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BACKGROUND: A recent study reported that papillary thyroid carcinoma (PTC) was associated with increased adrenergic nerve density. Meanwhile, emerging evidence suggested that tumor-innervating nerves might play a role in shaping the tumor microenvironment. We aimed to explore the potential interaction between neuronal markers and tumor microenvironmental signatures through a transcriptomic approach. METHODS: mRNA sequencing was conducted using five pairs of PTC and adjacent normal tissues. The Gene Set Variation Analysis (GSVA) was performed to calculate enrichment scores of gene sets related to tumor-infiltrating immune cells and the tumor microenvironment. The potential interaction was tested using the expression levels of a series of neuronal markers and gene set enrichment scores. RESULTS: PTC tissues were associated with increased enrichment scores of CD8 T cells, cancer-associated fibroblasts, mast cells, and checkpoint molecules. The neuronal marker for cholinergic neurons was positively correlated with CD8 T cell activation, while markers for serotonergic and dopaminergic neurons showed an inverse correlation. CONCLUSION: Distinct neuronal markers exerted different correlations with tumor microenvironmental signatures. Tumor-innervating nerves might play a role in the formation of the PTC microenvironment.
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Chemotherapy-induced peripheral neuropathy (CIPN)-mediated paresthesias are a common complication in cancer patients undergoing chemotherapy. There are currently no treatments available to prevent or reverse CIPN. Therefore, new therapeutic targets are urgently needed to develop more effective analgesics. However, the pathogenesis of CIPN remains unclear, and the prevention and treatment strategies of CIPN are still unresolved issues in medicine. More and more studies have demonstrated that mitochondrial dysfunction has become a major factor in promoting the development and maintenance of CIPN, and peroxisome proliferator-activated receptor gamma (PPARγ) coactivator 1α (PGC1α) plays a significant role in maintaining the mitochondrial function, protecting peripheral nerves, and alleviating CIPN. In this review, we highlight the core role of PGC1α in regulating oxidative stress and maintaining normal mitochondrial function and summarize recent advances in its therapeutic effects and mechanisms in CIPN and other forms of peripheral neuropathy. Emerging studies suggest that PGC1α activation may positively impact CIPN mitigation by modulating oxidative stress, mitochondrial dysfunction, and inflammation. Therefore, novel therapeutic strategies targeting PGC1α could be a potential therapeutic target in CIPN.
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The metastasis and recurrence of nasopharyngeal carcinoma (NPC) contribute to the poor prognosis of patients. Inhibiting epithelial-mesenchymal transition (EMT) is an effective strategy to obstruct metastasis. Therefore, this study aimed to explore the effects of Capn4 on the EMT of NPC cells and its specific mechanism of action. The mRNA and protein expression levels of objective genes in NPC cell lines (5-8F and CNE-2) were evaluated by qRT-PCR and western blotting methods. The subcellular localization of Capn4 was detected by immunofluorescence (IF). Migration and invasion abilities of NPC cells were examined via wound-healing and trans-well methods, and the linkage between Snail and its downstream effector gene (claudin-11) was validated by chromatin immunoprecipitation (ChIP), dual-luciferase, and the yeast one-hybrid assays in series. Over-expression of Capn4 activated the PI3K/AKT signaling pathway and improved the expression of Snail, thus promoting the migration and invasion abilities of NPC cells. Mechanically, claudin-11 is one of the target genes in NPC cells that Snail regulates in a transcriptional regulatory manner. By blocking the regulatory axis of CAPN4/AKT/Snail/claudin-11 can significantly inhibit the invasion and metastasis of NPC cells. Capn4 promoted the EMT of NPC cells by activating the PI3K/AKT/Snail/claudin-11 axis, thereby promoting the malignant development of NPC. The Capn4/PI3K/AKT/Snail/claudin-11 axis might be a novel target to prevent NPC progression.
