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1.
Genes Dev ; 34(3-4): 194-208, 2020 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-31919191

RESUMEN

Promoting axon regeneration in the central and peripheral nervous system is of clinical importance in neural injury and neurodegenerative diseases. Both pro- and antiregeneration factors are being identified. We previously reported that the Rtca mediated RNA repair/splicing pathway restricts axon regeneration by inhibiting the nonconventional splicing of Xbp1 mRNA under cellular stress. However, the downstream effectors remain unknown. Here, through transcriptome profiling, we show that the tubulin polymerization-promoting protein (TPPP) ringmaker/ringer is dramatically increased in Rtca-deficient Drosophila sensory neurons, which is dependent on Xbp1. Ringer is expressed in sensory neurons before and after injury, and is cell-autonomously required for axon regeneration. While loss of ringer abolishes the regeneration enhancement in Rtca mutants, its overexpression is sufficient to promote regeneration both in the peripheral and central nervous system. Ringer maintains microtubule stability/dynamics with the microtubule-associated protein futsch/MAP1B, which is also required for axon regeneration. Furthermore, ringer lies downstream from and is negatively regulated by the microtubule-associated deacetylase HDAC6, which functions as a regeneration inhibitor. Taken together, our findings suggest that ringer acts as a hub for microtubule regulators that relays cellular status information, such as cellular stress, to the integrity of microtubules in order to instruct neuroregeneration.


Asunto(s)
Anilidas/metabolismo , Axones/fisiología , Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Ácidos Hidroxámicos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Regeneración/genética , Animales , Proteínas de Drosophila/genética , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/genética , Unión Proteica , Empalme del ARN/genética , Células Receptoras Sensoriales/fisiología
2.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(1): 52-56, 2020 Jan 10.
Artículo en Chino | MEDLINE | ID: mdl-31922597

RESUMEN

OBJECTIVE: To delineate the clinical features,inheritance pattern, and genotype-phenotype correlation of a Chinese patient with a 17q25.3 duplication. METHODS: Whole exome sequencing(WES), chromosomal microarray analysis (CMA), chromosomal karyotyping and fluorescence in situ hybridization (FISH) were employed for the analysis of the proband and his family members. RESULTS: A 5.7 Mb duplication at 17q25.3→qter was identified by WES and CMA in the 4-year-old boy with multiple congenital anomalies, which was classified as a clinically pathogenic variant. This duplication was confirmed by FISH, and was inherited from his unaffected mother who carried a balanced translocation. Further study revealed that his grandmother also carried the balanced translocation but had gestated three healthy children and had no abortion history. His uncle also carried the balanced translocation, while his aunt was normal. CONCLUSION: Above results have enriched the clinical phenotypes of 17q25.3 duplication. Genetic counseling was provided for the family. P4HB, ACTG1, BAIAP2 and TBCD genes may underlie the clinical features for the 17q25.3 duplication.


Asunto(s)
Anomalías Múltiples , Duplicación Cromosómica , Cromosomas Humanos Par 17 , Discapacidades del Desarrollo , Anomalías Múltiples/genética , Adulto , Preescolar , China , Cromosomas Humanos Par 17/genética , Discapacidades del Desarrollo/genética , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Masculino , Proteínas Asociadas a Microtúbulos , Translocación Genética
3.
Toxicol Lett ; 320: 95-102, 2020 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-31760062

RESUMEN

Exposure to organic solvent in industry, including n-hexane is correlated with central-peripheral axonopathy, which is mediated by its active metabolite, 2,5-hexanedione (HD). However, the underlying mechanism is still largely unknown. Recently identified microRNAs (miRNAs) may play important roles in toxicant exposure and in the process of toxicant-induced neuropathys. To examine the role of miRNAs in HD-induced toxicity, neuropathic animal model was successfully built. miRNA microarray analysis revealed 105 differentially expressed miRNAs after HD exposure. Bioinformatics analysis showed that "Axon" and "Neurotrophin Signaling Pathway" was the top significant GO term and pathway, respectively. 7 miRNAs both related to "Axon" and "Neurotrophin Signaling Pathway" were screened out and further confirmed by Real-Time PCR. Correspondingly, the deregulation expression levels of proteins of four target genes (GSK3ß, Map3k1, BDNF and MAP1B) were further confirmed via western blot, verifying the results of gene target analysis. Taken together, our results showed that the axon-related miRNAs to be associated with MAP1B or neurotrophin signal pathways changed in nerve tissues following HD exposure. These miRNAs may play important roles in HD-induced neurotoxicity.


