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BACKGROUND AND AIMS: Liver fibrosis represents a global health burden, given the paucity of approved antifibrotic therapies. Liver sinusoidal endothelial cells (LSECs) play a major gatekeeping role in hepatic homeostasis and liver disease pathophysiology. In early tumorigenesis, runt-related transcription factor 3 (RUNX3) functions as a sentinel; however, its function in liver fibrosis in LSECs remains unclear. This study aimed to investigate the role of RUNX3 as an important regulator of the gatekeeping functions of LSECs and explore novel angiocrine regulators of liver fibrosis. APPROACH AND RESULTS: Mice with endothelial Runx3 deficiency develop gradual and spontaneous liver fibrosis secondary to LSEC dysfunction, thereby more prone to liver injury. Mechanistic studies in human immortalized LSECs and mouse primary LSECs revealed that IL-6/JAK/STAT3 pathway activation was associated with LSEC dysfunction in the absence of RUNX3. Single-cell RNA sequencing and quantitative RT-PCR revealed that leucine-rich alpha-2-glycoprotein 1 ( LRG1 ) was highly expressed in RUNX3-deficient and dysfunctional LSECs. In in vitro and coculture experiments, RUNX3-depleted LSECs secreted LRG1, which activated HSCs throughTGFBR1-SMAD2/3 signaling in a paracrine manner. Furthermore, circulating LRG1 levels were elevated in mouse models of liver fibrosis and in patients with fatty liver and cirrhosis. CONCLUSIONS: RUNX3 deficiency in the endothelium induces LSEC dysfunction, LRG1 secretion, and liver fibrosis progression. Therefore, endothelial RUNX3 is a crucial gatekeeping factor in LSECs, and profibrotic angiocrine LRG1 may be a novel target for combating liver fibrosis.
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BACKGROUND: Chaperon-mediated autophagy (CMA) has taken on a new emphasis in cancer biology. However, the roles of CMA in hypoxic tumours are poorly understood. We investigated the anti-tumour effects of the natural product ManA through the activation of CMA in tumour progression under hypoxia. METHODS: The effect of ManA on CMA activation was assessed in mouse xenograft models and cells. The gene expressions of HIF-1α, HSP90AA1, and transcription factor EB (TFEB) were analysed using The Cancer Genome Atlas (TCGA) datasets to assess the clinical relevance of CMA. RESULTS: ManA activates photoswitchable CMA reporter activity and inhibits Hsp90 chaperone function by disrupting the Hsp90/F1F0-ATP synthase complex. Hsp90 inhibition enhances the interaction between CMA substrates and LAMP-2A and TFEB nuclear localisation, suggesting CMA activation by ManA. ManA-activated CMA retards tumour growth and displays cooperative anti-tumour activity with anti-PD-1 antibody. TCGA datasets show that a combined expression of HSP90AA1High/HIF1AHigh or TFEBLow/HIF1AHigh is strongly correlated with poor prognosis in patients with lung cancer. CONCLUSIONS: ManA-induced CMA activation by modulating Hsp90 under hypoxia induces HIF-1α degradation and reduces tumour growth. Thus, inducing CMA activity by targeting Hsp90 may be a promising therapeutic strategy against hypoxic tumours.
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Autofagia Mediada por Chaperonas , Neoplasias Pulmonares , Camundongos , Animais , Humanos , Hipóxia , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares , Autofagia/genéticaRESUMO
BACKGROUND: Classically activated M1 macrophages, characterized by aberrant glycolysis and secretion of inflammatory cytokines, play pivotal roles in inflammatory diseases, including inflammatory bowel disease (IBD). Recently, sodium-glucose co-transporter 2 (SGLT2) inhibitors were shown to suppress Na+/H+ exchanger 1 (NHE1) and Na+/Ca2+ exchanger 1 (NCX1) activity, regulating downstream intracellular Ca2+ concentrations in cardiomyocytes. However, whether SGLT2 inhibitors regulate M1 macrophage polarization by downregulating NHE1 and NCX1 remains unknown. METHODS: We analyzed cellular responses to SGLT2 inhibitors using mouse bone marrow-derived macrophages and peritoneal macrophages treated with lipopolysaccharide (LPS). To induce IBD, we used a dextran sulfate sodium salt-induced colitis mouse model. RESULTS: We observed that NHE1 and NCX1 were overexpressed in LPS-treated macrophages, leading to M1 macrophage polarization. Mechanistically, NHE1 and NCX1-mediated Ca2+ accumulation in the macrophage resulted in enhanced glycolysis by promoting PI3K/AKT/mTORC1 signaling. SGLT2 inhibitors suppressed both the expression levels and activities of NHE1 and NCX1, and consequently downregulated PI3K/AKT/mTORC1 signaling and glycolysis in LPS-treated macrophages. We observed inhibition of LPS-stimulated M1 polarization and cytokine production by SGLT2 inhibitors in vitro, ex vivo, and in an IBD mouse model. CONCLUSIONS: NHE1 promotes M1 macrophage polarization and SGLT2 inhibitors are a novel strategy to treat M1 macrophage-mediated inflammatory diseases, including IBD.
