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Genome-wide association studies (GWAS) have identified genetic risk loci for age-related macular degeneration (AMD) on the chromosome 10q26 (Chr10) locus and are tightly linked: the A69S (G>T) rs10490924 single-nucleotide variant (SNV) and the AATAA-rich insertion-deletion (indel, del443/ins54), which are found in the age-related maculopathy susceptibility 2 (ARMS2) gene, and the G512A (G>A) rs11200638 SNV, which is found in the high-temperature requirement A serine peptidase 1 (HTRA1) promoter. The fourth variant is Y402H complement factor H (CFH), which directs CFH signaling. CRISPR manipulation of retinal pigment epithelium (RPE) cells may allow one to isolate the effects of the individual SNV and thus identify SNV-specific effects on cell phenotype. Clustered regularly interspaced short palindromic repeats (CRISPR) editing demonstrates that rs10490924 raised oxidative stress in induced pluripotent stem cell (iPSC)-derived retinal cells from patients with AMD. Sodium phenylbutyrate preferentially reverses the cell death caused by ARMS2 rs10490924 but not HTRA1 rs11200638. This study serves as a proof of concept for the use of patient-specific iPSCs for functional annotation of tightly linked GWAS to study the etiology of a late-onset disease phenotype. More importantly, we demonstrate that antioxidant administration may be useful for reducing reactive oxidative stress in AMD, a prevalent late-onset neurodegenerative disorder.
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Células Madre Pluripotentes Inducidas , Degeneración Macular , Humanos , Serina Peptidasa A1 que Requiere Temperaturas Altas/genética , Células Madre Pluripotentes Inducidas/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Proteínas/metabolismo , Serina Endopeptidasas/genética , Estudio de Asociación del Genoma Completo , Degeneración Macular/genética , Estrés Oxidativo , Polimorfismo de Nucleótido Simple , Factor H de Complemento/genética , GenotipoRESUMEN
Macular degeneration (MD) is characterized by the progressive deterioration of the macula and represents one of the most prevalent causes of blindness worldwide. Abnormal intracellular accumulation of lipid droplets and pericellular deposits of lipid-rich material in the retinal pigment epithelium (RPE) called drusen are clinical hallmarks of different forms of MD including Doyne honeycomb retinal dystrophy (DHRD) and age-related MD (AMD). However, the appropriate molecular therapeutic target underlying these disorder phenotypes remains elusive. Here, we address this knowledge gap by comparing the proteomic profiles of induced pluripotent stem cell (iPSC)-derived RPEs (iRPE) from individuals with DHRD and their isogenic controls. Our analysis and follow-up studies elucidated the mechanism of lipid accumulation in DHRD iRPE cells. Specifically, we detected significant downregulation of carboxylesterase 1 (CES1), an enzyme that converts cholesteryl ester to free cholesterol, an indispensable process in cholesterol export. CES1 knockdown or overexpression of EFEMP1R345W, a variant of EGF-containing fibulin extracellular matrix protein 1 that is associated with DHRD and attenuated cholesterol efflux and led to lipid droplet accumulation. In iRPE cells, we also found that EFEMP1R345W has a hyper-inhibitory effect on epidermal growth factor receptor (EGFR) signaling when compared to EFEMP1WT and may suppress CES1 expression via the downregulation of transcription factor SP1. Taken together, these results highlight the homeostatic role of cholesterol efflux in iRPE cells and identify CES1 as a mediator of cholesterol efflux in MD.
