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Nonalcoholic fatty liver disease (NAFLD) progresses to nonalcoholic steatohepatitis (NASH) in response to elevated endoplasmic reticulum (ER) stress. Whereas the onset of simple steatosis requires elevated de novo lipogenesis, progression to NASH is triggered by accumulation of hepatocyte-free cholesterol. We now show that caspase-2, whose expression is ER-stress inducible and elevated in human and mouse NASH, controls the buildup of hepatic-free cholesterol and triglycerides by activating sterol regulatory element-binding proteins (SREBP) in a manner refractory to feedback inhibition. Caspase-2 colocalizes with site 1 protease (S1P) and cleaves it to generate a soluble active fragment that initiates SCAP-independent SREBP1/2 activation in the ER. Caspase-2 ablation or pharmacological inhibition prevents diet-induced steatosis and NASH progression in ER-stress-prone mice. Caspase-2 inhibition offers a specific and effective strategy for preventing or treating stress-driven fatty liver diseases, whereas caspase-2-generated S1P proteolytic fragments, which enter the secretory pathway, are potential NASH biomarkers.
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Caspase 2/fisiologia , Lipogênese/fisiologia , Pró-Proteína Convertases/fisiologia , Serina Endopeptidases/fisiologia , Animais , Colesterol/metabolismo , Retículo Endoplasmático/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Fígado Gorduroso/fisiopatologia , Células HEK293 , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pró-Proteína Convertases/metabolismo , Serina Endopeptidases/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Triglicerídeos/metabolismoRESUMO
Hepatocellular carcinoma (HCC) is a slowly developing malignancy postulated to evolve from premalignant lesions in chronically damaged livers. However, it was never established that premalignant lesions actually contain tumor progenitors that give rise to cancer. Here, we describe isolation and characterization of HCC progenitor cells (HcPCs) from different mouse HCC models. Unlike fully malignant HCC, HcPCs give rise to cancer only when introduced into a liver undergoing chronic damage and compensatory proliferation. Although HcPCs exhibit a similar transcriptomic profile to bipotential hepatobiliary progenitors, the latter do not give rise to tumors. Cells resembling HcPCs reside within dysplastic lesions that appear several months before HCC nodules. Unlike early hepatocarcinogenesis, which depends on paracrine IL-6 production by inflammatory cells, due to upregulation of LIN28 expression, HcPCs had acquired autocrine IL-6 signaling that stimulates their in vivo growth and malignant progression. This may be a general mechanism that drives other IL-6-producing malignancies.
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Comunicação Autócrina , Regulação Neoplásica da Expressão Gênica , Interleucina-6/metabolismo , Neoplasias Hepáticas/patologia , Células-Tronco Neoplásicas/metabolismo , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Progressão da Doença , Hepacivirus , Hepatite C/genética , Hepatite C/metabolismo , Hepatite C/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
In this Article, the sentence: "After 7 months of HFD, MUP-uPA mice developed HCC15, which contained numerous (usually 50-100 per tumour) non-recurrent coding mutations in pathways that are mutated in human HCC (Fig. 2d and Extended Data Fig. 6a).", should have read: "After 7 months of HFD, MUP-uPA mice developed HCC15, which contained numerous (usually 50-100 per tumour) non-recurrent mutations in pathways that are mutated in human HCC (Fig. 2d and Extended Data Fig. 6a).". This has been corrected online. In Extended Data Fig. 6a and b, which show the number of point mutations identified per sample and the mutational signatures, all sequence variants (including non-coding mutations) are shown. Fig. 2d also presents all variants compared to human mutations. In the Supplementary Information to this Amendment, we now provide the comparisons of all variants and coding variants to human mutations.
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This corrects the article DOI: 10.1038/nature24302.
