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1.
Bioinformatics ; 40(6)2024 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-38857451

RESUMO

SUMMARY: The vast amount of publicly available genomic data requires analysis and visualization tools. Here, we present figeno, an application for generating publication-quality FIgures for GENOmics. Figeno particularly focuses on multi-region views across genomic breakpoints and on long reads with base modifications. In addition, we support epigenomic data including ATAC-seq, ChIP-seq or HiC, as well as whole genome sequencing data with copy numbers and structural variants. AVAILABILITY AND IMPLEMENTATION: Figeno is available as a python package with both a command line and graphical user interface. It can be installed via PyPI and the source code is available at https://github.com/CompEpigen/figeno.


Assuntos
Genômica , Software , Genômica/métodos , Humanos , Análise de Sequência de DNA/métodos
2.
Int J Cancer ; 152(5): 1025-1035, 2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36305646

RESUMO

Noninvasive detection of aberrant DNA methylation could provide invaluable biomarkers for earlier detection of triple-negative breast cancer (TNBC) which could help clinicians with easier and more efficient treatment options. We evaluated genome-wide DNA methylation data derived from TNBC and normal breast tissues, peripheral blood of TNBC cases and controls and reference samples of sorted blood and mammary cells. Differentially methylated regions (DMRs) between TNBC and normal breast tissues were stringently selected, verified and externally validated. A machine-learning algorithm was applied to select the top DMRs, which then were evaluated on plasma-derived circulating cell-free DNA (cfDNA) samples of TNBC patients and healthy controls. We identified 23 DMRs accounting for the methylation profile of blood cells and reference mammary cells and then selected six top DMRs for cfDNA analysis. We quantified un-/methylated copies of these DMRs by droplet digital PCR analysis in a plasma test set from TNBC patients and healthy controls and confirmed our findings obtained on tissues. Differential cfDNA methylation was confirmed in an independent validation set of plasma samples. A methylation score combining signatures of the top three DMRs overlapping with the SPAG6, LINC10606 and TBCD/ZNF750 genes had the best capability to discriminate TNBC patients from controls (AUC = 0.78 in the test set and AUC = 0.74 in validation set). Our findings demonstrate the usefulness of cfDNA-based methylation signatures as noninvasive liquid biopsy markers for the diagnosis of TNBC.


Assuntos
Ácidos Nucleicos Livres , Neoplasias de Mama Triplo Negativas , Humanos , Metilação de DNA , Neoplasias de Mama Triplo Negativas/diagnóstico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Biomarcadores Tumorais/genética , DNA , Ácidos Nucleicos Livres/genética , Marcadores Genéticos , Biópsia Líquida , Proteínas Associadas aos Microtúbulos/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética
3.
Semin Cancer Biol ; 51: 12-21, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29366906

RESUMO

Analogous to life on earth, tumor cells evolve through space and time and adapt to different micro-environmental conditions. As a result, tumors are composed of millions of genetically diversified cells at the time of diagnosis. Profiling these variants contributes to understanding tumors' clonal origins and might help to better understand response to therapy. However, even genetically homogenous cell populations show remarkable diversity in their response to different environmental stimuli, suggesting that genetic heterogeneity does not explain the full spectrum of tumor plasticity. Understanding epigenetic diversity across cancer cells provides important additional information about the functional state of subclones and therefore allows better understanding of tumor evolution and resistance to current therapies.


Assuntos
Evolução Clonal , Epigênese Genética , Heterogeneidade Genética , Neoplasias/genética , Animais , Humanos , Neoplasias/patologia
4.
Int J Sports Med ; 40(1): 62-70, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30508863

RESUMO

Positive effects of exercise on cancer prevention and progression have been proposed to be mediated by stimulating natural killer (NK) cells. Because NK cell receptors are regulated by epigenetic modifications, we investigated whether acute aerobic exercise and training change promoter DNA methylation and gene expression of the activating KIR2DS4 and the inhibiting KIR3DL1 gene. Sixteen healthy women (50-60 years) performed a graded exercise test (GXT) and were randomized into either a passive control group or an intervention group performing a four-week endurance exercise intervention. Blood samples (pre-, post-GXT and post-training) were used for isolation of DNA/RNA of NK cells to assess DNA promoter methylation by targeted deep-amplicon sequencing and gene expression by qRT-PCR. Potential changes in NK cell subsets were determined by flow cytometry. Acute and chronic exercise did not provoke significant alterations of NK cell proportions. Promoter methylation decreased and gene expression increased for KIR2DS4 after acute exercise. A high gene expression correlated with a low methylation of CpGs that were altered by acute exercise. Chronic exercise resulted in a minor decrease of DNA methylation and did not alter gene expression. Acute exercise provokes epigenetic modifications, affecting the balance between the activating KIR2DS4 and the inhibiting KIR3DL1, with potential benefits on NK cell function.


