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BACKGROUND: Severe asthma, characterized by inflammation and airway remodeling, involves fibroblast differentiation into myofibroblasts expressing α-SMA. This process leads to the production of fibronectin and connective tissue growth factor (CTGF), driven by factors such as transforming growth factor (TGF)-ß. Furthermore, the persistent presence of myofibroblasts is associated with resistance to apoptosis and mitochondrial dysfunction. The chemokine (C-X3-C motif) ligand 1 (CX3CL1) plays a role in tissue fibrosis. However, it is currently unknown whether neutralization of CX3CL1 decreases TGF-ß-induced fibroblast differentiation and mitochondrial dysfunction in normal human lung fibroblasts (NHLFs). METHODS: CX3CL1/C-X3-C motif chemokine receptor 1 (CX3CR1), CX3CL1 was analyzed by immunofluorescence (IF) or immunohistochemical (IHC) staining of ovalbumin-challenged mice. CX3CL1 release was detected by ELISA. TGF-ß-induced CTGF, fibronectin, and α-SMA expression were evaluated in NHLFs following neutralization of CX3CL1 (TP213) treatment for the indicated times by Western blotting or IF staining. Mitochondrion function was detected by a JC-1 assay and seahorse assay. Cell apoptosis was observed by a terminal uridine nick-end labeling (TUNEL) assay. RESULTS: An increase in CX3CL1 expression was observed in lung tissues from mice with ovalbumin-induced asthma by IF staining. CX3CR1 was increased in the subepithelial layer of the airway by IHC staining. Moreover, CX3CR1 small interfering (si)RNA downregulated TGF-ß-induced CTGF and fibronectin expression in NHLFs. CX3CL1 induced CTGF and fibronectin expression in NHLFs. TGF-ß-induced CX3CL1 secretion from NHLFs. Furthermore, TP213 decreased TGF-ß-induced CTGF, fibronectin, and α-SMA expression in NHLFs. Mitochondrion-related differentially expressed genes (DEGs) were examined after CX3CL1 neutralization in TGF-ß-treated NHLFs. TP213 alleviated TGF-ß-induced mitochondrial dysfunction and apoptosis resistance in NHLFs. CX3CL1 induced p65, IκBα, and IKKα phosphorylation in a time-dependent manner. Furthermore, CX3CL1-induced fibronectin expression and JC-1 monomer were decreased by p65 siRNA. TP213 reduced TGF-ß-induced p65 and α-SMA expression in NHLFs. CONCLUSIONS: These findings suggest that neutralizing CX3CL1 attenuates lung fibroblast activation and mitochondrial dysfunction. Understanding the impacts of CX3CL1 neutralization on fibroblast mitochondrial function could contribute to the development of therapeutic strategies for managing airway remodeling in severe asthma.
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Apoptosis , Receptor 1 de Quimiocinas CX3C , Diferenciación Celular , Quimiocina CX3CL1 , Factor de Crecimiento del Tejido Conjuntivo , Fibroblastos , Fibronectinas , Mitocondrias , Fibrosis Pulmonar , Factor de Crecimiento Transformador beta , Quimiocina CX3CL1/metabolismo , Quimiocina CX3CL1/genética , Animales , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Humanos , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/genética , Diferenciación Celular/efectos de los fármacos , Apoptosis/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Factor de Crecimiento Transformador beta/metabolismo , Receptor 1 de Quimiocinas CX3C/metabolismo , Receptor 1 de Quimiocinas CX3C/genética , Fibronectinas/metabolismo , Ratones , Actinas/metabolismo , Pulmón/patología , Pulmón/metabolismo , FN-kappa B/metabolismo , Transducción de Señal , Asma/metabolismo , Asma/patología , Modelos Animales de Enfermedad , Células Cultivadas , Miofibroblastos/metabolismo , Miofibroblastos/patología , Miofibroblastos/efectos de los fármacos , OvalbúminaRESUMEN
OBJECTIVES: Liver cirrhosis is a major risk factor for hepatocellular carcinoma (HCC), and all liver study societies recommend HCC surveillance in patients with cirrhosis. However, no ideal modality for HCC surveillance has been determined. The aim of this study is to assess the effectiveness of α-fetoprotein (AFP) measurement in HCC surveillance. METHODS: In this retrospective analysis, all patients with cirrhosis, who received HCC surveillance through ultrasound (US) and AFP measurement between January 2002 and July 2010, were followed up until June 2013. The performance effectiveness of surveillance using AFP, US, or both in HCC detection was compared. RESULTS: Overall, 1,597 patients were followed for a median duration of 4.75 (range 1.42-12) years. Over the 8563.25-person-year follow-up period, 363 patients (22.7%) developed HCCs. For HCC detection, the area under the receiver operator characteristic curve of surveillance AFP was 0.844 (95% confidence interval: 0.820-0.868, P<0.001). When the traditional cutoff value of 20 ng/ml was used, the sensitivity and specificity of AFP were 52.9% and 93.3%, respectively. US exhibited a sensitivity and specificity of 92.0% and 74.2%, respectively. A combination of US and AFP exhibited a sensitivity and specificity of 99.2% and 68.3%, respectively. By using cut-off at 20 ng/ml and AFP level increase ≥2 × from its nadir during the previous 1 year, the combination of US and AFP yielded a sensitivity of 99.2% and an improved specificity of 71.5%. CONCLUSIONS: The complementary use of AFP and US improved the effectiveness of HCC surveillance in patients with cirrhosis.
