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BACKGROUND: Human mesenchymal stem cells originating from umbilical cord matrix are a promising therapeutic resource, and their differentiated cells are spotlighted as a tissue regeneration treatment. However, there are limitations to the medical use of differentiated cells from human umbilical cord matrix-mesenchymal stem cells (hUCM-MSCs), such as efficient differentiation methods. METHODS: To effectively differentiate hUCM-MSCs into hepatocyte-like cells (HLCs), we used the ROCK inhibitor, fasudil, which is known to induce endoderm formation, and gelatin, which provides extracellular matrix to the differentiated cells. To estimate a differentiation efficiency of early stage according to combination of gelatin and fasudil, transcription analysis was conducted. Moreover, to demonstrate that organelle states affect differentiation, we performed transcription, tomographic, and mitochondrial function analysis at each stage of hepatic differentiation. Finally, we evaluated hepatocyte function based on the expression of mRNA and protein, secretion of albumin, and activity of CYP3A4 in mature HLCs. RESULTS: Fasudil induced endoderm-related genes (GATA4, SOX17, and FOXA2) in hUCM-MSCs, and it also induced lipid droplets (LDs) inside the differentiated cells. However, the excessive induction of LDs caused by fasudil inhibited mitochondrial function and prevented differentiation into hepatoblasts. To prevent the excessive LDs formation, we used gelatin as a coating material. When hUCM-MSCs were induced into hepatoblasts with fasudil on high-viscosity (1%) gelatin-coated dishes, hepatoblast-related genes (AFP and HNF4A) showed significant upregulation on high-viscosity gelatin-coated dishes compared to those treated with low-viscosity (0.1%) gelatin. Moreover, other germline cell fates, such as ectoderm and mesoderm, were repressed under these conditions. In addition, LDs abundance was also reduced, whereas mitochondrial function was increased. On the other hand, unlike early stage of the differentiation, low viscosity gelatin was more effective in generating mature HLCs. In this condition, the accumulation of LDs was inhibited in the cells, and mitochondria were activated. Consequently, HLCs originated from hUCM-MSCs were genetically and functionally more matured in low-viscosity gelatin. CONCLUSIONS: This study demonstrated an effective method for differentiating hUCM-MSCs into hepatic cells using fasudil and gelatin of varying viscosities. Moreover, we suggest that efficient hepatic differentiation and the function of hepatic cells differentiated from hUCM-MSCs depend not only on genetic changes but also on the regulation of organelle states.
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1-(5-Isoquinolinasulfonil)-2-Metilpiperazina , Diferenciação Celular , Gelatina , Hepatócitos , Células-Tronco Mesenquimais , Cordão Umbilical , Humanos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Diferenciação Celular/efeitos dos fármacos , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Gelatina/química , Gelatina/farmacologia , Hepatócitos/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/citologia , Cordão Umbilical/citologia , Viscosidade , Células Cultivadas , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacosRESUMO
Hepatitis B Virus (HBV) infection significantly elevates the risk of hepatocellular carcinoma (HCC), with the HBV X protein (HBx) playing a crucial role in cancer progression. Sorafenib, the primary therapy for advanced HCC, shows limited effectiveness in HBV-infected patients due to HBx-related resistance. Numerous studies have explored combination therapies to overcome this resistance. Sodium diethyldithiocarbamate (DDC), known for its anticancer effects and its inhibition of superoxide dismutase 1 (SOD1), is hypothesized to counteract sorafenib (SF) resistance in HBV-positive HCCs. Our research demonstrates that combining DDC with SF significantly reduces HBx and SOD1 expressions in HBV-positive HCC cells and human tissues. This combination therapy disrupts the PI3K/Akt/mTOR signalling pathway and promotes apoptosis by increasing reactive oxygen species (ROS) levels. These cellular changes lead to reduced tumour viability and enhanced sensitivity to SF, as evidenced by the synergistic suppression of tumour growth in xenograft models. Additionally, DDC-mediated suppression of SOD1 further enhances SF sensitivity in HBV-positive HCC cells and xenografted animals, thereby inhibiting cancer progression more effectively. These findings suggest that the DDC-SF combination could serve as a promising strategy for overcoming SF resistance in HBV-related HCC, potentially optimizing therapy outcomes.