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The role of adaptive immunity in early cancer development is controversial. Here we show that chronic inflammation and fibrosis in humans and mice with non-alcoholic fatty liver disease is accompanied by accumulation of liver-resident immunoglobulin-A-producing (IgA+) cells. These cells also express programmed death ligand 1 (PD-L1) and interleukin-10, and directly suppress liver cytotoxic CD8+ T lymphocytes, which prevent emergence of hepatocellular carcinoma and express a limited repertoire of T-cell receptors against tumour-associated antigens. Whereas CD8+ T-cell ablation accelerates hepatocellular carcinoma, genetic or pharmacological interference with IgA+ cell generation attenuates liver carcinogenesis and induces cytotoxic T-lymphocyte-mediated regression of established hepatocellular carcinoma. These findings establish the importance of inflammation-induced suppression of cytotoxic CD8+ T-lymphocyte activation as a tumour-promoting mechanism.
Assuntos
Carcinoma Hepatocelular/imunologia , Tolerância Imunológica/imunologia , Imunoglobulina A/imunologia , Inflamação/imunologia , Neoplasias Hepáticas/imunologia , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/imunologia , Animais , Antígeno B7-H1/metabolismo , Antígenos CD8/deficiência , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/patologia , Proliferação de Células , Células Clonais/citologia , Células Clonais/imunologia , Progressão da Doença , Feminino , Microbioma Gastrointestinal , Humanos , Imunoglobulina A/metabolismo , Inflamação/etiologia , Inflamação/patologia , Interleucina-10/metabolismo , Cirrose Hepática/complicações , Cirrose Hepática/imunologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/patologia , Ativação Linfocitária , Masculino , Camundongos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Plasmócitos/imunologia , Plasmócitos/metabolismo , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologiaRESUMO
Although hepatocellular cancer (HCC) usually occurs in the setting of liver fibrosis, the causal relationship between liver fibrosis and HCC is unclear. in vivo and in vitro models of HCC involving Colr/r mice (that produce a collagenase-resistant type I collagen) or wild-type (WT) mice were used to assess the relationship between type I collagen, liver fibrosis, and experimental HCC. HCC was either chemically induced in WT and Colr/r mice or Hepa 1-6 cells were engrafted into WT and Colr/r livers. The effect of hepatic stellate cells (HSCs) from WT and Colr/r mice on the growth of Hepa 1-6 cells was studied by using multicellular tumor spheroids and xenografts. Collagen type I deposition and fibrosis were increased in Colr/r mice, but they developed fewer and smaller tumors. Hepa 1-6 cells had reduced tumor growth in the livers of Colr/r mice. Although Colr/r HSCs exhibited a more activated phenotype, Hepa 1-6 growth and malignancy were suppressed in multicellular tumor spheroids and in xenografts containing Colr/r HSCs. Treatment with vitronectin, which mimics the presence of degraded collagen fragments, converted the Colr/r phenotype into a WT phenotype. Although Colr/r mice have increased liver fibrosis, they exhibited decreased HCC in several models. Thus, increased liver type I collagen does not produce increased experimental HCC.
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Carcinoma Hepatocelular/patologia , Colágeno Tipo I/metabolismo , Cirrose Hepática/patologia , Neoplasias Hepáticas Experimentais/patologia , Animais , Linhagem Celular Tumoral , Células Estreladas do Fígado/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND AND AIMS: In clinical and experimental NASH, the origin of the scar-forming myofibroblast is the HSC. We used foz/foz mice on a Western diet to characterize in detail the phenotypic changes of HSCs in a NASH model. APPROACH AND RESULTS: We examined the single-cell expression profiles (scRNA sequencing) of HSCs purified from the normal livers of foz/foz mice on a chow diet, in NASH with fibrosis of foz/foz mice on a Western diet, and in livers during regression of NASH after switching back to a chow diet. Selected genes were analyzed using immunohistochemistry, quantitative real-time PCR, and short hairpin RNA knockdown in primary mouse HSCs. Our analysis of the normal liver identified two distinct clusters of quiescent HSCs that correspond to their acinar position of either pericentral vein or periportal vein. The NASH livers had four distinct HSC clusters, including one representing the classic fibrogenic myofibroblast. The three other HSC clusters consisted of a proliferating cluster, an intermediate activated cluster, and an immune and inflammatory cluster. The livers with NASH regression had one cluster of inactivated HSCs, which was similar to, but distinct from, the quiescent HSCs. CONCLUSIONS: Analysis of single-cell RNA sequencing in combination with an interrogation of previous studies revealed an unanticipated heterogeneity of HSC phenotypes under normal and injured states.
