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1.
Food Chem ; 338: 127656, 2021 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-32798821

RESUMEN

Bisguaiacols are promising lignin-derivable alternatives to bisphenol A (BPA), but limited bioassay data are available on their estrogenic activity (EA). Herein, we investigated the estrogen receptor alpha (ERα)-mediated EA of six newly synthesized bisguaiacols, which differed in the number and location of methoxy substituents, through in vitro assays: MCF-7 cell proliferation and VM7Luc4E2 transactivation. The six bisguaiacols had undetectable EA at concentrations less than 10-7 M, most importantly, with significantly lower EA than BPA over an environmentally relevant range of 10-10-10-7 M. Adding a single methoxy group led to significant reduction in EA in all cases, relative to BPA and one petroleum-derived BPA analogue (bisphenol F, BPF), and the incorporation of more methoxy groups had subtler, but pronounced, impacts on either ERα binding or MCF-7 cell proliferation. In short, the six lignin-inspired bisguaiacols presented herein are viewed as promising sustainable alternatives to BPA and BPF.


Asunto(s)
Compuestos de Bencidrilo/química , Estrógenos/química , Lignina/química , Fenoles/química , Compuestos de Bencidrilo/farmacología , Proliferación Celular/efectos de los fármacos , Estrógenos/metabolismo , Estrógenos/farmacología , Humanos , Células MCF-7 , Oxidación-Reducción , Fenoles/farmacología , Unión Proteica , Relación Estructura-Actividad , Activación Transcripcional/efectos de los fármacos
2.
J Med Chem ; 64(1): 861-870, 2021 01 14.
Artículo en Inglés | MEDLINE | ID: mdl-33378197

RESUMEN

Retinoid X receptor (RXR) modulators (rexinoids) are considered to have therapeutic potential for multiple diseases, such as Alzheimer's disease and Parkinson's disease. To overcome various disadvantages of prior screening methods, we previously developed an RXR binding assay using a fluorescent RXR ligand, CU-6PMN (4). However, this ligand binds not only at the ligand-binding domain (LBD) but also at the dimer-dimer interface of hRXRα. Here, we present a new fluorescent RXR antagonist 6-[N-ethyl-N-(5-isobutoxy-4-isopropyl-2-(11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carboxamido)phenyl)amino]nicotinic acid (NEt-C343, 7), which emits strong fluorescence only when bound to the RXR-LBD. It allows us to perform a rapid, simple, and nonhazardous binding assay that does not require bound/free separation and uses a standard plate reader. The obtained Ki values of known compounds were correlated with the Ki values obtained using the standard [3H]9cis-retinoic acid assay. This assay should be useful for drug discovery as well as for research on endocrine disruptors, functional foods, and natural products.


Asunto(s)
Niacina/química , Receptores X Retinoide/antagonistas & inhibidores , Sitios de Unión , Humanos , Cinética , Ligandos , Simulación del Acoplamiento Molecular , Niacina/metabolismo , Niacina/farmacología , Unión Proteica , Receptores X Retinoide/genética , Receptores X Retinoide/metabolismo , Espectrometría de Fluorescencia , Activación Transcripcional/efectos de los fármacos
3.
Molecules ; 25(21)2020 Nov 02.
Artículo en Inglés | MEDLINE | ID: mdl-33147850

RESUMEN

Zebrafish has been a reliable model system for studying human viral pathologies. SARS-CoV-2 viral infection has become a global chaos, affecting millions of people. There is an urgent need to contain the pandemic and develop reliable therapies. We report the use of a humanized zebrafish model, xeno-transplanted with human lung epithelial cells, A549, for studying the protective effects of a tri-herbal medicine Coronil. At human relevant doses of 12 and 58 µg/kg, Coronil inhibited SARS-CoV-2 spike protein, induced humanized zebrafish mortality, and rescued from behavioral fever. Morphological and cellular abnormalities along with granulocyte and macrophage accumulation in the swim bladder were restored to normal. Skin hemorrhage, renal cell degeneration, and necrosis were also significantly attenuated by Coronil treatment. Ultra-high-performance liquid chromatography (UHPLC) analysis identified ursolic acid, betulinic acid, withanone, withaferine A, withanoside IV-V, cordifolioside A, magnoflorine, rosmarinic acid, and palmatine as phyto-metabolites present in Coronil. In A549 cells, Coronil attenuated the IL-1ß induced IL-6 and TNF-α cytokine secretions, and decreased TNF-α induced NF-κB/AP-1 transcriptional activity. Taken together, we show the disease modifying immunomodulatory properties of Coronil, at human equivalent doses, in rescuing the pathological features induced by the SARS-CoV-2 spike protein, suggesting its potential use in SARS-CoV-2 infectivity.


