RESUMEN
Photodynamic Therapy (PDT) is an effective treatment modality for cancers, with Protoporphyrin IX (PPIX)-based PDT being the most widely used to treat cancers in patients. However, PDT is limited to superficial, thin (few mm in depth) lesions that can be accessed by visible wavelength light. Interstitial light-delivery strategies have been developed to treat deep-seated lesions (i.e. prostate cancer). The most promising of these are X-ray-induced scintillation nanoparticles, which have shown potential benefits for PDT of deep-seated tumors. Herein, the design and use of a new nanoscintillator-based radiation-activated PDT (radioPDT) system is investigated in the treatment of deep-seated tumors. Poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) (PEG-PLGA) nanospheres were loaded with a scintillator (LaF3:Ce3+) and photosensitizer (PPIX) to effect radioPDT. UV-Vis spectroscopy and electron microscopy studies demonstrated efficient encapsulation of nanoscintillators and PPIX (>90% efficiency) into the PEG-PLGA nanospheres. The nanoparticles were uniform in size and approximately 100 nm in diameter. They were highly stable and functional for up to 24 h under physiological conditions and demonstrated slow release kinetics. In vitro and in vivo toxicity studies showed no appreciable drug toxicity to human skin fibroblast (GM38), prostate cancer cells (PC3), and to C57/BL mice. Cell uptake studies demonstrated accumulation of the nanoparticles in the cytoplasm of PC3 cells. When activated, fluorescent resonant energy transfer (FRET) was evident via fluorescent spectroscopy and singlet oxygen yield. Determination of stability revealed that the nanoparticles were stable for up to 4 weeks. The nanoparticle production was scaled-up with no change in properties. This nanoparticle represents a unique, optimally designed therapeutic and diagnostic agent (theranostic) agent for radioPDT with characteristics capable of potentially augmenting radiotherapy for deep-seated tumors and integrating into current cancer radiotherapy.
Asunto(s)
Nanopartículas , Nanosferas , Fotoquimioterapia , Línea Celular Tumoral , Humanos , Fármacos Fotosensibilizantes , Poliésteres , PolietilenglicolesRESUMEN
Angiogenesis is a dynamic process fundamental to the development of solid tumors. Epidermal growth factor-like domain 7 (EGFL7) is a protein whose expression is restricted to endothelial cells undergoing active remodeling that has emerged as a key mediator of this process. EGFL7 expression is associated with poor outcome in several cancers, making it a promising target for imaging or therapeutic strategies. Here, EGFL7 is explored as a molecular target for active neovascularization. Using a combinatorial peptide screening approach, we describe the discovery and characterization of a novel high affinity EGFL7-binding peptide, E7p72, that specifically targets human endothelial cells. Viral nanoparticles decorated with E7p72 peptides specifically target tumor-associated neovasculature with high specificity as assessed by intravital imaging. This work highlights the value of EGFL7 as a target for angiogenic vessels and opens the door for novel targeted therapeutic approaches.
RESUMEN
The flux of Ca(2+) from the endoplasmic reticulum (ER) to mitochondria regulates mitochondria metabolism. Within tumor tissue, mitochondria metabolism is frequently repressed, leading to chemotherapy resistance and increased growth of the tumor mass. Therefore, altered ER-mitochondria Ca(2+) flux could be a cancer hallmark, but only a few regulatory proteins of this mechanism are currently known. One candidate is the redox-sensitive oxidoreductase TMX1 that is enriched on the mitochondria-associated membrane (MAM), the site of ER-mitochondria Ca(2+) flux. Our findings demonstrate that cancer cells with low TMX1 exhibit increased ER Ca(2+), accelerated cytosolic Ca(2+) clearance, and reduced Ca(2+) transfer to mitochondria. Thus, low levels of TMX1 reduce ER-mitochondria contacts, shift bioenergetics away from mitochondria, and accelerate tumor growth. For its role in intracellular ER-mitochondria Ca(2+) flux, TMX1 requires its thioredoxin motif and palmitoylation to target to the MAM. As a thiol-based tumor suppressor, TMX1 increases mitochondrial ATP production and apoptosis progression.
