RESUMEN
Wounding is one of the most common healthcare problems. Bioactive hydrogels have attracted much attention in first-aid hemostasis and wound healing due to their excellent biocompatibility, antibacterial properties, and pro-healing bioactivity. However, their applications are limited by inadequate mechanical properties. In this study, we first prepared edible rose-derived exosome-like nanoparticles (ELNs) and used them to encapsulate antimicrobial peptides (AMP), abbreviated as ELNs(AMP). ELNs(AMP) showed superior intracellular antibacterial activity, 2.5 times greater than AMP, in in vitro cell infection assays. We then prepared and tested an FDA-approved fibrin-gel of fibrinogen and thrombin encapsulating ELNs(AMP) and novobiocin sodium salt (NB) (ELNs(AMP)/NB-fibrin-gels). The fibrin gel showed a sustained release of ELNs(AMP) and NB over the eight days of testing. After spraying onto the skin, the formulation underwent in situ gelation and developed a stable patch with excellent hemostatic performance in a mouse liver injury model with hemostasis in 31 s, only 35.6 % of the PBS group. The fibrin gel exhibited pro-wound healing properties in the mouse-infected skin defect model. The thickness of granulation tissue and collagen of the ELNs(AMP)/NB-fibrin-gels group was 4.00, 6.32 times greater than that of the PBS group. In addition, the ELNs(AMP)/NB-fibrin-gels reduced inflammation (decreased mRNA levels of TNF-α, IL-1ß, IL6, MCP1, and CXCL1) at the wound sites and demonstrated a biocompatible and biosafe profile. Thus, we have developed a hydrogel system with excellent hemostatic, antibacterial, and pro-wound healing properties, which may be a candidate for next-generation tissue regeneration with a wide clinical application for first-aid hemostasis and infected wound healing.
Asunto(s)
Antibacterianos , Exosomas , Fibrina , Hemostasis , Cicatrización de Heridas , Cicatrización de Heridas/efectos de los fármacos , Animales , Hemostasis/efectos de los fármacos , Ratones , Fibrina/química , Antibacterianos/farmacología , Antibacterianos/química , Exosomas/metabolismo , Péptidos Antimicrobianos/química , Péptidos Antimicrobianos/farmacología , Humanos , Infección de Heridas/tratamiento farmacológico , Nanopartículas/química , Geles/química , Hidrogeles/química , Hidrogeles/farmacología , MasculinoRESUMEN
The lysosome-targeting chimera (LYTAC) approach has shown promise for the targeted degradation of secreted and membrane proteins via lysosomes. However, there have been challenges in design, development, and targeting. Here, we have designed a genetically engineered transferrin receptor (TfR)-mediated lysosome-targeting chimera (TfR-LYTAC) that is efficiently internalized via TfR-mediate endocytosis and targets PD-L1 for lysosomal degradation in cultured cells but not in vivo due to short half-life and poor tumor targeting. A delivery platform was developed by fusing TfR-LYTAC to the surface of bacterial outer membrane vesicles (OMVs). The engineered OMV-LYTAC combines PD-1/PD-L1 pathway inhibition with LYTAC and immune activation by bacterial OMVs. OMV-LYTAC significantly reduced tumor growth in vivo. We have provided a modular and simple genetic strategy for lysosomal degradation as well as a delivery platform for in vivo tumor targeting. The study paves the way for the targeting and degradation of extracellular proteins using the TfR-LYTAC system.
Asunto(s)
Inmunoterapia , Lisosomas , Receptores de Transferrina , Receptores de Transferrina/metabolismo , Receptores de Transferrina/inmunología , Lisosomas/metabolismo , Humanos , Animales , Ratones , Membrana Externa Bacteriana/metabolismo , Femenino , Línea Celular Tumoral , Ratones Endogámicos BALB C , Antígeno B7-H1/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/químicaRESUMEN
Phosphatidic acid (PA), the simplest phospholipid, acts as a key metabolic intermediate and second messenger that impacts diverse cellular and physiological processes across species ranging from microbes to plants and mammals. The cellular levels of PA dynamically change in response to stimuli, and multiple enzymatic reactions can mediate its production and degradation. PA acts as a signalling molecule and regulates various cellular processes via its effects on membrane tethering, enzymatic activities of target proteins, and vesicular trafficking. Because of its unique physicochemical properties compared to other phospholipids, PA has emerged as a class of new lipid mediators influencing membrane structure, dynamics, and protein interactions. This review summarizes the biosynthesis, dynamics, and cellular functions and properties of PA.