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Doenças Inflamatórias Intestinais , Inibidores do Transportador 2 de Sódio-Glicose , Animais , Camundongos , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/metabolismo , Macrófagos/metabolismo , Modelos Animais de Doenças , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismoRESUMO
Oncogenic signals contribute to enhanced glycolysis and mTORC1 activity, leading to rapid cell proliferation in cancer. Regulation of glycolysis and mTORC1 by PI3K/Akt signaling is well established, but how KRAS-induced MEK signaling regulates these pathways remains poorly understood. Here, we report a role for MEK-driven lactate production in mTORC1 activation in KRAS-activated cells. KRAS/MEK-induced upregulation of the chicken ovalbumin upstream promoter transcriptional factor II (COUP-TFII) increases the expression of lactate dehydrogenase A (LDHA), resulting in lactate production and mTORC1 activation. Further, lactate inhibits the interaction of TSC2 and Rheb, leading to the cellular activation of mTORC1 irrespective of growth factor stimulation. These findings suggest that COUP-TFII is a novel oncogenic mediator, connecting KRAS signaling and glycolysis, and leading to mTORC1 activation and cellular growth.
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Fator II de Transcrição COUP/metabolismo , Ácido Láctico/biossíntese , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Fator II de Transcrição COUP/genética , Linhagem Celular Tumoral , Expressão Gênica , Técnicas de Silenciamento de Genes , Glicólise , Humanos , Modelos Biológicos , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismoRESUMO
Inflammation is considered the root cause of various inflammatory diseases, including cancers. Decursinol angelate (DA), a pyranocoumarin compound obtained from the roots of Angelica gigas, has been reported to exhibit potent anti-inflammatory effects. In this study, the anti-inflammatory effects of DA on the MAP kinase and NFκB signaling pathways and the expression of pro-inflammatory cytokines were investigated in phorbol 12-myristate 13-acetate (PMA)-activated human promyelocytic leukemia (HL-60) and lipopolysaccharide (LPS)-stimulated macrophage (Raw 264.7) cell lines. PMA induced the activation of the MAP kinase-NFκB pathway and the production of pro-inflammatory cytokines in differentiated monocytes. Treatment with DA inhibited the activation of MAP kinases and the translocation of NFκB, and decreased the expression and exogenous secretion of IL-1ß and IL-6. Furthermore, LPS-stimulated Raw 264.7 cells were found to have increased expression of M1 macrophage-associated markers, such as NADPH oxidase (NOX) and inducible nitric oxide synthase (iNOS), and the M2 macrophage-associated marker CD11b. LPS also activated pro-inflammatory cytokines and Erk-NFκB. Treatment with DA suppressed LPS-induced macrophage polarization and the inflammatory response by blocking Raf-ERK and the translocation of NFκB in Raw 264.7 cells. Treatment with DA also inhibited the expression of pro-inflammatory cytokines, such as IL-1ß and IL-6, NOX, and iNOS in Raw 264.7 cells. These results suggest that DA has the potential to inhibit macrophage polarization and inflammation by blocking the activation of pro-inflammatory signals. These anti-inflammatory effects of DA may contribute to its potential use as a therapeutic strategy against various inflammation-induced cancers.
