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1.
Nat Commun ; 11(1): 4458, 2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32895383

RESUMO

In rodent models of type 2 diabetes (T2D), sustained remission of hyperglycemia can be induced by a single intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1), and the mediobasal hypothalamus (MBH) was recently implicated as the brain area responsible for this effect. To better understand the cellular response to FGF1 in the MBH, we sequenced >79,000 single-cell transcriptomes from the hypothalamus of diabetic Lepob/ob mice obtained on Days 1 and 5 after icv injection of either FGF1 or vehicle. A wide range of transcriptional responses to FGF1 was observed across diverse hypothalamic cell types, with glial cell types responding much more robustly than neurons at both time points. Tanycytes and ependymal cells were the most FGF1-responsive cell type at Day 1, but astrocytes and oligodendrocyte lineage cells subsequently became more responsive. Based on histochemical and ultrastructural evidence of enhanced cell-cell interactions between astrocytes and Agrp neurons (key components of the melanocortin system), we performed a series of studies showing that intact melanocortin signaling is required for the sustained antidiabetic action of FGF1. These data collectively suggest that hypothalamic glial cells are leading targets for the effects of FGF1 and that sustained diabetes remission is dependent on intact melanocortin signaling.


Assuntos
Diabetes Mellitus Experimental/dietoterapia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Fator 1 de Crescimento de Fibroblastos/administração & dosagem , Hipoglicemiantes/administração & dosagem , Hipotálamo/efeitos dos fármacos , Proteínas Recombinantes/administração & dosagem , Proteína Relacionada com Agouti/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Glicemia/análise , Comunicação Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/patologia , Dieta Hiperlipídica/efeitos adversos , Sacarose na Dieta/administração & dosagem , Sacarose na Dieta/efeitos adversos , Humanos , Hipotálamo/citologia , Hipotálamo/patologia , Injeções Intraventriculares , Leptina/genética , Masculino , Melanocortinas/metabolismo , Hormônios Estimuladores de Melanócitos/administração & dosagem , Camundongos , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , RNA-Seq , Receptor Tipo 4 de Melanocortina/genética , Receptores de Melanocortina/antagonistas & inibidores , Receptores de Melanocortina/metabolismo , Indução de Remissão/métodos , Transdução de Sinais/efeitos dos fármacos , Análise de Célula Única , Técnicas Estereotáxicas , Transcriptoma/efeitos dos fármacos
2.
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
3.
Am J Physiol Heart Circ Physiol ; 319(2): H341-H348, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32618512

RESUMO

Progesterone exerts antihypertensive actions partially by modulating endothelial nitric oxide synthase (eNOS) activity. Here, we aimed to investigate the effects and mechanisms of progesterone on eNOS expression. First, human umbilical vein endothelial cells (HUVECs) were exposed to progesterone and then the eNOS transcription factor specificity protein-1 (SP-1) and progesterone receptor (PRA/B) expression were assessed by Western blotting and qRT-PCR. The interaction between SP-1 and PRA/B was next determined through coimmunoprecipitation assay. The chromatin immunoprecipitation assay and luciferase assay were used to investigate the relationship of PRA/B, SP-1, and eNOS promoter. At last, rats were intraperitoneally injected with progesterone receptor antagonist RU-486, and then the expression of eNOS and vasodilation function in thoracic aorta and mesenteric artery were measured. The results showed that progesterone could increase eNOS expression in HUVECs. Further study showed that progesterone increased PRA-SP-1 complex formation and facilitated PRA/B and SP-1 binding to eNOS promoter. Mutating SP-1 or PR-binding motif on eNOS promoter abolished the effect of progesterone on eNOS gene transcription. We also observed that progesterone receptor antagonist RU-486 reduced eNOS expression and impaired vasodilation in rats. Those results suggest that progesterone modulates eNOS expression through promoting PRA-SP-1 complex formation, and progesterone antagonist attenuates eNOS expression, leading to the loss of vascular relaxation.NEW & NOTEWORTHY Progesterone directly upregulated endothelial nitric oxide synthase (eNOS) expression in human endothelial cells. Progesterone augmented eNOS promoter activity through a progesterone receptor A- and specificity protein-1-dependent manner. Antagonism of the progesterone receptor reduced eNOS expression and impaired vasodilation in rats.