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Colesterol/metabolismo , Degeneración Macular/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Adolescente , Adulto , Hidrolasas de Éster Carboxílico/genética , Diferenciación Celular/genética , Citocinas/metabolismo , Receptores ErbB/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Inflamación/metabolismo , Metabolismo de los Lípidos , Degeneración Macular/patología , Persona de Mediana Edad , Drusas del Disco Óptico/congénito , Drusas del Disco Óptico/metabolismo , Proteómica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Epitelio Pigmentado de la Retina/patología , Transducción de Señal , Factor de Transcripción Sp1/metabolismo , Transcripción Genética , Respuesta de Proteína DesplegadaRESUMEN
BACKGROUND: As women with low ovarian reserve embark on the challenging journey of in-vitro fertilisation (IVF) treatment, the choice between natural and mildly stimulated cycles becomes a pivotal consideration. It is unclear which of these two regimens is superior for women with low ovarian reserve. Our study aims to assess the impact of natural cycles on embryo quality and pregnancy outcomes in women with low ovarian reserve undergoing IVF treatment compared to mildly stimulated cycles. METHODS: This retrospective study enrolled consecutive patients with low ovarian reserve who underwent IVF/intracytoplasmic sperm injection (ICSI) at Guangdong Second Provincial General Hospital between January 2017 and April 2021. The primary outcome for pregnancy rate of 478 natural cycles and 448 mild stimulated cycles was compared. Secondary outcomes included embryo quality and oocyte retrieval time of natural cycles. RESULTS: The pregnancy rate in the natural cycle group was significantly higher than that in the mildly stimulated cycle group (51.8% vs. 40.1%, p = 0.046). Moreover, natural cycles exhibited higher rates of available embryos (84.1% vs. 78.6%, p = 0.040), high-quality embryos (61.8% vs. 53.2%, p = 0.008), and utilisation of oocytes (73% vs. 65%, p = 0.001) compared to mildly stimulated cycles. Oocyte retrievals in natural cycles were predominantly performed between 7:00 and 19:00, with 94.9% occurring during this time frame. In natural cycles with high-quality embryos, 96.4% of oocyte retrievals were also conducted between 7:00 and 19:00. CONCLUSION: Natural cycles with appropriately timed oocyte retrieval may present a valuable option for patients with low ovarian reserve.
In the realm of in-vitro fertilisation (IVF) treatment, women with low ovarian reserve often face the crucial decision of opting for natural or mildly stimulated cycles. This retrospective study, conducted between January 2017 and April 2021 at Guangdong Second Provincial General Hospital, delves into the impact of these cycles on pregnancy outcomes. Examining 478 natural cycles and 448 mildly stimulated cycles, the study reveals a notably higher pregnancy rate in the natural cycle group (51.8% vs. 40.1%). Additionally, natural cycles demonstrated higher rates of available embryos, high-quality embryos, and oocyte utilisation compared to their mildly stimulated counterparts. The findings suggest that natural cycles, with proper oocyte retrieval timing, could be a favourable choice for those with low ovarian reserve seeking IVF treatment.
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Reserva Ovárica , Resultado del Embarazo , Femenino , Humanos , Masculino , Embarazo , Estudios de Cohortes , Estudios Retrospectivos , Semen , Recuperación del Oocito , Índice de EmbarazoRESUMEN
With the rapid change of people's lifestyle, more childbearing couples live with irregular schedules (i.e., staying up late) and suffer from decreased fertility and abortion, which can be caused by luteal phase defect (LPD). We used continuous light-exposed mice as a model to observe whether continuous light exposure may affect luteinization and luteal function. We showed that the level of progesterone in serum reduced (p < .001), the number of corpus luteum (CL) decreased (p < .01), and the expressions of luteinization-related genes (Lhcgr, Star, Ptgfr, and Runx2), clock genes (Clock and Per1), and Mt1 were downregulated (p < .05) in the ovaries of mice exposed to continuous light, suggesting that continuous light exposure induces defects in luteinization and luteal functions. Strikingly, injection of melatonin (3 mg/kg) could improve luteal functions in continuous light-exposed mice. Moreover, we found that, after 2 h of hCG injection, the level of pERK1/2 in the ovary decreased in the continuous light group, but increased in the melatonin administration group, suggesting that melatonin can improve LPD caused by continuous light exposure through activating the ERK1/2 pathway. In summary, our data demonstrate that continuous light exposure affects ovary luteinization and luteal function, which can be rescued by melatonin.