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Anti-Inflamatórios/farmacologia , Benzopiranos/farmacologia , Butiratos/farmacologia , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/citologia , NF-kappa B/metabolismo , Animais , Polaridade Celular/efeitos dos fármacos , Citocinas/metabolismo , Células HL-60 , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Ésteres de Forbol/farmacologia , Transporte Proteico/efeitos dos fármacos , Células RAW 264.7RESUMO
The proliferation and migration of vascular smooth muscle cells (VSMCs) have been implicated in the pathogenesis of atherosclerosis. Increased aerobic glycolysis is a key feature of cellular phenotypes including cancer and immune cells. However, the role of aerobic glycolysis in the atherogenic phenotype of VSMCs remains largely unknown. Here, we investigated the role of lactate dehydrogenase-A (LDHA), which is a key enzyme for glycolysis, in the proliferation and migration of VSMCs. Activation of primary rat VSMCs with fetal bovine serum (FBS) or platelet-derived growth factor (PDGF) increased their proliferation and migration, glycolytic activity, and expression of LDHA. Wound healing and transwell migration assays demonstrated that small interfering RNA-mediated knockdown of LDHA and pharmacological inhibition of LDHA by oxamate both effectively inhibited VSMC proliferation and migration. Inhibition of LDHA activity by oxamate reduced PDGF-stimulated glucose uptake, lactate production, and ATP production. Taken together, this study shows that enhanced glycolysis in PDGF- or FBS-stimulated VSMCs plays an important role in their proliferation and migration and suggests that LDHA is a potential therapeutic target to prevent vessel lumen constriction during the course of atherosclerosis and restenosis.
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Movimento Celular , Proliferação de Células , L-Lactato Desidrogenase/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Animais , Células Cultivadas , Isoenzimas/metabolismo , Lactato Desidrogenase 5 , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
During the past two decades, Runt domain transcription factors (RUNX1, 2, and 3) have been investigated in regard to their function, structural elements, genetic variants, and roles in normal development and pathological conditions. The Runt family proteins are evolutionarily conserved from Drosophila to mammals, emphasizing their physiological importance. A hypoxic microenvironment caused by insufficient blood supply is frequently observed in developing organs, growing tumors, and tissues that become ischemic due to impairment or blockage of blood vessels. During embryonic development and tumor growth, hypoxia triggers a stress response that overcomes low-oxygen conditions by increasing erythropoiesis and angiogenesis and triggering metabolic changes. This review briefly introduces hypoxic conditions and cellular responses, as well as angiogenesis and its related signaling pathways, and then describes our current knowledge on the functions and molecular mechanisms of Runx family proteins in hypoxic responses, especially in angiogenesis.
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Subunidades alfa de Fatores de Ligação ao Core/metabolismo , Hipóxia/metabolismo , Hipóxia/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Animais , Eritropoese/fisiologia , Humanos , Transdução de Sinais/fisiologiaRESUMO
Drug repositioning is the application of the existing drugs to new uses and has the potential to reduce the time and cost required for the typical drug discovery process. In this study, we repositioned thiopurine drugs used for the treatment of acute leukaemia as new tyrosinase inhibitors. Tyrosinase catalyses two successive oxidations in melanin biosynthesis: the conversions of tyrosine to dihydroxyphenylalanine (DOPA) and DOPA to dopaquinone. Continuous efforts are underway to discover small molecule inhibitors of tyrosinase for therapeutic and cosmetic purposes. Structure-based virtual screening predicted inhibitor candidates from the US Food and Drug Administration (FDA)-approved drugs. Enzyme assays confirmed the thiopurine leukaemia drug, thioguanine, as a tyrosinase inhibitor with the inhibitory constant of 52 µM. Two other thiopurine drugs, mercaptopurine and azathioprine, were also evaluated for their tyrosinase inhibition; mercaptopurine caused stronger inhibition than thioguanine did, whereas azathioprine was a poor inhibitor. The inhibitory constant of mercaptopurine (16 µM) was comparable to that of the well-known inhibitor kojic acid (13 µM). The cell-based assay using B16F10 melanoma cells confirmed that the compounds inhibit mammalian tyrosinase. Particularly, 50 µM thioguanine reduced the melanin content by 57%, without apparent cytotoxicity. Cheminformatics showed that the thiopurine drugs shared little chemical similarity with the known tyrosinase inhibitors.