Asunto(s)
Axones/efectos de los fármacos , Hexanonas/toxicidad , MicroARNs/metabolismo , Síndromes de Neurotoxicidad/etiología , Nervio Ciático/efectos de los fármacos , Solventes/toxicidad , Médula Espinal/efectos de los fármacos , Animales , Axones/metabolismo , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Bases de Datos Genéticas , Regulación de la Expresión Génica , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Quinasa 1 de Quinasa de Quinasa MAP/genética , Quinasa 1 de Quinasa de Quinasa MAP/metabolismo , Masculino , MicroARNs/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Síndromes de Neurotoxicidad/genética , Síndromes de Neurotoxicidad/metabolismo , Ratas Sprague-Dawley , Nervio Ciático/metabolismo , Transducción de Señal , Médula Espinal/metabolismo , Transcriptoma
4.
Nan Fang Yi Ke Da Xue Xue Bao ; 39(10): 1141-1148, 2019 Oct 30.
Artículo en Chino | MEDLINE | ID: mdl-31801720

RESUMEN

OBJECTIVE: To explore the molecular mechanism underlying the inhibitory effects of aspirin against human breast cancer cell proliferation through bioinformatics analysis. METHODS: Drug Bank 5.1.3 was searched to identify direct protein targets (DPTs) of aspirin, and the protein-protein interaction (PPI) network of the DPTs was constructed online using STRING and the signaling pathways involved were identified. The genetic alterations of 6 DPTs associated with human breast cancer was analyzed and visualized by cBio Portal and OncoPrint, respectively. The transcriptomic data of breast cancer and normal tissues were downloaded from TCGA database, and the overexpressed genes were analyzed by DECenter. The intersection between the genes associated with the DPTs obtained by STRING analysis and the differentially over-expressed genes in TCGA was determined to confirm the candidate DPTs as a potential target of aspirin, and GO functional enrichment analysis was performed using Gene Ontology. The potential targets of aspirin against the proliferation of human breast cancer cells were verified by Western blotting. RESULTS: Eleven DPTs of aspirin were identified. KEGG pathway enrichment indicated that 6 genes (EDNRA, IKBKB, NFKB2, NFKBIA, PTGS2 and TP53) were associated with the occurrence and development of cancer. A total of 10 220 differentially expressed genes were identified from the TCGA database, and among them 4 genes (CDC25C, TPX2, CDC20, PLK1) were found to be the potential targets for aspirin. These genes were involved mostly in the regulation of cell cycle and cell division. Western blotting showed that aspirin could down-regulate the expression levels of several pivotal proteins that regulated cell cycle and cell division, including CDC25C, TPX2, CDC20 and PLK1. CONCLUSIONS: CDC25C, TPX2, CDC20 and PLK1 may be potential targets for aspirin to inhibit the proliferation of human breast cancer cells, by affecting the progress of cell cycle and cell division.


Asunto(s)
Aspirina/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Biología Computacional , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas Cdc20 , Proteínas de Ciclo Celular , Proliferación Celular , Perfilación de la Expresión Génica , Humanos , Proteínas Asociadas a Microtúbulos , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas , Fosfatasas cdc25
5.
Anticancer Res ; 39(12): 6585-6593, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31810924

RESUMEN

BACKGROUND/AIM: Human lung adenocarcinoma PC14 cells without mutations in the epidermal growth factor receptor (EGFR) are less sensitive to gefitinib than PC9 cells with EGFR mutations. We report the involvement of tetrandrine in autophagy flux as a mechanism that enhances the sensitivity of PC14 cells to gefitinib. MATERIALS AND METHODS: Sensitivity to gefitinib was determined by a growth inhibition assay, and quantitative real-time PCR, western blotting, and fluorescent immunostaining were used to detect autophagy. RESULTS: In PC14 cells, combined treatment with gefitinib and tetrandrine caused a significant increase in gefitinib sensitivity and autophagy-related mRNAs and proteins (LC3, etc.), and the LC3 protein accumulated in lysosomes. Furthermore, an autophagy flux assay revealed that tetrandrine inhibited lysosomes and that gefitinib promoted autophagy. Finally, the sensitivity of PC14 cells to gefitinib was enhanced with chloroquine. CONCLUSION: Tetrandrine possibly increases the susceptibility of PC14 cells to gefitinib by lysosomal inhibition.