Asunto(s)
Antivirales/uso terapéutico , Infecciones por Coronavirus/tratamiento farmacológico , Extractos Vegetales/uso terapéutico , Neumonía Viral/tratamiento farmacológico , Glicoproteína de la Espiga del Coronavirus/antagonistas & inhibidores , Sacos Aéreos/efectos de los fármacos , Sacos Aéreos/virología , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Cromatografía Líquida de Alta Presión/métodos , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/fisiopatología , Modelos Animales de Enfermedad , Fiebre/tratamiento farmacológico , Fiebre/etiología , Hemorragia/prevención & control , Humanos , Interleucina-6/metabolismo , Riñón/efectos de los fármacos , Necrosis/patología , Necrosis/prevención & control , Pandemias , Fitoterapia , Neumonía Viral/patología , Neumonía Viral/fisiopatología , Mucosa Respiratoria/trasplante , Activación Transcripcional/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Pez Cebra
4.
Chem Biol Interact ; 331: 109284, 2020 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-33035518

RESUMEN

Glutathione S-transferases (GSTs) are a key enzyme superfamily involved in the detoxification and cytoprotection of a wide variety of xenobiotics, such as carcinogens, anticancer drugs, environmental toxicants, and endogenously produced free radicals. In the liver, the hGSTA1 isoenzyme is the most abundant and catalyzes the glutathione conjugation of a wide range of electrophiles and has been the principal GST responsible for xenobiotic detoxification. Given the critical role of this enzyme in several cellular processes, particularly cell detoxification, understanding the molecular mechanisms underlying the regulation of hGSTA1 expression is critical. Therefore, the aim of the present study was to investigate whether AHR is involved in the modulation of hGSTA1 gene expression and to characterize the molecular mechanism through which AHR exerts this regulation. Two xenobiotic response elements (XREs) were located at -602 bp and -1030 bp from the transcription start site at the hGSTA1 gene promoter. After treatment of HepG2 cells with beta-naphthoflavone (ß-NF), an AHR agonist, induction of hGSTA1 mRNA was observed. This effect was mediated by the recruitment of AHR to the hGSTA1 gene promoter and its transactivation, as indicated by the ChIP, EMSA and luciferase activity assays. The increase in hGSTA1 transcription regulated by AHR also resulted in enhanced levels of hGSTA1 protein and activity. Taken together, our data suggest that AHR ligands have the potential to modify xenobiotic and endobiotic metabolism mediated by hGSTA1, thereby altering the detoxification of xenobiotics, steroidogenesis and the efficacy of chemotherapeutic agents.


Asunto(s)
Glutatión Transferasa/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Secuencia de Bases , Ensayo de Cambio de Movilidad Electroforética , Inhibidores Enzimáticos/farmacología , Glutatión Transferasa/genética , Células Hep G2 , Humanos , Regiones Promotoras Genéticas , Receptores de Hidrocarburo de Aril/agonistas , Sitio de Iniciación de la Transcripción , Activación Transcripcional/efectos de los fármacos , beta-naftoflavona/farmacología
5.
Nat Commun ; 11(1): 4903, 2020 09 29.
Artículo en Inglés | MEDLINE | ID: mdl-32994412

RESUMEN

The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. The generation of a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene and its use in targeted ObLiGaRe results in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse allows robust and tunable genome editing granting flexibility, speed and uniformity at less cost, leading to high throughput and practical preclinical in vivo therapeutic testing.


Asunto(s)
Sistemas CRISPR-Cas/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Descubrimiento de Drogas/métodos , Edición Génica/métodos , Neoplasias Pulmonares/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Proteína 9 Asociada a CRISPR/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Doxiciclina/farmacología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Femenino , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Vectores Genéticos/genética , Células HEK293 , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Neoplasias Pulmonares/genética , Masculino , Ratones , Ratones Transgénicos , ARN Guia/genética , Recombinación Genética/efectos de los fármacos , Reproducibilidad de los Resultados , Activación Transcripcional/efectos de los fármacos , Transfección/métodos , Transgenes/genética
6.
Mol Pharmacol ; 98(5): 634-647, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32892155

RESUMEN

Long-term administration of some antiepileptic drugs often increases blood lipid levels. In this study, we investigated its molecular mechanism by focusing on the nuclear receptors constitutive active/androstane receptor (CAR) and peroxisome proliferator-activated receptor α (PPARα), which are key transcription factors for enzyme induction and lipid metabolism, respectively, in the liver. Treatment of mice with the CAR activator phenobarbital, an antiepileptic drug, increased plasma triglyceride levels and decreased the hepatic expression of PPARα target genes related to lipid metabolism. The increase in PPARα target gene expression induced by fenofibrate, a PPARα ligand, was inhibited by cotreatment with phenobarbital. CAR suppressed PPARα-dependent gene transcription in HepG2 cells but not in COS-1 cells. The mRNA level of peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), a coactivator for both CAR and PPARα, in COS-1 cells was much lower than in HepG2 cells. In reporter assays with COS-1 cells overexpressing PGC1α, CAR suppressed PPARα-dependent gene transcription, depending on the coactivator-binding motif. In mammalian two-hybrid assays, CAR attenuated the interaction between PGC1α and PPARα Chemical inhibition of PGC1α prevented phenobarbital-dependent increases in plasma triglyceride levels and the inhibition of PPARα target gene expression. These results suggest that CAR inhibits the interaction between PPARα and PGC1α, attenuating PPARα-dependent lipid metabolism. This might explain the antiepileptic drug-induced elevation of blood triglyceride levels. SIGNIFICANCE STATEMENT: Constitutive active/androstane receptor activated by antiepileptic drugs inhibits the peroxisome proliferator-activated receptor α-dependent transcription of genes related to lipid metabolism and upregulates blood triglyceride levels. The molecular mechanism of this inhibition involves competition between these nuclear receptors for coactivator peroxisome proliferator-activated receptor γ coactivator-1α binding.


