RESUMO
Disruption of sphingolipid homeostasis is known to cause neurological disorders, but the mechanisms by which specific sphingolipid species modulate pathogenesis remain unclear. The last step of de novo sphingolipid synthesis is the conversion of dihydroceramide to ceramide by dihydroceramide desaturase (human DEGS1; Drosophila Ifc). Loss of ifc leads to dihydroceramide accumulation, oxidative stress, and photoreceptor degeneration, whereas human DEGS1 variants are associated with leukodystrophy and neuropathy. In this work, we demonstrate that DEGS1/ifc regulates Rac1 compartmentalization in neuronal cells and that dihydroceramide alters the association of active Rac1 with organelle-mimicking membranes. We further identify the Rac1-NADPH oxidase (NOX) complex as the major cause of reactive oxygen species (ROS) accumulation in ifc-knockout (ifc-KO) photoreceptors and in SH-SY5Y cells with the leukodystrophy-associated DEGS1H132R variant. Suppression of Rac1-NOX activity rescues degeneration of ifc-KO photoreceptors and ameliorates oxidative stress in DEGS1H132R-carrying cells. Therefore, we conclude that DEGS1/ifc deficiency causes dihydroceramide accumulation, resulting in Rac1 mislocalization and NOX-dependent neurodegeneration.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Ácidos Graxos Dessaturases/genética , Proteínas de Membrana/genética , NADPH Oxidases/genética , Proteínas rac1 de Ligação ao GTP/genética , Animais , Linhagem Celular Tumoral , Ceramidas/metabolismo , Proteínas de Drosophila/deficiência , Drosophila melanogaster/metabolismo , Eletrorretinografia , Ácidos Graxos Dessaturases/antagonistas & inibidores , Ácidos Graxos Dessaturases/metabolismo , Regulação da Expressão Gênica , Humanos , Proteínas de Membrana/deficiência , NADPH Oxidases/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Estresse Oxidativo , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Invertebrados/patologia , Mutação Puntual , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Retina/metabolismo , Retina/patologia , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Proteasome inhibitors, such as bortezomib and carfilzomib, have shown efficacy in anti-cancer therapy in hematological diseases but not in solid cancers. Here, we found that liposarcomas (LPS) are susceptible to proteasome inhibition, and identified drugs that synergize with carfilzomib, such as selinexor, an inhibitor of XPO1-mediated nuclear export. Through quantitative nuclear protein profiling and phospho-kinase arrays, we identified potential mode of actions of this combination, including interference with ribosome biogenesis and inhibition of pro-survival kinase PRAS40. Furthermore, by assessing global protein levels changes, FADS2, a key enzyme regulating fatty acids synthesis, was found down-regulated after proteasome inhibition. Interestingly, SC26196, an inhibitor of FADS2, synergized with carfilzomib. Finally, to identify further combinational options, we performed high-throughput drug screening and uncovered novel drug interactions with carfilzomib. For instance, cyclosporin A, a known immunosuppressive agent, enhanced carfilzomib's efficacy in vitro and in vivo. Altogether, these results demonstrate that carfilzomib and its combinations could be repurposed for LPS clinical management.
Assuntos
Ácidos Graxos Dessaturases/genética , Carioferinas/genética , Lipossarcoma/tratamento farmacológico , Oligopeptídeos/farmacologia , Receptores Citoplasmáticos e Nucleares/genética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Bortezomib/farmacologia , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Ácidos Graxos Dessaturases/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Hidrazinas/farmacologia , Lipossarcoma/genética , Lipossarcoma/patologia , Piperazinas/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Triazóis/farmacologia , Proteína Exportina 1RESUMO
Cellular stress responses serve as crucial decision points balancing persistence or culling of hematopoietic stem cells (HSCs) for lifelong blood production. Although strong stressors cull HSCs, the linkage between stress programs and self-renewal properties that underlie human HSC maintenance remains unknown, particularly at quiescence exit when HSCs must also dynamically shift metabolic state. Here, we demonstrate distinct wiring of the sphingolipidome across the human hematopoietic hierarchy and find that genetic or pharmacologic modulation of the sphingolipid enzyme DEGS1 regulates lineage differentiation. Inhibition of DEGS1 in hematopoietic stem and progenitor cells during the transition from quiescence to cellular activation with N-(4-hydroxyphenyl) retinamide activates coordinated stress pathways that coalesce on endoplasmic reticulum stress and autophagy programs to maintain immunophenotypic and functional HSCs. Thus, our work identifies a linkage between sphingolipid metabolism, proteostatic quality control systems, and HSC self-renewal and provides therapeutic targets for improving HSC-based cellular therapeutics.