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Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/metabolismo , Detección Precoz del Cáncer/métodos , Cirrosis Hepática/metabolismo , Neoplasias Hepáticas/metabolismo , Hígado/metabolismo , alfa-Fetoproteínas/metabolismo , Anciano , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/etiología , Femenino , Humanos , Hígado/diagnóstico por imagen , Cirrosis Hepática/complicaciones , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/etiología , Masculino , Persona de Mediana Edad , Curva ROC , Estudios Retrospectivos , Sensibilidad y Especificidad , UltrasonografíaRESUMEN
BACKGROUND: Fibrotic interstitial lung disease (fILD) is characterised primarily by impaired lung function and quality of life. The present study investigated whether oxygen therapy could improve exercise capacity among patients with fILD. METHODS: Previously published randomised controlled trials (RCTs) were surveyed. A systematic review and meta-analysis was conducted to evaluate the effectiveness of oxygen therapy in improving the exertional capacity of patients with fILD. The primary outcome was peripheral oxygen saturation (SpO2) during exercise. The effects of oxygen therapy on fatigue, dyspnoea, heart rate, and exercise duration or distance were also analysed. RESULTS: Fourteen RCTs involving 370 patients were included. Oxygen therapy improved SpO2 during exercise (mean difference, MD = 6.26 %), exercise duration (MD = 122.15 s), fatigue (standard mean difference, SMD = -0.30), and dyspnoea (MD = -0.75 Borg score units). High-flow oxygen systems tended to be more effective than low-flow systems in improving exercising SpO2, duration, fatigue, dyspnoea, and heart rate. High-flow nasal cannulas (HFNCs) yielded better outcomes regarding SpO2 and fatigue than did high-flow Venturi masks (MD = 1.60 % and MD = -1.19 Borg score units, respectively). No major adverse events were reported. CONCLUSION: The evidence from RCTs supports the short-term use of oxygen supplementation to improve SpO2, exercise capacity, fatigue, and dyspnoea among patients with fILD. Further analyses demonstrates that HFNCs yield more favourable outcomes, yet not reaching statistical significance except for improving SpO2 and fatigue. However, the long-term effects of oxygen therapy on quality of life and mortality remain unclear.
