RESUMEN
The activity of sirtuin 1 (SIRT1, a member of the NAD+-dependent deacetylases family) decreases during aging as NAD+ levels naturally decline, thus increasing the risk of several age-associated diseases. Several sirtuin-activating compounds (STACs) have been developed to counteract the age-associated reduction in SIRT1 activity, and some of them are currently under development in clinical trials. STACs induce SIRT1 activation, either through allosteric activation of the enzyme in the presence of NAD+, or by increasing NAD+ levels by inhibiting its degradation or by supplying a key precursor in biosynthesis. In this study, we have identified (E)-2'-des-methyl sulindac analogues as a novel class of STACs that act also in the absence of NAD+, a peculiar behavior demonstrated through enzymatic and mass spectrometry experiments, both in vitro and in cell lines. The activation of the SIRT1 pathway was confirmed in vivo through gene expression and metabolomics analysis. Our data suggest that these compounds could serve as candidate leads for a novel therapeutic strategy aimed at addressing a key metabolic deficiency that may contribute to metabolic and age-associated diseases.
Asunto(s)
NAD , Sirtuina 1 , Sirtuina 1/metabolismo , NAD/metabolismo , Animales , Humanos , Activadores de Enzimas/farmacología , Línea Celular , Ratones , Masculino , Ratones Endogámicos C57BL , Descubrimiento de DrogasRESUMEN
All the different coronavirus SARS-CoV-2 variants isolated so far share the same mechanism of infection mediated by the interaction of their spike (S) glycoprotein with specific residues on their cellular receptor: the angiotensin converting enzyme 2 (ACE2). Therefore, the steric hindrance on this cellular receptor created by a bulk macromolecule may represent an effective strategy for the prevention of the viral spreading and the onset of severe forms of Corona Virus disease 19 (COVID-19). Here, we applied a systematic evolution of ligands by exponential enrichment (SELEX) procedure to identify two single strand DNA molecules (aptamers) binding specifically to the region surrounding the K353, the key residue in human ACE2 interacting with the N501 amino acid of the SARS-CoV-2 S. 3D docking in silico experiments and biochemical assays demonstrated that these aptamers bind to this region, efficiently prevent the SARS-CoV-2 S/human ACE2 interaction and the viral infection in the nanomolar range, regardless of the viral variant, thus suggesting the possible clinical development of these aptamers as SARS-CoV-2 infection inhibitors. Our approach brings a significant innovation to the therapeutic paradigm of the SARS-CoV-2 pandemic by protecting the target cell instead of focusing on the virus; this is particularly attractive in light of the increasing number of viral mutants that may potentially escape the currently developed immune-mediated neutralization strategies.
Asunto(s)
Enzima Convertidora de Angiotensina 2/antagonistas & inhibidores , Aptámeros de Nucleótidos/farmacología , Tratamiento Farmacológico de COVID-19 , Receptores Virales/antagonistas & inhibidores , SARS-CoV-2/patogenicidad , Internalización del Virus/efectos de los fármacos , Células A549 , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/metabolismo , COVID-19/enzimología , COVID-19/genética , COVID-19/virología , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Mutación , Receptores Virales/genética , Receptores Virales/metabolismo , SARS-CoV-2/genética , Técnica SELEX de Producción de AptámerosRESUMEN
BACKGROUND: Homozygotic mutations in the GBA gene cause Gaucher's disease; moreover, both patients and heterozygotic carriers have been associated with 20- to 30-fold increased risk of developing Parkinson's disease. In homozygosis, these mutations impair the activity of ß-glucocerebrosidase, the enzyme encoded by GBA, and generate a lysosomal disorder in macrophages, which changes morphology towards an engorged phenotype, considered the hallmark of Gaucher's disease. Notwithstanding the key role of macrophages in this disease, most of the effects in the brain have been attributed to the ß-glucocerebrosidase deficit in neurons, while a microglial phenotype for these mutations has never been reported. METHODS: We applied the bioluminescence imaging technology, immunohistochemistry and gene expression analysis to investigate the consequences of microglial ß-glucocerebrosidase inhibition in the brain of reporter mice, in primary neuron/microglia cocultures and in cell lines. The use of primary cells from reporter mice allowed for the first time, to discriminate in cocultures neuronal from microglial responses consequent to the ß-glucocerebrosidase inhibition; results were finally confirmed