Assuntos
Núcleo Celular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/biossíntese , Progesterona/farmacologia , Receptores de Progesterona/agonistas , Fator de Transcrição Sp1/metabolismo , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/enzimologia , Sítios de Ligação , Núcleo Celular/metabolismo , Células Cultivadas , Indução Enzimática , Feminino , Antagonistas de Hormônios/farmacologia , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/enzimologia , Óxido Nítrico Sintase Tipo III/genética , Regiões Promotoras Genéticas , Ratos Sprague-Dawley , Receptores de Progesterona/antagonistas & inibidores , Receptores de Progesterona/metabolismo , Transdução de Sinais , Vasodilatação/efeitos dos fármacos
4.
J Vis Exp ; (160)2020 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-32658191

RESUMO

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.


Assuntos
Fracionamento Celular/métodos , Núcleo Celular , Detergentes/farmacologia , Métodos Analíticos de Preparação de Amostras , Animais , Encéfalo/citologia , Núcleo Celular/efeitos dos fármacos , Células Cultivadas , Citometria de Fluxo , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Reprodutibilidade dos Testes , Peixe-Zebra
5.
J Vis Exp ; (159)2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32478749

RESUMO

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes more complex, the goal of targeting mitochondria using various compounds that inflict mitochondrial damage (so-called mitocans) is becoming quite popular. Unfortunately, many existing cytotoxicity assays, such as those based on tetrazolium salts or resazurin require functional mitochondrial enzymes for their performance. The damage inflicted by compounds that target mitochondria often compromises the accuracy of these assays. Here, we describe a modified protocol based on differential staining with two fluorescent dyes, one of which is cell-permeant (Hoechst 33342) and the other of which is not (propidium iodide). The difference in staining allows living and dead cells to be discriminated. The assay is amenable to automated microscopy and image analysis, which increases throughput and reduces bias. This also allows the assay to be used in high-throughput fashion using 96-well plates, making it a viable option for drug discovery efforts, particularly when the drugs in question have some level of mitotoxicity. Importantly, results obtained by Hoechst/PI staining assay show increased consistency, both with trypan blue exclusion results and between biological replicates when the assay is compared to other methods.


Assuntos
Bioensaio/métodos , Núcleo Celular/metabolismo , Mitocôndrias/metabolismo , Automação , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Reprodutibilidade dos Testes , Rotenona/farmacologia
6.
Nucleic Acids Res ; 48(11): 6210-6222, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32365182

RESUMO

The ribotoxin α-sarcin belongs to a family of ribonucleases that cleave the sarcin/ricin loop (SRL), a critical functional rRNA element within the large ribosomal subunit (60S), thereby abolishing translation. Whether α-sarcin targets the SRL only in mature 60S subunits remains unresolved. Here, we show that, in yeast, α-sarcin can cleave SRLs within late 60S pre-ribosomes containing mature 25S rRNA but not nucleolar/nuclear 60S pre-ribosomes containing 27S pre-rRNA in vivo. Conditional expression of α-sarcin is lethal, but does not impede early pre-rRNA processing, nuclear export and the cytoplasmic maturation of 60S pre-ribosomes. Thus, SRL-cleaved containing late 60S pre-ribosomes seem to escape cytoplasmic proofreading steps. Polysome analyses revealed that SRL-cleaved 60S ribosomal subunits form 80S initiation complexes, but fail to progress to the step of translation elongation. We suggest that the functional integrity of a α-sarcin cleaved SRL might be assessed only during translation.


