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1.
Bioelectrochemistry ; 131: 107350, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31518962

RESUMO

Curcumin (Cur), the yellow pigment of well-known turmeric (Curcuma longa L.) is effective in multiple cancers including triple negative breast cancer (TNBC). In combination with electrical pulses (EP), enhanced effects of curcumin (Cur + EP) are observed in TNBC cells. To gain insights into the mechanisms of enhanced anticancer effects of Cur + EP, we studied the proteins involved in the anticancer activity of Cur + EP in MDA-MB-231, human TNBC cells using high-throughput global proteomics. A curcumin dose of 50 µM was applied with eight, 1200 V/cm, 100 µs pulses, the most commonly used electrochemotherapy (ECT) parameter in clinics. Results show that the Cur + EP treatment reduced the clonogenic ability in MDA-MB-231 cells, with the induction of apoptosis. Proteomic analysis identified a total of 1456 proteins, of which 453 proteins were differentially regulated, including kinases, heat shock proteins, transcription factors, structural proteins, and metabolic enzymes. Eight key glycolysis proteins (ALDOA, ENO2, LDHA, LDHB, PFKP, PGM1, PGAM1 and PGK1) were downregulated in Cur + EP from Cur. There was a switch in the metabolism with upregulation of 10 oxidative phosphorylation pathway proteins and 8 tricarboxylic acid (TCA) cycle proteins in the Cur + EP sample, compared to curcumin. These results provide novel systematic insights into the mechanisms of ECT with curcumin.


Assuntos
Antineoplásicos/uso terapêutico , Curcumina/uso terapêutico , Eletroquimioterapia/métodos , Proteínas de Neoplasias/metabolismo , Proteômica , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Curcumina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Feminino , Glicólise , Humanos , Fosforilação Oxidativa , Via de Pentose Fosfato/efeitos dos fármacos , Reprodutibilidade dos Testes , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia
2.
Environ Toxicol ; 35(1): 78-86, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31486570

RESUMO

Di (2-ethylhexyl) phthalate (DEHP) is a plasticizer that gives flexibility to various polyvinyl chloride products. It is a pollutant easily released into the environment and can cause many adverse effects to living organisms including hepatotoxicity. The thioredoxin system is a determining factor in the redox balance maintaining in the liver, which is a vulnerable tissue of reactive oxygen species overproduction because of its high energy needs. In order to determine if the thioredoxin system is a target in the development of DEHP hepatotoxicity, Balb/c mice were administered with DEHP intraperitoneally daily for 30 days. Results demonstrated that after DEHP exposure, biochemical profile changes were observed. This phthalate causes oxidative damage through the induction of lipid peroxydation as well as the increase of superoxide dismutase and catalase activities. As new evidence provided in this study, we demonstrated that the DEHP affected the thioredoxin system by altering the expression and the activity of thioredoxin (Trx) and thioredoxin Reductase (TrxR1). The two enzyme activities of the oxidative phase of the pentose phosphate pathway: Glucose-6-phosphate dehydrogenase and 6-Phosphogluconate dehydrogenase were also affected by this phthalate. This leads to a decrease in the level of nicotinamide adenine dinucleotide phosphate used by the TrxR1 to maintain the regeneration of the reduced Trx. We also demonstrated that such effects can be responsible of DEHP-induced DNA damage.


Assuntos
Dietilexilftalato/toxicidade , Poluentes Ambientais/toxicidade , Fígado/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Via de Pentose Fosfato/efeitos dos fármacos , Plastificantes/toxicidade , Tiorredoxinas/metabolismo , Animais , Dano ao DNA , Relação Dose-Resposta a Droga , Glucosefosfato Desidrogenase/metabolismo , Injeções Intraperitoneais , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo
3.
Food Chem ; 305: 125439, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31499287

RESUMO

Compared to the control longans, hydrogen peroxide (H2O2)-treated longans exhibited higher index of pulp breakdown, higher fruit respiration rate, higher activities of pulp phosphohexose isomerase (PGI), succinate dehydrogenase (SDH), cytochrome C oxidase (CCO), ascorbic acid oxidase (AAO) and polyphenol oxidase (PPO), but lower activity of pulp nicotinamide adenine dinucleotide kinase (NADK). H2O2-treated longans also exhibited lower total activities of pulp glucose-6-phosphate dehydrogenase (G-6-PDH) and 6-phosphogluconate dehydrogenase (6-PGDH), lower levels of pulp NADP(H), but higher levels of pulp NAD(H). These data indicated that H2O2-stimulated longan pulp breakdown was owing to a decreased proportion of pentose phosphate pathway (PPP), the increased proportions of Embden-Meyerhof-Parnas pathway (EMP), tricarboxylic acid (TCA) cycle and cytochrome pathway (CCP) in total respiratory pathways. These findings further revealed that H2O2 could enhance respiration rate, and thus accelerate pulp breakdown occurrence and shorten the shelf life of longan fruit.


