Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 19.887
Filtrar
1.
Nat Commun ; 11(1): 4928, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004791

RESUMO

High-altitude adaptation of Tibetans represents a remarkable case of natural selection during recent human evolution. Previous genome-wide scans found many non-coding variants under selection, suggesting a pressing need to understand the functional role of non-coding regulatory elements (REs). Here, we generate time courses of paired ATAC-seq and RNA-seq data on cultured HUVECs under hypoxic and normoxic conditions. We further develop a variant interpretation methodology (vPECA) to identify active selected REs (ASREs) and associated regulatory network. We discover three causal SNPs of EPAS1, the key adaptive gene for Tibetans. These SNPs decrease the accessibility of ASREs with weakened binding strength of relevant TFs, and cooperatively down-regulate EPAS1 expression. We further construct the downstream network of EPAS1, elucidating its roles in hypoxic response and angiogenesis. Collectively, we provide a systematic approach to interpret phenotype-associated noncoding variants in proper cell types and relevant dynamic conditions, to model their impact on gene regulation.


Assuntos
Aclimatação/genética , Cromatina/metabolismo , Grupos Étnicos/genética , Redes Reguladoras de Genes , Modelos Genéticos , Altitude , Doença da Altitude/etnologia , Doença da Altitude/genética , Doença da Altitude/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Hipóxia Celular/genética , Células Cultivadas , Cromatina/genética , Sequenciamento de Cromatina por Imunoprecipitação , Resistência à Doença/genética , Feminino , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Oxigênio/metabolismo , Polimorfismo de Nucleotídeo Único , Gravidez , Cultura Primária de Células , RNA-Seq , Elementos Reguladores de Transcrição/genética , Seleção Genética , Tibet/etnologia , Fatores de Transcrição/metabolismo , Sequenciamento Completo do Genoma
2.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 34(9): 1163-1169, 2020 Sep 15.
Artigo em Chinês | MEDLINE | ID: mdl-32929911

RESUMO

Objective: To explore the feasibility and mechanism of inhibiting miR-429 to improve the permeability of the blood spinal cord barrier (BSCB) in vitro, and provide a new gene therapy target for enhancing the spinal cord microenvironment. Methods: First, the immortalized human brain microvascular endothelial cell line (hCMEC/D3) was transfected with the anti-miR-429 antagonist (antagomiR-429) and its negative control (antagomiR-429-NC), respectively. The miR-429 expression of hCMEC/D3 cells was observed by fluorescence microscopy and real-time fluorescence quantitative PCR to verify the transfection efficiency of antagomiR-429. Then the effect of miR-429 on BSCB permeability was observed in vitro. The experiment was divided into 4 groups. The blank control group (group A) was constructed of normal hCMEC/D3 cells and Ha-sc cells to prepare the BSCB model, the hypoxia-induced group (group B), the hypoxia-induced+antagomiR-429-NC group (group C), and the hypoxia-induced+antagomiR-429 group (group D) were constructed of normal, antagomiR-429-NC transfected, and antagomiR-429 transfected hCMEC/D3 cells and Ha-sc cells to prepare the BSCB models and hypoxia treatment for 12 hours. The permeability of BSCB in vitro was measured by horseradish peroxidase (HRP) permeability. Real-time fluorescence quantitative PCR, Western blot, and immunofluorescence staining were used to observe the expressions of ZO-1, Occludin, and Claudin-5. Results: The antagomiR-429 and antagomiR-429-NC were successfully transfected into hCMEC/D3 cells under a fluorescence microscope, and the transfection efficiency was about 90%. Real-time fluorescence quantitative PCR results showed that the relative expression of miR-429 in antagomiR-429 group was 0.109±0.013, which was significantly lower than that of antagomiR-429-NC group (0.956±0.004, P<0.05). HRP permeability measurement, real-time fluorescence quantitative PCR, and Western blot results showed that the HRP permeability of groups B and C were significantly higher than those of groups A and D ( P<0.05), and the relative expressions of ZO-1, Occludin, and Claudin-5 proteins and mRNAs were significantly lower in groups B and C than in groups A and D ( P<0.05) and in group D than in group A ( P<0.05); there was no significant difference between groups B and C ( P>0.05). Immunofluorescence staining showed that the immunofluorescence of ZO-1, Occudin, and Claudin-5 at the cell membrane boundary in group D were stronger than those in groups B and C, but not as strong as that in group A. Conclusion: Inhibition of miR-429 expression can promote the expressions of ZO-1, Occludin, and Claudin-5 proteins in microvascular endothelial cells, thereby improving the increased permeability of BSCB due to hypoxia.