Assuntos
Metilação de DNA , Exercício Físico/fisiologia , Células Matadoras Naturais/metabolismo , Regiões Promotoras Genéticas , Receptores KIR/genética , Desmetilação , Epigênese Genética , Teste de Esforço , Feminino , Expressão Gênica , Humanos , Pessoa de Meia-Idade
5.
Artigo em Alemão | MEDLINE | ID: mdl-28168353

RESUMO

Isoflavones (IFs) from soy and other legumes have weak estrogenic properties. Isolated IFs are available as dietary supplements and advertised to alleviate symptoms of menopause. The present chapter provides an overview of the occurrence, the chemical structure of IFs and their metabolites, the market situation and reviews the current evidence on the efficacy and safety of IF-containing dietary supplements.The biological effectiveness of IFs is attributable to the activation of the estrogen receptor (ER). Studies on the influence of IFs on endogenous estrogen levels in women show inconsistent results. So far, the European Food Safety Authority (EFSA) has rejected all submitted health claims for IFs due to insufficient scientific evidence for any of the postulated health effects. Based on the results of their recent risk assessment, the EFSA concluded that the available human studies did not support the hypothesis of adverse effects of isolated IFs on the human mammary gland, uterus or thyroid in healthy postmenopausal women. However, the assessment does not allow a general statement on the safety of IF-containing dietary supplements. Studies in animal models are often not comparable with the complex interactions in humans due to differences in the metabolism of IFs, in the developmental stage at time of consumption and in the temporarily restricted uptake of IFs during certain stages of life. CONCLUSION: So far, for none of the advertised functions is unequivocal scientific evidence available. On the basis of available data, potential unwanted side effects cannot be fully excluded. This holds particularly true for women with undiagnosed diseases, especially for those with undetected precancerous lesions in the mammary gland.


Assuntos
Suplementos Nutricionais/efeitos adversos , Fogachos/terapia , Isoflavonas/administração & dosagem , Isoflavonas/efeitos adversos , Fitoestrógenos/administração & dosagem , Fitoestrógenos/efeitos adversos , Medicina Baseada em Evidências , Feminino , Humanos , Resultado do Tratamento
6.
Arch Pharm (Weinheim) ; 349(6): 414-27, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27159630

RESUMO

New resveratrol (trans-3,4',5-trihydroxystilbene) analogs were synthesized and screened for their in vitro cancer chemopreventive potential using various bioassays relevant for the prevention of carcinogenesis in humans: two assays to detect modulators of carcinogen metabolism (Cyp1A inhibition; determination of NAD(P)H/quinone reductase (QR) activity), three assays to identify radical scavenging and antioxidant properties (DPPH, ORAC, superoxide anion radicals in differentiated HL-60 cells), four assays to determine anti-inflammatory and anti-hormonal effects (iNOS, Cox-1 and aromatase inhibition, anti-estrogenic potential). 3,4',5-Tri-O-methyl resveratrol 1a was about sevenfold more active than resveratrol in inhibiting Cyp1A activity, it was a potent inducer of QR activity, and it showed pure anti-estrogenic activity (whereas resveratrol is a known mixed estrogen (ant)agonist with both estrogenic and anti-estrogenic properties). Dual estrogen ant-/agonist activity was restored in the mono-O-benzyl-substituted derivatives 4b (4'-O-benzyl resveratrol) and 5b (3-O-benzyl resveratrol). With respect to aromatase inhibition (Cyp19), which provided the highest number of actives, the benzyl-substituted series was more potent than the methyl-substituted derivatives of resveratrol, and 3-O-benzyl resveratrol 5b was about eightfold more active than resveratrol. Overall, 3,4',5-tri-O-pivaloyl resveratrol oxide 7c was identified as a potent inducer of phase 2 enzymes concomitant with inhibition of LPS-mediated iNOS induction.