by pharmacological depletion of microglia from the brain of mice. RESULTS: Our data demonstrate the existence of a novel neuroprotective mechanism mediated by a direct microglia-to-neuron contact supported by functional actin structures. This cellular contact stimulates the nuclear factor erythroid 2-related factor 2 activity in neurons, a key signal involved in drug detoxification, redox balance, metabolism, autophagy, lysosomal biogenesis, mitochondrial dysfunctions, and neuroinflammation. The central role played by microglia in this neuronal response in vivo was proven by depletion of the lineage in the brain of reporter mice. Pharmacological inhibition of microglial ß-glucocerebrosidase was proven to induce morphological changes, to turn on an anti-inflammatory/repairing pathway, and to hinder the microglia ability to activate the nuclear factor erythroid 2-related factor 2 response, thus increasing the neuronal susceptibility to neurotoxins. CONCLUSION: This mechanism provides a possible explanation for the increased risk of neurodegeneration observed in carriers of GBA mutations and suggest novel therapeutic strategies designed to revert the microglial phenotype associated with ß-glucocerebrosidase inhibition, aimed at resetting the protective microglia-to-neuron communication.
Asunto(s)
Encéfalo/enzimología , Glucosilceramidasa/antagonistas & inhibidores , Microglía/enzimología , Neuronas/metabolismo , Neuroprotección/fisiología , Animales , Encéfalo/patología , Comunicación Celular/fisiología , Ratones , Microglía/patología , Neuronas/patologíaRESUMEN
BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) have been proposed as chemopreventive agents for many tumours; however, the mechanism responsible for their anti-neoplastic activity remains elusive and the side effects due to cyclooxygenase (COX) inhibition prevent this clinical application. METHODS: Molecular biology, in silico, cellular and in vivo tools, including innovative in vivo imaging and classical biochemical assays, were applied to identify and characterise the COX-independent anti-cancer mechanism of NSAIDs. RESULTS: Here, we show that tumour-protective functions of NSAIDs and exisulind (a sulindac metabolite lacking anti-inflammatory activity) occur through a COX-independent mechanism. We demonstrate these NSAIDs counteract carcinogen-induced proliferation by inhibiting the sirtuin 1 (SIRT1) deacetylase activity, augmenting acetylation and activity of the tumour suppressor p53 and increasing the expression of the antiproliferative gene p21. These properties are shared by all NSAIDs except for ketoprofen lacking anti-cancer properties. The clinical interest of the mechanism identified is underlined by our finding that p53 is activated in mastectomy patients undergoing intraoperative ketorolac, a treatment associated with decreased relapse risk and increased survival. CONCLUSION: Our study, for the first-time, links NSAID chemopreventive activity with direct SIRT1 inhibition and activation of the p53/p21 anti-oncogenic pathway, suggesting a novel strategy for the design of tumour-protective drugs.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Anticarcinógenos/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/efectos de los fármacos , Inhibidores de la Ciclooxigenasa/farmacología , Sirtuina 1/efectos de los fármacos , Sulindac/análogos & derivados , Proteína p53 Supresora de Tumor/efectos de los fármacos , Animales , Antiinflamatorios no Esteroideos/efectos adversos , Anticarcinógenos/efectos adversos , Línea Celular Tumoral , Simulación por Computador , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidores de la Ciclooxigenasa/efectos adversos , Humanos , Ketorolaco/efectos adversos , Ketorolaco/uso terapéutico , Ratones , Modelos Moleculares , Sirtuina 1/metabolismo , Sulindac/farmacología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Deciphering the etiology of complex pathologies at molecular level requires longitudinal studies encompassing multiple biochemical pathways (apoptosis, proliferation, inflammation, oxidative stress). In vivo imaging of current reporter animals enabled the spatio-temporal analysis of specific molecular events, however, the lack of a multiplicity of loci for the generalized and regulated expression of the integrated transgenes hampers the creation of systems for the simultaneous analysis of more than a biochemical pathways at the time. We here developed and tested an in vivo-based methodology for the identification of multiple insertional loci suitable for the generation of reliable reporter mice. The validity of the methodology was tested with the generation of novel mice useful to report on inflammation and oxidative stress.
