RESUMO
One of the emerging hallmarks of cancer illustrates the importance of metabolic reprogramming, necessary to synthesize the building blocks required to fulfill the high demands of rapidly proliferating cells. However, the proliferation-independent instructive role of metabolic enzymes in tumor plasticity is still unclear. Here, we provide evidence that glutathione peroxidase 8 (GPX8), a poorly characterized enzyme that resides in the endoplasmic reticulum, is an essential regulator of tumor aggressiveness. We found that GPX8 expression was induced by the epithelial-mesenchymal transition (EMT) program. Moreover, in breast cancer patients, GPX8 expression significantly correlated with known mesenchymal markers and poor prognosis. Strikingly, GPX8 knockout in mesenchymal-like cells (MDA-MB-231) resulted in an epithelial-like morphology, down-regulation of EMT characteristics, and loss of cancer stemness features. In addition, GPX8 knockout significantly delayed tumor initiation and decreased its growth rate in mice. We found that these GPX8 loss-dependent phenotypes were accompanied by the repression of crucial autocrine factors, in particular, interleukin-6 (IL-6). In these cells, IL-6 bound to the soluble receptor (sIL6R), stimulating the JAK/STAT3 signaling pathway by IL-6 trans-signaling mechanisms, so promoting cancer aggressiveness. We observed that in GPX8 knockout cells, this signaling mechanism was impaired as sIL6R failed to activate the JAK/STAT3 signaling pathway. Altogether, we present the GPX8/IL-6/STAT3 axis as a metabolic-inflammatory pathway that acts as a robust regulator of cancer cell aggressiveness.
Assuntos
Neoplasias da Mama/enzimologia , Interleucina-6/metabolismo , Janus Quinases/metabolismo , Peroxidases/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fenótipo , Transdução de SinaisRESUMO
BACKGROUND & AIMS: Primary liver cancers include hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (CCA) and combined HCC-CCA tumors (cHCC-CCA). It has been suggested, but not unequivocally proven, that hepatic progenitor cells (HPCs) can contribute to hepatocarcinogenesis. We aimed to determine whether HPCs contribute to HCC, cHCC-CCA or both types of tumors. METHODS: To trace progenitor cells during hepatocarcinogenesis, we generated Mdr2-KO mice that harbor a yellow fluorescent protein (YFP) reporter gene driven by the Foxl1 promoter which is expressed specifically in progenitor cells. These mice (Mdr2-KOFoxl1-CRE;RosaYFP) develop chronic inflammation and HCCs by the age of 14-16 months, followed by cHCC-CCA tumors at the age of 18 months. RESULTS: In this Mdr2-KOFoxl1-CRE;RosaYFP mouse model, liver progenitor cells are the source of cHCC-CCA tumors, but not the source of HCC. Ablating the progenitors, caused reduction of cHCC-CCA tumors but did not affect HCCs. RNA-sequencing revealed enrichment of the IL-6 signaling pathway in cHCC-CCA tumors compared to HCC tumors. Single-cell RNA-sequencing (scRNA-seq) analysis revealed that IL-6 is expressed by immune and parenchymal cells during senescence, and that IL-6 is part of the senescence-associated secretory phenotype. Administration of an anti-IL-6 antibody to Mdr2-KOFoxl1-CRE;RosaYFP mice inhibited the development of cHCC-CCA tumors. Blocking IL-6 trans-signaling led to a decrease in the number and size of cHCC-CCA tumors, indicating their dependence on this pathway. Furthermore, the administration of a senolytic agent inhibited IL-6 and the development of cHCC-CCA tumors. CONCLUSION: Our results demonstrate that cHCC-CCA, but not HCC tumors, originate from HPCs, and that IL-6, which derives in part from cells in senescence, plays an important role in this process via IL-6 trans-signaling. These findings could be applied to develop new therapeutic approaches for cHCC-CCA tumors. LAY SUMMARY: Combined hepatocellular carcinoma-cholangiocarcinoma is the third most prevalent type of primary liver cancer (i.e. a cancer that originates in the liver). Herein, we show that this type of cancer originates in stem cells in the liver and that it depends on inflammatory signaling. Specifically, we identify a cytokine called IL-6 that appears to be important in the development of these tumors. Our results could be used for the development of novel treatments for these aggressive tumors.