Assuntos
Autorrenovação Celular/genética , Ácidos Graxos Dessaturases/antagonistas & inibidores , Fenretinida/farmacologia , Células-Tronco Hematopoéticas/metabolismo , Proteostase/genética , Esfingolipídeos/metabolismo , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Autorrenovação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Feminino , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Células-Tronco Hematopoéticas/enzimologia , Humanos , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos NOD , Proteostase/efeitos dos fármacos , RNA Interferente Pequeno , RNA-Seq , Análise de Célula Única , Esfingolipídeos/química , Transplante HeterólogoRESUMO
Fluctuations in food availability and shifts in temperature are typical environmental changes experienced by animals. These environmental shifts sometimes portend more severe changes; e.g., chilly north winds precede the onset of winter. Such telltale signs may be indicators for animals to prepare for such a shift. Here we show that HEK293A cells, cultured under starvation conditions, can "memorize" a short exposure to cold temperature (15 °C), which was evidenced by their higher survival rate compared to cells continuously grown at 37 °C. We refer to this phenomenon as "cold adaptation". The cold-exposed cells retained high ATP levels, and addition of etomoxir, a fatty acid oxidation inhibitor, abrogated the enhanced cell survival. In our standard protocol, cold adaptation required linoleic acid (LA) supplementation along with the activity of Δ-6-desaturase (D6D), a key enzyme in LA metabolism. Moreover, supplementation with the LA metabolite arachidonic acid (AA), which is a high-affinity agonist of peroxisome proliferator-activated receptor-alpha (PPARα), was able to underpin the cold adaptation, even in the presence of a D6D inhibitor. Cold exposure with added LA or AA prompted a surge in PPARα levels, followed by the induction of D6D expression; addition of a PPARα antagonist or a D6D inhibitor abrogated both their expression, and reduced cell survival to control levels. We also found that the brief cold exposure transiently prevents PPARα degradation by inhibiting the ubiquitin proteasome system, and starvation contributes to the enhancement of PPARα activity by inhibiting mTORC1. Our results reveal an innate adaptive positive-feedback mechanism with a PPARα-D6D-AA axis that is triggered by a brief cold exposure in cells. "Cold adaptation" could have evolved to increase strength and resilience against imminent extreme cold temperatures.
Assuntos
PPAR alfa/metabolismo , Trifosfato de Adenosina/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Temperatura Baixa , Compostos de Epóxi/farmacologia , Ácidos Graxos Dessaturases/antagonistas & inibidores , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Células HEK293 , Humanos , Ácido Linoleico/metabolismo , Ácido Linoleico/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , PPAR alfa/agonistas , PPAR alfa/antagonistas & inibidores , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismoRESUMO
BACKGROUND: We previously demonstrated that knockdown of delta-5-desaturase via siRNA transfection together with dihomo-γ-linolenic acid supplementation inhibited colon cancer cell growth and migration, by promoting the production of the anti-cancer byproduct 8-hydroxyoctanoic acid from Cyclooxygenase-2-catalyzed dihomo-γ-linolenic acid peroxidation. Here, we extend our study to investigate the effects of delta-5-desaturase-knockdown and the resulting intensified dihomo-γ-linolenic acid peroxidation in xenograft tumor mice model. METHODS: Four-week old nude mice bearing the human colon cancer cell HCA-7/C29 vs. its delta-5-desaturase knockdown analog (via shRNA transfection) were subject to 4-week treatments of: vehicle control, dihomo-γ-linolenic acid supplementation, 5-Fluorouracil, and combination of dihomo-γ-linolenic acid and 5-Fluorouracil. Tumor growth was monitored during the treatment. At the endpoint, the mice were euthanized and the tumor tissues were collected for further mechanism analysis. RESULTS: Delta-5-desaturase knockdown (shRNA) together with dihomo-γ-linolenic acid supplementation increased 8-hydroxyoctanoic acid production to a threshold level in xenograft tumors, which consequently induced p53-dependent apoptosis and reduced tumors significantly. The promoted 8-hydroxyoctanoic acid formation was also found to suppress the tumors' metastatic potential via regulating MMP-2 and E-cadherin expressions. In addition, our in vivo data showed that delta-5-desaturase knockdown along with dihomo-γ-linolenic acid supplementation resulted in anti-tumor effects comparable to those of 5-Fluorouracil. CONCLUSIONS: We have demonstrated that our paradigm-shifting strategy of knocking down delta-5-desaturase and taking advantage of overexpressed Cyclooxygenase-2 in tumor cells can be used for colon cancer suppression. Our research outcome will lead us to develop a better and safer anti-cancer therapy for patients.
