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1.
Life Sci ; 271: 119173, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33556375

RESUMO

AIMS: Cell cycle arrest plays critical roles in preventing renal tubular epithelial cell (RTEC) injury and maladaptation after the onset of chronic kidney disease (CKD), but the underlying mechanism governing this arrest has not been fully elucidated. This study was designed to determine the underlying role of YB-1 in promoting cell cycle progression and nuclear translocation in HK-2 cells induced by trimethylamine N-oxide (TMAO). MAIN METHODS: YB-1 primarily accumulated in the cytoplasm in HK-2 cells after they were treated with TMAO for 30 min and 6 h. Gene expression was analysed using RNA sequencing in HK-2 cells treated with TMAO. Cell cycle progression was analysed via flow cytometry. Luciferase assay and ChIP-PCR were performed to determine the relationship between transcription factor YB-1 and Gadd45a promoter region. Additionally, mice were fed with TMAO to test renal dysfunction and measure the expression of YB-1, GADD45a and CCNA2 in the kidney sections through immunohistochemistry. KEY FINDINGS: YB-1 primarily accumulated in the cytoplasm in HK-2 cells after they were treated with TMAO for 30 min and 6 h. RNA sequencing analysis showed that the cell cycle checkpoint genes growth arrest and DNA damage (Gadd)45a, Gadd45g, cyclin (Ccn)a2, Ccnb1, Ccne1 and Ccnf were differentially expressed in HK-2 cells after treated with 400 µM TMAO for 30 min. Flow cytometry results demonstrated that cell cycle progression was blocked at the G2/M checkpoint. In animal models, elevated dietary TMAO directly led to progressive renal tubulointerstitial dysfunction and inhibited the expression of YB-1 in kidney. Moreover, YB-1 was determined to regulate Gadd45a expression by directly binding to its promoter region. YB-1 expression was negatively correlated with the expression of Gadd45a and Gadd45g but positively correlated with Ccna2, Ccnb1, Ccne1 and Ccnf in CKD. SIGNIFICANCE: YB-1 may be a reliable molecular target and an effective prognostic biomarker for CKD.


Assuntos
Proteínas de Ciclo Celular/biossíntese , Ciclo Celular/fisiologia , Regulação para Baixo/fisiologia , Metilaminas/toxicidade , Insuficiência Renal Crônica/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Expressão Gênica , Humanos , Túbulos Renais/citologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Camundongos Endogâmicos C57BL , Insuficiência Renal Crônica/induzido quimicamente , Insuficiência Renal Crônica/genética
2.
Nat Commun ; 12(1): 241, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33431824

RESUMO

Acute myeloid leukemia (AML) is a typically lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually, most AML retain wild-type TP53, encoding the pro-apoptotic tumor suppressor p53. MDM2 inhibitors (MDM2i), which activate wild-type p53, and BET inhibitors (BETi), targeting the BET-family co-activator BRD4, both show encouraging pre-clinical activity, but limited clinical activity as single agents. Here, we report enhanced toxicity of combined MDM2i and BETi towards AML cell lines, primary human blasts and mouse models, resulting from BETi's ability to evict an unexpected repressive form of BRD4 from p53 target genes, and hence potentiate MDM2i-induced p53 activation. These results indicate that wild-type TP53 and a transcriptional repressor function of BRD4 together represent a potential broad-spectrum synthetic therapeutic vulnerability for AML.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas de Ciclo Celular/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Terapia de Alvo Molecular , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Crise Blástica/patologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Mieloide Aguda/genética , Camundongos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/antagonistas & inibidores
3.
Nat Chem Biol ; 17(3): 280-290, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33462494

RESUMO

Although most acute skin wounds heal rapidly, non-healing skin ulcers represent an increasing and substantial unmet medical need that urgently requires effective therapeutics. Keratinocytes resurface wounds to re-establish the epidermal barrier by transitioning to an activated, migratory state, but this ability is lost in dysfunctional chronic wounds. Small-molecule regulators of keratinocyte plasticity with the potential to reverse keratinocyte malfunction in situ could offer a novel therapeutic approach in skin wound healing. Utilizing high-throughput phenotypic screening of primary keratinocytes, we identify such small molecules, including bromodomain and extra-terminal domain (BET) protein family inhibitors (BETi). BETi induce a sustained activated, migratory state in keratinocytes in vitro, increase activation markers in human epidermis ex vivo and enhance skin wound healing in vivo. Our findings suggest potential clinical utility of BETi in promoting keratinocyte re-epithelialization of skin wounds. Importantly, this novel property of BETi is exclusively observed after transient low-dose exposure, revealing new potential for this compound class.


