Leucine-Rich Diet Modulates the Metabolomic and Proteomic Profile of Skeletal Muscle during Cancer Cachexia

Background: Cancer-cachexia induces a variety of metabolic disorders, including skeletal muscle imbalance. Alternative therapy, as nutritional supplementation with leucine, shows a modulatory effect over tumour damage in vivo and in vitro. Method: Adult rats distributed into Control (C), Walker tumour-bearing (W), control fed a leucine-rich diet (L), and tumour-bearing fed a leucine-rich diet (WL) groups had the gastrocnemius muscle metabolomic and proteomic assays performed in parallel to in vitro assays. Results: W group presented an affected muscle metabolomic and proteomic profile mainly related to energy generation and carbohydrates catabolic processes, but leucine-supplemented group (WL) recovered the energy production. In vitro assay showed that cell proliferation, mitochondria number and oxygen consumption were higher under leucine effect than the tumour influence. Muscle proteomics results showed that the main affected cell component was mitochondria, leading to an impacted energy generation, including impairment in proteins of the tricarboxylic cycle and carbohydrates catabolic processes, which were modulated and improved by leucine treatment. Conclusion: In summary, we showed a beneficial effect of leucine upon mitochondria, providing information about the muscle glycolytic pathways used by this amino acid, where it can be associated with the preservation of morphometric parameters and consequent protection against the effects of cachexia

Structural basis of colchicine-site targeting acylhydrazones active against multidrug-resistant acute lymphoblastic leukemia

Tubulin is one of the best validated anti-cancer targets, but most anti-tubulin agents have unfavorable therapeutic indexes. Here, we characterized the tubulin-binding activity, the mechanism of action, and the in vivo anti-leukemia efficacy of three 3,4,5-trimethoxy-N-acylhydrazones. We show that all compounds target the colchicine-binding site of tubulin and that none is a substrate of ABC transporters. The crystal structure of the tubulin-bound N-(1′-naphthyl)-3,4,5-trimethoxybenzohydrazide (12) revealed steric hindrance on the T7 loop movement of β-tubulin, thereby rendering tubulin assembly incompetent. Using dose escalation and short-term repeated dose studies, we further report that this compound class is well tolerated to >100 mg/kg in mice. We finally observed that intraperitoneally administered compound 12 significantly prolonged the overall survival of mice transplanted with both sensitive and multidrug-resistant acute lymphoblastic leukemia (ALL) cells. Taken together, this work describes promising colchicine-site-targeting tubulin inhibitors featuring favorable therapeutic effects against ALL and multidrug-resistant cell2195109CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP305896/2013-0; 301596/2017-414/08247-8; 17/14737-6We thank Ganadería Fernando Díaz for calf brains for tubulin purification. The authors acknowledge networking contribution by the COST Action CM1407 “Challenging organic syntheses inspired by nature - from natural products chemistry to drug discovery.” J.F.D. is a member of the CIB Intramural Program “Molecular Machines for Better Life” (MACBET). N.M.C. was supported by a fellowship from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 14/08247-8, and 17/14737-6). J.A.Y. received a Productivity fellowship from the Brazilian National Counsel of Technological and Scientific Development (CNPq 305896/2013-0 and 301596/2017-4). This work was supported in part by grants BFU2016-75319-R (AEI/FEDER, UE) (J.F.D.) from Ministerio de Economía y Competitividad. The crystal structure work was supported by grants from the Swiss National Science Foundation (31003A_166608, to M.O.S.) and by the COST action CM1407 (to M.O.S.). Part of the in vivo work was supported by R01CA209829 and R01CA213912, Hyundai Hope On Wheels Scholar Grant, Bear Necessities Pediatric Cancer Foundation, Alex’s Lemonade Stand Foundation, the Four Diamonds Fund of the Pennsylvania State University College of Medicine, and the John Wawrynovic Leukemia Research Scholar Endowment (to S.D.

Addendum: Cruz, B., et al. c during Cancer Cachexia. Cancers 2020, 12, 1880

The authors wish to add the following statement in the Acknowledgements section of this paper [...