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Azatioprina/farmacologia , Reposicionamento de Medicamentos , Inibidores Enzimáticos/farmacologia , Melaninas/antagonistas & inibidores , Mercaptopurina/farmacologia , Monofenol Mono-Oxigenase/antagonistas & inibidores , Doença Aguda , Antimetabólitos Antineoplásicos/química , Antimetabólitos Antineoplásicos/uso terapêutico , Azatioprina/química , Domínio Catalítico , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Humanos , Leucemia/tratamento farmacológico , Leucemia/enzimologia , Leucemia/genética , Leucemia/patologia , Melaninas/biossíntese , Melaninas/genética , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/enzimologia , Melanoma Experimental/genética , Melanoma Experimental/patologia , Mercaptopurina/química , Simulação de Acoplamento Molecular , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Tioguanina/química , Tioguanina/uso terapêutico , Células Tumorais CultivadasRESUMO
The zebrafish (Danio rerio) is a popular animal model used for studies on vertebrate development and organogenesis. Recent research has shown a similarity of approximately 70% between the human and zebrafish genomes and about 84% of human disease-causing genes have common ancestry with that of the zebrafish genes. Zebrafish embryos have a number of desirable features, including transparency, a large size, and rapid embryogenesis. Protein phosphorylation is a well-known PTM, which can carry out various biological functions. Recent MS developments have enabled the study of global phosphorylation patterns by using MS-based proteomics coupled with titanium dioxide phosphopeptide enrichment. In the present study, we identified 3500 nonredundant phosphorylation sites on 2166 phosphoproteins and quantified 1564 phosphoproteins in developing embryos of zebrafish. The distribution of Ser/Thr/Tyr phosphorylation sites in zebrafish embryos was found to be 88.9, 10.2, and 0.9%, respectively. A potential kinase motif was predicted using Motif-X analysis, for 80% of the identified phosphorylation sites, with the proline-directed motif appearing most frequently, and 35 phosphorylation sites having the pSF motif. In addition, we created six phosphoprotein clusters based on their dynamic pattern during the development of zebrafish embryos. Here, we report the largest dataset of phosphoproteins in zebrafish embryos and our results can be used for further studies on phosphorylation sites or phosphoprotein dynamics in zebrafish embryos.
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Fosfoproteínas/análise , Proteoma/análise , Proteínas de Peixe-Zebra/análise , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Cromatografia Líquida , Dados de Sequência Molecular , Fosfopeptídeos/análise , Fosforilação , Proteômica , Espectrometria de Massas em TandemRESUMO
Recent studies have revealed that microRNAs (miRs) play important roles in the regulation of angiogenesis. In this study, we have characterized miR-382 upregulation by hypoxia and the functional relevance of miR-382 in tumor angiogenesis. miRs induced by hypoxia in MKN1 human gastric cancer cells were investigated using miRNA microarrays. We selected miR-382 and found that the expression of miR-382 was regulated by HIF-1α. Conditioned media (CM) from MKN1 cells transfected with a miR-382 inhibitor (antagomiR-382) under hypoxic conditions significantly decreased vascular endothelial cell (EC) proliferation, migration and tube formation. Algorithmic programs (Target Scan, miRanda and cbio) predicted that phosphatase and tensin homolog (PTEN) is a target gene of miR-382. Deletion of miR382-binding sequences in the PTEN mRNA 3'-untranslated region (UTR) diminished the luciferase reporter activity. Subsequent study showed that the overexpression of miR-382 or antagomiR-382 down- or upregulated PTEN and its downstream target AKT/mTOR signaling pathway, indicating that PTEN is a functional target gene of miR-382. In addition, PTEN inhibited miR-382-induced in vitro and in vivo angiogenesis as well as VEGF secretion, and the inhibition of miR-382 expression reduced xenograft tumor growth and microvessel density in tumors. Taken together, these results suggest that miR-382 induced by hypoxia promotes angiogenesis and acts as an angiogenic oncogene by repressing PTEN.