Assuntos
Antineoplásicos Fitogênicos/síntese química , Antineoplásicos Fitogênicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Ácidos Pentanoicos/síntese química , Ácidos Pentanoicos/farmacologia , Estilbenos/síntese química , Estilbenos/farmacologia , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/farmacologia , Antioxidantes/síntese química , Antioxidantes/farmacologia , Bioensaio , Indução Enzimática/efeitos dos fármacos , Moduladores de Receptor Estrogênico/síntese química , Moduladores de Receptor Estrogênico/farmacologia , Humanos , Resveratrol , Relação Estrutura-Atividade
7.
Org Biomol Chem ; 13(10): 3040-7, 2015 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-25622264

RESUMO

Inflammatory signaling pathways orchestrate the cellular response to infection and injury. These pathways are known to be modulated by compounds that alkylate cysteinyl thiols. One class of phytochemicals with strong thiol alkylating activity is the chalcones. In this study we tested fourteen chalcone derivatives, α-X-substituted 2',3,4,4'-tetramethoxychalcones (α-X-TMCs, X = H, F, Cl, Br, I, CN, Me, p-NO2-C6H4, Ph, p-OMe-C6H4, NO2, CF3, COOEt, COOH), for their ability to modulate inflammatory responses, as monitored by their influence on heme oxygenase-1 (HO-1) activity, inducible nitric oxide synthase (iNOS) activity, and cytokine expression levels. We confirmed that the transcriptional activity of Nrf2 was activated by α-X-TMCs while for NF-κB it was inhibited. For most α-X-TMCs, anti-inflammatory activity was positively correlated with thiol alkylating activity, i.e. stronger electrophiles (X = CF3, Br and Cl) being more potent. Notably, this correlation did not hold true for the strongest electrophiles (X = CN and NO2) which were found to be ineffective as anti-inflammatory compounds. These results emphasize the idea that chemical fine-tuning of electrophilicity is needed to achieve and optimize desired therapeutic effects.


Assuntos
Anti-Inflamatórios/química , Chalconas/química , Inflamação/metabolismo , Animais , Cisteamina/química , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Glutationa/química , Células HeLa , Heme Oxigenase-1/metabolismo , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipopolissacarídeos/química , Macrófagos/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Processos Fotoquímicos , Compostos de Sulfidrila/química , Transcrição Gênica
8.
Biol Chem ; 395(11): 1275-90, 2014 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-25153594

RESUMO

Prostate cancer (PCa) is the second most common cause of cancer-related deaths in men. Despite advances in the characterization of genomic and epigenetic aberrations contributing to PCa, the etiology of PCa is still far from being understood. Research over the past decade demonstrated the role of long non-coding RNAs (lncRNAs) in deregulation of target genes mainly through epigenetic mechanisms. In PCa, evidence accumulated that hundreds of lncRNAs are dysregulated. Functional analyses revealed their contribution to prostate carcinogenesis by targeting relevant pathways and gene regulation mechanisms including PTEN/AKT and androgen receptor signaling as well as chromatin remodeling complexes. Here we summarize our current knowledge on the roles of lncRNAs in PCa and their potential use as biomarkers for aggressive PCa and as novel therapeutic targets.


Assuntos
Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , RNA Longo não Codificante/genética , Animais , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Impressão Genômica , Humanos , Masculino , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/terapia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Longo não Codificante/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Transdução de Sinais
9.
Am J Pathol ; 181(2): 401-12, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22705054

RESUMO

The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene is often altered in prostate cancer. To determine the prevalence and clinical significance of the different mechanisms of PTEN inactivation, we analyzed PTEN deletions in TMAs containing 4699 hormone-naïve and 57 hormone-refractory prostate cancers using fluorescence in situ hybridization analysis. PTEN mutations and methylation were analyzed in subsets of 149 and 34 tumors, respectively. PTEN deletions were present in 20.2% (458/2266) of prostate cancers, including 8.1% heterozygous and 12.1% homozygous deletions, and were linked to advanced tumor stage (P < 0.0001), high Gleason grade (P < 0.0001), presence of lymph node metastasis (P = 0.0002), hormone-refractory disease (P < 0.0001), presence of ERG gene fusion (P < 0.0001), and nuclear p53 accumulation (P < 0.0001). PTEN deletions were also associated with early prostate-specific antigen recurrence in univariate (P < 0.0001) and multivariate (P = 0.0158) analyses. The prognostic impact of PTEN deletion was seen in both ERG fusion-positive and ERG fusion-negative tumors. PTEN mutations were found in 4 (12.9%) of 31 cancers with heterozygous PTEN deletions but in only 1 (2%) of 59 cancers without PTEN deletion (P = 0.027). Aberrant PTEN promoter methylation was not detected in 34 tumors. The results of this study demonstrate that biallelic PTEN inactivation, by either homozygous deletion or deletion of one allele and mutation of the other, occurs in most PTEN-defective cancers and characterizes a particularly aggressive subset of metastatic and hormone-refractory prostate cancers.