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Redes Reguladoras de Genes , Células Estreladas do Fígado/metabolismo , Fígado/patologia , Miofibroblastos/patologia , Hepatopatia Gordurosa não Alcoólica/patologia , Animais , Proteínas de Ciclo Celular/genética , Células Cultivadas , Dieta Ocidental/efeitos adversos , Modelos Animais de Doenças , Heterogeneidade Genética , Células Estreladas do Fígado/patologia , Humanos , Fígado/citologia , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Hepatopatia Gordurosa não Alcoólica/etiologia , Cultura Primária de Células , RNA-Seq , Análise de Célula ÚnicaRESUMO
Two-electron, one-proton reactions of a family of [CoCp(dxpe)(NCCH3)]2+ complexes (Cp = cyclopentadienyl, dxpe = 1,2-bis(di(aryl/alkyl)phosphino)ethane) form the corresponding hydride species [HCoCp(dxpe)]+ (dxpe = dppe (1,2-bis(diphenylphosphino)ethane), depe (1,2-bis(diethylphosphino)ethane), and dcpe (1,2-bis(dicyclohexylphosphino)ethane)) through a stepwise proton-coupled electron transfer process. For three [CoCp(dxpe)(NCCH3)]2+ complexes, peak shift analysis was employed to quantify apparent proton transfer rate constants from cyclic voltammograms recorded with acids ranging 22 pKa units. The apparent proton transfer rate constants correlate with the strength of the proton source for weak acids, but these apparent proton transfer rate constants curiously plateau (kpl) as the reaction becomes increasingly exergonic. The absolute apparent proton transfer rate constants across both these regions correlate with the steric bulk of the chelating diphosphine ligand, with bulkier ligands leading to slower kinetics (kplateau,depe = 3.5 × 107 M-1 s-1, kplateau,dppe = 1.7 × 107 M-1 s-1, kplateau,dcpe = 7.1 × 104 M-1 s-1). Mechanistic studies were conducted to identify the cause of the aberrant kPTapp-ΔpKa trends. When deuterated acids are employed, deuterium incorporation in the Cp ring is observed, indicating protonation of the CoCp(dxpe) species to form the corresponding hydride proceeds via initial ligand protonation. Digital simulations of cyclic voltammograms show ligand loss accompanying initial reduction gates subsequent PCET activity at higher driving forces. Together, these experiments reveal the details of the reaction mechanism: reduction of the Co(III) species is followed by dissociation of the bound acetonitrile ligand, subsequent reduction of the unligated Co(II) species to form a Co(I) species is followed by protonation, which occurs at the Cp ring, followed by tautomerization to generate the stable Co(III)-hydride product [HCoCp(dxpe)]+. Analysis as a function of chelating disphosphine ligand, solvent, and acid strength reveals that the ligand dissociation equilibrium is directly influenced by the steric bulk of the phosphine ligands and gates protonation, giving rise to the plateau of the apparent proton transfer rate constant with strong acids. The complexity of the reaction mechanism underpinning hydride formation, encompassing dynamic behavior of the entire ligand set, highlights the critical need to understand elementary reaction steps in proton-coupled electron transfer reactions.
