RESUMO
Cytotoxic stress activates stress-activated kinases, initiates adaptive mechanisms, including the unfolded protein response (UPR) and autophagy, and induces programmed cell death. Fatty acid unsaturation, controlled by stearoyl-CoA desaturase (SCD)1, prevents cytotoxic stress but the mechanisms are diffuse. Here, we show that 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol) [PI(18:1/18:1)] is a SCD1-derived signaling lipid, which inhibits p38 mitogen-activated protein kinase activation, counteracts UPR, endoplasmic reticulum-associated protein degradation, and apoptosis, regulates autophagy, and maintains cell morphology and proliferation. SCD1 expression and the cellular PI(18:1/18:1) proportion decrease during the onset of cell death, thereby repressing protein phosphatase 2 A and enhancing stress signaling. This counter-regulation applies to mechanistically diverse death-inducing conditions and is found in multiple human and mouse cell lines and tissues of Scd1-defective mice. PI(18:1/18:1) ratios reflect stress tolerance in tumorigenesis, chemoresistance, infection, high-fat diet, and immune aging. Together, PI(18:1/18:1) is a lipokine that links fatty acid unsaturation with stress responses, and its depletion evokes stress signaling.
Assuntos
Transdução de Sinais , Estearoil-CoA Dessaturase , Animais , Apoptose , Ácidos Graxos , Camundongos , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Resposta a Proteínas não DobradasRESUMO
Sorafenib represents the current standard of care for patients with advanced-stage hepatocellular carcinoma (HCC). However, acquired drug resistance occurs frequently during therapy and is accompanied by rapid tumor regrowth after sorafenib therapy termination. To identify the mechanism of this therapy-limiting growth resumption, we established robust sorafenib resistance HCC cell models that exhibited mitochondrial dysfunction and chemotherapeutic crossresistance. We found a rapid relapse of tumor cell proliferation after sorafenib withdrawal, which was caused by renewal of mitochondrial structures alongside a metabolic switch toward high electron transport system (ETS) activity. The translation-inhibiting antibiotic tigecycline impaired the biogenesis of mitochondrial DNA-encoded ETS subunits and limited the electron acceptor turnover required for glutamine oxidation. Thereby, tigecycline prevented the tumor relapse in vitro and in murine xenografts in vivo. These results offer a promising second-line therapeutic approach for advanced-stage HCC patients with progressive disease undergoing sorafenib therapy or treatment interruption due to severe adverse events.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Sorafenibe/farmacologia , Tigeciclina/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Feminino , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Camundongos SCID , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Recidiva Local de Neoplasia/prevenção & controle , Inibidores da Síntese de Proteínas/farmacologiaRESUMO
BACKGROUND: Urinary tract infections are among the most common types of infections and give rise to inflammation with pain as one of the main symptoms. The herbal medicinal product Canephron® N contains BNO 2103, a defined mixture of pulverized rosemary leaves, centaury herb, and lovage root, and has been used in the treatment of urinary tract infections for more than 25 years. PURPOSE: To test the hypothesis that BNO 2103 reduces pain in cystitis and prostatitis by virtue of anti-inflammatory properties, and to reveal potential mechanisms underlying the anti-inflammatory features. STUDY DESIGN: BNO 2103 was studied for anti-inflammatory and analgesic properties in three animal models in vivo, and the mode of action underlying the anti-inflammatory features was investigated in human leukocytes and cell-free assays in vitro. METHODS: To assess the anti-inflammatory and analgesic efficacy of BNO 2103 we employed cyclophosphamide-induced cystitis and carrageenan-induced prostatitis in rats, and zymosan-induced peritonitis in mice. Human neutrophils and monocytes as well as isolated human 5-lipoxygenase and microsomal prostaglandin E2 synthase-1-containing microsomes were utilized to assess inhibition of leukotriene and/or prostaglandin E2 production by HPLC and/or ELISA. RESULTS: When given orally, BNO 2103 reduced inflammation and hyperalgesia in experimental cystitis in rats, while individual components of BNO 2103 also reduced hyperalgesia. Furthermore, BNO 2103 reduced hyperalgesia in rats with carrageenan-induced prostatitis. Cell-based and cell-free studies implicate inhibition of prostaglandin E2 and leukotriene B4 biosynthesis as potential mechanisms underlying the analgesic and anti-inflammatory effects. CONCLUSION: Our data support the hypothesis that BNO 2103 reduces pain by virtue of its anti-inflammatory properties, possibly related to suppression of prostaglandin E2 and leukotriene B4 formation, and suggest that this combination has the potential to treat clinical symptoms such as inflammatory pain. Thus BNO 2103 may represent an alternative to reduce the use of antibiotics in urinary tract infections.
