Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Humanos , Neoplasias Bucais/diagnóstico , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço , RNA Mensageiro , Saliva , Biomarcadores Tumorais/genéticaRESUMO
Psychosocial stress promotes and links mood and cardiovascular disorders in a sex-specific manner. However, findings in animal models are equivocal, in some cases opposing human dimorphisms. We examined central nervous system (CNS), behavioral, endocrine, cardiac, and hepatic outcomes in male or female C57Bl/6 mice subjected to chronic social stress (56 days of social isolation, with intermittent social confrontation encounters twice daily throughout the final 20 days). Females exhibited distinct physiological and behavioral changes, including relative weight loss, and increases in coronary resistance, hepatic inflammation, and thigmotaxic behavior in the open field. Males evidence reductions in coronary resistance and cardiac ischemic tolerance, with increased circulating and hippocampal monoamine levels and emerging anhedonia. Shared CNS gene responses include reduced hippocampal Maoa and increased Htr1b expression, while unique responses include repression of hypothalamic Ntrk1 and upregulation of cortical Nrf2 and Htr1b in females; and repression of hippocampal Drd1 and hypothalamic Gabra1 and Oprm in males. Declining cardiac stress resistance in males was associated with repression of cardiac leptin levels and metabolic, mitochondrial biogenesis, and anti-inflammatory gene expression. These integrated data reveal distinct biological responses to social stress in males and females, and collectively evidence greater biological disruption or allostatic load in females (consistent with propensities to stress-related mood and cardiovascular disorders in humans). Distinct stress biology, and molecular to organ responses, emphasize the importance of sex-specific mechanisms and potential approaches to stress-dependent disease.
Assuntos
Ansiedade , Leptina , Animais , Ansiedade/psicologia , Comportamento Animal/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2 , Estresse Psicológico/psicologiaRESUMO
Arthritogenic alphaviruses are mosquito-borne arboviruses that include several re-emerging human pathogens, including the chikungunya (CHIKV), Ross River (RRV), Mayaro (MAYV), and o'nyong-nyong (ONNV) virus. Arboviruses are transmitted via a mosquito bite to the skin. Herein, we describe intradermal RRV infection in a mouse model that replicates the arthritis and myositis seen in humans with Ross River virus disease (RRVD). We show that skin infection with RRV results in the recruitment of inflammatory monocytes and neutrophils, which together with dendritic cells migrate to draining lymph nodes (LN) of the skin. Neutrophils and monocytes are productively infected and traffic virus from the skin to LN. We show that viral envelope N-linked glycosylation is a key determinant of skin immune responses and disease severity. RRV grown in mammalian cells elicited robust early antiviral responses in the skin, while RRV grown in mosquito cells stimulated poorer early antiviral responses. We used glycan mass spectrometry to characterize the glycan profile of mosquito and mammalian cell-derived RRV, showing deglycosylation of the RRV E2 glycoprotein is associated with curtailed skin immune responses and reduced disease following intradermal infection. Altogether, our findings demonstrate skin infection with an arthritogenic alphavirus leads to musculoskeletal disease and envelope glycoprotein glycosylation shapes disease outcome. IMPORTANCE Arthritogenic alphaviruses are transmitted via mosquito bites through the skin, potentially causing debilitating diseases. Our understanding of how viral infection starts in the skin and how virus systemically disseminates to cause disease remains limited. Intradermal arbovirus infection described herein results in musculoskeletal pathology, which is dependent on viral envelope N-linked glycosylation. As such, intradermal infection route provides new insights into how arboviruses cause disease and could be extended to future investigations of skin immune responses following infection with other re-emerging arboviruses.