Asunto(s)
Anticonvulsivantes/farmacología , PPAR alfa/metabolismo , PPAR gamma/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Triglicéridos/sangre , Animales , Línea Celular Tumoral , Inducción Enzimática/efectos de los fármacos , Fenofibrato/farmacología , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Fenobarbital/farmacología , Factores de Transcripción/metabolismo , Activación Transcripcional/efectos de los fármacos
7.
PLoS Genet ; 16(8): e1009005, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32841236

RESUMEN

Azole drugs are the most frequently used antifungal agents. The pathogenic yeast Candida glabrata acquires resistance to azole drugs via single amino acid substitution mutations eliciting a gain-of-function (GOF) hyperactive phenotype in the Pdr1 transcription factor. These GOF mutants constitutively drive high transcription of target genes such as the ATP-binding cassette transporter-encoding CDR1 locus. Previous characterization of Pdr1 has demonstrated that this factor is negatively controlled by the action of a central regulatory domain (CRD) of ~700 amino acids, in which GOF mutations are often found. Our earlier experiments demonstrated that a Pdr1 derivative in which the CRD was deleted gave rise to a transcriptional regulator that could not be maintained as the sole copy of PDR1 in the cell owing to its toxically high activity. Using a set of GOF PDR1 alleles from azole-resistant clinical isolates, we have analyzed the mechanisms acting to repress Pdr1 transcriptional activity. Our data support the view that Pdr1-dependent transactivation is mediated by a complex network of transcriptional coactivators interacting with the extreme C-terminal part of Pdr1. These coactivators include but are not limited to the Mediator component Med15A. Activity of this C-terminal domain is controlled by the CRD and requires multiple regions across the C-terminus for normal function. We also provide genetic evidence for an element within the transactivation domain that mediates the interaction of Pdr1 with coactivators on one hand while restricting Pdr1 activity on the other hand. These data indicate that GOF mutations in PDR1 block nonidentical negative inputs that would otherwise restrain Pdr1 transcriptional activation. The strong C-terminal transactivation domain of Pdr1 uses multiple different protein regions to recruit coactivators.


Asunto(s)
Candida glabrata/efectos de los fármacos , Candidiasis/tratamiento farmacológico , Farmacorresistencia Fúngica/genética , Proteínas Fúngicas/genética , Antifúngicos/efectos adversos , Antifúngicos/farmacología , Azoles/efectos adversos , Azoles/farmacología , Candida glabrata/genética , Candida glabrata/patogenicidad , Candidiasis/genética , Candidiasis/microbiología , Proteínas de Unión al ADN , Farmacorresistencia Fúngica/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Humanos , Activación Transcripcional/efectos de los fármacos
8.
Ecotoxicol Environ Saf ; 204: 111063, 2020 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-32791358

RESUMEN

The lipocalins genes have been assigned for involving in the responses of organisms to various stress factors. The function of lipocalins under PCB18 stress was addressed by pathway complementation in the Oryza sativa L. OsTIL-silenced mutant. The growth of wild type (WT) and OsTIL-silenced mutant (MT) callus were suppressed by PCB18, and MT varieties were inhibited more seriously than WT varieties. Meanwhile, only WT varieties showed "Hormesis" effect. Compared with WT (3 day > 90.0%, 6 day ≤45.5%), MT varieties kept high removing efficiency by HPLC analysis. Varied gene transcription after OsTIL silencing was demonstrated between two varieties, especially obvious under PCB stress. Silenced OsTIL induced more protective gene transcriptions by qPCR analysis, OsVDE at 3 day, OsCHL, OsZEP1, OsZEP2 and OsUN at 6 day and OsZEP2 at 9 day. PCB18 stress further irritated these genes transcription in MT varieties. The defense stagy in WT varieties was that the transcriptions of lipocalins were inhibited to reduce PCB18 accumulation and toxicity. OsTIL could effectively limit PCB18 accumulation and toxicity. After TIL lacking, OsCHL, OsZEP1, OsZEP2 and OsUN in mutant were strongly evoked to against PCB stress. Remarkably, OsUN and OsZEP2 gene expressions were responded to PCB18 stress in both two varieties.