Assuntos
Peróxido de Hidrogênio/farmacologia , Sapindaceae/efeitos dos fármacos , Aldeído Oxidase/metabolismo , Ciclo do Ácido Cítrico/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Armazenamento de Alimentos , Frutas/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Glicólise/efeitos dos fármacos , NAD/metabolismo , Via de Pentose Fosfato/efeitos dos fármacos , Sapindaceae/metabolismo
4.
Cancer Commun (Lond) ; 39(1): 17, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30947742

RESUMO

BACKGROUND: Internal tandem duplications (ITD) within the juxtamembrane domain of FMS-like tyrosine kinase 3 (FLT3) represent a poor prognostic indicator in acute myeloid leukemia (AML). Therapeutic benefits of tyrosine kinase inhibitors, such as sorafenib, are limited due to the emergence of drug resistance. While investigations have been conducted to improve the understanding of the molecular mechanisms underlying the resistance to this FLT3 inhibitor, a profile of cell functioning at the metabolite level and crosstalk between metabolic pathways has yet to be created. This study aimed to elucidate the alteration of metabolomic profile of leukemia cells resistant to the FLT3 inhibitor. METHODS: We established two sorafenib-resistant cell lines carrying FLT3/ITD mutations, namely the murine BaF3/ITD-R and the human MV4-11-R cell lines. We performed a global untargeted metabolomics and stable isotope-labeling mass spectrometry analysis to identify the metabolic alterations relevant to the therapeutic resistance. RESULTS: The resistant cells displayed fundamentally rewired metabolic profiles, characterized by a higher demand for glucose, accompanied by a reduction in glucose flux into the pentose phosphate pathway (PPP); and by an increase in oxidative stress, accompanied by an enhanced glutathione synthesis. We demonstrated that the highest scoring network of altered metabolites in resistant cells was related to nucleotide degradation. A stable isotope tracing experiment was performed and the results indicated a decrease in the quantity of glucose entering the PPP in resistant cells. Further experiment suggested that the inhibition of major enzymes in the PPP consist of glucose-6-phosphate dehydrogenase deficiency (G6PD) in the oxidative arm and transketolase (TKT) in the non-oxidative arm. In addition, we observed that chronic treatment with sorafenib resulted in an increased oxidative stress in FLT3/ITD-positive leukemia cells, which was accompanied by decreased cell proliferation and an enhanced antioxidant response. CONCLUSIONS: Our data regarding comparative metabolomics characterized a distinct metabolic and redox adaptation that may contribute to sorafenib resistance in FLT3/ITD-mutated leukemia cells.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Leucemia/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Sorafenibe/farmacologia , Tirosina Quinase 3 Semelhante a fms/genética , Animais , Apoptose/efeitos dos fármacos , Isótopos de Carbono , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Glucose/metabolismo , Glutationa/metabolismo , Humanos , Leucemia/tratamento farmacológico , Leucemia/genética , Espectrometria de Massas , Metabolômica , Camundongos , NADP/metabolismo , Oxirredução , Via de Pentose Fosfato/efeitos dos fármacos
5.
Food Chem ; 289: 112-120, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-30955592

RESUMO

Sucrose acts as a vital signal that modulates fruit ripening. In current study, 50 mM sucrose was applied in strawberry fruit to investigate the regulation of sucrose in anthocyanin synthesis after harvest. The results showed that sucrose treatment increased the contents of glucose, fructose and sucrose, which were 19.76%, 15.83% and 16.50% higher, respectively, compared with control at the end of storage. The increase of glucose and fructose contents resulted from the activation of acid invertase by sucrose treatment. In addition, sucrose treatment specifically increased four pelargonidin derivatives, pelargonidin 3-glucoside, pelargonidin 3-rutinoside, pelargonidin 3-malonylglucoside and pelargonidin 3-methylmalonyglucoside, during the storage. Further, transcriptional profiles and enzyme activities analysis revealed that the accumulation of pelargonidin derivatives was related to the activation of the pentose phosphate pathway, shikimate pathway, phenylpropanoid pathway, and flavonoid pathway. These results provided new insights into the regulation of sucrose on the accumulation of individual anthocyanins.