Asunto(s)
Neoplasias Nasofaríngeas , Proteínas Proto-Oncogénicas c-akt , Humanos , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Claudinas/genética , Claudinas/metabolismo , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Calpaína/metabolismo , Factores de Transcripción de la Familia Snail/metabolismoRESUMEN
Backgrounds: Papillary thyroid cancer is the most common pathological type of thyroid cancer. miR-96-5p, a member of the miR-183 family, constitute a polycistronic miRNA cluster. In breast cancer, miR-96-5p promotes cell invasion, migration, and proliferation in vitro by inhibiting PTPN9. Moreover, miR-96-5p was reported to function as an oncogene in many cancers. However, whether miR-96-5p is involved in the development of papillary thyroid cancers and its potential mechanism is still unknown. The present study aims to explore the relationship between miR-96-5p and GPC3 expression in the development of papillary thyroid cancers. Methods: Transcriptomic sequencing was carried out using six pairs of papillary thyroid cancer and adjacent normal tissues. Quantitative real-time polymerase chain reaction (PCR) experiments were performed to examine the expression of genes. Results: In total, there were 1588 up-regulated and 1803 down-regulated differentially expressed genes between papillary thyroid cancer and normal tissues. Gene ontology and Kyoto encyclopedia of genes and genomes analysis revealed that extracellular matrix structure and proteoglycans were mainly involved in papillary thyroid cancer. Among the cluster of proteoglycans, GPC3 was significantly down-regulated in papillary thyroid cancer and is a target of miR-96. Conclusion: miR-96-5p participates in the development of papillary thyroid cancer by regulating the expression of GPC3. Thus, targeting miR-96-5p may be a potential therapeutic approach for preventing and treating papillary thyroid cancer.
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The optimization of a series of thieno[3,2-b]thiophene-2-carboxylic acid derivatives for agonist activity against the GPR35 is reported. Compounds were optimized to achieve ß-arrestin-biased agonism for developing probe molecules that may be useful for elucidating the biology and physiology of GPR35. Compound 13 was identified to the most potent GPR35 agonist, and compounds 30 and 36 exhibited the highest efficacy to cause ß-arrestin translocation.
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Antiinflamatorios/síntesis química , Arrestinas/agonistas , Receptores Acoplados a Proteínas G/agonistas , Tiofenos/síntesis química , Antiinflamatorios/farmacología , Arrestinas/química , Arrestinas/genética , Bioensayo , Relación Dosis-Respuesta a Droga , Células HT29 , Humanos , Ligandos , Transporte de Proteínas/efectos de los fármacos , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusión/agonistas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Tiofenos/farmacología , beta-ArrestinasRESUMEN
Purine biosynthetic enzymes organize into dynamic cellular bodies called purinosomes. Little is known about the spatiotemporal control of these structures. Using super-resolution microscopy, we demonstrated that purinosomes colocalized with mitochondria, and these results were supported by isolation of purinosome enzymes with mitochondria. Moreover, the number of purinosome-containing cells responded to dysregulation of mitochondrial function and metabolism. To explore the role of intracellular signaling, we performed a kinome screen using a label-free assay and found that mechanistic target of rapamycin (mTOR) influenced purinosome assembly. mTOR inhibition reduced purinosome-mitochondria colocalization and suppressed purinosome formation stimulated by mitochondria dysregulation. Collectively, our data suggest an mTOR-mediated link between purinosomes and mitochondria, and a general means by which mTOR regulates nucleotide metabolism by spatiotemporal control over protein association.
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Mitocondrias/metabolismo , Purinas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Células HeLa , Humanos , Microscopía , Mitocondrias/ultraestructura , Transducción de SeñalRESUMEN
Fluorescent and luminescent probes are essential to both in vitro molecular assays and in vivo imaging techniques, and have been extensively used to measure biological function. However, little is known about the biological activity, thus potential interferences with the assay results, of these probe molecules. Here we show that D-luciferin, one of the most widely used bioluminescence substrates, is a partial agonist for G protein-coupled receptor-35 (GPR35). Label-free phenotypic profiling using dynamic mass redistribution (DMR) assays showed that D-luciferin led to a DMR signal in native HT-29 cells, whose characteristics are similar to those induced by known GPR35 agonists including zaprinast and pamoic acid. DMR assays further showed that D-luciferin is a partial agonist competitive to several known GPR35 agonists and antagonists. D-luciferin was found to cause the phosphorylation of ERK that was suppressed by known GPR35 antagonists, and also result in ß-arrestin translocation signal but with low efficacy. These results not only suggest that D-luciferin is a partial agonist of GPR35, but also will evoke careful interpretation of biological data obtained using molecular and in vivo imaging assays when these probe molecules are used.
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Benzotiazoles/farmacología , Receptores Acoplados a Proteínas G/agonistas , Benzotiazoles/química , Unión Competitiva , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células HT29 , Humanos , Ligandos , Fenotipo , FosforilaciónRESUMEN
Both kynurenic acid and 2-acyl lysophosphatidic acid have been postulated to be the endogenous agonists of GPR35. However, controversy remains whether alternative endogenous agonists exist. The molecular targets accounted for many nongenomic actions of thyroid hormones are mostly unknown. Here we report the agonist activity of multiple tyrosine metabolites at the GPR35. Tyrosine metabolism intermediates that contain carboxylic acid and/or catechol functional groups were first selected. Whole cell dynamic mass redistribution (DMR) assays enabled by label-free optical biosensor were then used to characterize their agonist activity in native HT-29. Molecular assays including ß-arrestin translocation, ERK phosphorylation and receptor internalization confirmed that GPR35 functions as a receptor for 5,6-dihydroxyindole-2-carboxylic acid, 3,3',5'-triiodothyronine, 3,3',5-triiodothyronine, gentisate, rosmarinate, and 3-nitrotyrosine. These results suggest that multiple tyrosine metabolites are alternative endogenous ligands of GPR35, and GPR35 may represent a druggable target for treating certain diseases associated with abnormality of tyrosine metabolism.