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Disnea , Tolerancia al Ejercicio , Enfermedades Pulmonares Intersticiales , Terapia por Inhalación de Oxígeno , Calidad de Vida , Ensayos Clínicos Controlados Aleatorios como Asunto , Humanos , Terapia por Inhalación de Oxígeno/métodos , Tolerancia al Ejercicio/fisiología , Enfermedades Pulmonares Intersticiales/terapia , Enfermedades Pulmonares Intersticiales/fisiopatología , Disnea/terapia , Disnea/etiología , Saturación de Oxígeno , Fatiga/terapia , Fatiga/etiología , Masculino , Femenino , Frecuencia Cardíaca/fisiología , Persona de Mediana Edad , Resultado del Tratamiento , AncianoRESUMEN
Lung cancer is the leading cause of cancer deaths, where the metastasis often causes chemodrug resistance and leads to recurrence after treatment. Desmethylclomipramine (DCMI), a bioactive metabolite of clomipramine, shows the therapeutic efficacy with antidepressive agency as well as potential cytostatic effects on lung cancer cells. Here, we demonstrated that DCMI effectively caused transforming growth factor (TGF)-ß1-mediated mesenchymal type of A549 cells to undergo mitochondrial death via myeloid cell leukemia-1 (Mcl-1) suppression and activation of truncated Bid (tBid). TGF-ß1 induced epithelial mesenchymal transition in A549 cells with the increase of fibronectin and decrease of E-cadherin, the activation of Akt/glycogen synthase kinase-3ß (GSK-ß)/Mcl-1 axis, and the hypo-responsiveness to cisplatin. DCMI initiated a dose-dependent cytotoxicity on TGF-ß1-mediated mesenchymal type of A549 cells through inactivating Akt/GSK-ß/Mcl-1 axis, in which mitochondria instability and caspase-9/3 activation also occurred concurrently. Pharmacological inhibition of caspase-8 and cathepsin B partly reversed tBid expression and mitochondrial damage to further attenuate DCMI-mediated cytotoxicity. Additionally, DCMI presented partial therapeutic effects in treating mesenchymal type of A549 tumor bearing nude mice through an acceleration of cancer cell death. Taken together, DCMI exerts antitumor effects via initiating the mechanisms of Akt/GSK-ß/Mcl-1 inactivation and cathepsin B/caspase-8-regulated mitochondrial death, which suggests its potential role in mesenchymal type of cancer cell therapy.
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Transición Epitelial-Mesenquimal , Neoplasias Pulmonares , Mitocondrias , Animales , Humanos , Ratones , Células A549 , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Ratones Endogámicos BALB C , Ratones Desnudos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cancer represents a profound challenge to healthcare systems and individuals worldwide. The development of multiple drug resistance is a major problem in cancer therapy and can result in progression of the disease. In our previous studies, we developed small-molecule inhibitors targeting ubiquitin-specific peptidase 24 (USP24) to combat drug-resistant lung cancer. Recently, we found that the USP24 inhibitor NCI677397 induced ferroptosis, a type of programmed cell death, in drug-resistant cancer cells by increasing lipid reactive oxygen species (ROS) levels. In the present study, we investigated the molecular mechanisms and found that the targeting of USP24 by NCI677397 increased gene expression of most lipogenesis-related genes, such as acyl-CoA synthetase long-chain family member 4 (ACSL4), and activated autophagy. In addition, the activity of several antioxidant enzymes, such as glutathione peroxidase 4 (GPX4) and dihydrofolate reductase (DHFR), was inhibited by NCI677397 treatment via an increase in protein degradation, thereby inducing lipid ROS production and lipid peroxidation. In summary, we demonstrated that NCI677397 induced a marked increase in lipid ROS levels, subsequently causing lipid peroxidation and leading to the ferroptotic death of drug-resistant cancer cells. Our study provides new insights into the clinical use of USP24 inhibitors as ferroptosis inducers (FINs) to block drug resistance during chemotherapy.
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Acute lung injury (ALI) is a severe inflammatory lung disease associated with macrophages. Somatic nuclear autoantigenic sperm protein (sNASP) is a negative regulator of Toll-like receptor (TLR) signaling that targets tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) in macrophages, which is required to maintain homeostasis of the innate immune response. In the present study, we generated a cell permeable PEP-sNASP peptide using the sNASP protein N-terminal domain, and examined its potential therapeutic effect in a mouse model of ALI induced by the intranasal administration of lipopolysaccharide (LPS) and elucidated the underlying molecular mechanisms in RAW 264.7 cells. In vivo, PEP-sNASP peptide treatment markedly ameliorated pathological injury, reduced the wet/dry (W/D) weight ratio of the lungs and the production of proinflammatory cytokines (interleukin (IL)-1ß, IL-6, and TNF-α). In vitro, we demonstrated that when the PEP-sNASP peptide was transduced into RAW 264.7 cells, it bound to TRAF6, which markedly decreased LPS-induced proinflammatory cytokines by inhibiting TRAF6 autoubiquitination, nuclear factor (NF)-κB activation, reactive oxygen species (ROS) and cellular nitric oxide (NO) levels. Furthermore, the PEP-sNASP peptide also inhibited NLR family pyrin domain containing 3 (NLRP3) inflammasome activation. Our results therefore suggest that the PEP-sNASP may provide a potential protein therapy against oxidative stress and pulmonary inflammation via selective TRAF6 signaling.
