RESUMO
BACKGROUND AND AIMS: Aberrant activation of fatty acid synthase (FASN) is a major metabolic event during the development of HCC. We evaluated the therapeutic efficacy of TVB3664, a FASN inhibitor, either alone or in combination, for HCC treatment. APPROACH AND RESULTS: The therapeutic efficacy and the molecular pathways targeted by TVB3664, either alone or with tyrosine kinase inhibitors or the checkpoint inhibitor anti-programmed death ligand 1 antibody, were assessed in human HCC cell lines and multiple oncogene-driven HCC mouse models. RNA sequencing was performed to elucidate the effects of TVB3664 on global gene expression and tumor metabolism. TVB3664 significantly ameliorated the fatty liver phenotype in the aged mice and AKT-induced hepatic steatosis. TVB3664 monotherapy showed moderate efficacy in NASH-related murine HCCs, induced by loss of phosphatase and tensin homolog and MET proto-oncogene, receptor tyrosine kinase (c-MET) overexpression. TVB3664, in combination with cabozantinib, triggered tumor regression in this murine model but did not improve the responsiveness to immunotherapy. Global gene expression revealed that TVB3664 predominantly modulated metabolic processes, whereas TVB3664 synergized with cabozantinib to down-regulate multiple cancer-related pathways, especially the AKT/mammalian target of rapamycin pathway and cell proliferation genes. TVB3664 also improved the therapeutic efficacy of sorafenib and cabozantinib in the FASN-dependent c-MYC-driven HCC model. However, TVB3664 had no efficacy nor synergistic effects in FASN-independent murine HCC models. CONCLUSIONS: This preclinical study suggests the limited efficacy of targeting FASN as monotherapy for HCC treatment. However, FASN inhibitors could be combined with other drugs for improved effectiveness. These combination therapies could be developed based on the driver oncogenes, supporting precision medicine approaches for HCC treatment.
Assuntos
Carcinoma Hepatocelular , Ácido Graxo Sintase Tipo I , Neoplasias Hepáticas , Anilidas , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Ácido Graxo Sintase Tipo I/antagonistas & inibidores , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Ácido Graxo Sintases/antagonistas & inibidores , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Mamíferos/metabolismo , Camundongos , Monoéster Fosfórico Hidrolases/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Piridinas , Sorafenibe/farmacologia , Serina-Treonina Quinases TOR , TensinasRESUMO
Altered metabolism of fatty acid synthesis is considered a hallmark characteristic of several malignancies, including acute lymphoblastic leukemia (ALL). To evaluate the impact of fatty acid synthase (FASN) on drug resistant ALL, bone marrow samples were collected from 65 pediatric ALLs, including 40 de novo and 25 relapsed patients. 22 non-cancer individuals were chosen as controls. Quantitative RT-PCR showed increased expression levels of FASN in drug resistant patients compared with the therapy responders. Single and combined treatment of malignant cells were analyzed using Annexin-V/PI double staining and MTT assays. Incubation of resistant primary cells with ginger showed simultaneous increased apoptosis rates and reduced FASN expression levels. Furthermore, docking studies demonstrated high affinity bindings between ginger derivatives and FASN thioesterase and ketosynthase domains, compared with their known inhibitors, fenofibrate and morin, respectively. Finally, combined treatment of in-house multidrug resistant T-ALL subline with ginger and dexamethasone induced drug sensitivity and down regulation of FASN expression, accordingly. To the best of our knowledge, this is the first study that introduces FASN upregulation as a poor prognostic factor for drug resistant childhood ALL. Moreover, it was revealed that FASN inhibition may be applied by ginger phytochemicals and overcome dexamethasone resistance, subsequently.