Assuntos
Antocianinas/biossíntese , Fragaria/metabolismo , Antocianinas/análise , Antocianinas/metabolismo , Flavonoides/metabolismo , Fragaria/química , Frutas/química , Frutas/metabolismo , Glucose/análise , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Espectrometria de Massas , Via de Pentose Fosfato/efeitos dos fármacos , Fenilalanina Amônia-Liase/metabolismo , Sacarose/farmacologia , Transcinamato 4-Mono-Oxigenase/metabolismo
6.
Biomed Pharmacother ; 111: 1353-1358, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30841449

RESUMO

Better understanding of the molecular mechanism involved in hepatocellular carcinoma (HCC) progression is essential for the development of therapeutic strategies to overcome chemoresistance in HCC patients. In this work, we show that 6-phosphogluconate dehydrogenase (6PGD), a key enzyme of the oxidative pentose phosphate pathway, is important for HCC growth and survival. Compared to normal liver tissues, we demonstrate that 6PGD expression is upregulated in HCC tissues. 6PGD overexpression increases 6PGD activity and promotes growth in normal liver cells. In contrast, targeting 6PGD using both genetic and pharmacological approaches inhibits HCC growth and survival. Combination of chemotherapeutic agents with 6PGD inhibition achieves greater efficacy in inhibiting HCC growth and survival than chemotherapeutic agent alone. We further show that inhibition of 6PGD activates AMP-activated protein kinase (AMPK) and acetyl-coenzyme A carboxylase 1 (ACC1), and decreases level of NADPH/NAD + and NADH in HCC, leading to SIRT1 activity reduction and oxidative stress. Conversely, AMPK depletion significantly abolishes the effects of physcion (a selective small-molecule 6PGD inhibitor) in decreasing NADPH/NAD + ratio, growth and survival, confirming the role of AMPK as the relevant upstream activator with 6PGD inhibition in HCC cells. Our work is the first to demonstrate the upregulation of 6PGD and its critical involvement in growth and survival in HCC. Our findings suggest 6PGD as a promising therapeutic target to overcome chemoresistance in HCC.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Fosfogluconato Desidrogenase/antagonistas & inibidores , Acetil-CoA Carboxilase/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Hep G2 , Humanos , NADP/metabolismo , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Via de Pentose Fosfato/efeitos dos fármacos , Sirtuína 1/metabolismo , Regulação para Cima/efeitos dos fármacos
7.
Metab Eng ; 52: 98-109, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30471359

RESUMO

The conversion of plant material into biofuels and high value products is a two-step process of hydrolysing plant lignocellulose and next fermenting the sugars produced. However, lignocellulosic hydrolysis not only frees sugars for fermentation it simultaneously generates toxic chemicals, including phenolic compounds which severely inhibit yeast fermentation. To understand the molecular basis of phenolic compound toxicity, we performed genome-wide chemogenomic screens in Saccharomyces cerevisiae to identify deletion mutants that were either hypersensitive or resistant to three common phenolic compounds found in plant hydrolysates: coniferyl aldehyde, ferulic acid and 4-hydroxybenzoic acid. Despite being similar in structure, our screen revealed that yeast utilizes distinct pathways to tolerate phenolic compound exposure. Furthermore, although each phenolic compound induced reactive oxygen species (ROS), ferulic acid and 4-hydroxybenzoic acid-induced a general cytoplasmic ROS distribution while coniferyl aldehyde-induced ROS partially localized to the mitochondria and to a lesser extent, the endoplasmic reticulum. We found that the glucose-6-phosphate dehydrogenase enzyme Zwf1, which catalyzes the rate limiting step of pentose phosphate pathway, is required for reducing the accummulation of coniferyl aldehyde-induced ROS, potentially through the sequestering of Zwf1 to sites of ROS accumulation. Our novel insights into biological impact of three common phenolic inhibitors will inform the engineering of yeast strains with improved efficiency of biofuel and biochemical production in the presence hydrolysate-derived phenolic compounds.


Assuntos
Acroleína/análogos & derivados , Ácidos Cumáricos/farmacologia , Fermentação , Redes e Vias Metabólicas/genética , Parabenos/farmacologia , Fenóis/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Acroleína/farmacologia , Estudo de Associação Genômica Ampla , Lignina , Via de Pentose Fosfato/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Triptofano/metabolismo
8.
Arthritis Rheumatol ; 71(5): 766-772, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30462889

RESUMO

OBJECTIVE: To clarify the significance of immunometabolism in systemic lupus erythematosus (SLE), and to determine the effect of calcium/calmodulin-dependent protein kinase 4 (CaMK4) on T cell metabolism. METHODS: Metabolomic profiling was performed using capillary electrophoresis mass spectrometry in naive T cells from MRL/lpr mice treated with anti-CD3/CD28 antibodies in the absence or presence of a CaMK4 inhibitor (KN-93). The expression of GLUT1 and CaMK4 in CD4+ T cells from healthy controls (n = 16), patients with inactive SLE (n = 13), and patients with active SLE (n = 14) was examined by flow cytometry and quantitative polymerase chain reaction. In vitro experiments were performed to determine the effect of KN-93 on the expression of GLUT1 during Th17 cell differentiation in T cells from patients with SLE. RESULTS: CaMK4 inhibition significantly decreased the levels of glycolytic intermediates such as glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, pyruvate, and lactate (P < 0.05), whereas it did not affect the levels of the pentose phosphate pathway intermediates such as 6-phospho-d-gluconate, ribulose-5-phosphate, ribose-5-phosphate, and phosphoribosyl pyrophosphate. The expression levels of GLUT1 and CaMK4 in effector memory CD4+ T cells were significantly higher in patients with active SLE compared to healthy controls (P < 0.01 and P < 0.05, respectively) and patients with inactive SLE (P < 0.05 and P < 0.01, respectively). A functional analysis revealed that CaMK4 inhibition decreased the expression of GLUT1 during Th17 cell differentiation (P < 0.01), followed by a reduction of interleukin-17 (IL-17) production (P < 0.05). CONCLUSION: The results of the study indicate that the activity of CaMK4 could be responsible for glycolysis, which contributes to the production of IL-17, and CaMK4 may contribute to aberrant expression of GLUT1 in T cells from patients with active SLE.