Assuntos
Infecções por Alphavirus , Artrite , Miosite , Polissacarídeos , Ross River virus , Pele , Infecções por Alphavirus/complicações , Infecções por Alphavirus/imunologia , Animais , Antivirais/imunologia , Artrite/complicações , Artrite/imunologia , Culicidae/virologia , Células Dendríticas , Modelos Animais de Doenças , Glicosilação , Humanos , Espectrometria de Massas , Camundongos , Monócitos , Miosite/complicações , Miosite/imunologia , Neutrófilos , Polissacarídeos/química , Polissacarídeos/imunologia , Ross River virus/imunologia , Pele/imunologia , Pele/virologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologiaRESUMO
Arthritogenic alphaviruses are mosquito-borne viruses that are a major cause of infectious arthropathies worldwide, and recent outbreaks of chikungunya virus and Ross River virus (RRV) infections highlight the need for robust intervention strategies. Alphaviral arthritis can persist for months after the initial acute disease, and is mediated by cellular immune responses. A common strategy to limit inflammation and pathology is to dampen the overwhelming inflammatory responses by modulating proinflammatory cytokine pathways. Here, we investigate the contribution of interleukin-17 (IL-17), a cytokine involved in arthropathies such as rheumatoid arthritis, in the development RRV-induced arthritis and myositis. IL-17 was quantified in serum from RRV-infected patients, and mice were infected with RRV and joints and muscle tissues collected to analyse cellular infiltrates, tissue mRNA, cytokine expression, and joint and muscle histopathology. IL-17 expression was increased in musculoskeletal tissues and serum of RRV-infected mice and humans, respectively. IL-17-producing T cells and neutrophils contributed to the cellular infiltrate in the joint and muscle tissue during acute RRV disease in mice. Blockade of IL-17A/F using a monoclonal antibody (mAb) reduced disease severity in RRV-infected mice and led to decreased proinflammatory proteins, cellular infiltration in synovial tissues and cartilage damage, without affecting viral titers in inflamed tissues. IL-17A/F blockade triggered a shift in transcriptional profile of both leukocyte infiltrates and musculoskeletal stromal cells by downregulating proinflammatory genes. This study highlights a previously uncharacterized role for an effector cytokine in alphaviral pathology and points towards potential therapeutic benefit in targeting IL-17 to treat patients presenting with RRV-induced arthropathy.
Assuntos
Artrite Reumatoide/imunologia , Imunidade Celular , Inflamação/imunologia , Interleucina-17/imunologia , Miosite/imunologia , Ross River virus/imunologia , Infecções por Alphavirus/imunologia , Infecções por Alphavirus/virologia , Animais , Artrite Reumatoide/virologia , Chlorocebus aethiops , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miosite/virologia , Células Vero , Carga ViralRESUMO
ABSTRACT: Horgan, BG, West, NP, Tee, N, Drinkwater, EJ, Halson, SL, Vider, J, Fonda, CJ, Haff, GG, and Chapman, DW. Acute inflammatory, anthropometric, and perceptual (muscle soreness) effects of postresistance exercise water immersion in junior international and subelite male volleyball athletes. J Strength Cond Res 36(12): 3473-3484, 2022-Athletes use water immersion strategies to recover from training and competition. This study investigated the acute effects of postexercise water immersion after resistance exercise. Eighteen elite and subelite male volleyball athletes participated in an intervention using a randomized cross-over design. On separate occasions after resistance exercise, subjects completed 1 of 4 15-minute interventions: control (CON), cold water immersion (CWI), contrast water therapy (CWT), or hot water immersion (HWI). Significance was accepted at p ≤ 0.05. Resistance exercise induced significant temporal changes (time effect) for inflammatory, anthropometric, perceptual, and performance measures. Serum creatine kinase was reduced ( g = 0.02-0.30) after CWI ( p = 0.007), CWT ( p = 0.006), or HWI ( p < 0.001) vs. CON, whereas it increased significantly ( g = 0.50) after CWI vs. HWI. Contrast water therapy resulted in significantly higher ( g = 0.56) interleukin-6 concentrations vs. HWI. Thigh girth increased ( g = 0.06-0.16) after CWI vs. CON ( p = 0.013) and HWI ( p < 0.001) and between CWT vs. HWI ( p = 0.050). Similarly, calf girth increased ( g = 0.01-0.12) after CWI vs. CON ( p = 0.039) and CWT ( p = 0.018), and HWI vs. CON ( p = 0.041) and CWT ( p = 0.018). Subject belief in a postexercise intervention strategy was associated with HSP72 ("believer">"nonbeliever," p = 0.026), muscle soreness ("believer">"nonbeliever," p = 0.002), and interleukin-4 ("nonbeliever">"believer," p = 0.002). There were no significant treatment × time (interaction effect) pairwise comparisons. Choice of postexercise water immersion strategy (i.e., cold, contrast, or hot) combined with a belief in the efficacy of that strategy to enhance recovery or performance improves biological and perceptual markers of muscle damage and soreness. On same or subsequent days where resistance exercise bouts are performed, practitioners should consider athlete beliefs when prescribing postexercise water immersion, to reduce muscle soreness.