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We report the discovery and characterization of natural phenols as G protein-coupled receptor-35 (GPR35) agonists. Pharmacological characterization using label-free dynamic mass redistribution and Tango ß-arrestin translocation assays revealed that GPR35-active natural phenols are divergent in their biased agonism.
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Stem cells hold great potential in drug discovery and development. However, challenges remain to quantitatively measure the functions of stem cells and their differentiated products. Here, we applied fluorescent imaging, quantitative real-time PCR, and label-free dynamic mass redistribution (DMR) assays to characterize the differentiation process of the ReNcell VM human neural progenitor stem cell. Immunofluorescence imaging showed that after growth factor withdrawal, the neuroprogenitor stem cell was differentiated into dopaminergic neurons, astrocytes, and oligodendrocytes, thus creating a neuronal cell system. High-performance liquid chromatography analysis showed that the differentiated cell system released dopamine upon depolarization with KCl. In conjunction with quantitative real-time PCR, DMR assays using a G-protein-coupled receptor agonist library revealed that a subset of receptors, including dopamine D(1) and D(4) receptors, underwent marked alterations in both receptor expression and signaling pathway during the differentiation process. These findings suggest that DMR assays can decode the differentiation process of stem cells at the cell system level.
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Imagen Molecular/métodos , Células-Madre Neurales/metabolismo , Neurogénesis , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Astrocitos/citología , Astrocitos/metabolismo , Células Cultivadas , Cromatografía Líquida de Alta Presión , Dopamina/análisis , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/metabolismo , Técnica del Anticuerpo Fluorescente/métodos , Humanos , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de los fármacos , Oligodendroglía/citología , Oligodendroglía/metabolismo , Cloruro de Potasio/farmacología , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D4/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiología , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
GPR35 is an orphan G protein-coupled receptor that is not well-characterized. Here we employ dynamic mass redistribution (DMR) assays to discover new GPR35 agonists. DMR assays identified tyrphostin analogs as GPR35 agonists, which were confirmed with receptor internalization, Tango ß-arrestin translocation, and extracellular-signal-regulated kinase phosphorylation assays. These agonists provide pharmacological tools to study the biology and function of GPR35.
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Descubrimiento de Drogas/métodos , Receptores Acoplados a Proteínas G/agonistas , Tirfostinos/farmacología , Arrestinas/metabolismo , Endocitosis , Fosforilación , Inhibidores de Proteínas Quinasas , Tirfostinos/química , beta-ArrestinasRESUMEN
Screening with dynamic mass redistribution (DMR) assays in a native cell line HT-29 led to identification of two novel series of chemical compounds, 2-(4-methylfuran-2(5H)-ylidene)malononitrile and thieno[3,2-b]thiophene-2-carboxylic acid derivatives, as GPR35 agonists. Of these, 2-(3-cyano-5-(3,4-dichlorophenyl)-4,5-dimethylfuran-2(5H)-ylidene)malononitrile (YE120) and 6-bromo-3-methylthieno[3,2-b]thiophene-2-carboxylic acid (YE210) were found to be the two most potent GPR35 agonists with an EC(50) of 32.5 ± 1.7 nM and 63.7 ± 4.1 nM, respectively. Both agonists exhibited better potency than that of zaprinast, a known GPR35 agonist. DMR antagonist assays, knockdown of GPR35 with interference RNA, receptor internalization assays, and Tango ß-arrestin translocation assays confirmed that the agonist activity of these ligands is specific to GPR35. The present study provides novel chemical series as a starting point for further investigations of GPR35 biology and pharmacology.