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Carcinoma Hepatocelular , Vírus da Hepatite B , Neoplasias Hepáticas , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Espécies Reativas de Oxigênio , Transdução de Sinais , Sorafenibe , Superóxido Dismutase-1 , Serina-Treonina Quinases TOR , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/virologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/virologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Superóxido Dismutase-1/metabolismo , Superóxido Dismutase-1/genética , Animais , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Camundongos , Vírus da Hepatite B/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Apoptose/efeitos dos fármacos , Hepatite B/complicações , Hepatite B/tratamento farmacológico , Hepatite B/virologia , Ditiocarb/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Camundongos Nus , Proliferação de Células/efeitos dos fármacos , Transativadores , Proteínas Virais Reguladoras e AcessóriasRESUMO
Nanomedicines hold promise for the treatment of various diseases. However, treating cancer metastasis remains highly challenging. In this study, we synthesized gold nanorods (AuNRs) containing (α-GC), an immune stimulator, for the treatment of primary cancer, metastasis, and recurrence of the cancer. Therefore, the AuNR were coated with lipid bilayers loaded with α-GC (α-LA). Upon irradiation with 808 nm light, α-LA showed a temperature increase. Intra-tumoral injection of α-LA in mice and local irradiation of the 4T1 breast cancer tumor effectively eliminated tumor growth. We found that the presence of α-GC in α-LA activated dendritic cells and T cells in the spleen, which completely blocked the development of lung metastasis. In mice injected with α-LA for primary breast cancer treatment, we observed antigen-specific T cell responses and increased cytotoxicity against 4T1 cells. We conclude that α-LA is promising for the treatment of both primary breast cancer and its metastasis.
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Angiogenesis is mainly regulated by the delivery of VEGF-dependent signaling to cells. However, the angiogenesis mechanism regulated by VEGF-induced miRNA is still not understood. After VEGF treatment in HUVECs, we screened the changed miRNAs through small-RNA sequencing and found VEGF-induced miR-4701-3p. Furthermore, the GFP reporter gene was used to reveal that TOB2 expression was regulated by miR-4701-3p, and it was found that TOB2 and miR-4701-3p modulation could cause angiogenesis in an in-vitro angiogenic assay. Through the luciferase assay, it was confirmed that the activation of the angiogenic transcription factor MEF2 was regulated by the suppression and overexpression of TOB2 and miR-4701-3p. As a result, MEF2 downstream gene mRNAs that induce angiogenic function were regulated. We used the NCBI GEO datasets to reveal that the expression of TOB2 and MEF2 was significantly changed in cardiovascular disease. Finally, it was confirmed that the expression of circulating miR-4701-3p in the blood of myocardial infarction patients was remarkably increased. In patients with myocardial infarction, circulating miR-4701-3p was increased regardless of age, BMI, and sex, and showed high AUC levels in specificity and sensitivity analysis (AUROC) (AUC = 0.8451, 95 % CI 0.78-0.90). Our data showed TOB2-mediated modulation of MEF2 and its angiogenesis by VEGF-induced miR-4701-3p in vascular endothelial cells. In addition, through bioinformatics analysis using GEO data, changes in TOB2 and MEF2 were revealed in cardiovascular disease. We suggest that circulating miR-4701-3p has high potential as a biomarker for myocardial infarction.