Assuntos
Neoplasias dos Ductos Biliares , Carcinoma Hepatocelular , Colangiocarcinoma , Neoplasias Hepáticas , Camundongos , Animais , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Células-Tronco , Transdução de Sinais , Carcinogênese , RNA , Ductos Biliares Intra-Hepáticos , Fatores de Transcrição ForkheadRESUMO
Acute liver injury can be secondary to a variety of causes, including infections, intoxication, and ischemia. All of these insults induce hepatocyte death and subsequent inflammation, which can make acute liver injury a life-threatening event. IL-22 is a dual natured cytokine which has context-dependent protective and pathogenic properties during tissue damage. Accordingly, IL-22 was shown to promote liver regeneration upon acute liver damage. However, other studies suggest pathogenic properties of IL-22 during chronic liver injury. IL-22 binding protein (IL-22BP, IL-22Ra2) is a soluble inhibitor of IL-22 that regulates IL-22 activity. However, the significance of endogenous IL-22BP in acute liver injury is unknown. We hypothesized that IL-22BP may play a role in acute liver injury. To test this hypothesis, we used Il22bp-deficient mice and murine models of acute liver damage induced by ischemia reperfusion and N-acetyl-p-aminophenol (acetaminophen) administration. We found that Il22bp-deficient mice were more susceptible to acute liver damage in both models. We used Il22 × Il22bp double-deficient mice to show that this effect is indeed due to uncontrolled IL-22 activity. We could demonstrate mechanistically increased expression of Cxcl10 by hepatocytes, and consequently increased infiltration of inflammatory CD11b+Ly6C+ monocytes into the liver in Il22bp-deficient mice upon liver damage. Accordingly, neutralization of CXCL10 reversed the increased disease susceptibility of Il22bp-deficient mice. In conclusion, our data indicate that IL-22BP plays a protective role in acute liver damage, via controlling IL-22-induced Cxcl10 expression.
Assuntos
Acetaminofen/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Fígado/irrigação sanguínea , Receptores de Interleucina/fisiologia , Traumatismo por Reperfusão/fisiopatologia , Animais , Movimento Celular , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Quimiocina CXCL10/antagonistas & inibidores , Quimiocina CXCL10/fisiologia , Constrição , Hepatectomia , Hepatócitos/metabolismo , Interleucinas/deficiência , Interleucinas/metabolismo , Isquemia/fisiopatologia , Fígado/fisiologia , Falência Hepática Aguda/etiologia , Falência Hepática Aguda/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/fisiologia , Receptores de Interleucina/deficiência , Receptores de Interleucina/genética , Regeneração , Traumatismo por Reperfusão/prevenção & controle , Interleucina 22RESUMO
Liver cancer, which typically develops on a background of chronic liver inflammation, is now the second leading cause of cancer mortality worldwide. For patients with liver cancer, surgical resection is a principal treatment modality that offers a chance of prolonged survival. However, tumor recurrence after resection, the mechanisms of which remain obscure, markedly limits the long-term survival of these patients. We have shown that partial hepatectomy in multidrug resistance 2 knockout (Mdr2-/- ) mice, a model of chronic inflammation-associated liver cancer, significantly accelerates hepatocarcinogenesis. Here, we explore the postsurgical mechanisms that drive accelerated hepatocarcinogenesis in Mdr2-/- mice by perioperative pharmacological inhibition of interleukin-6 (IL6), which is a crucial liver regeneration priming cytokine. We demonstrate that inhibition of IL6 signaling dramatically impedes tumorigenesis following partial hepatectomy without compromising survival or liver mass recovery. IL6 blockade significantly inhibited hepatocyte cell cycle progression while promoting a hypertrophic regenerative response, without increasing apoptosis. Mdr2-/- mice contain hepatocytes with a notable persistent DNA damage response (γH2AX, 53BP1) due to chronic inflammation. We show that liver regeneration in this microenvironment leads to a striking increase in hepatocytes bearing micronuclei, a marker of genomic instability, which is suppressed by IL6 blockade. CONCLUSION: Our findings indicate that genomic instability derived during the IL6-mediated liver regenerative response within a milieu of chronic inflammation links partial hepatectomy to accelerated hepatocarcinogenesis; this suggests a new therapeutic approach through the usage of an anti-IL6 treatment to extend the tumor-free survival of patients undergoing surgical resection. (Hepatology 2017;65:1600-1611).
Assuntos
Instabilidade Genômica , Hepatite Crônica/complicações , Interleucina-6/metabolismo , Neoplasias Hepáticas Experimentais/etiologia , Regeneração Hepática , Animais , Hepatectomia , Hiperplasia , Hipertrofia , Interleucina-6/antagonistas & inibidores , Fígado/patologia , Neoplasias Hepáticas Experimentais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Renal endothelin-converting enzyme (ECE)-1 is induced in experimental diabetes and following radiocontrast administration, conditions characterized by renal hypoxia, hypoxia-inducible factor (HIF) stabilization, and enhanced endothelin synthesis. Here we tested whether ECE-1 might be a HIF-target gene in vitro and in vivo. ECE-1 transcription and expression increased in cultured vascular endothelial and proximal tubular cell lines, subject to hypoxia, to mimosine or cobalt chloride. These interventions are known to stabilize HIF signaling by inhibition of HIF-prolyl hydroxylases. In rats, HIF-prolyl-hydroxylase inhibition by mimosine or FG-4497 increased HIF-1α immunostaining in renal tubules, principally in distal nephron segments. This was associated with markedly enhanced ECE-1 protein expression, predominantly in the renal medulla. A progressive and dramatic increase in ECE-1 immunostaining over time, in parallel with enhanced HIF expression, was also noted in conditional von Hippel-Lindau knockout mice. Since HIF and STAT3 are cross-stimulated, we triggered HIF expression by STAT3 activation in mice, transfected by or injected with a chimeric IL-6/IL-6-receptor protein, and found a similar pattern of enhanced ECE-1 expression. Chromatin immunoprecipitation sequence (ChIP-seq) and PCR analysis in hypoxic endothelial cells identified HIF binding at the ECE-1 promoter and intron regions. Thus, our findings suggest that ECE-1 may be a novel HIF-target gene.