Assuntos
Deleção de Genes , Proteínas de Fusão Oncogênica/metabolismo , PTEN Fosfo-Hidrolase/genética , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Transativadores/metabolismo , Idoso , Biomarcadores Tumorais/metabolismo , Cromossomos Humanos Par 10/genética , Metilação de DNA/genética , Análise Mutacional de DNA , Progressão da Doença , Epigênese Genética , Genoma Humano/genética , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Análise Multivariada , PTEN Fosfo-Hidrolase/metabolismo , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Modelos de Riscos Proporcionais , Recidiva , Regulador Transcricional ERG , Proteína Supressora de Tumor p53/metabolismo
10.
Top Curr Chem ; 329: 73-132, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-22955508

RESUMO

The term "epigenetics" refers to modifications in gene expression caused by heritable, but potentially reversible, changes in DNA methylation and chromatin structure. Epigenetic alterations have been identified as promising new targets for cancer prevention strategies as they occur early during carcinogenesis and represent potentially initiating events for cancer development. Over the past few years, nutriepigenetics - the influence of dietary components on mechanisms influencing the epigenome - has emerged as an exciting new field in current epigenetic research. During carcinogenesis, major cellular functions and pathways, including drug metabolism, cell cycle regulation, potential to repair DNA damage or to induce apoptosis, response to inflammatory stimuli, cell signalling, and cell growth control and differentiation become deregulated. Recent evidence now indicates that epigenetic alterations contribute to these cellular defects, for example epigenetic silencing of detoxifying enzymes, tumor suppressor genes, cell cycle regulators, apoptosis-inducing and DNA repair genes, nuclear receptors, signal transducers and transcription factors by promoter methylation, and modifications of histones and non-histone proteins such as p53, NF-κB, and the chaperone HSP90 by acetylation or methylation.The present review will summarize the potential of natural chemopreventive agents to counteract these cancer-related epigenetic alterations by influencing the activity or expression of DNA methyltransferases and histone modifying enzymes. Chemopreventive agents that target the epigenome include micronutrients (folate, retinoic acid, and selenium compounds), butyrate, polyphenols from green tea, apples, coffee, black raspberries, and other dietary sources, genistein and soy isoflavones, curcumin, resveratrol, dihydrocoumarin, nordihydroguaiaretic acid (NDGA), lycopene, anacardic acid, garcinol, constituents of Allium species and cruciferous vegetables, including indol-3-carbinol (I3C), diindolylmethane (DIM), sulforaphane, phenylethyl isothiocyanate (PEITC), phenylhexyl isothiocyanate (PHI), diallyldisulfide (DADS) and its metabolite allyl mercaptan (AM), cambinol, and relatively unexplored modulators of histone lysine methylation (chaetocin, polyamine analogs). So far, data are still mainly derived from in vitro investigations, and results of animal models or human intervention studies are limited that demonstrate the functional relevance of epigenetic mechanisms for health promoting or cancer preventive efficacy of natural products. Also, most studies have focused on single candidate genes or mechanisms. With the emergence of novel technologies such as next-generation sequencing, future research has the potential to explore nutriepigenomics at a genome-wide level to understand better the importance of epigenetic mechanisms for gene regulation in cancer chemoprevention.


Assuntos
Epigênese Genética , Nutrigenômica , Animais , Apoptose , Ciclo Celular , Diferenciação Celular , Divisão Celular , Metilação de DNA , Reparo do DNA , Histonas/metabolismo , MicroRNAs/metabolismo , NF-kappa B/metabolismo
11.
Curr Opin Clin Nutr Metab Care ; 16(4): 405-10, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23657153

RESUMO

PURPOSE OF REVIEW: There is growing evidence that cancer chemopreventive agents including isothiocyanates (ITCs) from cruciferous vegetables target epigenetic mechanisms. The present report will summarize novel findings of ITCs on histone deacetylase activity, DNA methylation, and short noncoding microRNAs, focusing on sulforaphane (SFN) from broccoli and phenethylisothiocyanate from watercress. RECENT FINDINGS: In a human intervention study, broccoli sprouts led to more efficient histone deacetylase inhibition in blood cells than a broccoli sprout supplement, correlating with higher levels of urinary ITC metabolites. A proteomics study with ¹4C-labeled ITCs revealed among others a direct interaction with histones and chromatin-modulating proteins. The well investigated Kelch-like erythroid-cell-derived protein with CNC homology-associated protein 1/nuclear factor erythroid 2-related factor 2/antioxidant-response element pathway is both affected by and mechanistically involved in epigenetic activities of ITCs. Accordingly, reduction of oxidative stress is shown to prevent hypertension-associated global hypomethylation in rats. Combination of SFN with (-)-epigallocatechin gallate as a demethylating agent is identified as an effective approach for re-expression of estrogen receptor in hormone negative breast cancer. Induction of miR-200c by SFN prevents epithelial-mesenchymal-transition and could be relevant for prevention of metastases. SUMMARY: The last year has identified interesting areas of ITCs affecting epigenetic mechanisms that will have implications for translational cancer (prevention) research once validated in animal studies and human intervention studies.