Asunto(s)
Señalización del Calcio , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Neoplasias/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Tiorredoxinas/metabolismo , Calcio/metabolismo , Proliferación Celular/efectos de los fármacos , Metabolismo Energético , Glucosa/farmacología , Células HeLa , Humanos , Lipoilación , Membranas Mitocondriales/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Recent evidence suggests that endoplasmic reticulum (ER) tubules mark the sites where the GTPase Drp1 promotes mitochondrial fission via a largely unknown mechanism. Here, we show that the SNARE protein syntaxin 17 (Syn17) is present on raft-like structures of ER-mitochondria contact sites and promotes mitochondrial fission by determining Drp1 localization and activity. The hairpin-like C-terminal hydrophobic domain, including Lys-254, but not the SNARE domain, is important for this regulation. Syn17 also regulates ER Ca(2+) homeostasis and interferes with Rab32-mediated regulation of mitochondrial dynamics. Starvation disrupts the Syn17-Drp1 interaction, thus favoring mitochondrial elongation during autophagy. Because we also demonstrate that Syn17 is an ancient SNARE, our findings suggest that Syn17 is one of the original key regulators for ER-mitochondria contact sites present in the last eukaryotic common ancestor. As such, Syn17 acts as a switch that responds to nutrient conditions and integrates functions for the ER and autophagosomes with mitochondrial dynamics.
Asunto(s)
Retículo Endoplásmico/metabolismo , Mitocondrias/metabolismo , Dinámicas Mitocondriales/fisiología , Proteínas Qa-SNARE/metabolismo , GTP Fosfohidrolasas/metabolismo , Células HeLa , Humanos , Proteínas Mitocondriales/metabolismo , Fagosomas/metabolismoRESUMEN
The endoplasmic reticulum (ER) is a multifunctional organelle that accommodates a large array of functions. Recent publications have shown that many of these functions are influenced by the ongoing oxidative folding of secretory and membrane proteins. Conversely, successful ER protein folding critically depends on the cellular redox state, but also the availability of Ca²âº. These findings suggest the existence of regulatory mechanisms that steer ER Ca²âº homeostasis according to the cellular redox state. Indeed, accumulating evidence demonstrates that ER Ca²âº uptake and release by sarco-endoplasmic reticulum Ca²âº transport ATPases (SERCAs), stromal interaction molecule 1 (STIM1), Orai1, inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) and ryanodine receptors (RyR) depends on redox modifications of these channels and pumps. In addition, ER chaperones and oxidoreductases moonlight as regulators of ER Ca²âº channels and pumps. Discrete redox conditions of channels, pumps and oxidoreductases exist that allow for opening and closing. Through these functions, redox regulation of ER Ca²âº influences signaling mechanisms governing cell growth and migration, apoptosis and mitochondrial energy production. Therefore, pharmacological intervention based on ER redox or on ER redox-sensitive chaperones and oxidoreductases is a promising strategy to influence all metabolic syndromes including cancer and neurodegeneration.
Asunto(s)
Señalización del Calcio/fisiología , Retículo Endoplásmico/metabolismo , Animales , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Mitocondrias/metabolismo , Oxidación-Reducción , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
The palmitoylation of calnexin serves to enrich calnexin on the mitochondria-associated membrane (MAM). Given a lack of information on the significance of this finding, we have investigated how this endoplasmic reticulum (ER)-internal sorting signal affects the functions of calnexin. Our results demonstrate that palmitoylated calnexin interacts with sarcoendoplasmic reticulum (SR) Ca(2+) transport ATPase (SERCA) 2b and that this interaction determines ER Ca(2+) content and the regulation of ER-mitochondria Ca(2+) crosstalk. In contrast, non-palmitoylated calnexin interacts with the oxidoreductase ERp57 and performs its well-known function in quality control. Interestingly, our results also show that calnexin palmitoylation is an ER-stress-dependent mechanism. Following a short-term ER stress, calnexin quickly becomes less palmitoylated, which shifts its function from the regulation of Ca(2+) signaling towards chaperoning and quality control of known substrates. These changes also correlate with a preferential distribution of calnexin to the MAM under resting conditions, or the rough ER and ER quality control compartment (ERQC) following ER stress. Our results have therefore identified the switch that assigns calnexin either to Ca(2+) signaling or to protein chaperoning.