Assuntos
Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Animais , Hipóxia Celular/fisiologia , Células Cultivadas , Cobalto/farmacologia , Dioxigenases/antagonistas & inibidores , Enzimas Conversoras de Endotelina , Células Endoteliais da Veia Umbilical Humana , Humanos , Íntrons , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Mimosina/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Inibidores de Prolil-Hidrolase/farmacologia , Regiões Promotoras Genéticas , Ratos , Ratos Sprague-Dawley , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transcrição Gênica , Doença de von Hippel-Lindau/genética , Doença de von Hippel-Lindau/metabolismoRESUMO
BACKGROUND/AIMS: Changes in renal oxygenation and perfusion have been identified as common pathways to the development and progression of renal disease. Recently, the sensitivity of hemodynamic response imaging (HRI) was demonstrated; this is a functional magnetic resonance imaging (MRI) method combined with transient hypercapnia and hyperoxia for the evaluation of renal perfusion and vascular reactivity. The aim of this study was to utilize HRI for the noninvasive evaluation of changes in renal hemodynamics and morphology during acute, chronic and acute-on-chronic renal failures. METHODS: Renal-HRI maps and true fast imaging with steady-state precession (True-FISP) images were used to evaluate renal perfusion, morphology and corticomedullary differentiation (CMD). MR images were acquired on two mouse models of kidney injury: adenine-induced chronic kidney disease (CKD) and rhabdomyolysis-induced acute kidney injury (AKI). Serum urea was measured from these mice in order to determine renal function. RESULTS: Renal-HRI maps revealed a blunted response to hypercapnia and hyperoxia with evolving kidney dysfunction in both models, reflecting hampered renal vascular reactivity and perfusion. True-FISP images showed a high sensitivity to renal morphological changes, with different patterns characterizing each model. Calculated data obtained from HRI and True-FISP during the evolution of renal failure and upon recovery, with and without protective intervention, closely correlated with the degree of renal impairment. CONCLUSIONS: This study suggests the potential combined usage of two noninvasive MRI methods, HRI and True-FISP, for the assessment of renal dysfunction without the potential risk associated with contrast-agents administration. HRI may also serve as a research tool in experimental settings, revealing the hemodynamic changes associated with kidney dysfunction.
Assuntos
Injúria Renal Aguda/patologia , Nefropatias/diagnóstico , Imageamento por Ressonância Magnética/métodos , Insuficiência Renal Crônica/patologia , Injúria Renal Aguda/diagnóstico , Adenina/química , Animais , Meios de Contraste/química , Hemodinâmica , Rim/patologia , Camundongos , Perfusão , Insuficiência Renal Crônica/diagnóstico , Rabdomiólise/complicações , Ureia/sangueRESUMO
Signal transducer and activator of transcription 3 (STAT3) is a point of convergence for numerous oncogenic signaling pathways. In breast cancer cell lines and xenograft models activated STAT3 participates in breast tumorigenesis, while studies in humans have demonstrated that phosphorylated (tyrosine705)-STAT3 is a marker of good prognosis in breast cancer. In order to resolve this paradox we hypothesized that in clinic, phospho-STAT3 has a predictive role of benefit from adjuvant chemotherapy; therefore the goal of this study was to determine the usefulness of phospho-STAT3 status as a predictor of benefit from adjuvant chemotherapy in breast cancer patients. Immunohistochemical analysis of phospho-STAT3 was performed on a tissue microarray of breast cancer specimens. The expression pattern of phospho-STAT3 was retrospectively correlated with pathological parameters and overall survival in patients who were or were not treated with adjuvant chemotherapy. Of 375 tissue specimens interpretable for phospho-STAT3, 134 (36 %) exhibited positive phospho-STAT3 nuclear expression. Among 234 patients who received adjuvant therapy, those with tumors displaying positive phospho-STAT3 nuclear expression had a better ten-year rate of overall survival than patients with tumors displaying negative phospho-STAT3 nuclear expression (P = 0.001). Among patients who did not received adjuvant chemotherapy, positive phospho-STAT3 nuclear status was not correlated with increased overall survival (P = 0.54). Positive phospho-STAT3 was correlated with improved overall survival only among patients who received adjuvant chemotherapy in a multivariate analysis adjusted for stage, grade, hormonal status, Her2 status, and age, irrespective of the chemotherapy regimen received (hazard ratio for death, 0.35 [95 % CI 0.188-0.667]; P = 0.001). These findings support the role of phospho-STAT3 as a marker of favorable outcome in breast cancer patients treated with adjuvant chemotherapy. Whether phospho-STAT3 has a predictive role of benefit from adjuvant chemotherapy has to be validated on prospective, randomized, controlled studies.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Processamento de Proteína Pós-Traducional , Fator de Transcrição STAT3/metabolismo , Tirosina/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Quimioterapia Adjuvante , Ciclofosfamida/uso terapêutico , Doxorrubicina/uso terapêutico , Feminino , Fluoruracila/uso terapêutico , Humanos , Estimativa de Kaplan-Meier , Metotrexato/administração & dosagem , Pessoa de Meia-Idade , Análise Multivariada , Fosforilação , Modelos de Riscos Proporcionais , Resultado do TratamentoRESUMO