Integrative analysis to select cancer candidate biomarkers to targeted validation

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOTargeted proteomics has flourished as the method of choice for prospecting for and validating potential candidate biomarkers in many diseases. However, challenges still remain due to the lack of standardized routines that can prioritize a limited number of proteins to be further validated in human samples. To help researchers identify candidate biomarkers that best characterize their samples under study, a well-designed integrative analysis pipeline, comprising MS-based discovery, feature selection methods, clustering techniques, bioinformatic analyses and targeted approaches was performed using discovery-based proteomic data from the secretomes of three classes of human cell lines (carcinoma, melanoma and non-cancerous). Three feature selection algorithms, namely, Beta-binomial, Nearest Shrunken Centroids (NSC), and Support Vector Machine-Recursive Features Elimination (SVM-RFE), indicated a panel of 137 candidate biomarkers for carcinoma and 271 for melanoma, which were differentially abundant between the tumor classes. We further tested the strength of the pipeline in selecting candidate biomarkers by immunoblotting, human tissue microarrays, label-free targeted MS and functional experiments. In conclusion, the proposed integrative analysis was able to pre-qualify and prioritize candidate biomarkers from discovery-based proteomics to targeted MS.Targeted proteomics has flourished as the method of choice for prospecting for and validating potential candidate biomarkers in many diseases. However, challenges still remain due to the lack of standardized routines that can prioritize a limited number of proteins to be further validated in human samples. To help researchers identify candidate biomarkers that best characterize their samples under study, a well-designed integrative analysis pipeline, comprising MS-based discovery, feature selection methods, clustering techniques, bioinformatic analyses and targeted approaches was performed using discovery-based proteomic data from the secretomes of three classes of human cell lines (carcinoma, melanoma and non-cancerous). Three feature selection algorithms, namely, Beta-binomial, Nearest Shrunken Centroids (NSC), and Support Vector Machine-Recursive Features Elimination (SVM-RFE), indicated a panel of 137 candidate biomarkers for carcinoma and 271 for melanoma, which were differentially abundant between the tumor classes. We further tested the strength of the pipeline in selecting candidate biomarkers by immunoblotting, human tissue microarrays, label-free targeted MS and functional experiments. In conclusion, the proposed integrative analysis was able to pre-qualify and prioritize candidate biomarkers from discovery-based proteomics to targeted MS6414363543652FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO2009/54067-3; 2010/19278-0; 2011/22421-2; 2009/53839-2470567/2009-0; 470549/2011-4; 301702/2011-0; 470268/2013-