Asunto(s)
Calnexina/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Lipoilación/fisiología , Membranas Mitocondriales/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Células 3T3 , Animales , Calcio/metabolismo , Señalización del Calcio , Línea Celular , Retículo Endoplásmico/metabolismo , Fibroblastos , Células HEK293 , Células HeLa , Humanos , Ratones , Mitocondrias/metabolismo , Proteína Disulfuro Isomerasas/metabolismoRESUMEN
More than a billion years ago, bacterial precursors of mitochondria became endosymbionts in what we call eukaryotic cells today. The true significance of the word "endosymbiont" has only become clear to cell biologists with the discovery that the endoplasmic reticulum (ER) superorganelle dedicates a special domain for the metabolic interaction with mitochondria. This domain, identified in all eukaryotic cell systems from yeast to man and called the mitochondria-associated membrane (MAM), has a distinct proteome, specific tethers on the cytosolic face and regulatory proteins in the ER lumen of the ER. The MAM has distinct biochemical properties and appears as ER tubules closely apposed to mitochondria on electron micrographs. The functions of the MAM range from lipid metabolism and calcium signaling to inflammasome formation. Consistent with these functions, the MAM is enriched in lipid metabolism enzymes and calcium handling proteins. During cellular stress situations, like an altered cellular redox state, the MAM alters its set of regulatory proteins and thus alters MAM functions. Notably, this set prominently comprises ER chaperones and oxidoreductases that connect protein synthesis and folding inside the ER to mitochondrial metabolism. Moreover, ER membranes associated with mitochondria also accommodate parts of the machinery that determines mitochondrial membrane dynamics and connect mitochondria to the cytoskeleton. Together, these exciting findings demonstrate that the physiological interactions between the ER and mitochondria are so bilateral that we are tempted to compare their relationship to the one of a married couple: distinct, but inseparable and certainly dependent on each other. In this paradigm, the MAM stands for the intracellular location where the two organelles tie the knot. Resembling "real life", the happy marriage between the two organelles prevents the onset of diseases that are characterized by disrupted metabolism and decreased lifespan, including neurodegeneration and cancer. This article is part of a Special Issue entitled: Mitochondrial dynamics and physiology.
Asunto(s)
Membrana Celular/fisiología , Retículo Endoplásmico/fisiología , Mitocondrias/fisiología , Dinámicas Mitocondriales/fisiología , Membranas Mitocondriales/fisiología , Animales , Señalización del Calcio/fisiología , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/fisiología , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Metabolismo de los Lípidos/fisiología , Masculino , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Membranas Mitocondriales/metabolismo , Modelos BiológicosRESUMEN
Platelet-derived microparticles (pMP) have been shown to be pro-aggregatory and retain most of their platelet membrane markers. Recent studies have correlated elevated pMP levels with pathogenesis of diabetes mellitus and cardiovascular disease. The pro-aggregatory effect of pMP has been largely attributed to their negatively charged outer surface and activation of factor X by membrane associated Tissue factor (TF). Here we sought to investigate whether, like platelets, protein disulfide isomerase (PDI) is present on the surface of pMP and, if so, to analyze its contribution to platelet hyperaggregability and insulin degradation. Using a fluorescent assay based upon a novel pseudo-substrate of PDI, flow cytometry and immunological techniques, we have demonstrated the presence of PDI on the surface of pMP (termed msPDI) and its ability to influence insulin-mediated Akt phosphorylation (Thr308) in 3T3-L1 fibroblasts. Moreover, pMP are shown to contain catalytically active PDI, capable of both promoting platelet aggregation and disrupting insulin signaling. pMP increased initial rates of aggregation by 4-fold and the pro-aggregatory activity of pMPs could be attenuated with an anti-PDI antibody. The pMP insulin-reductase activity was further attributed to PDI based on the ability of anti-PDI antibodies to block the degradation of insulin, thereby restoring insulin signaling. Plasma pMP counts were also obtained from diabetic (n=10) and non-diabetic individuals (n=10) and found to be elevated in the diabetic state. Detection of increased levels of PDI-containing microparticles in patients with T2D raises the possibility that platelet hypersensitivity and insulin desensitization observed in diabetes can partially be attributed to msPDI activity.
