RESUMO
Trypanosoma cruzi is a parasite with a high capacity to adapt to the host. Animal models have already demonstrated that the tropism of this parasite occurs not only in cardiac/digestive tissues but also in adipose tissue (AT). That said, the consequences ofT. cruziinfection for AT and the implications of treatment with Benzonidazole in this tissue are under discussion. Here, we tested the hypothesis that T. cruzi infection in adipose tissue upon treatment with Benzonidazole (Bz) and the interaction of mononuclear immune cells (PBMC) influences the relative expression of ACAT1, FASN, and PNPLA2 genes. Thus, stem cells derived from adipose tissue (ADSC) after adipogenic differentiation were indirectly cultivated with PBMC after infection with the T. cruzi Y strain and treatment with Bz. We use the TcSAT-IAM system and RT-qPCR to evaluate the parasite load and the relative quantification (ΔCt) of the ACAT1, FASN, and PNPLA2 genes. Our results demonstrate that treatment with Bz did not reduce adipocyte infection in the presence (p-value: 0.5796) or absence (p-value: 0.1854) of cultivation with PBMC. In addition, even though there is no statistical difference when compared to the control group (AT), T. cruzi induces the FASN expression (Rq: 14.00). However, treatment with Bz in AT suggests the increases of PNPLA2 expression levels (Rq: 12.58), even in the absence of T. cruzi infection. During indirect cultivation with PBMC, T. cruzi smooths the expression of PNPLA2 (Rq: 0.824) and instigates the expression of ACAT1 (Rq: 1.632) and FASN (Rq: 1.394). Furthermore, the treatment with Bz during infection induces PNPLA2 expression (Rq: 1.871), maintaining FASN expression levels (Rq: 1.334). Given this, our results indicate that treatment with Benzonidazole did not decrease T. cruzi infection in adipose tissue. However, treating the adipocyte cells with Bz during the interaction with PBMC cells influences the lipid pathways scenario, inducing lipolytic metabolism through the expression of PNPLA2.
Assuntos
Aciltransferases , Tecido Adiposo , Ácido Graxo Sintase Tipo I , Leucócitos Mononucleares , Lipase , Trypanosoma cruzi , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/parasitologia , Tecido Adiposo/parasitologia , Tecido Adiposo/metabolismo , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/genética , Lipase/genética , Lipase/metabolismo , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Acetil-CoA C-Acetiltransferase/genética , Acetil-CoA C-Acetiltransferase/metabolismo , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Doença de Chagas/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Carga Parasitária , Expressão Gênica , Células CultivadasRESUMO
Several genes are significantly mutated in breast cancer but only a small percentage of mutations are well-known to contribute to cancer development. FASN is involved in de novo lipogenesis and the regulation of ERα signaling. However, the effect of genetic mutations affecting FASN in breast cancer has not thoroughly studied. Therefore, we used the CRISPR/Cas9 system to edit the FASN locus in MCF-7 cells and evaluated its biological effect. We obtained four clones carrying mutations and frameshifts in the acyl-transferase domain of FASN. We found that clones had reduced proliferation, migration, viability, and showed alterations in cell cycle profiles. RNA-Seq analysis demonstrates that a lack of fully functional FASN may have a more significant role in proliferation-related genes than in lipid metabolism. We conclude that functional knockouts in FASN contributes to decrease the proliferation and migration of breast cancer cells contrary to point mutations in breast cancer patients.