Asunto(s)
Regulación de la Expresión Génica , Genes Reporteros , Sitios Genéticos , Ratones Transgénicos , Animales , Línea Celular , Electroporación , Células Madre Embrionarias/metabolismo , Femenino , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , Sustancias Luminiscentes , Mediciones Luminiscentes , Masculino , Ratones , Estrés Oxidativo , Regiones Promotoras Genéticas , Transgenes , Imagen de Cuerpo EnteroRESUMEN
The Parkinson's disease (PD) evolves over an extended period of time with the onset occurring long before clinical signs begin to manifest. Characterization of the molecular events underlying the PD onset is instrumental for the development of diagnostic markers and preventive treatments, progress in this field is hindered by technical limitations. We applied an imaging approach to demonstrate the activation of Nrf2 transcription factor as a hallmark of neurodegeneration in neurotoxin-driven models of PD. In dopaminergic SK-N-BE neuroblastoma cells, Nrf2 activation was detected in cells committed to die as proven by time lapse microscopy; in the substantia nigra pars compacta area of the mouse brain, the Nrf2 activation preceded dopaminergic neurodegeneration as demonstrated by in vivo and ex vivo optical imaging, a finding confirmed by co-localization experiments carried out by immunohistochemistry. Collectively, our results identify the Nrf2 signaling as an early marker of neurodegeneration, anticipating dopaminergic neurodegeneration and motor deficits.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Mediciones Luminiscentes/métodos , Factor 2 Relacionado con NF-E2/metabolismo , Imagen Óptica/métodos , Trastornos Parkinsonianos/diagnóstico por imagen , Trastornos Parkinsonianos/metabolismo , Animales , Muerte Celular/fisiología , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/patología , Humanos , Células MCF-7 , Masculino , Ratones , Ratones Transgénicos , Células 3T3 NIHRESUMEN
Recent studies have demonstrated that in mice, the estrogen receptor alpha (ERα) is expressed in the liver and has a direct effect on the regulation of the hepatic genes relevant for energy metabolism and drug metabolism. The sex-related differential expression of the hepatic ERα raises the questions as to whether this receptor is responsible for the sexual differences observed in the physiopathology of the liver.
Asunto(s)
Receptor alfa de Estrógeno/metabolismo , Estrógenos/metabolismo , Hígado/metabolismo , Caracteres Sexuales , Activación Metabólica/genética , Animales , Metabolismo Energético/genética , Receptor alfa de Estrógeno/genética , Femenino , Regulación de la Expresión Génica , Inactivación Metabólica/genética , Masculino , Ratones , Reproducción/genética , Factores Sexuales , Transducción de Señal , Transcripción GenéticaRESUMEN
Decades of studies provided a detailed view of the mechanism of estrogen receptor-α (ERα) regulated gene transcription and the physio-pathological relevance of the genetic programs controlled by this receptor in a variety of tissues. However, still limited is our knowledge on the regulation of ERα synthesis. Preliminary observations showed that the expression of ERα is cell cycle regulated. Here, we have demonstrated that a well described polymorphic sequence in the first intron of ERα (PvuII and XbaI) has a key role in regulating the ERα content in cycling cells. We have shown that the RNA Pol II (Pol II) elongation is blocked at the polymorphic site and that the proto-oncogene c-MYB modulates the release of the pausing polymerase. It is well known that the two SNPs are associated to an increased risk, progression, survival and mortality of endocrine-related cancers, here we have demonstrated that the c-MYB-dependent release of Pol II at a specific phase of the cell cycle is facilitated by the px haplotype, thus leading to a higher ERα mitogenic signal. In breast cancer, this mechanism is disrupted when the hormone refractory phenotype is established; therefore, we propose this oscillator as a novel target for the development of therapies aimed at sensitizing breast cancer resistant to hormonal treatments. Because PvuII and XbaI were associated to a broad range physio-pathological conditions beside neoplastic transformation, we expect that the ERα oscillator contributes to the regulation of the estrogen signal in several tissues.