Asunto(s)
Adenocarcinoma del Pulmón/metabolismo , Bencilisoquinolinas/farmacología , Gefitinib/farmacología , Neoplasias Pulmonares/metabolismo , Lisosomas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/genética , Autofagia , Bencilisoquinolinas/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Receptores ErbB/genética , Gefitinib/química , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Lisosomas/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/genética
6.
Anticancer Res ; 39(12): 6621-6633, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31810927

RESUMEN

BACKGROUND/AIM: The antidepressant duloxetine is known as a serotonin-norepinephrine reuptake inhibitor, used for treating depression and anxiety. TRAIL selectively induces cell death in a variety of tumor cells by binding to its membrane death receptor (DR). The aim of the study was to examine whether duloxetine affects TRAIL-mediated apoptosis. MATERIALS AND METHODS: Cell viability and apoptosis was measured by morphological image, crystal violet staining, MTT and LDH assay. Immunocytochemistry and western blotting techniques were applied to detect autophagy and apoptosis indicator proteins. TEM assay was used to determine the autophagy. RESULTS: Duloxetine treatment considerably sensitizes human lung adenocarcinoma cells to TRAIL-mediated apoptosis by targeting TRAIL-DR5. Treatment with duloxetine inhibited AMPK phosphorylation and resulted in increased p62 and microtubule-associated protein 1A/1B light chain 3B-II levels, indicating inhibition of autophagy flux. Blockade of DR5 with DR5-specific small-interfering RNA negatively regulated the apoptotic effect. CONCLUSION: Clinical administration of TRAIL in combination with duloxetine may serve as a therapeutic approach for the treatment of TRAIL-resistant lung cancer cells.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Apoptosis/efectos de los fármacos , Clorhidrato de Duloxetina/farmacología , Neoplasias Pulmonares , Inhibidores de Captación de Serotonina y Norepinefrina/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , L-Lactato Deshidrogenasa/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Proteínas Asociadas a Microtúbulos/metabolismo , Fosforilación/efectos de los fármacos , ARN Interferente Pequeño , Proteínas de Unión al ARN/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/antagonistas & inhibidores , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Regulación hacia Arriba
7.
Anticancer Res ; 39(12): 6711-6722, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31810936

RESUMEN

BACKGROUND/AIM: Autophagy is a cellular mechanism that recycles cellular components to maintain homeostasis. To investigate the clinical implication of autophagy in gastric cancer, the autophagy markers with autophagosome formation, LC3B and selective autophagy substrate p62/SQSTM1 (P62) were validated. MATERIALS AND METHODS: LC3B and p62 expression was examined using immunohistochemistry, western blot assays, and reverse-transcription polymerase chain reaction (RT-PCR). The relationship of LC3B and p62 expression in gastric adenocarcinomas with clinicopathological parameters, including patient survival, were analyzed. RESULTS: Normal gastric mucosae exhibit no LC3B and p62 expression, while tubular adenoma and gastric adenocarcinomas exhibit variable nuclear or cytoplasmic p62 expression. High LC3B, high cytoplasmic p62, and low nuclear p62 protein expression in gastric adenocarcinomas is positively correlated with poor prognostic factors including survival. CONCLUSION: Dynamic LC3B and p62 changes are suggested to be involved in gastric tumorigenesis and cancer progression. LC3B and p62 could be used as prognostic biomarkers and potential therapeutic targets for gastric adenocarcinomas.


Asunto(s)
Adenocarcinoma/metabolismo , Adenoma/metabolismo , Biomarcadores de Tumor/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína Sequestosoma-1/metabolismo , Neoplasias Gástricas/metabolismo , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Adenoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Femenino , Mucosa Gástrica/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Neoplasias Gástricas/mortalidad , Neoplasias Gástricas/patología , Adulto Joven
8.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(11): 973-978, 2019 Nov.
Artículo en Chino | MEDLINE | ID: mdl-31878992

RESUMEN

Objective To investigate the autophagy and the expression of CD40 and CD80 of dendritic cells (DCs) induced by hypoxia and lipopolysaccharide (LPS). Methods DCs were divided into normal group, hypoxia group, lipopolysaccharide (LPS) treatment group and hypoxia combined with LPS treatment group. Each group was treated for 24 hours, and the cell proliferation was detected by MTT assay. The protein levels of autophagy microtubule-associated protein 1 light chain 3I (LC3I), LC3II and beclin1 were detected by Western blot analysis. The formation of autophagosomes in DCs was observed under a transmission electron microscope after LPS treatment. The expression of CD40 and CD80 on DCs was detected by flow cytometry and immunofluorescence cytochemical staining. Results Hypoxia and LPS could induce autophagy in mouse DCs and increase the expression of CD40 and CD80 on their surface. After hypoxia combined with LPS treatment, autophagy in DCs was significantly enhanced; more autophagosomes were formed; and the expression of co-stimulatory molecules CD40 and CD80 were raised. Conclusion Hypoxia and LPS can induce autophagy and increase the expression of CD40 and CD80 in mouse DCs.