BACKGROUND: The clinical use of iodinated radiocontrast agents or gadolinium for renal perfusion imaging is limited in the presence of renal dysfunction. We have previously demonstrated the feasibility of hemodynamic response imaging (HRI), a functional magnetic resonance imaging (MRI) method combined with hypercapnia and hypercapnic-hyperoxia, for monitoring changes in liver perfusion and hemodynamics. The aim of the present study was to evaluate the utility of HRI for monitoring changes in renal perfusion and hemodynamics. METHODS: Renal HRI maps were acquired during graded hypercapnia (95% air + 5% CO2) and hypercapnic-hyperoxia (95% O2 + 5% CO2) in control mice. The utility of HRI for monitoring changes in renal perfusion and oxygenation was evaluated using pharmacological inhibition of nitric oxide synthase and cycloxygenase as well as in rhabdomyolysis-induced acute kidney injury (AKI) in mice. HRI results were further interpreted using Doppler ultrasound (US). RESULTS: Renal HRI maps revealed pronounced signal-intensity changes in response to both hypercapnia and hypercapnic-hyperoxia, reflecting intense vascular reactivity. These changes were significantly attenuated following the pharmacological intervention and during AKI, corresponding with hampered perfusion dynamics, as confirmed by Doppler US. CONCLUSIONS: The applicability of the non-invasive HRI method suggests its potential use for the evaluation of renal perfusion and vascular reactivity, excluding the need for contrast-agent administration.
Assuntos
Injúria Renal Aguda/patologia , Hemodinâmica , Hipercapnia/patologia , Imageamento por Ressonância Magnética , Rabdomiólise/complicações , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/metabolismo , Animais , Meios de Contraste , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Camundongos , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Oxigênio/metabolismo , Prostaglandina-Endoperóxido Sintases/química , Prostaglandina-Endoperóxido Sintases/metabolismo , Ultrassonografia DopplerRESUMO
In vitro studies suggest that combined activation of hypoxia-inducible factor (HIF) and signal transducer and activator of transcription 3 (STAT3) promotes the hypoxia response. However, their interrelationship in vivo remains poorly defined. The present study investigated the possible relationship between HIF-1 upregulation and STAT3 activation in the rodent kidney in vivo. Activation of HIF-1 and STAT3 was analysed by immunohistochemical staining and western blot analysis in: (i) models of hypoxia-associated kidney injury induced by radiocontrast media or rhabdomyolysis; (ii) following activation of STAT3 by the interleukin (IL)-6-soluble IL-6 receptor complex; or (iii) following HIF-1α stabilization using hypoxic and non-hypoxic stimuli (mimosine, FG-4497, CO, CoCl(2)) and in targeted von Hippel-Lindau-knockout mice. Western blot analysis and immunostaining revealed marked induction of both transcription factors under all conditions tested, suggesting that in vivo STAT3 can trigger HIF and vice versa. Colocalization of HIF-1α and phosphorylated STAT3 was detected in some, but not all, renal cell types, suggesting that in some cells a paracrine mechanism may be responsible for the reciprocal activation of the two transcription factors. Nevertheless, in several cell types spatial concordance was observed under the majority of conditions tested, suggesting that HIF-1 and STAT3 may act as cotranscription factors. These in vivo studies suggest that, in response to renal hypoxic-stress, upregulation of HIF-1 and activation of STAT3 may be both reciprocal and cell type dependent.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/metabolismo , Rim/metabolismo , Fator de Transcrição STAT3/metabolismo , Injúria Renal Aguda/genética , Injúria Renal Aguda/metabolismo , Animais , Hipóxia/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Oxigênio/farmacologia , Ratos , Ratos Sprague-Dawley , Regulação para Cima/efeitos dos fármacos , Proteína Supressora de Tumor Von Hippel-Lindau/genéticaRESUMO
Irradiation-induced alopecia and dermatitis (IRIAD) are two of the most visually recognized complications of radiotherapy, of which the molecular and cellular basis remains largely unclear. By combining scRNA-seq analysis of whole skin-derived irradiated cells with genetic ablation and molecular inhibition studies, we show that senescence-associated IL-6 and IL-1 signaling, together with IL-17 upregulation and CCR6+ -mediated immune cell migration, are crucial drivers of IRIAD. Bioinformatics analysis colocalized irradiation-induced IL-6 signaling with senescence pathway upregulation largely within epidermal hair follicles, basal keratinocytes, and dermal fibroblasts. Loss of cytokine signaling by genetic ablation in IL-6-/- or IL-1R-/- mice, or by molecular blockade, strongly ameliorated IRIAD, as did deficiency of CCL20/CCR6-mediated immune cell migration in CCR6-/- mice. Moreover, IL-6 deficiency strongly reduced IL-17, IL-22, CCL20, and CCR6 upregulation, whereas CCR6 deficiency reciprocally diminished IL-6, IL-17, CCL3, and MHC upregulation, suggesting that proximity-dependent cellular cross talk promotes IRIAD. Therapeutically, topical application of Janus kinase blockers or inhibition of T-cell activation by cyclosporine effectively reduced IRIAD, suggesting the potential of targeted approaches for the treatment of dermal side effects in radiotherapy patients.