Assuntos
Neoplasias da Mama/genética , Ácido Graxo Sintase Tipo I/genética , Transcriptoma , Neoplasias da Mama/patologia , Sistemas CRISPR-Cas , Movimento Celular , Proliferação de Células , Feminino , Humanos , Células MCF-7 , MutaçãoRESUMO
INTRODUCTION AND OBJECTIVES: Research in the last few years has proven that inhibition of fatty acid synthase (FASN) suppresses the migration and invasion of hepatoma carcinoma cells. This study aims to explore the effect of fatty acid synthase knockdown on the apoptosis and proliferation of HepG2 cells. MATERIALS AND METHODS: The human liver cancer cell line HepG2 was cultured and then transfected with FASN-specific siRNA and negative control RNAi. After 48h, cells and protein lysates were used for western blotting, CCK-8 (cell counting kit-8) assays, flow cytometry and other tests. To assess cell apoptosis, Bax, Bcl-2 and caspase-3 were detected; to assess proliferation, CDK4 (cyclin-dependent kinases 4) and P21 were detected; and to determine the signaling pathway involved, ß-catenin and C-myc were also detected. RESULTS: Inhibition of FASN in HepG2 cells can decrease proliferation and promote apoptosis. Flow cytometry and CCK-8 assays demonstrated that the apoptosis rate of FASN-specific siRNA-transfected cells was significantly increased compared to that of the control cells (p<0.01). In addition, the cell cycle analysis revealed that FASN-specific siRNA-transfected cells induced G1 phase arrest (p<0.05), but an increasing trend in G2 (p<0.05). Compared with expression in negative RNAi-transfected cells, the expression of Bcl-2 and CDK-4 was reduced and the expression of Bax, caspase-3 and P21 was increased in FASN-specific siRNA-transfected cells (p<0.05). Regarding the signaling pathway, the expression of ß-catenin and C-myc was significantly reduced when compared to that in negative control cells (p<0.05). CONCLUSIONS: Inhibition of FASN suppressed the cell survival of HepG2 cells by inhibiting the ß-catenin/C-myc pathway. This result suggests the potential treatment value of FASN for hepatoma carcinoma (HCC).
Assuntos
Apoptose/genética , Carcinoma Hepatocelular/genética , Proliferação de Células/genética , Ácido Graxo Sintase Tipo I/genética , Neoplasias Hepáticas/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , beta Catenina/metabolismo , Carcinoma Hepatocelular/metabolismo , Caspase 3/metabolismo , Sobrevivência Celular/genética , Quinase 4 Dependente de Ciclina/metabolismo , Técnicas de Silenciamento de Genes , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Proteína X Associada a bcl-2/metabolismoRESUMO
Fibrosis process in the liver is a clinical condition established in response to chronic lesions and may be reversible in many situations. In this process, hepatic stellate cells (HSCs) activate and produce extracellular matrix compounds. During fibrosis, the lipid metabolism is also altered and contributes to the transdifferentiation of the HSCs. Thus, controlling lipid metabolism in HSCs is suggested as a method to control or reverse the fibrotic condition. In the search for therapies that modulate lipid metabolism and treat liver diseases, silymarin has been identified as a relevant natural compound to treat liver pathologies. The present study aimed to evaluate the cellular and molecular effects of silymarin in the transdifferentiation process of HSCs (LX-2) from activated phenotype to a more quiesced-like cells , also focusing on understanding the modulatory effects of silymarin on lipid metabolism of HSCs. In our analyses, 100 µM of silymarin reduced the synthesis of actin filaments in activated cells, the synthesis of the protein level of α-SMA, and other pro-fibrotic factors such as CTGF and PFGF. The concentration of 150 µM silymarin did not reverse the activation aspects of LX-2 cells. However, both evaluated concentrations of the natural compound protected the cells from the negative effects of dimethyl sulfoxide (DMSO). Furthermore, we evaluated lipid-related molecules correlated to the transdifferentiation process of LX-2, and 100 µM of silymarin demonstrated to control molecules associated with lipid metabolism such as FASN, MLYCD, ACSL4, CPTs, among others. In contrast, cellular incubation with 150 µM of silymarin increased the synthesis of long-chain fatty acids and triglycerides, regarding the higher presence of DMSO (v/v) in the solvent. In conclusion, silymarin acts as a hepatoprotective agent and modulates the pro-fibrogenic stimuli of LX-2 cells, whose effects depend on stress levels in the cellular environment.