Metabolomics of methotrexate resistance in acute lymphoblastic leukemia

Orientadores: José Andrés Yunes, Ana Carolina de Mattos ZeriDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: O uso intensivo e combinado de diferentes quimioterápicos tem permitido a cura de 70-80% das leucemias linfóides agudas (LLA) da infância, sendo que a recaída da doença decorre em grande parte da resistência intrínseca das células leucêmicas à quimioterapia. Alguns dos quimioterápicos utilizados na LLA são inibidores metabólicos, como o metotrexato (MTX), antagonista do ácido fólico, que impede a divisão celular ao inibir a síntese de nucleotídeos. Em uma abordagem metabolômica, foi investigada a associação entre linhagens leucêmicas resistentes ou sensíveis ao MTX e metabólitos biondicadores de cada um destes fenótipos. Seis linhagens celulares B-derivadas e oito T-derivadas foram classificadas como sendo resistentes ou sensíveis ao MTX pelo método do 3-(4,5-dimetiltiazol-2il)-2,5-difenil brometo de tetrazolina (MTT) após 48h de co-cultura com diferentes concentrações da droga. Cinco linhagens foram classificadas como resistentes e nove como sensíveis ao MTX. Após 24h de cultura na presença ou ausência de MTX (ambos em triplicata), os metabólitos intracelulares das linhagens foram acessados por ressonância magnética nuclear (RMN) numa abordagem metabolômica. Ao total, oitenta e quatro metabólitos foram quantificados, dos quais 72 foram também identificados. A análise de componentes principais (PCA) não conseguiu segregar as amostras de acordo com sua resistência, ao passo que a análise discriminante por mínimos quadrados parciais (PLS-DA) foi efetiva nesta separação. Os metabólitos mais relevantes para a construção dos modelos de classificação quanto à resistência ao MTX, tanto para amostras tratadas quanto controles foram: ATP, dimetilglicina, fosfocolina e sarcosina (associados à resistência); carnitina, CB-09 (composto não identificado), colato, fumarato, glicocolato, lactato, malato e succinato (associados à sensibilidade ao MTX). A capacidade de classificar corretamente as amostras em sensíveis ou resistentes foi obtida com a construção de curvas da característica operativa do receptor (ROC) para os metabólitos individualmente. Os metabólitos com desempenho bom ou excelente na análise ROC (AUC>0,8) foram selecionados para comporem "testes diagnósticos" de classificação de amostras. De todas as combinações possíveis dentre os metabólitos selecionados, o teste que considerou a combinação de carnitina, sarcosina e succinato em amostras não tratadas com MTX apresentou sensibilidade de 100% (identificou todas as 15 amostras resistentes) e especificidade de 92,3% ao classificar corretamente 24 de 26 amostras sensíveis. O melhor teste diagnóstico para amostras tratadas com MTX considerou as concentrações de CB-MTX, glicocolato, sarcosina e succinato; apresentou sensibilidade de 100% (identificou as 15 amostras resistentes) e especificidade de 85,2%, equivocando-se na classificação de 4 dentre 27 amostras sensíveis. As concentrações metabólicas diferenciais apontaram para uma superativação dos metabolismos energético e de lipídeos em linhagens sensíveis ao MTX, ao passo que linhagens resistentes teriam superativado o metabolismo da glicina. As análises metabolômicas e de integração bioquímica dos metabólitos revelaram interações gênicas, enzimáticas e metabólicas que podem estar alteradas em linhagens sensíveis ou resistentes ao MTX, bem como permitiram a especulação sobre possíveis alvos moleculares que poderiam tornar sensíveis células resistentes ao quimioterápicoAbstract: The intensive use of different and combined chemotherapics has allowed curing 70-80% of pediatric acute lymphoblastic leukemia (ALL), and the relapse of the disease stems largely from the intrinsic resistance of leukemic cells to chemotherapy. Some of the chemotherapics used in ALL are metabolic inhibitors such as methotrexate (MTX), a folic acid antagonist, which prevents cell division by inhibiting the synthesis of nucleotides. The association between leukemic strains resistant or sensitive to MTX and the metabolites associated with each of these phenotypes were investigated. Six B- and eight T-derived cell lines were classified as resistant or sensitive to MTX by the 3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay, after 48h in co-culture with different concentrations of the drug. Five lineages were classified as resistant, and nine as sensitive to MTX. After 24 hours of culture in the presence or absence of MTX (both in triplicates), the intracellular metabolites of the lineages were assessed by nuclear magnetic resonance (NMR), in a metabolomic approach. In total, 84 metabolites were quantified, 72 of which were also identified. The principal component analysis (PCA) did not segregate the samples according to their resistance, whereas the supervised partial least square discriminate analysis (PLS-DA) was effective in this separation. ATP, dimethylglycine, sarcosine and phosphocholine were associated with MTX resistance in both models constructed for treated and untreated samples, whereas carnitine, CB-MTX (unidentified compound), cholate, fumarate, glycocholate, lactate, malate and succinate were associated with sensitivity to MTX. The ability to correctly classify the samples into sensitive or resistant groups was checked with the construction of the receiver operating characteristic (ROC) curves for metabolites individually. Metabolites with good or excellent performance in ROC analysis (AUC> 0.8) were selected to compose "diagnostic tests" for classifying samples. Of all the possible combinations among the selected metabolites, the test composed by the comination of carnitine, sarcosine and succinate in untreated samples exhibited sensitivity of 100% (identified all 15 resistant samples) and specificity of 92.3% in classifying correctly 24 of 26 sensitive samples. The best diagnostic test for samples treated with MTX took into consideration concentrations of CB-MTX, glycocholate, sarcosina, succinato. It had a sensitivity of 100% (identified 15 resistant samples) and specificity of 85.2%, classifying incorrectly 4 out of 27 sensitive samples. Differential metabolic concentrations pointed to an over activation of energy and lipids metabolism in MTX-sensitive strains, whereas resistant strains seemed to have overactive the glycine metabolism. Metabolomic and biochemical integration analysis revealed genetic, enzymatic and metabolic interactions that might be altered in strains sensitive or resistant to MTX, as well as allowed speculations about possible molecular targets on which intervention could make resistant cells susceptible to chemotherapyMestradoCiencias BiomedicasMestre em Ciências Médica