Asunto(s)
Plaquetas/fisiología , Hipoglucemiantes/inmunología , Insulina/inmunología , Agregación Plaquetaria/efectos de los fármacos , Proteína Disulfuro Isomerasas/fisiología , Adulto , Anciano , Fenómenos Biológicos , Plaquetas/enzimología , Diabetes Mellitus Tipo 2/sangre , Humanos , Hipoglucemiantes/metabolismo , Insulina/metabolismo , Persona de Mediana Edad , Tamaño de la Partícula , Proteína Disulfuro Isomerasas/sangre , Factores de TiempoRESUMEN
In this study, dieosin glutathione disulfide (Di-E-GSSG) was synthesized by the reaction of eosin isothiocyanate with GSSG. Di-E-GSSG had low fluorescence which increased approximately 70-fold on reduction of its disulfide bond. The substrate was used to monitor the disulfide reductase activity of PDI. Di-E-GSSG is the most sensitive pseudo substrate for PDI reductase activity reported to date. This probe was further used as an analytical reagent to develop an end point assay for measuring the redox state of PDI. The reduction of Di-E-GSSG by reduced enzyme was studied in the absence of reducing agents and the redox state of PDI was monitored as a function of the stoichiometric changes in the amount of eosin-glutathione (EGSH) generated by the active-site dithiols of PDI. The redox state of PDI was also studied under variable [GSH]/[GSSG] ratios. The results indicate that PDI is in approximately 1/2-reduced state where the [GSH]/[GSSG] ratio is between 1:1 and 3:1, conditions similar to the lumen of endoplasmic reticulum or in the extracellular environment. On the other hand, [GSH]/[GSSG] ratios of > or =8:1, such as in cytosol, all active-site thiols would be reduced. The study was extended to utilize Di-E-GSSG to investigate the effect of variable redox ratios on the platelet surface PDI reductase activity.
Asunto(s)
Eosina Amarillenta-(YS)/análogos & derivados , Colorantes Fluorescentes/química , Disulfuro de Glutatión/análogos & derivados , Proteína Disulfuro Isomerasas/química , Proteínas Recombinantes/química , Eosina Amarillenta-(YS)/síntesis química , Eosina Amarillenta-(YS)/química , Fluorescencia , Colorantes Fluorescentes/síntesis química , Glutatión/química , Disulfuro de Glutatión/síntesis química , Disulfuro de Glutatión/química , Humanos , Oxidación-Reducción , Proteína Disulfuro Isomerasas/genética , Proteínas Recombinantes/genéticaRESUMEN
The objectives of this study were to determine the relationships among Type II diabetes (T2DM)-dependent elevations in platelet-derived reactive oxygen species (ROS), platelet-surface protein disulfide isomerase (psPDI) NO-releasing activity, and platelet aggregation and to evaluate the efficacy of rosuvastatin in normalizing these parameters in primary cells derived from a hamster model of prediabetic insulin resistance induced by fructose feeding. Platelets from rosuvastatin-treated non-fructose-fed (NFF) and fructose-fed (FF) hamsters were analyzed for aggregability and psPDI-denitrosation activity. Platelets from NFF animals treated with xanthine/xanthine oxidase (X/XO) were assessed for the same parameters and primary aortic endothelial cells (AEC) cultivated with a range of [rosuvastatin] +/- mevalonate were analyzed for ROS production. Platelets from FF hamsters displayed statistically significant enhanced ROS production, diminished psPDI-mediated NO-releasing activity, and hyperaggregability. Suggestively, platelets from NFF animals treated with X/XO displayed characteristics similar to platelets from FF animals. Rosuvastatin elicited a normalizing effect on all parameters measured in platelets from FF animals. Further, ROS production in primary AEC from FF animals could be blunted to that of NFF animals by concentrations of rosuvastatin in the range of those achieved in the bloodstream. Diminished psPDI-dependent NO-releasing activity and increased initial aggregation rates of FF platelets may result from elevated vascular ROS production under conditions of insulin resistance. Normalization of ROS production and platelet aggregation by rosuvastatin indicates its potential use as a vasculoprotective agent.