Assuntos
Analgésicos/farmacologia , Anti-Inflamatórios/farmacologia , Cistite/complicações , Dor/tratamento farmacológico , Extratos Vegetais/farmacologia , Prostatite/complicações , Analgésicos/química , Animais , Anti-Inflamatórios/química , Carragenina/efeitos adversos , Ciclofosfamida/efeitos adversos , Cistite/induzido quimicamente , Medicamentos de Ervas Chinesas , Feminino , Humanos , Hiperalgesia/tratamento farmacológico , Hiperalgesia/etiologia , Inflamação/tratamento farmacológico , Inflamação/etiologia , Masculino , Camundongos , Monócitos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Dor/etiologia , Extratos Vegetais/química , Prostatite/induzido quimicamente , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: The understanding of lysosomes has been expanded in recent research way beyond their view as cellular trash can. Lysosomes are pivotal in regulating metabolism, endocytosis and autophagy and are implicated in cancer. Recently it was discovered that the lysosomal V-ATPase, which is known to induce apoptosis, interferes with lipid metabolism in cancer, yet the interplay between these organelles is poorly understood. METHODS: LC-MS/MS analysis was performed to investigate lipid distribution in cells. Cell survival and signaling pathways were analyzed by means of cell biological methods (qPCR, Western Blot, flow cytometry, CellTiter-Blue). Mitochondrial structure was analyzed by confocal imaging and electron microscopy, their function was determined by flow cytometry and seahorse measurements. RESULTS: Our data reveal that interfering with lysosomal function changes composition and subcellular localization of triacylglycerids accompanied by an upregulation of PGC1α and PPARα expression, master regulators of energy and lipid metabolism. Furthermore, cardiolipin content is reduced driving mitochondria into fission, accompanied by a loss of membrane potential and reduction in oxidative capacity, which leads to a deregulation in cellular ROS and induction of mitochondria-driven apoptosis. Additionally, cells undergo a metabolic shift to glutamine dependency, correlated with the fission phenotype and sensitivity to lysosomal inhibition, most prominent in Ras mutated cells. CONCLUSION: This study sheds mechanistic light on a largely uninvestigated triangle between lysosomes, lipid metabolism and mitochondrial function. Insight into this organelle crosstalk increases our understanding of mitochondria-driven cell death. Our findings furthermore provide a first hint on a connection of Ras pathway mutations and sensitivity towards lysosomal inhibitors.
Assuntos
Morte Celular , Metabolismo dos Lipídeos , Mitocôndrias/metabolismo , Muramidase/metabolismo , Linhagem Celular Tumoral , Humanos , Estresse Oxidativo , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidoresRESUMO
The plant Garcinia kola is used in African ethno-medicine to treat various oxidation- and inflammation-related diseases but its bioactive compounds are not well characterized. Garcinoic acid (GA) is one of the few phytochemicals that have been isolated from Garcinia kola. We investigated the anti-inflammatory potential of the methanol extract of Garcinia kola seeds (NE) and purified GA, as a major phytochemical in these seeds, in lipopolysaccharide (LPS)-activated mouse RAW264.7 macrophages and its anti-atherosclerotic potential in high fat diet fed ApoE-/- mice. This study outlines an optimized procedure for the extraction and purification of GA from Garcinia kola seeds with an increased yield and a purity of >99%. We found that LPS-induced upregulation of iNos and Cox2 expression, and the formation of the respective signaling molecules nitric oxide and prostanoids, were significantly diminished by both the NE and GA. In addition, GA treatment in mice decreased intra-plaque inflammation by attenuating nitrotyrosinylation. Further, modulation of lymphocyte sub-populations in blood and spleen have been detected, showing immune regulative properties of GA. Our study provides molecular insights into the anti-inflammatory activities of Garcinia kola and reveals GA as promising natural lead for the development of multi-target drugs to treat inflammation-driven diseases.
Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Benzopiranos/farmacologia , Garcinia kola/química , Nozes/química , Vitamina E/análogos & derivados , Vitamina E/farmacologia , Animais , Biomarcadores , Cromatografia Líquida , Mediadores da Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Células RAW 264.7 , Sementes , Transdução de Sinais , Espectrometria de Massas em TandemRESUMO
The epidithiodioxopiperazine gliotoxin is a virulence factor of Aspergillus fumigatus, the most important airborne fungal pathogen of humans. Gliotoxin suppresses innate immunity in invasive aspergillosis, particularly by compromising neutrophils, but the underlying molecular mechanisms remain elusive. Neutrophils are the first responders among innate immune cells recruited to sites of infection by the chemoattractant leukotriene (LT)B4 that is biosynthesized by 5-lipoxygenase and LTA4 hydrolase (LTA4H). Here, we identified gliotoxin as inhibitor of LTA4H that selectively abrogates LTB4 formation in human leukocytes and in distinct animal models. Gliotoxin failed to inhibit the formation of other eicosanoids and the aminopeptidase activity of the bifunctional LTA4H. Suppression of LTB4 formation by gliotoxin required the cellular environment and/or reducing conditions, and only the reduced form of gliotoxin inhibited LTA4H activity. Conclusively, gliotoxin suppresses the biosynthesis of the potent neutrophil chemoattractant LTB4 by direct interference with LTA4H thereby impairing neutrophil functions in invasive aspergillosis.
Assuntos
Aspergilose/imunologia , Aspergillus fumigatus/imunologia , Epóxido Hidrolases/imunologia , Gliotoxina/imunologia , Leucotrieno B4/imunologia , Animais , Aspergilose/microbiologia , Linhagem Celular , Feminino , Humanos , Imunidade Inata , Leucócitos/imunologia , Leucócitos/microbiologia , Masculino , Camundongos , Neutrófilos/imunologia , Neutrófilos/microbiologia , Ratos WistarRESUMO
Systemic vitamin E metabolites have been proposed as signaling molecules, but their physiological role is unknown. Here we show, by library screening of potential human vitamin E metabolites, that long-chain ω-carboxylates are potent allosteric inhibitors of 5-lipoxygenase, a key enzyme in the biosynthesis of chemoattractant and vasoactive leukotrienes. 13-((2R)-6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)-2,6,10-trimethyltridecanoic acid (α-T-13'-COOH) can be synthesized from α-tocopherol in a human liver-on-chip, and is detected in human and mouse plasma at concentrations (8-49 nM) that inhibit 5-lipoxygenase in human leukocytes. α-T-13'-COOH accumulates in immune cells and inflamed murine exudates, selectively inhibits the biosynthesis of 5-lipoxygenase-derived lipid mediators in vitro and in vivo, and efficiently suppresses inflammation and bronchial hyper-reactivity in mouse models of peritonitis and asthma. Together, our data suggest that the immune regulatory and anti-inflammatory functions of α-tocopherol depend on its endogenous metabolite α-T-13'-COOH, potentially through inhibiting 5-lipoxygenase in immune cells.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Inflamação/patologia , Vitamina E/metabolismo , Adolescente , Adulto , Idoso , Animais , Araquidonato 5-Lipoxigenase/química , Hiper-Reatividade Brônquica/patologia , Sobrevivência Celular/efeitos dos fármacos , Sistema Livre de Células , Humanos , Concentração Inibidora 50 , Leucócitos/metabolismo , Inibidores de Lipoxigenase/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Metaboloma , Camundongos , Pessoa de Meia-Idade , Peritonite/patologia , Proteínas Recombinantes/metabolismo , Vitamina E/química , Adulto JovemRESUMO
The enzyme acetyl-CoA carboxylase (ACC) plays a crucial role in fatty acid metabolism. In recent years, ACC has been recognized as a promising drug target for treating different diseases. However, the role of ACC in vascular endothelial cells (ECs) has been neglected so far. To characterize the role of ACC, we used the ACC inhibitor, soraphen A, as a chemical tool, and also a gene silencing approach. We found that ACC1 was the predominant isoform in human umbilical vein ECs as well as in human microvascular ECs and that soraphen A reduced the levels of malonyl-CoA. We revealed that ACC inhibition shifted the lipid composition of EC membranes. Accordingly, membrane fluidity, filopodia formation, and migratory capacity were reduced. The antimigratory action of soraphen A depended on an increase in the cellular proportion of PUFAs and, most importantly, on a decreased level of phosphatidylglycerol. Our study provides a causal link between ACC, membrane lipid composition, and cell migration in ECs. Soraphen A represents a useful chemical tool to investigate the role of fatty acid metabolism in ECs and ACC inhibition offers a new and valuable therapeutic perspective for the treatment of EC migration-related diseases.
