RESUMEN

Lung cancer is the malignancy with the highest morbidity and mortality worldwide. Approximately 60% of non-small cell lung cancer (NSCLC) presents driver alterations most of which are targetable. Nowadays, limited clinical data are available regarding the efficacy of epithelial growth factor receptor (EGFR) tyrosine kinase inhibitors in patients with NSCLC harboring uncommon EGFR mutations, considering their heterogeneity. Herein, we report a rare case of EGFR-mutated lung adenocarcinoma which has developed into squamous cell carcinoma with uncommon EGFR (Ex18) compound mutations and phosphatidylinositol 3-kinase mutation receiving afatinib at the forefront.

Asunto(s)

Adenocarcinoma del Pulmón , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Receptores ErbB/genética , Mutación , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/genética , Receptores de Factores de Crecimiento/genética

RESUMEN

BACKGROUND: Rat sarcoma virus mutational status guides first-line treatment in metastatic colorectal cancer. This study was a multi center, multi-country ambispective, observational study in the Middle East and North Africa assessing regional rat sarcoma virus testing practices in newly diagnosed patients. METHODS: The retrospective arm (2011-2014) included adults with metastatic colorectal cancer who had initiated first-line therapy with ≥1 post-baseline visit and survival data. The prospective arm (2014-2019) enrolled newly diagnosed patients with histologically proven metastatic colorectal cancer with ≥1 measurable lesion per Response Evaluation Criteria in Solid Tumors, and tissue availability for biomarker analysis. Data look-back and follow-up were 2 years; the rate of RAS mutation was evaluated. RESULTS: RAS testing was ordered for patients in retrospective (326/417) and prospective (407/500) studies. In the former, testing was typically prescribed after first-line treatment initiation, significantly more in patients with stage IV disease (P < .005), resulting in the addition of targeted therapy (41.8% anti-epidermal growth factor receptor, 30.2% anti-vascular endothelial growth factor) in wild-type metastatic colorectal cancer, and significantly impacted the treatment of left-sided tumors (P = .037). In the latter, 58.4% were RAS wild-type; 41.6% were RAS mutant. Non-prescription of RAS testing was attributed to test unavailability, financial, or medical rea sons; predictors of testing prescription were older age, primary tumor in ascending colon, and high tumor grade. RAS status knowledge resulted in the addition of anti-vascular endothelial growth factor (20.4%) or anti-epidermal growth factor receptor therapy (21.2%). CONCLUSION: Before 2014, RAS testing in patients with colorectal cancer in the Middle East and North Africa was often performed after first-line treatment. Testing is more routine in newly diagnosed patients, potentially shifting early treatment patterns.

Asunto(s)

Neoplasias del Colon , Neoplasias Colorrectales , Neoplasias del Recto , Humanos , Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias Colorrectales/genética , Factores de Crecimiento Endotelial/genética , Factores de Crecimiento Endotelial/uso terapéutico , Mutación , Estudios Prospectivos , Proteínas Proto-Oncogénicas p21(ras)/genética , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento/uso terapéutico , Estudios Retrospectivos , Sistema de Registros

RESUMEN

Lung adenocarcinoma is the most common form of lung cancer, accounting for 60% of non-small cell lung cancer (NSCLC) cases in Asian patients. Importantly, nearly half of these patients have epithelial growth factor receptor (EGFR) mutations. Though EGFR-tyrosine kinase inhibitors (EGFR-TKIs) are recommended as the first-line therapy for NSCLC patients, the development of resistance reduces their efficiency and limits their application. As the complicated and heterogeneous mechanism of acquired resistance among individuals, the efficiency of anti-angiogenesis therapy, immune checkpoint inhibitors, or chemo-radiotherapies is rather less promising. In this research, we investigated the role of the tumor stem cell marker DCLK1 in EGFR-TKI resistance of lung adenocarcinoma. We discovered that DCLK1 was critical in maintaining the stemness of tumor cells through the Wnt/ß-Catenin pathway, which was conducive to the development of EGFR-TKI resistance. Inhibiting DCLK1 activity restored the sensitivity of TKI-resistant tumor cells and organoids. Moreover, our study showed that DCLK1 inhibitor had a synergistic effect in controlling tumor growth when combined with EGFR-TKIs. Overall, our study provides new insights into EGFR-TKI resistant lung adenocarcinoma through inhibition of DCLK1 expression.