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Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , MicroRNAs/metabolismo , Neovascularização Patológica/genética , PTEN Fosfo-Hidrolase/metabolismo , Animais , Hipóxia Celular , Linhagem Celular Tumoral , Humanos , Camundongos Nus , MicroRNAs/biossíntese , Neovascularização Fisiológica/genética , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias Gástricas/irrigação sanguínea , Neoplasias Gástricas/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Glyceollins, a group of phytoalexins isolated from soybean, are known to exhibit anticancer, antiestrogenic, and antiangiogenic activities. However, whether glyceollins regulate tumor growth through regulation of hypoxia-inducible factor (HIF)-1α has not been investigated. We determined whether and how glyceollins regulate the synthesis and stability of HIF-1α. Quantitative real-time PCR revealed that glyceollins inhibited the expression of HIF-1-induced genes such as vascular endothelial growth factor (VEGF) in cancer cells. Enzyme-linked immunosorbent assay and reporter luciferase assay showed that glyceollins decreased VEGF secretion and its promoter activity, respectively. Treatment of various cancer cells with 0.5-100 µM glyceollins under hypoxic conditions reduced the expression of HIF-1α. Glyceollins blocked translation of HIF-1α by inhibiting the PI3K/AKT/mTOR pathway under hypoxic conditions. Glyceollins decreased the stability of HIF-1α after treatment with cycloheximide, a protein synthesis inhibitor, and increased the ubiquitination of HIF-1α after treatment with MG132, a proteasome inhibitor. Glyceollins blocked the interaction of Hsp90 with HIF-1α, as shown by immunoprecipitation assay. Chemical binding of Hsp90 with glyceollins, as confirmed by computational docking analysis, was stronger than that with geldanamycin at the HSP90 ATP-binding pocket. We found that glyceollins decreased microvessel density, as well as expression of phosphorylated AKT/mTOR and the Hsp90 client protein CDK4, in solid tumor tissues. Glyceollins potently inhibited HIF-1α synthesis and decreased its stability by blocking the PI3K/AKT/mTOR pathway and HSP90 binding activity, respectively. These results may provide new perspectives into potential therapeutic application of glyceollins for the prevention and treatment of hypervascularized diseases and into the mechanism of their anticancer activity.
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Proteínas de Choque Térmico HSP90/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pterocarpanos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Inibidores da Angiogênese/farmacologia , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Humanos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Exendin-4 (EX4), a glucagon-like peptide-1 receptor agonist, has been reported to attenuate myocardial ischemia and reperfusion injury and inflammatory or oxidative responses. The expression level of secretory group IIA phospholipase A2 (sPLA2-IIA) is elevated in inflammatory diseases. Lipopolysaccharide (LPS) upregulates the expression of sPLA2-IIA in human umbilical vein endothelial cells (HUVECs). Here, EX4 was examined for its effects on the expression and activity of sPLA2-IIA in HUVECs and mice. Pre-treatment of cells or mice with EX4 inhibited LPS-induced sPLA2-IIA expression and activity. Additionally, EX4 suppressed LPS-induced activation of cytosolic phospholipase A2 (cPLA2) and extracellular signal-regulated kinase (ERK) 1/2. Therefore, these results show that EX4 inhibited LPS-induced expression of sPLA2-IIA by suppressing cPLA2 and ERK 1/2.
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Peptídeos/fisiologia , Animais , Exenatida , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfolipases A2 , PeçonhasRESUMO
Sepsis and septic shock, which are conditions triggered by infection, occur with high incidence in emergency departments and are among the most common causes of death in hospitalized patients worldwide. Therefore, the identification of sepsis biomarkers for the rapid diagnosis is a major goal for researchers in the field of critical care. Endothelial cells play a pivotal role in orchestrating the inflammatory response triggered by sepsis. In this study, we used proteomics to investigate the secretome of EA.hy926 endothelial cells following lipopolysaccharide (LPS) stimulation with 1 µg/mL LPS for 12 or 24 h. SILAC in cell cultures and an online 2D-LC-MS/MS system were used to analyze the secretome dynamics in response to LPS. We found that 22 of the 77 secreted proteins identified in both the presence and absence of LPS and that 19 of the secreted proteins were quantified more strongly after LPS treatment for 24 h than after treatment for 12 h. By Gene Ontology and KEGG pathway analyses, we found that proteins related to the regulation of the actin cytoskeleton showed the highest secretion response to LPS stimulation. Out of the 19 candidate proteins, we focused on moesin, which is involved in the function of endothelial cells, and confirmed its amount in cellular lysates and media taken from primary human umbilical vein endothelial cells treated with LPS. To our knowledge, this study provides the first in-depth analysis of the LPS-induced secretome in human endothelial cells, and we propose 19 new biomarker candidates for sepsis, including moesin.