Assuntos
Mialgia , Voleibol , Masculino , Humanos , Mialgia/prevenção & controle , Água , Imersão , Atletas , Temperatura Baixa , Músculo Esquelético/fisiologiaRESUMO
AIM: Exercise is cardioprotective, though optimal interventions are unclear. We assessed duration dependent effects of exercise on myocardial ischemia-reperfusion (I-R) injury, kinase signaling and gene expression. METHODS: Responses to brief (2 day; 2EX), intermediate (7 and 14 day; 7EX and 14EX) and extended (28 day; 28EX) voluntary wheel running (VWR) were studied in male C57Bl/6 mice. Cardiac function, I-R tolerance and survival kinase signaling were assessed in perfused hearts. KEY FINDINGS: Mice progressively increased running distances and intensity, from 2.4 ± 0.2 km/day (0.55 ± 0.04 m/s) at 2-days to 10.6 ± 0.4 km/day (0.72 ± 0.06 m/s) after 28-days. Myocardial mass and contractility were modified at 14-28 days VWR. Cardioprotection was not 'dose-dependent', with I-R tolerance enhanced within 7 days and not further improved with greater VWR duration, volume or intensity. Protection was associated with AKT, ERK1/2 and GSK3ß phosphorylation, with phospho-AMPK selectively enhanced with brief VWR. Gene expression was duration-dependent: 7 day VWR up-regulated glycolytic (Pfkm) and down-regulated maladaptive remodeling (Mmp2) genes; 28 day VWR up-regulated caveolar (Cav3), mitochondrial biogenesis (Ppargc1a, Sirt3) and titin (Ttn) genes. Interestingly, I-R tolerance in 2EX/2SED groups improved vs. groups subjected to longer sedentariness, suggesting transient protection on transition to housing with running wheels. SIGNIFICANCE: Cardioprotection is induced with as little as 7 days VWR, yet not enhanced with further or faster running. This protection is linked to survival kinase phospho-regulation (particularly AKT and ERK1/2), with glycolytic, mitochondrial, caveolar and myofibrillar gene changes potentially contributing. Intriguingly, environmental enrichment may also protect via similar kinase regulation.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Regulação da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/metabolismo , Isquemia Miocárdica/prevenção & controle , Condicionamento Físico Animal , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Glicogênio Sintase Quinase 3 beta/genética , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genéticaRESUMO
OBJECTIVE: The present research aimed to investigate the anti-inflammatory potential of dietary anthocyanin (ACN) in type 2 diabetic (T2D), T2D-at-risk and healthy individuals. Furthermore, dietary inflammatory index (DII) was used to study the association of diet with biomarkers of inflammation. RESEARCH METHODS: An open-label clinical trial was conducted at Griffith University investigating the efficacy of 320 mg ACN supplementation per day over the course of 4 weeks. Diabetes-associated inflammatory biomarkers and relevant biochemical and physical parameters were tested pre-and post-intervention, and participants' dietary inflammatory potential was estimated. RESULTS: A significant reduction in the pro-inflammatory biomarkers' interleukin-6, interleukin-18, and tumour necrosis factor-α was observed in the T2D group. In addition, some, but not all, biochemical parameters including fasting blood glucose, low-density lipoprotein cholesterol and uric acid were significantly improved in T2D-at-risk group. Moreover, a significant difference was detected between the DII scores of the healthy and T2D groups. DII score for the T2D group was consistent with an anti-inflammatory diet. CONCLUSION: Anti-inflammatory potential of dietary ACN in T2D participants was evidenced in the present study. Although, anti-inflammatory dietary patterns of T2D participants may have accelerated the anti-inflammatory effect of the ACN capsules supplemented in this trial.
Assuntos
Antocianinas/administração & dosagem , Anti-Inflamatórios/administração & dosagem , Diabetes Mellitus Tipo 2/tratamento farmacológico , Suplementos Nutricionais , Adulto , Idoso , Biomarcadores/sangue , Glicemia/efeitos dos fármacos , LDL-Colesterol/sangue , Citocinas/sangue , Diabetes Mellitus Tipo 2/sangue , Humanos , Inflamação/sangue , Leptina/sangue , Pessoa de Meia-Idade , Ácido Úrico/sangueRESUMO
Whether dietary omega-3 (n-3) polyunsaturated fatty acid (PUFA) confers cardiac benefit in cardiometabolic disorders is unclear. We test whether dietary -linolenic acid (ALA) enhances myocardial resistance to ischemia-reperfusion (I-R) and responses to ischemic preconditioning (IPC) in type 2 diabetes (T2D); and involvement of conventional PUFA-dependent mechanisms (caveolins/cavins, kinase signaling, mitochondrial function, and inflammation). Eight-week male C57Bl/6 mice received streptozotocin (75 mg/kg) and 21 weeks high-fat/high-carbohydrate feeding. Half received ALA over six weeks. Responses to I-R/IPC were assessed in perfused hearts. Localization and expression of caveolins/cavins, protein kinase B (AKT), and glycogen synthase kinase-3 ß (GSK3ß); mitochondrial function; and inflammatory mediators were assessed. ALA reduced circulating leptin, without affecting body weight, glycemic dysfunction, or cholesterol. While I-R tolerance was unaltered, paradoxical injury with IPC was reversed to cardioprotection with ALA. However, post-ischemic apoptosis (nucleosome content) appeared unchanged. Benefit was not associated with shifts in localization or expression of caveolins/cavins, p-AKT, p-GSK3ß, or mitochondrial function. Despite mixed inflammatory mediator changes, tumor necrosis factor-a (TNF-a) was markedly reduced. Data collectively reveal a novel impact of ALA on cardioprotective dysfunction in T2D mice, unrelated to caveolins/cavins, mitochondrial, or stress kinase modulation. Although evidence suggests inflammatory involvement, the basis of this "un-conventional" protection remains to be identified.