Asunto(s)
Autofagia , Antígeno B7-1/metabolismo , Antígenos CD40/metabolismo , Células Dendríticas/citología , Animales , Beclina-1/metabolismo , Hipoxia de la Célula , Células Cultivadas , Lipopolisacáridos , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo
9.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(11): 1030-1034, 2019 Nov.
Artículo en Chino | MEDLINE | ID: mdl-31879000

RESUMEN

Objective To detect the mRNA and protein expression of microtubule-associated protein 1 light chain 3 (LC3) in peripheral blood mononuclear cells (PBMCs) of patients with rheumatoid arthritis (RA), and to investigate its relationship with RA. Methods Twenty-two patients with RA and 16 healthy subjects with matching gender and age as controls were included in the study. PBMCs were isolated by density gradient centrifugation. The level of LC3 mRNA in PBMCs was detected by real-time fluorescent quantitative PCR. The protein level of LC3 in PBMCs was detected by Western blot analysis. The expression of LC3 protein in PBMCs was detected by immunofluorescence staining. Pearson analysis was used to analyze the correlation between LC3 expression and clinical parameters of RA patients. Results Compared with the normal control group, the levels of LC3 mRNA and protein in PBMCs of RA patients went up significantly, and the expression of LC3 significantly increased in PBMCs. The mRNA expression level of LC3 was obviously positively correlated with erythrocyte sedimentation rate (ESR, r=0.7480), 28 joint disease activity (DAS28, r=0.5016), C-reactive protein (CRP, r=0.6518), and rheumatoid factor (RF, r=0.7232). Conclusion The expression of LC3 is up-regulated in RA patients and is associated with ESR, DAS28, CRP and RF.


Asunto(s)
Artritis Reumatoide/metabolismo , Leucocitos Mononucleares/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Artritis Reumatoide/patología , Sedimentación Sanguínea , Proteína C-Reactiva/análisis , Humanos , ARN Mensajero , Factor Reumatoide/análisis
10.
Nan Fang Yi Ke Da Xue Xue Bao ; 39(11): 1337-1343, 2019 Nov 30.
Artículo en Chino | MEDLINE | ID: mdl-31852639

RESUMEN

OBJECTIVE: To investigate the role of the protein-serine-threonine kinase (AKT)/glycogen synthase kinase 3ß (GSK3ß) signaling pathway in nicotinic acetylcholine receptors (nAChRs) aggregation disorder on skeletal muscle cell membranes induced by sepsis. METHODS: Mouse C2C12 myoblasts were differentiated into myotubes by horse serum, and then C2C12 myotubes were randomly divided into four groups: the Sham group treated with serum from sham-operated mice, the Sepsis group treated with serum from septic mice, the Sepsis+D group treated with serum from septic mice and dimethyl sulfoxide (DMSO), the Sepsis+SB group treated with serum from septic mice and GSK3ß inhibitor SB216763. Agrin was added into the cell culture to induce nAChRs aggregation before the treatment. After serum treatment for 5.5 h, the myotubes were examined for nAChRs clusters using Alexa Fluor 594-conjugated α-bungarotoxin (α- BTX). The expression levels of AKT, GSK3ß and CLIP- associated protein 2 (CLASP2) and the phosphorylation of AKT, GSK3ß were examined with Western blotting. The phosphorylation of CLASP2 and the interaction between CLASP2 and α-tubulin were detected with co-immunoprecipitation (Co-IP) assay. RESULTS: Compared with the serum from sham-operated mice, the serum from septic mice caused significant reduction in the area and density of nAChRs clusters on C2C12 myotubes, lowered the levels of phosphorylated AKT (p-AKT) and phosphorylated GSK3ß (p-GSK3ß), increased the expression of phosphorylated CLASP2 (p-CLASP2), and obviously reduced the binding between CLASP2 and α-tubulin. Compared with DMSO, SB216763 significantly increased the area and density of nAChRs clusters on C2C12 myotubes treated with serum from septic mice, decreased the expression of p-CLASP2, and enhanced the interaction between CLASP2 and α-tubulin. CONCLUSIONS: Septic mouse serum impairs nAChRs aggregation on C2C12 myotubes possibly by suppressing AKT/GSK3ß phosphorylation to cause reduced interaction between CLASP2 and α-tubulin.