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Cobalto/química , Complexos de Coordenação/síntese química , Elétrons , Hidrogênio/química , Prótons , Técnicas Eletroquímicas , Cinética , Ligantes , Oxirredução , Fosfinas/químicaRESUMO
Cancer-associated genetic alterations induce expression of tumour antigens that can activate CD8(+) cytotoxic T cells (CTLs), but the microenvironment of established tumours promotes immune tolerance through poorly understood mechanisms. Recently developed therapeutics that overcome tolerogenic mechanisms activate tumour-directed CTLs and are effective in some human cancers. Immune mechanisms also affect treatment outcome, and certain chemotherapeutic drugs stimulate cancer-specific immune responses by inducing immunogenic cell death and other effector mechanisms. Our previous studies revealed that B cells recruited by the chemokine CXCL13 into prostate cancer tumours promote the progression of castrate-resistant prostate cancer by producing lymphotoxin, which activates an IκB kinase α (IKKα)-BMI1 module in prostate cancer stem cells. Because castrate-resistant prostate cancer is refractory to most therapies, we examined B cell involvement in the acquisition of chemotherapy resistance. Here we focus on oxaliplatin, an immunogenic chemotherapeutic agent that is effective in aggressive prostate cancer. We show that mouse B cells modulate the response to low-dose oxaliplatin, which promotes tumour-directed CTL activation by inducing immunogenic cell death. Three different mouse prostate cancer models were refractory to oxaliplatin unless genetically or pharmacologically depleted of B cells. The crucial immunosuppressive B cells are plasmocytes that express IgA, interleukin (IL)-10 and programmed death ligand 1 (PD-L1), the appearance of which depends on TGFß receptor signalling. Elimination of these cells, which also infiltrate human-therapy-resistant prostate cancer, allows CTL-dependent eradication of oxaliplatin-treated tumours.
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Compostos Organoplatínicos/farmacologia , Plasmócitos/efeitos dos fármacos , Plasmócitos/imunologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Transferência Adotiva , Animais , Anticorpos Antineoplásicos/imunologia , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Antígeno B7-H1/metabolismo , Células Cultivadas , Quimiocina CXCL13/metabolismo , Humanos , Quinase I-kappa B/metabolismo , Imunoglobulina A/imunologia , Interleucina-10/imunologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neoplásicas/patologia , Compostos Organoplatínicos/administração & dosagem , Compostos Organoplatínicos/imunologia , Compostos Organoplatínicos/uso terapêutico , Oxaliplatina , Plasmócitos/citologia , Neoplasias da Próstata/patologia , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Linfócitos T Citotóxicos/citologia , Fator de Crescimento Transformador beta/imunologiaRESUMO
BACKGROUND & AIMS: Although there are associations among oxidative stress, reduced nicotinamide adenine dinucleotide phosphate oxidase (NOX) activation, and hepatocellular carcinoma (HCC) development, it is not clear how NOX contributes to hepatocarcinogenesis. We studied the functions of different NOX proteins in mice after administration of a liver carcinogen. METHODS: Fourteen-day-old Nox1-/- mice, Nox4-/- mice, Nox1-/-Nox4-/- (double-knockout) mice, and wild-type (WT) C57BL/6 mice were given a single intraperitoneal injection of diethylnitrosamine (DEN) and liver tumors were examined at 9 months. We also studied the effects of DEN in mice with disruption of Nox1 specifically in hepatocytes (Nox1ΔHep), hepatic stellate cells (Nox1ΔHep), or macrophages (Nox1ΔMac). Some mice were also given injections of the NOX1-specific inhibitor ML171. To study the acute effects of DEN, 8-12-week-old mice were given a single intraperitoneal injection, and liver and serum were collected at 72 hours. Liver tissues were analyzed by histologic examination, quantitative polymerase chain reaction, and immunoblots. Hepatocytes and macrophages were isolated from WT and knockout mice and analyzed by immunoblots. RESULTS: Nox4-/- mice and WT mice developed liver tumors within 9 months after administration of DEN, whereas Nox1-/- mice developed 80% fewer tumors, which were 50% smaller than those of WT mice. Nox1ΔHep and Nox1ΔHSC mice developed liver tumors of the same number and size as WT mice, whereas Nox1ΔMac developed fewer and smaller tumors, similar to Nox1-/- mice. After DEN injection, levels of tumor necrosis factor, interleukin 6 (IL6), and phosphorylated signal transducer and activator of transcription 3 were increased in livers from WT, but not Nox1-/- or Nox1ΔMac, mice. Conditioned medium from necrotic hepatocytes induced expression of NOX1 in cultured macrophages, followed by expression of tumor necrosis factor, IL6, and other inflammatory cytokines; this medium did not induce expression of IL6 or cytokines in Nox1ΔMac macrophages. WT mice given DEN followed by ML171 developed fewer and smaller liver tumors than mice given DEN followed by vehicle. CONCLUSIONS: In mice given injections of a liver carcinogen (DEN), expression of NOX1 by macrophages promotes hepatic tumorigenesis by inducing the production of inflammatory cytokines. We propose that upon liver injury, damage-associated molecular patterns released from dying hepatocytes activate liver macrophages to produce cytokines that promote tumor development. Strategies to block NOX1 or these cytokines might be developed to slow hepatocellular carcinoma progression.
