RESUMEN
Polyrotaxane is a supramolecular assembly consisting of multiple cyclic molecules threaded by a linear polymer. One of the unique properties of polyrotaxane is molecular mobility, cyclic molecules moving along the linear polymer. Molecular mobility of polyrotaxane surfaces affects cell spreading, differentiation, and other cell-related aspects through changing subcellular localization of yes-associated proteins (YAPs). Subcellular YAP localization is also related to cell senescence derived from oxidative stress, which is known to cause cancer, diabetes, and heart disease. Herein, the effects of polyrotaxane surface molecular mobility on subcellular YAP localization and cell senescence following H2 O2 -induced oxidative stress are evaluated in human mesenchymal stem cells (HMSCs) cultured on polyrotaxane surfaces with different molecular mobilities. Oxidative stress promotes cytoplasmic YAP localization in HMSCs on high-mobility polyrotaxane surfaces; however, low-mobility polyrotaxane surfaces more effectively maintain nuclear YAP localization, exhibiting lower senescence-associated ß-galactosidase activity and senescence-related gene expression and DNA damage than that seen with the high-mobility surfaces. These results suggest that the molecular mobility of polyrotaxane surfaces regulates subcellular YAP localization, thereby protecting HMSCs from oxidative stress-induced cell senescence. Applying the molecular mobility of polyrotaxane surfaces to implantable scaffolds can provide insights into the prevention and treatment of diseases caused by oxidative stress.
Asunto(s)
Senescencia Celular , Ciclodextrinas , Células Madre Mesenquimatosas , Estrés Oxidativo , Polímeros , Rotaxanos , Humanos , Senescencia Celular/efectos de los fármacos , Senescencia Celular/genética , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Polímeros/farmacología , Rotaxanos/farmacología , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Proteínas Señalizadoras YAP/metabolismo , beta-Galactosidasa/metabolismo , Daño del ADN/efectos de los fármacos , Andamios del Tejido/química , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Perfilación de la Expresión Génica , Ciclodextrinas/farmacologíaRESUMEN
Oxidative damage has been implicated in the pathogenesis of numerous disorders by affecting the normal functions of several tissues. Further, oxidative stress acts within cells to influence cell morphology and the behavior of cell migration. The movement and migration of cells are crucial during the development of organisms as they transition from embryo to adult, and for the homeostasis of adult tissues. Epicatechin (EC) is a natural flavonoid derived mostly from tea, chocolate, and red wine. We investigated the protective impact of EC on D-galactose(D-gal)/rotenone-injured NIH3T3 cells and found alterations in cell dynamics throughout the procedure. The results reveal that D-gal/rotenone stimulation can cause the cell area to expand and the number of cellular protrusions to increase. EC intervention can considerably minimize the oxidative damage of rotenone on NIH3T3 cells (p < 0.05) but showed little influence on cell damage induced by D-gal. Furthermore, the corrective ability of EC as an antioxidant is reflected in a dose-dependent effect on cell movement, including variations in movement speed and distance. Overall, from the perspective of cell morphology and cell motility, EC has a good protective impact on cells harmed by rotenone induced oxidative damage, as well as corrective properties as an antioxidant to balance intracellular oxidative stress, which allowing for a more comprehensive evaluation of antioxidant performance of EC.