Assuntos
Células Endoteliais , MicroRNAs , Barreira Hematoencefálica , Hipóxia Celular , Claudina-5 , Humanos , Ocludina , Permeabilidade , Medula Espinal , Proteína da Zônula de Oclusão-1
3.
Anticancer Res ; 40(9): 5071-5079, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32878795

RESUMO

BACKGROUND/AIM: Liver cancer has extremely poor prognosis. The cancerous tissues contain hypoxic regions, and the available drugs are poorly effective in hypoxic environments. NADPH oxidase 4 (NOX4), producing reactive oxygen species (ROS), may contribute to cancer malignancy under hypoxic conditions. However, its role in liver cancer has not been examined in detail. Our aim was to explore the effects of setanaxib, a recently developed selective NOX4 inhibitor, in liver cancer cells under hypoxic conditions. MATERIALS AND METHODS: Liver cancer cell lines (HepG2, HLE and Alexander) were treated with hypoxia-mimetic agent cobalt chloride. Cytotoxicity assays, immunoblot analysis and ROS detection assay were performed to detect the effect of setanaxib under hypoxic conditions. RESULTS: Setanaxib exhibited hypoxia-selective cytotoxicity and triggered apoptosis in cancer cells. Moreover, setanaxib caused mitochondrial ROS accumulation under hypoxic conditions. Treatment with antioxidants markedly attenuated setanaxib-induced cytotoxicity and apoptosis under hypoxic conditions. CONCLUSION: Setanaxib caused mitochondrial ROS accumulation in a hypoxia-selective manner and evoked cancer cell cytotoxicity by inducing apoptosis. Thus, setanaxib has a great potential as a novel anticancer compound under hypoxic conditions.


Assuntos
Antineoplásicos/farmacologia , Hipóxia Celular/efeitos dos fármacos , Hipóxia/metabolismo , Neoplasias Hepáticas/metabolismo , NADPH Oxidase 4/antagonistas & inibidores , Linhagem Celular Tumoral , Humanos , Mitocôndrias/metabolismo , NADPH Oxidases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
4.
Cells ; 9(9)2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32911794

RESUMO

All-trans-retinoic acid (atRA) is the essential derivative of vitamin A and is of interest due to its various biological key functions. As shown in the recent literature, atRA also plays a role in the failing heart during myocardial infarction, the leading cause of death globally. To date insufficient mechanistic information has been available on related hypoxia-induced cell damage and reperfusion injuries. However, it has been demonstrated that a reduction in cellular atRA uptake abrogates hypoxia-mediated cell and tissue damage, which may offer a new route for intervention. Consequently, in this study, the effect of the novel cardio-protective compound 5-methoxyleoligin (5ML) on cellular atRA uptake was tested in human umbilical-vein endothelial cells (HUVECs). For this purpose, a high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed to assess intra-cellular levels of the active substance and corresponding levels of vitamin A and its derivatives, including potential cis/trans isomers. This work also focused on light-induced isomerization and the stability of biological sample material to ensure sample integrity and avoid biased conclusions. This study provides evidence of the inhibitory effect of 5ML on cellular atRA uptake, a promising step toward a novel therapy for myocardial infarction.


Assuntos
Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Oxigênio/metabolismo , Tretinoína/metabolismo , Hipóxia Celular , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Lignanas/farmacologia
5.
Nat Commun ; 11(1): 4755, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958772

RESUMO

We hereby provide the initial portrait of lincNORS, a spliced lincRNA generated by the MIR193BHG locus, entirely distinct from the previously described miR-193b-365a tandem. While inducible by low O2 in a variety of cells and associated with hypoxia in vivo, our studies show that lincNORS is subject to multiple regulatory inputs, including estrogen signals. Biochemically, this lincRNA fine-tunes cellular sterol/steroid biosynthesis by repressing the expression of multiple pathway components. Mechanistically, the function of lincNORS requires the presence of RALY, an RNA-binding protein recently found to be implicated in cholesterol homeostasis. We also noticed the proximity between this locus and naturally occurring genetic variations highly significant for sterol/steroid-related phenotypes, in particular the age of sexual maturation. An integrative analysis of these variants provided a more formal link between these phenotypes and lincNORS, further strengthening the case for its biological relevance.