Assuntos
Ácido Graxo Sintase Tipo I/antagonistas & inibidores , Terapia de Alvo Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Extratos Vegetais/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Zingiber officinale/química , Apoptose/efeitos dos fármacos , Medula Óssea/enzimologia , Estudos de Casos e Controles , Criança , Dexametasona/farmacologia , Dexametasona/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Feminino , Fenofibrato/farmacologia , Flavonoides/farmacologia , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Modelos Moleculares , Simulação de Acoplamento Molecular , Extratos Vegetais/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimologia , Prognóstico , Conformação Proteica , Domínios Proteicos , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Células Tumorais CultivadasRESUMO
Human cancers require fatty acid synthase (FASN)-dependent de novo long-chain fatty acid synthesis for proliferation. FASN is therefore an attractive drug target, but fast technologies for reliable label-free cellular compound profiling are lacking. Recently, MALDI-mass spectrometry (MALDI-MS) has emerged as an effective technology for discovery of recombinant protein target inhibitors. Here we present an automated, mechanistic MALDI-MS cell assay, which monitors accumulation of the FASN substrate, malonyl-coenzyme A (CoA), in whole cells with limited sample preparation. Profiling of inhibitors, including unpublished compounds, identified compound 1 as the most potent FASN inhibitor (1 nM in A549 cells) discovered to date. Moreover, cellular MALDI-MS assays enable parallel profiling of additional pathway metabolites. Surprisingly, several compounds triggered cytidine 5'-diphosphocholine (CDP-choline) but not malonyl-CoA accumulation indicating that they inhibit diacylglycerol generation but not FASN activity. Taken together, our study suggests that MALDI-MS cell assays may become important tools in drug profiling that provide additional mechanistic insights concerning compound action on metabolic pathways.
Assuntos
Ácido Graxo Sintases/antagonistas & inibidores , Ácido Graxo Sintases/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Células A549 , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Ácido Graxo Sintase Tipo I/antagonistas & inibidores , Ácido Graxo Sintase Tipo I/metabolismo , Humanos , Concentração Inibidora 50 , Células K562 , Lipogênese , Malonil Coenzima A/metabolismo , Estudo de Prova de ConceitoRESUMO
PURPOSE: DNA methylation is one of the most extensively studied mechanisms within epigenetics, and it is suggested that diet-induced changes in methylation status could be involved in energy metabolism regulation. Conjugated linoleic acid (CLA) and calcium supplementation counteract body weight gain, particularly under a high-fat (HF) diet, in adult mice. The aim was to determine whether the modulation of DNA methylation pattern in target genes and tissues could be an underlying mechanism of action. METHODS: Mice (C57BL/6J) were divided into five groups according to diet and treatment: normal fat as the control group (12 % kJ content as fat), HF group (43 % kJ content as fat), HF + CLA (6 mg CLA/day), HF + calcium (12 g/kg of calcium) and HF with both compounds. Gene expression and methylation degree of CpG sites in promoter sequences of genes involved in fatty acid metabolism, including adiponectin (Adipoq), stearoyl-CoA desaturase (Scd1) and fatty acid synthase (Fasn), were determined by bisulphite sequencing in liver and epididymal white adipose tissue. RESULTS: Results showed that the methylation profile of promoters was significantly altered by dietary supplementation in a gene- and tissue-specific manner, whereas only slight changes were observed in the HF group. Furthermore, changes in specific CpG sites were also associated with an overall healthier metabolic profile, in particular for calcium-receiving groups. CONCLUSIONS: Both CLA and calcium were able to modify the methylation pattern of genes involved in energy balance in adulthood, which opens a novel area for increasing efficiency in body weight management strategies.