Asunto(s)
Antioxidantes , Catequina , Animales , Ratones , Antioxidantes/farmacología , Catequina/farmacología , Células 3T3 NIH , Estrés Oxidativo/efectos de los fármacos , Rotenona/farmacología , Galactosa/farmacología , Forma de la Célula/efectos de los fármacos , Extensiones de la Superficie Celular/efectos de los fármacos , Sustancias Protectoras/farmacologíaRESUMEN
Breast cancer is characterized by a hypoxic microenvironment inside the tumor mass, contributing to cell metastatic behavior. Hypoxia induces the expression of hypoxia-inducible factor (HIF-1α), a transcription factor for genes involved in angiogenesis and metastatic behavior, including the vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs), and integrins. Integrin receptors play a key role in cell adhesion and migration, being considered targets for metastasis prevention. We investigated the migratory behavior of hypoxia-cultured triple-negative breast cancer cells (TNBC) and endothelial cells (HUVEC) upon αvß3 integrin blocking with DisBa-01, an RGD disintegrin with high affinity to this integrin. Boyden chamber, HUVEC transmigration, and wound healing assays in the presence of DisBa-01 were performed in hypoxic conditions. DisBa-01 produced similar effects in the two oxygen conditions in the Boyden chamber and transmigration assays. In the wound healing assay, hypoxia abolished DisBa-01's inhibitory effect on cell motility and decreased the MMP-9 activity of conditioned media. These results indicate that αvß3 integrin function in cell motility depends on the assay and oxygen levels, and higher inhibitor concentrations may be necessary to achieve the same inhibitory effect as in normoxia. These versatile responses add more complexity to the role of the αvß3 integrin during tumor progression.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Células Endoteliales/metabolismo , Integrina alfaVbeta3/antagonistas & inhibidores , Integrina alfaVbeta3/metabolismo , Hipoxia Tumoral , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Venenos de Crotálidos/farmacología , Medios de Cultivo Condicionados/farmacología , Desintegrinas/farmacología , Células Endoteliales/patología , Femenino , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Metaloproteinasas de la Matriz/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Oxígeno , Subunidades de Proteína/metabolismo , Hipoxia Tumoral/efectos de los fármacosRESUMEN
The biocompatibility and the antioxidant activity of barium titanate (BaTiO3) and lithium niobate (LiNbO3) were investigated on a neuronal cell line, the PC12, to explore the possibility of using piezoelectric nanoparticles in the treatment of inner ear diseases, avoiding damage to neurons, the most delicate and sensitive human cells. The cytocompatibility of the compounds was verified by analysing cell viability, cell morphology, apoptotic markers, oxidative stress and neurite outgrowth. The results showed that BaTiO3 and LiNbO3 nanoparticles do not affect the viability, morphological features, cytochrome c distribution and production of reactive oxygen species (ROS) by PC12 cells, and stimulate neurite branching. These data suggest the biocompatibility of BaTiO3 and LiNbO3 nanoparticles, and that they could be suitable candidates to improve the efficiency of new implantable hearing devices without damaging the neuronal cells.
Asunto(s)
Antioxidantes/farmacología , Compuestos de Bario/farmacología , Materiales Biocompatibles/farmacología , Nanopartículas/química , Neuronas/efectos de los fármacos , Niobio/farmacología , Óxidos/farmacología , Titanio/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Supervivencia Celular , Citocromos c/metabolismo , Proyección Neuronal/efectos de los fármacos , Células PC12 , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is an effective anticancer agent, and when combined with other agents it shows superior activities. Vitamin B12 has been shown to contribute to increasing the effectiveness of anticancer drugs when used in combination. Thus, the current study aimed at investigating the anticancer potential of the combination of 1,25(OH)2D3 and vitamin B12. METHODS: MTT assay was used to determine the cytotoxic activity of combining 1,25(OH)2D3 and vitamin B12 against six different cancer cell lines and one normal cell line. The surviving fraction after clonogenic assay was measured, and the effects of 1,25(OH)2D3/B12 combination on the activity of different caspases, cell adhesion, actin cytoskeleton, cell morphology, and percentage of polarized cells were evaluated. RESULTS: Vitamin B12 did not cause cytotoxicity, however, it enhanced the cytotoxicity of 1,25(OH)2D3 against cancer cells. The cytotoxic effects of 1,25(OH)2D3 and its combination with vitamin B12 was not evident in the normal mammary MCF10A cell line indicating cancer cell-specificity. The cytotoxic effects of 1,25(OH)2D3/B12 combination occurred in a dose-dependent manner and was attributed to apoptosis induction which was mediated by caspase 4 and 8. Moreover, 1,25(OH)2D3/B12-treated cells showed enhanced inhibition of clonogenic tumor growth, reduced cell adhesion, reduced cell area, reduced percentage of cell polarization, and disorganized actin cytoskeleton resulting in reduced migratory phenotype when compared to cells treated with 1,25(OH)2D3 alone. CONCLUSION: 1,25(OH)2D3 and vitamin B12 exhibited synergistic anticancer effects against different cancer cell lines. The combination therapy of 1,25(OH)2D3 and vitamin B12 may provide a potential adjunctive treatment option for some cancer types.