Assuntos
Transdiferenciação Celular/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Cirrose Hepática/metabolismo , Substâncias Protetoras/farmacologia , Silimarina/farmacologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Linhagem Celular , Cromatografia Gasosa , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Dimetil Sulfóxido/toxicidade , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Células Estreladas do Fígado/enzimologia , Células Estreladas do Fígado/metabolismo , Humanos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/genética , Espectrometria de Massas , Triglicerídeos/metabolismoRESUMO
Iron (Fe) is an essential mineral for metabolism and plays a central role in a range of biochemical processes. Therefore, this study aimed to identify differentially expressed (DE) genes and metabolic pathways in Longissimus dorsi (LD) muscle from cattle with divergent iron content, as well as to investigate the likely role of these DE genes in biological processes underlying beef quality parameters. Samples for RNA extraction for sequencing and iron, copper, manganese, and zinc determination were collected from LD muscles at slaughter. Eight Nelore steers, with extreme genomic estimated breeding values for iron content (Fe-GEBV), were selected from a reference population of 373 animals. From the 49 annotated DE genes (FDR<0.05) found between the two groups, 18 were up-regulated and 31 down-regulated for the animals in the low Fe-GEBV group. The functional enrichment analyses identified several biological processes, such as lipid transport and metabolism, and cell growth. Lipid metabolism was the main pathway observed in the analysis of metabolic and canonical signaling pathways for the genes identified as DE, including the genes FASN, FABP4, and THRSP, which are functional candidates for beef quality, suggesting reduced lipogenic activities with lower iron content. Our results indicate metabolic pathways that are partially influenced by iron, contributing to a better understanding of its participation in skeletal muscle physiology.
Assuntos
Ferro/análise , Metabolismo dos Lipídeos/genética , Lipogênese/genética , Músculo Esquelético/metabolismo , Carne Vermelha/análise , Animais , Bovinos , Cobre/análise , Ácido Graxo Sintase Tipo I/genética , Proteínas de Ligação a Ácido Graxo/genética , Expressão Gênica , Perfilação da Expressão Gênica , Metabolismo dos Lipídeos/fisiologia , Lipogênese/fisiologia , Manganês/análise , RNA/biossíntese , Transdução de Sinais , Fatores de Transcrição/genética , Zinco/análiseRESUMO
DNA methylation is a stable epigenetic mark mediating gene expression. Methylation is crucial for diverse biological processes, including aging and embryo development. FASN (fatty acid synthase) plays an important role in de novo lipogenesis, through catalyzing the reductive synthesis of long-chain fatty acids. In this study, we investigated the FASN gene expression pattern and corresponding DNA methylation status in the inner layer of backfat from Jinhua pigs at different developmental stages. Our results showed that FASN gene expression increases with age and is positively associated with adipocyte volume (r = 0.98, P < 0.01). In addition, the DNA methylation level for the first exon (0.11, CGI 3) of the FASN gene is approximately 8-fold lower than levels for its promoter (0.94, CGI 1&2) (two-way ANOVA, PCGI < 0.01). The association analysis revealed that both promoter (r = -0.944, P < 0.01) and first exon methylation (r = -0.774, P < 0.01) are significantly and negatively correlated with FASN gene expression. Our results will benefit future investigations of the epigenetic mechanism underlying FASN gene expression.
Assuntos
Metilação de DNA , Éxons , Ácido Graxo Sintase Tipo I/genética , Suínos/genética , Tecido Adiposo , Animais , Epigenômica , Ácidos Graxos/genética , Regulação da Expressão Gênica , Lipogênese , Regiões Promotoras GenéticasRESUMO
Thyroid hormone responsive protein Spot 14 has been consistently associated with de novo fatty acid synthesis activity in multiple tissues, including the lactating mammary gland, which synthesizes large quantities of medium chain fatty acids (MCFAs) exclusively via FASN. However, the molecular function of Spot14 remains undefined during lactation. Spot14-null mice produce milk deficient in total triglyceride and de novo MCFA that does not sustain optimal neonatal growth. The lactation defect was rescued by provision of a high fat diet to the lactating dam. Transgenic mice overexpressing Spot14 in mammary epithelium produced total milk fat equivalent to controls, but with significantly greater MCFA. Spot14-null dams have no diminution of metabolic gene expression, enzyme protein levels, or intermediate metabolites that accounts for impaired de novo MCFA. When [(13)C] fatty acid products were quantified in vitro using crude cytosolic lysates, native FASN activity was 1.6-fold greater in control relative to Spot14-null lysates, and add back of Spot14 partially restored activity. Recombinant FASN catalysis increased 1.4-fold and C = 14:0 yield was enhanced 4-fold in vitro following addition of Spot14. These findings implicate Spot14 as a direct protein enhancer of FASN catalysis in the mammary gland during lactation when maximal MCFA production is needed.
