RESUMEN
Cancer is one of the leading causes of death worldwide, and its incidence and mortality are increasing each year. Improved therapeutic strategies against cancer have progressed, but remain insufficient to invert this trend. Along with several other risk factors, abnormal genetic and epigenetic regulations play a critical role in the initiation of cellular transformation, as well as tumorigenesis. The epigenetic regulator UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is a multidomain protein with oncogenic abilities overexpressed in most cancers. Through the coordination of its multiple domains and other epigenetic key players, UHRF1 regulates DNA methylation and histone modifications. This well-coordinated dialogue leads to the silencing of tumor-suppressor genes (TSGs) and facilitates tumor cells' resistance toward anticancer drugs, ultimately promoting apoptosis escape and uncontrolled proliferation. Several studies have shown that the downregulation of UHRF1 with natural compounds in tumor cells induces the reactivation of various TSGs, inhibits cell growth, and promotes apoptosis. In this review, we discuss the underlying mechanisms and the potential of various natural and synthetic compounds that can inhibit/minimize UHRF1's oncogenic activities and/or its expression.
Asunto(s)
Carcinogénesis , Transformación Celular Neoplásica , Humanos , Apoptosis , Ciclo Celular , Epigénesis Genética , Proteínas Potenciadoras de Unión a CCAAT/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
DNA methylation is an essential epigenetic modification, present in both unique DNA sequences and repetitive elements, but its exact function in repetitive elements remains obscure. Here, we describe the genome-wide comparative analysis of the 5mC, 5hmC, 5fC, and 5caC profiles of repetitive elements in mouse embryonic fibroblasts and mouse embryonic stem cells. We provide evidence for distinct and highly specific DNA methylation/oxidation patterns of the repetitive elements in both cell types, which mainly affect CA repeats and evolutionarily conserved mouse-specific transposable elements including IAP-LTRs, SINEs B1m/B2m, and L1Md-LINEs. DNA methylation controls the expression of these retroelements, which are clustered at specific locations in the mouse genome. We show that TDG is implicated in the regulation of their unique DNA methylation/oxidation signatures and their dynamics. Our data suggest the existence of a novel epigenetic code for the most recently acquired evolutionarily conserved repeats that could play a major role in cell differentiation.
Asunto(s)
Metilación de ADN , Epigénesis Genética , Fibroblastos/metabolismo , Genoma , Células Madre Embrionarias de Ratones/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animales , Diferenciación Celular , Citosina/análogos & derivados , Citosina/metabolismo , Elementos Transponibles de ADN , Fibroblastos/citología , Ratones , Células Madre Embrionarias de Ratones/citología , Cultivo Primario de Células , Timina ADN Glicosilasa/genética , Timina ADN Glicosilasa/metabolismoRESUMEN
During DNA replication, ubiquitin-like, containing PHD and RING fingers domainsâ 1 (UHRF1) plays key roles in the inheritance of methylation patterns to daughter strands by recognizing through its SET and RING-associated domain (SRA) the methylated CpGs and recruiting DNA methyltransferaseâ 1 (DNMT1). Herein, our goal is to identify UHRF1 inhibitors targeting the 5'-methylcytosine (5mC) binding pocket of the SRA domain to prevent the recognition and flipping of 5mC and determine the molecular and cellular consequences of this inhibition. For this, we used a multidisciplinary strategy combining virtual screening and molecular modeling with biophysical assays in solution and cells. We identified an anthraquinone compound able to bind to the 5mC binding pocket and inhibit the base-flipping process in the low micromolar range. We also showed in cells that this hit impaired the UHRF1/DNMT1 interaction and decreased the overall methylation of DNA, highlighting the critical role of base flipping for DNMT1 recruitment and providing the first proof of concept of the druggability of the 5mC binding pocket. The selected anthraquinone appears thus as a key tool to investigate the role of UHRF1 in the inheritance of methylation patterns, as well as a starting point for hit-to-lead optimizations.