Asunto(s)
Plaquetas/efectos de los fármacos , Diabetes Mellitus Tipo 2/prevención & control , Fluorobencenos/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Estado Prediabético/tratamiento farmacológico , Pirimidinas/farmacología , Sulfonamidas/farmacología , Animales , Antioxidantes , Cricetinae , Fructosa/efectos adversos , Mesocricetus , Óxido Nítrico/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Proteína Disulfuro Isomerasas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Rosuvastatina CálcicaRESUMEN
PDI (protein disulphide-isomerase) activity is generally monitored by insulin turbidity assay or scrambled RNase assay, both of which are performed by UV-visible spectroscopy. In this paper, we present a sensitive fluorimetric assay for continuous determination of disulphide reduction activity of PDI. This assay utilizes the pseudo-substrate diabz-GSSG [where diabz stands for di-(o-aminobenzoyl)], which is formed by the reaction of isatoic anhydride with the two free N-terminal amino groups of GSSG. The proximity of two benzoyl groups leads to quenching of the diabz-GSSG fluorescence by approx. 50% in comparison with its non-disulphide-linked form, abz-GSH (where abz stands for o-aminobenzoyl). Therefore the PDI-dependent disulphide reduction can be monitored by the increase in fluorescence accompanying the loss of proximity-quenching upon conversion of diabz-GSSG into abz-GSH. The apparent K(m) of PDI for diabz-GSSG was estimated to be approx. 15 muM. Unlike the insulin turbidity assay and scrambled RNase assay, the diabz-GSSG-based assay was shown to be effective in determining a single turnover of enzyme in the absence of reducing agents with no appreciable blank rates. The assay is simple to perform and very sensitive, with an estimated detection limit of approx. 2.5 nM PDI, enabling its use for the determination of platelet surface PDI activity in crude sample preparations.
Asunto(s)
Fluorescencia , Proteína Disulfuro Isomerasas/análisis , Plaquetas/enzimología , Disulfuros/química , Disulfuros/metabolismo , Glutatión/análogos & derivados , Glutatión/química , Glutatión/metabolismo , Disulfuro de Glutatión/química , Disulfuro de Glutatión/metabolismo , Humanos , Cinética , Proteínas de la Membrana/análisis , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Estructura Molecular , Oxidación-Reducción , Unión Proteica , Proteína Disulfuro Isomerasas/metabolismo , Sensibilidad y Especificidad , Especificidad por SustratoRESUMEN
S-nitrosoglutathione (GSNO) denitrosation activity of recombinant human protein disulfide isomerase (PDI) has been kinetically characterized by monitoring the loss of the S-NO absorbance, using a NO electrode, and with the aid of the fluorogenic NOx probe 2,3-diaminonaphthalene. The initial rates of denitrosation as a function of [GSNO] displayed hyperbolic behavior irrespective of the method used to monitor denitrosation. The Km values estimated for GSNO were 65 +/- 5 microm and 40 +/- 10 microm for the loss in the S-NO bond and NO production (NO electrode or 2,3-diaminonaphthalene), respectively. Hemoglobin assay provided additional evidence that the final product of PDI-dependent GSNO denitrosation was NO*. A catalytic mechanism, involving a nitroxyl disulfide intermediate stabilized by imidazole (His160 a-domain or His589 a'-domain), which after undergoing a one-electron oxidation decomposes to yield NO plus dithiyl radical, has been proposed. Evidence for the formation of thiyl/dithiyl radicals during PDI-catalyzed denitrosation was obtained with 4-((9-acridinecarbonyl)-amino)-2,2,6,6-tetramethylpiperidine-1-oxyl. Evidence has also been obtained showing that in a NO- and O2-rich environment, PDI can form N2O3 in its hydrophobic domains. This "NO-charged PDI" can perform intra- and intermolecular S-nitrosation reactions similar to that proposed for serum albumin. Interestingly, reduced PDI was able to denitrosate S-nitrosated PDI (PDI-SNO) resulting in the release of NO. PDI-SNO, once formed, is stable at room temperature in the absence of reducing agent over the period of 2 h. It has been established that PDI is continuously secreted from cells that are net producers of NO-like endothelial cells. The present demonstration that PDI can be S-nitrosated and that PDI-SNO can be denitrosated by PDI suggests that this enzyme could be intimately involved in the transport of intracellular NO equivalents to the cell surface as well as the previous demonstration of PDI in the transfer of S-nitrosothiol-bound NO to the cytosol.