Asunto(s)
Sepsis , Animales , Membrana Celular , Glucógeno Sintasa Quinasa 3 beta , Ratones , Proteínas Asociadas a Microtúbulos , Fibras Musculares Esqueléticas , Fosforilación , Proteínas Proto-Oncogénicas c-akt , Receptores Nicotínicos
11.
Zhonghua Bing Li Xue Za Zhi ; 48(11): 856-860, 2019 Nov 08.
Artículo en Chino | MEDLINE | ID: mdl-31775434

RESUMEN

Objective: To investigate the expression of LC3B, p-AMPKα and p27 in cortical tuberous sclerosis complex (TSC). Methods: Nineteen specimens of surgically resected TSC cortical tubers were collected at Xuanwu Hospital, Capital Medical University, from 2014 to 2017. The expression of the three proteins in the lesions and the adjacent relatively normal regions was detected by immunohistochemical staining (EnVision two-step method). Results: LC3B was mainly expressed in the dysmorphic neuron and giant cell in TSC cortical tubers and in the adjacent relatively normal neurons, and the expression was diffuse or perinuclear cytoplasmic. There was no significant difference in the average optical density between abnormal cells and neurons adjacent to the lesions (0.343±0.195 vs. 0.419±0.088, P>0.05). p-AMPKα was localized in the cytoplasm of dysmorphic neurons and giant cell in TSC cortical tubers. The average optical density of abnormal cells in the lesions was significantly higher than that of neurons adjacent to the lesions (0.306±0.123 vs. 0.233±0.654, P<0.05). P27 showed nuclear positivity, mainly expressed in the neurons and glial cells close to TSC cortical tubers, while the positive rate in the abnormal cells in TSC cortical tubers was low (15/19 vs. 7/19, P<0.05). Conclusion: There is no significant decrease in the level of autophagy in dysmorphic neurons and giant cells in TSC cortical tubers, which may be related to the compensatory mechanism of AMPK signaling pathway, but without activation of downstream p27.


Asunto(s)
Proteínas Relacionadas con la Autofagia/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Quinasas/metabolismo , Esclerosis Tuberosa/metabolismo , Corteza Cerebral/patología , Humanos , Neuroglía/metabolismo , Neuronas/metabolismo
12.
Nat Neurosci ; 22(12): 1961-1965, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31768057

RESUMEN

The exome sequences of approximately 8,000 children with autism spectrum disorder (ASD) and/or attention deficit hyperactivity disorder (ADHD) and 5,000 controls were analyzed, finding that individuals with ASD and individuals with ADHD had a similar burden of rare protein-truncating variants in evolutionarily constrained genes, both significantly higher than controls. This motivated a combined analysis across ASD and ADHD, identifying microtubule-associated protein 1A (MAP1A) as a new exome-wide significant gene conferring risk for childhood psychiatric disorders.


Asunto(s)
Trastorno por Déficit de Atención con Hiperactividad/genética , Trastorno del Espectro Autista/genética , Predisposición Genética a la Enfermedad/genética , Variación Genética/genética , Proteínas Asociadas a Microtúbulos/genética , Trastorno por Déficit de Atención con Hiperactividad/complicaciones , Trastorno del Espectro Autista/complicaciones , Estudios de Casos y Controles , Exoma/genética , Femenino , Humanos , Masculino
13.
J Photochem Photobiol B ; 201: 111653, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31710929

RESUMEN

Autophagy is an important process for maintaining intracellular homeostasis. Our previous study demonstrated that autophagy was down-regulated in ultraviolet B (UVB)-irradiated keratinocytes. Raffinose is a natural oligosaccharide that serves as a novel activator of autophagy and as a balancing agent to regulate the diversity of environmental stress. However, whether raffinose balances ultraviolet stress through the autophagy activation pathway has yet to be established. In this study, we found that raffinose treatment inhibited the LDH release and trypan blue staining in UVB-challenged human keratinocytes cell line HaCaT but did not affect the cleavage of apoptotic markers Caspase-3 and PARP, as well as translocation into nucleus of other cell death markers Endonuclease G and AIF. Moreover, we confirmed that raffinose treatment enhanced autophagy flux in an MTOR-independent manner in HaCaT cells. Importantly, decrease of LC3-II turnover in UVB-irradiated keratinocytes could be rescued by raffinose treatment, indicating that raffinose treatment increased autophagy in UVB-irradiated HaCaT cells. Furthermore, the effect on cell death by raffinose was inhibited when autophagy was suppressed with either a small interfering RNA targeting ATG5 (siATG5) or autophagic inhibitor wortmannin. In conclusion, we demonstrated that raffinose increases MTOR-independent autophagy and reduces cell death in UVB-irradiated keratinocytes. Our study indicated that the natural agent raffinose presents the potential value in opposing photodamage.