Asunto(s)
Antraquinonas/química , Proteínas Potenciadoras de Unión a CCAAT/antagonistas & inhibidores , Inhibidores Enzimáticos/química , 5-Metilcitosina/química , Sitios de Unión , ADN (Citosina-5-)-Metiltransferasa 1/química , Evaluación Preclínica de Medicamentos/métodos , Células HeLa , Humanos , Cinética , Metilación , Simulación del Acoplamiento Molecular , Estructura Molecular , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Transfección/métodos , Ubiquitina-Proteína LigasasRESUMEN
DNA methylation patterns, which are critical for gene expression, are replicated by DNA methyltransferase 1 (DNMT1) and ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) proteins. This replication is initiated by the recognition of hemimethylated CpG sites and further flipping of methylated cytosines (mC) by the Set and Ring Associated (SRA) domain of UHRF1. Although crystallography has shed light on the mechanism of mC flipping by SRA, tools are required to monitor in real time how SRA reads DNA and flips the modified nucleobase. To accomplish this aim, we have utilized two distinct fluorescent nucleobase surrogates, 2-thienyl-3-hydroxychromone nucleoside (3HCnt) and thienoguanosine (thG), incorporated at different positions into hemimethylated (HM) and nonmethylated (NM) DNA duplexes. Large fluorescence changes were associated with mC flipping in HM duplexes, showing the outstanding sensitivity of both nucleobase surrogates to the small structural changes accompanying base flipping. Importantly, the nucleobase surrogates marginally affected the structure of the duplex and its affinity for SRA at positions where they were responsive to base flipping, illustrating their promise as nonperturbing probes for monitoring such events. Stopped-flow studies using these two distinct tools revealed the fast kinetics of SRA binding and sliding to NM duplexes, consistent with its reader role. In contrast, the kinetics of mC flipping was found to be much slower in HM duplexes, substantially increasing the lifetime of CpG-bound UHRF1, and thus the probability of recruiting DNMT1 to faithfully duplicate the DNA methylation profile. The fluorescence-based approach using these two different fluorescent nucleoside surrogates advances the mechanistic understanding of the UHRF1/DNMT1 tandem and the development of assays for the identification of base flipping inhibitors.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Citosina/metabolismo , ADN/metabolismo , Termodinámica , Proteínas Potenciadoras de Unión a CCAAT/química , Citosina/química , ADN/química , Metilación de ADN , Replicación del ADN , Fluorescencia , Humanos , Cinética , Estructura Molecular , Ubiquitina-Proteína LigasasRESUMEN
UHRF1 plays a central role in the maintenance and transmission of epigenetic modifications by recruiting DNMT1 to hemimethylated CpG sites via its SET and RING-associated (SRA) domain, ensuring error-free duplication of methylation profiles. To characterize SRA-induced changes in the conformation and dynamics of a target 12 bp DNA duplex as a function of the methylation status, we labeled duplexes by the environment-sensitive probe 2-aminopurine (2-Ap) at various positions near or far from the central CpG recognition site containing either a nonmodified cytosine (NM duplex), a methylated cytosine (HM duplex), or methylated cytosines on both strands (BM duplex). Steady-state and time-resolved fluorescence indicated that binding of SRA induced modest conformational and dynamical changes in NM, HM, and BM duplexes, with only slight destabilization of base pairs, restriction of global duplex flexibility, and diminution of local nucleobase mobility. Moreover, significant restriction of the local motion of residues flanking the methylcytosine in the HM duplex suggested that these residues are more rigidly bound to SRA, in line with a slightly higher affinity of the HM duplex as compared to that of the NM or BM duplex. Our results are consistent with a "reader" role, in which the SRA domain scans DNA sequences for hemimethylated CpG sites without perturbation of the structure of contacted nucleotides.