Methotrexate resistance is directly associated with glutathione concentration in acute lymphoblastic leukemia cell lines

Orientador: José Andrés YunesTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: A leucemia linfoide aguda (LLA) é o tipo de câncer mais comum da infância, correspondendo a 25% de todos os casos de câncer nesta faixa etária. Um dos quimioterápicos utilizados na terapia da LLA (e de doenças autoimunes como a artrite reumatoide) é o metotrexato (MTX), um antagonista do ácido fólico (antifolato). O mecanismo de ação do MTX enquanto quimioterápico é primariamente atribuído à inibição da enzima dihidrofato redutase, que sintetiza tetrahidrofolato a partir de dihidrofolato ¿ etapa fundamental na síntese de novo de nucleotídeos purínicos utilizados na divisão celular. Em artrite reumatoide, doses menores de MTX inibem a enzima 5-aminoimidazole-4-ribonucleotídeo-carboxamida formiltransferase (ATIC), o que culmina com a produção de altos níveis de adenosina, um potente anti-inflamatório. No entanto, diversos trabalhos recentes continuam a apresentar mecanismos e efeitos até então desconhecidos por meio dos quais o MTX atua no ambiente celular, atestando que os mecanismos de ação do MTX parecem ser tão múltiplos quanto complexos. Utilizando diversas técnicas de biologia molecular, este trabalho procurou expandir o conhecimento existente da ação do MTX em LLA. Para isso, diversos parâmetros biológicos foram mensurados sob efeito ou não do MTX em um painel de 13 linhagens celulares de LLA. Foram realizados ensaios de proliferação, estudos metabolômicos, de sinergismo com drogas, quantificação da respiração celular e da produção de espécies reativas de oxigênio (ROS), além da medição da ativação da via de sinalização do NF-?B. A resistência das linhagens ao MTX em 48 h de tratamento (mas não em 96 h) mostrou-se relacionada ao tempo de duplicação celular. O tratamento com MTX alterou a concentração de 28 metabólitos intracelulares, com destaque para o consistente aumento da glicina. As concentrações intracelulares de asparagina, guanosina e glutationa ¿ inclusive a expressão de genes da via desta última ¿ se mostraram associadas com a resistência ao MTX. A suplementação do meio de cultura com N-acetilcisteína, um metabólito precursor de glutationa, promoveu proliferação e resistência ao MTX; entretanto, o tratamento das células com piperlongumina ou peróxido de hidrogênio, dois sequestradores de glutationa e promotores de ROS, não potencializou o efeito do MTX. O MTX induziu pequenas quantidades de ROS nas linhagens de LLA. Após 1 h com a droga, as células mais sensíveis ao antifolato produziram mais ROS que as mais resistentes; em 3 h essa dinâmica se inverteu e assim permaneceu até as 6 h. O consumo de oxigênio das linhagens não se mostrou associado com a resistência ao MTX e um teste preliminar mostrou que o MTX não alterou a respiração celular. O MTX ativou o fator de transcrição NF-?B em algumas linhagens de LLA e, curiosamente, a ativação deste fator de transcrição pelo fator de necrose tumoral alfa (TNF-?) mostrou-se positivamente correlacionada com a resistência das linhagens leucêmicas ao MTX. Uma vasta revisão bibliográfica permitiu tanto a integração dos resultados obtidos ao conhecimento mais atual sobre o assunto, quanto o apontamento de novos caminhos a serem explorados em etapas futurasAbstract: Acute lymphoblastic leukemia (ALL) is the most common type of childhood cancer, accounting for 25% of all cancers in this age group. One of the chemotherapeutics used in the therapy of ALL (and autoimmune diseases such as rheumatoid arthritis) is methotrexate (MTX), a folic acid antagonist (antifolate). As a chemotherapeutic agent, MTX's mechanism of action is primarily attributed to the inhibition of the dihydrophate reductase enzyme, which synthesizes tetrahydrofolate from dihydrofolate ¿ a key step in the de novo synthesis of purine nucleotides used in cell division. In rheumatoid arthritis, lower doses of MTX inhibit the 5-aminoimidazole-4-ribonucleotide-carboxamide formyltransferase (ATIC) enzyme, which culminates in the production of high levels of adenosine, a potent anti-inflammatory. However, recent works continue to present previously unknown mechanisms and effects through which MTX acts within the cell, attesting that MTX's mechanisms of action appear to be as multiple as complex. Using several techniques of molecular biology, this work sought to expand the existing knowledge of the action of MTX in ALL. For this purpose, several biological parameters were measured under or without MTX treatment in a panel of 13 ALL cell lines. Proliferation tests, metabolic studies, drug synergism, quantification of cellular respiration and the production of reactive oxygen species (ROS) were performed, as well as the measurement of the activation of the NF-?B signaling pathway. Resistance of the MTX strains within 48 h of treatment (but not 96 h) was related to the proliferation rate of the cells. Treatment with MTX altered the concentration of 28 intracellular metabolites, highlights for a consistent increase in glycine concentration. Intracellular concentrations of asparagine, guanosine and glutathione ¿ including the expression of genes from glutathione pathway ¿ were associated with MTX resistance. Supplementation of the culture medium with N-acetylcysteine, a precursor metabolite of glutathione, promoted proliferation and resistance to MTX; however, cell treatment with piperlongumine or hydrogen peroxide, two glutathione scavengers and ROS promoters, did not potentiate the effect of MTX. MTX produced small amounts of ROS in ALL cell lines. In 1 h treatment, MTX most sensitive cells produced more ROS than the most resistant ones; after 3 h this dynamics inverted and thus remained until 6 h. The oxygen uptake of the cell lines was not associated with MTX resistance and a preliminary test showed that MTX did not alter cellular respiration. MTX activated the transcription factor NF-?B in some ALL cell lines and, interestingly, the activation of this transcription factor by tumor necrosis factor alpha (TNF-?) was positively correlated with the resistance of leukemic lines to MTX. A wide bibliographic review allowed both the integration of the obtained results to the most current knowledge on the subject, and the identification of new paths to be explored in future stagesDoutoradoGenetica MedicaDoutor em Ciências2012/11952-0FAPES