Asunto(s)

Adenocarcinoma del Pulmón , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Quinasas Similares a Doblecortina , Resistencia a Antineoplásicos , Receptores ErbB/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/genética , Receptores de Factores de Crecimiento/genética , beta Catenina/genética

RESUMEN

Many small molecule kinase inhibitors (SMKIs), used predominantly in cancer therapy, have been implicated in serious clinical cardiac adverse events, which means that traditional preclinical drug development assays were not sufficient for identifying these cardiac liabilities. To improve clinical cardiac safety predictions, the effects of SMKIs targeting many different signaling pathways were studied using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) in combined assays designed for the detection of both electrophysiological (proarrhythmic) and non-electrophysiological (non-proarrhythmic) drug-induced cardiotoxicity. Several microplate-based assays were used to quantitate cell death, apoptosis, mitochondrial damage, energy depletion, and oxidative stress as mechanism-based non-electrophysiological cardiomyocyte toxicities. Microelectrode arrays (MEA) were used to quantitate in vitro arrhythmic events (iAEs), field potential duration (FPD) prolongation, and spike amplitude suppression (SAS) as electrophysiological effects. To enhance the clinical relevance, SMKI-induced cardiotoxicities were compared by converting drug concentrations into multiples of reported clinical maximum therapeutic plasma concentration, "FoldCmax", for each assay. The results support the conclusion that the combination of the hPSC-CM based electrophysiological and non-electrophysiological assays have significantly more predictive value than either assay alone and significantly more than the current FDA-recommended hERG assay. In addition, the combination of these assays provided mechanistic information relevant to cardiomyocyte toxicities, thus providing valuable information on potential drug-induced cardiotoxicities early in drug development prior to animal and clinical testing. We believe that this early information will be helpful to guide the development of safer and more cost-effective drugs.

Asunto(s)

Miocitos Cardíacos/efectos de los fármacos , Células Madre Pluripotentes/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Diferenciación Celular , Supervivencia Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Inhibidores de Proteínas Quinasas/química , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento/metabolismo

RESUMEN

The neurotrophin growth factors bind and activate two types of cell surface receptors: the tropomyosin receptor kinase (Trk) family and p75. TrkA, TrkB, and TrkC are bound preferentially by nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3 (NT3), respectively, to activate neuroprotective signals. The p75 receptors are activated by all neurotrophins, and paradoxically in neurodegenerative disease p75 is upregulated and mediates neurotoxic signals. To test neuroprotection strategies, we engineered NT3 to broadly activate Trk receptors (mutant D) or to reduce p75 binding (mutant RK). We also combined these features in a molecule that activates TrkA, TrkB, and TrkC but has reduced p75 binding (mutant DRK). In neurodegenerative disease mouse models in vivo, the DRK protein is a superior therapeutic agent compared with mutant D, mutant RK, and wild-type neurotrophins and protects a broader range of stressed neurons. This work rationalizes a therapeutic strategy based on the biology of each type of receptor, avoiding activation of p75 toxicity while broadly activating neuroprotection in stressed neuronal populations expressing different Trk receptors. SIGNIFICANCE STATEMENT: The neurotrophins nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3 each can activate a tropomyosin receptor kinase (Trk) A, TrkB, or TrkC receptor, respectively, and all can activate a p75 receptor. Trks and p75 mediate opposite signals. We report the engineering of a protein that activates all Trks, combined with low p75 binding, as an effective therapeutic agent in vivo.

Asunto(s)

Factores de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuroprotección/fisiología , Ingeniería de Proteínas/métodos , Receptor trkA/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Animales , Axotomía/efectos adversos , Neuropatías Diabéticas/tratamiento farmacológico , Neuropatías Diabéticas/genética , Neuropatías Diabéticas/metabolismo , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Factores de Crecimiento Nervioso/administración & dosificación , Factores de Crecimiento Nervioso/genética , Proteínas del Tejido Nervioso/genética , Neuroprotección/efectos de los fármacos , Nervio Óptico/efectos de los fármacos , Nervio Óptico/metabolismo , Receptor trkA/genética , Receptores de Factores de Crecimiento/genética

RESUMEN

Stromal-derived follicular dendritic cells (FDCs) are essential for germinal centers (GCs), the site where B cells maturate their antibodies. FDCs present native antigen to B cells and maintain a CXCL13 gradient to form the B cell follicle. Yet despite their essential role, the transcriptome of human FDCs remains undefined. Using single-cell RNA sequencing and microarray, we provided the transcriptome of these enigmatic cells as a comprehensive resource. Key genes were validated by flow cytometry and microscopy. Surprisingly, marginal reticular cells (MRCs) rather than FDCs expressed B cell activating factor (BAFF). Furthermore, we found that human FDCs expressed TLR4 and can alter antigen availability in response to pathogen-associated molecular patterns (PAMPs). High expression of PD-L1 and PD-L2 on FDCs activated PD1 on T cells. In addition, we found expression of genes related to T cell regulation, such as HLA-DRA, CD40, and others. These data suggest intimate contact between human FDCs and T cells.