Assuntos
Proteínas de Ciclo Celular/genética , Epiderme/efeitos dos fármacos , Reepitelização/efeitos dos fármacos , Úlcera Cutânea/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/genética , Ferimentos não Penetrantes/tratamento farmacológico , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Modelos Animais de Doenças , Epiderme/metabolismo , Epiderme/patologia , Transferência Ressonante de Energia de Fluorescência , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Cultura Primária de Células , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Precursores de Proteínas/antagonistas & inibidores , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Reepitelização/genética , Úlcera Cutânea/genética , Úlcera Cutânea/metabolismo , Úlcera Cutânea/patologia , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Transcrição Genética , Ferimentos não Penetrantes/genética , Ferimentos não Penetrantes/metabolismo , Ferimentos não Penetrantes/patologia
4.
Int J Mol Sci ; 22(2)2021 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-33477764

RESUMO

The Hippo signaling pathway plays a key role in regulating organ size and tissue homeostasis. Hippo and two of its main effectors, yes-associated protein (YAP) and WWTR1 (WW domain-containing transcription regulator 1, commonly listed as TAZ), play critical roles in angiogenesis. This study investigated the role of the Hippo signaling pathway in the pathogenesis of rosacea. We performed immunohistochemical analyses to compare the expression levels of YAP and TAZ between rosacea skin and normal skin in humans. Furthermore, we used a rosacea-like BALB/c mouse model induced by LL-37 injections to determine the roles of YAP and TAZ in rosacea in vivo. We found that the expression levels of YAP and TAZ were upregulated in patients with rosacea. In the rosacea-like mouse model, we observed that the clinical features of rosacea, including telangiectasia and erythema, improved after the injection of a YAP/TAZ inhibitor. Additionally, treatment with a YAP/TAZ inhibitor reduced the expression levels of YAP and TAZ and diminished vascular endothelial growth factor (VEGF) immunoreactivity in the rosacea-like mouse model. Our findings suggest that YAP/TAZ inhibitors can attenuate angiogenesis associated with the pathogenesis of rosacea and that both YAP and TAZ are potential therapeutic targets for patients with rosacea.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas de Ciclo Celular/antagonistas & inibidores , Rosácea/tratamento farmacológico , Transativadores/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/genética , Rosácea/genética , Rosácea/patologia , Transdução de Sinais/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/patologia , Transativadores/genética
5.
Nucleic Acids Res ; 49(4): 1914-1934, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33511417

RESUMO

During homologous recombination, Dbl2 protein is required for localisation of Fbh1, an F-box helicase that efficiently dismantles Rad51-DNA filaments. RNA-seq analysis of dbl2Δ transcriptome showed that the dbl2 deletion results in upregulation of more than 500 loci in Schizosaccharomyces pombe. Compared with the loci with no change in expression, the misregulated loci in dbl2Δ are closer to long terminal and long tandem repeats. Furthermore, the misregulated loci overlap with antisense transcripts, retrotransposons, meiotic genes and genes located in subtelomeric regions. A comparison of the expression profiles revealed that Dbl2 represses the same type of genes as the HIRA histone chaperone complex. Although dbl2 deletion does not alleviate centromeric or telomeric silencing, it suppresses the silencing defect at the outer centromere caused by deletion of hip1 and slm9 genes encoding subunits of the HIRA complex. Moreover, our analyses revealed that cells lacking dbl2 show a slight increase of nucleosomes at transcription start sites and increased levels of methylated histone H3 (H3K9me2) at centromeres, subtelomeres, rDNA regions and long terminal repeats. Finally, we show that other proteins involved in homologous recombination, such as Fbh1, Rad51, Mus81 and Rad54, participate in the same gene repression pathway.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Regulação Fúngica da Expressão Gênica , Inativação Gênica , Recombinação Homóloga , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiologia , Fatores de Transcrição/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Centrômero , Código das Histonas , Nucleossomos/metabolismo , Proteínas Repressoras/fisiologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores
6.
Cell Prolif ; 54(2): e12976, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33393124