Assuntos
Endorribonucleases/metabolismo , Proteínas Fúngicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/química , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Ricina/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Endorribonucleases/farmacologia , Proteínas Fúngicas/farmacologia , Biossíntese de Proteínas , RNA Ribossômico/metabolismo , Ricina/química , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento
7.
Mutat Res ; 850-851: 503147, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32247562

RESUMO

Bulbus Fritillariacirrhosa D. Don (BFC) has been widely used as an herbal medicament for respiratory diseases in China for over 2000 years. The ethnomedicinal effects of BFC have been scientifically verified, nevertheless its toxicity has not been completely studied. Previously, we have reported that the aqueous extract of BFC induces mitotic aberrations and chromosomal instability (CIN) in human colon epithelial NCM460 cells via dysfunctioning the mitotic checkpoint. Here, we extend this study and specifically focus on the influence of BFC on cytokinesis, the final step of cell division. One remarkable change in NCM460 cells following BFC treatment is the high incidence of binucleated cells (BNCs). More detailed investigation of the ana-telophases reveals that furrow ingression, the first stage of cytokinesis, is inhibited by BFC. Asynchronous cultures treatment demonstrates that furrow ingression defects induced by BFCs are highly associated with the formation of BNCs in ensuing interphase, indicating the BNCs phenotype after BFC treatment was resulted from cytokinesis failure. In line with this, the expression of genes involved in the regulation of furrow ingression is significantly de-regulated by BFC (e.g., LATS-1/2 and Aurora-B are upregulated, and YB-1 is downregulated). Furthermore, long-term treatment of BFC elucidates that the BNCs phenotype is transient and the loss of BNCs is associated with increased frequency of micronuclei and nuclear buds, two biomarkers of CIN. In supporting of these findings, the Nin Jiom Pei Pa Koa and Chuanbei Pipa Gao, two commercially available Chinese traditional medicines containing BFC, are able to induce multinucleation and CIN in NCM460 cells. Altogether, these data provide the first in vitro experimental evidence linking BFC to cytokinesis failure and suggest the resultant BNCs may be intermediates to produce CIN progenies.


Assuntos
Instabilidade Cromossômica/efeitos dos fármacos , Citocinese/efeitos dos fármacos , Fritillaria/química , Extratos Vegetais/farmacologia , Aurora Quinase B/genética , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Instabilidade Cromossômica/genética , Colo/efeitos dos fármacos , Colo/patologia , Citocinese/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mitose/efeitos dos fármacos , Extratos Vegetais/química , Raízes de Plantas/química , Proteínas Serina-Treonina Quinases/genética , Proteína 1 de Ligação a Y-Box/genética
8.
Cancer Cell ; 37(3): 387-402.e7, 2020 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-32142667

RESUMO

We report that neurofibromin, a tumor suppressor and Ras-GAP (GTPase-activating protein), is also an estrogen receptor-α (ER) transcriptional co-repressor through leucine/isoleucine-rich motifs that are functionally independent of GAP activity. GAP activity, in turn, does not affect ER binding. Consequently, neurofibromin depletion causes estradiol hypersensitivity and tamoxifen agonism, explaining the poor prognosis associated with neurofibromin loss in endocrine therapy-treated ER+ breast cancer. Neurofibromin-deficient ER+ breast cancer cells initially retain sensitivity to selective ER degraders (SERDs). However, Ras activation does play a role in acquired SERD resistance, which can be reversed upon MEK inhibitor addition, and SERD/MEK inhibitor combinations induce tumor regression. Thus, neurofibromin is a dual repressor for both Ras and ER signaling, and co-targeting may treat neurofibromin-deficient ER+ breast tumors.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Receptor alfa de Estrogênio/genética , Neurofibromina 1/genética , Motivos de Aminoácidos , Animais , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proteínas Correpressoras , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Células MCF-7 , Camundongos Nus , Camundongos SCID , Mutação , Neurofibromina 1/química , Neurofibromina 1/metabolismo , Transdução de Sinais , Tamoxifeno/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/metabolismo
9.
Nat Commun ; 11(1): 1270, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32152280