Assuntos
Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Glicólise/fisiologia , Lúpus Eritematoso Sistêmico/metabolismo , Células Th17/imunologia , Adulto , Animais , Benzilaminas/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Estudos de Casos e Controles , Diferenciação Celular/efeitos dos fármacos , Feminino , Frutosedifosfatos/metabolismo , Frutosefosfatos/metabolismo , Transportador de Glucose Tipo 1/efeitos dos fármacos , Glucose-6-Fosfato/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Memória Imunológica , Interleucina-17/imunologia , Ácido Láctico/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Metaboloma , Metabolômica , Camundongos , Camundongos Endogâmicos MRL lpr , Pessoa de Meia-Idade , Via de Pentose Fosfato/efeitos dos fármacos , Via de Pentose Fosfato/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Ácido Pirúvico/metabolismo , Sulfonamidas/farmacologia , Células Th17/efeitos dos fármacos
9.
Nat Commun ; 9(1): 4945, 2018 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-30470748

RESUMO

Oncogenic KRAS is the key driver of pancreatic ductal adenocarcinoma (PDAC). We previously described a role for KRAS in PDAC tumor maintenance through rewiring of cellular metabolism to support proliferation. Understanding the details of this metabolic reprogramming in human PDAC may provide novel therapeutic opportunities. Here we show that the dependence on oncogenic KRAS correlates with specific metabolic profiles that involve maintenance of nucleotide pools as key mediators of KRAS-dependence. KRAS promotes these effects by activating a MAPK-dependent signaling pathway leading to MYC upregulation and transcription of the non-oxidative pentose phosphate pathway (PPP) gene RPIA, which results in nucleotide biosynthesis. The use of MEK inhibitors recapitulates the KRAS-dependence pattern and the expected metabolic changes. Antagonizing the PPP or pyrimidine biosynthesis inhibits the growth of KRAS-resistant cells. Together, these data reveal differential metabolic rewiring between KRAS-resistant and sensitive cells, and demonstrate that targeting nucleotide metabolism can overcome resistance to KRAS/MEK inhibition.


Assuntos
Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Pirimidinas/biossíntese , Animais , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Via de Pentose Fosfato/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas p21(ras)/genética
10.
Cell Rep ; 25(8): 2044-2052.e5, 2018 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-30463003

RESUMO

Metabolic reprogramming has emerged as a crucial regulator of immune cell activation, but how systemic metabolism influences immune cell metabolism and function remains to be investigated. To investigate the effect of dyslipidemia on immune cell metabolism, we performed in-depth transcriptional, metabolic, and functional characterization of macrophages isolated from hypercholesterolemic mice. Systemic metabolic changes in such mice alter cellular macrophage metabolism and attenuate inflammatory macrophage responses. In addition to diminished maximal mitochondrial respiration, hypercholesterolemia reduces the LPS-mediated induction of the pentose phosphate pathway (PPP) and the Nrf2-mediated oxidative stress response. Our observation that suppression of the PPP diminishes LPS-induced cytokine secretion supports the notion that this pathway contributes to inflammatory macrophage responses. Overall, this study reveals that systemic and cellular metabolism are strongly interconnected, together dictating macrophage phenotype and function.


Assuntos
Hipercolesterolemia/metabolismo , Hipercolesterolemia/patologia , Inflamação/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Via de Pentose Fosfato , Animais , Ciclo do Ácido Cítrico/efeitos dos fármacos , Feminino , Glicólise/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/metabolismo , Via de Pentose Fosfato/efeitos dos fármacos
11.
Nutrients ; 10(10)2018 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-30279339