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Ácidos Graxos Ômega-3/farmacologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Ácido alfa-Linolênico/farmacologia , Animais , Caveolinas/genética , Caveolinas/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Precondicionamento Isquêmico Miocárdico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Miocárdio/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
This study aims to investigate the overexpression-induced properties of tumor suppressor FAM134B (family with sequence similarity 134, member B) in colon cancer, examine the potential gene regulators of FAM134B expression and its impact on mitochondrial function. FAM134B was overexpressed in colon cancer and non-neoplastic colonic epithelial cells. Various cell-based assays including apoptosis, cell cycle, cell proliferation, clonogenic, extracellular flux and wound healing assays were performed. Western blot analysis was used to confirm and identify potential interacting partners of FAM134B in vitro. Immunohistochemistry and qPCR were employed to determine the expressions of MIF and FAM134B, respectively, on 63 patients with colorectal carcinoma. Results showed that FAM134B is involved in the cell cycle and mitochondrial function of colon cancer. Overexpression of FAM134B was coupled with increased expression levels of APC, p53, and MIF. Increased expression of both APC and p53 further validates the potential role of tumor suppressor FAM134B in regulating cancer progression through the WNT/ß-catenin signaling pathway. In approximately 70% of the patients with colorectal cancer, FAM134B downregulation was correlated with MIF protein overexpression while the remaining 30% showed concurrent expression of FAM134B and MIF (P = .045). High expression of MIF coupled with low expression of FAM134B is associated with microsatellite instability status in colorectal carcinomas (P = .049). FAM134B may exert its tumor suppressive function through affecting cell cycle, mitochondrial function via potentially interacting with MIF and p53.
Assuntos
Neoplasias do Colo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Proteínas de Membrana/biossíntese , Apoptose/fisiologia , Proliferação de Células/fisiologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Feminino , Genes Supressores de Tumor , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas de Membrana/genética , TransfecçãoRESUMO
BACKGROUND: Digital multiplex gene expression profiling is overcoming the limitations of many tissue-processing and RNA extraction techniques for the reproducible and quantitative molecular classification of disease. We assessed the effect of different skin biopsy collection/storage conditions on mRNA quality and quantity and the NanoString nCounter™ System's ability to reproducibly quantify the expression of 730 immune genes from skin biopsies. METHODS: Healthy human skin punch biopsies (n = 6) obtained from skin sections from four patients undergoing routine abdominoplasty were subject to one of several collection/storage protocols, including: i) snap freezing in liquid nitrogen and transportation on dry ice; ii) RNAlater (ThermoFisher) for 24 h at room temperature then stored at - 80 °C; iii) formalin fixation with further processing for FFPE blocks; iv) DNA/RNA shield (Zymo) stored and shipped at room temperature; v) placed in TRIzol then stored at - 80 °C; vi) saline without RNAse for 24 h at room temperature then stored at - 80 °C. RNA yield and integrity was assessed following extraction via NanoDrop, QuantiFluor with RNA specific dye and a Bioanalyser (LabChip24, PerkinElmer). Immune gene expression was analysed using the NanoString Pancancer Immune Profiling Panel containing 730 genes. RESULTS: Except for saline, all protocols yielded total RNA in quantities/qualities that could be analysed by NanoString nCounter technology, although the quality of the extracted RNA varied widely. Mean RNA integrity was highest from samples that were placed in RNALater (RQS 8.2 ± 1.15), with integrity lowest from the saline stored sample (RQS < 2). There was a high degree of reproducibility in the expression of immune genes between all samples with the exception of saline, with the number of detected genes at counts < 100, between 100 and 1000 and > 10,000 similar across extraction protocols. CONCLUSIONS: A variety of processing methods can be used for digital immune gene expression profiling in mRNA extracted from skin that are comparable to snap frozen skin specimens, providing skin cancer clinicians greater opportunity to supply skin specimens to tissue banks. NanoString nCounter technology can determine gene expression in skin biopsy specimens with a high degree of sensitivity despite lower RNA yields and processing methods that may generate poorer quality RNA. The increased sensitivity of digital gene expression profiling continues to expand molecular pathology profiling of disease.