RESUMO

Prolonged cell survival occurs through the expression of specific protein isoforms generated by alternate splicing of mRNA precursors in cancer cells. How alternate splicing regulates tumor development and resistance to targeted therapies in cancer remain poorly understood. Here we show that RNF113A, whose loss-of-function causes the X-linked trichothiodystrophy, is overexpressed in lung cancer and protects from Cisplatin-dependent cell death. RNF113A is a RNA-binding protein which regulates the splicing of multiple candidates involved in cell survival. RNF113A deficiency triggers cell death upon DNA damage through multiple mechanisms, including apoptosis via the destabilization of the prosurvival protein MCL-1, ferroptosis due to enhanced SAT1 expression, and increased production of ROS due to altered Noxa1 expression. RNF113A deficiency circumvents the resistance to Cisplatin and to BCL-2 inhibitors through the destabilization of MCL-1, which thus defines spliceosome inhibitors as a therapeutic approach to treat tumors showing acquired resistance to specific drugs due to MCL-1 stabilization.


Assuntos
Proteínas de Ligação a DNA/genética , Genes Ligados ao Cromossomo X , Spliceossomos/metabolismo , Síndromes de Tricotiodistrofia/genética , Células A549 , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Processamento Alternativo/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/genética , Cisplatino/farmacologia , Citoproteção/efeitos dos fármacos , Dano ao DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Íntrons/genética , Camundongos Endogâmicos NOD , Camundongos SCID , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas de Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo
10.
PLoS One ; 15(3): e0230177, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32182273

RESUMO

Jasmonates (JAs) are key phytohormones involved in regulation of plant growth and development, stress responses, and secondary metabolism. It has been reported that treatments with JAs could increase the contents of Amaryllidaceae alkaloids in Amaryllidaceae plants. Jasmonate ZIM (zinc-finger inflorescence meristem) domain (JAZ) proteins are key components in JA signal processes. However, JAZ proteins have not been characterized in genus Lycoris. In this study, we identified and cloned seven differentially expressed JAZ genes (namely LaJAZ1-LaJAZ7) from Lycoris aurea. Bioinformatic analyses revealed that these seven LaJAZ proteins contain the ZIM domain and JA-associated (Jas, also named CCT_2) motif. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that these LaJAZ genes display different expression patterns in L. aurea tissues, and most of them are inducible when treated with methyl jasmonate (MeJA) treatment. Subcellular localization assay demonstrated that LaJAZ proteins are localized in the cell nucleus or cytoplasm. In addition, LaJAZ proteins could interact with each other to form homodimer and/or heterodimer. The findings in this study may facilitate further functional research of the LaJAZ genes, especially the potential regulatory mechanism of plant secondary metabolites including Amaryllidaceae alkaloids in L. aurea.


Assuntos
Regulação da Expressão Gênica de Plantas/genética , Lycoris/genética , Proteínas de Plantas/genética , Dedos de Zinco/genética , Acetatos/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Biologia Computacional/métodos , Ciclopentanos/farmacologia , Citoplasma/efeitos dos fármacos , Citoplasma/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Lycoris/efeitos dos fármacos , Oxilipinas/farmacologia , Reguladores de Crescimento de Planta/farmacologia , Domínios Proteicos/genética
11.
Biochem Biophys Res Commun ; 524(4): 895-902, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32057361

RESUMO

The transcription factor NRF2 plays a key role in the protection against environmental stress and maintaining cellular homeostasis. The acetyltransferase p300 is a known component of the NRF2 transcriptional complex and promotes its transcriptional activity. In this study we describe a novel mechanism by which p300 facilitates NRF2 activity. p300 physically interacts with NRF2 and interferes with NRF2-KEAP1 complex formation. In particular, p300 increases NRF2 protein abundance and stability, thereby promoting NRF2 nuclear localization. Notably, the acetyltransferase activity of p300 was indispensable for the stabilizing effects towards NRF2. Furthermore, overexpression of p300 protected HEK293T cells from oxidative stress and increased viability. Together our study uncovers a link between p300 and control of NRF2-KEAP1 signaling via regulation of NRF2 stability and this may act as a novel checkpoint on the adaptation to oxidative stress.