RESUMO

BACKGROUND: In mammals, early pregnancy is a critical vulnerable period during which complications may arise, including pregnancy failure. Establishment of a maternal endometrial acceptance phenotype is a prerequisite for semiheterogeneous embryo implantation, comprising the rate-limiting step of early pregnancy. METHODS: Confocal fluorescence, immunohistochemistry and western blot for nuclear and cytoplasmic protein were used to examine the activation of yes-associated protein (YAP) in uterine tissue and primary endometrial cells. The target binding between miR16a and YAP was verified by dual-luciferase reporter gene assay. The mouse pregnancy model and pseudopregnancy model were used to investigate the role of YAP in the maternal uterus during early pregnancy in vivo. RESULTS: We showed that YAP translocates into the nucleus in the endometrium of cattle and mice during early pregnancy. Mechanistically, YAP acts as a mediator of ECM rigidity and cell density, which requires the actomyosin cytoskeleton and is partially dependent on the Hippo pathway. Furthermore, we found that the soluble factor IFNτ, which is a ruminant pregnancy recognition factor, also induced activation of YAP by reducing the expression of miR-16a. CONCLUSIONS: This study revealed that activation of YAP is necessary for early pregnancy in bovines because it induced cell proliferation and established an immunosuppressive local environment that allowed conceptus implantation into the uterine epithelium.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Endométrio/metabolismo , Matriz Extracelular/metabolismo , Interferon Tipo I/metabolismo , Proteínas da Gravidez/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antagomirs/metabolismo , Bovinos , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Endométrio/citologia , Molécula de Adesão da Célula Epitelial/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Interferon Tipo I/farmacologia , Masculino , Camundongos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Musculares/metabolismo , Gravidez , Proteínas da Gravidez/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Útero/metabolismo , Útero/patologia
7.
Life Sci ; 266: 118886, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33310044

RESUMO

AIMS: Triple negative breast cancer (TNBC) has drawn more and more attention due to its high mitotic indices, high metastatic rate and poor prognosis. Gene therapy, especially RNA interference (RNAi), has become a promising targeted therapy. However, improvement of transfection efficiency and discovery of target genes are major problems for the delivery of small interfering RNAs (siRNA). MATERIALS AND METHODS: In the present study, we developed GALA- and CREKA-modified PEG-SS-PEI to deliver siRNAs targeting on EGFR and BRD4 for TNBC therapy. The PEG-SS-PEI/siRNA complexes were prepared by electrostatic interaction and characterized by dynamic light scattering (DLS) and transmission electron microscope (TEM). The release characteristic, stability, cellular uptake and intracellular localization of the complexes were also studied. The effect of the complexes on cell viability was measured in MDA-MB-231 and HUVEC cells. The in vitro anti-tumor activities of the complexes were analyzed by Transwell invasion assay and wound healing assay. The gene silencing effect was evaluated by quantitative real time-polymerase chain reaction (qRT-PCR) and western blot. KEY FINDINGS: The results revealed that the GALA- and CREKA-modified PEG-SS-PEI/siRNA complexes showed excellent transfection efficiency with redox-sensitive release profile and good biological compatibility. The complexes protected siRNA from the degradation of RNA enzymes. The complexes significantly inhibited the proliferation, invasion and migration of MDA-MB-231 cells via the synergistic inhibition of EGFR/PI3K/Akt and BRD4/c-Myc pathways. SIGNIFICANCE: Taken together, co-delivery of siEGFR and siBRD4 by GALA-PEG-SS-PEI and CREKA-PEG-SS-PEI may provide a more effective strategy for the treatment of TNBC.


