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1.
Molecules ; 25(21)2020 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-33147850

RESUMO

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.


Assuntos
Antivirais/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Sacos Aéreos/efeitos dos fármacos , Sacos Aéreos/virologia , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Cromatografia Líquida de Alta Pressão/métodos , Infecções por Coronavirus/patologia , Infecções por Coronavirus/fisiopatologia , Modelos Animais de Doenças , Febre/tratamento farmacológico , Febre/etiologia , Hemorragia/prevenção & controle , Humanos , Interleucina-6/metabolismo , Rim/efeitos dos fármacos , Necrose/patologia , Necrose/prevenção & controle , Pandemias , Fitoterapia , Pneumonia Viral/patologia , Pneumonia Viral/fisiopatologia , Mucosa Respiratória/transplante , Ativação Transcricional/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Peixe-Zebra
2.
Chem Biol Interact ; 331: 109284, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-33035518

RESUMO

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.


Assuntos
Glutationa Transferase/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Sequência de Bases , Ensaio de Desvio de Mobilidade Eletroforética , Inibidores Enzimáticos/farmacologia , Glutationa Transferase/genética , Células Hep G2 , Humanos , Regiões Promotoras Genéticas , Receptores de Hidrocarboneto Arílico/agonistas , Sítio de Iniciação de Transcrição , Ativação Transcricional/efeitos dos fármacos , beta-Naftoflavona/farmacologia
3.
Nat Commun ; 11(1): 4903, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994412

RESUMO

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.


Assuntos
Sistemas CRISPR-Cas/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Descoberta de Drogas/métodos , Edição de Genes/métodos , Neoplasias Pulmonares/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteína 9 Associada à CRISPR/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Doxiciclina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Neoplasias Pulmonares/genética , Masculino , Camundongos , Camundongos Transgênicos , RNA Guia/genética , Recombinação Genética/efeitos dos fármacos , Reprodutibilidade dos Testes , Ativação Transcricional/efeitos dos fármacos , Transfecção/métodos , Transgenes/genética
4.
Mol Pharmacol ; 98(5): 634-647, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32892155

RESUMO

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.


Assuntos
Anticonvulsivantes/farmacologia , PPAR alfa/metabolismo , PPAR gama/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Triglicerídeos/sangue , Animais , Linhagem Celular Tumoral , Indução Enzimática/efeitos dos fármacos , Fenofibrato/farmacologia , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenobarbital/farmacologia , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacos
5.
Ecotoxicol Environ Saf ; 204: 111063, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32791358

RESUMO

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.


Assuntos
Poluentes Ambientais/toxicidade , Lipocalinas/genética , Oryza/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Bifenilos Policlorados/toxicidade , Ativação Transcricional/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Genes de Plantas , Oryza/genética , Oryza/crescimento & desenvolvimento , Estresse Oxidativo/genética
6.
PLoS Genet ; 16(8): e1009005, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32841236

RESUMO

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.


Assuntos
Candida glabrata/efeitos dos fármacos , Candidíase/tratamento farmacológico , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Antifúngicos/efeitos adversos , Antifúngicos/farmacologia , Azóis/efeitos adversos , Azóis/farmacologia , Candida glabrata/genética , Candida glabrata/patogenicidade , Candidíase/genética , Candidíase/microbiologia , Proteínas de Ligação a DNA , Farmacorresistência Fúngica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Humanos , Ativação Transcricional/efeitos dos fármacos
7.
Nat Commun ; 11(1): 3501, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647108

RESUMO

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.


Assuntos
Processamento Alternativo , Regulação da Expressão Gênica , Oligonucleotídeos Antissenso/farmacologia , Alelos , Animais , Animais Recém-Nascidos , Encéfalo/metabolismo , Biologia Computacional , Éxons , Feminino , Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Íntrons , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Ativação Transcricional/efeitos dos fármacos , Regulação para Cima
8.
PLoS One ; 15(7): e0236403, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32716961

RESUMO

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.


Assuntos
Autofagia/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Ativação Transcricional/genética , Aminoácidos/deficiência , Autofagia/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Células HCT116 , Células HEK293 , Histonas/metabolismo , Humanos , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Sirolimo/farmacologia , Ativação Transcricional/efeitos dos fármacos , Proteína com Valosina/metabolismo
9.
Nat Commun ; 11(1): 3420, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647127

RESUMO

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.