Asunto(s)
Ciclo Celular/fisiología , Transformación Celular Neoplásica/metabolismo , Receptor alfa de Estrógeno/metabolismo , Regulación de la Expresión Génica/fisiología , ARN Polimerasa II/metabolismo , Análisis de Varianza , Inmunoprecipitación de Cromatina , Cartilla de ADN/genética , Receptor alfa de Estrógeno/genética , Genotipo , Humanos , Células MCF-7 , Plásmidos/genética , Polimorfismo de Nucleótido Simple , Proto-Oncogenes Mas , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
TFEB and TFE3 belong to the MiT/TFE family of transcription factors that bind identical DNA responsive elements in the regulatory regions of target genes. They are involved in regulating lysosomal biogenesis, function, exocytosis, autophagy, and lipid catabolism. Precise control of TFEB and TFE3 activity is crucial for processes such as senescence, stress response, energy metabolism, and cellular catabolism. Dysregulation of these factors is implicated in various diseases, thus researchers have explored pharmacological approaches to modulate MiT/TFE activity, considering these transcription factors as potential therapeutic targets. However, the physiological complexity of their functions and the lack of suitable in vivo tools have limited the development of selective MiT/TFE modulating agents. Here, we have created a reporter-based biosensor, named CLEARoptimized, facilitating the pharmacological profiling of TFEB- and TFE3-mediated transcription. This innovative tool enables the measurement of TFEB and TFE3 activity in living cells and mice through imaging and biochemical techniques. CLEARoptimized consists of a promoter with six coordinated lysosomal expression and regulation motifs identified through an in-depth bioinformatic analysis of the promoters of 128 TFEB-target genes. The biosensor drives the expression of luciferase and tdTomato reporter genes, allowing the quantification of TFEB and TFE3 activity in cells and in animals through optical imaging and biochemical assays. The biosensor's validity was confirmed by modulating MiT/TFE activity in both cell culture and reporter mice using physiological and pharmacological stimuli. Overall, this study introduces an innovative tool for studying autophagy and lysosomal pathway modulation at various biological levels, from individual cells to the entire organism.Abbreviations: CLEAR: coordinated lysosomal expression and regulation; MAR: matrix attachment regions; MiT: microphthalmia-associated transcription factor; ROI: region of interest; TBS: tris-buffered saline; TF: transcription factor; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; TH: tyrosine hydroxylase; TK: thymidine kinase; TSS: transcription start site.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Genes Reporteros , Lisosomas , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Ratones , Lisosomas/metabolismo , Humanos , Regiones Promotoras Genéticas/genética , Autofagia , Técnicas Biosensibles/métodos , Regulación de la Expresión Génica/efectos de los fármacosRESUMEN
The treatment of glioblastoma (GBM) faces significant challenges due to the difficulty of delivering drugs through the blood-brain barrier (BBB). Extracellular vesicles (EVs) have emerged as potential carriers for targeted drug delivery to brain tumors. However, their use and distribution in the presence of an intact BBB and their ability to target GBM tissue are still under investigation. This study explored the use of EVs for GBM targeting across the BBB. Canine plasma EVs from healthy dogs and dogs with glioma were isolated, characterized, and loaded with diagnostic agents. Biodistribution studies were conducted in healthy murine models and a novel intranasal model that preserved BBB integrity while initiating early-stage GBM growth. This model assessed EVs' potential for delivering the contrast agent gadoteric acid to intracranial tumors. Imaging techniques, such as bioluminescence and MRI, confirmed EVs' targeting and delivery capabilities thus revealing a selective accumulation of canine glioma-derived EVs in brain tissue under physiological conditions. In the model of brain tumor, MRI experiments demonstrated the ability of EVs to accumulate gadoteric acid within GBM to enhance contrast of the tumoral mass, even when BBB integrity is maintained. This study underscores the potential of EVs derived from glioma for the targeted delivery of drugs to glioblastoma. EVs from dogs with glioma showed capacity to traverse the BBB and selectively accumulate within the brain tumor. Overall, this research represents a foundation for the application of autologous EVs to precision glioblastoma treatment, addressing the challenge of BBB penetration and targeting specificity in brain cancer therapy.