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Células Endoteliais da Veia Umbilical Humana , Fatores de Transcrição MEF2 , MicroRNAs , Neovascularização Fisiológica , Humanos , Células Endoteliais da Veia Umbilical Humana/metabolismo , MicroRNAs/genética , MicroRNAs/sangue , MicroRNAs/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Masculino , Feminino , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/sangue , Fator A de Crescimento do Endotélio Vascular/genética , Transdução de Sinais , Infarto do Miocárdio/sangue , Infarto do Miocárdio/genética , Infarto do Miocárdio/diagnóstico , Células Cultivadas , Regulação da Expressão Gênica , Estudos de Casos e Controles , Pessoa de Meia-Idade , Bases de Dados Genéticas , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/sangue , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , AngiogêneseRESUMO
The importance of ß-glucan from S. cerevisiae in angiogenesis has not been well studied. We investigated whether ß-glucan induces angiogenesis through PI3K/Src and ERK1/2 signaling pathway in HUVECs. We identified that ß-glucan induced phosphorylation of PI3K, Src, Akt, eNOS, and ERK1/2. Subsequently, we found that this phosphorylation increased the viability of HUVECs. We also observed that stimulation of ß-glucan promoted the activity of MEF2 and MEF2-dependent pro-angiogenic genes, including EGR2, EGR3, KLF2, and KLF4. Additionally, the role of ß-glucan in angiogenesis was confirmed using in vitro and ex vivo experiments including cell migration, capillary-like tube formation and mouse aorta ring assays. To determine the effect of ß-glucan on the PI3K/Akt/eNOS and ERK1/2 signaling pathway, PI3K inhibitor wortmannin and ERK1/2 inhibitor SCH772984 were used. Through the Matrigel plug assay, we confirmed that ß-glucan significantly increased angiogenesis in vivo. Taken together, our study demonstrates that ß-glucan promotes angiogenesis via through PI3K and ERK1/2 signaling pathway.
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Células Endoteliais da Veia Umbilical Humana , Fator 4 Semelhante a Kruppel , Sistema de Sinalização das MAP Quinases , Neovascularização Fisiológica , Fosfatidilinositol 3-Quinases , beta-Glucanas , Quinases da Família src , beta-Glucanas/farmacologia , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Camundongos , Quinases da Família src/metabolismo , Movimento Celular/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , AngiogêneseRESUMO
BACKGROUND: Tumor-derived exosomes are critical elements of the cell-cell communication response to various stimuli. This study aims to reveal that the histone deacetylase 5 (HDAC5) and p53 interaction upon radiation in hepatocellular carcinoma intricately regulates the secretion and composition of exosomes. METHODS: We observed that HDAC5 and p53 expression were significantly increased by 2 Gy and 4 Gy radiation exposure in HCC. Normal- and radiation-derived exosomes released by HepG2 were purified to investigate the exosomal components. RESULTS: We found that in the radiation-derived exosome, exosomal Maspin was notably increased. Maspin is known as an anti-angiogenic gene. The expression of Maspin was regulated at the cellular level by HDAC5, and it was elaborately regulated and released in the exosome. Radiation-derived exosome treatment caused significant inhibition of angiogenesis in HUVECs and mouse aortic tissues. Meanwhile, we confirmed that miR-151a-3p was significantly reduced in the radiation-derived exosome through exosomal miRNA sequencing, and three HCC-specific exosomal miRNAs were also decreased. In particular, miR-151a-3p induced an anti-apoptotic response by inhibiting p53, and it was shown to induce EMT and promote tumor growth by regulating p53-related tumor progression genes. In the HCC xenograft model, radiation-induced exosome injection significantly reduced angiogenesis and tumor size. CONCLUSIONS: Our present findings demonstrated HDAC5 is a vital gene of the p53-mediated release of exosomes resulting in tumor suppression through anti-cancer exosomal components in response to radiation. Finally, we highlight the important role of exosomal Maspin and mi-151a-3p as a biomarker in enhancing radiation treatment sensitivity. Therapeutic potential of HDAC5 through p53-mediated exosome modulation in radiation treatment of hepatocellular carcinoma.