Assuntos
Homeostase , Oxigênio/metabolismo , RNA Longo não Codificante/fisiologia , Esteróis/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Colesterol/metabolismo , Estrogênios/metabolismo , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/metabolismo , Humanos , Células MCF-7 , Fenótipo , Polimorfismo de Nucleotídeo Único , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo
6.
PLoS One ; 15(8): e0235551, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833964

RESUMO

VPS34 is a key regulator of endomembrane dynamics and cargo trafficking, and is essential in cultured cell lines and in mice. To better characterize the role of VPS34 in cell growth, we performed unbiased cell line profiling studies with the selective VPS34 inhibitor PIK-III and identified RKO as a VPS34-dependent cellular model. Pooled CRISPR screen in the presence of PIK-III revealed endolysosomal genes as genetic suppressors. Dissecting VPS34-dependent alterations with transcriptional profiling, we found the induction of hypoxia response and cholesterol biosynthesis as key signatures. Mechanistically, acute VPS34 inhibition enhanced lysosomal degradation of transferrin and low-density lipoprotein receptors leading to impaired iron and cholesterol uptake. Excess soluble iron, but not cholesterol, was sufficient to partially rescue the effects of VPS34 inhibition on mitochondrial respiration and cell growth, indicating that iron limitation is the primary driver of VPS34-dependency in RKO cells. Loss of RAB7A, an endolysosomal marker and top suppressor in our genetic screen, blocked transferrin receptor degradation, restored iron homeostasis and reversed the growth defect as well as metabolic alterations due to VPS34 inhibition. Altogether, our findings suggest that impaired iron mobilization via the VPS34-RAB7A axis drive VPS34-dependence in certain cancer cells.


Assuntos
Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Ferro/metabolismo , Neoplasias/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Proliferação de Células , Colesterol/biossíntese , Colesterol/genética , Classe III de Fosfatidilinositol 3-Quinases/genética , Endossomos/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Receptores de LDL/metabolismo , Transferrina/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
7.
Gene ; 762: 145034, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32777521

RESUMO

Carbonic Anhydrase III (CAIII) belongs to a member of the alpha Carbonic Anhydrase (CA) family. Although some CA members are strongly up-regulated by HIF1-α, it is not known about the transcriptional regulation of CAIII in prostate cancer cells, PCa. Therefore, we aimed to identify regulatory regions important for the regulation of CAIII gene under hypoxic conditions in human prostate cancer cells (PC3). The present study, for the first time, demonstrated that the chemically mimicked hypoxic condition led to the induced CAIII mRNA and protein expression in prostate cancer cells. Transcriptional regulation of CAIII was investigated by transient transfection assay that indicates that the most active promoter activity was in the region of P2 -699/+86. Hypoxic condition also upregulates the basal activity of for P1;-941/+86 and P2;-699/+86 constructs containing putative Hypoxia Response Element (HRE) region located in -268/-252. EMSA analysis of HRE located in -268/-252 bases, showed one DNA-protein binding complexes. Competition assays indicated this complex is resulted from HIF1α interactions. In addition, site-directed mutagenesis of potential HIF1α binding sites diminished a DNA-protein complex. These findings suggest that CAIII is a hypoxia-regulated gene and valuable for targeting of prostate cancer tumors in hypoxic condition.


Assuntos
Anidrase Carbônica III/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias da Próstata/metabolismo , Anidrase Carbônica III/metabolismo , Hipóxia Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Masculino , Células PC-3 , Regiões Promotoras Genéticas , Regulação para Cima
8.
PLoS Comput Biol ; 16(8): e1008041, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32745136

RESUMO

Hypoxia-activated prodrugs (HAPs) present a conceptually elegant approach to not only overcome, but better yet, exploit intra-tumoural hypoxia. Despite being successful in vitro and in vivo, HAPs are yet to achieve successful results in clinical settings. It has been hypothesised that this lack of clinical success can, in part, be explained by the insufficiently stringent clinical screening selection of determining which tumours are suitable for HAP treatments. Taking a mathematical modelling approach, we investigate how tumour properties and HAP-radiation scheduling influence treatment outcomes in simulated tumours. The following key results are demonstrated in silico: (i) HAP and ionising radiation (IR) monotherapies may attack tumours in dissimilar, and complementary, ways. (ii) HAP-IR scheduling may impact treatment efficacy. (iii) HAPs may function as IR treatment intensifiers. (iv) The spatio-temporal intra-tumoural oxygen landscape may impact HAP efficacy. Our in silico framework is based on an on-lattice, hybrid, multiscale cellular automaton spanning three spatial dimensions. The mathematical model for tumour spheroid growth is parameterised by multicellular tumour spheroid (MCTS) data.