Assuntos
Dieta , Epigênese Genética , Isotiocianatos/farmacologia , Neoplasias/genética , Neoplasias/prevenção & controle , Animais , Anticarcinógenos/farmacologia , Brassica/química , Catequina/análogos & derivados , Catequina/farmacologia , Quimioprevenção , Metilação de DNA/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch , MicroRNAs/genética , MicroRNAs/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais , Sulfóxidos
12.
FASEB J ; 26(12): 4937-50, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22930747

RESUMO

Aberrant DNA methylation constitutes a well-established epigenetic marker for breast cancer. Changes in methylation early in cancer development may be clinically relevant for cancer detection and prognosis-based therapeutic decisions. In the present study, a combination of methyl-CpG immunoprecipitation (MCIp) and human CpG island (CGI) arrays was applied to compare genome-wide DNA methylation profiles in 10 low-grade in situ and invasive breast cancers against 10 normal breast samples. In total, 214 CGIs were found to be hypermethylated in ≥6 of 10 tumors. Functional term enrichment analyses revealed an overrepresentation of homeobox genes and genes involved in transcription and regulation of transcription. Significant hypermethylation of 11 selected genes in tumor vs. normal tissue was validated in two independent sample sets (45 tumors and 11 controls, 43 tumors and 8 controls) using quantitative EpiTyper technology. In tumors, median methylation levels of BCAN, HOXD1, KCTD8, KLF11, NXPH1, POU4F1, SIM1, and TCF7L1 were ≥30% higher than in normal samples, representing potential biomarkers for tumor diagnosis. Using the 90th percentile of methylation levels in normal tissue as cutoff value, 62-92% of in situ samples (n=13), 72-97% of invasive samples from the first validation set (n=32), and 86-100% of invasive samples from the second validation set (n=43) were classified as hypermethylated. Hypermethylation of KLF11 and SIM1 might also be associated with increased risk of developing metastases. In summary, early methylation changes are frequent in the low-grade pathway of breast cancer and may be useful in the development of differential diagnostic and possibly also prognostic markers.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Metilação de DNA , Estudo de Associação Genômica Ampla/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas Reguladoras de Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Neoplasias da Mama/patologia , Brevicam/genética , Proteínas de Ciclo Celular/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/genética , Proteínas de Homeodomínio/genética , Humanos , Células MCF-7 , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Neuropeptídeos/genética , Proteínas Repressoras/genética , Proteína 1 Semelhante ao Fator 7 de Transcrição/genética , Fator de Transcrição Brn-3A/genética
13.
Front Endocrinol (Lausanne) ; 14: 1134478, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37008919

RESUMO

Introduction: Beta cell dysfunction by loss of beta cell identity, dedifferentiation, and the presence of polyhormonal cells are main characteristics of diabetes. The straightforward strategy for curing diabetes implies reestablishment of pancreatic beta cell function by beta cell replacement therapy. Aristaless-related homeobox (Arx) gene encodes protein which plays an important role in the development of pancreatic alpha cells and is a main target for changing alpha cell identity. Results: In this study we used CRISPR/dCas9-based epigenetic tools for targeted hypermethylation of Arx gene promoter and its subsequent suppression in mouse pancreatic αTC1-6 cell line. Bisulfite sequencing and methylation profiling revealed that the dCas9-Dnmt3a3L-KRAB single chain fusion constructs (EpiCRISPR) was the most efficient. Epigenetic silencing of Arx expression was accompanied by an increase in transcription of the insulin gene (Ins2) mRNA on 5th and 7th post-transfection day, quantified by both RT-qPCR and RNA-seq. Insulin production and secretion was determined by immunocytochemistry and ELISA assay, respectively. Eventually, we were able to induce switch of approximately 1% of transiently transfected cells which were able to produce 35% more insulin than Mock transfected alpha cells. Conclusion: In conclusion, we successfully triggered a direct, transient switch of pancreatic alpha to insulin-producing cells opening a future research on promising therapeutic avenue for diabetes management.