Asunto(s)

Presentación de Antígeno , Linfocitos B/inmunología , Células Dendríticas Foliculares/fisiología , Inmunidad Adaptativa , Células Presentadoras de Antígenos/inmunología , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Cadenas alfa de HLA-DR/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células Jurkat , Proteína 2 Ligando de Muerte Celular Programada 1/genética , Proteína 2 Ligando de Muerte Celular Programada 1/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo

RESUMEN

Xenocoumacin (Xcn) 1 and 2 are the major antibiotics produced by the insect-pathogenic bacterium Xenorhabdus nematophila. Although the antimicrobial activity of Xcns has been explored, research regarding their action on mammalian cells is lacking. We aimed to investigate the action of Xcns in the context of inflammation and angiogenesis. We found that Xcns do not impair the viability of primary endothelial cells (ECs). Particularly Xcn2, but not Xcn1, inhibited the pro-inflammatory activation of ECs: Xcn2 diminished the interaction between ECs and leukocytes by downregulating cell adhesion molecule expression and blocked critical steps of the NF-κB activation pathway including the nuclear translocation of NF-κB p65 as well as the activation of inhibitor of κBα (IκBα) and IκB kinase ß (IKKß). Furthermore, the synthesis of pro-inflammatory mediators and enzymes, nitric oxide (NO) production and prostaglandin E2 (PGE2), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2), was evaluated in leukocytes. The results showed that Xcns reduced viability, NO release, and iNOS expression in activated macrophages. Beyond these anti-inflammatory properties, Xcn2 effectively hindered pro-angiogenic processes in HUVECs, such as proliferation, undirected and chemotactic migration, sprouting, and network formation. Most importantly, we revealed that Xcn2 inhibits de novo protein synthesis in ECs. Consequently, protein levels of receptors that mediate the inflammatory and angiogenic signaling processes and that have a short half-live are reduced by Xcn2 treatment, thus explaining the observed pharmacological activities. Overall, our research highlights that Xcn2 exhibits significant pharmacological in vitro activity regarding inflammation and angiogenesis, which is worth to be further investigated preclinically.

Asunto(s)

Inhibidores de la Angiogénesis/farmacología , Antiinflamatorios/farmacología , Benzopiranos/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Selectina E/genética , Selectina E/metabolismo , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Leucocitos/efectos de los fármacos , Leucocitos/fisiología , Ratones , FN-kappa B/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Receptores de Factores de Crecimiento/biosíntesis , Receptores de Factores de Crecimiento/genética , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismo

RESUMEN

Elevated glucocorticoid level in the early postnatal period is associated with glucocorticoid therapy prescribed at preterm delivery most often has severe long-lasting neurodevelopmental and behavioural effects. Detailed molecular mechanisms of such programming action of antenatal glucocorticoids on behaviour are still poorly understood. To address this question we studied neurotrophins: Bdnf, Nt-3, Ngf and their receptors: p75ngfr, Sorcs3 expression changes after subcutaneous dexamethasone (DEX) 0.2 mg/kg injection to P2 rat pups. Neurotrophins expression level was studied in the hippocampus (HPC). Disturbances in these brain regions have been implicated in the emergence of multiple psychopathologies. p75ngfr and Sorcs3 expression was studied in the brainstem-region where monoamine neurons are located. Immunohistochemically P75NTR protein level changes after DEX were investigated in the brainstem Locus Coereleus norepinephrine neurons (NE). In the first hours after DEX administration elevation of neurotrophins expression in HPC and decline of receptor's expression in the NE brainstem neurons were observed. Another critical time point during maturation is adolescence. Impact of elevated glucocorticoid level in the neonatal period and unpredictable stress (CMUS) at the end of adolescence on depressive-like behaviour was studied. Single neonatal DEX injection leads to decrease in depressive-like behaviour, observed in FST, independently from chronic stress. Neonatal DEX administration decreased Ntf3 and SorCS1 expression in the brainstem. Also Bdnf mRNA level in the brainstem of these animals didn't decrease after FST. CMUS at the end of adolescence changed p75ngfr and SorCS3 expression in the brainstem in the animals that received single neonatal DEX administration.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo/metabolismo , Depresión/etiología , Dexametasona/efectos adversos , Proteínas del Tejido Nervioso/metabolismo , Neurotrofina 3/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Animales , Animales Recién Nacidos , Tronco Encefálico/metabolismo , Factor Neurotrófico Derivado del Encéfalo/genética , Dexametasona/administración & dosificación , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Proteínas del Tejido Nervioso/genética , Neurotrofina 3/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores de Superficie Celular/genética , Receptores de Factores de Crecimiento/genética , Estrés Psicológico/etiología