Assuntos
Proteínas de Ciclo Celular/administração & dosagem , Peptídeos Penetradores de Células/química , Inativação Gênica , Polietilenoglicóis/química , Polietilenoimina/análogos & derivados , RNA Interferente Pequeno/administração & dosagem , Fatores de Transcrição/administração & dosagem , Neoplasias de Mama Triplo Negativas/terapia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proliferação de Células , Receptores ErbB/administração & dosagem , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Feminino , Terapia Genética , Humanos , Polietilenoimina/química , RNA Interferente Pequeno/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Células Tumorais Cultivadas
8.
Mol Pharmacol ; 99(1): 1-16, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33130557

RESUMO

Aberrant activation of Wnt/ß-catenin axis occurs in several gastrointestinal malignancies due to inactivating mutations of adenomatous polyposis coli (in colorectal cancer) or activating mutations of ß-catenin itself [in hepatocellular carcinoma (HCC)]. These lead to ß-catenin stabilization, increase in ß-catenin/T-cell factor (TCF)-mediated transcriptional activation, and target gene expression, many of which are involved in tumor progression. While studying pharmaceutical agents that can target ß-catenin in cancer cells, we observed that the plant compound berberine (BBR), a potent activator of AMP-activated protein kinase (AMPK), can reduce ß-catenin expression and downstream signaling in HCC cells in a dose-dependent manner. More in-depth analyses to understand the mechanism revealed that BBR-induced reduction of ß-catenin occurs independently of AMPK activation and does not involve transcriptional or post-translational mechanisms. Pretreatment with protein synthesis inhibitor cycloheximide antagonized BBR-induced ß-catenin reduction, suggesting that BBR affects ß-catenin translation. BBR treatment also antagonized mammalian target of rapamycin (mTOR) activity and was associated with increased recruitment of eukaryotic translation initiation factor 4E-binding protein (4E-BP) 1 in the translational complex, which was revealed by 7-methyl-cap-binding assays, suggesting inhibition of cap-dependent translation. Interestingly, knocking down 4E-BP1 and 4E-BP2 significantly attenuated BBR-induced reduction of ß-catenin levels and expression of its downstream target genes. Moreover, cells with 4E-BP knockdown were resistant to BBR-induced cell death and were resensitized to BBR after pharmacological inhibition of ß-catenin. Our findings indicate that BBR antagonizes ß-catenin pathway by inhibiting ß-catenin translation and mTOR activity and thereby reduces HCC cell survival. These also suggest that BBR could be used for targeting HCCs that express mutated/activated ß-catenin variants that are currently undruggable. SIGNIFICANCE STATEMENT: ß-catenin signaling is aberrantly activated in different gastrointestinal cancers, including hepatocellular carcinoma, which is currently undruggable. In this study we describe a novel mechanism of targeting ß-catenin translation via utilizing a plant compound, berberine. Our findings provide a new avenue of targeting ß-catenin axis in cancer, which can be utilized toward the designing of effective therapeutic strategies to combat ß-catenin-dependent cancers.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Berberina/farmacologia , Carcinoma Hepatocelular/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Neoplasias Hepáticas/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinoma Hepatocelular/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Fatores de Iniciação em Eucariotos/antagonistas & inibidores , Fatores de Iniciação em Eucariotos/genética , Células HEK293 , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/fisiologia , beta Catenina/antagonistas & inibidores , beta Catenina/genética
9.
Int J Mol Sci ; 21(24)2020 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-33348732

RESUMO

Renal ischemia reperfusion injury (IRI) is associated with inflammation, including neutrophil infiltration that exacerbates the initial ischemic insult. The molecular pathways involved are poorly characterized and there is currently no treatment. We performed an in silico analysis demonstrating changes in NFκB-mediated gene expression in early renal IRI. We then evaluated NFκB-blockade with a BRD4 inhibitor on neutrophil adhesion to endothelial cells in vitro, and tested BRD4 inhibition in an in vivo IRI model. BRD4 inhibition attenuated neutrophil adhesion to activated endothelial cells. In vivo, IRI led to increased expression of cytokines and adhesion molecules at 6 h post-IRI with sustained up-regulated expression to 48 h post-IRI. These effects were attenuated, in part, with BRD4 inhibition. Absolute neutrophil counts increased significantly in the bone marrow, blood, and kidney 24 h post-IRI. Activated neutrophils increased in the blood and kidney at 6 h post-IRI and remained elevated in the kidney until 48 h post-IRI. BRD4 inhibition reduced both total and activated neutrophil counts in the kidney. IRI-induced tubular injury correlated with neutrophil accumulation and was reduced by BRD4 inhibition. In summary, BRD4 inhibition has important systemic and renal effects on neutrophils, and these effects are associated with reduced renal injury.