RESUMO

Patterned hair loss (PHL) affects around 50% of the adult population worldwide. The negative impact that this condition exerts on people's life quality has boosted the appearance of over-the-counter products endowed with hair-promoting activity. Nutraceuticals enriched in polyphenols have been recently shown to promote hair growth and counteract PHL. Malus pumila Miller cv. Annurca is an apple native to Southern Italy presenting one of the highest contents of Procyanidin B2. We have recently shown that oral consumption of Annurca polyphenolic extracts (AAE) stimulates hair growth, hair number, hair weight and keratin content in healthy human subjects. Despite its activity, the analysis of the molecular mechanism behind its hair promoting effect is still partially unclear. In this work we performed an unprecedented metabolite analysis of hair follicles (HFs) in mice topically treated with AAE. The metabolomic profile, based on a high-resolution mass spectrometry approach, revealed that AAE re-programs murine HF metabolism. AAE acts by inhibiting several NADPH dependent reactions. Glutaminolysis, pentose phosphate pathway, glutathione, citrulline and nucleotide synthesis are all halted in vivo by the treatment of HFs with AAE. On the contrary, mitochondrial respiration, ß-oxidation and keratin production are stimulated by the treatment with AAE. The metabolic shift induced by AAE spares amino acids from being oxidized, ultimately keeping them available for keratin biosynthesis.


Assuntos
Biflavonoides/farmacologia , Catequina/farmacologia , Folículo Piloso/metabolismo , Queratinas/biossíntese , Malus/química , Fitoterapia/métodos , Extratos Vegetais/farmacologia , Polifenóis/farmacologia , Proantocianidinas/farmacologia , Alopecia/tratamento farmacológico , Aminoácidos/metabolismo , Animais , Folículo Piloso/efeitos dos fármacos , Humanos , Itália , Queratinas/efeitos dos fármacos , Espectrometria de Massas , Metabolômica , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução/efeitos dos fármacos , Via de Pentose Fosfato/efeitos dos fármacos
12.
Chem Biol Interact ; 294: 167-177, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30170107

RESUMO

6-Aminonicotinamide (6AN) is an antimetabolite used to inhibit the NADPH-producing pentose phosphate pathway (PPP) in many cellular systems, making them more susceptible to oxidative stress. It is converted by a NAD(P)+ glycohydrolase to 6-aminoNAD and 6-aminoNADP, causing the accumulation of PPP intermediates, due to their inability to participate in redox reactions. Some parasites like Plasmodium falciparum and Coccidia are highly sensitive but not all cell types showed a strong responsiveness to 6AN, probably due to the different targeted pathway. For instance, in bacteria the main target is the Preiss-Handler salvage pathway for NAD+ biosynthesis. We were interested in testing 6AN on the kinetoplastid protozoan Leishmania as another model to clarify the mechanisms of action of 6AN, by using metabolomics. Leishmania promastigotes, the life-cycle stage residing in the sandfly, demonstrated a three order of magnitude higher EC50 (mM) compared to P. falciparum and mammalian cells (µM), although pre-treatment with 100 µM 6AN prior to sub-lethal oxidative challenge induced a supra-additive cell kill in L. infantum. By metabolomics, we did not detect 6ANAD/P suggesting that NAD+ glycohydrolases in Leishmania may not be highly efficient in catalysing transglycosidation as happens in other microorganisms. Contrariwise to the reported effect on 6AN-treated cancer cells, we did not detect 6-phosphogluconate (6 PG) accumulation, indicating that 6ANADP cannot bind with high affinity to the PPP enzyme 6 PG dehydrogenase. By contrast, 6AN caused a profound phosphoribosylpyrophosphate (PRPP) decrease and nucleobases accumulation confirming that PPP is somehow affected. More importantly, we found a decrease in nicotinate production, evidencing the interference with the Preiss-Handler salvage pathway for NAD+ biosynthesis, most probably by inhibiting the reaction catalysed by nicotinamidase. Therefore, our combined data from Leishmania strains, though confirming the interference with PPP, also showed that 6AN impairs the Preiss-Handler pathway, underlining the importance to develop compounds targeting this last route.


Assuntos
6-Aminonicotinamida/farmacologia , Leishmania/metabolismo , Metabolômica , Via de Pentose Fosfato/efeitos dos fármacos , Aminoácidos/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Interações Hidrofóbicas e Hidrofílicas , Leishmania/efeitos dos fármacos , Leishmania/crescimento & desenvolvimento , Estágios do Ciclo de Vida/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Espectrometria de Massas , Metaboloma/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Fosfogluconato Desidrogenase/metabolismo
13.
Sci Rep ; 8(1): 13672, 2018 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-30209405

RESUMO

Doxorubicin is one of the most effective chemotherapy drugs used against solid tumors in the treatment of several cancer types. Two different mechanisms, (i) intercalation of doxorubicin into DNA and inhibition of topoisomerase II leading to changes in chromatin structure, (ii) generation of free radicals and oxidative damage to biomolecules, have been proposed to explain the mode of action of this drug in cancer cells. A genome-wide integrative systems biology approach used in the present study to investigate the long-term effect of doxorubicin in Saccharomyces cerevisiae cells indicated the up-regulation of genes involved in response to oxidative stress as well as in Rad53 checkpoint sensing and signaling pathway. Modular analysis of the active sub-network has also revealed the induction of the genes significantly associated with nucleosome assembly/disassembly and DNA repair in response to doxorubicin. Furthermore, an extensive re-wiring of the metabolism was observed. In addition to glycolysis, and sulfate assimilation, several pathways related to ribosome biogenesis/translation, amino acid biosynthesis, nucleotide biosynthesis, de novo IMP biosynthesis and one-carbon metabolism were significantly repressed. Pentose phosphate pathway, MAPK signaling pathway biological processes associated with meiosis and sporulation were found to be induced in response to long-term exposure to doxorubicin in yeast cells.