RESUMEN
D-2-Hydroxyglutarate (D-2HG) is an α-ketoglutarate-derived mitochondrial metabolite that causes D-2-hydroxyglutaric aciduria, a devastating developmental disorder. How D-2HG adversely affects mitochondria is largely unknown. Here, we report that in Caenorhabditis elegans, loss of the D-2HG dehydrogenase DHGD-1 causes D-2HG accumulation and mitochondrial damage. The excess D-2HG leads to a build-up of 3-hydroxypropionate (3-HP), a toxic metabolite in mitochondrial propionate oxidation, by inhibiting the 3-HP dehydrogenase HPHD-1. We demonstrate that 3-HP binds the MICOS subunit MIC60 (encoded by immt-1) and inhibits its membrane-binding and membrane-shaping activities. We further reveal that dietary and gut bacteria affect mitochondrial health by modulating the host production of 3-HP. These findings identify a feedback loop that links the toxic effects of D-2HG and 3-HP on mitochondria, thus providing important mechanistic insights into human diseases related to D-2HG and 3-HP.
Asunto(s)
Encefalopatías Metabólicas Innatas , Propionatos , Encefalopatías Metabólicas Innatas/metabolismo , Retroalimentación , Glutaratos/metabolismo , Humanos , Isocitrato Deshidrogenasa/metabolismo , Mitocondrias/metabolismo , Oxidorreductasas , Propionatos/metabolismoRESUMEN
The effectors of the Rab7 small GTPase play multiple roles in Rab7-dependent endosome-lysosome and autophagy-lysosome pathways. However, it is largely unknown how distinct Rab7 effectors coordinate to maintain the homeostasis of late endosomes and lysosomes to ensure appropriate endolysosomal and autolysosomal degradation. Here we report that WDR91, a Rab7 effector required for early-to-late endosome conversion, is essential for lysosome function and homeostasis. Mice lacking Wdr91 specifically in the central nervous system exhibited behavioral defects and marked neuronal loss in the cerebral and cerebellar cortices. At the cellular level, WDR91 deficiency causes PtdIns3P-independent enlargement and dysfunction of lysosomes, leading to accumulation of autophagic cargoes in mouse neurons. WDR91 competes with the VPS41 subunit of the HOPS complex, another Rab7 effector, for binding to Rab7, thereby facilitating Rab7-dependent lysosome fusion in a controlled manner. WDR91 thus maintains an appropriate level of lysosome fusion to guard the normal function and survival of neurons.
Asunto(s)
Autofagia , Corteza Cerebelosa/enzimología , Corteza Cerebral/enzimología , Lisosomas/metabolismo , Fusión de Membrana , Neuronas/enzimología , Proteínas de Unión al GTP rab/metabolismo , Animales , Conducta Animal , Corteza Cerebelosa/ultraestructura , Corteza Cerebral/ultraestructura , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lisosomas/ultraestructura , Proteínas de la Membrana/metabolismo , Ratones Noqueados , Microscopía Confocal , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/metabolismo , Actividad Motora , Neuronas/ultraestructura , Fosfatos de Fosfatidilinositol/metabolismo , Unión Proteica , Transporte de Proteínas , Proteolisis , Proteína Sequestosoma-1/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
Lysosomes are degradation and signaling organelles that adapt their biogenesis to meet many different cellular demands; however, it is unknown how lysosomes change their numbers for cell division. Here, we report that the cyclin-dependent kinases CDK4/6 regulate lysosome biogenesis during the cell cycle. Chemical or genetic inactivation of CDK4/6 increases lysosomal numbers by activating the lysosome and autophagy transcription factors TFEB and TFE3. CDK4/6 interact with and phosphorylate TFEB/TFE3 in the nucleus, thereby inactivating them by promoting their shuttling to the cytoplasm. During the cell cycle, lysosome numbers increase in S and G2/M phases when cyclin D turnover diminishes CDK4/6 activity. These findings not only uncover the molecular events that direct the nuclear export of TFEB/TFE3, but also suggest a mechanism that controls lysosome biogenesis in the cell cycle. CDK4/6 inhibitors promote autophagy and lysosome-dependent degradation, which has important implications for the therapy of cancer and lysosome-related disorders.