Asunto(s)
Contaminantes Ambientales/toxicidad , Lipocalinas/genética , Oryza/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Bifenilos Policlorados/toxicidad , Activación Transcripcional/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Genes de Plantas , Oryza/genética , Oryza/crecimiento & desarrollo , Estrés Oxidativo/genética
9.
PLoS One ; 15(7): e0236403, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32716961

RESUMEN

Autophagy, a self-degradative physiological process, is critical for homeostasis maintenance and energy source balancing in response to various stresses, including nutrient deprivation. It is a highly conserved catabolic process in eukaryotes and is indispensable for cell survival as it involves degradation of unessential or excessive components and their subsequent recycling as building blocks for the synthesis of necessary molecules. Although the dysregulation of autophagy has been reported to broadly contribute to various diseases, including cancers and neurodegenerative diseases, the molecular mechanisms underlying the epigenetic regulation of autophagy are poorly elucidated. Here, we report that the level of lysine demethylase 3B (KDM3B) increases in nutrient-deprived HCT116 cells, a colorectal carcinoma cell line, resulting in transcriptional activation of the autophagy-inducing genes. KDM3B was found to enhance the transcription by demethylating H3K9me2 on the promoter of these genes. Furthermore, we observed that the depletion of KDM3B inhibited the autophagic flux in HCT116 cells. Collectively, these data suggested the critical role of KDM3B in the regulation of autophagy-related genes via H3K9me2 demethylation and induction of autophagy in nutrient-starved HCT116 cells.


Asunto(s)
Autofagia/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Activación Transcripcional/genética , Aminoácidos/deficiencia , Autofagia/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Células HCT116 , Células HEK293 , Histonas/metabolismo , Humanos , Regiones Promotoras Genéticas , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteolisis/efectos de los fármacos , Sirolimus/farmacología , Activación Transcripcional/efectos de los fármacos , Proteína que Contiene Valosina/metabolismo
10.
Anticancer Res ; 40(8): 4687-4694, 2020 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-32727793

RESUMEN

BACKGROUND/AIM: The Japanese apricot "Prunus mume" is a traditional Japanese medicine. MK615, a compound extract from Prunus mume has been reported to have anti-tumor effects. Herein, we used 3D floating (3DF) culture to evaluate the anticancer effects of MK615 against human colorectal cancer (CRC) cells that contain mutant (mt) KRAS. MATERIALS AND METHODS: HKe3 cells exogenously expressing mtKRAS (HKe3-mtKRAS) were treated with MK615 in 3DF cultures. The protein levels of hypoxia-inducible factor 1 (HIF-1) and E-cadherin were quantified by western blotting. RESULTS: MtKRAS enhanced hypoxia tolerance via up-regulation of HIF-1. The expression of HIF-1 protein was suppressed by constitutive overexpression of E-cadherin in CRC HCT116 spheroids. MK615 increased the expression of E-cadherin and decreased the expression of HIF-1 in HKe3-mtKRAS. These results suggest that MK615 suppresses hypoxia tolerance by up-regulation of E-cadherin in CRC cells with mtKRAS. CONCLUSION: MK615 exhibits properties useful for the potential treatment of CRC patients with mtKRAS.


Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Hipoxia de la Célula/fisiología , Neoplasias del Colon/metabolismo , Neoplasias Colorrectales/metabolismo , Extractos Vegetales/farmacología , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Células HCT116 , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prunus/química , Activación Transcripcional/efectos de los fármacos
11.
Nat Commun ; 11(1): 3501, 2020 07 09.
Artículo en Inglés | MEDLINE | ID: mdl-32647108

RESUMEN

While most monogenic diseases are caused by loss or reduction of protein function, the need for technologies that can selectively increase levels of protein in native tissues remains. Here we demonstrate that antisense-mediated modulation of pre-mRNA splicing can increase endogenous expression of full-length protein by preventing naturally occurring non-productive alternative splicing and promoting generation of productive mRNA. Bioinformatics analysis of RNA sequencing data identifies non-productive splicing events in 7,757 protein-coding human genes, of which 1,246 are disease-associated. Antisense oligonucleotides targeting multiple types of non-productive splicing events lead to increases in productive mRNA and protein in a dose-dependent manner in vitro. Moreover, intracerebroventricular injection of two antisense oligonucleotides in wild-type mice leads to a dose-dependent increase in productive mRNA and protein in the brain. The targeting of natural non-productive alternative splicing to upregulate expression from wild-type or hypomorphic alleles provides a unique approach to treating genetic diseases.


Asunto(s)
Empalme Alternativo , Regulación de la Expresión Génica , Oligonucleótidos Antisentido/farmacología , Alelos , Animales , Animales Recién Nacidos , Encéfalo/metabolismo , Biología Computacional , Exones , Femenino , Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Intrones , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Activación Transcripcional/efectos de los fármacos , Regulación hacia Arriba
12.
Nat Commun ; 11(1): 3420, 2020 07 09.
Artículo en Inglés | MEDLINE | ID: mdl-32647127

RESUMEN

Remyelination of the peripheral and central nervous systems (PNS and CNS, respectively) is a prerequisite for functional recovery after lesion. However, this process is not always optimal and becomes inefficient in the course of multiple sclerosis. Here we show that, when acetylated, eukaryotic elongation factor 1A1 (eEF1A1) negatively regulates PNS and CNS remyelination. Acetylated eEF1A1 (Ac-eEF1A1) translocates into the nucleus of myelinating cells where it binds to Sox10, a key transcription factor for PNS and CNS myelination and remyelination, to drag Sox10 out of the nucleus. We show that the lysine acetyltransferase Tip60 acetylates eEF1A1, whereas the histone deacetylase HDAC2 deacetylates eEF1A1. Promoting eEF1A1 deacetylation maintains the activation of Sox10 target genes and increases PNS and CNS remyelination efficiency. Taken together, these data identify a major mechanism of Sox10 regulation, which appears promising for future translational studies on PNS and CNS remyelination.