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BACKGROUND: Small cell lung cancer (SCLC) has an exceptionally poor prognosis; as most of the cases are initially diagnosed as extensive disease with hematogenous metastasis. Therefore, the early diagnosis of SCLC is very important and may improve its prognosis. METHODS: To investigate the feasibility of early diagnosis of SCLC, we examined exosomal microRNAs (miRNAs) present in serum obtained from patients with SCLC. First, exosomes were isolated in serum from patients with SCLC and healthy individuals and were characterized using particle size and protein markers. Additionally, miRNA array was performed to define SCLC-specific exosomal miRNAs. Second, the obtained miRNAs were further validated employing a large cohort. Finally, the ability to diagnose SCLC was estimated by area under the curve (AUC), and intracellular mRNA change patterns were verified through validated miRNAs. RESULTS: From the miRNA array results, we selected 51-miRNAs based on p-values and top 10 differentially expressed genes, and 25-miRNAs were validated using quantitative reverse transcription-polymerase chain reaction. The 25-miRNAs were further validated employing a large cohort. Among them, 7-miRNAs showed significant differences. Furthermore, 6-miRNAs (miR-3565, miR-3124-5p, miR-200b-3p, miR-6515, miR-3126-3p and miR-9-5p) were up-regulated and 1-miRNA (miR-92b-5p) was down-regulated. The AUC value of each miRNA sets between 0.64 and 0.76, however the combined application of 3-miRNAs (miR-200b-3p, miR-3124-5p and miR-92b-5p) remarkably improved the diagnostic value (AUC = 0.93). Gene ontology analysis revealed that the 3-miRNA panel is linked to various oncogene pathways and nervous system development. When the 3-miRNAs were introduced to cells, the resulting changes in total mRNA expression strongly indicated the presence of lung diseases, including lung cancer. In addition, the 3-miRNA panel was significantly associated with a poorer prognosis, although individual miRNAs have not been validated as prognostic markers. CONCLUSION: Our study identified SCLC-specific exosomal miRNAs, and the 3-miRNAs panel (miR-200b-3p, miR-3124-5p and miR-92b-5p) may serve as a diagnostic and prognostic marker for SCLC.
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Although venous invasion (VI) is common in colorectal cancers (CRCs) and is associated with distant metastasis, the 3-dimensional (3D) microscopic features and associated mechanisms of VI are not well elucidated. To characterize the patterns of VI, 103 tissue slabs were harvested from surgically resected CRCs with ≥pT2. They were cleared using the modified immunolabeling-enabled 3D imaging of solvent-cleared organs method, labeled with multicolor fluorescent antibodies, including antibodies against cytokeratin 19, desmin, CD31, and E-cadherin, and visualized by confocal laser scanning microscopy. VI was classified as intravasation, intraluminal growth, and/or extravasation, and 2-dimensional and 3D microscopic features were compared. VI was detected more frequently in 3D (56/103 [54.4%]) than in conventional 2-dimensional hematoxylin and eosin-stained slides (33/103 [32%]; P < .001). When VI was present, it was most commonly in the form of intraluminal growth (51/56), followed by extravasation (13/56) and intravasation (5/56). The mean length of intraluminal growth was 334.0 ± 212.4 µm. Neoplastic cell projections extended from cancer cell clusters in the connective tissue surrounding veins, penetrated the smooth muscle layer, and then grew into and filled the venous lumen. E-cadherin expression changed at each invasion phase; intact E-cadherin expression was observed in the cancer cells in the venous walls, but its expression was lost in small clusters of intraluminal neoplastic cells. In addition, reexpression of E-cadherin was observed when cancer cells formed well-oriented tubular structures and accumulated and grew along the luminal side of the venous wall. In contrast, singly scattered cancer cells and cancer cells with poorly defined tubular structures showed loss of E-cadherin expression. E-cadherin expression was intact in the large cohesive clusters of extravasated cancer cells. However, singly scattered cells and smaller projections of neoplastic cells in the stroma outward of venous wall showed a loss of E-cadherin expression. In conclusion, VI was observed in more than half of the CRCs analyzed by 3D histopathologic image reconstruction. Once inside a vein, neoplastic cells can grow intraluminally. The epithelial-mesenchymal transition is not maintained during VI of CRCs.
Assuntos
Caderinas , Neoplasias Colorretais , Humanos , Caderinas/metabolismo , Transição Epitelial-Mesenquimal , Linhagem Celular Tumoral , Neoplasias Colorretais/cirurgia , Neoplasias Colorretais/patologiaRESUMO
C1q/tumor necrosis factor-related protein 9 (CTRP9) is an adipokine and has high potential as a therapeutic target. However, the role of CTRP9 in cardiovascular disease pathogenesis remains unclear. We found CTRP9 to induce HDAC7 and p38 MAPK phosphorylation via tight regulation of AMPK in vascular endothelial cells, leading to angiogenesis through increased MEF2 activity. The expression of CTRP9 and atheroprotective MEF2 was decreased in plaque tissue of atherosclerotic patients and the ventricle of post-infarction mice. CTRP9 treatment inhibited the formation of atherosclerotic plaques in ApoE KO and CTRP9 KO mice. In addition, CTRP9 induced significant ischemic injury prevention in the post-MI mice. Clinically, serum CTRP9 levels were reduced in patients with MI compared with healthy controls. In summary, CTRP9 induces a vasoprotective response via the AMPK/HDAC7/p38 MAPK pathway in vascular endothelial cells, whereas its absence can contribute to atherosclerosis and MI. Hence, CTRP9 may represent a valuable therapeutic target and biomarker in cardiovascular diseases.