Assuntos
Diabetes Mellitus , Células Secretoras de Glucagon , Camundongos , Animais , Fatores de Transcrição/metabolismo , Proteínas de Homeodomínio/genética , Insulina/metabolismo , Células Secretoras de Glucagon/metabolismo , Metilação de DNA , Diabetes Mellitus/metabolismo
14.
Nat Commun ; 13(1): 2042, 2022 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-35440565

RESUMO

Non-coding variants coordinate transcription factor (TF) binding and chromatin mark enrichment changes over regions spanning >100 kb. These molecularly coordinated regions are named "variable chromatin modules" (VCMs), providing a conceptual framework of how regulatory variation might shape complex traits. To better understand the molecular mechanisms underlying VCM formation, here, we mechanistically dissect a VCM-modulating noncoding variant that is associated with reduced chronic lymphocytic leukemia (CLL) predisposition and disease progression. This common, germline variant constitutes a 5-bp indel that controls the activity of an AXIN2 gene-linked VCM by creating a MEF2 binding site, which, upon binding, activates a super-enhancer-like regulatory element. This triggers a large change in TF binding activity and chromatin state at an enhancer cluster spanning >150 kb, coinciding with subtle, long-range chromatin compaction and robust AXIN2 up-regulation. Our results support a model in which the indel acts as an AXIN2 VCM-activating TF nucleation event, which modulates CLL pathology.


Assuntos
Cromatina , Leucemia Linfocítica Crônica de Células B , Cromatina/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Células Germinativas/metabolismo , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Fatores de Transcrição/metabolismo
15.
FASEB J ; 24(8): 2938-50, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20335224

RESUMO

Oxidative stress and increased release of reactive oxygen species (ROS) are associated with apoptosis induction. Here we report ROS-mediated induction of apoptosis by xanthohumol (XN) from hops. XN at concentrations of 1.6-25 microM induced an immediate and transient increase in superoxide anion radical (O(2)(-*)) formation in 3 human cancer cell lines (average+/-SD EC(50) of maximum O(2)(-*) induction=3.1+/-0.8 microM), murine macrophages (EC(50)=4.0+/-0.3 microM), and BPH-1 benign prostate hyperplasia cells (EC(50)=4.3+/-0.1 microM), as evidenced by the O(2)(-*)-specific indicator dihydroethidium. MitoSOX Red costaining and experiments using isolated mouse liver mitochondria (EC(50)=11.4+/-1.8 microM) confirmed mitochondria as the site of intracellular O(2)(-*) formation. Antimycin A served as positive control (EC(50)=12.4+/-0.9 microM). XN-mediated O(2)(-*) release was significantly reduced in BPH-1 rho(0) cells harboring nonfunctional mitochondria (EC(50)>25 microM) and by treatment of BPH-1 cells with vitamin C, N-acetylcysteine (NAC), or the superoxide dismutase mimetic MnTMPyP. In addition, we demonstrated a rapid 15% increase in oxidized glutathione and a dose-dependent overall thiol depletion within 6 h (IC(50)=24.3+/-11 microM). Respiratory chain complexes I-III were weakly inhibited by XN in bovine heart submitochondrial particles, but electron flux from complex I and II to complex III was significantly inhibited in BPH-1 cells, with IC(50) values of 28.1 +/- 2.4 and 24.4 +/- 5.2 microM, respectively. Within 15 min, intracellular ATP levels were significantly reduced by XN at 12.5 to 50 microM concentrations (IC(50)=26.7+/-3.7 microM). Concomitantly, XN treatment caused a rapid breakdown of the mitochondrial membrane potential and the release of cytochrome c, leading to apoptosis induction. Pre- or coincubation with 2 mM NAC and 50 microM MnTMPyP at various steps increased XN-mediated IC(50) values for cytotoxicity in BPH-1 cells from 6.7 +/- 0.2 to 12.2 +/- 0.1 and 41.4 +/- 7.6 microM, and it confirmed XN-induced O(2)(-*) as an essential trigger for apoptosis induction. In summary, we have identified mitochondria as a novel cellular target of XN action, resulting in increased O(2)(-*) production, disruption of cellular redox balance and mitochondrial integrity, and subsequent apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Flavonoides/farmacologia , Mitocôndrias Hepáticas/metabolismo , Neoplasias/tratamento farmacológico , Propiofenonas/farmacologia , Animais , Antineoplásicos/farmacologia , Bovinos , Linhagem Celular Tumoral , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Flavonoides/uso terapêutico , Glutationa/metabolismo , Humanos , Neoplasias/patologia , Propiofenonas/uso terapêutico , Espécies Reativas de Oxigênio , Compostos de Sulfidrila/metabolismo , Superóxidos/metabolismo
16.
Clin Epigenetics ; 13(1): 207, 2021 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-34789319