Assuntos
Antineoplásicos/farmacologia , Hipóxia Celular/fisiologia , Modelos Biológicos , Pró-Fármacos/farmacologia , Microambiente Tumoral/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Biologia Computacional , Simulação por Computador , Humanos , Radiação Ionizante , Radioterapia , Esferoides Celulares , Células Tumorais Cultivadas
9.
PLoS Comput Biol ; 16(8): e1007961, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32810174

RESUMO

Tumour spheroids are widely used as an in vitro assay for characterising the dynamics and response to treatment of different cancer cell lines. Their popularity is largely due to the reproducible manner in which spheroids grow: the diffusion of nutrients and oxygen from the surrounding culture medium, and their consumption by tumour cells, causes proliferation to be localised at the spheroid boundary. As the spheroid grows, cells at the spheroid centre may become hypoxic and die, forming a necrotic core. The pressure created by the localisation of tumour cell proliferation and death generates an cellular flow of tumour cells from the spheroid rim towards its core. Experiments by Dorie et al. showed that this flow causes inert microspheres to infiltrate into tumour spheroids via advection from the spheroid surface, by adding microbeads to the surface of tumour spheroids and observing the distribution over time. We use an off-lattice hybrid agent-based model to re-assess these experiments and establish the extent to which the spatio-temporal data generated by microspheres can be used to infer kinetic parameters associated with the tumour spheroids that they infiltrate. Variation in these parameters, such as the rate of tumour cell proliferation or sensitivity to hypoxia, can produce spheroids with similar bulk growth dynamics but differing internal compositions (the proportion of the tumour which is proliferating, hypoxic/quiescent and necrotic/nutrient-deficient). We use this model to show that the types of experiment conducted by Dorie et al. could be used to infer spheroid composition and parameters associated with tumour cell lines such as their sensitivity to hypoxia or average rate of proliferation, and note that these observations cannot be conducted within previous continuum models of microbead infiltration into tumour spheroids as they rely on resolving the trajectories of individual microbeads.


Assuntos
Modelos Biológicos , Esferoides Celulares , Células Tumorais Cultivadas , Animais , Fenômenos Biomecânicos , Morte Celular/fisiologia , Hipóxia Celular/fisiologia , Proliferação de Células/fisiologia , Biologia Computacional , Humanos , Esferoides Celulares/citologia , Esferoides Celulares/fisiologia , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/fisiologia
10.
J Cancer Res Clin Oncol ; 146(10): 2509-2517, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32620986

RESUMO

BACKGROUND: Colorectal cancer (CRC) is now a major human cancer, and B-cell translocation gene 3 (BTG3) has been reported as a tumor-suppressor in CRC, but its upstream regulator has not been identified. METHODS: Endogenous expression levels of BTG3 were compared between normal colorectal cell line CCD-18Co and two CRC cell lines SW480 and HT29, as well as between CRC patient tumor and adjacent normal tissues. Analysis of BTG3 genomic region was performed which identified a putative hypoxia response element (HRE). Effects of hypoxia condition, BTG3 overexpression, and their combination on the radiation sensitivity of CRC cell lines were assessed. RESULTS: BTG3 was downregulated in CRC cell lines and patient tumor samples, via the HRE in its promoter region. Hypoxia and BTG3 overexpression could both induce radiation resistance in CRC cells. Combining hypoxia with BTG3 overexpression effectively rendered the resistance of CRC cells to radiation to a level lower than hypoxia alone and higher than normoxia alone, indicating the essential role of BTG3 in hypoxia-induced radiation resistance of CRC cells. CONCLUSION: We therefore propose a novel signaling cascade involving hypoxia/BTG3 to be a potential risk factor for CRC patients undergoing radiation therapy, which could possibly serve as therapeutic targets among CRC patients with acquired radiotherapy resistance.


Assuntos
Proteínas de Ciclo Celular/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/radioterapia , Idoso , Apoptose/efeitos da radiação , Proteínas de Ciclo Celular/biossíntese , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Tolerância a Radiação , Elementos de Resposta
11.
J Cancer Res Clin Oncol ; 146(10): 2497-2507, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32620987