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Aterosclerose , Infarto do Miocárdio , Animais , Camundongos , Proteínas Angiogênicas , Adipocinas , Complemento C1q , Células Endoteliais , Proteínas Quinases Ativadas por AMP , Histona Desacetilases/genética , Proteínas Quinases p38 Ativadas por Mitógeno , Glicoproteínas , Adiponectina/genéticaRESUMO
We aimed to discover and validate urinary exosomal proteins as biomarkers for antibody-mediated rejection (ABMR) after kidney transplantation. Urine and for-cause biopsy samples from kidney transplant recipients were collected and categorized into the discovery cohort (n = 36) and a validation cohort (n = 65). Exosomes were isolated by stepwise ultra-centrifugation for proteomic analysis to discover biomarker candidates for ABMR (n = 12). Of 1820 exosomal proteins in the discovery cohort, four proteins were specifically associated with ABMR: cystatin C (CST3), serum paraoxonase/arylesterase 1, retinol-binding protein 4, and lipopolysaccharide-binding protein (LBP). In the validation cohort, the level of urinary exosomal LBP was significantly higher in the ABMR group (n = 25) compared with the T-cell-mediated rejection (TCMR) group and the no major abnormality (NOMOA) group. Urinary exosomal CST3 level was significantly higher in the ABMR group compared with the control and NOMOA groups. Immunohistochemical staining showed that LBP and CST3 in the glomerulus were more abundant in the ABMR group compared with other groups. The combined prediction probability of urinary exosomal LBP and CST3 was significantly correlated with summed LBP and CST3 intensity scores in the glomerulus and peritubular capillary as well as Banff g + ptc scores. Urinary exosomal CST3 and LBP could be potent biomarkers for ABMR after kidney transplantation.
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Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by the accumulation of amyloid-ß (Aß) aggregates in the brain. Clusterin (CLU), also known as apolipoprotein J, is a potent risk factor associated with AD pathogenesis, in which Aß aggregation is essentially involved. We observed close colocalization of CLU and Aß(1-42) (Aß42) in parenchymal amyloid plaques or vascular amyloid deposits in the brains of human amyloid precursor protein (hAPP)-transgenic Tg2576 mice. Therefore, to elucidate the binding interaction between CLU and Aß42 and its impact on amyloid aggregation and toxicity, the two synthetic proteins were incubated together under physiological conditions, and their structural and morphological variations were investigated using biochemical, biophysical, and microscopic analyses. Synthetic CLU spontaneously bound to different possible variants of Aß42 aggregates with very high affinity (Kd = 2.647 nM) in vitro to form solid CLU-Aß42 complexes. This CLU binding prevented further aggregation of Aß42 into larger oligomers or fibrils, enriching the population of smaller Aß42 oligomers and protofibrils and monomers. CLU either alleviated or augmented Aß42-induced cytotoxicity and apoptosis in the neuroblastoma-derived SH-SY5Y and N2a cells, depending on the incubation period and the molar ratio of CLU:Aß42 involved in the reaction before addition to the cells. Thus, the effects of CLU on Aß42-induced cytotoxicity were likely determined by the extent to which it bound and sequestered toxic Aß42 oligomers or protofibrils. These findings suggest that CLU could influence amyloid neurotoxicity and pathogenesis by modulating Aß aggregation.
