RESUMO
There are numerous studies that investigate the effects of static magnetic fields (SMFs) on osteoblasts and osteoclasts. However, although osteocytes are the most abundant cell type in bone tissue, there are few studies on the biological effects of osteocytes under magnetic fields. Iron is a necessary microelement that is involved in numerous life activities in cells. Studies have shown that high static magnetic fields (HiSMF) can regulate cellular iron metabolism. To illustrate the effect of HiSMF on activities of osteocytes, and whether iron is involved in this process, HiSMF of 16 tesla (T) was used, and the changes in cellular morphology, cytoskeleton, function-related protein expression, secretion of various cytokines, and iron metabolism in osteocytes under HiSMF were studied. In addition, the biological effects of HiSMF combined with iron preparation and iron chelator on osteocytes were also investigated. The results showed that HiSMF promoted cellular viability, decreased apoptosis, increased the fractal dimension of the cytoskeleton, altered the secretion of cytokines, and increased iron levels in osteocytes. Moreover, it was found that the biological effects of osteocytes under HiSMF are attenuated or enhanced by treatment with a certain concentration of iron. These data suggest that HiSMF-regulated cellular iron metabolism may be involved in altering the biological effects of osteocytes under HiSMF exposure.
Assuntos
Apoptose/genética , Sobrevivência Celular/genética , Ferro/metabolismo , Osteócitos/efeitos da radiação , Animais , Apoptose/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Citoesqueleto/genética , Citoesqueleto/metabolismo , Citoesqueleto/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Ferro/efeitos da radiação , Campos Magnéticos/efeitos adversos , Camundongos , Microtúbulos/genética , Microtúbulos/efeitos da radiação , Osteoblastos/metabolismo , Osteoblastos/efeitos da radiação , Osteoclastos/metabolismo , Osteoclastos/efeitos da radiação , Osteócitos/metabolismo , Células RAW 264.7RESUMO
Although cancer immunotherapy is effective against hematological malignancies, it is less effective against solid tumors due in part to significant metabolic challenges present in the tumor microenvironment (TME), where infiltrated CD8+ T cells face fierce competition with cancer cells for limited nutrients. Strong metabolic suppression in the TME is often associated with impaired T cell recruitment to the tumor site and hyporesponsive effector function via T cell exhaustion. Increasing evidence suggests that mitochondria play a key role in CD8+ T cell activation, effector function, and persistence in tumors. In this study, we showed that there was an increase in overall mitochondrial function, including mitochondrial mass and membrane potential, during both mouse and human CD8+ T cell activation. CD8+ T cell mitochondrial membrane potential was closely correlated with granzyme B and IFN-γ production, demonstrating the significance of mitochondria in effector T cell function. Additionally, activated CD8+ T cells that migrate on ICAM-1 and CXCL12 consumed significantly more oxygen than stationary CD8+ T cells. Inhibition of mitochondrial respiration decreased the velocity of CD8+ T cell migration, indicating the importance of mitochondrial metabolism in CD8+ T cell migration. Remote optical stimulation of CD8+ T cells that express our newly developed "OptoMito-On" successfully enhanced mitochondrial ATP production and improved overall CD8+ T cell migration and effector function. Our study provides new insight into the effect of the mitochondrial membrane potential on CD8+ T cell effector function and demonstrates the development of a novel optogenetic technique to remotely control T cell metabolism and effector function at the target tumor site with outstanding specificity and temporospatial resolution.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/efeitos da radiação , Optogenética/métodos , Trifosfato de Adenosina/metabolismo , Animais , Linfócitos T CD8-Positivos/metabolismo , Movimento Celular/efeitos da radiação , Citocinas/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/efeitos da radiação , Humanos , Imunoterapia , Ativação Linfocitária/efeitos da radiação , Potencial da Membrana Mitocondrial/genética , Potencial da Membrana Mitocondrial/efeitos da radiação , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Neoplasias/imunologia , Neoplasias/terapiaRESUMO