Assuntos
Ácido Graxo Sintase Tipo I/metabolismo , Lactação/fisiologia , Glândulas Mamárias Animais/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Catálise , Ácido Graxo Sintase Tipo I/genética , Feminino , Camundongos , Camundongos Mutantes , Proteínas Nucleares/genética , Fatores de Transcrição/genéticaRESUMO
Fatty acid synthase (FASN) is the biosynthetic enzyme responsible for the endogenous synthesis of fatty acids. It is downregulated in most normal cells, except in lipogenic tissues such as liver, lactating breast, fetal lung, and adipose tissue. Conversely, several human cancers, including head and neck squamous cell carcinomas (HNSCC), overexpress FASN, which has been associated with poor prognosis and recently suggested as a metabolic oncoprotein. Orlistat is an irreversible inhibitor of FASN activity with cytotoxic properties on several cancer cell lines that inhibits tumor progression and metastasis in prostate cancer xenografts and experimental melanomas, respectively. To explore whether the inhibition of FASN could impact oral tongue squamous cell carcinoma (OTSCC) metastatic spread, an orthotopic model was developed by the implantation of SCC-9 ZsGreen LN-1 cells into the tongue of BALB/c nude mice. These cells were isolated through in vivo selection, show a more invasive behavior in vitro than the parental cells, and generate orthotopic tumors that spontaneously metastasize to cervical lymph nodes in 10 to 15 days only. SCC-9 ZsGreen LN-1 cells also exhibit enhanced production of MMP-2, ERBB2, and CDH2. The treatment with orlistat reduced proliferation and migration, promoted apoptosis, and stimulated the secretion of VEGFA165b by SCC-9 ZsGreen LN-1 cells. In vivo, the drug was able to decrease both the volume and proliferation indexes of the tongue orthotopic tumors and, importantly, reduced the number of metastatic cervical lymph nodes by 43%. These results suggest that FASN is a potential molecular target for the chemotherapy of patients with OTSCC.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Ácido Graxo Sintase Tipo I/genética , Neoplasias da Língua/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Ácido Graxo Sintase Tipo I/antagonistas & inibidores , Humanos , Lactonas/administração & dosagem , Camundongos , Metástase Neoplásica , Orlistate , Neoplasias da Língua/genética , Neoplasias da Língua/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Copper is an essential micronutrient that functions as an enzymatic cofactor in a wide range of cellular processes. Although adequate Cu levels are essential for normal metabolism, excess Cu can be toxic to cells. Cellular responses to copper deficiency and overload involve changes in the expression of genes directly and indirectly involved in copper metabolism. However little is known on the effect of physiological copper concentration on gene expression changes. In the current study we aimed to establish whether the expression of genes encoding enzymes related to cholesterol (hmgcs1, hmgcr, fdft) and fatty acid biosynthesis and LDL receptor can be induced by an iso-physiological copper concentration. The iso-physiological copper concentration was determined as the bioavailable plasmatic copper in a healthy adult population. In doing so, two blood cell lines (Jurkat and THP-1) were exposed for 6 or 24 h to iso- or supraphysiological copper concentrations. Our results indicated that in cells exposed to an iso-physiological copper concentration the early induction of genes involved in lipid metabolism was not mediated by copper itself but by the modification of the cellular redox status. Thus our results contributed to understand the involvement of copper in the regulation of cholesterol metabolism under physiological conditions.