RESUMEN

The nerve growth factor precursor (proNGF) activates p75NTR receptor and promotes cell death in different tissues, yet this pathophysiological effect is not fully described in the bladder. The aim of this study was to identify the biological effect of proNGF/p75NTR activation on urothelial and smooth muscle (SM) cells of rodents' bladder. Cell viability was assessed by MTT assay which showed a significant reduction in urothelial viability after 24 h of incubation with proNGF in culture medium [5 or 10 nM], an effect not seen in SM cells. Western blot analysis on cellular protein extracts showed increased expression of the transmembrane TNF-α and activation of RhoA in urothelial cells exposed to proNGF with no evidence of a nuclear translocation of NF-κB assessed by western blotting on nuclear extracts and immunofluorescence. The activation of p75NTR-death domain related pathways in urothelial cells such as TNF-α or RhoA had a downstream effect on NO release and the junctional protein occludin, as estimated respectively by colorimetric and western blotting. On the other hand, proNGF did not induce TNF-α or RhoA expression in SM cells, but induced a significant NF-κB nuclear translocation. ProNGF had a different impact on SM as evidenced by a significant dose- and time-dependent increase in SM proliferation and migration examined by MTT test and cell migration assay. Together, our results indicate that activation of proNGF/p75NTR axis induces degenerative changes to the urothelial layer impacting its barrier and signaling integrity, while promoting adaptive proliferative changes in detrusor SM cells that can interfere with the contractile phenotype essential for proper bladder function.

Asunto(s)

Miocitos del Músculo Liso/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Precursores de Proteínas/metabolismo , Transducción de Señal , Vejiga Urinaria/metabolismo , Urotelio/metabolismo , Animales , Movimiento Celular , Proliferación Celular , Femenino , Miocitos del Músculo Liso/patología , Factores de Crecimiento Nervioso/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Precursores de Proteínas/genética , Ratas , Ratas Sprague-Dawley , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento/metabolismo , Vejiga Urinaria/patología , Urotelio/patología

RESUMEN

Endosomal trafficking of receptors and associated proteins plays a critical role in signal processing. Until recently, it was thought that trafficking was shut down during cell division. Thus, remarkably, the regulation of trafficking during division remains poorly characterized. Here we delineate the role of mitotic kinases in receptor trafficking during asymmetric division. Targeted perturbations reveal that Cyclin-dependent Kinase 1 (CDK1) and Aurora Kinase promote storage of Fibroblast Growth Factor Receptors (FGFRs) by suppressing endosomal degradation and recycling pathways. As cells progress through metaphase, loss of CDK1 activity permits differential degradation and targeted recycling of stored receptors, leading to asymmetric induction. Mitotic receptor storage, as delineated in this study, may facilitate rapid reestablishment of signaling competence in nascent daughter cells. However, mutations that limit or enhance the release of stored signaling components could alter daughter cell fate or behavior thereby promoting oncogenesis.

Asunto(s)

Aurora Quinasas/fisiología , Proteína Quinasa CDC2/fisiología , Mitosis/fisiología , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Animales , Animales Modificados Genéticamente , Aurora Quinasas/genética , Proteína Quinasa CDC2/genética , Proteínas de Ciclo Celular/metabolismo , Ciona intestinalis/embriología , Ciona intestinalis/genética , Embrión no Mamífero , Mitosis/genética , Transporte de Proteínas/genética , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento/metabolismo , Transducción de Señal/genética , Distribución Tisular/genética

RESUMEN

Ovarian response of collared peccaries (Pecari tajacu), after hormonal stimulation with gonadotropin association (eCG/hCG), was accessed by both gene expression and follicular development. Thus, collared peccaries (n = 8) were treated with the dose used for sows (swine dose, SWD) or with dose adjusted for peccary's weight (allometric dose, ALD). The gene expression of receptors was evaluated for both gonadotropins (FSHR and LHCGR) and growth factors (proteins codified by TGFßR-1, BMPR1-A and BMPR2 genes) in antral follicles, cortex and corpora haemorrhagica (CH). Five days after gonadotropin injection, all females presented CH. The ovulation rate was similar (p > .05) between SWD (4.00 ± 1.17) and ALD (2.50 ± 0.43) group. The total number of follicles per animal and amounts of small (<3 mm), medium (3-5 mm) and large (>5 mm) follicles was similar among groups. However, SWD produced large follicles heavier than ALD group, as accessed by weight of follicular wall biopsies. Ovarian follicles expressed both gonadotropin and growth factor receptors at levels which are independent from gonadotropin dose. In conclusion, the two gonadotropin doses (SWD and ALD) can be used for ovarian stimulation of collared peccary. Additionally, FSH and growth factors (TGFßR-1, BMPR1-A and BMPR2) receptors are more expressed in the early follicle development, while LH receptor seems to be more important in the final of follicular growth.