Asunto(s)
Neoplasias Encefálicas , Vesículas Extracelulares , Glioblastoma , Glioma , Perros , Animales , Ratones , Glioblastoma/diagnóstico por imagen , Barrera Hematoencefálica , Distribución Tisular , Neoplasias Encefálicas/diagnóstico por imagen , Quelantes , Medios de ContrasteRESUMEN
Microglia are heterogenous cells characterized by distinct populations each contributing to specific biological processes in the nervous system, including neuroprotection. To elucidate the impact of sex-specific microglia heterogenicity to the susceptibility of neuronal stress, we video-recorded with time-lapse microscopy the changes in shape and motility occurring in primary cells derived from mice of both sexes in response to pro-inflammatory or neurotoxic stimulations. With this morpho-functional analysis, we documented distinct microglia subpopulations eliciting sex-specific responses to stimulation: male microglia tended to have a more pro-inflammatory phenotype, while female microglia showed increased sensitivity to conduritol-B-epoxide (CBE), a small molecule inhibitor of glucocerebrosidase, the enzyme encoded by the GBA1 gene, mutations of which are the major risk factor for Parkinson's Disease (PD). Interestingly, glucocerebrosidase inhibition particularly impaired the ability of female microglia to enhance the Nrf2-dependent detoxification pathway in neurons, attenuating the sex differences observed in this neuroprotective function. This finding is consistent with the clinical impact of GBA1 mutations, in which the 1.5-2-fold reduced risk of developing idiopathic PD observed in female individuals is lost in the GBA1 carrier population, thus suggesting a sex-specific role for microglia in the etiopathogenesis of PD-GBA1.
Asunto(s)
Enfermedad de Parkinson , Animales , Femenino , Masculino , Ratones , Glucosilceramidasa/metabolismo , Microglía/metabolismo , Neuronas/metabolismo , Enfermedad de Parkinson/metabolismoRESUMEN
Detailed knowledge of drug metabolism is relevant information provided by preclinical drug development research. Oxidative enzymes such as those belonging to P450 family of cytochromes (CYP) play a prominent role in drug metabolism. Here, we propose an innovative method based on bioluminescence in vivo imaging which has the potential to simplify the in vivo measurement of CYP activity also providing a dynamic measure of the effects of a drug on a specific P450 enzyme complex in a living mouse. The method is based on a pro-luciferin which can be converted into the active luciferase substrate by a specific P450 activity. The pro-luciferin is administered to a luciferase reporter mouse which produces luminescent signals in relation to the cytochrome activity present in each tissue. The photon emission generated can be easily localized and quantified by optical imaging. To demonstrate the validity of the system, we pharmacologically induced hepatic Cyp3a in the reporter mouse and proved that pro-luciferin administration generates a Cyp3a selective signal in the chest area that can be efficiently detected by optical imaging. The kind of tool generated has the potential to be exploited for the study of additional CYPs.
Asunto(s)
Citocromo P-450 CYP3A/metabolismo , Acetales/metabolismo , Animales , Dexametasona/farmacología , Luciferina de Luciérnaga/análogos & derivados , Luciferina de Luciérnaga/metabolismo , Genes Reporteros , Hígado/efectos de los fármacos , Hígado/metabolismo , Luciferasas de Luciérnaga/genética , Luciferasas de Luciérnaga/metabolismo , Sustancias Luminiscentes/metabolismo , Mediciones Luminiscentes , Masculino , Ratones , Ratones Transgénicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Distribución TisularRESUMEN
In spite of the fact that women spend 1/3 of their lives in postmenopause, the search for appropriate therapies able to counteract the derangements associated with the menopause still represents a sort of sought after the "Holy Grail."Nowadays, the combination of estrogens and selective estrogen receptor modulators (SERMs), a class of compounds with a mixed agonist/antagonistic activity on the estrogen receptor (ER) in various tissues, represents the most promising approach to improve postmenopausal women's health, by preserving the benefits while avoiding the side effects of estrogen-based therapy.Given their complex mechanisms of action, the evaluation of SERM activity in combination with conjugated estrogens (CE) requires a multifactorial analysis that takes into account the multifaceted and dynamic effects of these compounds in target tissues, even in relation to the physiological/pathological status.To accomplish such a goal, we took advantage of the ERE-Luc model, a reporter mouse that allows the monitoring of ER transcriptional activity in a spatio-temporal dimension. Cluster analyses performed on in vivo/ex vivo bioluminescence (BLI) data and ex vivo luciferase activity enabled to sustain the combination of CE plus bazedoxifene (TSEC, tissue-selective estrogen complex) as a valuable option for the pharmacological treatment of the postmenopause.