Assuntos
Radiodermite , Receptores CCR6 , Animais , Interleucina-17/genética , Interleucina-17/metabolismo , Interleucina-6/genética , Camundongos , Receptores CCR6/genética , Receptores CCR6/metabolismo , TranscriptomaRESUMO
BACKGROUND & AIMS: Interleukin-6 (IL-6) is a crucial factor in liver regeneration following partial hepatectomy (PH); however, the role of IL-6 and IL-6 trans-signaling in particular, in hepatocyte mitosis remains controversial. IL-6 trans-signaling relies upon the release of the soluble IL-6R (sIL-6R), which binds IL-6 to form an agonistic IL-6/sIL-6R complex. Herein we have examined the hypothesis that IL-6 trans-signaling plays a crucial and distinct role in liver regeneration following PH. METHODS: The specific IL-6/sIL-6R antagonist, sgp130Fc, was expressed in mice and analyzed for its effect on hepatocyte mitosis following PH. Alternatively, we examined the effect of the IL-6/sIL-6R super-agonist, Hyper-IL-6, or IL-6 expressed either alone or in combination with hepatocyte growth factor (HGF) on hepatocyte mitosis in the absence of PH. RESULTS: Following PH, the dramatic rise of circulating IL-6 levels is accompanied by a concurrent â¼2-fold increase in circulating sIL-6R levels. Ectopic expression of sgp130Fc reduced hepatocyte mitosis by about 40% at early times following PH, while substantially reducing AKT, but not STAT3, activation. But, ectopic Hyper-IL-6 expression in mice without PH was not mitogenic to hepatocytes in vivo. Rather, Hyper-IL-6, but not IL-6, markedly increased HGF-induced hepatocyte mitosis. This cooperative effect correlated with greater resistance of HIL-6 than IL-6 to HGF-mediated reduction of AKT activation, rather than changes in STAT3 or MAPK signaling, and was completely blocked by PI3K inhibition. CONCLUSIONS: Following PH, IL-6/sIL-6R cooperates with growth factors, through a PI3K/AKT-dependent mechanism to promote entry of hepatocytes into the cell cycle.
Assuntos
Hepatectomia , Hepatócitos/fisiologia , Interleucina-6/metabolismo , Regeneração Hepática/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Expressão Gênica/fisiologia , Hepatectomia/métodos , Interleucina-6/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mitose/fisiologia , Receptores de Interleucina-6/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/fisiologia , TransfecçãoRESUMO
BACKGROUND: The possibility of restoring sevoflurane postconditioning (sevo-postC) cardioprotection in diabetic animals is uncertain. We hypothesized that attenuation of myocardial injury by sevo-postC might be hindered by inhibition of signal transducer and activator of transcription (STAT) 3-regulated activity of phosphatidylinositol 3-kinase (PI3K) in diabetic animals. To determine whether postC cardioprotection can be restored by normoglycemia, we treated rats with insulin. METHODS: Diabetic or nondiabetic rats were randomly subjected to 30-min ischemia/reperfusion, with ischemic postC or sevo-postC, with and without mitochondrial adenosine triphosphate-dependent potassium channel blocker 5-hydroxy decanoate sodium and PI3K antagonist wortmannin. The infarct area, phosphorylated STAT3, and apoptosis were examined. Studies were repeated after insulin treatment. RESULTS: Ischemic postC and sevo-postC significantly reduced infarct size by 50% in the nondiabetic rats (P < 0.002), a phenomenon completely reversed by 5-hydroxy decanoate sodium and wortmannin. Diabetes mellitus blocked the protective effect of postC, and insulin treatment to achieve normoglycemia did not restore cardioprotection. Phosphorylated STAT3 nuclear retention was significantly increased after ischemia-reperfusion and was further enhanced in response to ischemic postC (P < 0.05) but was significantly reduced in diabetic rats (by 43%; P < 0.01). CONCLUSIONS: The effective reduction in infarct size and apoptosis in the nondiabetic rat heart by postC was completely abrogated in diabetic rats. This inhibition is not relieved by insulin-induced normoglycemia. The PI3K pathway and mitochondrial adenosine triphosphate-dependent potassium channel activation are involved in the mechanism of postC. In diabetic rats, STAT3 activation was strongly reduced, as was postC cardioprotection, suggesting that the inability of insulin to restore postC may be attributed to diabetes-induced STAT3-mediated inhibition of PI3K signaling.