Asunto(s)
2-Aminopurina/química , Proteínas Potenciadoras de Unión a CCAAT/química , Islas de CpG , Metilación de ADN , ADN/química , 2-Aminopurina/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , ADN/genética , ADN/metabolismo , Humanos , Estructura Terciaria de Proteína , Ubiquitina-Proteína LigasasRESUMEN
Epigenetic inheritance in mammals is characterized by high-fidelity replication of CpG methylation patterns during development. UHRF1 (also known as ICBP90 in humans and Np95 in mouse) is an E3 ligase important for the maintenance of global and local DNA methylation in vivo. The preferential affinity of UHRF1 for hemi-methylated DNA over symmetrically methylated DNA by means of its SET and RING-associated (SRA) domain and its association with the maintenance DNA methyltransferase 1 (DNMT1) suggests a role in replication of the epigenetic code. Here we report the 1.7 A crystal structure of the apo SRA domain of human UHRF1 and a 2.2 A structure of its complex with hemi-methylated DNA, revealing a previously unknown reading mechanism for methylated CpG sites (mCpG). The SRA-DNA complex has several notable structural features including a binding pocket that accommodates the 5-methylcytosine that is flipped out of the duplex DNA. Two specialized loops reach through the resulting gap in the DNA from both the major and the minor grooves to read the other three bases of the CpG duplex. The major groove loop confers both specificity for the CpG dinucleotide and discrimination against methylation of deoxycytidine of the complementary strand. The structure, along with mutagenesis data, suggests how UHRF1 acts as a key factor for DNMT1 maintenance methylation through recognition of a fundamental unit of epigenetic inheritance, mCpG.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/química , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Metilación de ADN , ADN/química , ADN/metabolismo , 5-Metilcitosina/metabolismo , Sitios de Unión , Islas de CpG/genética , Cristalografía por Rayos X , ADN/genética , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Epigénesis Genética , Humanos , Modelos Moleculares , Conformación Molecular , Estructura Terciaria de Proteína , Ubiquitina-Proteína LigasasRESUMEN
HIRIP3 is a mammalian protein homologous to the yeast H2A.Z deposition chaperone Chz1. However, the structural basis underlying Chz's binding preference for H2A.Z over H2A, as well as the mechanism through which Chz1 modulates histone deposition or replacement, remains enigmatic. In this study, we aimed to characterize the function of HIRIP3 and to identify its interacting partners in HeLa cells. Our findings reveal that HIRIP3 is specifically associated in vivo with H2A-H2B dimers and CK2 kinase. While bacterially expressed HIRIP3 exhibited a similar binding affinity towards H2A and H2A.Z, the associated CK2 kinase showed a notable preference for H2A phosphorylation at serine 1. The recombinant HIRIP3 physically interacted with the H2A αC helix through an extended CHZ domain and played a crucial role in depositing the canonical core histones onto naked DNA. Our results demonstrate that mammalian HIRIP3 acts as an H2A histone chaperone, assisting in its selective phosphorylation by Ck2 kinase at serine 1 and facilitating its deposition onto chromatin.
Asunto(s)
Chaperonas de Histonas , Histonas , Animales , Humanos , Células HeLa , Chaperonas de Histonas/genética , Histonas/metabolismo , Mamíferos/metabolismo , Chaperonas Moleculares/metabolismo , Saccharomyces cerevisiae/metabolismo , Serina , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMEN
DNA methylation is one of the most important epigenetic mark involved in many physiologic cellular processes and pathologies. During mitosis, the transmission of DNA methylation patterns from a mother to the daughter cells is ensured through the action of the Ubiquitin-like, containing PHD and RING domains, 1/DNA methyltransferase 1 (UHRF1/DNMT1) tandem. UHRF1 is involved in the silencing of many tumor suppressor genes (TSGs) via mechanisms that remain largely to be deciphered. The present study investigated the role and the regulation of UHRF1 poly-ubiquitination induced by thymoquinone, a natural anti-cancer drug, known to enhance or re-activate the expression of TSGs. We found that the auto-ubiquitination of UHRF1, induced by TQ, is mediated by reactive oxygen species, and occurs following DNA damage. We demonstrated that the poly-ubiquitinated form of UHRF1 is K63-linked and can still silence the tumor suppressor gene p16INK4A/CDKN2A. We further showed that TQ-induced auto-ubiquitination is mediated via the activity of Tip60. Since this latter is known as a nuclear receptor co-factor, we investigated if the glucocorticoid receptor (GR) might be involved in the regulation of UHRF1 ubiquitination. Activation of the GR, with dexamethasone, did not influence auto-ubiquitination of UHRF1. However, we could observe that TQ induced a K48-linked poly-ubiquitination of GR, probably involved in the proteosomal degradation pathway. Mass-spectrometry analysis of FLAG-HA-tagged UHRF1 identified UHRF1 partners involved in DNA repair and showed that TQ increased their association with UHRF1, suggesting that poly-ubiquitination of UHRF1 is involved in the DNA repair process. We propose that poly-ubiquitination of UHRF1 serves as a scaffold to recruit the DNA repair machinery at DNA damage sites.