Assuntos
Perfilação da Expressão Gênica/métodos , Estabilidade de RNA , RNA Mensageiro/análise , Manejo de Espécimes/métodos , Preservação de Tecido/métodos , Biópsia , Humanos , PeleRESUMO
PURPOSE: Habitual intense exercise may increase the incidence of upper respiratory symptoms (URS) in elite athletes. This study investigated whether immune gene expression could identify gene markers that discriminate athletes with a higher prevalence of URS. METHODS: This cross-sectional analysis of elite Australian athletes from various sports investigated whether athletes retrospectively reporting URS for two days or more in a month (n=38), had an altered immune gene expression profile compared with asymptomatic athletes (n=33). Peripheral blood samples were collected during Olympic selection events with corresponding URS data collected for the one-month period before sampling. Digital immune gene expression analysis was undertaken using the NanoString PanCancer Immune Profiling panel. RESULTS: Fifty immune genes were differentially expressed between the groups (p<0.05) and approximately 78% of these genes were more highly expressed in athletes reporting URS. Many of these genes were interferon-stimulated genes or genes involved in the Jak/Stat signalling pathway. Only interferon alpha inducible protein 27 (IFI27), an interferon stimulated gene involved in viral response, remained significantly higher in athletes reporting URS (log2 fold-difference=2.49, odds ratio 1.02 per unit increase; p<0.01) post-adjustment and discriminated athletes reporting URS from asymptomatic athletes with 78% accuracy. CONCLUSIONS: Expression of IFI27 could differentiate athletes reporting URS from asymptomatic athletes, a gene that is upregulated in the immune response to viral infection. Upregulation of viral signalling pathways provides novel information on the potential aetiology of URS in elite Olympic athletes.
Assuntos
Atletas , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/genética , Transcriptoma , Austrália , Estudos Transversais , Humanos , Proteínas de Membrana/genética , Estudos RetrospectivosRESUMO
Arthritogenic alphaviruses such as Ross River and Chikungunya viruses cause debilitating muscle and joint pain and pose significant challenges in the light of recent outbreaks. How host immune responses are orchestrated after alphaviral infections and lead to musculoskeletal inflammation remains poorly understood. Here, we show that myositis induced by Ross River virus (RRV) infection is driven by CD11bhi Ly6Chi inflammatory monocytes and followed by the establishment of a CD11bhi Ly6Clo CX3CR1+ macrophage population in the muscle upon recovery. Selective modulation of CD11bhi Ly6Chi monocyte migration to infected muscle using immune-modifying microparticles (IMP) reduced disease score, tissue damage, and inflammation and promoted the accumulation of CX3CR1+ macrophages, enhancing recovery and resolution. Here, we detail the role of immune pathology, describing a poorly characterized muscle macrophage subset as part of the dynamics of alphavirus-induced myositis and tissue recovery and identify IMP as an effective immunomodulatory approach. Given the lack of specific treatments available for alphavirus-induced pathologies, this study highlights a therapeutic potential for simple immune modulation by IMP in infected individuals in the event of large alphavirus outbreaks.IMPORTANCE Arthritogenic alphaviruses cause debilitating inflammatory disease, and current therapies are restricted to palliative approaches. Here, we show that following monocyte-driven muscle inflammation, tissue recovery is associated with the accumulation of CX3CR1+ macrophages in the muscle. Modulating inflammatory monocyte infiltration using immune-modifying microparticles (IMP) reduced tissue damage and inflammation and enhanced the formation of tissue repair-associated CX3CR1+ macrophages in the muscle. This shows that modulating key effectors of viral inflammation using microparticles can alter the outcome of disease by facilitating the accumulation of macrophage subsets associated with tissue repair.
Assuntos
Infecções por Alphavirus/metabolismo , Infecções por Alphavirus/virologia , Receptor 1 de Quimiocina CX3C/genética , Monócitos/metabolismo , Miosite/etiologia , Miosite/metabolismo , Cicatrização , Infecções por Alphavirus/patologia , Animais , Biomarcadores , Biópsia , Receptor 1 de Quimiocina CX3C/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Imunomodulação/genética , Imunofenotipagem , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Camundongos Transgênicos , Monócitos/imunologia , Monócitos/virologia , Miosite/patologiaRESUMO
Dynamin-related protein-1 (DRP-1)-dependent mitochondrial fission may influence cardiac tolerance to ischemic or oxidative stress, presenting a potential "cardioprotective" target. Effects of dynamin inhibitors [mitochondrial division inhibitor 1 (MDIVI-1) and dynasore] on injury, mitochondrial function, and signaling proteins were assessed in distinct models: ischemia-reperfusion (I-R) in mouse hearts and oxidative stress in rat H9c2 cardiomyoblasts. Hearts exhibited substantial cell death [approx. 40 IU lactate dehydrogenase (LDH) efflux] and dysfunction (approx. 40 mmHg diastolic pressure, approx. 40% contractile recovery) following 25 minutes' ischemia. Pretreatment with 1 µM MDIVI-1 reduced dysfunction (30 mmHg diastolic pressure, approx. 