Assuntos
DNA Fúngico/efeitos dos fármacos , Doxorrubicina/farmacologia , Saccharomyces cerevisiae/metabolismo , Inibidores da Topoisomerase II/farmacologia , Transcrição Genética/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/biossíntese , Quinase do Ponto de Checagem 2/genética , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Reparo do DNA/genética , Fermentação/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Nucleossomos/metabolismo , Estresse Oxidativo/genética , Via de Pentose Fosfato/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética
14.
Bioprocess Biosyst Eng ; 41(10): 1509-1518, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30062600

RESUMO

Betaine can act as a stress protectant, methyl donor, or enzyme stabilizer in vitro for the biosynthesis of structurally complex compounds. The performances of betaine type and concentration on the metabolic processes of Escherichia coli JLTHR in a 5-L fermentor were investigated. The results showed that the maximum L-threonine production of 127.3 g/L and glucose conversion percentage of 58.12% was obtained fed with the glucose solution containing 2 g/L betaine hydrochloride, which increased by 14.5 and 6.87% more compared to that of the control, respectively. This study presents an analysis of the metabolic fluxes of E. coli JLTHR for the production of L-threonine with betaine supplementation. When betaine was fed into the fermentation culture medium, the metabolic flux entering into the pentose phosphate pathway (HMP) and biosynthesis route of L-threonine increased by 57.3 and 10.1%, respectively. In conclusion, exogenous addition of betaine was validated to be a feasible and efficacious approach to improve L-threonine production.


Assuntos
Betaína/farmacologia , Reatores Biológicos , Escherichia coli/crescimento & desenvolvimento , Via de Pentose Fosfato/efeitos dos fármacos , Treonina/biossíntese , Betaína/metabolismo
15.
Biochem Biophys Res Commun ; 503(2): 572-579, 2018 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-29885837

RESUMO

Transketolase-like 1 (TKTL1) plays an important role in the pentose phosphate pathway (PPP) branch. The main obstacle of ovarian cancer treatment is chemotherapeutic resistance. We investigated whether inhibiting TKTL1 in OC3/TAX300 cells could re-sensitize paclitaxel-resistant cells to paclitaxel and proposed a mechanism of action. Western blotting revealed that TKTL1 expression levels in OC3/Tax300 cells were significantly higher than those in OC3 cells. Inhibition of TKTL1 significantly decreased the cellular proliferation rate and IC50 for paclitaxel. Metabolomics revealed that NADPH levels were reduced in the si-TKTL1 group, whereas NADP+ was increased compared with the level in the negative si-TKTL1 group. A 2.2-fold increase in the ROS level and an obvious increase in the cell apoptosis rate were observed in the si-TKTL1+paclitaxel group compared with those in the negative si-TKTL1+paclitaxel and OC3/Tax300 + paclitaxel groups. Western blotting revealed that Bax and Caspase 3 proteins were up-regulated, whereas Bcl-2 expression was down-regulated. Quantitative RT-PCR revealed no changes in gst-π or mrp1 gene expression in the three groups, whereas GSH levels were reduced in the si-TKTL1 group as verified by metabolomics. TKTL1 inhibition also reduced tumor growth in vivo. Collectively, TKTL1 down-regulation sensitized paclitaxel-resistant OC3/Tax300 ovarian cancer cells to paclitaxel.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel/farmacologia , Transcetolase/genética , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Metabolômica , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Via de Pentose Fosfato/efeitos dos fármacos , Transcetolase/metabolismo , Regulação para Cima/efeitos dos fármacos
16.
Cell Death Dis ; 9(5): 572, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29760380

RESUMO

Pentose phosphate pathway (PPP) is a major glucose metabolism pathway, which has a fundamental role in cancer growth and metastasis. Even though PPP blockade has been pointed out as a very promising strategy against cancer, effective anti-PPP agents are not still available in the clinical setting. Here we demonstrate that the natural molecule polydatin inhibits glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of PPP. Polydatin blocks G6PD causing accumulation of reactive oxygen species and strong increase of endoplasmic reticulum stress. These effects are followed by cell cycle block in S phase, an about 50% of apoptosis, and 60% inhibition of invasion in vitro. Accordingly, in an orthotopic metastatic model of tongue cancer, 100 mg/kg polydatin induced an about 30% tumor size reduction with an about 80% inhibition of lymph node metastases and 50% reduction of lymph node size (p < 0.005). Polydatin is not toxic in animals up to a dose of 200 mg/kg and a phase II clinical trial shows that it is also well tolerated in humans (40 mg twice a day for 90 days). Thus, polydatin may be used as a reliable tool to limit human cancer growth and metastatic spread.