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Núcleo Celular/enzimología , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Lisosomas/enzimología , Biogénesis de Organelos , Transporte Activo de Núcleo Celular , Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Ciclo Celular , Núcleo Celular/genética , Proliferación Celular , Ciclina D1/metabolismo , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/genética , Células HCT116 , Células HeLa , Células Hep G2 , Humanos , Lisosomas/genética , Fosforilación , Proteolisis , Transducción de SeñalRESUMEN
Alzheimer disease (AD) is the most common neurodegenerative disease. An imbalance between the production and clearance of Aß (amyloid beta) is considered to be actively involved in AD pathogenesis. Macroautophagy/autophagy is a major cellular pathway leading to the removal of aggregated proteins, and upregulation of autophagy represents a plausible therapeutic strategy to combat overproduction of neurotoxic Aß. PPARA/PPARα (peroxisome proliferator activated receptor alpha) is a transcription factor that regulates genes involved in fatty acid metabolism and activates hepatic autophagy. We hypothesized that PPARA regulates autophagy in the nervous system and PPARA-mediated autophagy affects AD. We found that pharmacological activation of PPARA by the PPARA agonists gemfibrozil and Wy14643 induces autophagy in human microglia (HM) cells and U251 human glioma cells stably expressing the human APP (amyloid beta precursor protein) mutant (APP-p.M671L) and this effect is PPARA-dependent. Administration of PPARA agonists decreases amyloid pathology and reverses memory deficits and anxiety symptoms in APP-PSEN1ΔE9 mice. There is a reduced level of soluble Aß and insoluble Aß in hippocampus and cortex tissues from APP-PSEN1ΔE9 mice after treatment with either gemfibrozil or Wy14643, which promoted the recruitment of microglia and astrocytes to the vicinity of Aß plaques and enhanced autophagosome biogenesis. These results indicated that PPARA is an important factor regulating autophagy in the clearance of Aß and suggested gemfibrozil be assessed as a possible treatment for AD.Abbreviation: Aß: amyloid beta; ACTB: actin beta; ADAM10: ADAM metallopeptidase domain 10; AD: Alzheimer disease; AIF1/IBA1: allograft inflammatory factor 1; ANOVA: analysis of variance; APOE: apolipoprotein E; APP: amyloid beta precursor protein; APP-PSEN1ΔE9: APPswe/PSEN1dE9; BAFA1: bafilomycin A1; BDNF: brain derived neurotrophic factor; BECN1: beclin 1; CD68: CD68 molecule; CREB1: cAMP responsive element binding protein 1; DAPI: 4',6-diamidino-2-phenylindole; DLG4/PSD-95: discs large MAGUK scaffold protein 4; DMSO: dimethyl sulfoxide; ELISA: enzyme linked immunosorbent assay; FDA: U.S. Food and Drug Administration; FKBP5: FK506 binding protein 5; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; gemfibrozil: 5-(2,5-dimethylphenoxy)-2,2-dimethylpentanoic acid; GFAP: glial fibrillary acidic protein; GLI2/THP1: GLI family zinc finger 2; HM: human microglia; IL6: interleukin 6; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; NC: negative control; OQ: opposite quadrant; PPARA/PPARα, peroxisome proliferator activated receptor alpha; PSEN1/PS1: presenilin 1; SEM: standard error of the mean; SQSTM1: sequestosome 1; SYP: synaptophysin; TFEB: transcription factor EB; TNF/TNF-α: tumor necrosis factor; TQ: target quadrant; WT: wild type; Wy14643: 2-[4-chloro-6-(2,3-dimethylanilino)pyrimidin-2-yl]sulfanylacetic acid.