Asunto(s)
Benzoquinonas , Proteínas Potenciadoras de Unión a CCAAT , Reparación del ADN , Ubiquitina-Proteína Ligasas , Ubiquitinación , Humanos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Ubiquitinación/efectos de los fármacos , Benzoquinonas/farmacología , Reparación del ADN/efectos de los fármacos , Antineoplásicos/farmacología , Daño del ADN/efectos de los fármacosRESUMEN
Ubiquitin-like containing PHD and Ring finger 1 (UHRF1) contributes to silencing of tumor suppressor genes by recruiting DNA methyltransferase 1 (DNMT1) to their hemi-methylated promoters. Conversely, demethylation of these promoters has been ascribed to the natural anti-cancer drug, epigallocatechin-3-gallate (EGCG). The aim of the present study was to investigate whether the UHRF1/DNMT1 pair is an important target of EGCG action. Here, we show that EGCG down-regulates UHRF1 and DNMT1 expression in Jurkat cells, with subsequent up-regulation of p73 and p16(INK4A) genes. The down-regulation of UHRF1 is dependent upon the generation of reactive oxygen species by EGCG. Up-regulation of p16(INK4A) is strongly correlated with decreased promoter binding by UHRF1. UHRF1 over-expression counteracted EGCG-induced G1-arrested cells, apoptosis, and up-regulation of p16(INK4A) and p73. Mutants of the Set and Ring Associated (SRA) domain of UHRF1 were unable to down-regulate p16(INK4A) and p73, either in the presence or absence of EGCG. Our results show that down-regulation of UHRF1 is upstream to many cellular events, including G1 cell arrest, up-regulation of tumor suppressor genes and apoptosis.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Catequina/análogos & derivados , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Supresores de Tumor/efectos de los fármacos , Proteínas Nucleares/genética , Especies Reactivas de Oxígeno/metabolismo , Proteínas Supresoras de Tumor/genética , Apoptosis/efectos de los fármacos , Proteínas Potenciadoras de Unión a CCAAT/antagonistas & inhibidores , Catequina/farmacología , Ciclo Celular/efectos de los fármacos , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , Regulación hacia Abajo , Humanos , Células Jurkat , Regiones Promotoras Genéticas , Proteína Tumoral p73 , Ubiquitina-Proteína Ligasas , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Inherited defects in the base-excision repair gene MBD4 predispose individuals to adenomatous polyposis and colorectal cancer, which is characterized by an accumulation of C > T transitions resulting from spontaneous deamination of 5'-methylcytosine. METHODS: Here, we have investigated the potential role of MBD4 in regulating DNA methylation levels using genome-wide transcriptome and methylome analyses. Additionally, we have elucidated its function through a series of in vitro experiments. RESULTS: Here we show that the protein MBD4 is required for DNA methylation maintenance and G/T mismatch repair. Transcriptome and methylome analyses reveal a genome-wide hypomethylation of promoters, gene bodies and repetitive elements in the absence of MBD4 in vivo. Methylation mark loss is accompanied by a broad transcriptional derepression phenotype affecting promoters and retroelements with low methylated CpG density. MBD4 in vivo forms a complex with the mismatch repair proteins (MMR), which exhibits high bi-functional glycosylase/AP-lyase endonuclease specific activity towards methylated DNA substrates containing a G/T mismatch. Experiments using recombinant proteins reveal that the association of MBD4 with the MMR protein MLH1 is required for this activity. CONCLUSIONS: Our data identify MBD4 as an enzyme specifically designed to repair deaminated 5-methylcytosines and underscores its critical role in safeguarding against methylation damage. Furthermore, it illustrates how MBD4 functions in normal and pathological conditions.