Assuntos
Ácido 8,11,14-Eicosatrienoico/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias do Colo/tratamento farmacológico , Ácidos Graxos Dessaturases/genética , Animais , Caderinas/genética , Caprilatos/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Ciclo-Oxigenase 2/genética , Dessaturase de Ácido Graxo Delta-5 , Ácidos Graxos Dessaturases/antagonistas & inibidores , Fluoruracila/administração & dosagem , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Metaloproteinase 2 da Matriz/genética , Camundongos , Metástase Neoplásica , RNA Interferente Pequeno/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Many cancers and pre-cancerous lesions convert membrane-bound arachidonic acid (AA) to eicosanoids that promote the survival, growth, and spread of cancer. In contrast, the long-chain omega-3s eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can competitively inhibit AA's interaction with the enzymes that give rise to eicosanoids, while acting as precursors for alternative eicosanoids which oppose cancer development and growth. Hence, minimizing the AA content of cancer membranes, while boosting that of EPA and DHA, is a rational strategy for cancer prevention and control. The former goal can be achieved by eating a plant-based diet (inherently free of AA); by avoiding foods high in linoleic acid; by down-regulating the expression of delta-6-desaturase (D6D), rate-limiting for the conversion of linoleic acid to AA; and by competitively decreasing flux of linoleic acid through D6D with a high intake of alpha-linolenic acid (ALA) from flaxseed. ALA and DHA, potent agonists for the farnesoid X receptor, can be expected to suppress D6D transcription, and AMP-activated kinase (AMPK) activators and a cholesterol-free diet also have potential in this regard. Hence, a plant-based diet low in linoleic acid, complemented by an ample intake of flaxseed and supplemental fish oil, with or without metformin and other D6D-antagonist agents, may aid prevention and control of some cancers.
Assuntos
Ácido Araquidônico/análise , Membrana Celular/química , Neoplasias/prevenção & controle , Ácido Araquidônico/metabolismo , Ciclo-Oxigenase 2/fisiologia , Dieta , Ácidos Graxos Dessaturases/antagonistas & inibidores , Óleos de Peixe/administração & dosagem , Linho , Humanos , Ácido Linoleico/administração & dosagem , Ácido Linoleico/metabolismo , Neoplasias/química , Receptores Citoplasmáticos e Nucleares/fisiologiaRESUMO
The hepatitis C virus (HCV) life cycle is tightly linked to the host cell lipid metabolism with the endoplasmic reticulum-derived membranous web harboring viral RNA replication complexes and lipid droplets as virion assembly sites. To investigate HCV-induced changes in the lipid composition, we performed quantitative shotgun lipidomic studies of whole cell extracts and subcellular compartments. Our results indicate that HCV infection reduces the ratio of neutral to membrane lipids. While the amount of neutral lipids and lipid droplet morphology were unchanged, membrane lipids, especially cholesterol and phospholipids, accumulated in the microsomal fraction in HCV-infected cells. In addition, HCV-infected cells had a higher relative abundance of phosphatidylcholines and triglycerides with longer fatty acyl chains and a strikingly increased utilization of C18 fatty acids, most prominently oleic acid (FA [18:1]). Accordingly, depletion of fatty acid elongases and desaturases impaired HCV replication. Moreover, the analysis of free fatty acids revealed increased levels of polyunsaturated fatty acids (PUFAs) caused by HCV infection. Interestingly, inhibition of the PUFA synthesis pathway via knockdown of the rate-limiting Δ6-desaturase enzyme or by treatment with a high dose of a small-molecule inhibitor impaired viral progeny production, indicating that elevated PUFAs are needed for virion morphogenesis. In contrast, pretreatment with low inhibitor concentrations promoted HCV translation and/or early RNA replication. Taken together our results demonstrate the complex remodeling of the host cell lipid metabolism induced by HCV to enhance both virus replication and progeny production.