Asunto(s)
Enfermedad de Alzheimer/patología , Autofagia/fisiología , PPAR alfa/metabolismo , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Autofagia/genética , Disfunción Cognitiva/metabolismo , Modelos Animales de Enfermedad , Humanos , Ratones , Microglía/metabolismo , Enfermedades Neurodegenerativas/metabolismo , PPAR alfa/genética , Placa Amiloide/metabolismoRESUMEN
Alzheimer's disease is the most common neurodegenerative disease, and has a high level of genetic heritability and population heterogeneity. In this study, we performed the whole-exome sequencing of Han Chinese patients with familial and/or early-onset Alzheimer's disease, followed by independent validation, imaging analysis and function characterization. We identified an exome-wide significant rare missense variant rs3792646 (p.K420Q) in the C7 gene in the discovery stage (P = 1.09 × 10-6, odds ratio = 7.853) and confirmed the association in different cohorts and a combined sample (1615 cases and 2832 controls, Pcombined = 2.99 × 10-7, odds ratio = 1.930). The risk allele was associated with decreased hippocampal volume and poorer working memory performance in early adulthood, thus resulting in an earlier age of disease onset. Overexpression of the mutant p.K420Q disturbed cell viability, immune activation and ß-amyloid processing. Electrophysiological analyses showed that the mutant p.K420Q impairs the inhibitory effect of wild type C7 on the excitatory synaptic transmission in pyramidal neurons. These findings suggested that C7 is a novel risk gene for Alzheimer's disease in Han Chinese.
RESUMEN
Depression is one of the most frequent psychiatric symptoms observed in people during the development of Alzheimer's disease (AD). We hypothesized that genetic factors conferring risk of depression might affect AD development. In this study, we screened 31 genes, which were located in 19 risk loci for major depressive disorder (MDD) identified by two recent large genome-wide association studies (GWAS), in AD patients at the genomic and transcriptomic levels. Association analysis of common variants was performed by using summary statistics of the International Genomics of Alzheimer's Project (IGAP), and association analysis of rare variants was conducted by sequencing the entire coding region of the 31 MDD risk genes in 107 Han Chinese patients with early-onset and/or familial AD. We also quantified the mRNA expression alterations of these MDD risk genes in brain tissues of AD patients and AD mouse models, followed by protein-protein interaction network prediction to show their potential effects in AD pathways. We found that common and rare variants of L3MBTL2 were significantly associated with AD. mRNA expression levels of 18 MDD risk genes, in particular SORCS3 and OAT, were differentially expressed in AD brain tissues. 13 MDD risk genes were predicted to physically interact with core AD genes. The involvement of HACE1, NEGR1, and SLC6A15 in AD was supported by convergent lines of evidence. Taken together, our results showed that MDD risk genes might play an active role in AD pathology and supported the notion that depression might be the "common cold" of psychiatry.
Asunto(s)
Enfermedad de Alzheimer/genética , Trastorno Depresivo Mayor/genética , Predisposición Genética a la Enfermedad , Proteínas Nucleares/genética , Polimorfismo de Nucleótido Simple/genética , Factores de Transcripción/genética , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/patología , Sistemas de Transporte de Aminoácidos Neutros/genética , Animales , Encéfalo/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Trastorno Depresivo Mayor/complicaciones , Femenino , Proteínas Ligadas a GPI/genética , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Ratones , Proteínas del Tejido Nervioso/genética , Mapas de Interacción de Proteínas , ARN Mensajero/metabolismo , Receptores de Superficie Celular , Receptores de Neuropéptido/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
INTRODUCTION: Profiling the spatial-temporal expression pattern and characterizing the regulatory networks of brain tissues are vital for understanding the pathophysiology of Alzheimer's disease (AD). METHODS: We performed a systematic integrated analysis of expression profiles of AD-affected brain tissues (684 AD and 562 controls). A network-based convergent functional genomic approach was used to prioritize possible regulator genes during AD development, followed by functional characterization. RESULTS: We generated a complete list of differentially expressed genes and hub genes of the transcriptomic network in AD brain and constructed a Web server (www.alzdata.org) for public access. Seventeen hub genes active at the early stages, especially YAP1, were recognized as upstream regulators of the AD network. Cellular assays proved that early alteration of YAP1 could promote AD by influencing the whole transcriptional network. DISCUSSION: Early expression disturbance of hub genes is an important feature of AD development, and interfering with this process may reverse the disease progression.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/fisiopatología , Encéfalo/metabolismo , Expresión Génica/fisiología , Redes Reguladoras de Genes/fisiología , Fosfoproteínas/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Enfermedad de Alzheimer/diagnóstico por imagen , Enfermedad de Alzheimer/patología , Encéfalo/diagnóstico por imagen , Bases de Datos Genéticas/estadística & datos numéricos , Progresión de la Enfermedad , Femenino , Genómica , Humanos , Masculino , Fosfoproteínas/metabolismo , Factores de Transcripción , Proteínas Señalizadoras YAPRESUMEN