Asunto(s)
Reparación del ADN , Retroelementos , Humanos , Reparación de la Incompatibilidad de ADN , Proteínas Recombinantes/genética , Metilación de ADN , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismoRESUMEN
Histone modifications and DNA methylation represent two layers of heritable epigenetic information that regulate eukaryotic chromatin structure and gene activity. UHRF1 is a unique factor that bridges these two layers; it is required for maintenance DNA methylation at hemimethylated CpG sites, which are specifically recognized through its SRA domain and also interacts with histone H3 trimethylated on lysine 9 (H3K9me3) in an unspecified manner. Here we show that UHRF1 contains a tandem Tudor domain (TTD) that recognizes H3 tail peptides with the heterochromatin-associated modification state of trimethylated lysine 9 and unmodified lysine 4 (H3K4me0/K9me3). Solution NMR and crystallographic data reveal the TTD simultaneously recognizes H3K9me3 through a conserved aromatic cage in the first Tudor subdomain and unmodified H3K4 within a groove between the tandem subdomains. The subdomains undergo a conformational adjustment upon peptide binding, distinct from previously reported mechanisms for dual histone mark recognition. Mutant UHRF1 protein deficient for H3K4me0/K9me3 binding shows altered localization to heterochromatic chromocenters and fails to reduce expression of a target gene, p16(INK4A), when overexpressed. Our results demonstrate a novel recognition mechanism for the combinatorial readout of histone modification states associated with gene silencing and add to the growing evidence for coordination of, and cross-talk between, the modification states of H3K4 and H3K9 in regulation of gene expression.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/química , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Heterocromatina/química , Heterocromatina/metabolismo , Histonas/química , Histonas/metabolismo , Animales , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT/genética , Islas de CpG/fisiología , Cristalografía por Rayos X , Inhibidor p16 de la Quinasa Dependiente de Ciclina/química , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Metilación de ADN/fisiología , Regulación de la Expresión Génica/fisiología , Heterocromatina/genética , Histonas/genética , Humanos , Ratones , Ratones Noqueados , Resonancia Magnética Nuclear Biomolecular , Procesamiento Proteico-Postraduccional/fisiología , Estructura Terciaria de Proteína , Ubiquitina-Proteína LigasasRESUMEN
Aging is associated with blunted endothelium-dependent relaxations and vascular oxidative stress. Our previous study has indicated that daily intake of red wine polyphenols (RWPs) by young rats retards aging-related endothelial dysfunction in middle-aged rats. The aim of the present study is to determine whether intake of RWPs also improves an established endothelial dysfunction in middle-aged rats and, if so, to determine the underlying mechanism. Middle-aged rats (51 weeks) received either solvent (3% ethanol), RWPs extract (100mg/kg/day) or the antioxidant and NADPH oxidase inhibitor apocynin (100mg/kg/day) in the drinking water for 4 weeks. Vascular reactivity of mesenteric artery rings from control young (12 weeks) and middle-aged rats was assessed in organ chambers. The expression level of endothelial NO synthase (eNOS), arginase I, angiotensin II receptors (AT1R and AT2R), NADPH oxidase subunits and nitrotyrosines was assessed by immunohistochemistry, and the vascular formation of reactive oxygen species (ROS) by dihydroethidine. Aging is associated with blunted endothelium-dependent relaxations, an excessive vascular formation of ROS and peroxynitrites, and an up-regulation of eNOS, arginase I, NADPH oxidase subunits (nox-1, p22phox), and AT1R and AT2R expression. RWPs and apocynin treatments improved endothelial dysfunction, normalized oxidative stress and the expression of the different proteins in the mesenteric artery of middle-aged rats. The present findings indicate that aging is associated with blunted endothelium-dependent relaxations involving an increased oxidative stress, and that these responses are improved by the intake of RWPs or apocynin for 4weeks most likely by normalizing the expression of eNOS, arginase I, NADPH oxidase and angiotensin receptors.
Asunto(s)
Envejecimiento/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Polifenoles/administración & dosificación , Vasodilatación/efectos de los fármacos , Vino , Acetofenonas/administración & dosificación , Animales , Antioxidantes/administración & dosificación , Endotelio Vascular/fisiopatología , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/fisiopatología , NADPH Oxidasas/biosíntesis , Óxido Nítrico Sintasa de Tipo III/biosíntesis , Ratas , Ratas Wistar , Receptor de Angiotensina Tipo 1/biosíntesis , Receptor de Angiotensina Tipo 2/biosíntesis , Vasodilatación/fisiologíaRESUMEN