RESUMO

PURPOSE: Tumor explant culture systems can mimic the in vivo tumor microenvironment, proposing as a substitute for preclinical studies for prediction of individual treatment response. Therefore, our study evaluated the potential usefulness of ex vivo tumor explants culture assembled into the cell sheets by anticancer drug screening in patients with head and neck squamous cell carcinoma (HNSCC). METHODS: Our model included tumor explants incorporated into cell sheet composing of epithelium and subepithelial stroma using tumor and mucosal samples obtained from the HNSCC patients who underwent surgery. Cell growth, viability, and hypoxia were measured by cell counting kit-8, live/dead assay, propidium iodide, and LOX-1 staining, and were compared among the different treatment groups with vehicle, cisplatin or docetaxel. RESULTS: Tumor explants stably survived in the cell sheet over 10 days after explantation, whereas most of the explants in non-matrix culture became nonviable within 5-8 days with the significant daily decrease of viability. The live tissue areas of tumor explants in the cell sheet maintained over 30 days without significant changes although hypoxic cell areas gradually increased up to 5 days. Tissue viability and live cancer tissue areas significantly decreased after the treatment of cisplatin or docetaxel in the dose and time-dependent manners. CONCLUSION: Our cell sheet-based tumor explants model might be applied to the reliable ex vivo screening for anticancer chemotherapeutics for HNSCC.


Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Hipóxia Celular/fisiologia , Sobrevivência Celular/fisiologia , Cisplatino/farmacologia , Docetaxel/farmacologia , Relação Dose-Resposta a Droga , Neoplasias de Cabeça e Pescoço/sangue , Humanos , Técnicas de Cultura de Órgãos/métodos , Carcinoma de Células Escamosas de Cabeça e Pescoço/sangue , Células Tumorais Cultivadas
12.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 40(7): 942-948, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32701238

RESUMO

OBJECTIVE: To investigate the effect of periostin on hypoxia-induced oxidative stress and apoptosis in human periodontal ligament fibroblasts and the molecular mechanism involved. METHODS: In vitro cultured human periodontal ligament fibroblasts were placed in an anaerobic gas-producing bag for hypoxia treatment for 48 h followed by treatment with periostin at low (25 ng/mL), moderate (50 ng/mL) or high (100 ng/mL) doses. MTT assay was used to measure the cell viability, and the cell apoptosis rate was determined using flow cytometry. The contents of IL-1ß, IL-6 and TNF-α in the cells were determined with ELISA, and ROS levels were measured using a fluorescent plate reader. The intracellular SOD activity was detected using ELISA. The expressions of HIF-1α, P21, cyclin D1, Bax, cleaved caspase-3, Bcl-2, P38MAPK and p-p38 MAPK proteins in the cells were detected with Western blotting. RESULTS: Hypoxia treatment significantly reduced the cell viability (P < 0.05), increased P21, Bax, and cleaved caspase-3 protein levels (P < 0.05), promoted cell apoptosis (P < 0.05), and decreased cyclin D1 and Bcl-2 protein levels (P < 0.05) in the cells. Compared with the hypoxic group, the cells treated with periostin at different concentrations showed significantly increased cell viability (P < 0.05) with significantly lowered apoptotic rates (P < 0.05) and decreased expression levels of Bax and cleaved caspase-3 (P < 0.05) but significantly increased expression levels of cyclin D1 and Bcl-2 (P < 0.05). Hypoxic exposure of the cells resulted in significantly increased expression levels of HIF-1α and p-p38 MAPK (P < 0.05) and increased levels of IL-1ß, IL-6, TNF-α and ROS (P < 0.05) but decreased SOD activity (P < 0.05). Periostin treatment at different concentrations significantly lowered the expression levels of HIF-1α and p-p38 MAPK (P < 0.05) and the levels of IL-1ß, IL-6, TNF-α and ROS (P < 0.05) and significantly increased SOD activity in the hypoxic cells (P < 0.05). CONCLUSIONS: Periostin promotes the proliferation, inhibits apoptosis, enhances cellular antioxidant capacity, and reduces inflammatory damage in human periodontal ligament fibroblasts exposed to hypoxia possibly by inhibiting the activation of the p38 MAPK signaling pathway.


Assuntos
Apoptose , Moléculas de Adesão Celular , Estresse Oxidativo , Transdução de Sinais , Apoptose/efeitos dos fármacos , Moléculas de Adesão Celular/administração & dosagem , Hipóxia Celular , Fibroblastos/efeitos dos fármacos , Humanos , Estresse Oxidativo/efeitos dos fármacos , Ligamento Periodontal/citologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno
13.
Surg Clin North Am ; 100(4): 757-776, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32681875

RESUMO

This review of the literature concerning bacteria, antibiotics and tissue repair shows there are extensive data supporting microbial interference with wound healing once bacterial burden exceeds 104 CFU per unit of measure, The mechanism of bacterial interference lies largely in prolonging the inflammatory phase of tissue repair. Reducing the microbial bioburden allows tissue repair to continue. Systemic and topical antimicrobials appear critical to reducing the bioburden and facilitating repair. The current controversy over the use of antimicrobials in patients with chronically infected wounds, in particular, revolves around the definition of infection. The reliance on classic clinical signs of inflammation to support antimicrobial use in these patients is tenuous due to the lack of correlation of these signs with the microbial burden known to impair tissue repair.