Assuntos
Diabetes Mellitus/metabolismo , Insulina/farmacologia , Pós-Condicionamento Isquêmico , Éteres Metílicos/farmacologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fator de Transcrição STAT3/metabolismo , Androstadienos/farmacologia , Animais , Diabetes Mellitus/tratamento farmacológico , Coração/efeitos dos fármacos , Coração/fisiologia , Insulina/uso terapêutico , Pós-Condicionamento Isquêmico/métodos , Masculino , Éteres Metílicos/uso terapêutico , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Fosfatidilinositol 3-Quinase/fisiologia , Fosforilação/fisiologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Sevoflurano , WortmaninaRESUMO
Background: Most of the ischemia-reperfusion injury (IR-I) occurs during reperfusion and is mediated by the immune system. In this study we determined whether immunomodulation with hyper-Interleukin-6 (a recombinant designer cytokine composed of interleukin-6 linked to its soluble receptor) is protective against IR-I in mice kidneys. Methods: Hyper-Interleukin-6 (HIL-6) was administered by in vivo plasmid DNA transfection to 10 male mice. Twenty-four hours later, unilateral nephrectomy was done. IR-I immediately followed by closure of the remaining kidney vascular pedicle for 40 min. Seven mice transfected with non-coding control plasmid served as the control group. The functional and morphological effects of IR-I and its effect on mice longevity were explored. This was done by serial blood tests and by histopathology done upon sacrifice of the animals at post-operative day 7. Findings: Mice pretreated with HIL-6 had a mean creatinine level at post-operative day 1 of 35.45 ± 4.03 µmol/l and mean Urea level was 14.18 ± 2.69 mmol/l, whereas mean creatinine was 89.33 ± 69.27 µmol/l (P = 0.025), and mean urea was 38.17 ± 20.77 mmol/l (P = 0.0024) in the control group. Histological changes in the control group included inflammatory infiltration, tubular damage, and architectural distortion. These were not seen in the treatment group. Seven days post-operatively the survival rate of treated mice was 100% compared to 50% in the control group (P = 0.015). Interpretation: In this single kidney mouse model, pretreatment with HIL-6 administration effectively protected against IR-I both morphologically and functionally. Further studies are needed to better understand the mechanism and feasibility of using this immunomodulator.
RESUMO
Hepatocellular carcinoma (HCC) typically develops on a background of chronic hepatitis for which the proinflammatory cytokine IL6 is conventionally considered a crucial driving factor. Paradoxically, IL6 also acts as a hepatoprotective factor in chronic liver injury. Here we used the multidrug-resistant gene 2 knockout (Mdr2-/-) mouse model to elucidate potential roles of IL6 in chronic hepatitis-associated liver cancer. Long-term analysis of three separate IL6/Stat3 signaling-deficient Mdr2-/- strains revealed aggravated liver injury with increased dysplastic nodule formation and significantly accelerated tumorigenesis in all strains. Tumorigenesis in the IL6/Stat3-perturbed models was strongly associated with enhanced macrophage accumulation and hepatosteatosis, phenotypes of nonalcoholic steatohepatitis (NASH), as well as with significant reductions in senescence and the senescence-associated secretory phenotype (SASP) accompanied by increased hepatocyte proliferation. These findings reveal a crucial suppressive role for IL6/Stat3 signaling in chronic hepatitis-associated hepatocarcinogenesis by impeding protumorigenic NASH-associated phenotypes and by reinforcing the antitumorigenic effects of the SASP. SIGNIFICANCE: These findings describe a context-dependent role of IL6 signaling in hepatocarcinogenesis and predict that increased IL6-neutralizing sgp130 levels in some patients with NASH may herald early HCC development.See related commentary by Huynh and Ernst, p. 4671.