Asunto(s)
Citoesqueleto de Actina/efectos de los fármacos , Antineoplásicos/farmacología , Calcitriol/farmacología , Caspasas Iniciadoras/metabolismo , Vitamina B 12/farmacología , Apoptosis/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Supervivencia Celular , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , HumanosRESUMEN
Freshly isolated primary cardiomyocytes (CM) are indispensable for cardiac research. Experimental CM research is generally incompatible with life of the donor animal, while human heart samples are usually small and scarce. CM isolation from animal hearts, traditionally performed by coronary artery perfusion of enzymes, liberates millions of cells from the heart. However, due to progressive cell remodeling following isolation, freshly isolated primary CM need to be used within 4-8 h post-isolation for most functional assays, meaning that the majority of cells is essentially wasted. In addition, coronary perfusion-based isolation cannot easily be applied to human tissue biopsies, and it does not straightforwardly allow for assessment of regional differences in CM function within the same heart. Here, we provide a method of multi-day CM isolation from one animal heart, yielding calcium-tolerant ventricular and atrial CM. This is based on cell isolation from cardiac tissue slices following repeated (usually overnight) storage of the tissue under conditions that prolong CM viability beyond the day of organ excision by two additional days. The maintenance of cells in their near-native microenvironment slows the otherwise rapid structural and functional decline seen in isolated CM during attempts for prolonged storage or culture. Multi-day slice-based CM isolation increases the amount of useful information gained per animal heart, improving reproducibility and reducing the number of experimental animals required in basic cardiac research. It also opens the doors to novel experimental designs, including exploring same-heart regional differences.
Asunto(s)
Investigación Biomédica , Atrios Cardíacos/citología , Ventrículos Cardíacos/citología , Miocitos Cardíacos/citología , Animales , Calcio/farmacología , Separación Celular , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Conejos , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacosRESUMEN
Polyetheretherketone (PEEK) is a potential substitute for conventional metallic biomedical implants owing to its superior mechanical and chemical properties, as well as biocompatibility. However, its inherent bio-inertness and poor osseointegration limit its use in clinical applications. Herein, thin titanium films were deposited on the PEEK substrate by plasma sputtering, and porous nanonetwork structures were incorporated on the PEEK surface by alkali treatment (PEEK-TNS). Changes in the physical and chemical characteristics of the PEEK surface were analyzed to establish the interactions with cell behaviors. The osteoimmunomodulatory properties were evaluated using macrophage cells and osteoblast lineage cells. The functionalized nanostructured surface of PEEK-TNS effectively promoted initial cell adhesion and proliferation, suppressed inflammatory responses, and induced macrophages to anti-inflammatory M2 polarization. Compared with PEEK, PEEK-TNS provided a more beneficial osteoimmune environment, including increased levels of osteogenic, angiogenic, and fibrogenic gene expression, and balanced osteoclast activities. Furthermore, the crosstalk between macrophages and osteoblast cells showed that PEEK-TNS could provide favorable osteoimmunodulatory environment for bone regeneration. PEEK-TNS exhibited high osteogenic activity, as indicated by alkaline phosphatase activity, osteogenic factor production, and the osteogenesis/osteoclastogenesis-related gene expression of osteoblasts. The study establishes that the fabrication of titanate nanonetwork structures on PEEK surfaces could extract an adequate immune response and favorable osteogenesis for functional bone regeneration. Furthermore, it indicates the potential of PEEK-TNS in implant applications.
Asunto(s)
Benzofenonas/farmacología , Factores Inmunológicos/farmacología , Nanopartículas/química , Osteogénesis , Polímeros/farmacología , Titanio/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Calcificación Fisiológica/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Forma de la Célula/efectos de los fármacos , Forma de la Célula/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Inmunidad/efectos de los fármacos , Inflamación/patología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Ratones , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Células RAW 264.7 , Propiedades de SuperficieRESUMEN
Over the years, natural-based scaffolds have presented impressive results for bone tissue engineering (BTE) application. Further, outstanding interactions have been observed during the interaction of graphene oxide (GO)-reinforced biomaterials with both specific cell cultures and injured bone during in vivo experimental conditions. This research hereby addresses the potential of fish gelatin/chitosan (GCs) hybrids reinforced with GO to support in vitro osteogenic differentiation and, further, to investigate its behavior when implanted ectopically. Standard GCs formulation was referenced against genipin (Gp) crosslinked blend and 0.5 wt.% additivated GO composite (GCsGp/GO 0.5 wt.%). Pre-osteoblasts were put in contact with these composites and induced to differentiate in vitro towards mature osteoblasts for 28 days. Specific bone makers were investigated by qPCR and immunolabeling. Next, CD1 mice models were used to assess de novo osteogenic potential by ectopic implantation in the subcutaneous dorsum pocket of the animals. After 4 weeks, alkaline phosphate (ALP) and calcium deposits together with collagen synthesis were investigated by biochemical analysis and histology, respectively. Further, ex vivo materials were studied after surgery regarding biomineralization and morphological changes by means of qualitative and quantitative methods. Furthermore, X-ray diffraction and Fourier-transform infrared spectroscopy underlined the newly fashioned material structuration by virtue of mineralized extracellular matrix. Specific bone markers determination stressed the osteogenic phenotype of the cells populating the material in vitro and successfully differentiated towards mature bone cells. In vivo results of specific histological staining assays highlighted collagen formation and calcium deposits, which were further validated by micro-CT. It was observed that the addition of 0.5 wt.% GO had an overall significant positive effect on both in vitro differentiation and in vivo bone cell recruitment in the subcutaneous region. These data support the GO bioactivity in osteogenesis mechanisms as being self-sufficient to elevate osteoblast differentiation and bone formation in ectopic sites while lacking the most common osteoinductive agents.