Asunto(s)
Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Rafinosa/farmacología , Rayos Ultravioleta , Apoptosis/efectos de la radiación , Autofagia/efectos de la radiación , Proteína 5 Relacionada con la Autofagia/antagonistas & inhibidores , Proteína 5 Relacionada con la Autofagia/genética , Proteína 5 Relacionada con la Autofagia/metabolismo , Caspasa 3/metabolismo , Línea Celular , Humanos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo
14.
Medicine (Baltimore) ; 98(48): e18228, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31770286

RESUMEN

BACKGROUND: Dynamin-related protein 1 (Drp1) plays important roles in tumorigenesis, including lung cancer. However, the effect of Drp1 in lung cancer remains unclear. The present study was aimed to investigate the clinical significance and effect of Drp1 on prognosis of lung cancer. METHODS: Oncomine and The Cancer Genome Atlas (TCGA) databases were selected to predict the differential expression levels of Drp1 in lung cancer. Then, 70 cases of lung cancer and normal tissues were collected and immunohistochemistry was used to detect the expression of Drp1. In addition, Kaplan-Meier Plotter database and TCGA database were used to verify the correlation between Drp1 expression and the clinical prognosis in lung cancer patients. RESULTS: Drp1 was significantly overexpressed in lung cancer tissues based on Oncomine and TCGA databases (P < .05). Moreover, results from immunohistochemistry showed that Drp1 protein level in lung cancer was also significantly higher than that in the matched normal tissues (P < .05). Prognostic analysis from Kaplan-Meier Plotter database with the chosen probe IDs of 203105_s_at suggested that Drp1 was negatively correlated to overall survival (OS) of lung cancer patients (HR = 1.16, 95% CI: 1.02-1.31; P = .025), but not in the probe IDs of 226154_at (HR = 0.86, 95% CI: 0.73-1.01; P = .069). However, prognosis from TCGA database showed inconsistent results in which high expression of Drp1 was correlated with worse survival probability of all, male, female in lung adenocarcinoma (P < .05), but not in LUSC (P > .05). CONCLUSION: Drp1 was highly expressed in lung cancer based on bioinformatics analysis and tissue microarray, but there was a lot of inconsistency in prognosis depending on different levels of Drp1 from the bioinformatics analysis.


Asunto(s)
GTP Fosfohidrolasas , Neoplasias Pulmonares , Proteínas Asociadas a Microtúbulos , Proteínas Mitocondriales , China/epidemiología , Biología Computacional/métodos , Femenino , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/epidemiología , Neoplasias Pulmonares/genética , Masculino , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Persona de Mediana Edad , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Pronóstico
15.
Life Sci ; 237: 116944, 2019 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-31604108

RESUMEN

AIMS: Endoplasmic reticulum stress (ERS) is an evolutionarily conserved cell stress response. Recently, it was found that ERS induces not only apoptosis but also endoplasmic reticulophagy (ER-phagy). A previous study demonstrated that inhibition of ER-phagy alleviates cell injury. The purpose of this study was to investigate the involvement of the protein kinase R-like ER kinase (PERK)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in ERS-induced ER-phagy in H9c2 cardiomyoblasts. To address this aim, cells were treated with ERS inhibitors and a Nrf2 inhibitor before establishment of thapsigargin (TG)- or tunicamycin (TM)-induced ERS models in H9c2 cardiomyoblasts. MAIN METHODS: Transmission electron microscopy and immunofluorescence staining were used to detect ER-phagy. Western blotting was employed to detect the levels of calreticulin (CRT), total and phosphorylated PERK, nuclear Nrf2, activated transcription factor 4 (ATF4), light chain 3B (LC3B)-II and Beclin 1. Immunofluorescence staining was used to assess subcellular location of Nrf2. KEY FINDING: TG or TM induced H9c2 cell injury and ER-phagy and upregulated CRT expression, PERK phosphorylation, Nrf2 nuclear translocation, and expression of ATF4, Beclin 1, and LC3B-II compared with control cells. Treatment with ERS inhibitors decreased TG- or TM-induced ER-phagy, downregulated CRT expression, PERK phosphorylation, Nrf2 nuclear translocation and the expression of ATF4, Beclin 1 and LC3B-II. Moreover, a Nrf2 inhibitor downregulated the expression of ATF4, Beclin 1 and LC3B-II and alleviated TG- or TM-induced ER-phagy and H9c2 cell injury. SIGNIFICANCE: These findings suggest that the PERK/Nrf2 pathway mediates upregulation of ER-phagy, thereby inducing cell injury in H9c2 cardiomyoblasts.