RNA editing was first discovered in mitochondrial RNA molecular. However, whether adenosine-to-inosine (A-to-I) RNA editing has functions in nuclear genes involved in mitochondria remains elusive. Here, we retrieved 707,246 A-to-I RNA editing sites in Macaca mulatta leveraging massive transcriptomes of 30 different tissues and genomes of nine tissues, together with the reported data, and found that A-to-I RNA editing occurred frequently in nuclear genes that have functions in mitochondria. The mitochondrial structure, the level of ATP production, and the expression of some key genes involved in mitochondrial function were dysregulated after knocking down the expression of ADAR1 and ADAR2, the key genes encoding the enzyme responsible for RNA editing. When investigating dynamic changes of RNA editing during brain development, an amino-acid-changing RNA editing site (I234/V) in MFN1, a mediator of mitochondrial fusion, was identified to be significantly correlated with age, and could influence the function of MFN1. When studying transcriptomes of brain disorder, we found that dysregulated RNA editing sites in autism were also enriched within genes having mitochondrial functions. These data indicated that RNA editing had a significant function in mitochondria via their influence on nuclear genes.
RESUMEN
The immune response is highly active in Alzheimer's disease (AD). Identification of genetic risk contributed by immune genes to AD may provide essential insight for the prognosis, diagnosis, and treatment of this neurodegenerative disease. In this study, we performed a genetic screening for AD-related top immune genes identified in Europeans in a Chinese cohort, followed by a multiple-stage study focusing on Complement Factor H (CFH) gene. Effects of the risk SNPs on AD-related neuroimaging endophenotypes were evaluated through magnetic resonance imaging scan, and the effects on AD cerebrospinal fluid biomarkers (CSF) and CFH expression changes were measured in aged and AD brain tissues and AD cellular models. Our results showed that the AD-associated top immune genes reported in Europeans (CR1, CD33, CLU, and TREML2) have weak effects in Chinese, whereas CFH showed strong effects. In particular, rs1061170 (P(meta)=5.0 × 10(-4)) and rs800292 (P(meta)=1.3 × 10(-5)) showed robust associations with AD, which were confirmed in multiple world-wide sample sets (4317 cases and 16 795 controls). Rs1061170 (P=2.5 × 10(-3)) and rs800292 (P=4.7 × 10(-4)) risk-allele carriers have an increased entorhinal thickness in their young age and a higher atrophy rate as the disease progresses. Rs800292 risk-allele carriers have higher CSF tau and Aß levels and severe cognitive decline. CFH expression level, which was affected by the risk-alleles, was increased in AD brains and cellular models. These comprehensive analyses suggested that CFH is an important immune factor in AD and affects multiple pathological changes in early life and during disease progress.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/inmunología , Encéfalo/patología , Predisposición Genética a la Enfermedad , Anciano , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Pueblo Asiatico , China , Factor H de Complemento/genética , Factor H de Complemento/metabolismo , Endofenotipos , Femenino , Pruebas Genéticas , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , ARN Mensajero/metabolismo , Factores de RiesgoRESUMEN
Autophagy is involved in the pathogenesis of neurodegenerative diseases including Parkinson disease (PD). However, little is known about the regulation of autophagy in neurodegenerative process. In this study, we characterized aberrant activation of autophagy induced by neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) and demonstrated that melatonin has a protective effect on neurotoxicity. We found an excessive activation of autophagy in monkey brain tissues and C6 cells, induced by MPTP, which is mediated by CDK5 (cyclin-dependent kinase 5). MPTP treatment significantly reduced total dendritic length and dendritic complexity of cultured primary cortical neurons and melatonin could reverse this effect. Decreased TH (tyrosine hydroxylase)-positive cells and dendrites of dopaminergic neurons in the substantia nigra pars compacta (SNc) were observed in MPTP-treated monkeys and mice. Along with decreased TH protein level, we observed an upregulation of CDK5 and enhanced autophagic activity in the striatum of mice with MPTP injection. These changes could be salvaged by melatonin treatment or knockdown of CDK5. Importantly, melatonin or knockdown of CDK5 reduced MPTP-induced SNCA/α-synuclein aggregation in mice, which is widely thought to trigger the pathogenesis of PD. Finally, melatonin or knockdown of CDK5 counteracted the PD phenotype in mice induced by MPTP. Our findings uncover a potent role of CDK5-mediated autophagy in the pathogenesis of PD, and suggest that control of autophagic pathways may provide an important clue for exploring potential target for novel therapeutics of PD.