Assuntos
Acetil-CoA Carboxilase/metabolismo , Movimento Celular , Células Endoteliais/metabolismo , Fosfolipídeos/metabolismo , Acetil-CoA Carboxilase/antagonistas & inibidores , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Humanos , Macrolídeos/farmacologiaRESUMO
Proinflammatory leukotrienes (LTs) are produced by 5-lipoxygenase (5-LO) aided by 5-LO-activating protein (FLAP). LT biosynthesis inhibitors are currently under clinical investigation as treatments for respiratory and cardiovascular diseases. Here, we have revealed a sex bias in the efficiency of clinically relevant LT biosynthesis inhibitors, showing that their effects are superior in females. We found that androgens cause these sex differences by impeding the LT-biosynthetic 5-LO/FLAP complex assembly. Lower doses of the FLAP inhibitor MK886 were required to reduce LTB4 levels in exudates of female versus male mice and rats. Following platelet-activating factor-induced shock, MK886 increased survival exclusively in female mice, and this effect was abolished by testosterone administration. FLAP inhibitors and the novel-type 5-LO inhibitors licofelone and sulindac sulfide exhibited higher potencies in human blood from females, and bioactive 5-LO/FLAP complexes were formed in female, but not male, human and murine leukocytes. Supplementation of female blood or leukocytes with 5α-dihydrotestosterone abolished the observed sex differences. Our data suggest that females may benefit from anti-LT therapy to a greater extent than males, prompting consideration of sex issues in LT modifier development.
Assuntos
Androgênios/metabolismo , Leucotrienos/biossíntese , Fatores Sexuais , Testosterona/administração & dosagem , Proteínas Ativadoras de 5-Lipoxigenase/metabolismo , Animais , Araquidonato 5-Lipoxigenase/metabolismo , Di-Hidrotestosterona/metabolismo , Feminino , Humanos , Hidroxiureia/análogos & derivados , Hidroxiureia/farmacologia , Leucócitos/metabolismo , Inibidores de Lipoxigenase/farmacologia , Masculino , Camundongos , Pirróis/administração & dosagem , Ratos , Ratos Wistar , Sulindaco/administração & dosagem , Sulindaco/análogos & derivados , Testosterona/metabolismoRESUMO
Protein kinases, including the serine/threonine kinase Akt, mediate manifold bioactivities of vitamin A, although the mechanisms behind the sustained kinase activation are diffuse. To investigate the role of cellular lipids as targetable factors in Akt signaling, we combined mass spectrometry-based lipidomics with immunologic detection of Akt (Ser473) phosphorylation. A screening campaign revealed retinol (vitamin A alcohol) and all-trans retinoic acid (vitamin A acid) (RA) as hits that time-dependently (≥24 h) deplete phosphatidylcholine-bound polyunsaturated fatty acids (PUFA-PCs) from NIH-3T3 mouse fibroblasts while inducing Akt activation (EC50 ≈ 0.1-1 µM). Other mitogenic and stress-regulated kinases were hardly affected. Organized in a coregulated phospholipid subcluster, PUFA-PCs compensated for the RA-induced loss of cellular PUFA-PCs and diminished Akt activation when supplemented. The counter-regulation of phospholipids and Akt by RA was mimicked by knockdown of lysophosphatidylcholine acyltransferase-3 or the selective retinoid X receptor (RXR) agonist bexarotene and prevented by the selective RXR antagonist Hx531. Treatment of mice with retinol decreased the tissue ratio of PUFA-PC and enhanced basal Akt activation preferentially in brain, which was attributed to astrocytes in dissociated cortical cultures. Together, our findings show that RA regulates the long-term activation of Akt by changes in the phospholipid composition.-Pein, H., Koeberle, S. C., Voelkel, M., Schneider, F., Rossi, A., Thürmer, M., Loeser, K., Sautebin, L., Morrison, H., Werz, O., Koeberle, A. Vitamin A regulates Akt signaling through the phospholipid fatty acid composition.
Assuntos
Ácidos Graxos/metabolismo , Fosfolipídeos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tretinoína/farmacologia , Vitamina A/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Camundongos , Fosforilação , Receptores X de Retinoides/metabolismo , Tretinoína/metabolismo , Vitamina A/farmacologiaRESUMO
There is huge number of oligomeric proteins that show allosteric behaviour as soon as their allosteric effector is provided. Thermus sp. GH5 methylglyoxal synthase is also a homohexameric protein, which displays cooperative behaviour when phosphate concentration increases. Previous studies on this enzyme have indicated that binding of phosphate leads to formation of specific interactions which makes the enzyme capable of displaying allosteric behaviour when its substrate is bound. In this study, it has been shown that a single mutation, independent of phosphate, provides the requirements for showing such cooperative behaviour. However, it is proposed that the allosteric mechanism triggered by phosphate is different from that applied by the mutation. These findings point towards the fact that allostery can be acquired, modulated or eliminated by any alteration in structure and/or dynamics of the proteins and all proteins are potentially capable of showing cooperative behaviour as soon as their prerequisites for this phenomenon are provided by the allosteric effector.