Asunto(s)
Factor 1 de Elongación Peptídica/metabolismo , Remielinización/genética , Activación Transcripcional/genética , Acetilación , Envejecimiento/metabolismo , Animales , Desdiferenciación Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/metabolismo , Lisina Acetiltransferasa 5/metabolismo , Ratones , Modelos Biológicos , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Sistema Nervioso Periférico/efectos de los fármacos , Sistema Nervioso Periférico/fisiología , Recuperación de la Función/efectos de los fármacos , Remielinización/efectos de los fármacos , Factores de Transcripción SOXE/metabolismo , Factor de Transcripción STAT3/metabolismo , Células de Schwann/efectos de los fármacos , Células de Schwann/metabolismo , Teofilina/farmacología , Transactivadores/metabolismo , Activación Transcripcional/efectos de los fármacos
13.
Proc Natl Acad Sci U S A ; 117(28): 16292-16301, 2020 07 14.
Artículo en Inglés | MEDLINE | ID: mdl-32601208

RESUMEN

Notch pathway signaling is implicated in several human cancers. Aberrant activation and mutations of Notch signaling components are linked to tumor initiation, maintenance, and resistance to cancer therapy. Several strategies, such as monoclonal antibodies against Notch ligands and receptors, as well as small-molecule γ-secretase inhibitors (GSIs), have been developed to interfere with Notch receptor activation at proximal points in the pathway. However, the use of drug-like small molecules to target the downstream mediators of Notch signaling, the Notch transcription activation complex, remains largely unexplored. Here, we report the discovery of an orally active small-molecule inhibitor (termed CB-103) of the Notch transcription activation complex. We show that CB-103 inhibits Notch signaling in primary human T cell acute lymphoblastic leukemia and other Notch-dependent human tumor cell lines, and concomitantly induces cell cycle arrest and apoptosis, thereby impairing proliferation, including in GSI-resistant human tumor cell lines with chromosomal translocations and rearrangements in Notch genes. CB-103 produces Notch loss-of-function phenotypes in flies and mice and inhibits the growth of human breast cancer and leukemia xenografts, notably without causing the dose-limiting intestinal toxicity associated with other Notch inhibitors. Thus, we describe a pharmacological strategy that interferes with Notch signaling by disrupting the Notch transcription complex and shows therapeutic potential for treating Notch-driven cancers.


Asunto(s)
Receptores Notch/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Activación Transcripcional/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Sitios de Unión , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Drosophila , Resistencia a Antineoplásicos/efectos de los fármacos , Células HeLa , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/química , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Ratones , Mutación , Fenotipo , Multimerización de Proteína , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/uso terapéutico
14.
Am J Physiol Gastrointest Liver Physiol ; 319(1): G63-G73, 2020 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-32538139

RESUMEN

Hyaluronic acid (HA), a glycosaminoglycan in the extracellular matrix, binds to CD44 and Toll-like receptor 4 (TLR4). We previously demonstrated that both CD44 and TLR4, but predominately TLR4, mediated HA stimulation of Lgr5+ stem cell proliferation, crypt fission, and intestinal growth in postnatal mice. Here we address the questions of which cell type expresses the relevant TLR4 in driving intestinal growth and what are the downstream events from TLR4 activation. Studies were done in 14-day-old mice: wild type (WT), mice deficient in cyclooxygenase 2 (COX2), mice deficient in myeloid cell TLR4, and mice deficient in epithelial cell epidermal growth factor receptor (EGFR). Biological end points included crypt fission and Lgr5 cell proliferation. In WT mice, treatment with NS-398 (a COX2 inhibitor), clodronate (a macrophage-depleting agent), or tyrphostin (an EGFR inhibitor) resulted in 30% reductions in crypt fission and Lgr5+ stem cell proliferation compared with control mice. Mice deficient in COX2 or myeloid TLR4 or epithelial cell EGFR all had 30% reductions in crypt fission and Lgr5+ stem cell proliferation compared with WT mice. Administration of dimethyl PGE2, a stable PGE2 analog, increased crypt fission and Lgr5+ stem cell proliferation. Administration of dimethyl PGE2 reversed the effects of NS-398, clodronate, COX2 deficiency, and myeloid TLR4 deficiency but had no effect on mice treated with tyrphostin or mice deficient in epithelial cell EGFR. We conclude that, in postnatal mice, ~30% of intestinal growth as manifested by crypt fission and Lgr5+ stem cell proliferation is driven by a novel pathway: Extracellular HA binds TLR4 on pericryptal macrophages, inducing the production of PGE2 through COX2. PGE2 transactivates EGFR in Lgr5+ epithelial stem cells, resulting in Lgr5+ stem cell proliferation and crypt fission.NEW & NOTEWORTHY This study, in newborn mice, describes a novel molecular pathway regulating Lgr5+ epithelial stem cell proliferation and normal intestinal elongation, as assessed by crypt fission. In this pathway, endogenous extracellular hyaluronic acid binds to Toll-like receptor 4 on pericryptal macrophages releasing PGE2 which binds to epidermal growth factor receptor on Lgr5+ stem cells resulting in proliferation. Lgr5+ stem cell proliferation leads to crypt fission and intestinal elongation. The demonstration that normal growth requires microbial-independent Toll-like receptor activation is novel.