Assuntos
Adesão Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/imunologia , Proteínas Nucleares/antagonistas & inibidores , Traumatismo por Reperfusão/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Rim/citologia , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Neutrófilos/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Traumatismo por Reperfusão/imunologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo
10.
Nat Commun ; 11(1): 4766, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958778

RESUMO

Germline telomere maintenance defects are associated with an increased incidence of inflammatory diseases in humans, yet whether and how telomere dysfunction causes inflammation are not known. Here, we show that telomere dysfunction drives pATM/c-ABL-mediated activation of the YAP1 transcription factor, up-regulating the major pro-inflammatory factor, pro-IL-18. The colonic microbiome stimulates cytosolic receptors activating caspase-1 which cleaves pro-IL-18 into mature IL-18, leading to recruitment of interferon (IFN)-γ-secreting T cells and intestinal inflammation. Correspondingly, patients with germline telomere maintenance defects exhibit DNA damage (γH2AX) signaling together with elevated YAP1 and IL-18 expression. In mice with telomere dysfunction, telomerase reactivation in the intestinal epithelium or pharmacological inhibition of ATM, YAP1, or caspase-1 as well as antibiotic treatment, dramatically reduces IL-18 and intestinal inflammation. Thus, telomere dysfunction-induced activation of the ATM-YAP1-pro-IL-18 pathway in epithelium is a key instigator of tissue inflammation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Inflamação/patologia , Telômero/patologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antibacterianos/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Caspase 1/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Criança , Colo/metabolismo , Colo/microbiologia , Colo/patologia , Gastroenteropatias/patologia , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/fisiologia , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/microbiologia , Interleucina-18/genética , Interleucina-18/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Mutantes , Fosforilação , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Transdução de Sinais , Telomerase/genética , Telomerase/metabolismo
11.
Nat Commun ; 11(1): 4053, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792481

RESUMO

A significant proportion of patients with oestrogen receptor (ER) positive breast cancers (BC) develop resistance to endocrine treatments (ET) and relapse with metastatic disease. Here we perform whole exome sequencing and gene expression analysis of matched primary breast tumours and bone metastasis-derived patient-derived xenografts (PDX). Transcriptomic analyses reveal enrichment of the G2/M checkpoint and up-regulation of Polo-like kinase 1 (PLK1) in PDX. PLK1 inhibition results in tumour shrinkage in highly proliferating CCND1-driven PDX, including different RB-positive PDX with acquired palbociclib resistance. Mechanistic studies in endocrine resistant cell lines, suggest an ER-independent function of PLK1 in regulating cell proliferation. Finally, in two independent clinical cohorts of ER positive BC, we find a strong association between high expression of PLK1 and a shorter metastases-free survival and poor response to anastrozole. In conclusion, our findings support clinical development of PLK1 inhibitors in patients with advanced CCND1-driven BC, including patients progressing on palbociclib treatment.


Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina D1/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sequenciamento Completo do Exoma/métodos , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Ciclina D1/genética , Variações do Número de Cópias de DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Humanos , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Camundongos Nus , Piperazinas/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Pteridinas/uso terapêutico , Piridinas/uso terapêutico
12.
SAR QSAR Environ Res ; 31(7): 547-570, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32657160