Assuntos
Hepacivirus/metabolismo , Hepatócitos/metabolismo , Interações Hospedeiro-Patógeno , Metabolismo dos Lipídeos/genética , Metaboloma , Vírion/metabolismo , Replicação Viral/fisiologia , Acetiltransferases/antagonistas & inibidores , Acetiltransferases/genética , Acetiltransferases/metabolismo , Linhagem Celular Tumoral , Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Ácidos Graxos Dessaturases/antagonistas & inibidores , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Elongases de Ácidos Graxos , Ácidos Graxos Insaturados/metabolismo , Regulação da Expressão Gênica , Hepacivirus/crescimento & desenvolvimento , Hepatócitos/química , Hepatócitos/virologia , Humanos , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/virologia , Microssomos/metabolismo , Microssomos/virologia , Ácido Oleico/metabolismo , Fosfatidilcolinas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Viral/biossíntese , RNA Viral/genética , Triglicerídeos/metabolismo , Vírion/crescimento & desenvolvimento , Montagem de Vírus/fisiologiaRESUMO
Polyunsaturated fatty acids (PUFAs) are important for immune function. Limited evidence indicates that immune cell activation involves endogenous PUFA synthesis, but this has not been characterised. To address this, we measured metabolism of 18:3n-3 in quiescent and activated peripheral blood mononuclear cells (PBMCs), and in Jurkat T cell leukaemia. PBMCs from men and women (n = 34) were incubated with [1-13C]18:3n-3 with or without Concanavalin A (Con. A). 18:3n-3 conversion was undetectable in unstimulated PBMCs, but up-regulated when stimulated. The main products were 20:3n-3 and 20:4n-3, while 18:4n-3 was undetectable, suggesting initial elongation and Δ8 desaturation. PUFA synthesis was 17.4-fold greater in Jurkat cells than PBMCs. The major products of 18:3n-3 conversion in Jurkat cells were 20:4n-3, 20:5n-3, and 22:5n-3. 13C Enrichment of 18:4n-3 and 20:3n-3 suggests parallel initial elongation and Δ6 desaturation. The FADS2 inhibitor SC26196 reduced PBMC, but not Jurkat cell, proliferation suggesting PUFA synthesis is involved in regulating mitosis in PBMCs. Con. A stimulation increased FADS2, FADS1, ELOVL5 and ELOVL4 mRNA expression in PBMCs. A single transcript corresponding to the major isoform of FADS2, FADS20001, was detected in PBMCs and Jurkat cells. PBMC activation induced hypermethylation of a 470bp region in the FADS2 5'-regulatory sequence. This region was hypomethylated in Jurkat cells compared to quiescent PBMCs. These findings show that PUFA synthesis involving initial elongation and Δ8 desaturation is involved in regulating PBMC proliferation and is regulated via transcription possibly by altered DNA methylation. These processes were dysregulated in Jurkat cells. This has implications for understanding the regulation of mitosis in normal and transformed lymphocytes.
Assuntos
DNA/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Insaturados/metabolismo , Leucemia/metabolismo , Leucócitos Mononucleares/fisiologia , Proliferação de Células , Senescência Celular , Metilação de DNA , Dessaturase de Ácido Graxo Delta-5 , Ácidos Graxos Dessaturases/antagonistas & inibidores , Ácidos Graxos Dessaturases/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Células Jurkat , Ativação Linfocitária , Piperazinas/farmacologia , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
The aim of our study was to compare the influence of diet supplementation with pomegranate seed oil - as conjugated linolenic acids (CLnA) source, or conjugated linoleic acids (CLA) and to examine the mechanism of their activity. The content of fatty acids, levels of biomarkers of lipids' oxidation and the activity of key enzymes catalyzing lipids metabolism were measured. Obtained results revealed that conjugated fatty acids significantly decrease the activity of Δ5-desaturase (p=0.0001) and Δ6-desaturase (p=0.0008) and pomegranate seed oil reduces their activity in the most potent way. We confirmed that diet supplementation with pomegranate seed oil - a rich source of punicic acid leads to the increase of cis-9, trans-11 CLA content in livers (p=0.0003). Lack of side effects and beneficial influence on desaturases activity and fatty acids profile claim pomegranate seed oil to become interesting alternative for CLA as functional food.
Assuntos
Ácidos Graxos Dessaturases/antagonistas & inibidores , Ácidos Graxos/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Lythraceae/química , Óleos de Plantas/farmacologia , Sementes/química , Animais , Feminino , Fígado/enzimologia , Oxirredução/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Dihydroceramide desaturase 1 (Des1) catalyzes the last step of ceramide synthesis de novo, thus regulating the physiologically relevant balance between dihydrosphingolipids and sphingolipids. Here we report on the configurational preference of Des1 towards isomeric Δ6-unsaturated dihydroceramide analogs and the discovery of a potent Des1 inhibitor.