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Doença de Alzheimer , Neuroblastoma , Síndromes Neurotóxicas , Doença de Alzheimer/patologia , Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Clusterina , Humanos , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidadeRESUMO
Semiconductor nanocrystals (SNCs) are an essential optical tool in life sciences. Application of SNCs to living systems requires that their surfaces be covered with biocompatible molecules. The surface capping of SNCs by glutathione (GSH) is an effective means to prepare biocompatible SNCs and involves replacement of the initial surface ligands with GSH. However, molecular insight into such ligand-exchange reactions remains elusive. Molecular insight into this process is important, because surface ligands significantly impact physical properties such as the stability and quantum yield of SNCs. In this study, we investigate the ligand-exchange reaction of GSH on rod-shaped CdSe/CdS SNCs by Fourier-transform infrared (FTIR) absorption spectroscopy. The structure and interactions of GSH on SNC surfaces are clarified. Quantitative determination of the GSH molar fraction on SNC surfaces reveals that â¼3% of the initial trioctylphosphine oxide (TOPO) ligand is retained. Concentration-dependent experiments show that the surface molar fraction of GSH impacts the physical properties, solubilization yields, and quantum yields of SNCs in a linear manner.
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Compostos de Cádmio , Pontos Quânticos , Compostos de Selênio , Compostos de Cádmio/química , Glutationa , Ligantes , Pontos Quânticos/química , Compostos de Selênio/química , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Chemotherapy is one of the most effective treatments for cancer. However, intracellular delivery of many anticancer drugs is hindered by their hydrophobicity and low molecular weight. Here, we describe highly biocompatible and biodegradable amphiphilic vitamin conjugates comprising hydrophobic vitamin E and hydrophilic vitamin B labeled with dual pH and glutathione-responsive degradable linkages. Vitamin-based micelles (vitamicelles), formed by self-assembly in aqueous solutions, were optimized based on their stability after encapsulation of doxorubicin (DOX). The resulting vitamicelles have great potential as vehicles for anticancer drugs because they show excellent biocompatibility (>94% after 48 h of incubation) and rapid biodegradability (>90% after 2.5 h). Compared with free DOX, DOX-loaded vitamicelles showed a markedly enhanced anticancer effect as they released the drug rapidly and inhibited drug efflux out of cells efficiently. By exploiting these advantages, this study not only provides a promising strategy for circumventing existing challenges regarding the delivery of anticancer drugs but also extends the utility of current DOX-induced chemotherapy.
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Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Portadores de Fármacos/química , Micelas , Nanopartículas/administração & dosagem , Neoplasias/tratamento farmacológico , Vitaminas/química , Antibióticos Antineoplásicos/química , Apoptose , Proliferação de Células , Doxorrubicina/química , Células Hep G2 , Humanos , Células MCF-7 , Nanopartículas/química , Neoplasias/patologiaRESUMO
In vertebrates, nucleostemin (NS) is an important marker of proliferation in several types of stem and cancer cells, and it can also interact with the tumor-suppressing transcription factor p53. In the present study, the intra-nuclear diffusional dynamics of native NS tagged with GFP and two GFP-tagged NS mutants with deleted guanosine triphosphate (GTP)-binding domains were analyzed by fluorescence correlation spectroscopy. Free and slow binding diffusion coefficients were evaluated, either under normal culture conditions or under treatment with specific cellular proliferation inhibitors actinomycin D (ActD), 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), or trichostatin A (TSA). When treated with ActD, the fractional ratio of the slow diffusion was significantly decreased in the nucleoplasm. The decrease was proportional to ActD treatment duration. In contrast, DRB or TSA treatment did not affect NS diffusion. Interestingly, it was also found that the rate of diffusion of two NS mutants increased significantly even under normal conditions. These results suggest that the mobility of NS in the nucleoplasm is related to the initiation of DNA or RNA replication, and that the GTP-binding motif is also related to the large change of mobility.
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Núcleo Celular/metabolismo , Dactinomicina/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Nucleares/metabolismo , Inibidores da Síntese de Ácido Nucleico/farmacologia , Transcrição Gênica , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/genética , Células HeLa , Humanos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genéticaRESUMO
Tumor-derived exosomes (TEXs) contain enriched miRNAs, and exosomal miRNAs can affect tumor growth, including cell proliferation, metastasis, and drug resistance through cell-to-cell communication. We investigated the role of exosomal miR-1260b derived from non-small cell lung cancer (NSCLC) in tumor progression. Exosomal miR-1260b induced angiogenesis by targeting homeodomain-interacting protein kinase-2 (HIPK2) in human umbilical vein endothelial cells (HUVECs). Furthermore, exosomal miR-1260b or suppression of HIPK2 led to enhanced cellular mobility and cisplatin resistance in NSCLC cells. In patients with NSCLC, the level of HIPK2 was significantly lower in tumor tissues than in normal lung tissues, while that of miR-1260b was higher in tumor tissues. HIPK2 and miR-1260b expression showed an inverse correlation, and this correlation was strong in distant metastasis. Finally, the expression level of exosomal miR-1260b in plasma was higher in patients with NSCLC than in healthy individuals, and higher levels of exosomal miR-1260b were associated with high-grade disease, metastasis, and poor survival. In conclusion, exosomal miR-1260b can promote angiogenesis in HUVECs and metastasis of NSCLC by regulating HIPK2 and may serve as a prognostic marker for lung cancers.