Maritime pine bark is a rich source of polyphenolic compounds and is commonly employed as a herbal supplement worldwide. This study was designed to check the potential of maritime pine tannin extract (MPTE) in anticancer therapy and to determine the underlying mechanism of action. Our results showed that MPTE, containing procyanidin oligomers and lanostane type terpenoids, has an inhibitory effect on cancer cell proliferation through cell cycle arrest in the G2/M phase. Treatment with MPTE also induced apoptosis in a concentration-dependent manner in human cancer cell lines (HeLa and U2OS), as evidenced by the enhanced activation of caspase 3 and the cleavage of PARP along with the downregulation of the antiapoptotic protein Bcl-2. Interestingly, human non-cancerous fibroblasts are much less sensitive to MPTE, suggesting that it preferentially targets cancer cells. MPTE played a pro-oxidant role in cancer cells and promoted the expression of the p73 tumor suppressor gene in p53-deficient cells. It also downregulated the protooncogenic proteins UHRF1 and DNMT1, mediators of the DNA methylation machinery, and reduced the global methylation levels in HeLa cells. Overall, our results show that maritime pine tannin extract can play a favorable role in cancer treatment, and can be further explored by the pharmaceutical industry.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Potenciadoras de Unión a CCAAT , Epigénesis Genética/efectos de los fármacos , Pinus/química , Taninos/farmacología , Ubiquitina-Proteína Ligasas , Apoptosis/efectos de los fármacos , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Células HeLa , Humanos , Corteza de la Planta/química , Extractos Vegetales/farmacología , Proteína Tumoral p73/genética , Proteína Tumoral p73/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
BACKGROUND & AIMS: Advanced stages of portal hypertension are characterized by generalized vasodilatation and a hyperdynamic syndrome that leads to complications such as hepatopulmonary syndrome. We assessed the endothelial function--particularly the formation of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF)--in rats following common bile duct ligation (CBDL) to determine the underlying mechanisms of these processes. METHODS: Reactivity of mesenteric artery rings from male Wistar rats was determined in organ chambers. The expression levels of connexins (Cx) (Cx37, Cx40, Cx43), intermediate and small conductance Ca(2+)-activated K(+) channels (IK(Ca), SK(Ca)), endothelial NO synthase (eNOS), NADPH oxidase subunits, and nitrotyrosines were assessed by immunohistochemistry in mesenteric and pulmonary arteries. The vascular formation of reactive oxygen species (ROS) was evaluated using dihydroethidine. Control rats or those that had undergone CBDL were given either the NADPH oxidase inhibitor apocynin or the angiotensin II receptor type 1 antagonist losartan. RESULTS: Decreased EDHF-mediated relaxations to acetylcholine and red wine polyphenols were observed in CBDL rats, compared with controls, whereas the level of NO-mediated relaxation was similar. Impaired EDHF-mediated relaxations were associated with reduced vascular expression of Cx37, Cx40, Cx43, IK(Ca) and SK(Ca); increased expression of eNOS and NADPH oxidase subunits; and increased vascular formation of ROS and peroxynitrites. These effects were prevented by exposure to apocynin or losartan. CONCLUSIONS: CBDL is associated with reduced EDHF-mediated relaxations in the mesenteric artery, whereas NO-mediated relaxations persisted. These findings indicate that impaired EDHF-mediated relaxation involves an excessive vascular oxidative stress, most likely following activation of angiotensin II type 1 receptors.
Asunto(s)
Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Endotelio Vascular/fisiopatología , Hipertensión Portal/fisiopatología , Losartán/farmacología , Arterias Mesentéricas/fisiopatología , Acetofenonas/farmacología , Animales , Factores Biológicos/fisiología , Conexinas/análisis , Hipertensión Portal/patología , Masculino , NADPH Oxidasas/genética , Óxido Nítrico Sintasa de Tipo III/genética , Oxidación-Reducción , Estrés Oxidativo , Canales de Potasio/análisis , Ratas , Ratas Wistar , Vasodilatación/efectos de los fármacosRESUMEN
Aging is associated with oxidative stress-mediated endothelial dysfunction and decline in physical performance, which promote cardiovascular diseases. This study examined whether chronic intake of red wine polyphenols (RWPs), a rich source of natural antioxidants, prevents aging-related impairment of vascular function and physical exercise capacity. Vascular reactivity from 12, 20 and 40 week-old rats was assessed in organ chambers. Rats received from week 16 to 40 either solvent, RWPs or the antioxidant and NADPH oxidase inhibitor, apocynin. Aging was associated with blunted endothelium-dependent relaxations, oxidative stress (dihydroethidine staining), and an upregulation of eNOS, arginase I, NADPH oxidase p22phox and nox1 subunits, and AT1 and AT2 receptors (assessed by immunohistochemistry) in the mesenteric artery. RWPs and apocynin improved the endothelial dysfunction, normalized oxidative stress and the expression of the different proteins. RWPs also improved aging-related decline in physical exercise. Thus, intake of RWPs protects against aging-induced endothelial dysfunction and decline in physical performance. These effects likely involve the ability of RWPs to normalize oxidative stress and the expression of proteins involved in the formation of NO and the angiotensin II pathway.