Assuntos
Fármacos Antiobesidade/uso terapêutico , Cálcio da Dieta/uso terapêutico , Metilação de DNA , Suplementos Nutricionais , Epigênese Genética , Ácidos Linoleicos Conjugados/uso terapêutico , Obesidade/prevenção & controle , Adiponectina/antagonistas & inibidores , Adiponectina/genética , Adiponectina/metabolismo , Tecido Adiposo Branco/enzimologia , Tecido Adiposo Branco/metabolismo , Animais , Ilhas de CpG , Dieta Hiperlipídica/efeitos adversos , Metabolismo Energético , Ácido Graxo Sintase Tipo I/antagonistas & inibidores , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Obesidade/metabolismo , Especificidade de Órgãos , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Estearoil-CoA Dessaturase/química , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Aumento de PesoRESUMO
AIM: To investigate long-term effects of Garcinia Cambogia (GC), weight-loss supplement, on adiposity and non-alcoholic fatty liver disease in obese mice. METHODS: Obesity-prone C57BL/6J mice were fed a high-fat diet (HFD, 45 kcal% fat) with or without GC (1%, w/w) for 16 wk. The HFD contained 45 kcal% fat, 20 kcal% protein and 35 kcal% carbohydrate. They were given free access to food and distilled water, and food consumption and body weight were measured daily and weekly, respectively. Data were expressed as the mean ± SE. Statistical analyses were performed using the statistical package for the social science software program. Student's t test was used to assess the differences between the groups. Statistical significance was considered at P < 0.05. RESULTS: There were no significant changes in body weight and food intake between the groups. However, the supplementation of GC significantly lowered visceral fat accumulation and adipocyte size via inhibition of fatty acid synthase activity and its mRNA expression in visceral adipose tissue, along with enhanced enzymatic activity and gene expression involved in adipose fatty acid ß-oxidation. Moreover, GC supplementation resulted in significant reductions in glucose intolerance and the plasma resistin level in the HFD-fed mice. However, we first demonstrated that it increased hepatic collagen accumulation, lipid peroxidation and mRNA levels of genes related to oxidative stress (superoxide dismutase and glutathione peroxidase) and inflammatory responses (tumor necrosis factor-α and monocyte chemoattractant protein-1) as well as plasma alanine transaminase and aspartate transaminase levels, although HFD-induced hepatic steatosis was not altered. CONCLUSION: GC protects against HFD-induced obesity by modulating adipose fatty acid synthesis and ß-oxidation but induces hepatic fibrosis, inflammation and oxidative stress.
Assuntos
Adiposidade/efeitos dos fármacos , Fármacos Antiobesidade/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Fígado Gorduroso/induzido quimicamente , Garcinia cambogia , Gordura Intra-Abdominal/efeitos dos fármacos , Cirrose Hepática Experimental/induzido quimicamente , Fígado/efeitos dos fármacos , Obesidade/tratamento farmacológico , Extratos Vegetais/toxicidade , Animais , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/sangue , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Colágeno/metabolismo , Citocinas/sangue , Dieta Hiperlipídica , Modelos Animais de Doenças , Ácido Graxo Sintase Tipo I/antagonistas & inibidores , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Fígado Gorduroso/sangue , Fígado Gorduroso/genética , Fígado Gorduroso/imunologia , Fígado Gorduroso/patologia , Regulação da Expressão Gênica , Intolerância à Glucose/sangue , Intolerância à Glucose/tratamento farmacológico , Intolerância à Glucose/etiologia , Mediadores da Inflamação/sangue , Insulina/sangue , Gordura Intra-Abdominal/metabolismo , Gordura Intra-Abdominal/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática Experimental/sangue , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/imunologia , Cirrose Hepática Experimental/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Hepatopatia Gordurosa não Alcoólica , Obesidade/sangue , Obesidade/etiologia , Obesidade/genética , Obesidade/imunologia , Obesidade/patologia , Estresse Oxidativo/efeitos dos fármacos , Fitoterapia , Plantas Medicinais , RNA Mensageiro/metabolismo , Resistina/sangue , Fatores de TempoRESUMO