RESUMO

BACKGROUND: A shift in the proportions of blood immune cells is a hallmark of cancer development. Here, we investigated whether methylation-derived immune cell type ratios and methylation-derived neutrophil-to-lymphocyte ratios (mdNLRs) are associated with triple-negative breast cancer (TNBC). METHODS: Leukocyte subtype-specific unmethylated/methylated CpG sites were selected, and methylation levels at these sites were used as proxies for immune cell type proportions and mdNLR estimation in 231 TNBC cases and 231 age-matched controls. Data were validated using the Houseman deconvolution method. Additionally, the natural killer (NK) cell ratio was measured in a prospective sample set of 146 TNBC cases and 146 age-matched controls. RESULTS: The mdNLRs were higher in TNBC cases compared with controls and associated with TNBC (odds ratio (OR) range (2.66-4.29), all Padj. < 1e-04). A higher neutrophil ratio and lower ratios of NK cells, CD4 + T cells, CD8 + T cells, monocytes, and B cells were associated with TNBC. The strongest association was observed with decreased NK cell ratio (OR range (1.28-1.42), all Padj. < 1e-04). The NK cell ratio was also significantly lower in pre-diagnostic samples of TNBC cases compared with controls (P = 0.019). CONCLUSION: This immunomethylomic study shows that a shift in the ratios/proportions of leukocyte subtypes is associated with TNBC, with decreased NK cell showing the strongest association. These findings improve our knowledge of the role of the immune system in TNBC and point to the possibility of using NK cell level as a non-invasive molecular marker for TNBC risk assessment, early detection, and prevention.


Assuntos
Contagem de Leucócitos/estatística & dados numéricos , Neoplasias de Mama Triplo Negativas/genética , Adulto , Estudos de Casos e Controles , Metilação de DNA/genética , Metilação de DNA/imunologia , Epigenômica/métodos , Epigenômica/estatística & dados numéricos , Feminino , Humanos , Contagem de Leucócitos/classificação , Contagem de Leucócitos/métodos , Modelos Logísticos , Pessoa de Meia-Idade , Razão de Chances , Modelos de Riscos Proporcionais , Neoplasias de Mama Triplo Negativas/sangue , Neoplasias de Mama Triplo Negativas/imunologia
17.
Clin Epigenetics ; 11(1): 148, 2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31640781

RESUMO

BACKGROUND: The clinical course of prostate cancer (PCa) is highly variable, demanding an individualized approach to therapy. Overtreatment of indolent PCa cases, which likely do not progress to aggressive stages, may be associated with severe side effects and considerable costs. These could be avoided by utilizing robust prognostic markers to guide treatment decisions. RESULTS: We present a random forest-based classification model to predict aggressive behaviour of prostate cancer. DNA methylation changes between PCa cases with good or poor prognosis (discovery cohort with n = 70) were used as input. DNA was extracted from formalin-fixed tumour tissue, and genome-wide DNA methylation differences between both groups were assessed using Illumina HumanMethylation450 arrays. For the random forest-based modelling, the discovery cohort was randomly split into a training (80%) and a test set (20%). Our methylation-based classifier demonstrated excellent performance in discriminating prognosis subgroups in the test set (Kaplan-Meier survival analyses with log-rank p value < 0.0001). The area under the receiver operating characteristic curve (AUC) for the sensitivity analysis was 95%. Using the ICGC cohort of early- and late-onset prostate cancer (n = 222) and the TCGA PRAD cohort (n = 477) for external validation, AUCs for sensitivity analyses were 77.1% and 68.7%, respectively. Cancer progression-related DNA hypomethylation was frequently located in 'partially methylated domains' (PMDs)-large-scale genomic areas with progressive loss of DNA methylation linked to mitotic cell division. We selected several candidate genes with differential methylation in gene promoter regions for additional validation at the protein expression level by immunohistochemistry in > 12,000 tissue micro-arrayed PCa cases. Loss of ZIC2 protein expression was associated with poor prognosis and correlated with significantly shorter time to biochemical recurrence. The prognostic value of ZIC2 proved to be independent from established clinicopathological variables including Gleason grade, tumour stage, nodal stage and prostate-specific-antigen. CONCLUSIONS: Our results highlight the prognostic relevance of methylation loss in PMD regions, as well as of several candidate genes not previously associated with PCa progression. Our robust and externally validated PCa classification model either directly or via protein expression analyses of the identified top-ranked candidate genes will support the clinical management of prostate cancer.