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Estrógenos Conjugados (USP) , Receptores de Estrógenos , Animales , Estrógenos/farmacología , Estrógenos Conjugados (USP)/efectos adversos , Femenino , Humanos , Menopausia , Ratones , Receptores de Estrógenos/genética , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Moduladores Selectivos de los Receptores de Estrógeno/uso terapéuticoRESUMEN
Objective: To conduct a systematic review of the currently available literature on the use of ICG to guide surgical dissection in gastrointestinal (GI) cancer surgery. Background: Real-time indocyanine green (ICG) fluorescence-guided surgery has the potential to enhance surgical outcomes by increasing patient-tailored oncological precision. Methods: MEDLINE, PubMed, EMBASE, and Google Scholar were searched for publications on the use of ICG as a contrast agent in GI cancer surgery until December 2020. Perfusion studies were excluded. Quality of the studies was assessed with the Methodological Index for nonrandomized Studies or Jadad scale for randomized controlled trials. A narrative synthesis of the results was provided, with descriptive statistics when appropriate. Results: Seventy-eight studies were included. ICG was used for primary tumor and metastases localization, for sentinel lymph node detection, and for lymph flow mapping. The detection rate for primary colorectal and gastric tumors was 100% after preoperative ICG endoscopic injection. For liver lesions, the detection rate after intravenous ICG infusion was 80% and up to 100% for lesions less than 8 mm from the liver surface. The detection rate for sentinel lymph nodes was 89.8% for esophageal, 98.6% for gastric, 87.4% for colorectal, and 83.3% for anal tumors, respectively. In comparative studies, ICG significantly increases the quality of D2 lymphadenectomy in oncological gastrectomy. Conclusion: The use of ICG as a guiding tool for dissection in GI surgery is promising. Further evidence from high-quality studies on larger sample sizes is needed to assess whether ICG-guided surgery may become standard of care.
RESUMEN
Extracellular vesicles (EVs) are a complex and heterogeneous population of nanoparticles involved in cell-to-cell communication. Recently, numerous studies have indicated the potential of EVs as therapeutic agents, drug carriers and diagnostic tools. However, the results of these studies are often difficult to evaluate, since different characterization methods are used to assess the purity, physical and biochemical characteristics of the EV samples. In this study, we compared four methods for the EV sample characterization and purity assessment: i) the particle-to-protein ratio based on particle analyses with nanoparticle tracking and protein concentration by bicinchoninic acid assay, ii) Western Blot analysis for specific EV biomarkers, iii) two spectroscopic lipid-to-protein ratios by either the attenuated total reflection Fourier transform infrared (ATR-FTIR) or Raman spectroscopy. The results confirm the value of Raman and ATR-FTIR spectroscopy as robust, fast and operator independent tools that require only a few microliters of EV sample. We propose that the spectroscopic lipid-to-protein (Li/Pr) ratios are reliable parameters for the purity assessment of EV preparations. Moreover, apart from determining protein concentrations, we show that ATR-FTIR spectroscopy can also be used for indirect measurements of EV concentrations. Nevertheless, the Li/Pr ratios do not represent full characterization of the EV preparations. For a complete characterization of selected EV preparations, we recommend also additional use of particle size distribution and EV biomarker analysis.