Asunto(s)
Envejecimiento/efectos de los fármacos , Antioxidantes/administración & dosificación , Endotelio Vascular/efectos de los fármacos , Flavonoides/administración & dosificación , NADPH Oxidasas/fisiología , Estrés Oxidativo/efectos de los fármacos , Fenoles/administración & dosificación , Vino , Acetofenonas/administración & dosificación , Envejecimiento/metabolismo , Envejecimiento/fisiología , Angiotensina II/metabolismo , Animales , Arginasa/metabolismo , Endotelio Vascular/enzimología , Endotelio Vascular/fisiología , Inhibidores Enzimáticos/administración & dosificación , Actividad Motora/efectos de los fármacos , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , NADH NADPH Oxidorreductasas/fisiología , NADPH Oxidasa 1 , NADPH Oxidasas/antagonistas & inhibidores , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Polifenoles , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Cancer stem cells are expected to be responsible for tumor initiation and metastasis. These cells are therefore potential targets for innovative anticancer therapies. However, the absence of bona fide cancer stem cell lines is a real problem for the development of such approaches. Since teratocarcinoma cells are totipotent stem cells with a high degree of malignancy, we used them as a model of cancer stem cells in order to evaluate the anticancer chemopreventive activity of red wine polyphenols (RWPs) and to determine the underlying cellular and molecular mechanisms. We therefore investigated the effects of RWPs on the embryonal carcinoma (EC) cell line P19 which was grown in the same culture conditions as the most appropriate normal cell line counterpart, the pluripotent embryonic fibroblast cell line NIH/3T3. The present study indicates that RWPs selectively inhibited the proliferation of P19 EC cells and induced G1 cell cycle arrest in a dose-dependent manner. Moreover, RWPs treatment specifically triggered apoptosis of P19 EC cells in association with a dramatic upregulation of the tumor suppressor gene p53 and caspase-3 activation. Our findings suggest that the chemopreventive activity of RWPs on tumor initiation and development is related to a growth inhibition and a p53-dependent induction of apoptosis in teratocarcinoma cells. In addition, this study also shows that the EC cell line is a convenient source for studying the responses of cancer stem cells to new potential anticancer agents.
Asunto(s)
Apoptosis/efectos de los fármacos , Flavonoides/farmacología , Modelos Biológicos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Fenoles/farmacología , Teratocarcinoma/patología , Vino , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Flavonoides/química , Fase G1/efectos de los fármacos , Ratones , Células 3T3 NIH , Fenoles/química , Polifenoles , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
This study evaluated the in vivo antitumor activity of grape-derived polyphenols. BALB/c mice were subcutaneously implanted with C26 colon carcinoma cells, and 2 d later they received either solvent or red wine polyphenols (RWPs) (100 mg/kg/d, human equivalent dose approximately 500 mg/d) in the drinking water for 25 d. Wistar rats received either solvent or RWPs (100 mg/kg/d, human equivalent dose approximately 1000 mg/d) in the drinking water 1 wk before injection of azoxymethane and were studied 10 wk later. In mice, RWPs inhibited tumor growth by 31%, reduced tumor vascularization and the number of lung metastases, decreased proliferation as indicated by down-regulation of Ki67, cyclin D1, and UHRF1, and increased apoptosis as indicated by TUNEL staining and active caspase-3 levels in tumor cells. RWPs reduced expression of VEGF, matrix metalloproteinase (MMP)-2, MMP-9, and cyclooxygenase-2 and increased expression of tumor suppressor genes p16(INK4A), p53, and p73 in tumor cells. In rats, RWPs reduced by 49% the number of azoxymethane-induced aberrant crypt foci (preneoplastic lesions) in colon. Thus, RWPs effectively reduced the development of colon carcinoma tumors in vivo by blunting tumor vascularization and by inhibiting proliferation and promoting apoptosis of tumor cells subsequent to an up-regulation of tumor suppressor genes.
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Inhibidores de la Angiogénesis/farmacología , Apoptosis/efectos de los fármacos , Flavonoides/farmacología , Fenoles/farmacología , Vitis/química , Animales , Azoximetano/toxicidad , Línea Celular Tumoral , Neoplasias del Colon/inducido químicamente , Neoplasias del Colon/metabolismo , Femenino , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Endogámicos BALB C , Polifenoles , Distribución Aleatoria , Ratas , Ratas WistarRESUMEN
Silencing of tumor suppressor genes (TSGs) through epigenetic mechanisms, mainly via abnormal promoter DNA methylation, is considered a main mechanism of tumorigenesis. The abnormal DNA methylation profiles are transmitted from the cancer mother cell to the daughter cells through the involvement of a macromolecular complex in which the ubiquitin-like containing plant homeodomain (PHD), and an interesting new gene (RING) finger domains 1 (UHRF1), play the role of conductor. Indeed, UHRF1 interacts with epigenetic writers, such as DNA methyltransferase 1 (DNMT1), histone methyltransferase G9a, erasers like histone deacetylase 1 (HDAC1), and functions as a hub protein. Thus, targeting UHRF1 and/or its partners is a promising strategy for epigenetic cancer therapy. The natural compound thymoquinone (TQ) exhibits anticancer activities by targeting several cellular signaling pathways, including those involving UHRF1. In this review, we highlight TQ as a potential multitarget single epidrug that functions by targeting the UHRF1/DNMT1/HDAC1/G9a complex. We also speculate on the possibility that TQ might specifically target UHRF1, with subsequent regulatory effects on other partners.