Assuntos
Regulação da Expressão Gênica , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Fator 2 Relacionado a NF-E2/genética , Fatores de Transcrição de p300-CBP/genética , Adaptação Fisiológica , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Peróxido de Hidrogênio/farmacologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Ligação Proteica , Estabilidade Proteica , Transporte Proteico , Transdução de Sinais , Transcrição Genética , Fatores de Transcrição de p300-CBP/deficiência
12.
Int J Nanomedicine ; 15: 675-704, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32103936

RESUMO

With the advent of nanotechnology, various modes of traditional treatment strategies have been transformed extensively owing to the advantageous morphological, physiochemical, and functional attributes of nano-sized materials, which are of particular interest in diverse biomedical applications, such as diagnostics, sensing, imaging, and drug delivery. Despite their success in delivering therapeutic agents, several traditional nanocarriers often end up with deprived selectivity and undesired therapeutic outcome, which significantly limit their clinical applicability. Further advancements in terms of improved selectivity to exhibit desired therapeutic outcome toward ablating cancer cells have been predominantly made focusing on the precise entry of nanoparticles into tumor cells via targeting ligands, and subsequent delivery of therapeutic cargo in response to specific biological or external stimuli. However, there is enough room intracellularly, where diverse small-sized nanomaterials can accumulate and significantly exert potentially specific mechanisms of antitumor effects toward activation of precise cancer cell death pathways that can be explored. In this review, we aim to summarize the intracellular pathways of nanoparticles, highlighting the principles and state of their destructive effects in the subcellular structures as well as the current limitations of conventional therapeutic approaches. Next, we give an overview of subcellular performances and the fate of internalized nanoparticles under various organelle circumstances, particularly endosome or lysosome, mitochondria, nucleus, endoplasmic reticulum, and Golgi apparatus, by comprehensively emphasizing the unique mechanisms with a series of interesting reports. Moreover, intracellular transformation of the internalized nanoparticles, prominent outcome and potential affluence of these interdependent subcellular components in cancer therapy are emphasized. Finally, we conclude with perspectives with a focus on the contemporary challenges in their clinical applicability.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/uso terapêutico , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Núcleo Celular/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Humanos , Lisossomos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neoplasias/patologia
13.
Chem Commun (Camb) ; 56(20): 3019-3022, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-32048647

RESUMO

Nucleus-targeting NPs based on RuO2 (RuO2NPs) were developed by controlling the size and the surface charge of nanoparticles (NPs). This study not only demonstrates a facile approach for the fabrication of ultrasmall CS-RuO2NPs with good biocompatibility and excellent photothermal properties but also their unique potential for the nucleus-targeted low-temperature PTT.


Assuntos
Nanopartículas/química , Imagem Óptica , Óxidos/química , Técnicas Fotoacústicas , Rutênio/química , Temperatura , Núcleo Celular/efeitos dos fármacos , Humanos , Raios Infravermelhos , Células MCF-7 , Óxidos/farmacologia , Tamanho da Partícula , Fototerapia , Rutênio/farmacologia , Propriedades de Superfície
14.
Cell Rep ; 30(4): 1117-1128.e5, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31995753

RESUMO

Prion-like proteins form multivalent assemblies and phase separate into membraneless organelles. Heterogeneous ribonucleoprotein D-like (hnRNPDL) is a RNA-processing prion-like protein with three alternative splicing (AS) isoforms, which lack none, one, or both of its two disordered domains. It has been suggested that AS might regulate the assembly properties of RNA-processing proteins by controlling the incorporation of multivalent disordered regions in the isoforms. This, in turn, would modulate their activity in the downstream splicing program. Here, we demonstrate that AS controls the phase separation of hnRNPDL, as well as the size and dynamics of its nuclear complexes, its nucleus-cytoplasm shuttling, and amyloidogenicity. Mutation of the highly conserved D378 in the disordered C-terminal prion-like domain of hnRNPDL causes limb-girdle muscular dystrophy 1G. We show that D378H/N disease mutations impact hnRNPDL assembly properties, accelerating aggregation and dramatically reducing the protein solubility in the muscle of Drosophila, suggesting a genetic loss-of-function mechanism for this muscular disorder.