Asunto(s)
1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Autofagia/efectos de los fármacos , Quinasa 5 Dependiente de la Ciclina/metabolismo , Neuronas Dopaminérgicas/efectos de los fármacos , Melatonina/farmacología , alfa-Sinucleína/metabolismo , Animales , Autofagia/fisiología , Modelos Animales de Enfermedad , Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Haplorrinos , Ratones , Neurotoxinas/farmacología , Enfermedad de Parkinson/metabolismoRESUMEN
Recent advances in nanomedicine provide promising alternatives for cancer treatment that may improve the survival of patients with metastatic disease. The goal of the present study was to evaluate graphene oxide (GO) as a potential anti-metastatic agent. For this purpose, GO was modified with polyethylene glycol (PEG) to form PEG-modified GO (PEG-GO), which improves its aqueous stability and biocompatibility. We show here that PEG-GO exhibited no apparent effects on the viability of breast cancer cells (MDA-MB-231, MDA-MB-436, and SK-BR-3) or non-cancerous cells (MCF-10A), but inhibited cancer cell migration in vitro and in vivo. Analysis of cellular energy metabolism revealed that PEG-GO significantly impaired mitochondrial oxidative phosphorylation (OXPHOS) in breast cancer cells; however, PEG-GO showed no effect on OXPHOS in non-cancerous cells. To explore the underlying mechanisms, a SILAC (Stable Isotope Labeling by Amino acids in Cell culture) labeling strategy was used to quantify protein expression in PEG-GO-exposed breast cancer versus non-cancerous cells. The results indicated that PEG-GO selectively down-regulated PGC-1α in breast cancer cells and thus modified the expression of diverse energy generation-related proteins, which accounts for the inhibition of OXPHOS. The inhibition of OXPHOS by PEG-GO significantly reduced ATP production and impaired assembly of the F-actin cytoskeleton in breast cancer cells, which is required for the migratory and invasive phenotype of cancer cells. Taken together, these effects of PEG-GO on cancer cell metastasis may allow the development of a new approach to treat metastatic breast cancer.
Asunto(s)
Neoplasias de la Mama/patología , Metabolismo Energético/efectos de los fármacos , Grafito/farmacología , Metástasis de la Neoplasia/prevención & control , Óxidos/farmacología , Polietilenglicoles/farmacología , Mama/efectos de los fármacos , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Grafito/química , Humanos , Óxidos/química , Polietilenglicoles/químicaRESUMEN
Graphene and its derivatives have become important nanomaterials worldwide and have potential medical applications including in vivo diagnosis, drug delivery, and photothermal therapy of cancer. However, little is known about their effect on the metastasis of cancer cells, which is the cause of over 90% of patient deaths. In the present investigation, we provide direct evidence that low concentrations of pristine graphene and graphene oxide show no apparent influence on the viability of MDA-MB-231 human breast cancer cells, PC3 human prostate cancer cells, as well as B16F10 mouse melanoma cells. However, both pristine graphene and graphene oxide can effectively inhibit the migration and invasion of these cancer cells. Further studies indicate that exposure of cells to graphene led to the direct inhibition of the electron transfer chain complexes I, II, III and IV, most likely by disrupting electron transfer between iron-sulfur centers, which is due to its stronger ability to accept electrons compared to iron-sulfur clusters through theoretical calculations. The decreased electron transfer chain activity caused a reduction in the production of ATP and subsequent impairment of F-actin cytoskeleton assembly, which is crucial for the migration and invasion of metastatic cancer cells. The inhibition of cancer cell metastasis by graphene and graphene oxide might provide new insights into specific cancer treatment.