55% recovery) and delayed without reducing overall cell death, whereas 5 µM MDIVI-1 reduced overall death at the same time paradoxically exaggerating dysfunction. Postischemic expression of mitochondrial DRP-1 and phospho-activation of ERK1/2 were reduced by MDIVI-1. Conversely, 1 µM dynasore worsened cell death and reduced nonmitochondrial DRP-1. Postischemic respiratory fluxes were unaltered by MDIVI-1, although a 50% fall in complex-I flux control ratio was reversed. In H9c2 myoblasts stressed with 400 µM H2O2, treatment with 50 µM MDIVI-1 preserved metabolic (MTT assay) and mitochondrial (basal respiration) function without influencing survival. This was associated with differential signaling responses, including reduced early versus increased late phospho-activation of ERK1/2, increased phospho-activation of protein kinase B (AKT), and differential changes in determinants of autophagy [reduced microtubule-associated protein 1 light chain 3b (LC3B-II/I) vs. increased Parkinson juvenile disease protein 2 (Parkin)] and apoptosis [reduced poly-(ADP-ribose) polymerase (PARP) cleavage vs. increased BCL2-associated X (BAX)/B-cell lymphoma 2 (BCL2)]. These data show MDIVI-1 (not dynasore) confers some benefit during I-R/oxidative stress. However, despite mitochondrial and metabolic preservation, MDIVI-1 exerts mixed effects on cell death versus dysfunction, potentially reflecting differential changes in survival kinase, autophagy, and apoptosis pathways. SIGNIFICANCE STATEMENT: Inhibition of mitochondrial fission is a novel approach to still elusive cardioprotection. Assessing effects of fission inhibitors on responses to ischemic or oxidative stress in hearts and cardiomyoblasts reveals mitochondrial division inhibitor 1 (MDIVI-1) and dynasore induce complex effects and limited cardioprotection. This includes differential impacts on death and dysfunction, survival kinases, and determinants of autophagy and apoptosis. Although highlighting the interconnectedness of fission and these key processes, results suggest MDIVI-1 and dynasore may be of limited value in the quest for effective cardioprotection.
Assuntos
Cardiotônicos/farmacologia , Dinaminas/metabolismo , Mitocôndrias Cardíacas/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Miócitos Cardíacos/metabolismo , Estresse Oxidativo , Quinazolinonas/farmacologia , Animais , Apoptose , Autofagia , Cardiotônicos/uso terapêutico , Linhagem Celular , Células Cultivadas , Dinaminas/antagonistas & inibidores , Coração/efeitos dos fármacos , Hidrazonas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinazolinonas/uso terapêutico , RatosRESUMO
BACKGROUND: West Nile virus (WNV) circulates across Australia and was referred to historically as Kunjin virus (WNVKUN). WNVKUN has been considered more benign than other WNV strains circulating globally. In 2011, a more virulent form of the virus emerged during an outbreak of equine arboviral disease in Australia. METHODS: To better understand the emergence of this virulent phenotype and the mechanism by which pathogenicity is manifested in its host, cells were infected with either the virulent strain (NSW2012), or less pathogenic historical isolates, and their innate immune responses compared by digital immune gene expression profiling. Two different cell systems were used: a neuroblastoma cell line (SK-N-SH cells) and neuronal cells derived from induced pluripotent stem cells (iPSCs). RESULTS: Significant innate immune gene induction was observed in both systems. The NSW2012 isolate induced higher gene expression of two genes (IL-8 and CCL2) when compared with cells infected with less pathogenic isolates. Pathway analysis of induced inflammation-associated genes also indicated generally higher activation in infected NSW2012 cells. However, this differential response was not paralleled in the neuronal cultures. CONCLUSION: NSW2012 may have unique genetic characteristics which contributed to the outbreak. The data herein is consistent with the possibility that the virulence of NSW2012 is underpinned by increased induction of inflammatory genes.
Assuntos
Surtos de Doenças , Imunidade Inata/genética , Inflamação/genética , Febre do Nilo Ocidental/epidemiologia , Vírus do Nilo Ocidental/genética , Austrália/epidemiologia , Linhagem Celular Tumoral , Quimiocina CCL2/genética , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Interleucina-8/genética , Neurônios/virologia , Fenótipo , Virulência , Vírus do Nilo Ocidental/patogenicidadeRESUMO
Gene amplified in esophageal cancer 1 (GAEC1) expression and copy number changes are frequently associated with the pathogenesis of colorectal carcinomas. The current study aimed to identify the pathway and its transcriptional factors with which GAEC1 interacts within colorectal cancer, to gain a better understanding of the mechanics by which this gene exercises its effect on colorectal cancer. Two colonic adenocarcinoma cell lines (SW48 and SW480) and a nonneoplastic colon epithelial cell line (FHC) were transfected with GAEC1 to assess the oncogenic potential of GAEC1 overexpression. Multiple in vitro assays, including cell proliferation, wound healing, clonogenic, apoptosis, cell cycle, and extracellular flux, were performed. Western blot analysis was performed to identify potential gene-interaction partners of GAEC1 in vitro. Results showed that the overexpression of GAEC1 significantly increased cell proliferation, migration, and clonogenic potential ( P < 0.05) of colonic adenocarcinoma. Furthermore, GAEC1 portrayed its ability to influence mitochondrial respiration changes. The observations were in tandem with a significant increase in the expression of phosphorylated protein kinase B, forkhead box O3, and matrix metallopeptidase 9. Thus, GAEC1 has a role in regulating gene pathways, potentially in the Akt pathway. This could help in developing targeted therapies in the future.