Assuntos
Proteínas Amiloidogênicas/metabolismo , Núcleo Celular/metabolismo , Drosophila/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Distrofia Muscular do Cíngulo dos Membros/genética , Agregação Patológica de Proteínas/metabolismo , Processamento Alternativo , Proteínas Amiloidogênicas/genética , Proteínas Amiloidogênicas/ultraestrutura , Animais , Núcleo Celular/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Dactinomicina/farmacologia , Drosophila/metabolismo , Técnicas de Inativação de Genes , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/ultraestrutura , Humanos , Cinética , Microscopia Eletrônica de Transmissão , Células Musculares/metabolismo , Células Musculares/patologia , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Mutação , Agregação Patológica de Proteínas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/ultraestrutura
15.
Mol Cell ; 77(5): 970-984.e7, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31982308

RESUMO

Cytosolic caspase-8 is a mediator of death receptor signaling. While caspase-8 expression is lost in some tumors, it is increased in others, indicating a conditional pro-survival function of caspase-8 in cancer. Here, we show that tumor cells employ DNA-damage-induced nuclear caspase-8 to override the p53-dependent G2/M cell-cycle checkpoint. Caspase-8 is upregulated and localized to the nucleus in multiple human cancers, correlating with treatment resistance and poor clinical outcome. Depletion of caspase-8 causes G2/M arrest, stabilization of p53, and induction of p53-dependent intrinsic apoptosis in tumor cells. In the nucleus, caspase-8 cleaves and inactivates the ubiquitin-specific peptidase 28 (USP28), preventing USP28 from de-ubiquitinating and stabilizing wild-type p53. This results in de facto p53 protein loss, switching cell fate from apoptosis toward mitosis. In summary, our work identifies a non-canonical role of caspase-8 exploited by cancer cells to override the p53-dependent G2/M cell-cycle checkpoint.


Assuntos
Caspase 8/metabolismo , Núcleo Celular/enzimologia , Proliferação de Células , Pontos de Checagem da Fase G2 do Ciclo Celular , Neoplasias/enzimologia , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina Tiolesterase/metabolismo , Antineoplásicos/farmacologia , Apoptose , Caspase 8/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Núcleo Celular/patologia , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HeLa , Humanos , Células MCF-7 , Masculino , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Células PC-3 , Estabilidade Proteica , Transdução de Sinais , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Ubiquitina Tiolesterase/genética
16.
J Steroid Biochem Mol Biol ; 199: 105594, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31968225

RESUMO

Breast cancer is currently the leading cause of cancer death among women worldwide. AP-1 (c-Fos/c-Jun) is associated with proliferation and survival, while cytoplasmic c-Fos activates phospholipid synthesis in cells induced to differentiate or grow. Estrogen receptor α 46 (ERα46) is a splice variant of full-length ERα66 and it is known that it has an inhibitory role in cancer cell growth. We investigated c-Fos localization, its relationship to AP-1, the non genomic pathway of phospho-Tyr537-ERα66, as well as ERα46 and ERα66 isoforms in rat mammary gland development and carcinogenic transformation, and in mammary tumors. Female rats were injected: a) saline solution (Control mammary gland, CMG) or b) N-Nitroso-N-methyl urea (NMU), and samples were taken at 60, 90, 120 and 150 days of life. In addition, we analyzed hormone-dependent (HD) and independent (HI) tumors in ovariectomized rats, and intact tumors (IT) in non-ovariectomized ones. Our results show that, in CMG, nuclear c-Fos and proliferation decreased with age, AP-1 content was low, and nuclear ERα46/ERα66 ratio was higher than 1. In NMU, nuclear c-Fos and proliferation increased with carcinogenic transformation, AP-1 content was high, and nuclear ERα46/ERα66 was below 1. As tumor grade increased, proliferation, nuclear c-Fos and AP-1 expression were negatively associated to nuclear ERα46/ERα66 in IT. In HD, nuclear ERα46/ERα66, nuclear c-Fos expression, AP-1 levels and proliferation were lower than in HI, whose growth is estrogen-independent. Phospho-Tyr537-ERα66 content and ERK1/2 activation were associated with AP-1 levels and cell proliferation. Collectively, our findings support the notion that variant detection and ERα46/ERα66 ratio could shed light on the role of ERα isoforms in mammary gland transformation and the behavior of ERα positive mammary tumors.