Asunto(s)
Dinoprostona/metabolismo , Receptores ErbB/efectos de los fármacos , Ácido Hialurónico/farmacología , Receptor Toll-Like 4/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Ciclooxigenasa 2/metabolismo , Receptores ErbB/metabolismo , Ácido Hialurónico/antagonistas & inhibidores , Intestinos/efectos de los fármacos , Ratones Noqueados , Receptor Toll-Like 4/metabolismo , Activación Transcripcional/efectos de los fármacos
15.
Proc Natl Acad Sci U S A ; 117(23): 13044-13055, 2020 06 09.
Artículo en Inglés | MEDLINE | ID: mdl-32434920

RESUMEN

Epstein-Barr virus (EBV) is associated with epithelial and lymphoid malignancies, establishes latent infection in memory B cells, and intermittently produces infectious virions through lytic replication. Released virions play a key role in latent reservoir maintenance and transmission. Lytic EBV transcription differs from cellular transcription in requiring a virus-encoded preinitiation complex that binds to TATT motifs unique to EBV late lytic promoters. Expression of 15 late lytic genes that are important for virion production and infectivity is particularly dependent on the EBV SM protein, a nuclear protein expressed early during lytic reactivation that binds to viral RNAs and enhances RNA stability. We recently discovered that spironolactone blocks EBV virion production by inhibiting EBV SM function. Since spironolactone causes degradation of xeroderma pigmentosum group B-complementing protein (XPB), a component of human transcription factor TFIIH, in both B lymphocytes and epithelial cells, we hypothesized that SM utilizes XPB to specifically activate transcription of SM target promoters. While EBV SM has been thought to act posttranscriptionally, we provide evidence that SM also facilitates EBV gene transcription. We demonstrate that SM binds and recruits XPB to EBV promoters during lytic replication. Depletion of XPB protein, by spironolactone treatment or by siRNA transfection, inhibits SM-dependent late lytic gene transcription but not transcription of other EBV genes or cellular genes. These data indicate that SM acts as a transcriptional activator that has co-opted XPB to specifically target 15 EBV promoters that have uniquely evolved to require XPB for activity, providing an additional mechanism to differentially regulate EBV gene expression.


Asunto(s)
ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Herpesvirus Humano 4/genética , Interacciones Huésped-Patógeno/genética , Fosfoproteínas/metabolismo , Transactivadores/metabolismo , Línea Celular Tumoral , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Infecciones por Virus de Epstein-Barr/tratamiento farmacológico , Infecciones por Virus de Epstein-Barr/virología , Regulación Viral de la Expresión Génica/efectos de los fármacos , Humanos , Regiones Promotoras Genéticas/genética , Proteolisis/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Espironolactona/farmacología , Espironolactona/uso terapéutico , Activación Transcripcional/efectos de los fármacos , Virión/efectos de los fármacos , Virión/metabolismo
16.
Chem Biol Interact ; 325: 109129, 2020 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-32418914

RESUMEN

Alcohol has been classified as carcinogenic to humans by the International Agency for Research on Cancer (IARC). Studies have demonstrated that alcohol intake increases the risk of breast cancer, and alcohol also stimulates breast cancer cell growth. Deregulation of Pol III genes is tightly associated with tumour development. Transcription factor II-B (TFIIB)-related factor 1 (Brf1) is a transcription factor that specifically regulates Pol III gene transcription. Our in vivo and in vitro studies have indicated that alcohol enhances the transcription of Pol III genes to cause an alteration of cellular phenotypes, which is closely related with human breast cancer. Betaine is a vegetable alkaloid and has antitumor functions. Most reports about betaine show that the consumption level of betaine is inversely associated with a risk of breast cancer. Although different mechanisms of betaine against tumour have been investigated, nothing has been reported on the effect of betaine on the deregulation of Brf1 and Pol III genes. In this study, we determine the role of betaine in breast cancer cell growth and colony formation and explore its mechanism. Our results indicate that alcohol increases the rates of growth and colony formation of breast cancer cells, whereas betaine is able to significantly inhibit the effects of alcohol on these cell phenotypes. Betaine decreases the induction of Brf1 expression and Pol III gene transcription caused by ethanol to reduce the rates of cell growth and colony formation. Together, these studies provide novel insights into the role of betaine in alcohol-caused breast cancer cell growth and deregulation of Brf1 and Pol III genes. These results suggest that betaine consumption is able to prevent alcohol-associated human cancer development.