Assuntos
Ceramidas/farmacologia , Ácidos Graxos Dessaturases/antagonistas & inibidores , Linhagem Celular Tumoral , Ceramidas/síntese química , Ceramidas/química , Química Click , Ensaios Enzimáticos , Ácidos Graxos Dessaturases/química , Humanos , Isomerismo , Cinética , Especificidade por SubstratoRESUMO
Obesity is now recognized as a state of chronic low-grade inflammation and is called as metabolic inflammation. Delta-5 desaturase (D5D) is an enzyme that metabolizes dihomo-γ-linolenic acid (DGLA) to arachidonic acid (AA). Thus, D5D inhibition increases DGLA (precursor to anti-inflammatory eicosanoids) while decreasing AA (precursor to pro-inflammatory eicosanoids), and could result in synergistic improvement in the low-grade inflammatory state. Here, we demonstrate reduced insulin resistance and the anti-obesity effect of a D5D selective inhibitor (compound-326), an orally active small-molecule, in a high-fat diet-induced obese (DIO) mouse model. In vivo D5D inhibition was confirmed by determining changes in blood AA/DGLA profiles. In DIO mice, chronic treatment with compound-326 lowered insulin resistance and caused body weight loss without significant impact on cumulative calorie intake. Decreased macrophage infiltration into adipose tissue was expected from mRNA analysis. Increased daily energy expenditure was also observed following administration of compound-326, in line with sustained body weight loss. These data indicate that the novel D5D selective inhibitor, compound-326, will be a new class of drug for the treatment of obese and diabetic patients.
Assuntos
Peso Corporal/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Ácidos Graxos Dessaturases/antagonistas & inibidores , Resistência à Insulina , Obesidade/prevenção & controle , Pirimidinonas/farmacologia , Pirrolidinonas/farmacologia , Ácido 8,11,14-Eicosatrienoico/sangue , Ácido 8,11,14-Eicosatrienoico/metabolismo , Adiponectina/genética , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Ácido Araquidônico/sangue , Ácido Araquidônico/metabolismo , Dessaturase de Ácido Graxo Delta-5 , Dieta Hiperlipídica/efeitos adversos , Metabolismo Energético/efeitos dos fármacos , Ácidos Graxos Dessaturases/metabolismo , Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/prevenção & controle , Leptina/genética , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Obesidade/fisiopatologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Redução de Peso/efeitos dos fármacosRESUMO
Cyclooxygenase (COX), commonly overexpressed in cancer cells, is a major lipid peroxidizing enzyme that metabolizes polyunsaturated fatty acids (ω-3s and ω-6s). The COX-catalyzed free radical peroxidation of arachidonic acid (ω-6) can produce deleterious metabolites (e.g. 2-series prostaglandins) that are implicated in cancer development. Thus, COX inhibition has been intensively investigated as a complementary therapeutic strategy for cancer. However, our previous study has demonstrated that a free radical-derived byproduct (8-hydroxyoctanoic acid) formed from COX-catalyzed peroxidation of dihomo-γ-linolenic acid (DGLA, the precursor of arachidonic acid) can inhibit colon cancer cell growth. We thus hypothesize that the commonly overexpressed COX in cancer (~90% of colon cancer patients) can be taken advantage to suppress cell growth by knocking down delta-5-desaturase (D5D, a key enzyme that converts DGLA to arachidonic acid). In addition, D5D knockdown along with DGLA supplement may enhance the efficacy of chemotherapeutic drugs. After knocking down D5D in HCA-7 colony 29 cells and HT-29 cells (human colon cancer cell lines with high and low COX levels, respectively), the antitumor activity of DGLA was significantly enhanced along with the formation of a threshold range (~0.5-1.0µM) of 8-hydroxyoctanoic acid. In contrast, DGLA treatment did not inhibit cell growth when D5D was not knocked down and only limited amount of 8-hydroxyoctanoic acid was formed. D5D knockdown along with DGLA treatment also enhanced the cytotoxicities of various chemotherapeutic drugs, including 5-fluorouracil, regorafenib, and irinotecan, potentially through the activation of pro-apoptotic proteins, e.g. p53 and caspase 9. For the first time, we have demonstrated that the overexpressed COX in cancer cells can be utilized in suppressing cancer cell growth. This finding may provide a new option besides COX inhibition to optimize cancer therapy. The outcome of this translational research will guide us to develop a novel ω-6-based diet-care strategy in combination with current chemotherapy for colon cancer prevention and treatment.