Assuntos
Transformação Celular Neoplásica/metabolismo , Senescência Celular , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Interleucina-6/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Biomarcadores , Transformação Celular Neoplásica/genética , Senescência Celular/genética , Modelos Animais de Doenças , Progressão da Doença , Fígado Gorduroso/patologia , Feminino , Imuno-Histoquímica , Interleucina-6/genética , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
The oncofetal long noncoding RNA (lncRNA) H19 is postnatally repressed in most tissues, and re-expressed in many cancers, including hepatocellular carcinoma (HCC). The role of H19 in carcinogenesis is a subject of controversy. We aimed to examine the role of H19 in chronic inflammation-mediated hepatocarcinogenesis using the Mdr2/Abcb4 knockout (Mdr2-KO) mouse, a well-established HCC model. For this goal, we have generated Mdr2-KO/H19-KO double knockout (dKO) mice and followed spontaneous tumor development in the dKO and control Mdr2-KO mice. Cellular localization of H19 and effects of H19 loss in the liver were determined in young and old Mdr2-KO mice. Tumor incidence and tumor load were both significantly decreased in the liver of dKO versus Mdr2-KO females. The expression levels of H19 and Igf2 were variable in nontumor liver tissues of Mdr2-KO females and were significantly downregulated in most matched tumors. In nontumor liver tissue of aged Mdr2-KO females, H19 was expressed mainly in hepatocytes, and hepatocyte proliferation was increased compared to dKO females. At an early age, dKO females displayed lower levels of liver injury and B-cell infiltration, with higher percentage of binuclear hepatocytes. In human samples, H19 expression was higher in females, positively correlated with cirrhosis (in nontumor liver samples) and negatively correlated with CTNNB1 (beta-catenin) mutations and patients' survival (in tumors). Our data demonstrate that the lncRNA H19 is pro-oncogenic during the development of chronic inflammation-mediated HCC in the Mdr2-KO mouse model, mainly by increasing liver injury and decreasing hepatocyte polyploidy in young mice.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Carcinoma Hepatocelular/patologia , Fibrose/genética , Neoplasias Hepáticas/patologia , RNA Longo não Codificante/genética , beta Catenina/genética , Animais , Carcinoma Hepatocelular/genética , Feminino , Fibrose/complicações , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Masculino , Camundongos , Camundongos Knockout , Caracteres Sexuais , Carga Tumoral , Regulação para Cima , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
The response to tissue injury involves the coordination of inflammatory and repair processes. IL-6 expression correlates with the onset and severity of acute kidney injury (AKI), but its contribution to pathogenesis remains unclear. This study established a critical role for IL-6 in both the inflammatory response and the resolution of AKI. IL-6-deficient mice were resistant to HgCl2-induced AKI compared with wild-type mice. The accumulation of peritubular neutrophils was lower in IL-6-deficient mice than in wild-type mice, and neutrophil depletion before HgCl2 administration in wild-type mice significantly reduced AKI; these results demonstrate the critical role of IL-6 signaling in the injurious inflammatory process in AKI. Renal IL-6 expression and STAT3 activation in renal tubular epithelial cells significantly increased during the development of injury, suggesting active IL-6 signaling. Although a lack of renal IL-6 receptors (IL-6R) precludes the activation of classical signaling pathways, IL-6 can stimulate target cells together with a soluble form of the IL-6R (sIL-6R) in a process termed trans-signaling. During injury,serum sIL-6R levels increased three-fold, suggesting a possible role for IL-6 trans-signaling in AKI. Stimulation of IL-6 trans-signaling with an IL-6/sIL-6R fusion protein activated STAT3 in renal tubular epithelium and prevented AKI. IL-6/sIL-6R reduced lipid peroxidation after injury, suggesting that its protective effect may be largely mediated through amelioration of oxidative stress. In summary, IL-6 simultaneously promotes an injurious inflammatory response and, through a mechanism of trans-signaling, protects the kidney from further injury.
Assuntos
Injúria Renal Aguda/etiologia , Interleucina-6/fisiologia , Receptores de Interleucina-6/fisiologia , Animais , CamundongosRESUMO
Trans-signaling of the major pro- and anti-inflammatory cytokines Interleukin (IL)-6 and IL-11 has the unique feature to virtually activate all cells of the body and is critically involved in chronic inflammation and regeneration. Hyper-IL-6 and Hyper-IL-11 are single chain designer trans-signaling cytokines, in which the cytokine and soluble receptor units are trapped in one complex via a flexible peptide linker. Albeit, Hyper-cytokines are essential tools to study trans-signaling in vitro and in vivo, the superior potency of these designer cytokines are accompanied by undesirable stress responses. To enable tailor-made generation of Hyper-cytokines, we developed inactive split-cytokine-precursors adapted for posttranslational reassembly by split-intein mediated protein trans-splicing (PTS). We identified cutting sites within IL-6 (E134/S135) and IL-11 (G116/S117) and obtained inactive split-Hyper-IL-6 and split-Hyper-IL-11 cytokine precursors. After fusion with split-inteins, PTS resulted in reconstitution of active Hyper-cytokines, which were efficiently secreted from transfected cells. Our strategy comprises the development of a background-free cytokine signaling system from reversibly inactivated precursor cytokines.