Asunto(s)
Apoptosis , Estrés del Retículo Endoplásmico , Retículo Endoplásmico/patología , Miocitos Cardíacos/patología , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo , eIF-2 Quinasa/metabolismo , Factor de Transcripción Activador 4/metabolismo , Animales , Beclina-1/metabolismo , Células Cultivadas , Retículo Endoplásmico/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Miocitos Cardíacos/metabolismo , Fosforilación , Ratas , Transducción de Señal
16.
Cell Physiol Biochem ; 53(5): 747-759, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31622062

RESUMEN

BACKGROUND/AIMS: Angiotensin II (Ang II) induces podocyte injury resulting in apoptosis in vitro and in vivo. However, the relationship between autophagy and apoptosis in Ang II-induced podocyte injury is unknown and the role of Ang II-induced autophagy in podocyte survival or death remains unclear. We investigated the sequential relationship between autophagy and apoptosis in Ang II-induced podocytes as well as the role of phosphatidylinositide 3-kinase (PI3-kinase). METHODS: Mouse podocytes were incubated in media containing various concentrations of Ang II and at different incubation times. The changes of podocyte autophagy and apoptosis were observed by electron microscopy, confocal imaging, western blotting, and FACS assay according to the presence of Ang II. RESULTS: Ang II enhanced the podocyte expression of the autophagic proteins, LC3A/B-II and beclin-1, and also increased the number of autophagosomes compared with control cells at early phase of 12 hours in a dose-dependent manner. This effect was inhibited by pretreatment with 3-methyladenine (3-MA), a PI3-kinase class III inhibitor. Thereafter, the Ang II-induced enhancement in autophagy decreased, whereas, podocyte apoptosis appeared later at 24 hours in concentration- and time-dependent manners in FACS and TUNEL assays. 3-MA and LY294002, a pan PI3-kinase inhibitor, further increased Ang II-induced podocyte apoptosis. Suppression of autophagy by Atg5 siRNA could induce podocyte apoptosis and further augment high-dose Ang II-induced podocyte apoptosis. CONCLUSION: These findings suggest that Ang II promotes autophagy in podocytes before apoptosis as an early adaptive cytoprotective mechanism for podocyte survival after Ang II treatment, and the transitional imbalance between autophagy and apoptosis causes podocyte injury.


Asunto(s)
Angiotensina II/farmacología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Adenina/análogos & derivados , Adenina/farmacología , Animales , Autofagosomas/metabolismo , Proteína 5 Relacionada con la Autofagia/antagonistas & inhibidores , Proteína 5 Relacionada con la Autofagia/genética , Proteína 5 Relacionada con la Autofagia/metabolismo , Beclina-1/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Podocitos/citología , Podocitos/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Regulación hacia Arriba/efectos de los fármacos
17.
Plant Sci ; 289: 110248, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31623783

RESUMEN

Microtubule arrays play notable roles in cell division, cell movement, cell morphogenesis and signal transduction. Due to their important regulation of microtubule dynamic instability and array-ordering processes, microtubule-associated proteins have been a cutting-edge issue in research. Here, a new maize microtubule-associated protein, ZmGLR (Zea mays glutamic acid- and lysine-rich), was found. ZmGLR bundles microtubules in vitro and targets the cell membrane through an interaction between 24 conserved N-terminal amino acids and specific phosphatidylinositol phosphates (PtdInsPs). Increased Ca2+ levels in the cytoplasm lead to ZmGLR partially dissociating from the cell membrane and moving into the cytoplasm to associate with microtubule. Overexpression and RNAi of ZmGLR both resulted in misoriented microtubule arrays, which led to dwarf maize plants and curved leaves. In addition, the expression of ZmGLR was regulated by BR and auxin through ZmBES1 and ZmARF9, respectively. This study reveals that the microtubule-associated protein ZmGLR plays a crucial role in cortical microtubule reorientation and maize leaf morphogenesis.


Asunto(s)
Proteínas Asociadas a Microtúbulos/genética , Hojas de la Planta/crecimiento & desarrollo , Proteínas de Plantas/genética , Zea mays/genética , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Filogenia , Hojas de la Planta/genética , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alineación de Secuencia , Zea mays/crecimiento & desarrollo , Zea mays/metabolismo
18.
Cell Physiol Biochem ; 53(4): 656-686, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31573152