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BACKGROUND: Small blood stem cells (SB cells), isolated from human peripheral blood, demonstrated the ability to benefit bone regeneration and osseointegration. The primary goal of our study is to examine the safety and tolerability of SB cells in dental implantation for human patients with severe bone defects. METHODS: Nine patients were enrolled and divided into three groups with SB cell treatment doses of 1 × 105, 1 × 106, and 1 × 107 SB cells, and then evaluated by computed tomography (CT) scans to assess bone mineral density (BMD) by Hounsfield units (HU) scoring. Testing was conducted before treatment and on weeks 4, 6, 8, and 12 post dental implantation. Blood and comprehensive chemistry panel testing were also performed. RESULTS: No severe adverse effects were observed for up to 6-month trial. Grade 1 leukocytosis, anemia, and elevated liver function were observed, but related with the patient's condition or the implant treatment itself and not the transplantation of SB cells. The levels of cytokines and chemokines were detected by a multiplex immunological assay. Elevated levels of eotaxin, FGF2, MCP-1, MDC, and IL17a were found among patients who received SB cell treatment. This observation suggested SB cells triggered cytokines and chemokines for local tissue repair. To ensure the efficacy of SB cells in dental implantation, the BMD and maximum stresses via stress analysis model were measured through CT scanning. All patients who suffered from severe bone defect showed improvement from D3 level to D1 or D2 level. The HU score acceleration can be observed by week 2 after guided bone regeneration (GBR) and prior to dental implantation. CONCLUSIONS: This phase I study shows that treatment of SB cells for dental implantation is well tolerated with no major adverse effects. The use of SB cells for accelerating the osseointegration in high-risk dental implant patients warrants further phase II studies. TRIAL REGISTRATION: Taiwan Clinical Trial Registry ( SB-GBR001 ) and clinical trial registry of the United States ( NCT04451486 ).
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Implantes Dentales , Oseointegración , Densidad Ósea , Regeneración Ósea , Humanos , Células MadreRESUMEN
BACKGROUND: Precise sperm-oocyte interaction is a critical event for successful fertilization. However, the identity of molecules involved in this process in humans remains largely unknown. This report describes the identification and characterization of a novel cell-surface protein and its potential role in human sperm-oocyte interaction. METHODS AND RESULTS: We previously identified an orphan guanylyl cyclase receptor (mouse GC-G) highly enriched in mouse testis and involved in sperm activation. By using a comparative genomic approach, we found the homologue gene in human (hGC-G) composed of 21 exons, spanning a minimum of 48 kb on chromosome 10q25. Real-time RT-PCR analysis revealed hGC-G mRNA selectively expressed in testis but with low or no expression in all other tissues examined. Compared with mGC-G, the hGC-G transcript contains three 1-bp deletions and two in-frame termination codons, which results in a short putative receptor-like polypeptide. Western blot analysis with an anti-hGC-G-specific antibody confirmed the protein expression of hGC-G in human sperm lysate. Flow cytometry and confocal immunofluorescence analysis demonstrated the localization of hGC-G protein on the acrosome cap and equatorial segment of mature human sperm. In addition, an integrin-binding Arg-Gly-Asp (RGD) motif was found in the extracellular domain of hGC-G. Pre-incubation of the hGC-G RGD peptide with zona pellucida-free oocytes greatly decreased the binding of human sperm to hamster oocytes, which suggests a role for hGC-G role in sperm-oocyte interaction. CONCLUSIONS: hGC-G is a novel surface protein on human sperm and potentially mediates sperm-oocyte interaction through its RGD-containing motif.