Ultrasound is considered a safe and non-invasive tool in regenerative medicine and has been used in the clinic for more than twenty years for applications in bone healing after the approval of the Exogen device, also known as low-intensity pulsed ultrasound (LIPUS). Beyond its effects on bone health, LIPUS has also been investigated for wound healing of soft tissues, with positive results for various cell processes including cell proliferation, migration and angiogenesis. As LIPUS has the potential to treat chronic skin wounds, we sought to evaluate the effects produced by a conventional therapeutic ultrasound device at low intensities (also considered LIPUS) on the migration capacity of mouse and human skin mesenchymal precursors (s-MPs). Cells were stimulated for 3 days (20 minutes per day) using a traditional ultrasound device with the following parameters: 100 mW/cm2 with 20% duty cycle and frequency of 3 MHz. At the parameters used, ultrasound failed to affect s-MP proliferation, with no evident changes in morphology or cell groupings, and no changes at the cytoskeletal level. Further, the migration and invasion ability of s-MPs were unaffected by the ultrasound protocol, and no major changes were detected in the gene/protein expression of ROCK1, integrin ß1, laminin ß1, type I collagen and transforming growth factor ß1. Finally, RNA-seq analysis revealed that only 10 genes were differentially expressed after ultrasound stimulation. Among them, 5 encode for small nuclear RNAs and 2 encode for proteins belonging to the nuclear pore complex. Considering the results overall, while the viability of s-MPs was not affected by ultrasound stimulation and no changes were detected in proliferation/migration, RNA-seq analysis would suggest that s-MPs do respond to ultrasound. The use of 100 mW/cm2 intensity or conventional therapeutic ultrasound devices might not be optimal for the stimulation the properties of cell populations. Future studies should investigate the potential application of ultrasound using variations of the tested parameters.
Assuntos
Células-Tronco Mesenquimais/efeitos da radiação , Terapia por Ultrassom , Ondas Ultrassônicas , Animais , Western Blotting , Movimento Celular/efeitos da radiação , Citoesqueleto/efeitos da radiação , Humanos , Camundongos , Microscopia de Fluorescência , Reação em Cadeia da Polimerase em Tempo Real , Transcriptoma/efeitos da radiação , Terapia por Ultrassom/efeitos adversos , Terapia por Ultrassom/métodos , Ondas Ultrassônicas/efeitos adversos , Cicatrização/efeitos da radiaçãoRESUMO
The biological effects of circularly polarized light on living cells are considered to be negligibly weak. Here, we show that the differentiation of neural stem cells into neurons can be accelerated by circularly polarized photons when DNA-bridged chiral assemblies of gold nanoparticles are entangled with the cells' cytoskeletal fibres. By using cell-culture experiments and plasmonic-force calculations, we demonstrate that the nanoparticle assemblies exert a circularly-polarized-light-dependent force on the cytoskeleton, and that the light-induced periodic mechanical deformation of actin nanofibres with a frequency of 50 Hz stimulates the differentiation of neural stem cells into the neuronal phenotype. When implanted in the hippocampus of a mouse model of Alzheimer's disease, neural stem cells illuminated following a polarity-optimized protocol reduced the formation of amyloid plaques by more than 70%. Our findings suggest that circularly polarized light can guide cellular development for biomedical use.
Assuntos
Diferenciação Celular , Nanopartículas Metálicas/química , Nanoestruturas/química , Células-Tronco Neurais , Animais , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos da radiação , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/efeitos da radiação , DNA/química , Ouro/química , Hipocampo/citologia , Humanos , Camundongos , Camundongos Transgênicos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/efeitos da radiação , Proteínas Recombinantes de Fusão , EstereoisomerismoRESUMO
PURPOSE: Radiation therapy (RT) affects tumor-infiltrating immune cells, cooperatively driving tumor growth inhibition. However, there is still no absolute consensus on whether the homing ability of dendritic cells (DCs) is affected by direct x-ray irradiation. Most importantly, the underlying mechanisms are poorly understood. METHODS AND MATERIALS: Using noninvasive imaging, we systematically examined the dose effect of RT on the in vivo homing and distribution of bone marrow-derived DCs and elucidated the detailed mechanisms underlying these events. After exposure to 2, 5, 10, 15, and 20 Gy, DCs were analyzed for maturation, in vivo homing ability, and T cell priming. RESULTS: At ranges of 2 to 20 Gy, irradiation did not cause direct cellular apoptosis or necrosis, but it induced mitochondrial damage in DCs independent of dose. In addition, upregulation of CD40, CD80, CD86, CXCR4, and CCR7 were detected on irradiated DCs. Secretion of IL-1ß and IL-12p70 remained unchanged, whereas decreased secretion of IL-6 and promotion of tumor necrosis factor α secretion were observed. In particular, the homing ability of both the local residual and blood circulating DCs to lymphoid tissues was significantly higher in groups that received ≥5 Gy radiation than in the group that received 2 Gy. Furthermore, improved homing ability was associated with rearrangement of the cytoskeleton, which was regulated by reactive oxygen species accumulation through the RhoA/ROCK1 signaling pathway. Finally, more robust T cell activation was observed in mice inoculated with 20 Gy-treated DCs than in those inoculated with 2 Gy-irradiated DCs, and T cell activation also correlated with reactive oxygen species production. CONCLUSIONS: An RT dose ≥5 Gy has distinct advantages over 2 Gy in facilitating DC homing to lymph nodes and cross-priming T cells.