Assuntos
Fator 1 de Elongação de Peptídeos/metabolismo , Remielinização/genética , Ativação Transcricional/genética , Acetilação , Envelhecimento/metabolismo , Animais , Desdiferenciação Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Lisina Acetiltransferase 5/metabolismo , Camundongos , Modelos Biológicos , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Sistema Nervoso Periférico/efeitos dos fármacos , Sistema Nervoso Periférico/fisiologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Remielinização/efeitos dos fármacos , Fatores de Transcrição SOXE/metabolismo , Fator de Transcrição STAT3/metabolismo , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Teofilina/farmacologia , Transativadores/metabolismo , Ativação Transcricional/efeitos dos fármacos
10.
Anticancer Res ; 40(8): 4687-4694, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727793

RESUMO

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.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Hipóxia Celular/fisiologia , Neoplasias do Colo/metabolismo , Neoplasias Colorretais/metabolismo , Extratos Vegetais/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Regulação para Cima/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Células HCT116 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prunus/química , Ativação Transcricional/efeitos dos fármacos
11.
Proc Natl Acad Sci U S A ; 117(28): 16292-16301, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601208

RESUMO

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.


Assuntos
Receptores Notch/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Ativação Transcricional/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Sítios de Ligação , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Drosophila , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células HeLa , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/química , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Camundongos , Mutação , Fenótipo , Multimerização Proteica , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/uso terapêutico
12.
Am J Physiol Gastrointest Liver Physiol ; 319(1): G63-G73, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32538139

RESUMO

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.


Assuntos
Dinoprostona/metabolismo , Receptores ErbB/efeitos dos fármacos , Ácido Hialurônico/farmacologia , Receptor 4 Toll-Like/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Receptores ErbB/metabolismo , Ácido Hialurônico/antagonistas & inibidores , Intestinos/efeitos dos fármacos , Camundongos Knockout , Receptor 4 Toll-Like/metabolismo , Ativação Transcricional/efeitos dos fármacos
13.
Proc Natl Acad Sci U S A ; 117(23): 13044-13055, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32434920

RESUMO

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.


Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Herpesvirus Humano 4/genética , Interações Hospedeiro-Patógeno/genética , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Linhagem Celular Tumoral , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Infecções por Vírus Epstein-Barr/virologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Humanos , Regiões Promotoras Genéticas/genética , Proteólise/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Espironolactona/farmacologia , Espironolactona/uso terapêutico , Ativação Transcricional/efeitos dos fármacos , Vírion/efeitos dos fármacos , Vírion/metabolismo
14.
Chem Biol Interact ; 325: 109129, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32418914

RESUMO

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.


Assuntos
Betaína/farmacologia , Etanol/antagonistas & inibidores , Etanol/farmacologia , RNA Polimerase II/genética , Ativação Transcricional/efeitos dos fármacos , Neoplasias da Mama/induzido quimicamente , Neoplasias da Mama/patologia , Neoplasias da Mama/prevenção & controle , Proliferação de Células/efeitos dos fármacos , Humanos , Cinética , Células MCF-7 , Risco
15.
Nucleic Acids Res ; 48(9): 4877-4890, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32297950

RESUMO

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.


Assuntos
Inibidores de Histona Desacetilases/farmacologia , Histonas/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Ativação Transcricional , Acetilação , Animais , Linhagem Celular , Cromatina/metabolismo , Drosophila , Células HEK293 , Humanos , Elongação da Transcrição Genética , Transcrição Genética/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos
16.
Sci Rep ; 10(1): 6560, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32300202

RESUMO

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.


Assuntos
Neoplasias Mamárias Animais/patologia , Ácido Oleanólico/análogos & derivados , Receptores Estrogênicos/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Citocinas/biossíntese , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Neoplasias Mamárias Animais/imunologia , Camundongos Endogâmicos C57BL , Ácido Oleanólico/farmacologia , Baço/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Microambiente Tumoral/imunologia
17.
Phytomedicine ; 69: 153211, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32259676

RESUMO

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.


Assuntos
Acetaminofen/efeitos adversos , Antioxidantes/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Limoninas/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Animais , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Espécies Reativas de Oxigênio/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Ativação Transcricional/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
18.
Sci Rep ; 10(1): 4337, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32152395