Assuntos
Adenocarcinoma/genética , Carcinogênese/genética , Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica/genética , Proteínas Nucleares/genética , Adenocarcinoma/patologia , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias do Colo/patologia , Variações do Número de Cópias de DNA/genética , Células Epiteliais/patologia , Proteína Forkhead Box O3/biossíntese , Humanos , Metaloproteinase 9 da Matriz/biossíntese , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-akt/biossíntese , TransfecçãoRESUMO
BACKGROUND: This study aimed to investigate the impact of miR-451 on the biological behaviours of colon cancer cells along with its targets interactions. METHOD: The levels of miR-451 were tested in colon cancer cell lines (SW480 and SW48). Multiple functional and immunological assays were performed to analyse miR-451 induced growth changes in-vitro and downstream effects on target proteins. RESULTS: Overexpression of miR-451 in colon cancer cells led to reduced cell proliferation, increased apoptosis and decrease accumulation of the cells at the G0/G1 phase of the cell cycle. In addition, a significant increase in the number of the cells was noted in the G2-M phase of cell cycle. Moreover, miR-451 reduced the expression of Oct-4, Sox-2 and Snail indicating its role in stem cell and epithelial-mesenchymal transition (EMT) regulation. An inverse correlation between miR-451 and macrophage migration inhibitory protein (MIF) protein expression occurred in colon cancer cells. Furthermore, restoration the level of miR-451 in colon cancer cells inhibits tumour spheres formation. CONCLUSION: miR-451 has tumour suppressor effects in vitro, which can inhibit the cancer-related signalling pathways in colon cancer.
Assuntos
Neoplasias do Colo/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Neoplasias do Colo/imunologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Transição Epitelial-Mesenquimal , Genes Supressores de Tumor , Humanos , Oxirredutases Intramoleculares/genética , Fatores Inibidores da Migração de Macrófagos/genética , Macrófagos/imunologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Transdução de SinaisRESUMO
BACKGROUND: The clinical pathological features, as well as the cellular mechanisms of miR-195, have not been investigated in thyroid carcinoma. OBJECTIVE: The aim of this study is to identify the interactions of vascular endothelial growth factor (VEGF), p53 and miR-195 in thyroid carcinoma. The clinical and pathological features of miR-195 were also investigated. METHODS: The expression levels of miR-195 were identified in 123 primary thyroid carcinomas, 40 lymph nodes with metastatic papillary thyroid carcinomas and seven non-neoplastic thyroid tissues (controls) as well as two thyroid carcinoma cell lines, B-CPAP (from metastasizing human papillary thyroid carcinoma) and MB-1 (from anaplastic thyroid carcinoma), by the real-time polymerase chain reaction. Using Western blot and immunofluorescence, the effects of exogenous miR-195 on VEGF-A and p53 protein expression levels were examined. Then, cell cycle and apoptosis assays were performed to evaluate the roles of miR-195 in cell cycle progression and apoptosis. RESULTS: The expression of miR-195 was downregulated in majority of the papillary thyroid carcinoma tissue as well as in cells. Introduction of exogenous miR-195 resulted in downregulation of VEGF-A and upregulation of p53 protein expressions. Upregulation of miR-195 in thyroid carcinoma cells resulted in cell cycle arrest. Moreover, we demonstrated that miR-195 inhibits cell cycle progression by induction of apoptosis in the thyroid carcinoma cells. CONCLUSION: Our findings showed for the first time that miR-195 acts as a tumour suppressor and regulates cell cycle progression and apoptosis by targeting VEGF-A and p53 in thyroid carcinoma. The current study exhibited that miR-195 might represent a potential therapeutic target for patients with thyroid carcinomas having aggressive clinical behaviour.