Assuntos
Glucosefosfato Desidrogenase/antagonistas & inibidores , Glucosídeos/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/enzimologia , Via de Pentose Fosfato/efeitos dos fármacos , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Estilbenos/farmacologia , Animais , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Humanos , Células MCF-7 , Masculino , Camundongos Nus , Metástase Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Pontos de Checagem da Fase S do Ciclo Celular/genética , Ensaios Antitumorais Modelo de Xenoenxerto
17.
ASN Neuro ; 10: 1759091418775562, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29768946

RESUMO

Oxidative stress plays an important role in the onset and progression of Parkinson disease. Although released dopamine at the synaptic terminal is mostly reabsorbed by dopaminergic neurons, some dopamine is presumably taken up by astroglia. This study examined the dopamine-induced astroglial protective function through the activation of the pentose-phosphate pathway (PPP) to reduce reactive oxygen species (ROS). In vitro experiments were performed using striatal neurons and cortical or striatal astroglia prepared from Sprague-Dawley rats or C57BL/6 mice. The rates of glucose phosphorylation in astroglia were evaluated using the [14C]deoxyglucose method. PPP activity was measured using [1-14C]glucose and [6-14C]glucose after acute (60 min) or chronic (15 hr) exposure to dopamine. ROS production was measured using 2',7'-dichlorodihydrofluorescein diacetate. The involvement of the Kelch-like ECH-associated protein 1 (Keap1) or nuclear factor-erythroid-2-related factor 2 (Nrf2) system was evaluated using Nrf2 gene knockout mice, immunohistochemistry, and quantitative reverse transcription polymerase chain reaction analysis for heme oxygenase-1. Acute exposure to dopamine elicited increases in astroglial glucose consumption with lactate release. PPP activity in astroglia was robustly enhanced independently of Na+-dependent monoamine transporters. In contrast, chronic exposure to dopamine induced moderate increases in PPP activity via the Keap1/Nrf2 system. ROS production from dopamine increased gradually over 12 hr. Dopamine induced neuronal cell damage that was prevented by coculturing with astroglia but not with Nrf2-deficient astroglia. Dopamine-enhanced astroglial PPP activity in both acute and chronic manners may possibly reduce neuronal oxidative stress.


Assuntos
Astrócitos/efeitos dos fármacos , Dopamina/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Via de Pentose Fosfato/efeitos dos fármacos , Animais , Encéfalo/citologia , Células Cultivadas , Técnicas de Cocultura , Dopamina/metabolismo , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Glucose/metabolismo , Peróxido de Hidrogênio/farmacologia , Lactatos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Relacionado a NF-E2/deficiência , Fator 2 Relacionado a NF-E2/genética , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio
18.
Redox Biol ; 17: 338-347, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29793167

RESUMO

Over the last decades, the silent-killer carbon monoxide (CO) has been shown to also be an endogenous cytoprotective molecule able to inhibit cell death and modulate mitochondrial metabolism. Neuronal metabolism is mostly oxidative and neurons also use glucose for maintaining their anti-oxidant status by generation of reduced glutathione (GSH) via the pentose-phosphate pathway (PPP). It is established that neuronal differentiation depends on reactive oxygen species (ROS) generation and signalling, however there is a lack of information about modulation of the PPP during adult neurogenesis. Thus, the main goal of this study was to unravel the role of CO on cell metabolism during neuronal differentiation, particularly by targeting PPP flux and GSH levels as anti-oxidant system. A human neuroblastoma SH-S5Y5 cell line was used, which differentiates into post-mitotic neurons by treatment with retinoic acid (RA), supplemented or not with CO-releasing molecule-A1 (CORM-A1). SH-SY5Y cell differentiation supplemented with CORM-A1 prompted an increase in neuronal yield production. It did, however, not alter glycolytic metabolism, but increased the PPP. In fact, CORM-A1 treatment stimulated (i) mRNA expression of 6-phosphogluconate dehydrogenase (PGDH) and transketolase (TKT), which are enzymes for oxidative and non-oxidative phases of the PPP, respectively and (ii) protein expression and activity of glucose 6-phosphate dehydrogenase (G6PD) the rate-limiting enzyme of the PPP. Likewise, whenever G6PD was knocked-down CO-induced improvement on neuronal differentiation was reverted, while pharmacological inhibition of GSH synthesis did not change CO's effect on the improvement of neuronal differentiation. Both results indicate the key role of PPP in CO-modulation of neuronal differentiation. Furthermore, at the end of SH-SY5Y neuronal differentiation process, CORM-A1 supplementation increased the ratio of reduced and oxidized glutathione (GSH/GSSG) without alteration of GSH metabolism. These data corroborate with PPP stimulation. In conclusion, CO improves neuronal differentiation of SH-S5Y5 cells by stimulating the PPP and modulating the GSH system.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios/metabolismo , Via de Pentose Fosfato/efeitos dos fármacos , Monóxido de Carbono/farmacologia , Glucose/metabolismo , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Tretinoína/farmacologia
19.
Redox Biol ; 17: 180-191, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29702405