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Melatonina , Ovario , Femenino , Embarazo , Ratones , Animales , Ovario/metabolismo , Ratones Endogámicos ICR , Melatonina/farmacología , Melatonina/metabolismo , Cuerpo Lúteo/metabolismo , Progesterona/metabolismo , LuteinizaciónRESUMEN
PURPOSE: Antiangiogenic therapy is crucial for advanced hepatocellular carcinoma (HCC) treatment. Interleukin (IL)-6 is an inflammatory response mediator that can promote angiogenesis. We explored its prognostic role in patients with advanced HCC. METHODS: We had two patient cohorts, both comprising patients who received sorafenib-containing therapy as the first-line treatment for advanced HCC. We explored the best cut point for pretreatment plasma IL-6 levels in the exploration cohort and then confirmed it in the validation cohort. RESULTS: In total, 55 and 73 patients constituted the exploration and validation cohorts, respectively. In the exploration cohort, a cut point of 4.28 pg/ml was the best for defining high and low IL-6 levels because it could most effectively differentiate overall survival (OS). On application of this cut point to the validation cohort, patients with high plasma IL-6 levels, compared with patients with low IL-6 levels, exhibited significantly poorer OS (median, 8.0 vs 13.9 months, P = 0.031) but similar progression-free survival or treatment response. After adjusting for patient demographics and tumor characteristics, a high plasma IL-6 level remained an independent predictor of poor OS (hazard ratio 2.594, P = 0.005). CONCLUSION: High pretreatment plasma IL-6 levels were associated with poor prognosis of patients with advanced HCC.
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Carcinoma Hepatocelular/sangre , Interleucina-6/sangre , Neoplasias Hepáticas/sangre , Niacinamida/análogos & derivados , Compuestos de Fenilurea/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Estudios de Cohortes , Supervivencia sin Enfermedad , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Persona de Mediana Edad , Niacinamida/administración & dosificación , Niacinamida/uso terapéutico , Compuestos de Fenilurea/administración & dosificación , Pronóstico , Inhibidores de Proteínas Quinasas/administración & dosificación , Sorafenib , Adulto JovenRESUMEN
Regulatory factor X-1 (RFX-1) is a transcription factor that has been linked to negative regulation of tumor progression; however, its biological function and signaling cascades are unknown. Here, we performed several studies to elucidate the roles of RFX-1 in the regulation of SHP-1 in hepatocellular carcinoma (HCC) cells. Overexpression of RFX-1 resulted in the activation of SHP-1 and repressed colony formation of HCC cells. In addition, by a mouse xenograft model, we demonstrated that RFX-1 overexpression also inhibited the tumor growth of HCC cells in vivo, suggesting that RFX-1 is of potential interest for small-molecule-targeted therapy. We also found that SC-2001, a bipyrrole molecule, induced apoptosis in HCC cells through activating RFX-1 expression. SC-2001 induced RFX-1 translocation from the cytosol to nucleus, bound to the SHP-1 promoter, and activated SHP-1 transcription. In a xenograft model, knockdown of RFX-1 reversed the antitumor effect of SC-2001. Notably, SC-2001 is much more potent than sorafenib, a clinically approved drug for HCC, in in vitro and in vivo assays. Our study confirmed that RFX-1 acts as a tumor suppressor in HCC and might be a new target for HCC therapy. The findings of this study also provide a new lead compound for targeted therapy via the activation of the RFX-1/SHP-1 pathway.