RESUMO

It is well known that bromodomain-containing protein 4 (BRD4) has been thought as a promising target utilized for treating various human diseases, such as inflammatory disorders, malignant tumours, acute myelogenous leukaemia (AML), bone diseases, etc. For this study, molecular dynamics (MD) simulations, binding free energy calculations, and principal component analysis (PCA) were integrated together to uncover binding modes of inhibitors 8P9, 8PU, and 8PX to BRD4(1). The results obtained from binding free energy calculations show that van der Waals interactions act as the main regulator in bindings of inhibitors to BRD4(1). The information stemming from PCA reveals that inhibitor associations extremely affect conformational changes, internal dynamics, and movement patterns of BRD4(1). Residue-based free energy decomposition method was wielded to unveil contributions of independent residues to inhibitor bindings and the data signify that hydrogen bonding interactions and hydrophobic interactions are decisive factors affecting bindings of inhibitors to BRD4(1). Meanwhile, eight residues Trp81, Pro82, Val87, Leu92, Leu94, Cys136, Asn140, and Ile146 are recognized as the common hot interaction spots of three inhibitors with BRD4(1). The results from this work are expected to provide a meaningfully theoretical guidance for design and development of effective inhibitors inhibiting of the activity of BRD4.


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/química , Simulação de Dinâmica Molecular , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/química , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Análise de Componente Principal , Ligação Proteica
13.
Nucleic Acids Res ; 48(14): 7844-7855, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32652013

RESUMO

The catalytic activity of human AURORA-A kinase (AURKA) regulates mitotic progression, and its frequent overexpression in major forms of epithelial cancer is associated with aneuploidy and carcinogenesis. Here, we report an unexpected, kinase-independent function for AURKA in DNA replication initiation whose inhibition through a class of allosteric inhibitors opens avenues for cancer therapy. We show that genetic depletion of AURKA, or its inhibition by allosteric but not catalytic inhibitors, blocks the G1-S cell cycle transition. A catalytically inactive AURKA mutant suffices to overcome this block. We identify a multiprotein complex between AURKA and the replisome components MCM7, WDHD1 and POLD1 formed during G1, and demonstrate that allosteric but not catalytic inhibitors prevent the chromatin assembly of functional replisomes. Indeed, allosteric but not catalytic AURKA inhibitors sensitize cancer cells to inhibition of the CDC7 kinase subunit of the replication-initiating factor DDK. Thus, our findings define a mechanism essential for replisome assembly during DNA replication initiation that is vulnerable to inhibition as combination therapy in cancer.


Assuntos
Aurora Quinase A/fisiologia , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Complexos Multienzimáticos/metabolismo , Regulação Alostérica , Aurora Quinase A/antagonistas & inibidores , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular , Pontos de Checagem da Fase G1 do Ciclo Celular , Células HeLa , Humanos , Interfase/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Origem de Replicação
15.
Oncogene ; 39(27): 5015-5030, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32523092

RESUMO

Triple-negative breast cancer (TNBC) is extremely aggressive and lacks effective therapy. SAM and SH3 domain containing1 (SASH1) has been implicated in TNBC as a candidate tumor suppressor; however, the mechanisms of action of SASH1 in TNBC remain underexplored. Here, we show that SASH1 was significantly downregulated in TNBC patients samples compared with other subtypes of breast cancer. Ectopic SASH1 expression inhibited, while depletion of SASH1 enhanced, the invasive phenotype of TNBC cells, accompanied by deregulated expression of MMP2 and MMP9. The functional effects of SASH1 depletion were confirmed in the chicken chorioallantoic membrane and mouse xenograft models. Mechanistically, SASH1 knockdown downregulated the phosphorylation levels of the Hippo kinase LATS1 and its effector YAP (Yes associated protein), thereby upregulating YAP accumulation together with its downstream target CYR61. Consistently, forced SASH1 expression exhibited opposite effects. Pharmacological inhibition of YAP or knockdown of YAP reversed the enhanced cell invasion of TNBC cells following SASH1 depletion. Furthermore, SASH1-induced YAP signaling was LATS1-dependent, which in reverse enhanced phosphorylation of SASH1. The SASH1 S407A mutant (phosphorylation deficient) failed to rescue the altered YAP signaling by SASH1 knockdown. Notably, SASH1 depletion upregulated ARHGAP42 levels via YAP-TEAD and the YAP-ARHGAP42-actin axis contributed to SASH1-regulated TNBC cell invasion. Therefore, our findings uncover a new mechanism for the tumor-suppressive activity of SASH1 in TNBC, which may serve as a novel target for therapeutic intervention.