Asunto(s)
Biopolímeros/farmacología , Diferenciación Celular , Grafito/farmacología , Osteogénesis , Células 3T3 , Fosfatasa Alcalina/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Osteogénesis/efectos de los fármacos , Porosidad , Espectroscopía Infrarroja por Transformada de Fourier , Tejido Subcutáneo/efectos de los fármacos , Andamios del Tejido/química , Difracción de Rayos X , Microtomografía por Rayos XRESUMEN
Epidemiological studies demonstrate that arsenic exposure is associated with cognitive dysfunction. Experimental arsenic exposure models showed learning and memory deficits and molecular changes resembling the functional and pathologic neurodegeneration features. The present work focuses on hippocampal pathological changes in Wistar rats induced by continuous arsenic exposure from in utero up to 12 months of age, evaluated by magnetic resonance imaging along with immunohistochemistry. Diffusion-weighted images revealed age-related lower fractional anisotropy and higher radial-axial and mean diffusivity at 6 and 12 months, indicating that arsenic exposure leads to hippocampal demyelination. These structural alterations were paralleled by immunohistochemical changes that showed a significant loss of myelin basic protein in CA1 and CA3 regions accompanied by increased glial fibrillary acidic protein expression at all time-points studied. Concomitantly, arsenic exposure induced an altered morphology of astrocytes at all studied ages, whereas increased synaptogenesis was only observed at two months of age. These results suggest that environmental arsenic exposure is linked to impaired hippocampal connectivity and perhaps early glial senescence, which together might resemble a premature aging phenomenon leading to cognitive deficits.
Asunto(s)
Arsénico/farmacología , Astrocitos/efectos de los fármacos , Hipocampo/efectos de los fármacos , Sustancia Blanca/efectos de los fármacos , Animales , Astrocitos/citología , Forma de la Célula/efectos de los fármacos , Hipocampo/citología , Hipocampo/diagnóstico por imagen , Imagen por Resonancia Magnética , Masculino , Ratas , Ratas Wistar , Sustancia Blanca/citología , Sustancia Blanca/diagnóstico por imagenRESUMEN
OBJECTIVES: Aging and common diseases alter the stiffness of bone tissue, causing changes to the microenvironment of the mechanosensitive bone cells. Osteoclasts, the sole bone-resorbing cells, play a vital role in bone remodeling. This study was performed to elucidate the mechanism through which osteoclasts sense and react to substrate stiffness signals. MATERIALS AND METHODS: We fabricated polydimethylsiloxane (PDMS) substrates of different stiffness degrees for osteoclast formation progressed from osteoclast precursors including bone marrow-derived macrophages (BMMs) and RAW264.7 monocytes. Osteoclast differentiation in response to the stiffness signals was determined by examining the cell morphology, fusion/fission activities, transcriptional profile, and resorption function. Cytoskeletal changes and mechanosensitive adhesion molecules were also assessed. RESULTS: Stiffer PDMS substrates accelerated osteoclast differentiation, firstly observed by variations in their morphology and fusion/fission activities. Upregulation of canonical osteoclast markers (Nfatc1, Acp5, Ctsk, Camk2a, Mmp9, Rela, and Traf6) and the fusion master regulator DC-stamp were detected on stiffer substrates, with similar increases in their bone resorption functions. Additionally, the activation of cytoskeleton-associated adhesion molecules, including fibronectin and integrin αvß3, followed by biochemical signaling cascades of paxillin, FAK, PKC, and RhoA, was detected on the stiffer substrates. CONCLUSIONS: This is the first study to provide evidence proving that extracellular substrate stiffness is a strong determinant of osteoclast differentiation and functions. Higher stiffness upregulated the differentiation profile and activity of osteoclasts, revealing the mechanical regulation of osteoclast activity in bone homeostasis and diseases.