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Biomarcadores/análise , Lipopolissacarídeos/farmacologia , Proteínas/análise , Proteômica/métodos , Sepse/metabolismo , Biomarcadores/metabolismo , Células Cultivadas , Cromatografia Líquida/métodos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Proteínas dos Microfilamentos/metabolismo , Proteínas/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
Cancer cells use multiple mechanisms to evade the effects of glutamine metabolism inhibitors. The pathways that govern responses to alterations in glutamine availability within the tumor may represent therapeutic targets for combinatorial strategies with these inhibitors. Here, we showed that targeting glutamine utilization stimulated Yes-associated protein (YAP) signaling in cancer cells by reducing cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)-dependent phosphorylation of large tumor suppressor (LATS). Elevated YAP activation induced extracellular matrix (ECM) deposition by increasing secretion of connective tissue growth factor (CTGF) that promoted production of fibronectin and collagen by surrounding fibroblasts. Consequently, inhibiting YAP synergized with inhibition of glutamine utilization to effectively suppress tumor growth in vivo, along with a concurrent decrease in ECM deposition. Blocking ECM remodeling also augmented the tumor suppressive effects of the glutamine utilization inhibitor. Collectively, these data reveal mechanisms by which targeting glutamine utilization increases ECM accumulation and identify potential strategies to reduce ECM levels and increase the efficacy of glutamine metabolism inhibitors.
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Oligodendrocytes are myelinating glial cells in the CNS and are essential for proper neuronal function. During development, oligodendrocyte progenitor cells (OPCs) are specified from the motor neuron precursor domain of the ventral spinal cord and differentiate into myelinating oligodendrocytes after migration to the white matter of the neural tube. Cell cycle control of OPCs influences the balance between immature OPCs and myelinating oligodendrocytes, but the precise mechanism regulating the differentiation of OPCs into myelinating oligodendrocytes is unclear. To understand the mechanisms underlying oligodendrocyte differentiation, an N-ethyl-N-nitrosourea-based mutagenesis screen was performed and a zebrafish leo1 mutant, dalmuri (dal(knu6)) was identified in the current study. Leo1 is a component of the evolutionarily conserved RNA polymerase II-associated factor 1 complex (PAF1C), which is a positive regulator of transcription elongation. The dal(knu6) mutant embryos specified motor neurons and OPCs normally, and at the appropriate time, but OPCs subsequently failed to differentiate into myelinating oligodendrocytes and were eliminated by apoptosis. A loss-of-function study of cdc73, another member of PAF1C, showed the same phenotype in the CNS, indicating that PAF1C function is required for oligodendrocyte differentiation. Interestingly, inhibition of positive transcription elongation factor b (p-TEFb), rescued downregulated gene expression and impaired oligodendrocyte differentiation in the dal(knu6) mutant and Cdc73-deficient embryos. Together, these results indicate that antagonistic regulation of gene expression by PAF1C and p-TEFb plays a crucial role in oligodendrocyte development in the CNS.