Assuntos
Citoesqueleto/efeitos da radiação , Células Dendríticas/citologia , Células Dendríticas/efeitos da radiação , Doses de Radiação , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T/citologia , Linfócitos T/efeitos da radiação , Diferenciação Celular/efeitos da radiação , Linhagem Celular , Movimento Celular/efeitos da radiação , Citoesqueleto/metabolismo , Relação Dose-Resposta à Radiação , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Quinases Associadas a rho/metabolismoRESUMO
Photobiomodulation (PBM) has been shown to improve cell proliferation and cell migration. Many cell types have been investigated, with most studies using deep penetrating red light irradiation. Considering the interest of surface biostimulation of oral mesenchymal cells after surgical wound, the present study aimed to assess green light irradiation effects on Dental Pulp Stem Cells' (DPSC) proliferation and migration. To understand the mechanisms underlying these effects, we investigated cytoskeleton organization and subsequent cell shape and stiffness. A 532-nm wavelength Nd:YAG laser (30 mW) was applied between 30 and 600 s on DPSC in vitro. Cell proliferation was analyzed at 24, 48, and 72 h after irradiation, by cell counting and enzymatic activity quantification (paranitrophenylphosphate phosphatase (pNPP) test). A wound healing assay was used to study cell migration after irradiation. Effects of PBM on cytoskeleton organization and cell shape were assessed by actin filaments staining. Elasticity changes after irradiation were quantified in terms of Young's modulus measured using Atomic Force Microscopy (AFM) force spectroscopy. Green light significantly improved DPSC proliferation with a maximal effect obtained after 300-s irradiation (energy fluence 5 J/cm2). This irradiation had a significant impact on cell migration, improving wound healing after 24 h. These results were concomitant with a decrease of cells' Young's modulus after irradiation. This cell softening was explained by actin cytoskeleton reorganization, with diminution of cell circularity and more abundant pseudopodia. This study highlights the interest of green laser PMB for the proliferation and migration of mesenchymal stem cells, with encouraging results for clinical application, especially for surgical wound healing procedures.
Assuntos
Citoesqueleto/efeitos da radiação , Polpa Dentária/citologia , Terapia com Luz de Baixa Intensidade , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos da radiação , Cicatrização/efeitos da radiação , Adolescente , Adulto , Fenômenos Biomecânicos/efeitos da radiação , Movimento Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Forma Celular/efeitos da radiação , Células Cultivadas , Humanos , Adulto JovemRESUMO
Optically controlled chemical reagents, termed "photopharmaceuticals," are powerful tools for precise spatiotemporal control of proteins particularly when genetic methods, such as knockouts or optogenetics are not viable options. However, current photopharmaceutical scaffolds, such as azobenzenes are intolerant of GFP/YFP imaging and are metabolically labile, posing severe limitations for biological use. We rationally designed a photoswitchable "SBT" scaffold to overcome these problems, then derivatized it to create exceptionally metabolically robust and fully GFP/YFP-orthogonal "SBTub" photopharmaceutical tubulin inhibitors. Lead compound SBTub3 allows temporally reversible, cell-precise, and even subcellularly precise photomodulation of microtubule dynamics, organization, and microtubule-dependent processes. By overcoming the previous limitations of microtubule photopharmaceuticals, SBTubs offer powerful applications in cell biology, and their robustness and druglikeness are favorable for intracellular biological control in in vivo applications. We furthermore expect that the robustness and imaging orthogonality of the SBT scaffold will inspire other derivatizations directed at extending the photocontrol of a range of other biological targets.