Assuntos
Ácido 8,11,14-Eicosatrienoico/administração & dosagem , Neoplasias do Colo/tratamento farmacológico , Ciclo-Oxigenase 2/genética , Ácidos Graxos Dessaturases/genética , Ácido 8,11,14-Eicosatrienoico/metabolismo , Ácido Araquidônico/metabolismo , Caprilatos/metabolismo , Caspase 9/genética , Proliferação de Células/genética , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Dessaturase de Ácido Graxo Delta-5 , Ácidos Graxos Dessaturases/antagonistas & inibidores , Ácidos Graxos Ômega-6/metabolismo , Fluoruracila/administração & dosagem , Radicais Livres/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HT29 , Humanos , Proteína Supressora de Tumor p53/genéticaRESUMO
Cystic fibrosis patients and model systems exhibit consistent abnormalities in metabolism of polyunsaturated fatty acids that appear to play a role in disease pathophysiology. Recent in vitro studies have suggested that these changes are due to overexpression of fatty acid desaturases that can be reversed by supplementation with the long-chain polyunsaturated fatty acids docosahexaenoate and eicosapentaenoate. However, these findings have not been tested in vivo. The current study aimed to test these results in an in vivo model system, the CFTR(-/-) knockout mouse. When compared with wild-type mice, the knockout mice exhibited fatty acid abnormalities similar to those seen in cystic fibrosis patients and other model systems. The abnormalities were confined to lung, ileum and pancreas, tissues that are affected by the disease. Similar to in vitro models, these fatty acid changes correlated with increased expression of Δ5- and Δ6-desaturases and elongase 5. Dietary supplementation with high-dose free docosahexaenoate or a combination of lower-dose docosahexaenoate and eicosapentaenoate in triglyceride form corrected the fatty acid abnormalities and reduced expression of the desaturase and elongase genes in the ileum and liver of knockout mice. Only the high-dose docosahexaenoate reduced histologic evidence of disease, reducing mucus accumulation in ileal sections. These results provide in vivo support for the hypothesis that fatty acid abnormalities in cystic fibrosis result from abnormal expression and activity of metabolic enzymes in affected cell types. They further demonstrate that these changes can be reversed by dietary n-3 fatty acid supplementation, highlighting the potential therapeutic benefit for cystic fibrosis patients.
Assuntos
Fibrose Cística/tratamento farmacológico , Suplementos Nutricionais , Ácidos Docosa-Hexaenoicos/administração & dosagem , Ácido Eicosapentaenoico/administração & dosagem , Ácidos Graxos Dessaturases/antagonistas & inibidores , Animais , Relação Dose-Resposta a Droga , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Íleo/efeitos dos fármacos , Íleo/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos CFTR , Camundongos Knockout , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismoRESUMO
The aim of this study was to assess the influence of diet supplementation of pregnant and breast-feeding female Sprague-Dawley rats with conjugated linoleic acids (CLA) on the Δ6- and Δ5-desaturase activity in hepatic microsomes as well as on fatty acids profile and lipids peroxidation in liver and hepatic microsomes of the progeny with chemically induced mammary tumors. Rats were divided into two groups with different diet supplementation (vegetable oil (which did not contain CLA) or CLA). Their female offspring was divided within these groups into two subgroups: (1)--fed the same diet as mothers (K1 - oil, 01 - CLA), and (2)--fed the standard fodder (K2, O2). At 50th day of life, the progeny obtained carcinogenic agent (7,12-dimethylbenz[a]anthracene). Higher supply of CLA in diet of mothers resulted in lower susceptibility to chemically induced mammary tumors in their offspring (p = 0.0322). It also influenced the fatty acids profile in livers and in hepatic microsomes, especially polyunsaturated n3 and n6 fatty acids. CLA inhibited the activity of the desaturases, which confirmed that CLA can reduce the level of arachidonic acid directly, reducing linoleic acid content in membranes, or indirectly, through the regulation of its metabolism. We were unable to confirm or deny the antioxidative properties of CLA. Our results indicate that the higher supply of CLA in mothers' diet during pregnancy and breastfeeding causes their incorporation into tissues of children, changes the efficiency of fatty acids metabolism and exerts health-promoting effect in their adult life reducing the breast cancer risk.
Assuntos
9,10-Dimetil-1,2-benzantraceno , Fenômenos Fisiológicos da Nutrição Animal , Anticarcinógenos/administração & dosagem , Neoplasias da Mama/prevenção & controle , Suplementos Nutricionais , Inibidores Enzimáticos/administração & dosagem , Ácidos Graxos Dessaturases/antagonistas & inibidores , Ácidos Linoleicos Conjugados/administração & dosagem , Fígado/efeitos dos fármacos , Fenômenos Fisiológicos da Nutrição Materna , Animais , Neoplasias da Mama/enzimologia , Dessaturase de Ácido Graxo Delta-5 , Modelos Animais de Doenças , Ácidos Graxos Dessaturases/metabolismo , Feminino , Lactação , Linoleoil-CoA Desaturase/antagonistas & inibidores , Linoleoil-CoA Desaturase/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/enzimologia , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
Membrane biomechanical properties are critical in modulating nutrient and metabolite exchange as well as signal transduction. Biological membranes are predominantly composed of lipids, cholesterol and proteins, and their fluidity is tightly regulated by cholesterol and lipid desaturases. To determine whether such membrane fluidity regulation occurred in mammalian cells under pressure, we investigated the effects of pressure on membrane lipid order of mouse chondrogenic ATDC5 cells and desaturase gene expression. Hydrostatic pressure linearly increased membrane lipid packing and simultaneously repressed lipid desaturase gene expression. We also showed that cholesterol mimicked and cholesterol depletion reversed those effects, suggesting that desaturase gene expression was controlled by the membrane physical state itself. This study demonstrates a new effect of hydrostatic pressure on mammalian cells and may help to identify the molecular mechanisms involved in hydrostatic pressure sensing in chondrocytes.