Asunto(s)
Grafito/farmacología , Mitocondrias/efectos de los fármacos , Invasividad Neoplásica/prevención & control , Metástasis de la Neoplasia/prevención & control , Citoesqueleto de Actina/metabolismo , Animales , Línea Celular Tumoral , Humanos , Proteínas Hierro-Azufre/metabolismo , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Mitocondrias/fisiología , Neoplasias/enzimología , Neoplasias/metabolismo , Neoplasias/patología , Porfirinas/metabolismo , Succinato Deshidrogenasa/antagonistas & inhibidoresRESUMEN
Upon apoptotic stimuli, lysosomal proteases, including cathepsins and chymotrypsin, are released into cytosol due to lysosomal membrane permeabilization (LMP), where they trigger apoptosis via the lysosomal-mitochondrial pathway of apoptosis. Herein, the mechanism of LMP was investigated. We found that caspase 8-cleaved Bid (tBid) could result in LMP directly. Although Bax or Bak might modestly enhance tBid-triggered LMP, they are not necessary for LMP. To study this further, large unilamellar vesicles (LUVs), model membranes mimicking the lipid constitution of lysosomes, were used to reconstitute the membrane permeabilization process in vitro. We found that phosphatidic acid (PA), one of the major acidic phospholipids found in lysosome membrane, is essential for tBid-induced LMP. PA facilitates the insertion of tBid deeply into lipid bilayers, where it undergoes homo-oligomerization and triggers the formation of highly curved nonbilayer lipid phases. These events induce LMP via pore formation mechanisms because encapsulated fluorescein-conjugated dextran (FD)-20 was released more significantly than FD-70 or FD-250 from LUVs due to its smaller molecular size. On the basis of these data, we proposed tBid-PA interactions in the lysosomal membranes form lipidic pores and result in LMP. We further noted that chymotrypsin-cleaved Bid is more potent than tBid at binding to PA, inserting into the lipid bilayer, and promoting LMP. This amplification mechanism likely contributes to the culmination of apoptotic signaling.
Asunto(s)
Apoptosis , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/antagonistas & inhibidores , Lisosomas/metabolismo , Ácidos Fosfatidicos/metabolismo , Animales , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Ratones , Mitocondrias/metabolismo , Permeabilidad , Ratas , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Graphene may have attractive properties for some biomedical applications, but its potential adverse biological effects, in particular, possible modulation of immune responses, require further investigation. Macrophages are one of the most important effector cells of the innate immune system, and play pivotal roles in the response to graphene exposure. We have previously reported that exposure of macrophages to high concentrations of graphene triggers cell death via MAPK- and TGF-related pathways. However, little is known about the influence of exposure to low concentrations of graphene on the function of macrophages. In the present investigation, we demonstrate the biological effects of sub-cytotoxic concentrations of commercial pristine graphene on both primary murine macrophages and immortalized macrophages. Graphene significantly stimulates the secretion of Th1/Th2 cytokines including IL-1α, IL-6, IL-10, TNF-α and GM-CSF as well as chemokines such as MCP-1, MIP-1α, MIP-1ß and RANTES, probably by activating TLR-mediated and NF-κB-dependent transcription. Furthermore, these graphene-induced factors alter the morphology of naïve macrophages by remodeling their actin assembly, decreasing their ability to adhere to the extracellular matrix, and attenuating their phagocytosis. This negative feedback of the immune response of macrophages by graphene-induced factors may play an important role in the prevention of their over-activation after graphene exposure. These findings shed light on the interaction of graphene and macrophages in vitro. Further research is needed to systematically assess the biological responses of graphene, both to improve its safety and to contribute to the design of new biological applications.