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Carcinoma Hepatocelular/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Hepáticas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Factor de Transcripción STAT3/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Animales , Antineoplásicos/farmacología , Western Blotting , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Citometría de Flujo , Humanos , Técnicas para Inmunoenzimas , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Luciferasas/metabolismo , Masculino , Ratones , Ratones Desnudos , Niacinamida/análogos & derivados , Niacinamida/farmacología , Compuestos de Fenilurea/farmacología , Regiones Promotoras Genéticas/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Pirroles/farmacología , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción del Factor Regulador X , Factor Regulador X1 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Sorafenib , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND & AIMS: Nintedanib, a triple angiokinase inhibitor, is currently being evaluated against advanced HCC in phase I/II clinical trials. Here, we report the underlying molecular mechanism by which nintedanib (BIBF-1120) induces an anti-HCC effect. METHODS: To further elucidate whether the effect of nintedanib on SHP-1 is dependent on its angiokinase inhibition activity, we developed a novel kinase-independent derivative of nintedanib, ΔN. HCC cell lines were treated with nintedanib or its derivative (ΔN) and apoptosis, signal transduction, and phosphatase activity were analyzed. Purified SHP-1 proteins or HCC cells expressing deletion N-SH2 domain or D61A point mutants were used to investigate the potential effect of nintedanib on SHP-1. In vivo efficacy was determined in nude mice with HCC subcutaneous xenografts (n⩾8 mice). RESULTS: Nintedanib induced anti-proliferation in HCC cell lines by targeting STAT3. Ectopic STAT3 abolished nintedanib-mediated apoptosis in HCC cells. Nintedanib further activated SHP-1 in purified SHP-1 proteins suggesting that nintedanib directly affects SHP-1 for STAT3 inhibition. HCC cells or recombinant SHP-1 proteins expressing deletion of N-SH2 domain or D61A mutants restored the activity of nintedanib suggesting that the auto-inhibition structure of SHP-1 was relieved by nintedanib. Although ΔN only retained the backbone of nintedanib without kinase activity, ΔN still induced substantial anti-HCC activity in vitro and in vivo by targeting STAT3. CONCLUSIONS: Nintedanib induced significant anti-HCC activity independent of angiokinase inhibition activity in a preclinical HCC model by relieving autoinhibition of SHP-1. Our findings provide new mechanistic insight into the inhibition of HCC growth by nintedanib.
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Antineoplásicos/uso terapéutico , Carcinoma Hepatocelular/tratamiento farmacológico , Indoles/uso terapéutico , Neoplasias Hepáticas/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Sitios de Unión , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Desnudos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformación Proteica , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteína Tirosina Fosfatasa no Receptora Tipo 6/química , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/química , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Patient-specific induced pluripotent stem cell-derived (iPSC-derived) cell lines allow for therapies to be tailored to individual patients, increasing therapeutic precision and efficiency. Bietti crystalline dystrophy (BCD) is a rare blinding disease estimated to affect about 67,000 individuals worldwide. Here, we used iPSC-derived retinal pigment epithelium (iRPE) cells from patients with BCD to evaluate adeno-associated virus-mediated (AAV-mediated) gene augmentation therapy strategies. We found that BCD iRPE cells were vulnerable to blue light-induced oxidative stress and that cellular phenotype can be quantified using 3 robust biomarkers: reactive oxygen species (ROS), 4-hydroxy 2-nonenal (4-HNE) levels, and cell death rate. Additionally, we demonstrated that AAV-mediated gene therapy can significantly reduce light-induced cell death in BCD iRPE cells. This is the first proof-of-concept study to our knowledge to show that AAV-CYP4V2 gene therapy can be used to treat light-induced RPE damage in BCD. Furthermore, we observed significant variability in cellular phenotypes among iRPE from patients with BCD of divergent mutations, which outlined genotype-phenotype correlations in BCD patient-specific cell disease models. Our results reveal that patient-specific iRPE cells retained personalized responses to AAV-mediated gene therapy. Therefore, this approach can advance BCD therapy and set a precedent for precision medicine in other diseases, emphasizing the necessity for personalization in healthcare to accommodate individual diversity.