Pristimerin is a naturally occurring triterpenoid that causes cytotoxicity in several cancer cell lines. However, the mechanism of action for the cytotoxic effect of pristimerin has not been unexplored. The purpose of this study was to investigate the effect of pristimerin on cytotoxicity using the epidermal growth factor receptor 2 (HER2)-positive SKBR3 human breast cancer cell line. Pristimerin inhibited proliferation in dose- and time-dependent manners in cells. We found it to be effective for suppressing HER2 protein and mRNA expression. Fatty acid synthase (FASN) expression and FASN activity were downregulated by pristimerin. Adding of exogenous palmitate, the end product of de novo fatty acid synthesis, reduced the proliferation activity of pristimerin. The changes in HER2 and FASN expression induced by pristimerin altered the levels of Akt and mitogen-activated protein kinase (MAPK) phosphorylation (Erk1/2, p38, and c-Jun N-terminal kinase (JNK)). Pristimerin lowered the levels of phosphorylated mammalian target of rapamycin (mTOR) and its downstream targets such as phosphoprotein 70 ribosomal protein S6 kinase and 4E binding protein1. Pristimerin inhibited migration and invasion of cells, and co-treatment with the mTOR inhibitor rapamycin additionally suppressed these activities. Pristimerin-induced apoptosis was evaluated using Western blotting for caspase-3, -8, -9, and poly (ADP-ribose) polymerase expression and flow cytometric analysis for propidium iodide labeling. These results suggest that pristimerin is a novel HER2-downregulated compound that is able to decrease fatty acid synthase and modulate the Akt, MAPK, and mTOR signaling pathways to influence metastasis and apoptosis. Pristimerin may be further evaluated as a chemotherapeutic agent for HER2-positive breast cancers.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Receptor ErbB-2/metabolismo , Triterpenos/farmacologia , Antineoplásicos Fitogênicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ácido Graxo Sintase Tipo I/antagonistas & inibidores , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Triterpenos Pentacíclicos , Extratos Vegetais , Raízes de Plantas , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Mensageiro/metabolismo , Receptor ErbB-2/genética , Salacia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Triterpenos/isolamento & purificação , CicatrizaçãoRESUMO
AIM: To study, in intact male transgenic mice, the effects of three diets based on olive oil and olive oil diet supplemented with lovastatin and orlistat on hepatic lipogenic enzymes expression, considered markers of cell proliferation. METHODS: Forty Apc(Min/+) mice were randomly divided into 4 groups and fed for 10 wk: olive oil (OO) group, n = 10 animals received a diet with olive oil 12%; olive oil plus lovastatin (LOVA) group, n = 10 animals received the same diet with olive oil supplemented with lovastatin 5 mg/kg; olive oil plus orlistat (OR) group, n = 10 animals fed the diet with olive oil supplemented with orlistat 50 mg/kg and SD group, n = 10 animals fed a standard diet. The activity of lipogenic enzymes and their gene expression were evaluated by radiometric and real-time reverse transcription-polymerase chain reaction assay, respectively. RESULTS: After 10 wk of dietary treatment, the body weight was no different among animal groups (21.3 ± 3.1 g for standard group, 22.1 ± 3.6 g for OO group, 22.0 ± 3.2 g for LOVA group and 20.7 ± 3.4 g for OR group, data expressed as mean ± SD), observing a generalized well-being in all animals. All the dietary managed treated groups presented significantly reduced hepatic levels of fatty acid synthase, farnesyl pyrophosphate synthase and 3-hydroxyl-3-methyl-glutaryl CoA reductase activity and gene expression when compared with the mice fed the standard diet. To evaluate cell proliferation in the liver of treated mice, the levels of cyclin E mRNA have been measured, demonstrating a significant reduction of cyclin E gene expression in all treated groups. Evidence of reduced hepatic cell proliferation was present overall in OO group mice. CONCLUSION: We confirm the role of lipogenic enzymes as markers of cell proliferation, suggesting that appropriate dietary management alone or with drugs can be a feasible approach to counteract hepatic cell proliferation in mice.