RESUMEN
Proline, the only proteinogenic secondary amino acid, is metabolized by its own family of enzymes responding to metabolic stress and participating in metabolic signaling. Collagen in extracellular matrix, connective tissue, and bone is an abundant reservoir for proline. Matrix metalloproteinases degrading collagen are activated during stress to make proline available, and proline oxidase, the first enzyme in proline degradation, is induced by p53, peroxisome proliferator-activated receptor gamma (PPARgamma) and its ligands, and by AMP-activated protein kinase downregulating mTOR. Metabolism of proline generates electrons to produce ROS and initiates a variety of downstream effects, including blockade of the cell cycle, autophagy, and apoptosis. The electrons can also enter the electron transport chain to produce adenosine triphosphate for survival under nutrient stress. Pyrroline-5-carboxylate, the product of proline oxidation, is recycled back to proline with redox transfers or is sequentially converted to glutamate and alpha-ketoglutarate. The latter augments the prolyl hydroxylation of hypoxia-inducible factor-1alpha and its proteasomal degradation. These effects of proline oxidase, as well as its decreased levels in tumors, support its role as a tumor suppressor. The mechanism for its decrease is mediated by a specific microRNA. The metabolic signaling by proline oxidase between oxidized low-density lipoproteins and autophagy provides a functional link between obesity and increased cancer risk.
Asunto(s)
Colágeno/metabolismo , Regulación Enzimológica de la Expresión Génica , Metaloproteinasas de la Matriz/metabolismo , Prolina Oxidasa/metabolismo , Prolina/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Apoptosis/fisiología , Autofagia/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , PPAR gamma/metabolismo , Prolina Oxidasa/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Epidemiological studies showed that high levels of oxidized low-density lipoproteins (oxLDLs) are associated with increased cancer risk. We examined the direct effect of physiologic concentrations oxLDL on cancer cells. OxLDLs were cytotoxic and activate both apoptosis and autophagy. OxLDLs have ligands for peroxisome proliferator-activated receptor gamma and upregulated proline oxidase (POX) through this nuclear receptor. We identified 7-ketocholesterol (7KC) as a main component responsible for the latter. To elucidate the role of POX in oxLDL-mediated cytotoxicity, we knocked down POX via small interfering RNA and found that this (i) further reduced viability of cancer cells treated with oxLDL; (ii) decreased oxLDL-associated reactive oxygen species generation; (iii) decreased autophagy measured via beclin-1 protein level and light-chain 3 protein (LC3)-I into LC3-II conversion. Using POX-expressing cell model, we established that single POX overexpression was sufficient to activate autophagy. Thus, it led to autophagosomes accumulation and increased conversion of LC3-I into LC3-II. Moreover, beclin-1 gene expression was directly dependent on POX catalytic activity, namely the generation of POX-dependent superoxide. We conclude that POX is critical in the cellular response to the noxious effects of oxLDL by activating protective autophagy.
Asunto(s)
Autofagia/fisiología , Carcinoma/patología , Lipoproteínas LDL/farmacología , Prolina Oxidasa/biosíntesis , Especies Reactivas de Oxígeno/metabolismo , Autofagia/efectos de los fármacos , Carcinoma/metabolismo , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Inducción Enzimática/efectos de los fármacos , Femenino , Humanos , Masculino , Malondialdehído/análisis , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/fisiología , PPAR gamma/fisiología , Prolina Oxidasa/antagonistas & inhibidores , Prolina Oxidasa/genética , Prolina Oxidasa/fisiología , Regiones Promotoras Genéticas , Interferencia de ARN , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/fisiología , Sustancias Reactivas al Ácido Tiobarbitúrico/análisis , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Our recent findings establish a functional link between foreign nanosized bodies and autophagy. We find that nanoparticles (NP) within a certain size range act as potent autophagy activators, and that autophagic flux is an underlying physiological process of the cellular clearance of the NP. Therefore, NP may be used to study and to monitor autophagy. We provide a detailed description of laboratory protocols designed for studying NP-mediated autophagy. In addition, we review available methods of nanotechnology, which may benefit autophagy research.
Asunto(s)
Autofagia/fisiología , Puntos Cuánticos , Western Blotting , Citometría de Flujo , Hibridación in Situ , Microscopía FluorescenteRESUMEN
Proline is metabolized by its own specialized enzymes with their own tissue and subcellular localizations and mechanisms of regulation. The central enzyme in this metabolic system is proline oxidase, a flavin adenine dinucleotide-containing enzyme which is tightly bound to mitochondrial inner membranes. The electrons from proline can be used to generate ATP or can directly reduce oxygen to form superoxide. Although proline may be derived from the diet and biosynthesized endogenously, an important source in the microenvironment is from degradation of extracellular matrix by matrix metalloproteinases. Previous studies showed that proline oxidase is a p53-induced gene and its overexpression can initiate proline-dependent apoptosis by both intrinsic and extrinsic pathways. Another important factor regulating proline oxidase is peroxisome proliferator activated receptor gamma (PPARgamma). Importantly, in several cancer cells, proline oxidase may be an important mediator of the PPARgamma-stimulated generation of ROS and induction of apoptosis. Knockdown of proline oxidase expression by antisense RNA markedly decreased these PPARgamma-stimulated effects. These findings suggest an important role in the proposed antitumor effects of PPARgamma. Moreover, it is possible that proline oxidase may contribute to the other metabolic effects of PPARgamma.