Assuntos
Antibacterianos/uso terapêutico , Infecções Bacterianas/tratamento farmacológico , Cicatrização/fisiologia , Infecção dos Ferimentos/tratamento farmacológico , Infecções Bacterianas/fisiopatologia , Carga Bacteriana/fisiologia , Biofilmes , Hipóxia Celular/fisiologia , Humanos , Neutrófilos/fisiologia , Infecção dos Ferimentos/fisiopatologia
14.
Anticancer Res ; 40(8): 4687-4694, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727793

RESUMO

BACKGROUND/AIM: The Japanese apricot "Prunus mume" is a traditional Japanese medicine. MK615, a compound extract from Prunus mume has been reported to have anti-tumor effects. Herein, we used 3D floating (3DF) culture to evaluate the anticancer effects of MK615 against human colorectal cancer (CRC) cells that contain mutant (mt) KRAS. MATERIALS AND METHODS: HKe3 cells exogenously expressing mtKRAS (HKe3-mtKRAS) were treated with MK615 in 3DF cultures. The protein levels of hypoxia-inducible factor 1 (HIF-1) and E-cadherin were quantified by western blotting. RESULTS: MtKRAS enhanced hypoxia tolerance via up-regulation of HIF-1. The expression of HIF-1 protein was suppressed by constitutive overexpression of E-cadherin in CRC HCT116 spheroids. MK615 increased the expression of E-cadherin and decreased the expression of HIF-1 in HKe3-mtKRAS. These results suggest that MK615 suppresses hypoxia tolerance by up-regulation of E-cadherin in CRC cells with mtKRAS. CONCLUSION: MK615 exhibits properties useful for the potential treatment of CRC patients with mtKRAS.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Hipóxia Celular/fisiologia , Neoplasias do Colo/metabolismo , Neoplasias Colorretais/metabolismo , Extratos Vegetais/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Regulação para Cima/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Células HCT116 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prunus/química , Ativação Transcricional/efeitos dos fármacos
15.
Zhongguo Zhong Yao Za Zhi ; 45(12): 2960-2965, 2020 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-32627473

RESUMO

The aim of this paper was to investigate whether the mechanism of salvianolic acid B in protecting H9 c2 cardiomyocytes from hypoxia/reoxygenation injury is related to the regulation of mitochondrial autophagy mediated by NIX. H9 c2 cardiomyocytes were cultured in vitro and divided into normal group, model group and salvianolic acid B group(50 µmol·L~(-1)). Hypoxia/reoxygenation injury model was established by hypoxia for 4 h and reoxygenation for 2 h. In normal group, high glucose DMEM medium was used for culture. Those in model group were cultured with DMEM medium without glucose and oxygen, and no drugs for hypoxia and reoxyge-nation. In salvianolic acid B group, salvianolic acid B prepared by glucose-free DMEM medium was added during hypoxia, and the other process was as same as the model group. The cell viability was evaluated by CCK-8 assay. The leakage of lactate dehydrogenase(LDH) was detected by microplate method. The levels of intracellular reactive oxygen species(ROS) and mitochondrial membrane potential(ΔΨm) were measured by chemical fluorescence method. The level of intracellular adenosine triphosphate(ATP) was mea-sured by fluorescein enzyme method. The autophagy related proteins LC3-Ⅰ, LC3-Ⅱ, apoptosis related protein cleaved caspase-3 and mitochondrial autophagy receptor protein NIX were detected by Western blot. As compared with the normal group, the activity of H9 c2 cardiomyocytes and ATP level were decreased(P<0.05); LDH leakage and ROS production were increased(P<0.01); ΔΨm was decreased(P<0.01); LC3-Ⅱ/LC3-Ⅰ ratio, cleaved caspase-3 and NIX protein expression levels were increased(all P<0.05) in the model group. As compared with the model group, the activity of cells and ΔΨm were significantly increased(P<0.01); ATP level was increased(P<0.05); LDH leakage and ROS generation were decreased(P<0.01); LC3-Ⅱ/LC3-Ⅰ ratio was decreased(P<0.01); cleaved caspase-3 and NIX expression levels were decreased(P<0.05) in the salvianolic acid B group. The protective effect of salvianolic acid B on hypoxia/reoxygenation injury of H9 c2 cardiomyocytes may be associated with inhibiting mitochondrial auto-phagy. The specific mechanism may be related to inhibiting the activation of mitochondrial autophagy mediated by NIX, increasing ΔΨm, reducing ROS production, reducing the expression of cleaved caspase-3, LC3-Ⅱ, and increasing cell viability.