Asunto(s)

Artiodáctilos/fisiología , Gonadotropina Coriónica/farmacología , Ovario/efectos de los fármacos , Animales , Peso Corporal , Gonadotropina Coriónica/administración & dosificación , Femenino , Folículo Ovárico/efectos de los fármacos , Ovulación/efectos de los fármacos , Receptores de Gonadotropina/genética , Receptores de Gonadotropina/metabolismo , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento/metabolismo

RESUMEN

OBJECTIVE: To investigate the effects of silencing P75 neurotrophin receptor (P75NTR) and nerve growth factor (NGF) overexpression on the proliferative activity and ectopic osteogenesis ability of bone marrow mesenchymal stem cells (BMSCs) combined with demineralized bone matrix for heterotopic osteogenesis. METHODS: BMSCs of Sprague Dawley (SD) rats were cultured and passaged by adherent isolation method. The third generation BMSCs were transfected with lentivirus mediated P75NTR gene silencing (group B), NGF overexpression gene (group C), P75NTR silencing and NGF overexpression double genes (group D), respectively, and untransfected cells as control (group A). After 7 days of transfection, the expression of fluorescent protein of the target gene was observed by fluorescence microscope; cell counting kit 8 method was used to detect the cells activity for 8 days after transfection; the expressions of P75NTR and NGF proteins in each group were detected by Western blot. The adhesion of BMSCs to demineralized bone matrix (DBM) was observed by inverted phase contrast microscope and scanning electron microscope after transfection of p75NTR silencing and NGF overexpression double genes. After transfection, BMSCs and DBM were co-cultured to prepare 4 groups of tissue engineered bone, which were respectively placed in the dorsal subcutaneous tissue of 8-week-old SD rats to construct subcutaneous ectopic osteogenesis model ( n=6). HE staining was performed at 4 and 8 weeks after operation. ALP staining was used to observe the formation of calcium nodules at 8 weeks after operation. The expressions of Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN) were detected by real-time fluorescent quantitative PCR. RESULTS: At 7 days after transfection, there was no fluorescence expression in group A, red fluorescence expression was seen in group B, green fluorescence expression in group C, and red-green compound fluorescence expression in group D. The fluorescence expression rate of target gene was about 70%. Western blot detection showed that the relative expression of P75NTR protein in groups A and C was significantly higher than that in groups B and D, and the relative expression of NGF protein in groups C and D was significantly higher than that in groups A and B ( P<0.05). With the passage of time, the cell proliferation activity increased in all groups, especially in group D, which was significantly higher than that in group A at 3-8 days ( P<0.05). The results of inverted phase contrast microscope and scanning electron microscope showed that BMSCs could adhere well to DBM. In the subcutaneous ectopic osteogenesis experiment, HE staining showed that at 4 and 8 weeks after operation, the more bone tissue was formed in group D than in the other 3 groups. ALP staining showed that group D had the highest ALP activity and better osteogenic expression. Compared with group A, the relative expressions of Runx2, ALP, and OCN mRNAs in group D were significantly higher than those in group A ( P<0.05). CONCLUSION: Silencing P75NTR and NGF overexpression double genes co-transfected BMSCs with DBM to construct tissue engineered bone has good ectopic osteogenic ability. By increasing NGF level and closing P75NTR apoptosis channel, it can not only improve cell activity, but also promote bone tissue regeneration.

Asunto(s)

Células Madre Mesenquimatosas , Proteínas del Tejido Nervioso , Receptores de Factores de Crecimiento , Animales , Células de la Médula Ósea , Matriz Ósea , Diferenciación Celular , Células Cultivadas , Silenciador del Gen , Lentivirus , Factor de Crecimiento Nervioso/genética , Factor de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Osteogénesis , Ratas , Ratas Sprague-Dawley , Receptor de Factor de Crecimiento Nervioso , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento/metabolismo , Transfección