Assuntos
Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologia , Células A549 , Animais , Compostos Azo/química , Compostos Azo/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/efeitos da radiação , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/efeitos da radiação , Imagem Óptica , Optogenética , Processos Fotoquímicos , Ratos WistarRESUMO
Epidemiological studies on workers employed at the Mayak plutonium enrichment plant have demonstrated an association between external gamma ray exposure and an elevated risk of ischemic heart disease (IHD). In a previous study using fresh-frozen post mortem samples of the cardiac left ventricle of Mayak workers and non-irradiated controls, we observed radiation-induced alterations in the heart proteome, mainly downregulation of mitochondrial and structural proteins. As the control group available at that time was younger than the irradiated group, we could not exclude age as a confounding factor. To address this issue, we have now expanded our study to investigate additional samples using archival formalin-fixed paraffin-embedded (FFPE) tissue. Importantly, the control group studied here is older than the occupationally exposed (>500 mGy) group. Label-free quantitative proteomics analysis showed that proteins involved in the lipid metabolism, sirtuin signaling, mitochondrial function, cytoskeletal organization, and antioxidant defense were the most affected. A histopathological analysis elucidated large foci of fibrotic tissue, myocardial lipomatosis and lymphocytic infiltrations in the irradiated samples. These data highlight the suitability of FFPE material for proteomics analysis. The study confirms the previous results emphasizing the role of adverse metabolic changes in the radiation-associated IHD. Most importantly, it excludes age at the time of death as a confounding factor.
Assuntos
Isquemia Miocárdica/metabolismo , Plutônio/efeitos adversos , Proteoma/metabolismo , Proteoma/efeitos da radiação , Cromatografia Líquida , Citoesqueleto/metabolismo , Citoesqueleto/efeitos da radiação , Formaldeído/química , Humanos , Metabolismo dos Lipídeos/efeitos da radiação , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Isquemia Miocárdica/epidemiologia , Isquemia Miocárdica/etiologia , Isquemia Miocárdica/patologia , Exposição Ocupacional , Inclusão em Parafina , Análise de Componente Principal , Mapas de Interação de Proteínas , Proteômica/métodos , Radiação Ionizante , Transdução de Sinais/efeitos da radiação , Sirtuínas/metabolismo , Espectrometria de Massas em Tandem , Fixação de TecidosRESUMO
PURPOSE: The aim of the present study was to investigate the duality of irradiation effect on osteoclastogenesis, particularly on the cytoskeleton and expression of lytic enzymes in osteoclast precursors. Therefore, the present study may serve as a useful reference for the prevention and treatment of radiation-induced bone loss in the clinic. MATERIALS AND METHODS: Two typical osteoclast precursors, murine RAW 264.7 macrophage cells and mouse bone marrow-derived macrophages (BMMs), were exposed to radiation in the order of 0.25-8 Gy, and the effects on cell viability, TRAP activity and bone resorption were subsequently investigated. Furthermore, changes in the cytoskeleton, cell apoptosis, and expression of lytic enzymes in osteoclasts were examined to elucidate the molecular mechanism of the duality of irradiation on osteoclastogenesis. RESULTS: Morphological changes and impaired viability were observed in RAW 264.7 cells and BMMs treated with 1-8 Gy irradiation with or without RANKL. However, the cell fusion tendency of osteoclasts was enhanced after 2 Gy irradiation, and an increased number of fused giant osteoclasts and enhanced F-actin ring formation were observed. Consistently, the bone resorption activity and the enzyme expression of TRAP, cathepsin K, matrix metalloproteinase 9, activator protein 1, and Caspase 9 were increased following irradiation with 2 Gy. Furthermore, intracellular ROS production and apoptosis of osteoclast precursors were increased. CONCLUSIONS: Irradiation with 2 Gy inhibited the viability of osteoclast precursors, but increased osteoclastogenesis by enhancing cell fusion and increasing the secretion of lytic enzymes, which may be an important mechanism of radiation-induced bone loss.