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Distrofias Hereditarias de la Córnea , Dependovirus , Terapia Genética , Células Madre Pluripotentes Inducidas , Medicina de Precisión , Epitelio Pigmentado de la Retina , Humanos , Medicina de Precisión/métodos , Terapia Genética/métodos , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Células Madre Pluripotentes Inducidas/metabolismo , Distrofias Hereditarias de la Córnea/terapia , Distrofias Hereditarias de la Córnea/genética , Distrofias Hereditarias de la Córnea/metabolismo , Distrofias Hereditarias de la Córnea/patología , Dependovirus/genética , Estrés Oxidativo/genética , Familia 4 del Citocromo P450/genética , Familia 4 del Citocromo P450/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Enfermedades de la Retina/terapia , Enfermedades de la Retina/genética , Enfermedades de la Retina/patología , Aldehídos/metabolismo , MasculinoRESUMEN
OBJECTIVE: In this study, we aimed to identify long non-coding RNAs (lncRNAs) that play important roles in starvation stress, analyze their functions, and discover potential molecular targets to alleviate starvation stress to provide a theoretical reference for subsequent in-depth research. METHODS: We generated a piglet starvation stress animal model. Nine Yorkshire weaned piglets were randomly divided into a long-term starvation stress group (starved for 72 h), short-term starvation stress group (starved for 48 h), and the control group. LncRNA libraries were constructed using high-throughput sequencing of piglet ileums. RESULTS: We obtained 11,792 lncRNAs, among which, 2,500 lncRNAs were novel. In total, 509 differentially expressed (DE)lncRNAs were identified in this study. Target genes of DElncRNAs were predicted via cis and trans interactions, and functional and pathway analyses were performed. Gene ontology functions and Kyoto encyclopedia of genes and genomes analysis revealed that lncRNA-targeted genes mainly participated in metabolic pathways, cellular processes, immune system processes, digestive systems, and transport activities. To reveal the mechanism underlying starvation stress, the interaction network between lncRNAs and their targets was constructed based on 26 DElncRNAs and 72 DEmRNAs. We performed an interaction network analysis of 121 DElncRNA-DEmRNA pairs with a Pearson correlation coefficient greater than 0.99. CONCLUSION: We found that MSTRG.19894.13, MSTRG.16726.3, and MSTRG.12176.1 might play important roles in starvation stress. This study not only generated a library of enriched lncRNAs in piglets, but its outcomes also provide a strong foundation to screen key lncRNAs involved in starvation stress and a reference for subsequent in-depth research.
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Impairment in glucocerebrosidase (GCase) is strongly associated with the development of Parkinson's disease (PD), yet the regulators responsible for its impairment remain elusive. In this paper, we identify the E3 ligase Thyroid Hormone Receptor Interacting Protein 12 (TRIP12) as a key regulator of GCase. TRIP12 interacts with and ubiquitinates GCase at lysine 293 to control its degradation via ubiquitin proteasomal degradation. Ubiquitinated GCase by TRIP12 leads to its functional impairment through premature degradation and subsequent accumulation of α-synuclein. TRIP12 overexpression causes mitochondrial dysfunction, which is ameliorated by GCase overexpression. Further, conditional TRIP12 knockout in vitro and knockdown in vivo promotes the expression of GCase, which blocks α-synuclein preformed fibrils (α-syn PFFs)-provoked dopaminergic neurodegeneration. Moreover, TRIP12 accumulates in human PD brain and α-synuclein-based mouse models. The identification of TRIP12 as a regulator of GCase provides a new perspective on the molecular mechanisms underlying dysfunctional GCase-driven neurodegeneration in PD.
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Proteínas Portadoras/metabolismo , Glucosilceramidasa , Enfermedad de Parkinson , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Glucosilceramidasa/genética , Glucosilceramidasa/metabolismo , Ratones , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Ubiquitinación , alfa-Sinucleína/metabolismoRESUMEN
Essential tremor (ET) is one of the most common movement disorders and the prototypical disorder for abnormal rhythmic movements. However, the pathophysiology of tremor generation in ET remains unclear. Here, we used autoptic cerebral tissue from patients with ET, clinical data, and mouse models to report that synaptic pruning deficits of climbing fiber (CF)-to-Purkinje cell (PC) synapses, which are related to glutamate receptor delta 2 (GluRδ2) protein insufficiency, cause excessive cerebellar oscillations and might be responsible for tremor. The CF-PC synaptic pruning deficits were correlated with the reduction in GluRδ2 expression in the postmortem ET cerebellum. Mice with GluRδ2 insufficiency and CF-PC synaptic pruning deficits develop ET-like tremor that can be suppressed with viral rescue of GluRδ2 protein. Step-by-step optogenetic or pharmacological inhibition of neuronal firing, axonal activity, or synaptic vesicle release confirmed that the activity of the excessive CF-to-PC synapses is required for tremor generation. In vivo electrophysiology in mice showed that excessive cerebellar oscillatory activity is CF dependent and necessary for tremor and optogenetic-driven PC synchronization was sufficient to generate tremor in wild-type animals. Human validation by cerebellar electroencephalography confirmed that excessive cerebellar oscillations also exist in patients with ET. Our findings identify a pathophysiologic contribution to tremor at molecular (GluRδ2), structural (CF-to-PC synapses), physiological (cerebellar oscillations), and behavioral levels (kinetic tremor) that might have clinical applications for treating ET.