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Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Transformação Celular Neoplásica/genética , Hepatite/genética , Hepatócitos/patologia , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Macrófagos/enzimologia , NADPH Oxidase 1/genética , NADPH Oxidase 4/genética , Alarminas/metabolismo , Animais , Carcinoma Hepatocelular/induzido quimicamente , Proliferação de Células/fisiologia , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Dietilnitrosamina , Inibidores Enzimáticos/farmacologia , Células Estreladas do Fígado , Hepatócitos/fisiologia , Humanos , Interleucina-6/metabolismo , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas Experimentais/induzido quimicamente , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 1/metabolismo , Necrose , Fator de Transcrição STAT3/metabolismo , Carga Tumoral , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Proinflammatory cytokine TNFα plays critical roles in promoting malignant cell proliferation, angiogenesis, and tumor metastasis in many cancers. However, the mechanism of TNFα-mediated tumor development remains unclear. Here, we show that IKKα, an important downstream kinase of TNFα, interacts with and phosphorylates FOXA2 at S107/S111, thereby suppressing FOXA2 transactivation activity and leading to decreased NUMB expression, and further activates the downstream NOTCH pathway and promotes cell proliferation and tumorigenesis. Moreover, we found that levels of IKKα, pFOXA2 (S107/111), and activated NOTCH1 were significantly higher in hepatocellular carcinoma tumors than in normal liver tissues and that pFOXA2 (S107/111) expression was positively correlated with IKKα and activated NOTCH1 expression in tumor tissues. Therefore, dysregulation of NUMB-mediated suppression of NOTCH1 by TNFα/IKKα-associated FOXA2 inhibition likely contributes to inflammation-mediated cancer pathogenesis. Here, we report a TNFα/IKKα/FOXA2/NUMB/NOTCH1 pathway that is critical for inflammation-mediated tumorigenesis and may provide a target for clinical intervention in human cancer.
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Carcinoma Hepatocelular/metabolismo , Transformação Celular Neoplásica/metabolismo , Fator 3-beta Nuclear de Hepatócito/genética , Quinase I-kappa B/metabolismo , Neoplasias Hepáticas/metabolismo , Receptor Notch1/metabolismo , Animais , Carcinoma Hepatocelular/patologia , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Fator 3-beta Nuclear de Hepatócito/metabolismo , Humanos , Neoplasias Hepáticas Experimentais/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Modelos Biológicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fosforilação , Receptor Notch1/genética , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The ability to identify a specific cancer using minimally invasive biopsy holds great promise for improving the diagnosis, treatment selection, and prediction of prognosis in cancer. Using whole-genome methylation data from The Cancer Genome Atlas (TCGA) and machine learning methods, we evaluated the utility of DNA methylation for differentiating tumor tissue and normal tissue for four common cancers (breast, colon, liver, and lung). We identified cancer markers in a training cohort of 1,619 tumor samples and 173 matched adjacent normal tissue samples. We replicated our findings in a separate TCGA cohort of 791 tumor samples and 93 matched adjacent normal tissue samples, as well as an independent Chinese cohort of 394 tumor samples and 324 matched adjacent normal tissue samples. The DNA methylation analysis could predict cancer versus normal tissue with more than 95% accuracy in these three cohorts, demonstrating accuracy comparable to typical diagnostic methods. This analysis also correctly identified 29 of 30 colorectal cancer metastases to the liver and 32 of 34 colorectal cancer metastases to the lung. We also found that methylation patterns can predict prognosis and survival. We correlated differential methylation of CpG sites predictive of cancer with expression of associated genes known to be important in cancer biology, showing decreased expression with increased methylation, as expected. We verified gene expression profiles in a mouse model of hepatocellular carcinoma. Taken together, these findings demonstrate the utility of methylation biomarkers for the molecular characterization of cancer, with implications for diagnosis and prognosis.