RESUMEN

The carotid body may undergo plasticity changes during development/ageing and in response to environmental (hypoxia and hyperoxia), metabolic, and inflammatory stimuli. The different cell types of the carotid body express a wide series of growth factors and corresponding receptors, which play a role in the modulation of carotid body function and plasticity. In particular, type I cells express nerve growth factor, brain-derived neurotrophic factor, neurotrophin 3, glial cell line-derived neurotrophic factor, ciliary neurotrophic factor, insulin-like-growth factor-I and -II, basic fibroblast growth factor, epidermal growth factor, transforming growth factor-α and -ß, interleukin-1ß and -6, tumor necrosis factor-α, vascular endothelial growth factor, and endothelin-1. Many specific growth factor receptors have been identified in type I cells, indicating autocrine/paracrine effects. Type II cells may also produce growth factors and express corresponding receptors. Future research will have to consider growth factors in further experimental models of cardiovascular, metabolic, and inflammatory diseases and in human (normal and pathologic) samples. From a methodological point of view, microarray and/or proteomic approaches would permit contemporary analyses of large groups of growth factors. The eventual identification of physical interactions between receptors of different growth factors and/or neuromodulators could also add insights regarding functional interactions between different trophic mechanisms.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo/genética , Cuerpo Carotídeo/metabolismo , Hiperoxia/genética , Hipoxia/genética , Factor de Crecimiento Nervioso/genética , Receptores de Factores de Crecimiento/genética , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Cuerpo Carotídeo/citología , Factor Neurotrófico Ciliar/genética , Factor Neurotrófico Ciliar/metabolismo , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Regulación de la Expresión Génica , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Humanos , Hiperoxia/metabolismo , Hiperoxia/patología , Hipoxia/metabolismo , Hipoxia/patología , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Neurotrofina 3/genética , Neurotrofina 3/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador alfa/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

The tropomyosin-related kinase (Trk) family consists of three receptor tyrosine kinases (RTKs) called TrkA, TrkB, and TrkC. These RTKs are regulated by the neurotrophins, a class of secreted growth factors responsible for the development and function of neurons. The Trks share a high degree of homology and utilize overlapping signaling pathways, yet their signaling is associated with starkly different outcomes in certain cancers. For example, in neuroblastoma, TrkA expression and signaling correlates with a favorable prognosis, whereas TrkB is associated with poor prognoses. To begin to understand how activation of the different Trks can lead to such distinct cellular outcomes, we investigated differences in kinase activity and duration of autophosphorylation for the TrkA and TrkB tyrosine kinase domains (TKDs). We find that the TrkA TKD has a catalytic efficiency that is ∼2-fold higher than that of TrkB, and becomes autophosphorylated in vitro more rapidly than the TrkB TKD. Studies with mutated TKD variants suggest that a crystallographic dimer seen in many TrkA (but not TrkB) TKD crystal structures, which involves the kinase-insert domain, may contribute to this enhanced TrkA autophosphorylation. Consistent with previous studies showing that cellular context determines whether TrkB signaling is sustained (promoting differentiation) or transient (promoting proliferation), we also find that TrkB signaling can be made more transient in PC12 cells by suppressing levels of p75NTR. Our findings shed new light on potential differences between TrkA and TrkB signaling, and suggest that subtle differences in signaling dynamics can lead to substantial shifts in the cellular outcome.

Asunto(s)

Neuroblastoma/metabolismo , Receptor trkA/metabolismo , Receptor trkB/metabolismo , Transducción de Señal/genética , Animales , Factor Neurotrófico Derivado del Encéfalo/farmacología , Dominio Catalítico , Diferenciación Celular/genética , Proliferación Celular/genética , Técnicas de Silenciamiento del Gen , Cinética , Mutación , Factores de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuroblastoma/enzimología , Neuroblastoma/genética , Células PC12 , Fosforilación , Dominios Proteicos , ARN Interferente Pequeño , Ratas , Receptor trkA/química , Receptor trkA/genética , Receptor trkB/química , Receptor trkB/genética , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento/metabolismo , Proteínas Recombinantes , Transducción de Señal/efectos de los fármacos

RESUMEN

OBJECTIVE: Pharmacological inhibition of GABAergic synapses could represent a potent neuromodulation strategy to activate hippocampal neurons and increase neurotrophic factor gene expression, thus exerting a beneficial effect on post-stroke cognitive impairment (PSCI). The objective of this study was to assess the effects of low-level inhibition of GABAergic synapses on hippocampal gene expressions related to neuroplasticity using the middle cerebral artery occlusion surgery (MCAO) ischemic stroke rat model. METHODS: The animals were randomly assigned to three experimental groups-(1) a sham operated group (SHAM), (2) a control group (CON), and (3) a bicuculline group (BIC). MCAO was performed in the CON and BIC groups. A non-epileptic dose of bicuculline (0.25 mg/kg) was intraperitoneally administered every day for two weeks, starting three days after surgery, to the rats in the BIC group. The mRNA expression of brain-derived neurotrophic factor (BDNF), tropomyosin-related kinase B (TrkB), in relation to neurotrophic intracellular signal, p75, in relation to apoptosis, and synaptophysin (SYP) and PSD-95, synaptic markers, were assessed in the hippocampus ipsilateral to the ischemic site. RESULTS: MCAO increased the gene expression of TrkB. Furthermore, MCAO plus bicuculline administration increased the expression ratio of TrkB to p75 and SYP gene expression. CONCLUSION: Therefore, this study showed that administration of bicuculline after stroke beneficially modulated the expression of crucial genes for neuroplasticity, including BDNF receptors and SYP, in the ipsilateral hippocampus, suggesting that low-level inhibition of GABAergic synapses could lead to beneficial neuromodulation in the hippocampus after stroke.