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Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Transporte/antagonistas & inibidores , Exossomos/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , MicroRNAs/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Apoptose/genética , Sequência de Bases , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Cisplatino/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , MicroRNAs/genética , Invasividade Neoplásica , Metástase Neoplásica , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Transmembrane 4 L six family member 5 (TM4SF5) translocates intracellularly and promotes cell migration, but how subcellular TM4SF5 traffic is regulated to guide cellular migration is unknown. We investigated the influences of the extracellular environment and intracellular signaling on the TM4SF5 traffic with regard to migration directionality. Cell adhesion to fibronectin (FN) but not poly-l-lysine enhanced the traffic velocity and straightness of the TM4SF5WT (but not palmitoylation-deficient mutant TM4SF5Pal- ) toward the leading edges, depending on tubulin acetylation. Acetylated-microtubules in SLAC2B-positive cells reached mostly the juxtanuclear regions, but reached-out toward the leading edges upon SLAC2B suppression. TM4SF5 expression caused SLAC2B not to be localized at the leading edges. TM4SF5 colocalization with HDAC6 depended on paxillin expression. The trimeric complex consisting of TM4SF5, HDAC6, and SLAC2B might, thus, be enriched at the perinuclear cytosols toward the leading edges. More TM4SF5WT translocation to the leading edges was possible when acetylated-microtubules reached the frontal edges following HDAC6 inhibition by paxillin presumably at new cell-FN adhesions, leading to persistent cell migration. Collectively, this study revealed that cell-FN adhesion and microtubule acetylation could control intracellular traffic of TM4SF5 vesicles to the leading edges via coordinated actions of paxillin, SLAC2B, and HDAC6, leading to TM4SF5-dependent cell migration.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Membrana Celular/metabolismo , Matriz Extracelular/fisiologia , Proteínas de Membrana/metabolismo , Microtúbulos/metabolismo , Acetilação , Adesão Celular , Movimento Celular , Fibronectinas/fisiologia , Células Hep G2 , Desacetilase 6 de Histona/fisiologia , Humanos , Paxilina/fisiologia , Transporte ProteicoRESUMO
Adult hippocampal neurogenesis supports the structural and functional plasticity of the brain, while its decline is associated with neurodegeneration common in Alzheimer's disease (AD). Although the dysregulation of certain microRNAs (miRNAs) in AD have been observed, the effects of miRNAs on hippocampal neurogenesis are largely unknown. In this study, we demonstrated miR-351-5p as a causative factor in hippocampal neural progenitor cell death through modulation of the mitochondrial guanosine triphosphatase (GTPase), Miro2. Downregulation of Miro2 by siMiro2 induced cell death, similar to miR-351-5p, whereas ectopic Miro2 expression using an adenovirus abolished these effects. Excessively fragmented mitochondria and dysfunctional mitochondria were indexed by decreased mitochondrial potential, and increased reactive oxygen species were identified in miR-351-5p-induced cell death. Moreover, subsequent induction of mitophagy via Pink1 and Parkin was observed in the presence of miR-351-5p and siMiro2. The suppression of mitochondrial fission by Mdivi-1 completely inhibited cell death by miR-351-5p. miR-351-5p expression increased whereas the level of Miro2 decreased in the hippocampus of AD model mice, emulating expression in AD patients. Collectively, the data indicate the mitochondrial fission and accompanying mitophagy by miR-351-5p/Miro2 axis as critical in hippocampal neural progenitor cell death, and a potential therapeutic target in AD.