Asunto(s)
Diferenciación Celular , Citoesqueleto/metabolismo , Dimetilpolisiloxanos/farmacología , Osteoclastos/citología , Animales , Biomarcadores/metabolismo , Resorción Ósea/genética , Resorción Ósea/patología , Diferenciación Celular/efectos de los fármacos , Fusión Celular , Forma de la Célula/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Elasticidad , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Integrinas/metabolismo , Ratones , Modelos Biológicos , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Células RAW 264.7 , Transducción de Señal/efectos de los fármacos , Especificidad por SustratoRESUMEN
The present study reports a cost-effective, environmentally friendly method to increase the bioavailability and bio-efficacy of B. rufescens stem bark extract in the biological system via functional modification as B. rufescens stem bark nanoparticles (BR-TO2-NPs). The biosynthesis of BR- -NPs was confirmed by UV-visible (UV-vis) and Fourier-transform infrared (FT-IR) spectroscopy, transmission electron microscopy (TEM), and X-ray diffraction analyses. The shifts in FT-IR stretching vibrations of carboxylic and nitro groups (1615 cm-1), the O-H of phenolics or carboxylic acids (3405 cm-1), alkanes, and alkyne groups (2925 and 2224 cm-1) of the plant extract and lattice (455) indicated successful biosynthesis of BR- -NPs. Compared with the stem bark extract, 40 ng/dL dose of BR- -NPs led to a reduction in adipogenesis and an increase in mitochondrial biogenesis-related gene expressions, adiponectin-R1, PPARγC1α, UCP-1, and PRDM16, in maturing-adipocytes. This confirmed the intracellular uptake, bioavailability, and bio-efficiency of BR-TiO2-NPs. The lipid-lowering capacity of BR-TiO2-NPs effectively inhibited the metabolic inflammation-related gene markers, IL-6, TNF-α, LTB4-R, and Nf-κb. Further, BR-TiO2-NPs stimulating mitochondrial thermogenesis capacity was proven by the significantly enhanced CREB-1 and AMPK protein levels in adipocytes. In conclusion, BR-TiO2-NPs effectively inhibited lipid accumulation and proinflammatory adipokine levels in maturing adipocytes; it may help to overcome obesity-associated comorbidities.
Asunto(s)
Adipocitos/citología , Adipocitos/metabolismo , Adipoquinas/metabolismo , Bauhinia/química , Metabolismo de los Lípidos , Nanopartículas del Metal/química , Corteza de la Planta/química , Titanio/farmacología , Adipogénesis/efectos de los fármacos , Adipogénesis/genética , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Forma de la Célula/efectos de los fármacos , Cromatografía de Gases y Espectrometría de Masas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Lipólisis/efectos de los fármacos , Lipólisis/genética , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Nanopartículas del Metal/ultraestructura , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Estrés Oxidativo/efectos de los fármacos , Tallos de la Planta/química , Termogénesis/efectos de los fármacos , Termogénesis/genéticaRESUMEN
The liver is among the principal organs for glucose homeostasis and metabolism. Studies of liver metabolism are limited by the inability to expand primary hepatocytes in vitro while maintaining their metabolic functions. Human hepatic three-dimensional (3D) organoids have been established using defined factors, yet hepatic organoids from adult donors showed impaired expansion. We examined conditions to facilitate the expansion of adult donor-derived hepatic organoids (HepAOs) and HepG2 cells in organoid cultures (HepGOs) using combinations of growth factors and small molecules. The expansion dynamics, gluconeogenic and HNF4α expression, and albumin secretion are assessed. The conditions tested allow the generation of HepAOs and HepGOs in 3D cultures. Nevertheless, gluconeogenic gene expression varies greatly between conditions. The organoid expansion rates are limited when including the TGFß inhibitor A8301, while are relatively higher with Forskolin (FSK) and Oncostatin M (OSM). Notably, expanded HepGOs grown in the optimized condition maintain detectable gluconeogenic expression in a spatiotemporal distribution at 8 weeks. We present optimized conditions by limiting A8301 and incorporating FSK and OSM to allow the expansion of HepAOs from adult donors and HepGOs with gluconeogenic competence. These models increase the repertoire of human hepatic cellular tools available for use in liver metabolic assays.