Assuntos
Metilação de DNA , Epigenômica/métodos , Perfilação da Expressão Gênica/métodos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Progressão da Doença , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Teóricos , Gradação de Tumores , Medicina de Precisão , Prognóstico , Regiões Promotoras Genéticas , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Análise de Sobrevida , Análise Serial de Tecidos
18.
J Nat Prod ; 71(11): 1793-9, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18939864

RESUMO

Investigations of the marine-derived fungus Monodictys putredinis led to the isolation of two novel dimeric chromanones (1, 2) that consist of two uniquely modified xanthone-derived units. The structures were elucidated by extensive spectroscopic measurements including NOE experiments and CD analysis to deduce the configuration. The compounds (1, 2) were examined for their cancer chemopreventive potential and shown to inhibit cytochrome P450 1A activity with IC(50) values of 5.3 and 7.5 µM, respectively. In addition, both compounds displayed moderate activity as inducers of NAD(P)H:quinone reductase (QR) in cultured mouse Hepa 1c1c7 cells, with CD values (concentration required to double the specific activity of QR) of 22.1 and 24.8 µM, respectively. Compound 1 was slightly less potent than compound 2 in inhibiting aromatase activity, with IC(50) values of 24.4 and 16.5 µM.


Assuntos
Anticarcinógenos/isolamento & purificação , Inibidores da Aromatase/isolamento & purificação , Ascomicetos/química , Cromonas/isolamento & purificação , Xantonas/isolamento & purificação , Animais , Anticarcinógenos/química , Anticarcinógenos/farmacologia , Inibidores da Aromatase/química , Inibidores da Aromatase/farmacologia , Clorófitas/microbiologia , Cromonas/química , Cromonas/farmacologia , Indução Enzimática/efeitos dos fármacos , Concentração Inibidora 50 , Biologia Marinha , Camundongos , Estrutura Molecular , NAD(P)H Desidrogenase (Quinona)/biossíntese , NAD(P)H Desidrogenase (Quinona)/efeitos dos fármacos , Espanha , Xantonas/química , Xantonas/farmacologia
19.
Artigo em Inglês | MEDLINE | ID: mdl-29685968

RESUMO

Within the past decade, epigenetic mechanisms and their modulation by natural products have gained increasing interest. Dietary bioactive compounds from various sources, including green tea, soya, fruit and berries, cruciferous vegetables, whole grain foods, fish and others, have been shown to target enzymes involved in epigenetic gene regulation, including DNA methyltransferases, histone acetyltransferases, deacetylases and demethylases in vitro and in cell culture. Also, many dietary agents were shown to alter miRNA expression. In vivo studies in animal models and humans are still limited. Recent research has indicated that the gut microbiota and gut microbial metabolites might be important mediators of diet-epigenome interactions. Inter-individual differences in the gut microbiome might affect release, metabolism and bioavailability of dietary agents and explain variability in response to intervention in human studies. Only a few microbial metabolites, including folate, phenolic acids, S-(-)equol, urolithins, isothiocyanates, and short- and long-chain fatty acids have been tested with respect to their potential to influence epigenetic mechanisms. Considering that a complex mixture of intermediary and microbial metabolites is present in human circulation, a more systematic interdisciplinary investigation of nutri-epigenetic activities and their impact on human health is called for.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.


Assuntos
Dieta , Epigênese Genética , Microbioma Gastrointestinal/fisiologia , Metagenoma/fisiologia , Humanos
20.
Oxid Med Cell Longev ; 2018: 1617202, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29576843

RESUMO

Obesity is characterized by excess body fat accumulation due to an increase in the size and number of differentiated mature adipocytes. Adipocyte differentiation is regulated by genetic and environmental factors, and its inhibition could represent a strategy for obesity prevention and treatment. The current study was designed with two aims: (i) to evaluate the changes in the expression of adipogenic markers (C/EBPα, PPARγ variant 1 and variant 2, and GLUT4) in 3T3-L1 murine preadipocytes at four stages of the differentiation process and (ii) to compare the effectiveness of sulforaphane, genistein, and docosahexaenoic acid in reducing lipid accumulation and modulating C/EBPα, PPARγ1, PPARγ2, and GLUT4 mRNA expression in mature adipocytes. All bioactive compounds were shown to suppress adipocyte differentiation, although with different effectiveness. These results set the stage for further studies considering natural food constituents as important agents in preventing or treating obesity.


Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Genisteína/farmacologia , Isotiocianatos/farmacologia , Obesidade/tratamento farmacológico , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Proteínas Estimuladoras de Ligação a CCAAT/genética , Diferenciação Celular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Transportador de Glucose Tipo 4/biossíntese , Transportador de Glucose Tipo 4/genética , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologia , PPAR gama/biossíntese , PPAR gama/genética , Sulfóxidos
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