Assuntos
Receptor alfa de Estrogênio/genética , Genes fos/genética , Neoplasias Mamárias Animais/genética , Isoformas de Proteínas/genética , Animais , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Proliferação de Células/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citoplasma/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Metilnitrosoureia/farmacologia , Isoformas de Proteínas/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição AP-1/genética
17.
Am J Respir Cell Mol Biol ; 62(4): 479-492, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31944822

RESUMO

Idiopathic pulmonary fibrosis is a lung disease with limited therapeutic options that is characterized by pathological fibroblast activation and aberrant lung remodeling with scar formation. YAP (Yes-associated protein) is a transcriptional coactivator that mediates mechanical and biochemical signals controlling fibroblast activation. In this study, we developed a high-throughput small-molecule screen for YAP inhibitors in primary human lung fibroblasts. Multiple HMG-CoA (hydroxymethylglutaryl-coenzyme A) reductase inhibitors (statins) were found to inhibit YAP nuclear localization via induction of YAP phosphorylation, cytoplasmic retention, and degradation. We further show that the mevalonate pathway regulates YAP activation, and that simvastatin treatment reduces fibrosis markers in activated human lung fibroblasts and in the bleomycin mouse model of pulmonary fibrosis. Finally, we show that simvastatin modulates YAP in vivo in mouse lung fibroblasts. Our results highlight the potential of small-molecule screens for YAP inhibitors and provide a mechanism for the antifibrotic activity of statins in idiopathic pulmonary fibrosis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas de Ciclo Celular/antagonistas & inibidores , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Fibrose Pulmonar/tratamento farmacológico , Acil Coenzima A/metabolismo , Animais , Biomarcadores/metabolismo , Bleomicina/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Ácido Mevalônico/metabolismo , Camundongos , Fosfoproteínas/metabolismo , Fibrose Pulmonar/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sinvastatina/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia
18.
Asian Pac J Cancer Prev ; 21(1): 87-92, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31983169

RESUMO

OBJECTIVE: The main aim of this study is to evaluate the micronuclei scoring as a biomarker for early detection and screening of genotoxic effect of cigarette smoking in the peripheral blood T- lymphocytes. METHODS: A total number of eligible 148 individuals have participated in the study; 78 Current smokers and 70 never smokers. Cytokinesis-block micronucleus assay was performed for all the participants in the peripheral blood T-lymphocytes. Assessment of the smoking status of the participants was conducted through the detailed smoking history, Fagerström test for nicotine dependence (FTND) scoring, and determination of the urinary cotinine creatinine ratio (CCR). RESULT: A significantly higher frequency of  micronuclei  in the binucleated T-lymphocytes(BMNi) was identified in the smokers group as compared to the nonsmokers; OR=4.9, 95% CI=1.9-12.5), P-value=0.006. Both of the pack years and the smoking duration of the smokers could significantly predict the BMNi scoring; P-value=0.001, 0.002 respectively. CONCLUSION: Our results indicate the association between BMNi and cigarette smoking, suggesting that BMNi Scoring can be a useful biomarker for early detection and screening of the genotoxic effect of cigarette smoking as a primary preventive measure for various smoking induced cancers.
.