Assuntos
Regiões Constantes de Imunoglobulina , Interleucina-11 , Interleucina-6 , Proteínas Recombinantes de Fusão , Trans-Splicing , Animais , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Regiões Constantes de Imunoglobulina/biossíntese , Regiões Constantes de Imunoglobulina/genética , Interleucina-11/biossíntese , Interleucina-11/genética , Interleucina-6/biossíntese , Interleucina-6/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genéticaRESUMO
The long-term prognosis after resection of hepatocellular carcinoma (HCC), which is one of the treatment options for early-stage HCC, remains unsatisfactory as a result of a high incidence of disease recurrence. Recent studies performed in murine models revealed a link between liver regeneration under chronic inflammation and hepatic tumorigenesis. Sorafenib is a potent drug for advanced HCC with multikinase inhibition activity. We propose that inhibition of signal transduction pathways which are activated during hepatectomy, using Sorafenib, will reduce accelerated tumorigenesis. To test this hypothesis, we studied the Mdr2-knockout (KO) mouse strain, a model of inflammation-associated cancer, which underwent partial hepatectomy (PHx) at three months of age, with or without Sorafenib.Here we show that Sorafenib treatment during PHx inhibited different signal transduction pathways at the multikinase levels, but did not result in increased morbidity or mortality. At the early stages after PHx, Sorafenib treatment had no effect on the course of proliferation, apoptosis and DNA repair in the regenerating liver, but resulted in decreased stellate cells activation and inflammatory response. Finally, we show that Sorafenib treatment during PHx at three months of age resulted in decreased fibrosis and tumor formation at 8.5 months.In conclusion our study indicates that short-term Sorafenib treatment during PHx is safe and effective in inhibiting inflammation-associated cancer, and is therefore a potential strategy for recurrence prevention in patients with early-stage HCC treated with PHx.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/fisiologia , Carcinoma Hepatocelular/prevenção & controle , Transformação Celular Neoplásica/efeitos dos fármacos , Modelos Animais de Doenças , Hepatectomia , Inflamação/complicações , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Animais , Antineoplásicos/farmacologia , Apoptose , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/patologia , Proliferação de Células , Hepatite/complicações , Técnicas Imunoenzimáticas , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/prevenção & controle , Regeneração Hepática/efeitos dos fármacos , Camundongos , Camundongos Knockout , Niacinamida/farmacologia , Análise Serial de Proteínas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sorafenibe , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
Head and neck cancer patients treated by radiation commonly suffer from a devastating side effect known as dry-mouth syndrome, which results from the irreversible loss of salivary gland function via mechanisms that are not completely understood. In this study, we used a mouse model of radiation-induced salivary hypofunction to investigate the outcomes of DNA damage in the head and neck region. We demonstrate that the loss of salivary function was closely accompanied by cellular senescence, as evidenced by a persistent DNA damage response (γH2AX and 53BP1) and the expression of senescence-associated markers (SA-ßgal, p19ARF, and DcR2) and secretory phenotype (SASP) factors (PAI-1 and IL6). Notably, profound apoptosis or necrosis was not observed in irradiated regions. Signs of cellular senescence were also apparent in irradiated salivary glands surgically resected from human patients who underwent radiotherapy. Importantly, using IL6 knockout mice, we found that sustained expression of IL6 in the salivary gland long after initiation of radiation-induced DNA damage was required for both senescence and hypofunction. Additionally, we demonstrate that IL6 pretreatment prevented both senescence and salivary gland hypofunction via a mechanism involving enhanced DNA damage repair. Collectively, these results indicate that cellular senescence is a fundamental mechanism driving radiation-induced damage in the salivary gland and suggest that IL6 pretreatment may represent a promising therapeutic strategy to preserve salivary gland function in head and neck cancer patients undergoing radiotherapy.
Assuntos
Senescência Celular , Neoplasias de Cabeça e Pescoço/radioterapia , Interleucina-6/farmacologia , Glândulas Salivares/efeitos da radiação , Animais , Apoptose/efeitos da radiação , Dano ao DNA , Reparo do DNA , Feminino , Histonas/análise , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Interleucina-6/fisiologia , Glândulas Salivares/fisiologiaRESUMO
The liver is a unique organ, and first in line, the hepatocytes encounter the potential to proliferate during cell mass loss. This phenomenon is tightly controlled and resembles in some way the embryonal co-inhabitant cell lineage of the liver, the embryonic hematopoietic system. Interestingly, both the liver and hematopoietic cell proliferation and growth are controlled by various growth factors and cytokines. IL-6 and its signaling cascade inside the cells through STAT3 are both significantly important for liver regeneration as well as for hematopoietic cell proliferation. The process of liver regeneration is very complex and is dependent on the etiology and extent of liver damage and the genetic background. In this review we will initially describe the clinical relevant condition, portraying a number of available animal models with an emphasis on the relevance of each one to the human condition of fulminant hepatic failure (FHF). The discussion will then be focused on the role of cytokines in liver failure and regeneration, and suggest potential new therapeutic modalities for FHF. The recent findings on the role of IL-6 in liver regeneration and the activity of the designer IL-6/sIL-6R fusion protein, hyper-IL-6, in particular, suggest that this molecule could significantly enhance liver regeneration in humans, and as such could be a useful treatment for FHF in patients.