Assuntos
Autofagia , Miócitos Cardíacos , Apoptose , Benzofuranos , Hipóxia Celular , Sobrevivência Celular , Humanos , Hipóxia
16.
Adv Cancer Res ; 148: 27-67, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32723566

RESUMO

Vascular mimicry is induced by a wide array of genes with functions related to cancer stemness, hypoxia, angiogenesis and autophagy. Vascular mimicry competent (VM-competent) cells that form de novo blood vessels are common in solid tumors facilitating tumor cell survival and metastasis. VM-competent cells display increased levels of vascular mimicry selecting for stem-like cells in an O2-gradient-dependent manner in deeply hypoxic tumor regions, while also aiding in maintaining tumor cell metabolism and stemness. Three of the principal drivers of vascular mimicry are EphA2, Nodal and HIF-1α, however, directly or indirectly many of these molecules affect VE-Cadherin (VE-Cad), which forms gap-junctions to bind angiogenic blood vessels together. During vascular mimicry, the endothelial-like functions of VM-competent cancer stem cells co-opt VE-Cad to bind cancer cells together to create cancer cell-derived blood conducting vessels. This process potentially compensates for the lack of access to blood and nutrient in avascular tumors, simultaneously providing nutrients and enhancing cancer invasion and metastasis. Current evidence also supports that vascular mimicry promotes cancer malignancy and metastasis due to the cooperation of oncogenic signaling molecules driving cancer stemness and autophagy. While a number of currently used cancer therapeutics are effective inhibitors of vascular mimicry, developing a new class of vascular mimicry specific inhibitors could allow for the treatment of angiogenesis-resistant tumors, inhibit cancer metastasis and improve patient survival. In this review, we describe the principal vascular mimicry pathways in addition to emphasizing the roles of hypoxia, autophagy and select proangiogenic oncogenes in this process.


Assuntos
Neoplasias/irrigação sanguínea , Neoplasias/patologia , Animais , Autofagia/fisiologia , Hipóxia Celular/fisiologia , Modelos Animais de Doenças , Humanos , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia
17.
PLoS One ; 15(7): e0232072, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645038

RESUMO

The vasculature within a tumor is highly disordered both structurally and functionally. Endothelial cells that comprise the vasculature are poorly connected causing vessel leakage and exposing the endothelium to a hypoxic microenvironment. Therefore, most anti-angiogenic therapies are generally inefficient and result in acquired resistance to increased hypoxia due to elimination of the vasculature. Recent studies have explored the efficacy of targeting metabolic pathways in tumor cells in combination with anti-angiogenic therapy. However, the metabolic alterations of endothelial cells in response to hypoxia have been relatively unexplored. Here, we measured polar metabolite levels in microvascular endothelial cells exposed to short- and long-term hypoxia with the goal of identifying metabolic vulnerabilities that can be targeted to normalize tumor vasculature and improve drug delivery. We found that many amino acid-related metabolites were altered by hypoxia exposure, especially within alanine-aspartate-glutamate, serine-threonine, and cysteine-methionine metabolism. Additionally, there were significant changes in de novo pyrimidine synthesis as well as glutathione and taurine metabolism. These results provide key insights into the metabolic alterations that occur in endothelial cells in response to hypoxia, which serve as a foundation for future studies to develop therapies that lead to vessel normalization and more efficient drug delivery.


Assuntos
Hipóxia Celular , Células Endoteliais/metabolismo , Redes e Vias Metabólicas , Aminoácidos/metabolismo , Ácido Aspártico/metabolismo , Linhagem Celular , Cisteína/metabolismo , Células HEK293 , Humanos , Microvasos/metabolismo , Nucleotídeos/metabolismo
18.
Gen Physiol Biophys ; 39(3): 259-268, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32525819

RESUMO

Ischemic stroke is one of the most common public health problems worldwide. The aim of the present study was to investigate the role of miR-19a and its possible target genes in SK-N-SH cells subjected to oxygen-glucose deprivation/re-oxygenation (OGD/R) injury. SK-N-SH cells are a suitable model for host transfection. SK-N-SH cells were transfected with miR-19a mimic or inhibitor and PTEN-small interfering (si) RNA in order to alter the expression of miR-19a, PTEN and AKT. The expression changes in acute cerebral ischemic injury (ACII) were verified using RT-qPCR and Western blotting. Expression changes and the association between miR-19a and PTEN following OGD/R were also assessed using a double luciferase analysis. In addition, cell viability and apoptosis were measured using an MTT and flow cytometry. miR-19a was downregulated; however, PTEN was markedly increased following OGD/R injury. miR-19a mimics increased cell viability, decreased cell apoptosis of SK-N-SH cells following OGD/R, which effects was similar to PTEN siRNA; however, miR-19a inhibitor had the opposite roles with miR-19a mimics. The present study provides novel information about the cell apoptosis and invasion mechanisms associated with the miR-19a/PTEN/AKT pathway and may present a potential therapeutic approach for OGD/R injury.