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Proteínas de la Membrana/fisiología , Receptores Acoplados a la Guanilato-Ciclasa/fisiología , Espermatozoides/metabolismo , Acrosoma/metabolismo , Reacción Acrosómica , Secuencias de Aminoácidos , Sitios de Unión , Western Blotting , Mapeo Cromosómico , Cromosomas Humanos Par 10 , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , ARN Mensajero/metabolismo , Receptores Acoplados a la Guanilato-Ciclasa/química , Receptores Acoplados a la Guanilato-Ciclasa/genética , Análisis de Secuencia de ADN , Capacitación Espermática , Interacciones Espermatozoide-Óvulo , Testículo/metabolismoRESUMEN
Recent studies indicate that neonatal spermatogonial stem cells (SSCs) possess pluripotency. However, the mechanisms that regulate the pluripotent differentiation capacity of SSCs remain unclear. Here, we describe a new method to clonally derive pluripotent SSCs from neonatal mouse testis. By coculturing with testicular stromal cells, SSCs can be maintained and expanded in serum-free conditions. Unlike endogenous SSCs, these in vitro expanded SSCs showed strong alkaline phosphatase (AP) activity and displayed characteristics of embryonic stem cells and primordial germ cells, which were therefore designated as AP(+) germline stem cells (AP(+)GSCs). The pluripotency of AP(+)GSCs was confirmed by in vitro differentiation toward hepatic and neuronal lineages and formation of embryonic chimeras after injection into blastocysts. Further investigation revealed that insulin-like growth factor-1 (IGF-1) secreted from Leydig cells was a key factor involved in maintaining the pluripotency of AP(+)GSCs. The blockage of IGF-1 receptor phosphorylation and its downstream PI3K pathway by PPP or LY294002 dramatically reduced their AP activity and expression of pluripotent genes, such as Oct-4, Blimp1, and Nanog. In conclusion, the present study demonstrated that IGF-1 secreted by testicular Leydig cells plays an important role in maintaining the pluripotency of SSCs in culture, which provides an insight into the molecular mechanism underlying germ cell pluripotency.
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Factor I del Crecimiento Similar a la Insulina/metabolismo , Células Madre Pluripotentes/citología , Espermatogonias/citología , Animales , Animales Recién Nacidos , Linaje de la Célula , Factor I del Crecimiento Similar a la Insulina/fisiología , Células Intersticiales del Testículo/metabolismo , Masculino , Ratones , Comunicación Paracrina , Testículo/citologíaRESUMEN
In the original publication of this article [1], labelling within Fig. 7a was incorrect. The updated figure is shown below, with 'DMT1' now corrected to read 'DNMT1'.
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Current guidelines recommend antibiotic prophylaxis for all patients with various degrees of cirrhosis and upper gastrointestinal (UGI) bleeding. This study assessed the need for antibiotic prophylaxis in patients with low Child-Pugh scores. We retrospectively screened all patients with cirrhosis who underwent upper endoscopies for UGI bleeding in a referral hospital in Taiwan between 2003 and 2014, from which 913 patients were enrolled after excluding patients with active bacterial infections, recent antibiotic use, early death, and Child-Pugh class C cirrhosis. Among them, 73 (8%) received prophylactic antibiotics, and 45 (4.9%) exhibited 14-day bacterial infection. Neither Child-Pugh score nor model for end stage liver disease score were optimal for predicting bacterial infection because their areas under the curves were 0.610 (95% confidence interval [CI]: 0.529-0.691) and 0.666 (95% CI: 0.591-0.742), respectively. Antibiotic prophylaxis did not reduce the risks of 14-day bacterial infection (relative risk [RR]: 0.932, 95% CI: 0.300-2.891, P = 0.902), 14-day rebleeding (RR: 0.791, 95% CI: 0.287-2.181, P = 0.650), or 42-day mortality (RR: 2.710, 95% CI: 0.769-9.524, P = 0.121). The results remained similar after propensity score adjustment. On-demand antibiotic treatment might suffice for patients with Child-Pugh class A/B cirrhosis and UGI bleeding.