Assuntos
Células da Medula Óssea/citologia , Citoesqueleto/efeitos da radiação , Macrófagos/efeitos da radiação , Osteoclastos/citologia , Osteoclastos/efeitos da radiação , Animais , Apoptose/efeitos da radiação , Reabsorção Óssea/patologia , Sobrevivência Celular/efeitos da radiação , Citoesqueleto/metabolismo , Macrófagos/citologia , Camundongos , Osteoclastos/metabolismo , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismoRESUMO
Cell biology is moving from observing molecules to controlling them in real time, a critical step towards a mechanistic understanding of how cells work. Initially developed from light-gated ion channels to control neuron activity, optogenetics now describes any genetically encoded protein system designed to accomplish specific light-mediated tasks. Recent photosensitive switches use many ingenious designs that bring spatial and temporal control within reach for almost any protein or pathway of interest. This next generation optogenetics includes light-controlled protein-protein interactions and shape-shifting photosensors, which in combination with live microscopy enable acute modulation and analysis of dynamic protein functions in living cells. We provide a brief overview of various types of optogenetic switches. We then discuss how diverse approaches have been used to control cytoskeleton dynamics with light through Rho GTPase signaling, microtubule and actin assembly, mitotic spindle positioning and intracellular transport and highlight advantages and limitations of different experimental strategies.
Assuntos
Citoesqueleto/metabolismo , Optogenética , Animais , Citoesqueleto/efeitos da radiação , Humanos , Luz , Transdução de Sinais/efeitos da radiação , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
The purpose of this study was to investigate the effects that photobiomodulation therapy might produce in cells, in particular, related to their structure. Thus, this paper presents the results of morphological changes in fibroblasts following low-intensity light illumination. Mouse fibroblasts were grown on glass coverslips on either 4 kPa or 16 kPa gels, to mimic normal tissue conditions. Cells were photo-irradiated with laser light at either 625 nm or 808 nm (total energies ranging from 34 to 47 J). Cells were fixed at 5 min, 1 h, or 24 h after photo-irradiation, stained for both actin filaments and the cell nucleus, and imaged by confocal microscopy. A non-light exposed group was also imaged. A detailed analysis of the images demonstrated that the total polymerized actin and number of actin filaments decrease, while the nucleus area increases in treated cells shortly after photo-irradiation, regardless of substrate and wavelength. This experiment indicated that photobiomodulation therapy could change the morphological properties of cells and affect their cytoskeleton. Further investigations are required to determine the specific mechanisms involved and how this phenomenon is related to the photobiomodulation therapy mechanisms of action.
Assuntos
Fibroblastos/efeitos da radiação , Terapia com Luz de Baixa Intensidade , Células 3T3 , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/efeitos da radiação , Animais , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Citoesqueleto/metabolismo , Citoesqueleto/efeitos da radiação , Fibroblastos/citologia , Camundongos , Microscopia ConfocalRESUMO
In children, ketamine sedation is often used during radiological procedures. Combined exposure of ketamine and radiation at doses that alone did not affect learning and memory induced permanent cognitive impairment in mice. The aim of this study was to elucidate the mechanism behind this adverse outcome. Neonatal male NMRI mice were administered ketamine (7.5 mg kg-1) and irradiated (whole-body, 100 mGy or 200 mGy, 137Cs) one hour after ketamine exposure on postnatal day 10. The control mice were injected with saline and sham-irradiated. The hippocampi were analyzed using label-free proteomics, immunoblotting, and Golgi staining of CA1 neurons six months after treatment. Mice co-exposed to ketamine and low-dose radiation showed alterations in hippocampal proteins related to neuronal shaping and synaptic plasticity. The expression of brain-derived neurotrophic factor, activity-regulated cytoskeleton-associated protein, and postsynaptic density protein 95 were significantly altered only after the combined treatment (100 mGy or 200 mGy combined with ketamine, respectively). Increased numbers of basal dendrites and branching were observed only after the co-exposure, thereby constituting a possible reason for the displayed alterations in behavior. These data suggest that the risk of radiation-induced neurotoxic effects in the pediatric population may be underestimated if based only on the radiation dose.