Asunto(s)
Betaína/farmacología , Etanol/antagonistas & inhibidores , Etanol/farmacología , ARN Polimerasa II/genética , Activación Transcripcional/efectos de los fármacos , Neoplasias de la Mama/inducido químicamente , Neoplasias de la Mama/patología , Neoplasias de la Mama/prevención & control , Proliferación Celular/efectos de los fármacos , Humanos , Cinética , Células MCF-7 , Riesgo
17.
Phytomedicine ; 69: 153211, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-32259676

RESUMEN

BACKGROUND: Limonin, a bioactive compound from citrus plants, exerts antioxidant activities, however its therapeutic potential in acetaminophen (APAP)-induced hepatotoxicity remains unclear. PURPOSE: Our study aims to investigate the protective effect of limonin on APAP-induced hepatotoxicity and illuminate the underlying mechanisms. STUDY: design In vitro, we chose L-02 cells to establish in vitro APAP-induced liver injury model. L-02 cells were treated with APAP (7.5 mM) for 24 h after pre-incubation with limonin (10, 25, 50 µM) or NAC (250 µM) for 2 h. In vivo, we used C57BL/6 mice as an in vivo APAP-induced liver injury model. C57BL/6 mice with pre-treatment of limonin (40, 80 mg/kg) or NAC (150 mg/kg) for 1 h, were given with a single dose of APAP (300 mg/kg). METHODS: After pre-incubation with limonin (10, 25, 50 µM) for 2 h, L-02 cells were treated with APAP (7.5 mM) for 24 h.The experiments in vitro included MTT assay, Annexin V/PI staining, measurement of reactive oxygen species (ROS), quantitative real-time PCR analysis, Western blot analysis, immunofluorescence microscopy and analysis of LDH activity. Transfection of Nrf2 or Sirt1 siRNA was also conducted in vitro. In vivo, C57BL/6 mice with pre-treatment of limonin (40, 80 mg/kg) or NAC (150 mg/kg) for 1 h, were given with a single dose of APAP (300 mg/kg). Mice were sacrificed at 4, 12 h after APAP poisoning, and analysis of ALT and AST in serum, GSH level in liver tissues, liver histological observation and immunohistochemistry were performed. RESULTS: Limonin increased the cell viability and alleviated APAP-induced apoptosis in hepatocytes. Limonin also inhibited APAP-induced mitochondrial-mediated apoptosis by decreasing the ratio of Bax/Bcl-2, recovery of mitochondrial membrane potential (MMP), inhibiting ROS production and cleavage of caspase-3 in L-02 cells. Moreover, limonin induced activation of Nrf2 and increased protein expression and mRNA levels of its downstream targets, including HO-1, NQO1 and GCLC/GCLM. The inhibition of limonin on apoptosis and promotion on Nrf2 antioxidative pathway were lessened after the application of Nrf2 siRNA. In addition, limonin inhibited NF-κB transcriptional activation, NF-κB-regulated genes and protein expression of inflammatory related proteins iNOS and COX2. Furthermore, limonin increased the protein expression of Sirt1. Sirt1 siRNA transfection confirmed that limonin activated Nrf2 antioxidative pathway and inhibited NF-κB inflammatory response by upregulating Sirt1. Finally, we established APAP-induced liver injury in vivo and demonstrated that limonin alleviated APAP-induced hepatotoxicity by activating Nrf2 antioxidative signals and inhibiting NF-κB inflammatory response via upregulating Sirt1. CONCLUSION: In summary, this study documented that limonin mitigated APAP-induced hepatotoxicity by activating Nrf2 antioxidative pathway and inhibiting NF-κB inflammatory response via upregulating Sirt1, and demonstrated that limonin had therapeutic promise in APAP-induced liver injury.


Asunto(s)
Acetaminofén/efectos adversos , Antioxidantes/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Limoninas/farmacología , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/metabolismo , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Especies Reactivas de Oxígeno/metabolismo , Sirtuina 1/genética , Sirtuina 1/metabolismo , Activación Transcripcional/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos
18.
Sci Rep ; 10(1): 6560, 2020 04 16.
Artículo en Inglés | MEDLINE | ID: mdl-32300202

RESUMEN

The tumor microenvironment (TME) is an essential contributor to the development and progression of malignancy. Within the TME, tumor associated macrophages (TAMs) mediate angiogenesis, metastasis, and immunosuppression, which inhibits infiltration of tumor-specific cytotoxic CD8+ T cells. In previous work, we demonstrated that the synthetic triterpenoid CDDO-methyl ester (CDDO-Me) converts breast TAMs from a tumor-promoting to a tumor-inhibiting activation state in vitro. We show now that CDDO-Me remodels the breast TME, redirecting TAM activation and T cell tumor infiltration in vivo. We demonstrate that CDDO-Me significantly attenuates IL-10 and VEGF expression but stimulates TNF production, and reduces surface expression of CD206 and CD115, markers of immunosuppressive TAMs. CDDO-Me treatment redirects the TAM transcriptional profile, inducing signaling pathways associated with immune stimulation, and inhibits TAM tumor infiltration, consistent with decreased expression of CCL2. In CDDO-Me-treated mice, both the absolute number and proportion of splenic CD4+ T cells were reduced, while the proportion of CD8+ T cells was significantly increased in both tumors and spleen. Moreover, mice fed CDDO-Me demonstrated significant reductions in numbers of CD4+ Foxp3+ regulatory T cells within tumors. These results demonstrate for the first time that CDDO-Me relieves immunosuppression in the breast TME and unleashes host adaptive anti-tumor immunity.