Assuntos
Biomarcadores Tumorais/metabolismo , MicroRNAs/genética , Neovascularização Patológica , Câncer Papilífero da Tireoide/secundário , Neoplasias da Glândula Tireoide/patologia , Proteína Supressora de Tumor p53/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Apoptose , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Pontos de Checagem do Ciclo Celular , Proliferação de Células , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Prognóstico , Câncer Papilífero da Tireoide/genética , Câncer Papilífero da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
OBJECTIVES: In this study, we aimed to investigate the expression pattern, clinicopathological significance and tumour suppressive properties of miR-15a in patients with colorectal carcinomas. METHODS: Tissue samples from 87 patients with primary colorectal carcinomas, 50 matched metastatic lymph node and 37 non-neoplastic colon (control) were prospectively recruited. The expression level of miR-15a was measured by quantitative real-time polymerase chain reaction. Restoration/overexpression of the miR-15a was achieved by exogenous transfection. Four colon cancer cell lines (SW480, CaCO2, SW48 and HCT116) and a non-cancer colon cell line (FHC) were also used for examining the miR-15a induced tumour suppression properties using various in-vitro and immunological assays. RESULTS: Downregulation of miR-15a was noted in ~ 62% of the colorectal carcinoma tissues and it was positively correlated with the presence of cancer recurrence in patients with colorectal carcinomas (pâ¯=â¯0.05). Also, these patients with low miR-15a expression showed relatively shorter survival time when compared to those with miR-15a overexpression. Following miR-15a exogenous overexpression, colon cancer cells showed reduced cell proliferation, low colony formation, less cell invasion properties and mitochondrial respiration when compared to control cells. In addition, BCL2 and SOX2 proteins showed a significant downregulation following miR-15a overexpression suggesting its regulatory role in cancer growth, apoptosis and stemness. CONCLUSION: This study has confirmed the tumour suppressor properties of miR-15a in colorectal cancers. Therefore, its modulation has potential implications in controlling various biological and pathogenic processes in colon carcinogenesis via targeting its downstream proteins such as BCL2 and SOX2.
Assuntos
Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Fatores de Transcrição SOXB1/genética , Apoptose/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Neoplasias Colorretais/patologia , Feminino , Genes Supressores de Tumor , Humanos , Masculino , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Cell growth and survival depend on a delicate balance between energy production and synthesis of metabolites. Here, we provide evidence that an alternative mitochondrial complex II (CII) assembly, designated as CIIlow, serves as a checkpoint for metabolite biosynthesis under bioenergetic stress, with cells suppressing their energy utilization by modulating DNA synthesis and cell cycle progression. Depletion of CIIlow leads to an imbalance in energy utilization and metabolite synthesis, as evidenced by recovery of the de novo pyrimidine pathway and unlocking cell cycle arrest from the S-phase. In vitro experiments are further corroborated by analysis of paraganglioma tissues from patients with sporadic, SDHA and SDHB mutations. These findings suggest that CIIlow is a core complex inside mitochondria that provides homeostatic control of cellular metabolism depending on the availability of energy.
Assuntos
Complexo II de Transporte de Elétrons/metabolismo , Metabolismo Energético/fisiologia , Mitocôndrias/metabolismo , Paraganglioma/patologia , Estresse Fisiológico , Animais , Vias Biossintéticas/fisiologia , Linhagem Celular Tumoral , Complexo II de Transporte de Elétrons/genética , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutação , Paraganglioma/genética , RNA Interferente Pequeno/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVES: miR-142-5p was noted aberrantly expressed and plays important roles in different pathophysiological conditions in human. The present study aims to examine the expression of miR-142-5p and its association with clinicopathological factors in a large cohort of patients with colorectal cancer. In addition, the cellular effects of miR-142-5p and its interacting targets in colon cancer cells were investigated. METHODS: Expression of miR-142-5p in colorectal cancer tissues (n=125) and colon cancer cell lines were analysed using real-time polymerase chain reaction. In vitro assays (cell proliferation, wound healing and colony formation) were used to study the miR-142-5p induced cellular effects. Western blots were used to examine the modulation of FAM134B, KRAS, EPAS1 and KLF6 proteins expression followed by miR-142-5p expression-manipulation. RESULTS: Significant high expression of miR-142-5p was noted in cancer tissues and cells when compared to the controls (p<0.001). Overexpression of miR-142-5p in patients with colorectal cancer was common (72%; 90/125). miR-142-5p overexpression was associated with cancer in the proximal colorectum and with B-raf positive patients (p=0.05). Exogenous overexpression of miR-142-5p resulted in significantly increased cell proliferation, colony formation, and wound healing capacities, whereas inhibition of endogenous miR-142-5p led reduced cancer growth properties. The cellular effects of miR-142-5p were mediated by the modulation of tumour suppressor KLF6 expression, as the expression of miR-142-5p and KLF6 protein are inversely correlated in colon cancer cells. CONCLUSION: High miR-142-5p expression was associated with the biological aggressiveness of cancer. Thus, suppression of miR-142-5p could be a therapeutic strategy for patients with colorectal cancers.