Assuntos
Região CA1 Hipocampal/patologia , Ketamina/toxicidade , Neurônios/patologia , Neurônios/efeitos da radiação , Radiação Ionizante , Animais , Animais Recém-Nascidos , Forma Celular/efeitos dos fármacos , Forma Celular/efeitos da radiação , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/efeitos da radiação , Masculino , Camundongos , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/efeitos da radiação , Neurônios/efeitos dos fármacos , Proteoma/metabolismoRESUMO
Optogenetics provides promising tools for the precise control of receptor-mediated cell behaviors in a spatiotemporal manner. Yet, most photoreceptors require extensive genetic manipulation and respond only to ultraviolet or visible light, which are suboptimal for in vivo applications because they do not penetrate thick tissues. Here we report a novel near-infrared light-activated DNA agonist (NIR-DA) nanodevice for nongenetic manipulation of cell signaling and phenotype in deep tissues. This nanodevice is prepared by conjugating a preinactivated DNA agonist onto the gold nanorods (AuNRs). Upon NIR light treatment, the DNA agonist is released through the localized surface plasmon resonance (LSPR)-based photothermal effect of AuNRs and becomes active. The active DNA agonist dimerizes the DNA-modified chimeric or native receptor tyrosine kinase (RTK) on cell surfaces and activates downstream signal transduction in live cells. Such NIR-DA activation of RTK signaling enables the control of cytoskeletal remodeling, cell polarization, and directional migration. Furthermore, we demonstrate that the NIR-DA system can be used in vivo to mediate RTK signaling and skeletal muscle satellite cell migration and myogenesis, which are critical cellular behaviors in the process of skeletal muscle regeneration. Thus, the NIR-DA system offers a powerful and versatile platform for exogenous modulation of deep tissues for purposes such as regenerative medicine.
Assuntos
Materiais Biocompatíveis/farmacologia , Comunicação Celular/efeitos dos fármacos , DNA/genética , Receptores Proteína Tirosina Quinases/genética , Materiais Biocompatíveis/química , Comunicação Celular/efeitos da radiação , Movimento Celular/efeitos dos fármacos , Movimento Celular/efeitos da radiação , Polaridade Celular/efeitos dos fármacos , Polaridade Celular/efeitos da radiação , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/efeitos da radiação , DNA/agonistas , DNA/química , DNA/efeitos dos fármacos , Ouro/química , Humanos , Raios Infravermelhos , Nanotubos/química , Receptores Proteína Tirosina Quinases/química , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Ressonância de Plasmônio de SuperfícieRESUMO
The mechanobiology is providing novel perspectives in the study of cancer and is contributing to evaluate the cancer responses, from a biophysical point of view, to classical therapeutic approaches- radiotherapy and chemotherapy. Here we have explored the effects of two doses (4 and 8 Gy) of 6 MeV photons on spreading, focal adhesions, migration and mechanical properties of BALB/c 3T3 and their SV40 transformed equivalent, SVT2. Cell biophysical responses to 4 and 8 Gy were analysed and compared with those reported in previous published work when lower doses (1 and 2 Gy) were administered Panzetta et al. (Effects of high energy X-rays on cell morphology and functions. Proc. Book 2017;16:116). We observed that the range of sensitivity to ionising radiations profoundly changes depending on the patho-physiological state of cells. In particular, we found that X-rays induce morphological and functional variations in both cell lines (decreased motility, increased adhesion and increased cytoskeleton stiffness). These changes were slightly dependent on doses in the case of SVT2 cells and may indicate a possible mechanical normalisation in their phenotype. Nevertheless, the responses of BALB/c 3T3 were negligible only for the low dose of 1 Gy and increased significantly in a dose-dependent manner with higher doses. We believe that the characterisation of X-rays effects on the cell mechanobiology could shed new light in the design and customisation of radiotherapy treatments.
Assuntos
Biofísica/métodos , Células Cultivadas/efeitos da radiação , Linhagem Celular , Citoesqueleto/efeitos da radiação , Relação Dose-Resposta à Radiação , Fótons , Eficiência Biológica Relativa , Raios XRESUMO
Exposure to ionizing radiation is unavoidable for noncancerous cells during the external radiotherapy process. Increasing the dose delivery fraction times leads to increasing the endothelial cell damage. Vascular abnormalities are commonly associated with the alternation of endothelium biomechanical properties. The goal of the present study was to quantify the elastic and viscoelastic properties of human umbilical vein endothelial cells (HUVECs) using the micropipette aspiration technique in conjunction with a theoretical model while an 8 Gy dose was given in four fractions. Confocal imaging was performed for evaluation of cytoskeletal changes during fractionation 60Co radiotherapy. The results indicated an increase in elastic modulus from 29.87 ± 1.04 Pa to 46.69 ± 1.17 Pa while the fractional doses increased from 0 Gy to 8 Gy along with the obvious cytoskeletal changes. Moreover, in the creep behavior of radiated groups, a significant decrease was shown in the time constant and viscoelastic properties. On the other hand, it was observed that the change in the biomechanical properties of the cells while applying a single fraction of 8 Gy was not exactly the same as that in the properties of the radiation-exposed cells while delivering an 8 Gy dose at 2 Gy per fraction. The observed differences in the biomechanical behavior of endothelium provide a quantitative description of radiobiological effects for evaluating the dose-response relationship as a biological dosimetry procedure.