Asunto(s)
Neoplasias Mamarias Animales/patología , Ácido Oleanólico/análogos & derivados , Receptores Estrogénicos/metabolismo , Microambiente Tumoral/efectos de los fármacos , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Citocinas/biosíntesis , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/patología , Neoplasias Mamarias Animales/inmunología , Ratones Endogámicos C57BL , Ácido Oleanólico/farmacología , Bazo/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética , Microambiente Tumoral/inmunología
19.
Nucleic Acids Res ; 48(9): 4877-4890, 2020 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-32297950

RESUMEN

A correlation between histone acetylation and transcription has been noted for a long time, but little is known about what step(s) in the transcription cycle is influenced by acetylation. We have examined the immediate transcriptional response to histone deacetylase (HDAC) inhibition, and find that release of promoter-proximal paused RNA polymerase II (Pol II) into elongation is stimulated, whereas initiation is not. Although histone acetylation is elevated globally by HDAC inhibition, less than 100 genes respond within 10 min. These genes are highly paused, are strongly associated with the chromatin regulators NURF and Trithorax, display a greater increase in acetylation of the first nucleosomes than other genes, and become transcriptionally activated by HDAC inhibition. Among these rapidly up-regulated genes are HDAC1 (Rpd3) and subunits of HDAC-containing co-repressor complexes, demonstrating feedback regulation upon HDAC inhibition. Our results suggest that histone acetylation stimulates transcription of paused genes by release of Pol II into elongation, and that increased acetylation is not a consequence of their enhanced expression. We propose that HDACs are major regulators of Pol II pausing and that this partly explains the presence of HDACs at active genes.


Asunto(s)
Inhibidores de Histona Desacetilasas/farmacología , Histonas/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Activación Transcripcional , Acetilación , Animales , Línea Celular , Cromatina/metabolismo , Drosophila , Células HEK293 , Humanos , Elongación de la Transcripción Genética , Transcripción Genética/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos
20.
Aquat Toxicol ; 222: 105472, 2020 May.
Artículo en Inglés | MEDLINE | ID: mdl-32203794

RESUMEN

ß-carotene is an efficient antioxidant and its accumulation is an oxidative response to stressors. Dunaliella salina strain GY-H13 is rich in ß-carotene under environmental stresses, which was selected as material to understand the molecular mechanism underlying ß-carotene biosynthesis. Seven full length cDNA sequences in ß-carotene biosynthesis pathway were cloned, including geranylgeranyl pyrophosphate synthase (GGPS), phytoene synthase (PSY), phytoene desaturase (PDS), 15-cis-zeta-carotene isomerase (ZISO), zeta-carotene desaturase (ZDS), prolycopene isomerase (CRTISO), lycopene beta-cyclase (LCYb). The seven protein sequences from the strain GY-H13 showed the highest similarity with other D. salina strains. Especially, PSY, PDS and LCYb protein sequences shared 100 % identity. Phylogenetic analysis indicated all proteins from GY-H13 firstly clustered with those from other D. salina strains with a bootstrap of 100 %. Multiple alignment indicated several distinct conserved motifs such as aspartate-rich domain (ARD), dinucleotide binding domain (DBD), and carotene binding domain (CBD). These motifs are located near ligand-binding pocket, which may be required for the activity of enzyme. Expression levels of these genes and ß-carotene content were measured over 24-h cycle, showing clear daily dynamics. All genes were dramatically up-regulated in the morning but the highest accumulation of ß-carotene was observed at noon, suggesting a lag-effect between gene transcription and biological response. Furthermore, the accumulation of ß-carotene increased under nitrogen deficiency, Cd exposure and high light and decreased under high salinity in a time-dependent manner. No gene of ß-carotene biosynthesis was up-regulated by high salinity while most genes were activated by the other stresses at the beginning stage of exposure. Growth inhibition and oxidative damage were also observed under high salinity. Overall, transcription activation of ß-carotene biosynthetic genes at the initial stage of stress exposure is a determinant of the increased accumulation of ß-carotene in microalgae, which help their survive under harsh environments. The newly isolated D. salina strain GY-H13 would be a promising microalgae model for investigating the molecular mechanism of stress-induced ß-carotene biosynthesis.


Asunto(s)
Cadmio/toxicidad , Microalgas/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , beta Caroteno/biosíntesis , Secuencia de Aminoácidos , Antioxidantes/metabolismo , Liasas Intramoleculares/genética , Microalgas/genética , Microalgas/metabolismo , Oxidorreductasas/genética , Filogenia , Salinidad , beta Caroteno/genética
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