RESUMEN

BACKGROUND/AIMS: Despite recent advances in melanoma drug discovery, the average overall survival of patients with late stage metastatic melanoma is approximately 3 years, suggesting a need for approaches that identify new melanoma targets. We have previously reported a discovery of novel anti-melanoma compound 2155-14 (Onwuha-Ekpete et al., J Med Chem. 2014 Feb 27; 57(4):1599-608). In the report presented herein we aim to identify its target(s) and mechanism of action. METHODS: We utilized biotinylated analog of 2155-14 to pull down its targets from melanoma cells. Proteomics in combination with western blot were used to identify the targets. Mechanism of action of 2155-14 was determined using flow cytometry, RT-PCR, microscopy, western blot, and enzymatic activity assays. Where applicable, one-way analysis of variance (ANOVA) was used followed by Dunnett post hoc test. RESULTS: In the present study, we identified ATP-dependent RNA helicase DDX1 and heterogeneous nuclear ribonucleoproteins (hnRNPs) H1, H2 and A2/B1 as targets of anti-melanoma compound 215514. To the best of our knowledge, this is a first report suggesting that these proteins could be targeted for melanoma therapy. Mechanistic investigations showed that 2155-14 induces ER stress leading to potentiation of basal autophagy resulting in melanoma cell death in BRAF and NRAS mutated melanoma cells. CONCLUSION: Identification of mode of action of 2155-14 may provide insight into novel therapies against a broad range of melanoma subtypes. These studies were enabled by the novel probe derived from a mixture-based library, an important class of chemical biology tools for discovering novel targets.


Asunto(s)
Apoptosis , Autofagia , ARN Helicasas DEAD-box/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Línea Celular Tumoral , ARN Helicasas DEAD-box/antagonistas & inhibidores , ARN Helicasas DEAD-box/genética , Evaluación Preclínica de Medicamentos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Ribonucleoproteínas Nucleares Heterogéneas/antagonistas & inhibidores , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Humanos , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Melanoma/patología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal/efectos de los fármacos
19.
BMC Complement Altern Med ; 19(1): 274, 2019 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-31638961

RESUMEN

BACKGROUND: Recent research has suggested that autophagy can provide a better mechanism for inducing cell death than current therapeutic strategies. This study investigated the effects of using an ethanol extract of Chrysanthemum zawadskii Herbich (ECZ) to induce apoptosis and autophagy associated with reliable signal pathways in mouse colon cancer CT-26 cells. METHODS: Using ECZ on mouse colon cancer CT-26 cells, cell viability, annexin V/propidium iodide staining, acridine orange staining, reactive oxygen species (ROS) and western blotting were assayed. RESULTS: ECZ exhibited cytotoxicity in CT-26 cells in a dose-dependent manner. ECZ induced apoptosis was confirmed by caspase-3 activation, poly (ADP-ribose) polymerase cleavage, and increased production of reactive oxygen species (ROS). Furthermore, it was shown that ECZ induced autophagy via the increased conversion of microtubule-associated protein 1 light chain 3II, the degradation of p62, and the formation of acidic vesicular organelles. The inhibition of ROS production by N-Acetyl-L-cysteine resulted in reduced ECZ-induced apoptosis and autophagy. Furthermore, the inhibition of autophagy by 3-methyladenine resulted in enhanced ECZ-induced apoptosis via increased ROS generation. CONCLUSION: These findings confirmed that ECZ induced ROS-mediated autophagy and apoptosis in colon cancer cells. Therefore, ECZ may serve as a novel potential chemotherapeutic candidate for colon cancer.


Asunto(s)
Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Chrysanthemum/química , Neoplasias del Colon/fisiopatología , Extractos Vegetales/farmacología , Especies Reactivas de Oxígeno/metabolismo , Animales , Caspasa 3/genética , Caspasa 3/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Extractos Vegetales/aislamiento & purificación , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo
20.
APMIS ; 127(12): 746-752, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31520423

RESUMEN

Meningiomas are common intracranial tumors, and most exhibit optimal prognosis; however, some meningiomas still recur and even develop malignant transformation in the following years, regardless of initial pathological grade. During these years, autophagy raises its significance in tumorigenesis and tumor suppression, both important for tumor development. The aim of this study was to elucidate the relationship between two autophagy markers, LC3B and beclin 1, with clinical and pathological parameters in patients with meningiomas. A total of 77 thin-sectioned slides, retrospectively collected from meningioma patients, were analyzed and correlated with clinicopathological parameters. We found that expression of beclin 1 rather than LC3B correlated to better prognosis, lower pathological grade, and longer survival. Furthermore, intensity of beclin 1 was also found to be significantly related to the pathological grade. These findings indicated that beclin 1 as a protective factor predicts better prognosis and plays the role of tumor suppression in meningiomas.


Asunto(s)
Beclina-1/genética , Beclina-1/metabolismo , Neoplasias Meníngeas/fisiopatología , Meningioma/fisiopatología , Femenino , Humanos , Inmunohistoquímica , Masculino , Neoplasias Meníngeas/metabolismo , Neoplasias Meníngeas/mortalidad , Meningioma/metabolismo , Meningioma/mortalidad , Proteínas Asociadas a Microtúbulos/metabolismo , Persona de Mediana Edad , Pronóstico , Estudios Retrospectivos , Tasa de Supervivencia
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