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Proteínas de Transporte/fisiologia , Diferenciação Celular/fisiologia , Regulação da Expressão Gênica/fisiologia , Oligodendroglia/fisiologia , Células-Tronco/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Proteínas de Transporte/genética , Sistema Nervoso Central/citologia , Sistema Nervoso Central/fisiologia , Técnicas de Silenciamento de Genes/métodos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Oligodendroglia/citologia , Fator B de Elongação Transcricional Positiva/metabolismo , Células-Tronco/citologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The regulation of hypoxia-inducible factor-1α (HIF-1α) during endochondral bone formation is not fully understood. Here, we investigated the cross-talk between HIF-1α and Runt-related transcription factor 2 (Runx2) in the growth plate. Runx2 caused the accumulation of HIF-1α protein in ATDC5 chondrocytes and HEK293 cells under normoxic conditions. Runx2 also increased the nuclear translocation of HIF-1α when coexpressed in HEK293 cells and interacted with HIF-1α at the oxygen-dependent degradation domain (ODDD). In addition, Runx2 competed with von Hippel-Lindau tumor suppressor protein by directly binding to ODDD-HIF-1α and significantly inhibited the ubiquitination of HIF-1α, even though Runx2 did not change the hydroxylation status of HIF-1α. Furthermore, overexpression of Runx2 resulted in the significant enhancement of vascular endothelial growth factor (VEGF) promoter reporter activity and protein secretion. Runx2 significantly increased angiogenic activity in human umbilical vein endothelial cells in vitro. In wild-type mice, HIF-1α and Runx2 were colocalized in hypertrophic chondrocytes in which the cluster of differentiation 31 (CD31) protein was expressed at embryonic day 15.5 (E15.5). In contrast, the expression of HIF-1α was markedly reduced in areas of CD31 expression in Runx2(-/-) mice. These results suggest that Runx2 stabilizes HIF-1α by binding to ODDD to block the interaction between von Hippel-Lindau protein and HIF-1α. In conclusion, Runx2, HIF-1α, and VEGF may regulate vascular angiogenesis spatially and temporally in the hypertrophic zone of the growth plate during endochondral bone formation.
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Condrócitos/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neovascularização Fisiológica , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/fisiologia , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Condrócitos/citologia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Células HEK293 , Humanos , Hidroxilação/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Camundongos Knockout , Ligação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genéticaRESUMO
PURPOSE: Tumor microenvironment consists of various kind of cells, forming complex interactions and signal transductions for tumor growth. Due to this complexity, targeting multiple kinases could yield improved clinical outcomes. In this study, we aimed to investigate the potential of myriocin, from Mycelia sterilia, as a novel dual-kinase inhibitor and suggest myriocin as a candidate for combined chemotherapy. METHODS: We initially evaluated the anti-tumor and anti-metastatic effect of myriocin in mouse allograft tumor models. We examined the effects of myriocin on angiogenesis and tumor vasculature using in vitro, in vivo, and ex vivo models, and also tested the anti-migration effect of myriocin in in vitro models. Next, we explored the effects of myriocin alone and in combination with cisplatin on tumor growth and vascular normalization in mouse models. RESULTS: We found that myriocin inhibited tumor growth and lung metastasis in mouse allograft tumor models. Myriocin induced normalization of the tumor vasculature in the mouse models. We also found that myriocin suppressed angiogenesis through the VEGFR2/PI3K/AKT pathway in endothelial cells (ECs), as well as cancer cell migration by blocking the IκBα/NF-κB(p65)/MMP-9 pathway. Finally, we found that myriocin enhanced the drug delivery efficacy of cisplatin by increasing the integrity of tumor vasculature in the mouse models, which synergistically increased the anti-tumor activity of cisplatin. CONCLUSION: We suggest that myriocin is a novel potent anti-cancer agent that dually targets both VEGFR2 in ECs and IκBα in cancer cells, and exerts more pronounced anti-tumor effects than with either kinase being inhibited alone.
Assuntos
Cisplatino , Neoplasias Pulmonares , Camundongos , Animais , Cisplatino/farmacologia , Inibidor de NF-kappaB alfa , Células Endoteliais , Fosfatidilinositol 3-Quinases , Proliferação de Células , Neovascularização Patológica , Linhagem Celular Tumoral , Microambiente TumoralRESUMO
Macrophages within the tumor microenvironment (TME), referred to as tumor-associated macrophages (TAMs), are involved in various aspects of tumor progression including initiation, angiogenesis, metastasis, immunosuppression, and resistance to therapy. Myriocin, a natural compound isolated from Mycelia sterilia, is an immunosuppressant that inhibits tumor growth and angiogenesis. However, the mechanisms underlying the effects of myriocin on TAMs and TAM-mediated tumor growth have not yet been elucidated. In this study, we determined the effects of myriocin on TAMs and the underlying mechanism in vitro and in vivo. Myriocin significantly suppressed monocyte-macrophage differentiation and M2 polarization of macrophages but not M1 polarization. In addition, myriocin inhibited the expression of anti-inflammatory cytokines and secretion of proangiogenic factors, such as vascular endothelial growth factor, in M2 macrophages as well as M2-induced endothelial cell permeability. Myriocin also inhibited the PI3K/Akt/mTOR signaling pathway in M2 macrophages. Myriocin reduced the population of M2-like TAMs within the tumor tissue of a mouse allograft tumor model but not that of M1-like TAMs. Moreover, combined treatment with myriocin and cisplatin synergistically suppressed tumor growth and enhanced survival rate in mice by reducing the population of M2-like TAMs. Overall, these results suggest that myriocin inhibits tumor growth by remodeling the TME through suppression of differentiation and polarization of M2-like TAMs via the PI3K/Akt/mTOR signaling pathway.