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Metilação de DNA , Neoplasias/diagnóstico , Neoplasias/genética , Alelos , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Estudos de Casos e Controles , Estudos de Coortes , Neoplasias do Colo/diagnóstico , Neoplasias do Colo/genética , Ilhas de CpG , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Masculino , Metástase Neoplásica , Neoplasias/mortalidade , Prognóstico , Risco , Fatores de TempoRESUMO
The kinetics and mechanism(s) of the reactions of [K(Krypt)][LCuO2] (Krypt = 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane, L = a bis(arylcarboxamido)pyridine ligand) with 2,2,6,6-tetramethylpiperdine- N-hydroxide (TEMPOH) and the para-substituted phenols XArOH (X = para substituent NO2, CF3, Cl, H, Me, tBu, OMe, or NMe2) at low temperatures were studied. The reaction with TEMPOH occurs rapidly ( k = 35.4 ± 0.3 M-1 s-1) by second-order kinetics to yield TEMPO⢠and [LCuOOH]- on the basis of electron paramagnetic resonance spectroscopy, the production of H2O2 upon treatment with protic acid, and independent preparation from reaction of [NBu4][LCuOH] with H2O2 ( Keq = 0.022 ± 0.007 for the reverse reaction). The reactions with XArOH also follow second-order kinetics, and analysis of the variation of the k values as a function of phenol properties (Hammett σ parameter, O-H bond dissociation free energy, p Ka, E1/2) revealed a change in mechanism across the series, from proton transfer/electron transfer for X = NO2, CF3, Cl to concerted-proton/electron transfer (or hydrogen-atom transfer) for X = OMe, NMe2 (data for X = H, Me, tBu are intermediate between the extremes). Thermodynamic analysis and comparisons to previous results for LCuOH, a different copper-oxygen intermediate with the same supporting ligand, and literature for other [CuO2]+ complexes reveal significant differences in proton-coupled electron-transfer mechanisms that have implications for understanding oxidation catalysis by copper-containing enzymes and abiological catalysts.
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Cobre/química , Compostos Organometálicos/química , Fenóis/química , Prótons , Superóxidos/química , Transporte de Elétrons , Cinética , Estrutura Molecular , TermodinâmicaRESUMO
BACKGROUND & AIMS: Chronic liver injury often results in the activation of hepatic myofibroblasts and the development of liver fibrosis. Hepatic myofibroblasts may originate from 3 major sources: hepatic stellate cells (HSCs), portal fibroblasts (PFs), and fibrocytes, with varying contributions depending on the etiology of liver injury. Here, we assessed the composition of hepatic myofibroblasts in multidrug resistance gene 2 knockout (Mdr2-/-) mice, a genetic model that resembles primary sclerosing cholangitis in patients. METHODS: Mdr2-/- mice expressing a collagen-GFP reporter were analyzed at different ages. Hepatic non-parenchymal cells isolated from collagen-GFP Mdr2-/- mice were sorted based on collagen-GFP and vitamin A. An NADPH oxidase (NOX) 1/4 inhibitor was administrated to Mdr2-/- mice aged 12-16â¯weeks old to assess the therapeutic approach of targeting oxidative stress in cholestatic injury. RESULTS: Thy1+ activated PFs accounted for 26%, 51%, and 54% of collagen-GFP+ myofibroblasts in Mdr2-/- mice at 4, 8, and 16â¯weeks of age, respectively. The remaining collagen-GFP+ myofibroblasts were composed of activated HSCs, suggesting that PFs and HSCs are both activated in Mdr2-/- mice. Bone-marrow-derived fibrocytes minimally contributed to liver fibrosis in Mdr2-/- mice. The development of cholestatic liver fibrosis in Mdr2-/- mice was associated with early recruitment of Gr1+ myeloid cells and upregulation of pro-inflammatory cytokines (4â¯weeks). Administration of a NOX inhibitor to 12-week-old Mdr2-/- mice suppressed the activation of myofibroblasts and attenuated the development of cholestatic fibrosis. CONCLUSIONS: Activated PFs and activated HSCs contribute to cholestatic fibrosis in Mdr2-/- mice, and serve as targets for antifibrotic therapy. LAY SUMMARY: Activated portal fibroblasts and hepatic stellate cells, but not fibrocytes, contributed to the production of the fibrous scar in livers of Mdr2-/- mice, and these cells can serve as targets for antifibrotic therapy in cholestatic injury. Therapeutic inhibition of the enzyme NADPH oxidase (NOX) in Mdr2-/- mice reversed cholestatic fibrosis, suggesting that targeting NOXs may be an effective strategy for the treatment of cholestatic fibrosis.
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Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Fibroblastos/metabolismo , Células Estreladas do Fígado/metabolismo , Cirrose Hepática Biliar/metabolismo , Veia Porta/patologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática Biliar/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirazolonas , Piridinas/farmacologia , Piridinas/uso terapêutico , Piridonas , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
A longstanding research goal has been to understand the nature and role of copper-oxygen intermediates within copper-containing enzymes and abiological catalysts. Synthetic chemistry has played a pivotal role in highlighting the viability of proposed intermediates and expanding the library of known copper-oxygen cores. In addition to the number of new complexes that have been synthesized since the previous reviews on this topic in this journal (Mirica, L. M.; Ottenwaelder, X.; Stack, T. D. P. Chem. Rev. 2004, 104, 1013-1046 and Lewis, E. A.; Tolman, W. B. Chem. Rev. 2004, 104, 1047-1076), the field has seen significant expansion in the (1) range of cores synthesized and characterized, (2) amount of mechanistic work performed, particularly in the area of organic substrate oxidation, and (3) use of computational methods for both the corroboration and prediction of proposed intermediates. The scope of this review has been limited to well-characterized examples of copper-oxygen species but seeks to provide a thorough picture of the spectroscopic characteristics and reactivity trends of the copper-oxygen cores discussed.
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Cobre/química , Oxigênio/química , Análise Espectral/métodos , Estrutura MolecularRESUMO
With the goal of understanding how distal charge influences the properties and hydrogen atom transfer (HAT) reactivity of the [CuOH]2+ core proposed to be important in oxidation catalysis, the complexes [M]3[SO3LCuOH] (M = [K(18-crown-6)]+ or [K(crypt-222)]+) and [NMe3LCuOH]X (X = BArF4- or ClO4-) were prepared, in which SO3- or NMe3+ substituents occupy the para positions of the flanking aryl rings of the supporting bis(carboxamide)pyridine ligands. Structural and spectroscopic characterization showed that the [CuOH]+ cores in the corresponding complexes were similar, but cyclic voltammetry revealed the E1/2 value for the [CuOH]2+/[CuOH]+ couple to be nearly 0.3 V more oxidizing for the [NMe3LCuOH]2+ than the [SO3LCuOH]- species, with the latter influenced by interactions between the distal -SO3- substituents and K+ or Na+ counterions. Chemical oxidations of the complexes generated the corresponding [CuOH]2+ species as evinced by UV-vis spectroscopy. The rates of HAT reactions of these species with 9,10-dihydroanthracene to yield the corresponding [Cu(OH2)]2+ complexes and anthracene were measured, and the thermodynamics of the processes were evaluated via determination of the bond dissociation enthalpies (BDEs) of the product O-H bonds. The HAT rate for [SO3LCuOH]- was found to be â¼150 times faster than that for [NMe3LCuOH]2+, despite finding approximately the same BDEs for the product O-H bonds. Rationales for these observations and new insights into the roles of supporting ligand attributes on the properties of the [CuOH]2+ unit are presented.