Assuntos
MicroRNAs/genética , Neuroproteção , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Apoptose , Hipóxia Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Glucose , Humanos , Oxigênio
19.
Life Sci ; 257: 117919, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32585247

RESUMO

AIM: This study is undertaken to investigate the role and molecular mechanisms of miR-18a-5p in regulating pulmonary arterial hypertension (PAH) pathogenesis. METHODS: Gene expression and protein levels were determined by qRT-PCR and western blot, respectively; Cell counting kti-8 and Transwell migration assays were used to determine the biological functions of miR-18a-5p in pulmonary arterial smooth muscle cells (PASMCs); bioinformatics analysis, luciferase reporter assays were used to elucidate the mechanisms of miR-18a-5p. RESULTS: MiR-18a-5p was up-regulated in the clinical samples from PAH patients. PASMCs treated with hypoxia exhibited enhanced proliferative ability and upregulated miR-18a-5p expression. Knockdown of miR-18a-5p attenuated hypoxia-induced hyper-proliferation and enhanced migratory potential of PASMCs; while miR-18a-5p overexpression promoted PASMC proliferation and migration. Further mechanistic studies showed that Notch2 was a direct target of miR-18a-5p and was repressed by miR-18a-5p overexpression. The rescue studies indicated that Notch2 overexpression counteracted the enhanced proliferation and migration induced by miR-18a-5p mimics in PASMCs. Similarly, Notch2 overexpression also block the effects caused by hypoxia in PASMCs. Moreover, Notch2 expression was down-regulated in the PAH patients and was negatively correlated with miR-18a-5p expression. In vivo animal studies further revealed the up-regulation of miR-18a-5p and the down-regulation of Notch2 in the PAH rats. CONCLUSIONS: Collectively, this study identified the up-regulated miR-18a-5p in the PAH patients; our data suggest that miR-18a-5p contributes to the enhanced proliferation and migration of PASMCs via repressing Notch2 expression.


Assuntos
MicroRNAs/genética , Hipertensão Arterial Pulmonar/genética , Receptor Notch2/metabolismo , Animais , Apoptose/fisiologia , Hipóxia Celular/fisiologia , Movimento Celular/genética , Proliferação de Células/genética , Células Cultivadas , China , Hipertensão Pulmonar Primária Familiar/patologia , Feminino , Humanos , Hipertensão Pulmonar/metabolismo , Hipóxia/fisiopatologia , Masculino , MicroRNAs/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Ratos , Receptor Notch2/genética , Transdução de Sinais
20.
PLoS One ; 15(6): e0225485, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32484831

RESUMO

Mesenchymal stem cells (MSC)-spheroid models favor maintenance of stemness, ex vivo expansion and transplantation efficacy. Spheroids may also be considered as useful surrogate models of the hematopoietic niche. However, accessibility to primary cells, from bone marrow (BM) or adipose tissues, may limit their experimental use and the lack of consistency in methods to form spheroids may affect data interpretation. In this study, we aimed to create a simple model by examining the ability of cell lines, from human (HS-27a and HS-5) and murine (MS-5) BM origins, to form spheroids, compared to primary human MSCs (hMSCs). Our protocol efficiently allowed the spheroid formation from all cell types within 24 hours. Whilst hMSC-spheroids began to shrink after 24 hours, the size of spheroids from cell lines remained constant during three weeks. The difference was partially explained by the balance between proliferation and cell death, which could be triggered by hypoxia and induced oxidative stress. Our results demonstrate that, like hMSCs, MSC cell lines make reproductible spheroids that are easily handled. Thus, this model could help in understanding mechanisms involved in MSC functions and may provide a simple model by which to study cell interactions in the BM niche.


Assuntos
Células-Tronco Mesenquimais/citologia , Esferoides Celulares/citologia , Animais , Agregação Celular , Morte Celular , Desdiferenciação Celular , Hipóxia Celular , Linhagem Celular , Proliferação de Células , Humanos , Camundongos , Estresse Oxidativo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA