RESUMEN
Accumulation of senescent cells with age leads to tissue dysfunction and related diseases. Their detection in vivo still constitutes a challenge in aging research. We describe the generation of a fluorogenic probe (sulfonic-Cy7Gal) based on a galactose derivative, to serve as substrate for ß-galactosidase, conjugated to a Cy7 fluorophore modified with sulfonic groups to enhance its ability to diffuse. When administered to male or female mice, ß-galactosidase cleaves the O-glycosidic bond, releasing the fluorophore that is ultimately excreted by the kidneys and can be measured in urine. The intensity of the recovered fluorophore reliably reflects an experimentally controlled load of cellular senescence and correlates with age-associated anxiety during aging and senolytic treatment. Interestingly, our findings with the probe indicate that the effects of senolysis are temporary if the treatment is discontinued. Our strategy may serve as a basis for developing fluorogenic platforms designed for easy longitudinal monitoring of enzymatic activities in biofluids.
Asunto(s)
Envejecimiento , Senescencia Celular , Masculino , Femenino , Ratones , Animales , Envejecimiento/fisiología , Senescencia Celular/fisiología , beta-Galactosidasa , Riñón , Colorantes FluorescentesRESUMEN
Despite advances in breast cancer treatment, it remains the leading cause of cancer-related death in women worldwide. In this context, microRNAs have emerged as potential therapeutic targets but still present some limitations for in vivo applications. Particularly, miR-200c-3p is a well-known tumor suppressor microRNA that inhibits tumor progression and metastasis in breast cancer through downregulating ZEB1 and ZEB2. Based on the above, we describe the design and validation of a nanodevice using mesoporous silica nanoparticles for miR-200c-3p delivery for breast cancer treatment. We demonstrate the biocompatibility of the synthesized nanodevices as well as their ability to escape from endosomes/lysosomes and inhibit tumorigenesis, invasion, migration, and proliferation of tumor cells in vitro. Moreover, tumor targeting and effective delivery of miR-200c-3p from the nanoparticles in vivo are confirmed in an orthotopic breast cancer mouse model, and the therapeutic efficacy is also evidenced by a decrease in tumor size and lung metastasis, while showing no signs of toxicity. Overall, our results provide evidence that miR-200c-3p-loaded nanoparticles are a potential strategy for breast cancer therapy and a safe and effective system for tumor-targeted delivery of microRNAs.
Asunto(s)
Neoplasias Pulmonares , MicroARNs , Nanopartículas , Femenino , Ratones , Animales , Dióxido de Silicio , MicroARNs/genética , Neoplasias Pulmonares/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Movimiento Celular/genética , Proliferación Celular/genéticaRESUMEN
Kidney damage generates changes at the phenotypic and genotypic levels that allow its monitoring using different biomarkers in blood, urine or serum. Among these biomarkers, kidney failure causes the urine overrepresentation of the alanine aminopeptidase (APN) enzyme. Here, we describe the design of a molecular probe (NB-ALA) based on the Nile Blue fluorophore (NB), which can detect the APN enzyme in urine by simple fluorometric measurements.
Asunto(s)
Antígenos CD13 , Colorantes Fluorescentes , Biomarcadores , Riñón , Sondas Moleculares , Enfermedades Renales/diagnósticoRESUMEN
Cellular senescence is a stable cell cycle arrest in response to stress or other damage stimuli to maintain tissue homeostasis. However, the accumulation of senescent cells can lead to the progression of various senescence-related disorders. In this paper, we describe the development of a ß-galactosidase-activatable near-infrared (NIR) senoprobe, NBGal, for the detection of senescent cells based on the use of the FDA-approved Nile blue (NB) fluorophore. NBGal was validated in chemotherapeutic-induced senescence cancer models in vitro using SK-Mel 103 and 4T1 cell lines. In vivo monitoring of cellular senescence was evaluated in orthotopic triple-negative breast cancer-bearing mice treated with palbociclib to induce senescence. In all cases, NBGal exhibited a selective tracking of senescent cells mainly ascribed to the overexpressed ß-galactosidase enzyme responsible for hydrolyzing the NBGal probe generating the highly emissive NB fluorophore. In this way, NBGal has proven to be a qualitative, rapid, and minimally invasive probe that allows the direct detection of senescent cells in vivo.
Asunto(s)
Senescencia Celular , Ratones , Animales , Puntos de Control del Ciclo Celular/fisiología , Línea Celular , beta-Galactosidasa/metabolismoRESUMEN
Triple-negative breast cancer (TNBC) is a very aggressive subtype of breast cancer with a poor prognosis and limited effective therapeutic options. Induction of senescence, arrest of cell proliferation, has been explored as an effective method to limit tumor progression in metastatic breast cancer. However, relapses occur in some patients, possibly as a result of the accumulation of senescent tumor cells in the body after treatment, which promote metastasis. In this study, we explored the combination of senescence induction and the subsequent removal of senescent cells (senolysis) as an alternative approach to improve outcomes in TNBC patients. We demonstrate that a combination treatment, using the senescence-inducer palbociclib and the senolytic agent navitoclax, delays tumor growth and reduces metastases in a mouse xenograft model of aggressive human TNBC (hTNBC). Furthermore, considering the off-target effects and toxicity derived from the use of navitoclax, we propose a strategy aimed at minimizing the associated side effects. We use a galacto-conjugated navitoclax (nav-Gal) as a senolytic prodrug that can preferentially be activated by ß-galactosidase overexpressed in senescent cells. Concomitant treatment with palbociclib and nav-Gal in vivo results in the eradication of senescent hTNBC cells with consequent reduction of tumor growth, while reducing the cytotoxicity of navitoclax. Taken together, our results support the efficacy of combination therapy of senescence-induction with senolysis for hTNBC, as well as the development of a targeted approach as an effective and safer therapeutic opportunity.
Asunto(s)
Neoplasias de la Mama Triple Negativas , Humanos , Animales , Ratones , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Senoterapéuticos , Recurrencia Local de Neoplasia , Modelos Animales de Enfermedad , Línea Celular TumoralRESUMEN
A new method for senescent cell detection is described, which is based on lipofuscin labeling with a fluorescent reporter through a biorthogonal strain-promoted azide-alkyne cycloaddition. The sensing protocol involves a first step where the interaction of lipofuscin with a Sudan Black B derivative containing an azide moiety (SBB-N3 ) is carried out. In the final step, the azide moiety reacts with a fluorophore containing a cyclooctene ring (BODIPY). The efficacy of this two-step protocol is assessed in senescent melanoma SK-MEL-103 cells, senescent triple-negative breast cancer MDA-MB-231 cells and senescent WI-38 fibroblasts. In all cases, a clear fluorescence pattern was observed in senescent cells, compared to proliferative cells, only when the SBB-N3 -BODIPY probe was formed. Our results provide an alternative tool for the detection of senescent cells, based on an in situ bio-orthogonal reaction for lipofuscin labeling.
Asunto(s)
Azidas , Lipofuscina , Alquinos , Reacción de Cicloadición , Colorantes Fluorescentes , Senescencia CelularAsunto(s)
MicroARNs , Neoplasias de la Mama Triple Negativas , Humanos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Ciclooxigenasa 2 , MicroARNs/genética , Regulación Neoplásica de la Expresión Génica , Doxorrubicina/farmacología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Proteínas de NeoplasiasRESUMEN
Many anticancer agents used in clinics induce premature senescence in healthy tissues generating accelerated aging processes and adverse side-effects in patients. Cardiotoxicity is a well-known limiting factor of anticancer treatment with doxorubicin (DOX), a very effective anthracycline widely used as antitumoral therapy in clinical practice, that leads to long-term morbidity and mortality. DOX exposure severely affects the population of cardiac cells in both mice and human hearts by inducing premature senescence, which may represent the molecular basis of DOX-induced cardiomyopathy. Here, we demonstrate that senescence induction in the heart contributes to impaired cardiac function in mice upon DOX treatment. Concomitant elimination of senescent cells with the senolytic Navitoclax in different formulations produces a significant decrease in senescence and cardiotoxicity markers together with the restoration of the cardiac function in mice followed by echocardiography. These results evidence the potential clinical use of senolytic therapies to alleviate cardiotoxicities induced in chemotherapy-treated patients.
Asunto(s)
Cardiomiopatías , Cardiotoxicidad , Animales , Antibióticos Antineoplásicos/toxicidad , Cardiomiopatías/inducido químicamente , Cardiomiopatías/prevención & control , Cardiotoxicidad/tratamiento farmacológico , Doxorrubicina/efectos adversos , Humanos , Ratones , Miocitos Cardíacos , SenoterapéuticosRESUMEN
ß-Galactosidase (ß-Gal) is a widely used enzyme as a reporter gene in the field of molecular biology which hydrolyzes the ß-galactosides into monosaccharides. ß-Gal is an essential enzyme in humans and its deficiency or its overexpression results in several rare diseases. Cellular senescence is probably one of the most relevant physiological disorders that involve ß-Gal enzyme. In this review, we assess the progress made to date in the design of molecular-based probes for the detection of ß-Gal both in vitro and in vivo. Most of the reported molecular probes for the detection of ß-Gal consist of a galactopyranoside residue attached to a signalling unit through glycosidic bonds. The ß-Gal-induced hydrolysis of the glycosidic bonds released the signalling unit with remarkable changes in color and/or emission. Additional examples based on other approaches are also described. The wide applicability of these probes for the rapid and in situ detection of de-regulation ß-Gal-related diseases has boosted the research in this fertile field.
Asunto(s)
Colorantes Fluorescentes/química , Galactosa/análogos & derivados , beta-Galactosidasa/análisis , Animales , Senescencia Celular , Colorimetría/métodos , Pruebas de Enzimas/métodos , Galactósidos/química , Humanos , Hidrólisis , Sondas Moleculares/químicaRESUMEN
Cellular senescence is a state of stable cell cycle arrest that can negatively affect the regenerative capacities of tissues and can contribute to inflammation and the progression of various aging-related diseases. Advances in the in vivo detection of cellular senescence are still crucial to monitor the action of senolytic drugs and to assess the early onset or accumulation of senescent cells. Here, we describe a naphthalimide-styrene-based probe (HeckGal) for the detection of cellular senescence both in vitro and in vivo. HeckGal is hydrolyzed by the increased lysosomal ß-galactosidase activity of senescent cells, resulting in fluorescence emission. The probe was validated in vitro using normal human fibroblasts and various cancer cell lines undergoing senescence induced by different stress stimuli. Remarkably, HeckGal was also validated in vivo in an orthotopic breast cancer mouse model treated with senescence-inducing chemotherapy and in a renal fibrosis mouse model. In all cases, HeckGal allowed the unambiguous detection of senescence in vitro as well as in tissues and tumors in vivo. This work is expected to provide a potential technology for senescence detection in aged or damaged tissues.
Asunto(s)
Naftalimidas , Estireno , Animales , Senescencia Celular , Fibroblastos , Ratones , FotonesRESUMEN
Benzene is a highly toxic aromatic hydrocarbon. Inhaling benzene can cause dizziness, vertigo, headaches, aplasia, mutations and, in the most extreme cases, cancer. Trans,trans-muconic acid (t,t-MA) is one of the metabolization products of benzene. Although different analytical methods have been reported for the determination of t,t-MA, these are often expensive, require trained personnel, are not suitable for on-site measurements, and use hazardous organic solvents. For these reasons, the development of reliable, selective and sensitive methods for rapid and in situ detection of t,t-MA are of importance. Addressing this challenge, a nanodevice for the selective and sensitive quantification of t,t-MA in urine is reported. The nanodevice used is achieved using mesoporous silica nanoparticles loaded with a dye reporter and capped with a dicopper(II) azacryptand. Pore opening and payload release is induced rapidly (10â min) and selectively with t,t-MA in urine, using a simple fluorimeter without sample pretreatment.
Asunto(s)
Benceno , Nanopartículas , Biomarcadores , Dióxido de Silicio/química , Ácido Sórbico/análogos & derivados , Ácido Sórbico/química , Ácido Sórbico/metabolismoRESUMEN
The characteristics and electromechanical properties of conductive polymers together to their biocompatibility have boosted their application as a suitable tool in regenerative medicine and tissue engineering. However, conducting polymers as drug release materials are far from being ideal. A possibility to overcome this drawback is to combine conducting polymers with on-command delivery particles with inherent high-loading capacity. In this scenario, we report here the preparation of conduction polymers containing gated mesoporous silica nanoparticles (MSN) loaded with a cargo that is delivered on command by electro-chemical stimuli increasing the potential use of conducting polymers as controlled delivery systems. MSNs are loaded with Rhodamine B (Rh B), anchored to the conductive polymer poly(3,4-ethylenedioxythiophene) (PEDOT) doped with poly[(4-styrenesulfonic acid)-co-(maleic acid)], functionalized with a bipyridinium derivative and pores are capped with heparin (P3) by electrostatic interactions. P3 releases the entrapped cargo after the application of -640â¯mV voltage versus the saturated calomel electrode (SCE). Pore opening in the nanoparticles and dye delivery is ascribed to both (i) the reduction of the grafted bipyridinium derivative and (ii) the polarization of the conducting polymer electrode to negative potentials that induce detachment of positively charged heparin from the surface of the nanoparticles. Biocompatibility and cargo release studies were carried out in HeLa cells cultures.
Asunto(s)
Nanopartículas , Dióxido de Silicio , Compuestos Bicíclicos Heterocíclicos con Puentes , Células HeLa , Humanos , Polímeros , PorosidadRESUMEN
Inâ vivo detection of cellular senescence is accomplished by using mesoporous silica nanoparticles loaded with the NIR-FDA approved Nile blue (NB) dye and capped with a galactohexasaccharide (S3). NB emission at 672â nm is highly quenched inside S3, yet a remarkable emission enhancement is observed upon cap hydrolysis in the presence of ß-galactosidase and dye release. The efficacy of the probe to detect cellular senescence is tested inâ vitro in melanoma SK-Mel-103 and breast cancer 4T1 cells and inâ vivo in palbociclib-treated BALB/cByJ mice bearing breast cancer tumor.
Asunto(s)
Senescencia Celular/inmunología , Colorantes Fluorescentes/uso terapéutico , Animales , Femenino , Humanos , Ratones , OxazinasRESUMEN
Pharmacologically active compounds with preferential cytotoxic activity for senescent cells, known as senolytics, can ameliorate or even revert pathological manifestations of senescence in numerous preclinical mouse disease models, including cancer models. However, translation of senolytic therapies to human disease is hampered by their suboptimal specificity for senescent cells and important toxicities that narrow their therapeutic windows. We have previously shown that the high levels of senescence-associated lysosomal ß-galactosidase (SA-ß-gal) found within senescent cells can be exploited to specifically release tracers and cytotoxic cargoes from galactose-encapsulated nanoparticles within these cells. Here, we show that galacto-conjugation of the BCL-2 family inhibitor Navitoclax results in a potent senolytic prodrug (Nav-Gal), that can be preferentially activated by SA-ß-gal activity in a wide range of cell types. Nav-Gal selectively induces senescent cell apoptosis and has a higher senolytic index than Navitoclax (through reduced activation in nonsenescent cells). Nav-Gal enhances the cytotoxicity of standard senescence-inducing chemotherapy (cisplatin) in human A549 lung cancer cells. Concomitant treatment with cisplatin and Nav-Gal in vivo results in the eradication of senescent lung cancer cells and significantly reduces tumour growth. Importantly, galacto-conjugation reduces Navitoclax-induced platelet apoptosis in human and murine blood samples treated ex vivo, and thrombocytopenia at therapeutically effective concentrations in murine lung cancer models. Taken together, we provide a potentially versatile strategy for generating effective senolytic prodrugs with reduced toxicities.
Asunto(s)
Compuestos de Anilina/farmacología , Antineoplásicos/farmacología , Plaquetas/efectos de los fármacos , Galactosa/farmacología , Profármacos/farmacología , Sulfonamidas/farmacología , Compuestos de Anilina/química , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Galactosa/química , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Profármacos/síntesis química , Profármacos/química , Sulfonamidas/química , Células Tumorales CultivadasRESUMEN
Metal oxide nanoparticles supported on graphene exhibit high catalytic activity for oxidation, reduction and coupling reactions. Here we show that pyrolysis at 900 °C under inert atmosphere of copper(II) nitrate embedded in chitosan films affords 1.1.1 facet-oriented copper nanoplatelets supported on few-layered graphene. Oriented (1.1.1) copper nanoplatelets on graphene undergo spontaneous oxidation to render oriented (2.0.0) copper(I) oxide nanoplatelets on few-layered graphene. These films containing oriented copper(I) oxide exhibit as catalyst turnover numbers that can be three orders of magnitude higher for the Ullmann-type coupling, dehydrogenative coupling of dimethylphenylsilane with n-butanol and C-N cross-coupling than those of analogous unoriented graphene-supported copper(I) oxide nanoplatelets.
RESUMEN
Copper nanoparticles (NPs) supported on a series of undoped and doped graphene materials (Gs) have been obtained by pyrolysis of alginate or chitosan biopolymers, modified or not with boric acid, containing Cu(2+) ions at 900 °C under inert atmosphere. The resulting Cu-G materials containing about 17â wt % Cuâ NPs (from 10 to 200â nm) exhibit high catalytic activity for the dehydrogenative coupling of silanes with alcohols. The optimal material consisting on Cu-(B)G is more efficient than Cuâ NPs on other carbon supports.