Assuntos
Biomarcadores/metabolismo , Núcleo Celular/efeitos dos fármacos , Fumar Cigarros/efeitos adversos , Mutagênicos/efeitos adversos , Fumar/efeitos adversos , Tabaco/efeitos adversos , Adulto , Biomarcadores/urina , Cotinina/urina , Estudos Transversais , Dano ao DNA/efeitos dos fármacos , Detecção Precoce de Câncer/métodos , Feminino , Humanos , Masculino , Testes para Micronúcleos/métodos , Fumantes
19.
Int J Mol Sci ; 21(2)2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31952110

RESUMO

Maresin-1 (MaR1) is a specialized pro-resolving mediator, derived from omega-3 fatty acids, whose functions are to decrease the pro-inflammatory and oxidative mediators, and also to stimulate cell division. We investigated the hepatoprotective actions of MaR1 in a rat model of liver ischemia-reperfusion (IR) injury. MaR1 (4 ng/gr body weight) was administered prior to ischemia (1 h) and reperfusion (3 h), and controls received isovolumetric vehicle solution. To analyze liver function, transaminases levels and tissue architecture were assayed, and serum cytokines TNF-α, IL-6, and IL-10, mitotic activity index, and differential levels of NF-κB and Nrf-2 transcription factors, were analyzed. Transaminase, TNF-α levels, and cytoarchitecture were normalized with the administration of MaR1 and associated with changes in NF-κB. IL-6, mitotic activity index, and nuclear translocation of Nrf-2 increased in the MaR1-IR group, which would be associated with hepatoprotection and cell proliferation. Taken together, these results suggest that MaR1 alleviated IR liver injury, facilitated by the activation of hepatocyte cell division, increased IL-6 cytokine levels, and the nuclear localization of Nrf-2, with a decrease of NF-κB activity. All of them were related to an improvement of liver injury parameters. These results open the possibility of MaR1 as a potential therapeutic tool in IR and other hepatic pathologies.


Assuntos
Proliferação de Células/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Traumatismo por Reperfusão/prevenção & controle , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citocinas/sangue , Citocinas/metabolismo , Ácidos Docosa-Hexaenoicos/química , Ácidos Graxos Ômega-3/química , Hepatócitos/citologia , Hepatócitos/metabolismo , Fígado/irrigação sanguínea , Fígado/fisiopatologia , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Substâncias Protetoras/farmacologia , Ratos Sprague-Dawley , Traumatismo por Reperfusão/fisiopatologia , Transaminases/metabolismo
20.
Expert Opin Pharmacother ; 21(4): 399-408, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31957504

RESUMO

Introduction: Despite unprecedented advances in the treatment of multiple myeloma (MM), almost all patients develop a disease that is resistant to the five most commonly used and active anti-MM agents. The prognosis for this patient population is particularly poor resulting in an unmet need for additional therapeutic options. Exportin-1 (XPO-1) is a major nuclear export protein of macromolecular cargo frequently overexpressed in MM. Selinexor is a first-in-class, oral Selective-Inhibitor-of-Nuclear-Export (SINE) compound that impedes XPO-1. Based on results of the STORM-trial, selinexor in combination with dexamethasone was granted accelerated FDA approval for patients with penta-refractory MM in July 2019.Areas covered: This article summarizes our up-to-date knowledge on the pathophysiologic role of XPO-1 in MM. Furthermore, it reviews the most recent clinical data on selinexor in combination with dexamethasone and other anti-MM agents; and discusses its safety profile, management strategies; and potential future developments.Expert opinion: Selinexor represents a next-generation-novel agent with an innovative mechanism of action that marks a significant advance in the treatment of heavily pretreated MM patients. Ongoing studies investigate its therapeutic potential also in earlier lines of therapy. Additional data is needed to confirm that selinexor and other SINE compounds are a valuable addition to our current therapeutic armamentarium.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Hidrazinas/uso terapêutico , Carioferinas/antagonistas & inibidores , Mieloma Múltiplo/tratamento farmacológico , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Triazóis/uso terapêutico , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Ensaios Clínicos como Assunto , Dexametasona/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Humanos , Hidrazinas/administração & dosagem , Hidrazinas/efeitos adversos , Hidrazinas/farmacocinética , Carioferinas/genética , Mieloma Múltiplo/metabolismo , Prognóstico , Receptores Citoplasmáticos e Nucleares/genética , Triazóis/administração & dosagem , Triazóis/efeitos adversos , Triazóis/farmacocinética
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