RESUMEN
The oxidized low-density lipoprotein (oxLDL)-dependent activation of the lectin-like oxLDL receptor-1 (LOX-1) triggers apoptosis in vascular cells and appears to be involved in atherosclerosis. Autophagy might be an alternate to apoptosis in endothelial cells. The EA.hy926 endothelial cell line has been reported to undergo necrosis under oxLDL stimulation. For this reason, we studied the expression of LOX-1 and its oxLDL-dependent function in EA.hy926 cells under serum starvation. Untreated and oxLDL-treated cells expressed the LOX-1 protein at similar levels 6h after starvation. After 24h without oxLDL and with native LDL (nLDL), statistically significant higher levels were found in LOX-1 than in the oxLDL-treated probes. The oxLDL cultures with low LOX-1 expression displayed stronger features of autophagy than those with nLDL as there were remodelling of actin filaments, disrupture of adherens junctions (immunofluorescence staining), and autophagosomes with the characteristic double membrane at the ultrastructural level. For the advanced oxLDL exposure times (18 and 24 h), autophagic vacuoles/autophagolysosomes were morphologically identified accompanied by a decrease in lysosomes. The autophagosome marker protein MAP LC3-II (Western blotting) was significantly augmented 6 and 18 h after oxLDL treatment compared with cultures treated with nLDL and medium alone. Signs of apoptosis were undetectable in cultures under oxLDL exposure, yet present under staurosporin (apoptosis inducer), i.e. presence of apoptotic bodies and cleaved caspase 3. We conclude that serum starvation upregulates LOX-1 in EA.hy926 cells, whereas the additional oxLDL treatment downregulates the receptor and intensifies autophagy probably by increase in oxidative stress.
Asunto(s)
Autofagia , Endotelio Vascular/efectos de los fármacos , Lipoproteínas LDL/farmacología , Receptores Depuradores de Clase E/metabolismo , Citoesqueleto de Actina/metabolismo , Apoptosis/efectos de los fármacos , Western Blotting , Cadherinas/metabolismo , Células Cultivadas , Medio de Cultivo Libre de Suero , Endotelio Vascular/metabolismo , Humanos , Lisosomas/metabolismo , Estrés Oxidativo , Fagocitosis , Fagosomas , Estaurosporina/farmacología , Venas Umbilicales/citología , Venas Umbilicales/metabolismo , Regulación hacia ArribaRESUMEN
Nano-sized objects exist as engineered tools as well as natural or anthropogenic environmental factors. Recent progress in the field of nanotechnology allows for a deeper understanding of their impact on organisms. Recently, we showed that the size-dependent cell interaction with quantum dots is autophagy-mediated. The potential role of other endo- and exogenous nanoparticles in terms of autophagy is discussed here. Their physical properties should be taken into consideration while constructing delivery systems. Furthermore, we propose several models of targeted nanoparticles delivery. Autophagy can be considered as an additional mechanism providing intracellular selectivity for introduced nanoparticles.
Asunto(s)
Autofagia , Nanopartículas , Animales , Línea Celular , Sistemas de Liberación de Medicamentos , Ambiente , Humanos , Ratones , Nanopartículas/administración & dosificación , Nanopartículas/química , Puntos CuánticosRESUMEN
Lately certain cytotoxicity of quantum dots (QDs) and some deleterious effects of labeling procedure on stem cells differentiation abilities were shown. In the present study we compared cytotoxicity and intracellular processing of two different-sized protein-conjugated QDs after labeling of the human mesenchymal stem cells (hMSC). An asymmetrical intracellular uptake of red (605 nm) and green (525 nm) quantum dots was observed. We describe for the first time a size-dependent activation of autophagy, caused by nanoparticles.
Asunto(s)
Autofagia , Células Madre Mesenquimatosas/fisiología , Puntos Cuánticos , Diferenciación Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Supervivencia Celular , Células Cultivadas , Técnicas de Cocultivo , Fluorescencia , Humanos , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/ultraestructura , Microscopía Electrónica de Transmisión , Proteínas Asociadas a Microtúbulos/metabolismoRESUMEN
The LOX-1 receptor, identified on endothelial cells, mediates the uptake of oxidized low-density lipoprotein (oxLDL). The oxLDL-dependent LOX-1 activation causes endothelial cell apoptosis. We here investigated the presence of LOX-1 in granulosa cells from patients under in vitro fertilization therapy. We were interested in the oxLDL-dependent LOX-1 receptor biology, in particular in the induction of apoptosis. In the human ovary, LOX-1 was localized in regressing antral follicles. In granulosa cell cultures, oxLDL-induced mRNA expression of LOX-1 in a time- and dose-dependent manner. The LOX-1 inhibitors (anti-LOX-1 antibody and kappa-carrageenan) abrogated the up-regulation of LOX-1. The oxLDL (100 microg/ml) treatment caused the autophagy form of programmed cell death: 1) reorganization of the actin cytoskeleton at the 6-h time point; 2) uptake of YO-PRO, a marker for the early step of programmed cell death, before propidium iodide staining to signify necrosis; 3) absence of apoptotic bodies and cleaved caspase-3; 4) abundant vacuole formation at the ultrastructural level; and 5) decrease of the autophagosome marker protein MAP LC3-I at the 6-h time point indicative of autophagosome formation. We conclude that follicular atresia is not under the exclusive control of apoptosis. The LOX-1-dependent autophagy represents an alternate form of programmed cell death. Obese women with high blood levels of oxLDL may display an increased rate of autophagic granulosa cell death.