RESUMO
Reaction of [NBu4][LCuIIOH] with excess ROOH (R = cumyl or tBu) yielded [NBu4][LCuIIOOR], the reversible one-electron oxidation of which generated novel species with [CuOOR]2+ cores (formally CuIIIOOR), identified by spectroscopy and theory for the case R = cumyl. This species reacts with weak O-H bonds in TEMPO-H and 4-dimethylaminophenol (NMe2PhOH), the latter yielding LCu(OPhNMe2), which was also prepared independently. With the identification of [CuOOR]2+ complexes, the first precedent for this core in enzymes is provided, with implications for copper monooxygenase mechanisms.
Assuntos
Complexos de Coordenação/metabolismo , Cobre/metabolismo , Oxigenases de Função Mista/química , Peróxidos/metabolismo , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Cobre/química , Cristalografia por Raios X , Oxigenases de Função Mista/metabolismo , Modelos Moleculares , Peróxidos/químicaRESUMO
The stretching frequency, ν(Cu-O), of the [CuOH]2+ core in the complexes LCuOH (L = N,N'-bis(2,6-diisopropyl-4-R-phenyl)pyridine-2,6-dicarboxamide, R = H or NO2, or N,N'-bis(2,6-diisopropylphenyl)-1-methylpiperidine-2,6-dicarboxamide) was determined to be â¼630 cm-1 by resonance Raman spectroscopy and verified by isotopic labeling. In efforts to use Badger's rule to estimate the bond distance corresponding to ν(Cu-O), a modified version of the rule was developed through use of stretching frequencies normalized by dividing by the appropriate reduced masses. The modified version was found to yield excellent fits of normalized frequencies to bond distances for >250 data points from theory and experiment for a variety of M-X and X-X bond distances in the range â¼1.1-2.2 Å (root mean squared errors for the predicted bond distances of 0.03 Å). Using the resulting general equation, the Cu-O bond distance was predicted to be â¼1.80 Å for the reactive [CuOH]2+ core. Limitations of the equation and its use in predictions of distances in a variety of moieties for which structural information is not available were explored.
Assuntos
Cobre/química , Hidróxidos/química , Teoria Quântica , Cristalografia por Raios X , Modelos Moleculares , Conformação Molecular , Análise Espectral RamanRESUMO
E-cadherin is an important adhesion molecule whose loss is associated with progression and poor prognosis of liver cancer. However, it is unclear whether the loss of E-cadherin is a real culprit or a bystander in liver cancer progression. In addition, the precise role of E-cadherin in maintaining liver homeostasis is also still unknown, especially in vivo. Here we demonstrate that liver-specific E-cadherin knockout mice develop spontaneous periportal inflammation via an impaired intrahepatic biliary network, as well as periductal fibrosis, which resembles primary sclerosing cholangitis. Inducible gene knockout studies identified E-cadherin loss in biliary epithelial cells as a causal factor of cholangitis induction. Furthermore, a few of the E-cadherin knockout mice developed spontaneous liver cancer. When knockout of E-cadherin is combined with Ras activation or chemical carcinogen administration, E-cadherin knockout mice display markedly accelerated carcinogenesis and an invasive phenotype associated with epithelial-mesenchymal transition, up-regulation of stem cell markers, and elevated ERK activation. Also in human hepatocellular carcinoma, E-cadherin loss correlates with increased expression of mesenchymal and stem cell markers, and silencing of E-cadherin in hepatocellular carcinoma cell lines causes epithelial-mesenchymal transition and increased invasiveness, suggesting that E-cadherin loss can be a causal factor of these phenotypes. Thus, E-cadherin plays critical roles in maintaining homeostasis and suppressing carcinogenesis in the liver.