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Benzoquinonas/farmacología , Proteínas Potenciadoras de Unión a CCAAT/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Carcinogénesis/efectos de los fármacos , Carcinogénesis/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Metilación de ADN/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacosRESUMEN
OBJECTIVE: Macrophage migration inhibitory factor (MIF) is an inflammatory and neurorendocrine mediator that counterregulates glucocorticoid immunosuppression. MIF polymorphisms, which comprise a variant promoter microsatellite (-794 CATT5-8 ), are linked genetically to autoimmune disease severity and to glucocorticoid resistance. While invasive stimuli increase MIF expression, MIF also is up-regulated by glucocorticoids, which serve as a physiologic regulator of inflammatory responses. This study was undertaken to define interactions between the MIF promoter, the glucocorticoid receptor (GR), and the transcription factor inverted CCAAT box binding protein 90 kd (ICBP90) (also referred to as UHRF1), which binds to the promoter in a -794 CATT5-8 length-dependent manner, to regulate MIF transcription. METHODS: Interactions of ICBP90, GR, and activator protein 1 (AP-1) with MIF -794 CATT5-8 promoter constructs were assessed by coimmunoprecipitation, Western blotting, and genetic knockdown. Nuclear colocalization studies were performed using anti-transcription factor antibodies and confocal microscopy of glucocorticoid-treated cells. MIF transcription was studied in CEM-C7 T cells, and the impact of the MIF -794 CATT5-8 microsatellite variation confirmed in peripheral blood T cells and in rheumatoid synovial fibroblasts of defined MIF genotype. Functional interactions were quantified by apoptosis and apoptotic signaling in high- and low-genotypic MIF-expressing human cells. RESULTS: We defined functional interactions between the transcription factors ICBP90, the GR, and AP-1 that up-regulated MIF transcription in a -794 CATT5-8 length-dependent manner. Experimental reduction of ICBP90, GR, or AP-1 decreased MIF expression and increased glucocorticoid sensitivity, leading to enhanced apoptosis in T lymphocytes and in rheumatoid synovial fibroblasts. CONCLUSION: These findings suggest a mechanism for genetic variation of glucocorticoid-regulated MIF transcription, with implications for autoimmune disease severity and glucocorticoid responsiveness.
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Apoptosis/genética , Proteínas Potenciadoras de Unión a CCAAT/genética , Glucocorticoides/administración & dosificación , Oxidorreductasas Intramoleculares/genética , Factores Inhibidores de la Migración de Macrófagos/genética , Receptores de Glucocorticoides/genética , Ubiquitina-Proteína Ligasas/genética , Alelos , Apoptosis/efectos de los fármacos , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Línea Celular , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Sitios Genéticos , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Oxidorreductasas Intramoleculares/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Regiones Promotoras Genéticas , Receptores de Glucocorticoides/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The Rett syndrome protein MeCP2 was described as a methyl-CpG-binding protein, but its exact function remains unknown. Here we show that mouse MeCP2 is a microsatellite binding protein that specifically recognizes hydroxymethylated CA repeats. Depletion of MeCP2 alters chromatin organization of CA repeats and lamina-associated domains and results in nucleosome accumulation on CA repeats and genome-wide transcriptional dysregulation. The structure of MeCP2 in complex with a hydroxymethylated CA repeat reveals a characteristic DNA shape, with considerably modified geometry at the 5-hydroxymethylcytosine, which is recognized specifically by Arg133, a key residue whose mutation causes Rett syndrome. Our work identifies MeCP2 as a microsatellite DNA binding protein that targets the 5hmC-modified CA-rich strand and maintains genome regions nucleosome-free, suggesting a role for MeCP2 dysfunction in Rett syndrome.