Asunto(s)
Bioensayo/métodos , Técnicas de Cultivo de Célula , Hepatocitos/metabolismo , Hígado/metabolismo , Organoides/metabolismo , Adulto , Albúminas/metabolismo , Biomarcadores/metabolismo , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Medios de Cultivo/farmacología , Congelación , Glucosa-6-Fosfatasa/metabolismo , Células Hep G2 , Factor Nuclear 4 del Hepatocito/metabolismo , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Humanos , Organoides/efectos de los fármacos , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismoRESUMEN
Chronic airway inflammation and oxidative stress play crucial roles in the pathogenesis of chronic inflammatory lung diseases, with airway inflammation being a key driving mechanism of oxidative stress in the lungs. Inflammatory responses in the lungs activate neutrophils and/or eosinophils, leading to the generation of hypohalous acids (HOX). These HOX oxidants can damage the extracellular matrix (ECM) structure and may influence cell-ECM interactions. The ECM of the lung provides structural, mechanical, and biochemical support for cells and determines the airway structure. One of the critical cells in chronic respiratory disease is the fibroblast. Thus, we hypothesised that primary human lung fibroblasts (PHLF) exposed to an oxidised cell-derived ECM will result in functional changes to the PHLF. Here, we show that PHLF adhesion, proliferation, and inflammatory cytokine secretion is affected by exposure to HOX-induced oxidisation of the cell-derived ECM. Furthermore, we investigated the impact on fibroblast function from the presence of haloamines in the ECM. Haloamines are chemical by-products of HOX and, like the HOX, haloamines can also modify the ECM. In conclusion, this study revealed that oxidising the cell-derived ECM might contribute to functional changes in PHLF, a key mechanism behind the pathogenesis of inflammatory lung diseases.
Asunto(s)
Bromatos/farmacología , Matriz Extracelular/metabolismo , Fibroblastos/citología , Granulocitos/metabolismo , Ácido Hipocloroso/farmacología , Pulmón/citología , Oxidantes/farmacología , Adhesión Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Colágeno Tipo I/metabolismo , Citocinas/metabolismo , Matriz Extracelular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Granulocitos/efectos de los fármacos , Humanos , Mediadores de Inflamación/metabolismo , Persona de Mediana Edad , Oxidación-ReducciónRESUMEN
Factor quinolinone inhibitors are promising anti-cancer compounds, initially characterized as specific inhibitors of the oncogenic transcription factor LSF (TFCP2). These compounds exert anti-proliferative activity at least in part by disrupting mitotic spindles. Herein, we report additional interphase consequences of the initial lead compound, FQI1, in two telomerase immortalized cell lines. Within minutes of FQI1 addition, the microtubule network is disrupted, resulting in a substantial, although not complete, depletion of microtubules as evidenced both by microtubule sedimentation assays and microscopy. Surprisingly, this microtubule breakdown is quickly followed by an increase in tubulin acetylation in the remaining microtubules. The sudden breakdown and partial depolymerization of the microtubule network precedes FQI1-induced morphological changes. These involve rapid reduction of cell spreading of interphase fetal hepatocytes and increase in circularity of retinal pigment epithelial cells. Microtubule depolymerization gives rise to FH-B cell compaction, as pretreatment with taxol prevents this morphological change. Finally, FQI1 decreases the rate and range of locomotion of interphase cells, supporting an impact of FQI1-induced microtubule breakdown on cell motility. Taken together, our results show that FQI1 interferes with microtubule-associated functions in interphase, specifically cell morphology and motility.
Asunto(s)
Benzodioxoles/farmacología , Microtúbulos/efectos de los fármacos , Quinolonas/farmacología , Antineoplásicos/farmacología , Línea Celular , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Forma de la Célula/efectos de los fármacos , Forma de la Célula/fisiología , Proteínas de Unión al ADN/antagonistas & inhibidores , Hepatocitos/efectos de los fármacos , Hepatocitos/fisiología , Hepatocitos/ultraestructura , Humanos , Interfase , Microtúbulos/fisiología , Microtúbulos/ultraestructura , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/fisiología , Epitelio Pigmentado de la Retina/ultraestructura , Factores de Transcripción/antagonistas & inhibidores , Tubulina (Proteína)/metabolismoRESUMEN
Platelets are functionally versatile blood cells involved in thrombosis, hemostasis, atherosclerosis, and immune response. Platelet interaction with the immediate microenvironment in blood, vasculature, and tissues alters platelet morphology. The quantification of platelet morphodynamics by geometrical parameters (morphometry) can provide important insights into how platelets sense and respond to stimulatory cues in their vicinity. However, the extraction of platelet shapes from phase contrast microscopy images by conventional image processing is difficult. Here, we used a convolutional neural network (CNN) to develop a deep-learning-based approach for the unbiased extraction of information on platelet morphodynamics by phase contrast microscopy. We then investigated the effect of normal and oxidized low-density lipoproteins (LDL, oxLDL) on platelet morphodynamics, spreading, and haptotactic migration. Exposure of platelets to oxLDL led to a decreased spreading area and rate on fibrinogen, accompanied by increased formation of filopodia and impaired formation of lamellipodia. Haptotactic platelet migration was affected by both LDL and oxLDL in terms of decreased migration velocity and reduced directional persistence. Our results demonstrate the use of deep learning in investigating platelet morphodynamics and reveal differential effects of LDL and oxLDL on platelet morphology and platelet-matrix interaction.