Asunto(s)
Vesículas Extracelulares , Espectrometría Raman , Portadores de Fármacos/metabolismo , Vesículas Extracelulares/metabolismo , Proteínas/análisis , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Computed tomography (CT) is a diagnostic medical imaging modality commonly used to detect disease and injury. Contrast agents containing iodine, such as iohexol, are frequently used in CT examinations to more clearly differentiate anatomic structures and to detect and characterize abnormalities, including tumors. However, these contrast agents do not have a specific tropism for cancer cells, so the ability to detect tumors is severely limited by the degree of vascularization of the tumor itself. Identifying delivery systems allowing enrichment of contrast agents at the tumor site would increase the sensitivity of detection of tumors and metastases, potentially in organs that are normally inaccessible to contrast agents, such as the CNS. Recent work from our laboratory has identified cancer patient-derived extracellular vesicles (PDEVs) as effective delivery vehicles for targeting diagnostic drugs to patients' tumors. Based on this premise, we explored the possibility of introducing iohexol into PDEVs for targeted delivery to neoplastic tissue. Here, we provide preclinical proof-of-principle for the tumor-targeting ability of iohexol-loaded PDEVs, which resulted in an impressive accumulation of the contrast agent selectively into the neoplastic tissue, significantly improving the ability of the contrast agent to delineate tumor boundaries.
RESUMEN
Through intracellular receptors, estrogens control growth, differentiation and function of not only reproductive tissues, but also other systems. Estrogen receptors are ligand-dependent transcription factors whose activity is modulated either by estrogens, or by alternative intracellular signaling pathways downstream of growth factors and neurotransmitters. To determine the dynamics of estrogen receptor activity and the dependence of estrogen receptor on 17beta-estradiol in vivo, we generated a transgenic mouse that expresses a luciferase reporter gene under the control of activated estrogen receptors. As expected, luciferase activity, monitored with a cooled charged coupled device camera, paralleled circulating estrogen levels in reproductive tissues and in liver, indicating that the peak transcriptional activity of the estrogen receptor occurred at proestrus. In contrast, in tissues such as bone and brain, the peak activity of estrogen receptors was observed at diestrus. These tissue-specific responses are masked when mice undergo conventional hormone treatment. We also demonstrate that estrogen receptors are active in immature mice before gonadal production of sex hormones as well as in ovariectomized adult mice. These findings emphasize the importance of hormone-independent activation of the estrogen receptor, and have implications for the therapeutic use of estrogens, such as hormone replacement therapy.
Asunto(s)
Estradiol/metabolismo , Receptores de Estrógenos/metabolismo , Transcripción Genética , Animales , Diagnóstico por Imagen , Ciclo Estral/fisiología , Femenino , Genes Reporteros , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ovariectomía , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , Distribución TisularRESUMEN
From the past decade, extracellular vesicles (EVs) have attracted considerable attention as tools for the selective delivery of anti-neoplastic drugs to cancer tissues. Compared to other nanoparticles, EVs display interesting unique features including immune compatibility, low toxicity and the ability to encapsulate a large variety of small- and macro-molecules. However, in virtually all studies, investigations on EVs have been focused on fully transformed cancers: the possibility to apply EV technology also to early-stage tumors has never been explored. Methods: Herein, we studied the ability of cancer-derived EVs to recognize and deliver their cargo also to incipient cancers. To this purpose, EV biodistribution was studied in MMTV-NeuT genetically modified mice during early mammary transformation, in fully developed breast tumors and in the normal gland of wild type syngeneic mice. EVs were loaded with indocyanine green (ICG), a near-infrared (NIR) dye together with oncolytic viruses and i.v. injected in mice. The nanoparticle biodistribution was assayed by in vivo and ex vivo optical imaging (detecting the ICG) and semiquantitative real-time PCR (measuring the adenoviral genome) in different tissues. Results: Our results demonstrate the ability of cancer-derived EVs to recognize early-stage neoplastic tissues opening the possibility to selectively deliver theranostics also for tumor prevention. Conclusions: Taken together our study demonstrates the ability of EVs to recognize and deliver diagnostic and therapeutic agents not only to fully transformed tissues but also to early stage tumors. These findings pave the way for the synthesis of "universal" EVs-based formulation for targeted cancer therapy.