Assuntos
Actinas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Transcrição/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Embrião de Galinha , Proteína Rica em Cisteína 61/metabolismo , Humanos , Camundongos , Invasividade Neoplásica/genética , Fosforilação/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Ensaios Antitumorais Modelo de Xenoenxerto
16.
SAR QSAR Environ Res ; 31(5): 373-398, 2020 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-32496901

RESUMO

Emerging evidences indicate bromodomain-containing proteins 2 and 4 (BRD2 and BRD4) play critical roles in cancers, inflammations, cardiovascular diseases and other pathologies. Multiple short molecular dynamics (MSMD) simulations combined with molecular mechanics generalized Born surface area (MM-GBSA) method were applied to investigate the binding selectivity of three inhibitors 87D, 88M and 89G towards BRD2 over BRD4. The root-mean-square fluctuation (RMSF) analysis indicates that the structural flexibility of BRD4 is stronger than that of BRD2. Moreover the calculated distances between the Cα atoms in the centres of the ZA_loop and BC_loop of BRD4 are also bigger than that of BRD2. The rank of binding free energies calculated using MM-GBSA method agrees well with that determined by experimental data. The results show that 87D can bind more favourably to BRD2 than BRD4, while 88M has better selectivity on BRD4 over BRD2. Residue-based free-energy decomposition method was utilized to estimate the inhibitor-residue interaction spectrum and the results not only identify the hot interaction spots of inhibitors with BRD2 and BRD4, but also demonstrate that several common residues, including (W370, W374), (P371, P375), (V376, V380) and (L381, L385) belonging to (BRD2, BRD4), generate significant binding difference of inhibitors to BRD2 and BRD4.


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Simulação de Dinâmica Molecular , Fatores de Transcrição/antagonistas & inibidores , Proteínas de Ciclo Celular/química , Ligação Proteica , Fatores de Transcrição/química
17.
Gene ; 754: 144891, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32535048

RESUMO

Characterized by ankyrin repeat motifs, the feminization-1 (fem-1) gene plays an essential role in sex determination/differentiation in Caenorhabditis elegans. However, there are only a few reports on fem-1 in crustaceans. In this study, a fem-1 gene (Mrfem-1) was first isolated from the giant freshwater prawn Macrobrachium rosenbergii. The full-length cDNA of Mrfem-1 was 2607 bp long, containing an open reading frame encoding 615 amino acids, and presenting eight ankyrin repeats. The full-length cDNA has been submitted to GenBank with the accession no. MT160093. According to the RT-PCR results, Mrfem-1 was exclusively expressed in the ovary. The expression level of Mrfem-1 had increased with ovarian maturation and reached the highest peak at vitellogenic stage. In situ hybridization results showed that positive signals were concentrated in the cytoplasm of previtellogenic stage, and scattered in the cytoplasm and follicular cells at vitellogenic stage, suggesting that Mrfem-1 might be associated with ovarian maturation. Moreover, two effective siRNAs targeting Mrfem-1 were found and their effectiveness verified in vitro. These results on Mrfem-1 will help us to better understand the fem family and provide a new resource for subsequent investigations of siRNA-mediated regulation on ovarian development in M. rosenbergii.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Regulação da Expressão Gênica , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Palaemonidae/metabolismo , RNA Interferente Pequeno/genética , Animais , Sequência de Bases , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Feminino , Especificidade de Órgãos , Palaemonidae/genética , Filogenia
18.
Nat Commun ; 11(1): 2109, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32355159

RESUMO

Repair of double strand DNA breaks (DSBs) can result in gene disruption or gene modification via homology directed repair (HDR) from donor DNA. Altering cellular responses to DSBs may rebalance editing outcomes towards HDR and away from other repair outcomes. Here, we utilize a pooled CRISPR screen to define host cell involvement in HDR between a Cas9 DSB and a plasmid double stranded donor DNA (dsDonor). We find that the Fanconi Anemia (FA) pathway is required for dsDonor HDR and that other genes act to repress HDR. Small molecule inhibition of one of these repressors, CDC7, by XL413 and other inhibitors increases the efficiency of HDR by up to 3.5 fold in many contexts, including primary T cells. XL413 stimulates HDR during a reversible slowing of S-phase that is unexplored for Cas9-induced HDR. We anticipate that XL413 and other such rationally developed inhibitors will be useful tools for gene modification.