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Furanos/síntesis química , Nitrilos/síntesis química , Receptores Acoplados a Proteínas G/agonistas , Tiofenos/síntesis química , Arrestinas/genética , Arrestinas/metabolismo , Línea Celular , Furanos/química , Furanos/farmacología , Genes Reporteros , Ensayos Analíticos de Alto Rendimiento , Humanos , Nitrilos/química , Nitrilos/farmacología , Transporte de Proteínas/efectos de los fármacos , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Relación Estructura-Actividad , Tiofenos/química , Tiofenos/farmacología , beta-ArrestinasRESUMEN
Mutant huntingtin lowered steady-state levels of DARPP-32 mRNA in the brain but not kidney of R6 transgenic HD mice by repressing transcription from one of two promoters. The activity of DARPP-32 promoter deletion constructs were lower in the presence of mutant huntingtin in immortalized striatal cell lines but no difference in transcription factor binding to the promoter was detected. The activity of CMV, TK and HPRT promoters was also affected by mutant huntingtin in these cell lines. Transient transfection experiments demonstrated that short-term expression of mutant huntingtin exerted a cell- and promoter-specific transcriptional repression. In in vitro experiments, transcription of the CMV promoter was reduced in the presence of striatal proteins and mutant huntingtin. It is likely that select combinations of trans-acting factors, co-activators and components of the Pol II holoenzyme acting in concert provide the basis for both the gene- and tissue-specific effects of mutant huntingtin.
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Encéfalo/metabolismo , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Regulación de la Expresión Génica , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Transcripción Genética , Animales , Encéfalo/citología , Línea Celular , Citomegalovirus/genética , Modelos Animales de Enfermedad , Fosfoproteína 32 Regulada por Dopamina y AMPc/genética , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Enfermedad de Huntington/fisiopatología , Hipoxantina Fosforribosiltransferasa/genética , Hibridación in Situ , Riñón/citología , Riñón/metabolismo , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Timidina Quinasa/genéticaRESUMEN
Huntington's disease (HD) is caused by the inheritance of a copy of the gene encoding mutant huntingtin with an expanded CAG repeat. Phosphodiesterase 10A (PDE10A) mRNA decreases in transgenic HD mice expressing exon 1 of the human huntingtin gene (HD). The mouse PDE10A mRNA is expressed through alternative splicing and polyadenylation in a tissue-specific manner and that transcription of striatal PDE10A mRNA is driven by two promoters. PDE10A2 is the predominant isoform of the gene is expressed in the striatum. Using in situ hybridization and quantitative RT-PCR, we determined that decreased steady-state levels of PDE10A2 mRNA were caused by an altered transcription initiation rate rather than by post-transcriptional mRNA instability in HD mice. Transcription from three initiation sites located within a 50-bp region in the PDE10A2-specific promoter was differentially affected by the presence of the mutant huntingtin transgene. The mouse and human PDE10A2 promoters are highly conserved with respect to the relative position of cis-regulatory elements. Several transcription factors that have been shown to interact with mutant huntingtin, including Sp1, neuron restrictive silencing factor, TATA-binding protein and cAMP-response element binding protein, are unlikely to be involved in mutant huntingtin-induced PDE10A2 transcriptional dysregulation.
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Cuerpo Estriado/enzimología , Enfermedad de Huntington/genética , Mutación , Proteínas del Tejido Nervioso/fisiología , Proteínas Nucleares/fisiología , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Transcripción Genética/fisiología , Empalme Alternativo/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Humanos , Proteína Huntingtina , Isoenzimas/biosíntesis , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Especificidad por Sustrato/genéticaRESUMEN
Loss of cannabinoid receptors (CB1) occurs prior to neurodegeneration in Huntington's disease (HD). The levels and distribution of CB1 RNA were equivalent in 3-week-old mice regardless of genotype demonstrating that the specific factors and appropriate chromatin structure that lead to the transcription of CB1 were present in the striatum of young R6/2 and R6/1 transgenic HD mice. The expression of the mutant HD transgene led progressively to decreased steady-state levels of CB1 mRNA in neurons of the lateral striatum, which was dependent on the size of the CAG repeat and relative expression of the gene encoding mutant huntingtin (HD). Although it is known that the coding region of CB1 is contained within a single exon in mice, rats and humans, the 5'-untranslated region of the mouse gene remained to be defined. CB1 mRNA is encoded by two exons separated by an 18.4-kb intron. Transcription of CB1 occurred at multiple sites within a GC-rich promoter region upstream of exon 1 encoding the 5'-UTR of CB1. There was no difference in the selection of specific transcription initiation sites associated with higher levels of CB1 expression in the striatum compared to the cortex or between the striata of wild-type and HD transgenic mice. The progressive decline in CB1 mRNA levels in R6 compared to wild-type mice was due to decreased transcription, which is consistent with the hypothesis that mutant huntingtin exerts its effects by altering transcription factor activity. The cell-specific conditions that allow for increased transcription of CB1 in the lateral striatum compared to other forebrain regions from all transcription start sites were affected by the expression of mutant huntingtin in a time-dependent manner.