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Cerebelo/metabolismo , Temblor/metabolismo , Temblor/patología , Animales , Humanos , Ratones , Células de Purkinje/metabolismo , Células de Purkinje/patología , Receptores de Glutamato/metabolismo , Sinapsis/metabolismo , Sinapsis/patologíaRESUMEN
Alterations in cell cycle regulators are common in hepatocellular carcinoma (HCC). We tested the efficacy of composite inhibition of CDKs 1, 2, 5, and 9 through dinaciclib on HCC. In vitro, dinaciclib exhibited potent antiproliferative activities in HCC cell lines regardless of Rb or c-myc expression levels. Dinaciclib significantly downregulated the phosphorylation of Rb (target of CDKs 1 and 2), ataxia telangiectasia mutated kinase (target of CDK5), and RNA polymerase II (target of CDK9) in the HCC cells. In xenograft studies, mice receiving dinaciclib tolerated the treatment well without significant body weight changes and exhibited a significantly slower tumor growth rate than the mice receiving vehicles. RNA interference (RNAi) of CDKs 1 and 9 was more effective in inhibiting the cell proliferation of HCC cells than RNAi of CDKs 2 and 5. Overexpression of CDK9 significantly reduced the efficacy of dinaciclib in HCC cells, but overexpression of CDK1 did not. In conclusion, composite inhibition of CDKs 1, 2, 5, and 9 through dinaciclib exhibited potent in vitro and in vivo activity against HCC. CDK9 inhibition might be the crucial mechanism.
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Background: Tremor is the most common movement disorder; however, the pathophysiology of tremor remains elusive. While several neuropathological alterations in tremor disorders have been observed in post-mortem studies of human brains, a full understanding of the relationship between brain circuitry alterations and tremor requires testing in animal models. Additionally, tremor animal models are critical for our understanding of tremor pathophysiology, and/or to serve as a platform for therapy development. Methods: A PubMed search was conducted in May 2018 to identify published papers for review. Results: The methodology used in most studies on animal models of tremor lacks standardized measurement of tremor frequency and amplitude; instead, these studies are based on the visual inspection of phenotypes, which may fail to delineate tremor from other movement disorders such as ataxia. Of the animal models with extensive tremor characterization, harmaline-induced rodent tremor models provide an important framework showing that rhythmic and synchronous neuronal activities within the olivocerebellar circuit can drive action tremor. In addition, dopamine-depleted monkey and mouse models may develop rest tremor, highlighting the role of dopamine in rest tremor generation. Finally, other animal models of tremor have involvement of the cerebellar circuitry, leading to altered Purkinje cell physiology. Discussion: Both the cerebellum and the basal ganglia are likely to play a role in tremor generation. While the cerebellar circuitry can generate rhythmic movements, the nigrostriatal system is likely to modulate the tremor circuit. Tremor disorders are heterogeneous in nature. Therefore, each animal model may represent a subset of tremor disorders, which collectively can advance our understanding of tremor.
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Encéfalo/patología , Modelos Animales de Enfermedad , Temblor , Animales , Encéfalo/fisiopatología , Humanos , PubMed/estadística & datos numéricos , Temblor/patología , Temblor/fisiopatologíaRESUMEN
Increased angiogenic activity has been demonstrated in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC), but the mechanism was unclear. To study the role of HCV core protein, we used tube formation and Matrigel plug assays to assess the proangiogenic activity of an HCC cell line, HuH7, and 2 of its stable clones-HuH7-core-high and HuH7-core-low, with high and low HCV core protein expression, respectively. In both assays, HuH7-core-high and HuH7-core-low cells dose-dependently induced stronger angiogenesis than control cells. HuH7 cells with HCV core protein expression showed increased mRNA and protein expression of vascular endothelial growth factor (VEGF). VEGF inhibition by bevacizumab reduced the proangiogenic activity of HuH7-core-high cells. The promotor region of VEGF contains the binding site of activator protein-1 (AP-1). Compared with controls, HuH7-core-high cells had an increased AP-1 activity and nuclear localization of phospho-c-jun. AP-1 inhibition using either RNA knockdown or AP-1 inhibitors reduced the VEGF mRNA expression and the proangiogenic activity of HuH7-core-high cells. Among 131 tissue samples from HCC patients, HCV-related HCC revealed stronger VEGF expression than did hepatitis B virus-related HCC. In conclusion, increased VEGF expression through AP-1 activation is a crucial mechanism underlying the proangiogenic activity of the HCV core protein in HCC cells.