RESUMO

The two vasoactive hormones, angiotensin II (ANG II; vasoconstrictive) and atrial natriuretic peptide (ANP; vasodilatory) antagonize the biological actions of each other. ANP acting through natriuretic peptide receptor-A (NPRA) lowers blood pressure and blood volume. We tested hypothesis that ANG II plays critical roles in the transcriptional repression of Npr1 (encoding NPRA) and receptor function. ANG II significantly decreased NPRA mRNA and protein levels and cGMP accumulation in cultured mesangial cells and attenuated ANP-mediated relaxation of aortic rings ex vivo. The transcription factors, cAMP-response element-binding protein (CREB) and heat-shock factor-4a (HSF-4a) facilitated the ANG II-mediated repressive effects on Npr1 transcription. Tyrosine kinase (TK) inhibitor, genistein and phosphatidylinositol 3-kinase (PI-3K) inhibitor, wortmannin reversed the ANG II-dependent repression of Npr1 transcription and receptor function. ANG II enhanced the activities of Class I histone deacetylases (HDACs 1/2), thereby decreased histone acetylation of H3K9/14ac and H4K8ac. The repressive effect of ANG II on Npr1 transcription and receptor signaling seems to be transduced by TK and PI-3K pathways and modulated by CREB, HSF-4a, HDACs, and modified histones. The current findings suggest that ANG II-mediated repressive mechanisms of Npr1 transcription and receptor function may provide new molecular targets for treatment and prevention of hypertension and cardiovascular diseases.


Assuntos
Angiotensina II/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição de Choque Térmico/metabolismo , Histona Desacetilases/metabolismo , Receptores do Fator Natriurético Atrial/genética , Receptores do Fator Natriurético Atrial/metabolismo , Acetilação , Angiotensina II/farmacologia , Animais , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Fatores de Transcrição de Choque Térmico/genética , Histonas/metabolismo , Camundongos , Modelos Biológicos , Ligação Proteica , Ativação Transcricional/efeitos dos fármacos
19.
Biochim Biophys Acta Mol Cell Res ; 1867(7): 118692, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32135176

RESUMO

Prostate apoptosis response-4 (Par-4) is a tumor suppressor protein that selectively induces apoptosis in cancer cells. Although the mechanism of Par-4-mediated induction of apoptosis has been well studied, the involvement of Par-4 in other mechanisms of cell death such as autophagy is unclear. We investigated the mechanism involved in Par-4-mediated autophagic cell death in human malignant glioma. We demonstrate for the first time that the tumor suppressor lipid, ceramide (Cer), causes Par-4 induction, leading to autophagic cell death in human malignant glioma. Furthermore, we identified the tumor suppressor protein p53 and BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) as downstream targets of Par-4 during Cer-mediated autophagic cell death. RNAi-mediated down-regulation of Par-4 blocks Cer-induced p53-BNIP3 activation and autophagic cell death, while upregulation of Par-4 augmented p53-BNIP3 activation and autophagic cell death. Remarkably, in many instances, Par-4 overexpression alone was sufficient to induce cell death which is associated with features of autophagy. Interestingly, similar results were seen when glioma cells were exposed to classical autophagy inducers such as serum starvation, arsenic trioxide, and curcumin. Collectively, the novel Par-4-p53-BNIP3 axis plays a crucial role in autophagy-mediated cell death in human malignant glioma.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Glioma/genética , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas/genética , Proteína Supressora de Tumor p53/genética , Apoptose/efeitos dos fármacos , Trióxido de Arsênio/farmacologia , Morte Celular Autofágica/efeitos dos fármacos , Morte Celular Autofágica/genética , Linhagem Celular Tumoral , Curcumina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/patologia , Humanos , Ativação Transcricional/efeitos dos fármacos
20.
Sci Adv ; 6(12): eaax7945, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32219156

RESUMO

Because of its key role in cancer development and progression, STAT3 has become an attractive target for developing new cancer therapeutics. While several STAT3 inhibitors have progressed to advanced stages of development, their underlying biology and mechanisms of action are often more complex than would be expected from specific binding to STAT3. Here, we have identified and optimized a series of compounds that block STAT3-dependent luciferase expression with nanomolar potency. Unexpectedly, our lead compounds did not bind to cellular STAT3 but to another prominent anticancer drug target, TrxR1. We further identified that TrxR1 inhibition induced Prx2 and STAT3 oxidation, which subsequently blocked STAT3-dependent transcription. Moreover, previously identified inhibitors of STAT3 were also found to inhibit TrxR1, and likewise, established TrxR1 inhibitors block STAT3-dependent transcriptional activity. These results provide new insights into the complexities of STAT3 redox regulation while highlighting a novel mechanism to block aberrant STAT3 signaling in cancer cells.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Tiorredoxina Redutase 1/antagonistas & inibidores , Antineoplásicos/química , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Fator 2 Relacionado a NF-E2/agonistas , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Ativação Transcricional/efeitos dos fármacos
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