Assuntos
Condrócitos/enzimologia , Ácidos Graxos Dessaturases/antagonistas & inibidores , Ácidos Graxos Dessaturases/genética , Fluidez de Membrana/fisiologia , Lipídeos de Membrana/antagonistas & inibidores , Células-Tronco/enzimologia , Animais , Linhagem Celular Tumoral , Colesterol/biossíntese , Colesterol/deficiência , Colesterol/metabolismo , Condrócitos/patologia , Regulação para Baixo/genética , Ácidos Graxos Dessaturases/biossíntese , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Pressão Hidrostática , Lipídeos de Membrana/genética , Camundongos , Células-Tronco/patologia , Regulação para Cima/genéticaRESUMO
A multiplexed cell assay has been optimized to measure the activities of fatty acyl-CoA elongase, delta-5 desaturase (Delta5D), delta-6 desaturase (Delta6D), and delta-9 desaturase (Delta9D) together using (14)C-labeled tracers in HepG2 cells, which express the human stearoyl-CoA desaturase-1 isoform (SCD1) exclusively. The Delta5 and Delta9 desaturase activities are indexed by the efficient conversion of [1-(14)C]-eicosatrienoic acid (C20:3, cis-8,11,14) to (14)C-arachidonic acid (C20:4, cis-5,8,11,14) and the conversion of [1-(14)C]-stearic acid to (14)C-oleic acid (C18:1, cis-9), respectively. CP-74006 potently blocks the Delta5D activity with an IC(50) value of 20 nM and simplifies the metabolism of [1-(14)C]-alpha-linolenate (C18:3, cis-9,12,15) by accumulating (14)C-eicosatetraenoic acid (C20:4, cis-8,11,14,17) as the major (14)C-eicosatrienoic acid (C20:3, cis-11,14,17) and (14)C-docosatetraenoic acid (C22:4, cis-10,13,16,19) as the minor metabolites through Delta6 desaturation and elongation. This simplified metabolite spectrum enables the delineation of the Delta6D activity by comparing the combined Delta6D/elongase activity index of the (14)C-(C20:4/C18:3) ratio with the corresponding elongation index of the (14)C-(C20:3/C18:3) ratio following compound treatment. SC-26196 and sterculic acid specifically inhibit the Delta6D and Delta9D activities with an IC(50) value of 0.1 microM and 0.9 microM, respectively. This medium-throughput cell assay provides an efficient tool in the identification of specific desaturase and elongase inhibitors.
Assuntos
Acetiltransferases/antagonistas & inibidores , Acil Coenzima A/antagonistas & inibidores , Bioensaio , Ácidos Graxos Dessaturases/antagonistas & inibidores , Linoleoil-CoA Desaturase/antagonistas & inibidores , Acetiltransferases/química , Acetiltransferases/metabolismo , Acil Coenzima A/química , Acil Coenzima A/metabolismo , Radioisótopos de Carbono , Dessaturase de Ácido Graxo Delta-5 , Ácidos Graxos Dessaturases/química , Ácidos Graxos Dessaturases/metabolismo , Elongases de Ácidos Graxos , Células Hep G2 , Humanos , Concentração Inibidora 50 , Cinética , Linoleoil-CoA Desaturase/química , Linoleoil-CoA Desaturase/metabolismo , Modelos Biológicos , Modelos QuímicosRESUMO
Relative to carbon, hydrogen, nitrogen and oxygen, very little is currently known about boron in therapeutics. In addition, there are very few boron-containing natural products identified to date to serve as leads for medicinal chemists. Perceived risks of using boron and lack of synthetic methods to handle boron-containing compounds have caused the medicinal chemistry community to shy away from using the atom. However, physical, chemical and biological properties of boron offer medicinal chemists a rare opportunity to explore and pioneer new areas of drug discovery. Boron therapeutics are emerging that show different modes of inhibition against a variety of biological targets. With one boron-containing therapeutic agent on the market and several more in various stages of clinical trials, the occurrence of this class of compound is likely to grow over the next decade and boron could become widely accepted as a useful element in future drug discovery.