Assuntos
Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Animais , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Linhagem Celular Tumoral , Serina-Treonina Quinases TOR/metabolismo , Macrófagos , Microambiente TumoralRESUMO
BACKGROUND: Hypoxia is a characteristic feature of many solid tumors. As an adaptive response to hypoxia, tumor cells activate hypoxia-inducible factor-1α (HIF-1α). Under hypoxic conditions, angiogenesis mediated by HIF-1α is involved in the growth and metastasis of tumor cells. During the angiogenic process, differentiated tip endothelial cells (ECs) characterized by high expression of DLL4 promote angiogenic germination through filopodia. Inhibitors of HIF-1α or DLL4 have been widely studied PURPOSE: We tried to find inhibitors targeting both HIF-1α and DLL4 in tumor which have not yet been developed. STUDY DESIGN: In this study, we examined a natural compound that inhibits sprouting angiogenesis and tumor growth by targeting both HIF-1α and DLL4 under hypoxic conditions. METHODS: After examining cell viability of 70 selected natural compounds, we assessed the effects of compounds on HIF-1α and DLL4 transcriptional activity using a dual-luciferase reporter assay. Western blot analysis, immunofluoresecnt assay and real-time qPCR were performed to identify expression of proteins, such as HIF-1α and DLL4, as well as HIF-1α target genes under hypoxic conditions. In vitro angiogenesis assay and in vivo allograft tumor experiment were performed to investigate inhibition of tumor growth through anti-angiogenic activity. RESULTS: Among these compounds, steppogenin, which is extracted from the root bark of Morus alba l, respectively inhibited the transcriptional activity of HIF-1α under hypoxic conditions in HEK293T cells and vascular endothelial growth factor (VEGF)-induced DLL4 expression in vascular ECs in a dose-dependent manner. In tumor cells and retinal pigment epithelial cells, steppogenin significantly suppressed HIF-1α protein levels under hypoxic conditions as well as VEGF-induced DLL4 expression in ECs. Furthermore, steppogenin suppressed hypoxia-induced vascular EC proliferation and migration as well as VEGF-induced sprouting of EC spheroids. CONCLUSION: These results suggest that the natural compound steppogenin could potentially be used to treat angiogenic diseases, such as those involving solid tumors, because of its dual inhibition of HIF-1α and DLL4.
Assuntos
Neoplasias , Fator A de Crescimento do Endotélio Vascular , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Células Endoteliais/metabolismo , Endotélio/metabolismo , Endotélio/patologia , Células HEK293 , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Androgen deprivation therapy (ADT) is a systemic therapy for advanced prostate cancer (PCa); although most patients initially respond to ADT, almost all cancers eventually develop castration-resistant PCa (CRPC). Currently, most research focuses on castration-resistant tumors, and the role of tumors in remission is almost completely ignored. Here, we report that odorant-binding protein (OBP2A) released from tumors in remission during ADT catches survival factors, such as CXCL15/IL8, to promote PCa cell androgen-independent growth and enhance the infiltration of myeloid-derived suppressor cells (MDSCs) into tumor microenvironment, leading to the emergence of castration resistance. OBP2A knockdown significantly inhibits CRPC and metastatic CRPC development and improves therapeutic efficacy of CTLA-4/PD-1 antibodies. Treatment with OBP2A-binding ligand α-pinene interrupts the function of OBP2A and suppresses CRPC development. Furthermore, α-pinene-conjugated doxorubicin/docetaxel can be specifically delivered to tumors, resulting in improved anticancer efficacy. Thus, our studies establish a novel concept for the emergence of PCa castration resistance and provide new therapeutic strategies for advanced PCa.