Asunto(s)

Bicuculina/farmacología , Antagonistas de Receptores de GABA-A/farmacología , Neuronas GABAérgicas/efectos de los fármacos , Hipocampo/efectos de los fármacos , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/genética , Inhibición Neural/efectos de los fármacos , Plasticidad Neuronal/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Modelos Animales de Enfermedad , Homólogo 4 de la Proteína Discs Large/genética , Homólogo 4 de la Proteína Discs Large/metabolismo , Neuronas GABAérgicas/metabolismo , Neuronas GABAérgicas/patología , Regulación de la Expresión Génica , Hipocampo/metabolismo , Hipocampo/patología , Hipocampo/fisiopatología , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/fisiopatología , Masculino , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Ratas Sprague-Dawley , Receptor trkB/genética , Receptor trkB/metabolismo , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento/metabolismo , Sinaptofisina/genética , Sinaptofisina/metabolismo

RESUMEN

SARS-CoV-2 infections are rapidly spreading around the globe. The rapid development of therapies is of major importance. However, our lack of understanding of the molecular processes and host cell signaling events underlying SARS-CoV-2 infection hinders therapy development. We use a SARS-CoV-2 infection system in permissible human cells to study signaling changes by phosphoproteomics. We identify viral protein phosphorylation and define phosphorylation-driven host cell signaling changes upon infection. Growth factor receptor (GFR) signaling and downstream pathways are activated. Drug-protein network analyses revealed GFR signaling as key pathways targetable by approved drugs. The inhibition of GFR downstream signaling by five compounds prevents SARS-CoV-2 replication in cells, assessed by cytopathic effect, viral dsRNA production, and viral RNA release into the supernatant. This study describes host cell signaling events upon SARS-CoV-2 infection and reveals GFR signaling as a central pathway essential for SARS-CoV-2 replication. It provides novel strategies for COVID-19 treatment.

Asunto(s)

Antivirales/uso terapéutico , Betacoronavirus/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/genética , Fosfatidilinositol 3-Quinasa/genética , Receptores de Factores de Crecimiento/genética , Proteínas Virales/genética , Corticoesteroides/uso terapéutico , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Anticuerpos Neutralizantes/uso terapéutico , Betacoronavirus/inmunología , Betacoronavirus/patogenicidad , Células CACO-2 , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/genética , Humanos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Receptores de Factores de Crecimiento/antagonistas & inhibidores , Receptores de Factores de Crecimiento/metabolismo , SARS-CoV-2 , Transducción de Señal , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacos

RESUMEN

The neurotrophin receptor p75NTR plays crucial roles in neuron development and regulates important neuronal processes like degeneration, apoptosis and cell survival. At the same time the detailed mechanism of signal transduction is unclear. One of the main hypotheses known as the snail-tong mechanism assumes that in the inactive state, the death domains interact with each other and in response to ligand binding there is a conformational change leading to their exposure. Here, we show that neither rat nor human p75NTR death domains homodimerize in solution. Moreover, there is no interaction between the death domains in a more native context: the dimerization of transmembrane domains in liposomes and the presence of activating mutation in extracellular juxtamembrane region do not lead to intracellular domain interaction. These findings suggest that the activation mechanism of p75NTR should be revised. Thus, we propose a novel model of p75NTR functioning based on interaction with "helper" protein.

Asunto(s)

Mutación , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Receptores de Factores de Crecimiento/química , Receptores de Factores de Crecimiento/metabolismo , Receptores de Factor de Crecimiento Nervioso/química , Receptores de Factor de Crecimiento Nervioso/metabolismo , Animales , Humanos , Ligandos , Liposomas/metabolismo , Modelos Moleculares , Proteínas del Tejido Nervioso/genética , Unión Proteica , Conformación Proteica , Dominios Proteicos , Multimerización de Proteína , Ratas , Receptores de Factores de Crecimiento/genética , Receptores de Factor de Crecimiento Nervioso/genética