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Plaquetas/citología , Movimiento Celular , Forma de la Célula , Aprendizaje Profundo , Lipoproteínas LDL/farmacología , Movimiento Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Humanos , Seudópodos/efectos de los fármacos , Seudópodos/metabolismo , TactoRESUMEN
Chronic skin ulcers are a primary global health problem. Velvet antler polypeptide (VAP) regulates endothelial cell migration and angiogenic sprout. Adipose-derived stem cells (ADSCs) are reported to make pivotal impacts upon wound healing. This study aimed to explore the role of VAP combined with ADSCs in wound healing of chronic skin ulcers. The effect of VAP on phenotypes of ADSCs, and VAP (PLGA microspheres) combining with ADSCs on wound healing of chronic skin ulcers in vivo was evaluated. VAP generally promoted the proliferation, migration and invasion of ADSCs, and ADSC-induced angiogenesis in human umbilical vein endothelial cells (HUVECs) through PI3K/Akt/HIF-1α pathway. VAP-PLGA (PLGA microspheres) enhanced the promoting effect of ADSCs on wound healing, pathological changes, and angiogenesis in chronic skin ulcers in vivo. VAP-PLGA intensified the effect of ADSCs on up-regulating the levels of p-PI3K/PI3K, p-Akt/Akt, HIF-1α, vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 (SDF-1), C-X-C motif chemokine receptor 4 (CXCR4), angiopoietin-4 (Ang-4), VEGF receptor (VEGFR), and transforming growth factor-ß1 (TGF-ß1), and down-regulating the levels of interleukin-1 ß (IL-1ß), IL-18 and IL-6 in wound tissues in chronic skin ulcers in vivo. Collectively, VAP promoted the growth, migration, invasion, and angiogenesis of ADSCs through activating PI3K/Akt/HIF-1α pathway, and VAP-PLGA enhanced the function of ADSCs in promoting wound healing in vivo, which was associated with angiogenesis, inflammation inhibition, and dermal collagen synthesis.
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Tejido Adiposo/citología , Microesferas , Péptidos/farmacología , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Transducción de Señal , Úlcera Cutánea/terapia , Células Madre/citología , Cicatrización de Heridas , Animales , Cuernos de Venado , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Cromonas/farmacología , Enfermedad Crónica , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Ratones , Morfolinas/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Fenotipo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Background: Several pre-clinical and clinical reports suggest that HIV-1 protease inhibitors, in addition to the antiretroviral properties, possess pleiotropic pharmacological effects including anticancer action. Therefore, we investigated the pro-apoptotic activity in tumor cells of two molecules, RDD-19 and RDD-142, which are hydroxyethylamine derivatives' precursors of darunavir and several HIV-1 protease inhibitors. Methods: Three hepatoma cell lines and one non-pathological cell line were treated with RDD-19 and RDD-142, and cell viability was assessed. The expression levels of several markers for ER stress, autophagy, cellular ubiquitination, and Akt activation were quantified in HepG2 cells treated with RDD-19 and RDD-142 to evaluate apoptotic and non-apoptotic cell death. Results: RDD-19 and RDD-142 showed a greater dose-dependent cytotoxicity towards the hepatic tumor cell line HepG2 compared to the non-pathological hepatic cell line IHH. Both molecules caused two types of cell death, a caspase-dependent apoptosis, which was ascertained by a series of biochemical and morphological assays, and a caspase-independent death that was characterized by the induction of ER stress and autophagy. The strong increase of ubiquitinated proteins inside the cells suggested that the target of these molecules could be the proteasome and in silico molecular docking analysis that was used to support the plausibility of this hypothesis. Furthermore, cells treated with the two compounds displayed decreased levels of p-AKT, which interferes with cell survival and proliferation. Conclusions: These findings demonstrate that two compounds, RDD-19 and RDD-142, have pleiotropic effects and that they may represent promising anticancer candidates.
