prostate TRAMP tumor and in TRAMP C1 cells but not in normal prostate or TRAMP C3 cells, as shown by bisulfite genomic sequencing. Reporter assays indicated that methylation of these CpG sites dramatically inhibited the transcriptional activity of the Nrf2 promoter. Chromatin immunopreceipitation (ChIP) assays revealed increased binding of the methyl-CpG-binding protein 2 (MBD2) and trimethyl-histone H3 (Lys9) proteins to these CpG sites in the TRAMP C1 cells as compared to TRAMP C3 cells. In contrast, the binding of RNA Pol II and acetylated histone H3 to the Nrf2 promoter was decreased. Furthermore, treatment of TRAMP C1 cells with DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-aza) and histone deacetylase (HDAC) inhibitor trichostatin A (TSA) restored the expression of Nrf2 as well as the induction of NQO1 in TRAMP C1 cells. Taken together, these results indicate that the expression of Nrf2 is suppressed epigenetically by promoter methylation associated with MBD2 and histone modifications in the prostate tumor of TRAMP mice. Our present findings reveal a novel mechanism by which Nrf2 expression is suppressed in TRAMP prostate tumor, shed new light on the role of Nrf2 in carcinogenesis and provide potential new directions for the detection and prevention of prostate cancer.
Assuntos
Epigênese Genética , Fator 2 Relacionado a NF-E2/genética , Neoplasias da Próstata/genética , Membro 25 de Receptores de Fatores de Necrose Tumoral/fisiologia , Animais , Azacitidina/farmacologia , Ilhas de CpG , Metilação de DNA , Masculino , Camundongos , Membro 25 de Receptores de Fatores de Necrose Tumoral/genética , Transcrição Gênica , Tricosantina/farmacologiaRESUMO
Previously, phenethyl isothiocyanate (PEITC) and dibenzoylmethane (DBM) had been shown to inhibit intestinal carcinogenesis in Apc(Min/+) mice. In this study, we investigated the chemopreventive efficacy of PEITC and DBM in the azoxymethane (AOM)-initiated and dextran sodium sulfate (DSS)-promoted colon cancer mouse model and to compare their potential in vivo mechanisms leading to chemoprevention. The mice were fed with diet supplemented with 0.05% PEITC or 1% DBM before or after AOM initiation. Our results showed that AOM/DSS mice fed with PEITC- or DBM-supplemented diet had lower tumor incidence, lower colon tumor multiplicities and smaller polyps as compared with mice fed with the standard AIN-76A diet. PEITC was effective even after AOM initiation, whereas DBM was not as effective when fed after AOM initiation. Hematoxylin and eosin staining showed that mice fed with PEITC or DBM had attenuated loss of crypt, a marker of inflammation. To examine potential in vivo mechanisms involved in chemoprevention, western blotting was performed and showed that inhibition of growth of adenomas by PEITC was associated with an increase of apoptosis (increased cleaved caspase-3 and-7) and cell cycle arrest (increased p21). In contrast DBM's effect on cell cycle arrest and apoptosis markers was not as substantial as PEITC. Instead, DBM showed increased induction of NF-E2-related factor-2 (Nrf2) transcription factor and phase II detoxifying enzymes, which appears to correlate with in vitro cell lines results that DBM is a more potent Nrf2 activator than PEITC. In summary, our present study shows that PEITC and DBM are potent natural dietary compounds for chemoprevention of colon cancer induced by AOM/DSS and appears to be associated with different in vivo mechanism of actions. PEITC's chemopreventive effect appears to be due to induction of apoptosis and cell cycle arrest, whereas DBM's effect is due to prevention of AOM initiation via induction of Nrf2 and phase II detoxifying enzymes.
Assuntos
Anticarcinógenos/farmacologia , Azoximetano/toxicidade , Chalconas/farmacologia , Neoplasias Colorretais/prevenção & controle , Sulfato de Dextrana/toxicidade , Isotiocianatos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/patologia , Mucosa Intestinal/patologia , Pólipos Intestinais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Development of cancer is a long-term and multistep process which comprises initiation, progression, and promotion stages of carcinogenesis. Conceivably, it can be targeted and interrupted along these different stages. In this context, many naturally occurring dietary compounds from our daily consumption of fruits and vegetables have been shown to possess cancer preventive effects. Phenethyl isothiocyanate (PEITC) and sulforaphane (SFN) are two of the most widely investigated isothiocyanates from the crucifers. Both have been found to be very potent chemopreventive agents in numerous animal carcinogenesis models as well as cell culture models. They exert their chemopreventive effects through regulation of diverse molecular mechanisms. In this review, we will discuss the molecular targets of PEITC and SFN potentially involved in cancer chemoprevention. These include the regulation of drug-metabolizing enzymes phase I cytochrome P450s and phase II metabolizing enzymes. In addition, the signaling pathways including Nrf2-Keap 1, anti-inflammatory NFkappaB, apoptosis, and cell cycle arrest as well as some receptors will also be discussed. Furthermore, we will also discuss the similarities and their potential differences in the regulation of these molecular targets by PEITC and SFN.
Assuntos
Anticarcinógenos/farmacologia , Isotiocianatos/farmacologia , Neoplasias/prevenção & controle , Tiocianatos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Inibidores das Enzimas do Citocromo P-450 , Dieta , Indução Enzimática , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Isotiocianatos/administração & dosagem , Proteína 1 Associada a ECH Semelhante a Kelch , Sistema de Sinalização das MAP Quinases , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Sulfóxidos , Tiocianatos/administração & dosagemRESUMO
PURPOSE: Accumulating evidence from epidemiologic and clinical studies indicates that chronic inflammatory disorders harbor an increased risk of cancer development. Curcumin (CUR) has been strongly linked to the anti-inflammatory effect. On the other hand, isothiocyanates such as sulforaphane (SFN) and phenethyl isothiocyanate (PEITC) are strong phase-II detoxifying/antioxidant enzymes inducer. Therefore it is interesting to see if combination of these drugs can inhibit inflammation with higher combined efficacies. METHODS: We used nitric oxide (NO) assay to assess the synergism of the different combinations of CUR, SFN and PEITC. The inflammatory markers, e.g. iNOS, COX-2, prostaglandin E2 (PGE2), tumor necrosis factor (TNF) and interleukin-1 (IL-1) levels were determined using RT-PCR, Western blot and ELISA assays. RESULTS: We report that combination of PEITC + SFN or CUR + SFN has a synergistic effect in down-regulating inflammation markers like TNF, IL-1, NO, PGE2. The synergism is probably due to the synergistic induction of phase II/antioxidant enzymes including heme-oxygenase1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO-1). CONCLUSIONS: Our data suggest that CUR + SFN and PEITC + SFN combinations could be more effective than used alone in preventing inflammation and possibly its associated diseases including cancer.