Assuntos
Compostos de Boro/química , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Aminoacil-tRNA Sintetases/metabolismo , Compostos de Boro/farmacologia , Ácidos Borônicos/química , Ácidos Borônicos/farmacologia , Ácidos Borônicos/uso terapêutico , Ensaios Clínicos como Assunto , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Ácidos Graxos Dessaturases/antagonistas & inibidores , Ácidos Graxos Dessaturases/metabolismo , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/enzimologia , Hepatite C/tratamento farmacológico , Humanos , Serina Proteases/química , Serina Proteases/metabolismo , Inibidores de beta-Lactamases , beta-Lactamases/metabolismoRESUMO
All higher plants contain at least one microsomal omega-6 desaturase (FAD2), which inserts a double bond between the carbons 12 and 13 of monounsaturated oleic acid to generate polyunsaturated linoleic acid and controls most of the polyunsaturated lipid synthesis in plant cells. RNA interference can be used to silence endogenous genes by effective degradation of target transcripts. To investigate development-related silencing of the FAD2, fatty acid composition was analyzed in the context of leaf expansion in FAD2-silenced tobacco lines obtained by RNA interference technology. We observed that the increased oleate level in unexpanded leaves due to FAD2-silencing receded significantly in fully expanded leaves. The mechanism involved in this interesting phenomenon was investigated by analyses of individual lipid proportion, fatty acid composition of individual lipids, and FAD2 transcript level in the transgenic leaves at different expansion stages. Data revealed that the expansion-related FAD2-silencing effect was not due to rebound of FAD2 transcript, but rather probably due to chloroplast development with leaf expansion.
Assuntos
Ácidos Graxos Dessaturases/antagonistas & inibidores , Microssomos/metabolismo , Nicotiana/genética , Ácido Oleico/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/metabolismo , Interferência de RNA , Ácidos Graxos Dessaturases/genética , Metabolismo dos Lipídeos , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , RNA Mensageiro/metabolismo , Sementes/metabolismo , Nicotiana/crescimento & desenvolvimento , Transformação GenéticaRESUMO
The influence of cytoskeleton integrity on the metabolism of saturated and unsaturated FA was studied in surface cultures and cell suspensions of human Hep G2 hepatoma cells. We found that colchicine (COL), nocodazol, and vinblastin produced a significant inhibition in the incorporation of labeled saturated FA, whereas incorporation of the unsaturated FA remained unaltered. These microtubule-disrupting drugs also diminished Delta9-, Delta5-, and Delta6-desaturase capacities. The effects produced by COL were dose (0-50 microM) and time (0-300 min) dependent, and were antagonized by stabilizing agents (phalloidin and DMSO). Dihydrocytochalasin B (20 microM) was tested as a microfilament-disrupting drug and produced no changes in either the incorporation of [14C] FA or the desaturase conversion of the substrates. We hypothesized that the interactions between cytoskeleton and membrane proteins such as FA desaturases may explain the functional organization, facilitating both substrate channeling and regulation of unsaturated FA biosynthesis.
Assuntos
Ácidos Graxos Insaturados/metabolismo , Ácidos Graxos/metabolismo , Microtúbulos/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Colchicina/farmacologia , Citocalasina B/análogos & derivados , Citocalasina B/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Dimetil Sulfóxido/farmacologia , Relação Dose-Resposta a Droga , Ácidos Graxos Dessaturases/antagonistas & inibidores , Ácidos Graxos Dessaturases/metabolismo , Humanos , Nocodazol/farmacologia , Tubulina (Proteína)/metabolismo , Vimblastina/farmacologiaRESUMO
Maternal smoking during pregnancy has been associated with a reduced content of n-3 long-chain PUFA (LC-PUFA) in breast milk, thereby reducing the intake of key nutrients by the infants. We postulated that the mammary gland is affected by maternal smoking in the process of n-3 LC-PUFA secretion into milk. This prompted us to investigate the effects of cigarette smoke on the synthesis of n-3 LC-PUFA in vitro by using a line of healthy epithelial cells from the human mammary gland, MCF-10A. Cells were exposed to cigarette smoke under controlled conditions by adding to the medium aliquots of horse serum containing smoke components, as analyzed by GC-MS. The major findings concern the inhibition of both the conversion of the precursor 14C-ALA (alpha-linolenic acid) to n-3 LC-PUFA and of the A5 desaturation step (assessed by HPLC analysis with radiodetection of n-3 FAME) following exposure to minimal doses of smoke-enriched serum, and the dose-dependent relationship of these effects. The data indicate that exposure to cigarette smoke negatively affects the synthesis of n-3 LC-PUFA from the precursor in mammary gland cells.