Assuntos
Sistemas CRISPR-Cas , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Reparo de DNA por Recombinação , Quebras de DNA de Cadeia Dupla , Edição de Genes , Engenharia Genética/métodos , Células HCT116 , Células HEK293 , Células HeLa , Recombinação Homóloga , Humanos , Células K562 , Fenótipo , RNA Guia/metabolismo , Fase S
19.
Cell Physiol Biochem ; 54(2): 303-320, 2020 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-32259417

RESUMO

BACKGROUND/AIMS: Chromosomal instability is a well-known factor in the progression of different types of cancer, including colorectal cancer. Chromosomal instability results in severely rearranged karyotypes and aneuploidy. Tetraploidy constitutes an intermediate phase during the polyploidy/aneuploidy cascade in oncogenesis, and tetraploid cells are particularly resistant to chemotherapy. Whether inhibition of the mitotic protein polo-like kinase 1 (PLK1) prevents the survival of tetraploid colon cancer cells is unknown. METHODS: Diploid and tetraploid cells were transfected with siPLK1 or treated with PLK1 inhibitor Bi2536 in combination with spindle poison. Cell toxicity was assessed via crystal violet staining and clonogenic assay. Flow cytometry assessment analyzed numerous cell apoptotic parameters and cell cycle phases. Synergistic activity between Bi2536 and paclitaxel, vincristine or colchicine was calculated using the CompuSyn software. RESULTS: Inhibition or abrogation of PLK1 prevented the survival of colon cancer cells, specifically tetraploid cells. The cell death induced by PLK inhibition was due to mitotic slippage, followed by the activation of the intrinsic pathway of apoptosis. We further demonstrated that co-treatment of the tetraploid colon cancer cells with a PLK1 inhibitor and the microtubule polymerisation inhibitor vincristine or colchicine, but not the microtubule depolymerisation inhibitor paclitaxel, provoked a lethal synergistic effect. CONCLUSION: PLK1 inhibition together with microtubule-targeting chemicals, serve as a potent therapeutic strategy for targeting tetraploid cancer cells.


Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Neoplasias do Colo/tratamento farmacológico , Mitose/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pteridinas/toxicidade , Tetraploidia , Antimitóticos/farmacologia , Antimitóticos/toxicidade , Antineoplásicos Fitogênicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Colchicina/farmacologia , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Sinergismo Farmacológico , Humanos , Microtúbulos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Paclitaxel/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Pteridinas/farmacologia , RNA Interferente Pequeno , Moduladores de Tubulina/farmacologia , Vincristina/farmacologia
20.
Nat Commun ; 11(1): 1935, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32321921

RESUMO

Although cellular senescence acts primarily as a tumour suppression mechanism, the accumulation of senescent cells in vivo eventually exerts deleterious side effects through inflammatory/tumour-promoting factor secretion. Thus, the development of new drugs that cause the specific elimination of senescent cells, termed senolysis, is anticipated. Here, by an unbiased high-throughput screening of chemical compounds and a bio-functional analysis, we identify BET family protein degrader (BETd) as a promising senolytic drug. BETd provokes senolysis through two independent but integrated pathways; the attenuation of non-homologous end joining (NHEJ), and the up-regulation of autophagic gene expression. BETd treatment eliminates senescent hepatic stellate cells in obese mouse livers, accompanied by the reduction of liver cancer development. Furthermore, the elimination of chemotherapy-induced senescent cells by BETd increases the efficacy of chemotherapy against xenograft tumours in immunocompromised mice. These results reveal the vulnerability of senescent cells and open up possibilities for its control.


Assuntos
Antineoplásicos/administração & dosagem , Autofagia/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Neoplasias/fisiopatologia , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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