Assuntos
Módulo de Elasticidade/efeitos da radiação , Raios gama , Radioisótopos de Cobalto/química , Citoesqueleto/efeitos da radiação , Células Endoteliais da Veia Umbilical Humana , Humanos , Microscopia Confocal , Doses de RadiaçãoRESUMO
PURPOSE: Although the microvascular system is a significant target for radiation-induced effects, the lymphatic response to radiation has not been extensively investigated. This is one of the first investigations characterizing the lymphatic endothelial response to ionizing radiation. MATERIALS AND METHODS: Rat mesenteric lymphatic endothelial cells (RMLECs) were exposed to X-ray doses of 0, 0.5, 1, 1.5, and 2 Gy. RMLEC cellular response was assessed 24 and 72-h post-irradiation via measures of cellular morphometry and junctional adhesion markers. RMLEC functional response was characterized through permeability experiments. RESULTS: Cell morphometry showed radiation sensitivity at all doses. Notably, there was a loss of cell-to-cell adhesion with irradiated cells increasing in size and cellular roundness. This was coupled with decreased ß-catenin and VE-cadherin intensity and altered F-actin anisotropy, leading to a loss of intercellular contact. RMLEC monolayers demonstrated increased permeability at all doses 24 h post-irradiation and at 2-Gy 72 h post-irradiation. CONCLUSIONS: In summary, lymphatics show radiation sensitivity in the context of these cell culture experiments. Our results may have functional implications of lymphatics in tissue, with endothelial barrier dysfunction due to loss of cell-cell adhesion leading to leaky vessels and lymphedema. These preliminary experiments will build the framework for future investigations towards lymphatic radiation exposure response.
Assuntos
Endotélio Linfático/efeitos da radiação , Junções Aderentes/metabolismo , Junções Aderentes/efeitos da radiação , Animais , Adesão Celular/efeitos da radiação , Citoesqueleto/metabolismo , Citoesqueleto/efeitos da radiação , Relação Dose-Resposta à Radiação , Endotélio Linfático/irrigação sanguínea , Endotélio Linfático/citologia , Endotélio Linfático/metabolismo , Masculino , Microvasos/efeitos da radiação , Permeabilidade/efeitos da radiação , Ratos , Ratos Sprague-Dawley , Raios X/efeitos adversosRESUMO
Radiation therapy has been widely utilized as an effective method to eliminate malignant tumors and cancerous cells. However, subjection of healthy tissues and the related networks of blood vessels adjacent to the tumor area to irradiation is inevitable. The aim of this study was to investigate the consequent effects of fractionation radiotherapy on the mechanical characteristics of human umbilical vein endothelial cells (HUVECs) through alterations in cytoskeleton organization and cell and nucleus morphology. In order to simulate the clinical condition of radiotherapy, the HUVECs were exposed to the specific dose of 2â¯Gy for 1-4â¯times among four groups with incremental total dose from 2â¯Gy up to 8â¯Gy. Fluorescence staining was performed to label F-actin filaments and nuclei. Micropipette aspiration and standard linear solid model were employed to evaluate the elastic and viscoelastic characteristics of the HUVECs. Radiotherapy significantly increased cell elastic moduli. Due to irradiation, instantaneous and equilibrium Young's modulus were also increased. Radiotherapy diminished HUVECs viscoelastic behavior and shifted their creep compliance curves downward. Furthermore, gamma irradiation elevated the nuclei sizes and to a lesser extent the cells sizes resulting in the accumulation of F-actin filaments within the rest of cell body. Endothelial stiffening correlates with endothelial dysfunction, hence the results may be helpful when the consequent effects of radiotherapy are the focus of concern.