RESUMEN

Rupture of weakened blood vessels could lead to severe intracerebral hemorrhage (ICH) and brain injuries. This study was designed to explore the roles of p75 neurotrophin receptor (p75NTR) in neuronal autophagy in ICH rats. An ICH rat model was established, and then gain and loss of functions of p75NTR in rat tissues were performed. Then, the pathologic morphology, water content, and inflammation in brain tissues were assessed. Western blot analysis was applied to detect the levels of inflammatory proteins, apoptosis- and autophagy-related proteins, and the mammalian target of rapamycin (mTOR) pathway-related proteins. Neuronal autophagy was further measured with mTOR activated. In vitro experiments were also performed on brain microvascular endothelial cells (BMECs) and astrocytes. Consequently, we found p75NTR knockdown improved the pathologic morphology with reduced neuron damage, water content, permeability of blood-brain barrier and inflammation in ICH rat brain tissues. Besides, Knockdown of p75NTR decreased neuronal apoptosis and inactivated mTOR signaling pathway, but it elevated the levels of autophagy-related proteins. In vivo results were reproduced in in vitro experiments. This study demonstrated that knockdown of p75NTR could promote neuronal autophagy and reduce neuronal apoptosis via inactivating the mTOR pathway. We hope these findings could provide new therapeutic options for ICH treatment.

Asunto(s)

Autofagia/genética , Hemorragia Cerebral/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Apoptosis/genética , Astrocitos/metabolismo , Barrera Hematoencefálica/metabolismo , Células Cultivadas , Hemorragia Cerebral/enzimología , Hemorragia Cerebral/genética , Hemorragia Cerebral/patología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Técnicas de Silenciamiento del Gen , Inflamación/genética , Inflamación/metabolismo , Masculino , Proteínas del Tejido Nervioso/genética , Neuronas/enzimología , Neuronas/patología , Ratas , Ratas Sprague-Dawley , Receptores de Factores de Crecimiento/genética , Transducción de Señal/genética , Regulación hacia Arriba

RESUMEN

Chronic obstructive pulmonary disease (COPD) is characterised by an accelerated decline in airway function with age compared to age-matched non-smokers. There is increasing evidence that this is due to small airway disease rather than from emphysema, especially in the early stages of the disease. Small airways (<2 mm internal diameter) are narrowed in COPD with thickening and distortion of the airway wall and peribronchiolar fibrosis. In addition, loss of elasticity in alveolar attachments and mucus hypersecretion contribute to the airway narrowing and closure, leading to air trapping. The mechanisms of peribronchiolar fibrosis are poorly understood and small airway fibroblasts have not been characterised. In small airways of COPD patients the fibroblasts are profibrotic, pro-inflammatory and senescent. There is a reduction in the anti-ageing molecules sirtuin-1 and -6, which are regulated by specific microRNAs that are increased in COPD cells. It is plausible that extracellular vesicles from senescent airway epithelium transmit senescent signals to airway fibroblasts to stimulate fibrosis and inflammation. Small airways fibrosis is a target for new drug development that inhibit growth factor receptors, new antioxidants and particularly those that are targeted to mitochondria and inhibitors of cellular senescence or senolytic therapies.

Asunto(s)

Enfermedad Pulmonar Obstructiva Crónica/genética , Enfisema Pulmonar/genética , Fibrosis Pulmonar/genética , Sistema Respiratorio/metabolismo , Sirtuina 1/genética , Sirtuinas/genética , Antiinflamatorios/uso terapéutico , Antioxidantes/uso terapéutico , Senescencia Celular , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Vesículas Extracelulares/efectos de los fármacos , Vesículas Extracelulares/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Regulación de la Expresión Génica , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/patología , Enfisema Pulmonar/tratamiento farmacológico , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/patología , Fibrosis Pulmonar/tratamiento farmacológico , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento/metabolismo , Sistema Respiratorio/efectos de los fármacos , Sistema Respiratorio/patología , Transducción de Señal , Sirtuina 1/metabolismo , Sirtuinas/metabolismo

RESUMEN

Retinoic acid receptor (RAR) signaling plays an important role in embryonic development and homeostasis of many tissues. At the cellular level, activation of RAR signaling often induces cell cycle arrest, differentiation, and apoptosis in many types of cells. Consequently, loss of normal RAR function in the presence of physiological levels of retinoic acid (RA) is often observed in cancers, and pharmacological reactivation of RAR signaling has been considered a promising strategy for cancer therapy and prevention. One of important mechanisms that regulate RAR activity in cancer cells is cross-talk with growth factor signaling, where activation of extracellular signal-regulated kinase (ERK) plays a major role in suppressing RAR transcriptional activity downstream of growth factor receptors. Conversely, strong activation of RAR can induce suppression of ERK activity by inducing expression of a phosphatase specific for ERK to exert tumor-suppressive activity in colorectal cancer. Here, we describe the basic methods to analyze interactions between RAR and ERK signaling in colorectal cancer cells.

Asunto(s)

Neoplasias Colorrectales/genética , Receptores de Factores de Crecimiento/genética , Receptores de Ácido Retinoico/metabolismo , Células CACO-2 , Neoplasias Colorrectales/metabolismo , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Sistema de Señalización de MAP Quinasas