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Profilaxis Antibiótica/métodos , Hemorragia Gastrointestinal/tratamiento farmacológico , Cirrosis Hepática/tratamiento farmacológico , Anciano , Antibacterianos/uso terapéutico , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/microbiología , Femenino , Hemorragia Gastrointestinal/microbiología , Mortalidad Hospitalaria , Humanos , Cirrosis Hepática/microbiología , Pruebas de Función Hepática , Masculino , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
BACKGROUND: The inflammatory cytokine interleukin-6 (IL-6) is critical for the expression of octamer-binding transcription factor 4 (OCT4), which is highly associated with early tumor recurrence and poor prognosis of hepatocellular carcinomas (HCC). DNA methyltransferase (DNMT) family is closely linked with OCT4 expression and drug resistance. However, the underlying mechanism regarding the interplay between DNMTs and IL-6-induced OCT4 expression and the sorafenib resistance of HCC remains largely unclear. METHODS: HCC tissue samples were used to examine the association between DNMTs/OCT4 expression levels and clinical prognosis. Serum levels of IL-6 were detected using ELISA assays (n = 144). Gain- and loss-of-function experiments were performed in cell lines and mouse xenograft models to determine the underlying mechanism in vitro and in vivo. RESULTS: We demonstrate that levels of DNA methyltransferase 3 beta (DNMT3b) are significantly correlated with the OCT4 levels in HCC tissues (n = 144), and the OCT4 expression levels are positively associated with the serum IL-6 levels. Higher levels of IL-6, DNMT3b, or OCT4 predicted early HCC recurrence and poor prognosis. We show that IL-6/STAT3 activation increases DNMT3b/1 and OCT4 in HCC. Activated phospho-STAT3 (STAT-Y640F) significantly increased DNMT3b/OCT4, while dominant negative phospho-STAT3 (STAT-Y705F) was suppressive. Inhibiting DNMT3b with RNA interference or nanaomycin A (a selective DNMT3b inhibitor) effectively suppressed the IL-6 or STAT-Y640F-induced increase of DNMT3b-OCT4 and ALDH activity in vitro and in vivo. The fact that OCT4 regulates the DNMT1 expressions were further demonstrated either by OCT4 forced expression or DNMT1 silence. Additionally, the DNMT3b silencing reduced the OCT4 expression in sorafenib-resistant Hep3B cells with or without IL-6 treatment. Notably, targeting DNMT3b with nanaomycin A significantly increased the cell sensitivity to sorafenib, with a synergistic combination index (CI) in sorafenib-resistant Hep3B cells. CONCLUSIONS: The DNMT3b plays a critical role in the IL-6-mediated OCT4 expression and the drug sensitivity of sorafenib-resistant HCC. The p-STAT3 activation increases the DNMT3b/OCT4 which confers the tumor early recurrence and poor prognosis of HCC patients. Findings from this study highlight the significance of IL-6-DNMT3b-mediated OCT4 expressions in future therapeutic target for patients expressing cancer stemness-related properties or sorafenib resistance in HCC.
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Antineoplásicos/uso terapéutico , Carcinoma Hepatocelular/tratamiento farmacológico , ADN (Citosina-5-)-Metiltransferasas/biosíntesis , Interleucina-6/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Factor 3 de Transcripción de Unión a Octámeros/biosíntesis , Factor de Transcripción STAT3/metabolismo , Sorafenib/farmacología , Animales , Antineoplásicos/farmacología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos , Femenino , Células Hep G2 , Xenoinjertos , Humanos , Interleucina-6/sangre , Interleucina-6/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Persona de Mediana Edad , Factor 3 de Transcripción de Unión a Octámeros/genética , Pronóstico , ADN Metiltransferasa 3BRESUMEN
Rationale: Cancer cells reprogram cellular metabolism to fulfill their needs for rapid growth and metastasis. However, the mechanism controlling this reprogramming is poorly understood. We searched for upregulated signaling in metastatic colorectal cancer and investigated the mechanism by which Glut3 promotes tumor metastasis. Methods: We compared RNA levels and glycolytic capacity in primary and metastatic colon cancer. The expression and association of Glut3 with clinical prognosis in colon cancer tissues was determined by immunohistochemistry. Glut3 gain-of-function and loss-of-function were established using colon cancer HCT116, HT29, and metastatic 116-LM cells, and tumor invasiveness and stemness properties were evaluated. Metabolomic profiles were analyzed by GC/MS and CE-TOF/MS. The metastatic burden in mice fed a high-fat sucrose diet was assessed by intravenous inoculation with Glut3 knockdown 116-LM cells. Results: Upregulation of glycolytic genes and glycolytic capacity was detected in metastatic colorectal cancer cells. Specifically, Glut3 overexpression was associated with metastasis and poor survival in colorectal cancer patients. Mechanistically, Glut3 promoted invasiveness and stemness in a Yes-associated protein (YAP)-dependent manner. Activation of YAP in turn transactivated Glut3 and regulated a group of glycolytic genes. Interestingly, the expression and phosphorylation of PKM2 were concomitantly upregulated in metastatic colorectal cancer, and it was found to interact with YAP and enhance the expression of Glut3. Importantly, a high-fat high-sucrose diet promoted tumor metastasis, whereas the inhibition of either Glut3 or YAP effectively reduced the metastatic burden. Conclusion: Activation of the Glut3-YAP signaling pathway acts as a master activator to reprogram cancer metabolism and thereby promotes metastasis. Our findings reveal the importance of metabolic reprogramming in supporting cancer metastasis as well as possible therapeutic targets.