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BACKGROUND: Genome-wide association studies have revealed a link between essential tremor (ET) and the gene SLC1A2, which encodes excitatory amino acid transporter type 2 (EAAT2). We explored EAAT biology in ET by quantifying EAAT2 and EAAT1 levels in the cerebellar dentate nucleus, and expanded our prior analysis of EAAT2 levels in the cerebellar cortex. OBJECTIVE: To quantify EAAT2 and EAAT1 levels in the cerebellar dentate nucleus and cerebellar cortex of ET cases vs. METHODS: We used immunohistochemistry to quantify EAAT2 and EAAT1 levels in the dentate nucleus of a discovery cohort of 16 ET cases and 16 controls. Furthermore, we quantified EAAT2 levels in the dentate nucleus in a replicate cohort (61 ET cases, 25 controls). Cortical EAAT2 levels in all 77 ET cases and 41 controls were quantified. RESULTS: In the discovery cohort, dentate EAAT2 levels were 1.5-fold higher in 16 ET cases vs. 16 controls (p = 0.007), but EAAT1 levels did not differ significantly (p = 0.279). Dentate EAAT2 levels were 1.3-fold higher in 61 ET cases vs. 25 controls in the replicate cohort (p = 0.022). Cerebellar cortical EAAT2 levels were 20% and 40% lower in ET cases vs. controls in the discovery and the replicate cohorts (respective p values = 0.045 and < 0.001). CONCLUSION: EAAT2 expression is enhanced in the ET dentate nucleus, in contrast to differentially reduced EAAT2 levels in the ET cerebellar cortex, which might reflect a compensatory mechanism to maintain excitation-inhibition balance in cerebellar nuclei.
Asunto(s)
Corteza Cerebelosa/metabolismo , Núcleos Cerebelosos/metabolismo , Temblor Esencial/patología , Transportador 1 de Aminoácidos Excitadores/metabolismo , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Polimorfismo de Nucleótido Simple/genética , Anciano , Anciano de 80 o más Años , Diagnóstico , Transportador 1 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores , Femenino , Estudio de Asociación del Genoma Completo , Genotipo , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Glutamato Sintasa/metabolismo , Humanos , Masculino , Estadísticas no ParamétricasRESUMEN
Hepatocellular carcinoma is the fifth most common solid cancer worldwide. Sorafenib, a small multikinase inhibitor, is the only approved therapy for advanced HCC. The clinical benefit of sorafenib is offset by the acquisition of sorafenib resistance. Understanding of the molecular mechanism of STAT3 overexpression in sorafenib resistance is critical if the clinical benefits of this drug are to be improved. In this study, we explored our hypothesis that loss of RFX-1/SHP-1 and further increase of p-STAT3 as a result of sorafenib treatment induces sorafenib resistance as a cytoprotective response effect, thereby, limiting sorafenib sensitivity and efficiency. We found that knockdown of RFX-1 protected HCC cells against sorafenib-induced cell apoptosis and SHP-1 activity was required for the process. SC-2001, a molecule with similar structure to obatoclax, synergistically suppressed tumor growth when used in combination with sorafenib in vitro and overcame sorafenib resistance through up-regulating RFX-1 and SHP-1 resulting in tumor suppression and mediation of dephosphorylation of STAT3. In addition, sustained sorafenib treatment in HCC led to increased p-STAT3 which was a key mediator of sorafenib sensitivity. The combination of SC-2001 and sorafenib strongly inhibited tumor growth in both wild-type and sorafenib-resistant HCC cell bearing xenograft models. These results demonstrate that inactivation of RFX/SHP-1 induced by sustained sorafenib treatment confers sorafenib resistance to HCC through p-STAT3 up-regulation. These effects can be overcome by SC-2001 through RFX-1/SHP-1 dependent p-STAT3 suppression. In conclusion, the use of SC-2001 in combination with sorafenib may constitute a new strategy for HCC therapy.