RESUMO
BACKGROUND AND AIMS: Mutations in XIAP can lead to the development of treatment-refractory severe paediatric Crohn's disease [CD], for which haematopoietic stem cell transplantation is the primary therapeutic option. The interpretation of variants of uncertain significance [VUSs] in XIAP needs to be scrutinized. METHODS: Targeted next-generation sequencing was performed for 33 male paediatric patients with refractory CD admitted at a tertiary referral hospital. To obtain functional data, biomolecular cell assays and supercomputing molecular dynamics simulations were performed. RESULTS: Nine unrelated male patients harboured hemizygous XIAP variants. Four known pathogenic variants and one novel pathogenic variant [p.Lys168Serfs*12] were identified in five patients, and two novel VUSs [p.Gly205del and p.Pro260Ser] and one known VUS [p.Glu350del] were identified in the remaining four. Among children with VUSs, only the subject with p.Gly205del exhibited defective NOD2 signalling. Using molecular dynamics simulation, we determined that the altered backbone torsional energy of C203 in XIAP of p.G205del was ~2 kcal/mol, suggesting loss of zinc binding in the mutant XIAP protein and poor coordination between the mutant XIAP and RIP2 proteins. Elevated auto-ubiquitination of zinc-depleted p.G205del XIAP protein resulted in XIAP protein deficiency. CONCLUSION: A high prevalence of XIAP deficiency was noted among children with refractory CD. Advanced functional studies decreased the subjectivity in the case-level interpretation of XIAP VUSs and directed consideration of haematopoietic stem cell transplantation.
Assuntos
Doença de Crohn/genética , Mutação , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Povo Asiático , Criança , Hemizigoto , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , República da Coreia , Transdução de Sinais , Falha de TratamentoRESUMO
Uniformly parallel Au-coated ZnO nanorods have previously been shown to amplify local Raman signals, providing increased sensitivity to disease markers in the detection of inflammation and cancer. However, practical and cost-effective fabrication methods of substrates for surface-enhanced Raman spectroscopy (SERS) fail to produce highly uniform surfaces. Here, the feasibility of Raman enhancement on less-uniform substrates is assessed. ZnO nanorod structures were fabricated by hydrothermal synthesis, starting from spin-coated seed substrates. Following analysis, the nanostructures were coated with Au to create stochastically variant substrates. The non-uniformity of the fabricated Au-coated ZnO nanorod structures is confirmed morphologically by FE-SEM and structurally by X-ray diffraction, and characterized by the angular distributions of the nanorods. Monte Carlo finite element method simulations matching the measured angular distributions and separations predicted only moderate increases in the overall Raman enhancement with increasing uniformity. Highly variant substrates exhibited approximately 76% of the Raman enhancement of more uniform substrates in simulations and experiments. The findings suggest that, although highly inhomogeneous Au-coated ZnO nanorod substrates may not attain the same Raman enhancement as more uniform substrates, the relaxation of fabrication tolerances may be economically viable.
RESUMO
Breast cancer (BC) is one of the most common cancers in women. TNBC (Triple-negative breast cancer) has limited treatment options and still lacks viable molecular targets, leading to poor outcomes. Recently, RNA-binding proteins (RBPs) have been shown to play crucial roles in human cancers, including BC, by modulating a number of oncogenic phenotypes. This suggests that RBPs represent potential molecular targets for BC therapy. Methods: We employed genomic data to identify RBPs specifically expressed in TNBC. NONO was silenced in TNBC cell lines to examine cell growth, colony formation, invasion, and migration. Gene expression profiles in NONO-silenced cells were generated and analyzed. A high-throughput screening for NONO-targeted drugs was performed using an FDA-approved library. Results: We found that the NONO RBP is highly expressed in TNBC and is associated with poor patient outcomes. NONO binds to STAT3 mRNA, increasing STAT3 mRNA levels in TNBC. Surprisingly, NONO directly interacts with STAT3 protein increasing its stability and transcriptional activity, thus contributing to its oncogenic function. Importantly, high-throughput drug screening revealed that auranofin is a potential NONO inhibitor and inhibits cell growth in TNBC. Conclusions: NONO is an RBP upstream regulator of both STAT3 RNA and protein levels and function. It represents an important and clinically relevant promoter of growth and resistance of TNBCs. NONO is also therefore a potential therapeutic target in TNBC.