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Proteínas Adaptadoras Transductoras de Señales/genética , Adenocarcinoma/genética , Transformación Celular Neoplásica/genética , Neoplasias del Colon/genética , Regulación Neoplásica de la Expresión Génica , Transportador de Glucosa de Tipo 3/genética , Factores de Transcripción/genética , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenocarcinoma/diagnóstico , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Neoplasias del Colon/diagnóstico , Neoplasias del Colon/mortalidad , Neoplasias del Colon/patología , Dieta Alta en Grasa/efectos adversos , Transportador de Glucosa de Tipo 3/agonistas , Transportador de Glucosa de Tipo 3/antagonistas & inhibidores , Transportador de Glucosa de Tipo 3/metabolismo , Glucólisis/genética , Células HCT116 , Células HT29 , Humanos , Metástasis Linfática , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Desnudos , Pronóstico , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Análisis de Supervivencia , Hormonas Tiroideas/genética , Hormonas Tiroideas/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Señalizadoras YAP , Proteínas de Unión a Hormona TiroideRESUMEN
BACKGROUND: Melanin is the pigment responsible for skin color. Melanin synthesis occurs with the participation of the tyrosinase (TYR) family of proteins including TYR, tyrosinase-related protein 1 (TRP1), and tyrosinase-related protein 2(TRP2/DCT). OBJECTIVE: The effect of a newly isolated natural compound that inhibits hyperpigmentation on the regulation of the TYR family of proteins was examined. METHODS: The natural compound, anemonin, was isolated from Clematis crassifolia Benth and was used to inhibit cellular TYR activity; it was found to have a low cytotoxicity (cell viability > 80%) in human melanocytes. RESULTS: In human melanocytes, anemonin showed both time- and dose-dependent inhibition (IC(50) 43.5 microM) of TYR. Western blot analysis and immunocytochemical staining revealed that expression of TYR, TRP1, and TRP2 was decreased in anemonin-treated melanocytes. Additionally, reverse transcription and quantitative real-time polymerase chain reaction analyses revealed that expression of mRNAs for MITF, TYR, TYRP1, and TYRP2 was also suppressed by anemonin. CONCLUSION: The natural compound, anemonin, an active compound of C. crassifolia, inhibits pigmentation synthesis in human melanocytes. Anemonin inhibits melanin synthesis by inhibiting the transcription of the genes encoding MITF, TYR, TRP1, and TRP2. This natural compound may be a candidate for cosmetic use.
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Clematis , Inhibidores Enzimáticos/farmacología , Furanos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Oxidorreductasas Intramoleculares/metabolismo , Melanocitos/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Monofenol Monooxigenasa/antagonistas & inhibidores , Oxidorreductasas/metabolismo , ARN Mensajero/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Clematis/química , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Inhibidores Enzimáticos/aislamiento & purificación , Inhibidores Enzimáticos/toxicidad , Furanos/aislamiento & purificación , Furanos/toxicidad , Humanos , Concentración 50 Inhibidora , Oxidorreductasas Intramoleculares/genética , Melaninas/metabolismo , Melanocitos/enzimología , Glicoproteínas de Membrana/genética , Factor de Transcripción Asociado a Microftalmía/metabolismo , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/metabolismo , Oxidorreductasas/genética , Extractos Vegetales/farmacología , Hojas de la Planta , Factores de Tiempo , Transcripción Genética/efectos de los fármacosRESUMEN
Autologous cell transplantation is a promising approach for cartilage repair, but the expansion of chondrocytes in a monolayer, a common approach to amplifying the cell number, inevitably leads to cell de-differentiation. To explore whether porous alginate sponges could be utilized for chondrocyte expansion and investigate the effects of seeding densities, the porcine chondrocytes were seeded to porous alginate sponges at low (5 x 10(5) cells per 40 sponges), medium (5 x 10(6) cells per 40 sponges), or high (2 x 10(7) cells per 40 sponges) density. After 4-week perfusion culture, all three groups resulted in chondrocyte proliferation, maintenance of chondrocytic gene (collagen II, Sox 9 and aggrecan) expression, and formation of cell clusters resembling cartilaginous tissues. The higher the seeding density, the higher the final cell density and GAGs production and, accordingly, the larger the cell clusters. Strikingly, the cumulative expansion ratios achieved by the low-density group ( approximately 150-fold) significantly exceeded those achieved by the medium (approximately 21-fold) and high (approximately 4.7-fold) density groups, as well as those achieved using other scaffolds. In conclusion, seeding chondrocytes to the alginate sponges at a low density, combined with perfusion culture, represents a drastic improvement in expanding autologous chondrocytes.