RESUMO

Breast cancer is one of the most lethal tumors in the world, among which 15% are triple-negative breast cancers (TNBCs) with higher metastasis and lower survival rate. Anoikis resistance is a key process during tumor metastasis, which is usually accompanied with metabolism reprogram. In this study, we established an anchorage independent growth model for MDA-MB-231 cells and investigated the changes in metabolism and redox homeostasis. Results showed that during detached-growth, MDA-MB-231 cells tend to generate ATP through fatty acid oxidation (FAO), instead of glycolysis. Amount of glucose was used for pentose phosphate pathway (PPP) to keep redox balance. Moreover, we discovered that a synthesized flavonoid derivative GL-V9, exhibited a potent inhibitory effect on the anchorage independent growth of TNBCs in vitro and anti-metastasis effect in vivo. In terms of the mechanism, GL-V9 could promote the expression and activity of AMPK, leading to the decrease of G6PD and the increase of p-ACC. Thus, the level of PPP was suppressed, whereas FAO was highly enhanced. The reprogram of glycolipid metabolism destroyed the redox balance ultimately and induced cell death. This paper indicated a novel regulating mechanism of redox homeostasis involving with glycolipid metabolism, and provided a potential candidate for the anti-metastatic therapy of TNBCs.


Assuntos
Proteínas Quinases Ativadas por AMP/genética , Neoplasias da Mama/genética , Glicolipídeos/genética , Estresse Oxidativo/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Flavonoides/administração & dosagem , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Glucosefosfato Desidrogenase/genética , Glicólise/efeitos dos fármacos , Humanos , Metabolismo dos Lipídeos/genética , Oxirredução , Via de Pentose Fosfato/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
20.
Microb Cell Fact ; 17(1): 10, 2018 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-29357933

RESUMO

BACKGROUND: Azotobacter vinelandii is a bacterium that produces alginate and polyhydroxybutyrate (P3HB); however, the role of NAD(P)H/NAD(P)+ ratios on the metabolic fluxes through biosynthesis pathways of these biopolymers remains unknown. The aim of this study was to evaluate the NAD(P)H/NAD(P) + ratios and the metabolic fluxes involved in alginate and P3HB biosynthesis, under oxygen-limiting and non-limiting oxygen conditions. RESULTS: The results reveal that changes in the oxygen availability have an important effect on the metabolic fluxes and intracellular NADPH/NADP+ ratio, showing that at the lowest OTR (2.4 mmol L-1 h-1), the flux through the tricarboxylic acid (TCA) cycle decreased 27.6-fold, but the flux through the P3HB biosynthesis increased 6.6-fold in contrast to the cultures without oxygen limitation (OTR = 14.6 mmol L-1 h-1). This was consistent with the increase in the level of transcription of phbB and the P3HB biosynthesis. In addition, under conditions without oxygen limitation, there was an increase in the carbon uptake rate (twofold), as well as in the flux through the pentose phosphate (PP) pathway (4.8-fold), compared to the condition of 2.4 mmol L-1 h-1. At the highest OTR condition, a decrease in the NADPH/NADP+ ratio of threefold was observed, probably as a response to the high respiration rate induced by the respiratory protection of the nitrogenase under diazotrophic conditions, correlating with a high expression of the uncoupled respiratory chain genes (ndhII and cydA) and induction of the expression of the genes encoding the nitrogenase complex (nifH). CONCLUSIONS: We have demonstrated that changes in oxygen availability affect the internal redox state of the cell and carbon metabolic fluxes. This also has a strong impact on the TCA cycle and PP pathway as well as on alginate and P3HB biosynthetic fluxes.


Assuntos
Azotobacter vinelandii/metabolismo , Análise do Fluxo Metabólico , NADP/análise , NAD/análise , Oxigênio/metabolismo , Alginatos/metabolismo , Biomassa , Vias Biossintéticas/efeitos dos fármacos , Carbono/metabolismo , Ciclo do Ácido Cítrico/efeitos dos fármacos , Meios de Cultura/química , NAD/efeitos dos fármacos , NAD/metabolismo , NADP/efeitos dos fármacos , NADP/metabolismo , Oxirredução , Oxigênio/farmacologia , Via de Pentose Fosfato/efeitos dos fármacos
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