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Proteínas de Unión al ADN/metabolismo , Resistencia a Antineoplásicos , Neoplasias Hepáticas/metabolismo , Niacinamida/análogos & derivados , Compuestos de Fenilurea/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Pirroles/farmacología , Factor de Transcripción STAT3/metabolismo , Factores de Transcripción/metabolismo , Animales , Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Masculino , Ratones , Niacinamida/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Factores de Transcripción del Factor Regulador X , Factor Regulador X1 , Sorafenib , Factores de Transcripción/genética , Ensayo de Tumor de Célula Madre , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Sorafenib is the first approved targeted therapeutic reagent for hepatocellular carcinoma. Here, we report that SC-60, a dimer-based sorafenib derivative, overcomes the resistance of sorafenib and shows a better anti-hepatocellular carcinoma effect in vitro and in vivo. SC-60 substantially increased SH2 domain-containing phosphatase 1 (SHP-1) phosphatase activity in hepatocellular carcinoma cells and purified SHP-1 proteins, suggesting that SC-60 affects SHP-1 directly. Molecular docking and truncated mutants of SHP-1 further confirmed that SC-60 interferes with the inhibitory N-SH2 domain to relieve the closed catalytic protein tyrosine phosphatase domain of SHP-1. Deletion of N-SH2 domain (dN1) or point mutation (D61A) of SHP-1 abolished the effect of SC-60 on SHP-1, p-STAT3, and apoptosis. Importantly, SC-60 exhibited significant survival benefits compared with sorafenib in a hepatocellular carcinoma orthotopic model via targeting the SHP-1/STAT3-related signaling pathway. In summary, dimer derivative of sorafenib, SC-60, is a SHP-1 agonist and may be a potent reagent for hepatocellular carcinoma-targeted therapy.
Asunto(s)
Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Niacinamida/análogos & derivados , Compuestos de Fenilurea/administración & dosificación , Apoptosis , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Dominio Catalítico/genética , Línea Celular Tumoral , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Niacinamida/administración & dosificación , Niacinamida/química , Compuestos de Fenilurea/química , Mutación Puntual , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Sorafenib , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Here, we aim to investigate the molecular mechanism of regorafenib and verify the potential druggable target for the treatment of hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: HCC cell lines (PLC5, HepG2, Hep3B, SK-Hep1, and HA59T) were used to investigate the in vitro effect of regorafenib. Phosphatase activity was analyzed in HCC cells and purified SHP-1 proteins. PLC5-bearing mice were used to test the therapeutic efficiency of 20 and 40 mg/kg/d treatment with regorafenib ([Formula: see text] mice). The clinical relevance of STAT3 signaling was investigated with 142 tumor samples from different patients with HCC. Descriptive statistical analysis was used to compare the baseline characteristics of patients and the expression of p-STAT3. RESULTS: Regorafenib inhibited STAT3-related signaling in a dose-dependent manner and was a more potent inhibitor of STAT3 than sorafenib. Regorafenib increased SHP-1 phosphatase activity in purified SHP-1 protein directly. N-SH2 domain deletion and D61A mutants mimicking open-form SHP-1 partially abolished regorafenib-induced STAT3 inhibition and apoptosis. Importantly, a higher level of expression of STAT3 was found in patients with advanced clinical stages (P = 0.009) and poorly differentiated tumors (P = 0.035). CONCLUSIONS: Regorafenib induced significant tumor inhibition by relieving the autoinhibited N-SH2 domain of SHP-1 directly and inhibiting p-STAT3 signals. STAT3 may be suitable as a prognostic marker of HCC development, and may be a druggable target for HCC-targeted therapy using regorafenib.