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Carcinoma Hepatocelular/patología , Darunavir/farmacología , VIH-1/efectos de los fármacos , Neoplasias Hepáticas/patología , Inhibidores de Proteasas/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Respuesta de Proteína Desplegada , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Sitios de Unión , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Hep G2 , Humanos , Simulación del Acoplamiento Molecular , Inhibidores de Proteasas/química , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
We have investigated motility in breast cancer cell lines in association with the expression of Transglutaminase type 2 (TG2) as well as upon the administration of Doxorubicin (Dox), an active cytotoxic agent that is employed in chemotherapy. The exposure of MCF-7 cells to the drug increased TG2 levels, triggering epithelial-mesenchymal transition (EMT), thereby supporting cell motility. The effects of Dox on the movement of MCF-7 cells were counteracted by treatment with NC9, a TG2 inhibitor, which induced morphological changes and also reduced the migration of MDA-MB-231 cells exhibiting high levels of TG2. The physical association of TG2 with the cytoskeletal component vimentin appeared pivotal both in drug-treated MCF-7 and in MDA-MB-231 cells and seemed to be independent of the catalytic activity of TG2. NC9 altered the subcellular distribution of TG2 and, consequently, the co-localization of TG2 with vimentin. Furthermore, NC9 induced a nuclear accumulation of TG2 as a prelude to TG2-dependent gene expression modifications. Since enzyme activity can affect both motility and nuclear functions, targeting of this protein could represent a method to improve therapeutic interventions in breast tumors, particularly those to control progression and to limit drug resistance.
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Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Movimiento Celular , Espacio Intracelular/metabolismo , Mesodermo/patología , Proteína Glutamina Gamma Glutamiltransferasa 2/metabolismo , Cadherinas/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Forma de la Célula/efectos de los fármacos , Citoesqueleto/metabolismo , Doxorrubicina/farmacología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Femenino , Fluorescencia , Humanos , Invasividad Neoplásica , Proteína Glutamina Gamma Glutamiltransferasa 2/genética , Transcripción Genética , Vimentina/metabolismoRESUMEN
Adipose-derived stem cells (ASCs) are an abundant and easily accessible multipotent stem cell source with potential application in smooth muscle regeneration strategies. In 3D collagen hydrogels, we investigated whether sustained release of growth factors (GF) PDGF-AB and TGF-ß1 from GF-loaded microspheres could induce a smooth muscle cell (SMC) phenotype in ASCs, and if the addition of uniaxial cyclic stretch could enhance the differentiation level. This study demonstrated that the combination of cyclic stretch and GF release over time from loaded microspheres potentiated the differentiation of ASCs, as quantified by protein expression of early to late SMC differentiation markers (SMA, TGLN and smooth muscle MHC). The delivery of GFs via microspheres produced large ASCs with a spindle-shaped, elongated SMC-like morphology. Cyclic strain produced the largest, longest, and most spindle-shaped cells regardless of the presence or absence of growth factors or the growth factor delivery method. Protein expression and cell morphology data confirmed that the sustained release of GFs from GF-loaded microspheres can be used to promote the differentiation of ASCs into SMCs and that the addition of uniaxial cyclic stretch significantly enhances the differentiation level, as quantified by intermediate and late SMC markers and a SMC-like elongated cell morphology.
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Tejido Adiposo/citología , Péptidos y Proteínas de Señalización Intercelular/farmacología , Miocitos del Músculo Liso/citología , Células Madre/citología , Estrés Mecánico , Adulto , Biomarcadores/metabolismo , Reactores Biológicos , Diferenciación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Reactivos de Enlaces Cruzados/química , Gelatina/química , Geles/química , Humanos , Iridoides/química , Microesferas , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Fenotipo , Células Madre/efectos de los fármacos , Células Madre/metabolismoRESUMEN
Anhydrosafflor yellow B (AHSYB) is a major active water-soluble pigment in Safflower, but it has not received enough attention yet. In this study, high-speed counter-current chromatography (HSCCC) was used to prepare AHSYB from safflower. The parameters of the separation process were optimized by response surface methodology for the first time. The entropy weight method (EWM) was applied to calculate the information entropy and the weight of five indexes, and then figure out a comprehensive index of the HSCCC separation effect. Under the optimized separation conditions, a HSCCC apparatus speed of 850 rpm, a flow rate of 2 mL min-1 for the mobile phase and a separation temperature of 40 °C for AHSYB were achieved with a purity of 98%. Furthermore, AHSYB was found to have cardio-protective effects by inhibiting apoptosis via the mitochondrial-mediated pathway in oxygen-glucose deprivation/reoxygenation-induced H9c2 cells. This research provides good method guides for the rapid and efficient separation of active compounds from food-grade Chinese herb medicines.