Asunto(s)
Alginatos/química , Condrocitos/fisiología , Animales , Cartílago/citología , Cartílago/crecimiento & desarrollo , Cartílago/fisiología , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Condrocitos/metabolismo , Condrocitos/ultraestructura , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Inmunohistoquímica , Articulaciones/metabolismo , Microscopía Electrónica de Rastreo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Porcinos , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
Hypoxia cooperates with endocrine signaling to maintain the symmetric self-renewal proliferation and migration of embryonic germline stem cells (GSCs). However, the lack of an appropriate in vitro cell model has dramatically hindered the understanding of the mechanism underlying this cooperation. Here, using a serum-free system, we demonstrated that hypoxia significantly induced the GSC mesenchymal transition, increased the expression levels of the pluripotent transcription factor OCT4 and migration-associated proteins (SDF-1, CXCR4, IGF-1, and IGF-1R), and activated the cellular expression and translocalization of the CXCR4-downstream proteins ARP3/pFAK. The underlying mechanism involved significant IGF-1/IGF-1R activation of OCT4/CXCR4 expression through HIF-2α regulation. Picropodophyllin-induced inhibition of IGF-1R phosphorylation significantly suppressed hypoxia-induced SDF-1/CXCR4 expression and cell migration. Furthermore, transactivation between IGF-1R and CXCR4 was involved. In summary, we demonstrated that niche hypoxia synergistically cooperates with its associated IGF-1R signaling to regulate the symmetric division (self-renewal proliferation) and cell migration of alkaline phosphatase-positive GSCs through HIF-2α-OCT4/CXCR4 during embryogenesis.
Asunto(s)
Hipoxia de la Célula/genética , Células Germinales Embrionarias/citología , Factor 3 de Transcripción de Unión a Octámeros/genética , Receptor IGF Tipo 1/genética , Factores de Transcripción/genética , Animales , Movimiento Celular/genética , Proliferación Celular/genética , Autorrenovación de las Células/genética , Quimiocina CXCL12/genética , Células Germinales Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Ratones , Fosforilación , Receptores CXCR4/genética , Transducción de Señal , Nicho de Células Madre/genéticaRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0149897.].
RESUMEN
The expression of cancer stemness is believed to reduce the efficacy of current therapies against hepatocellular carcinoma (HCC). Understanding of the stemness-regulating signaling pathways incurred by a specific etiology can facilitate the development of novel targets for individualized therapy against HCC. Niche environments, such as virus-induced inflammation, may play a crucial role. However, the mechanisms linking inflammation and stemness expression in HCC remain unclear. Here we demonstrated the distinct role of inflammatory mediators in expressions of stemness-related properties involving the pluripotent octamer-binding transcription factor 4 (OCT4) in cell migration and drug resistance of hepatitis B virus-related HCC (HBV-HCC). We observed positive immunorecognition for macrophage chemoattractant protein 1 (MCP-1)/CD68 and OCT4/NANOG in HBV-HCC tissues. The inflammation-conditioned medium (inflamed-CM) generated by lipopolysaccharide-stimulated U937 human leukemia cells significantly increased the mRNA and protein levels of OCT4/NANOG preferentially in HBV-active (HBV+HBsAg+) HCC cells. The inflamed-CM also increased the side population (SP) cell percentage, green fluorescent protein (GFP)-positive cell population, and luciferase activity of OCT4 promoter-GFP/luciferase in HBV-active HCC cells. Furthermore, the inflamed-CM upregulated the expressions of insulin-like growth factor-I (IGF-I)/IGF-I receptor (IGF-IR) and activated IGF-IR/Akt signaling in HBV-HCC. The IGF-IR phosphorylation inhibitor picropodophyllin (PPP) suppressed inflamed-CM-induced OCT4 and NANOG levels in HBV+HBsAg+ Hep3B cells. Forced expression of OCT4 significantly increased the secondary sphere formation and cell migration, and reduced susceptibility of HBV-HCC cells to cisplatin, bleomycin, and doxorubicin. Taking together, our results show that niche inflammatory mediators play critical roles in inducing the expression of stemness-related properties involving IGF-IR activation, and the upregulation of OCT4 contributes to cancer migration and drug resistance of HBV-HCC cells. Findings in this paper would provide potential targets for a therapeutic strategy targeting on inflammatory environment for HBV-HCC.