Assuntos
Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos da radiação , Fenômenos Mecânicos/efeitos da radiação , Radioterapia/efeitos adversos , Fenômenos Biomecânicos/efeitos da radiação , Núcleo Celular/efeitos da radiação , Tamanho Celular/efeitos da radiação , Citoesqueleto/metabolismo , Citoesqueleto/efeitos da radiação , Fracionamento da Dose de Radiação , Elasticidade/efeitos da radiação , Humanos , Viscosidade/efeitos da radiaçãoRESUMO
Endothelial nitric oxide synthase (eNOS), a constitutive enzyme expressed in vascular endothelial cells, is the main source of nitric oxide (NO), which plays key roles in diverse biological functions, including regulation of vascular tone. Exposure to radiation has been known to generate nitric oxide from eNOS; however, the precise mechanism of its generation and function is not known. The goal of this study was to determine the involvement of radiation-induced DNA damage response (DDR) on eNOS transcription and its effect on cell survival after irradiation. Irradiated bovine aortic endothelial cells showed increased eNOS transcription and NO generation through upregulation of ataxia-telangiectasia mutated (ATM) kinase. Radiation exposure induced NO inhibited cell death, as well as induced cellular senescence postirradiation. This study established that radiation-induced DDR uses ATM kinase to upregulate eNOS transcription and NO generation, leading to cellular senescence, which may play a critical role in radiation-mediated cardiovascular injury.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/efeitos da radiação , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Óxido Nítrico Sintase Tipo III/genética , Tolerância a Radiação , Animais , Bovinos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Senescência Celular/efeitos dos fármacos , Senescência Celular/efeitos da radiação , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/efeitos da radiação , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Tolerância a Radiação/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/efeitos da radiaçãoRESUMO
The linker of nucleoskeleton and cytoskeleton (LINC) complex is a multifunctional protein complex that is involved in various processes at the nuclear envelope, including nuclear migration, mechanotransduction, chromatin tethering and DNA damage response. We recently showed that a nuclear envelope protein, Sad1 and UNC84 domain protein 1 (SUN1), a component of the LINC complex, has a critical function in cell migration. Although ionizing radiation activates cell migration and invasion in vivo and in vitro, the underlying molecular mechanism remains unknown. Here, we examined the involvement of the LINC complex in radiation-enhanced cell migration and invasion. A sublethal dose of X-ray radiation promoted human breast cancer MDA-MB-231 cell migration and invasion, whereas carbon ion beam radiation suppressed these processes in a dose-dependent manner. Depletion of SUN1 and SUN2 significantly suppressed X-ray-enhanced cell migration and invasion. Moreover, depletion or overexpression of each SUN1 splicing variant revealed that SUN1_888 containing 888 amino acids of SUN1 but not SUN1_916 was required for X-ray-enhanced migration and invasion. In addition, the results suggested that X-ray irradiation affected the expression level of SUN1 splicing variants and a SUN protein binding partner, nesprins. Taken together, our findings supported that the LINC complex contributed to photon-enhanced cell migration and invasion.
Assuntos
Movimento Celular/fisiologia , Movimento Celular/efeitos da radiação , Citoesqueleto/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Matriz Nuclear/metabolismo , Linhagem Celular Tumoral , Citoesqueleto/efeitos da radiação , Humanos , Mecanotransdução Celular/fisiologia , Mecanotransdução Celular/efeitos da radiação , Proteínas de Membrana/metabolismo , Invasividade Neoplásica/patologia , Membrana Nuclear/metabolismo , Matriz Nuclear/efeitos da radiação , Proteínas Nucleares/metabolismo , Ligação Proteica/efeitos da radiação , Splicing de RNA/efeitos da radiação , Raios XRESUMO
Actin, cytoskeleton protein forming microfilaments, play a crucial role in cellular motility. Here we show that exposure to very low levels of polarized light guide their orientation in-vivo within the live cell. Using a simple model to describe the role of actin-filament orientation in directional cellular motion, we demonstrate that the actin polymerization/depolymerization mechanism develops primarily along this direction and, under certain conditions, can lead to guidance of the cell movement. Our results also show a dose dependent increase in actin activity in direct correspondence to the level of laser irradiance. We found that total expression of Tau protein, which stabilize microtubules, was decreased by the irradiance, indicating that exposure to the light may change the activity of kinase, leading to increased cell activity.