The Expression and Activation of the NF-κB Pathway Correlate with Methotrexate Resistance and Cell Proliferation in Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Although its prognosis continually improves with time, a significant proportion of patients still relapse from the disease because of the leukemia’s resistance to therapy. Methotrexate (MTX), a folic-acid antagonist, is a chemotherapy agent commonly used against ALL and as an immune-system suppressant for rheumatoid arthritis that presents multiple and complex mechanisms of action and resistance. Previous studies have shown that MTX modulates the nuclear factor kappa B (NF-κB) pathway, an important family of transcription factors involved in inflammation, immunity, cell survival, and proliferation which are frequently hyperactivated in ALL. Using a gene set enrichment analysis of publicly available gene expression data from 161 newly diagnosed pediatric ALL patients, we found the Tumor necrosis factor α (TNF-α) signaling pathway via NF-κB to be the most enriched Cancer Hallmark in MTX-poor-responder patients. A transcriptomic analysis using a panel of ALL cell lines (six B-cell precursor acute lymphoblastic leukemia and seven T-cell acute lymphoblastic leukemia) also identified the same pathway as differentially enriched among MTX-resistant cell lines, as well as in slowly dividing cells. To better understand the crosstalk between NF-κB activity and MTX resistance, we genetically modified the cell lines to express luciferase under an NF-κB-binding-site promoter. We observed that the fold change in NF-κB activity triggered by TNF-α (but not MTX) treatment correlated with MTX resistance and proliferation across the lines. At the individual gene level, NFKB1 expression was directly associated with a poorer clinical response to MTX and with both an increased TNF-α-triggered NF-κB activation and MTX resistance in the cell lines. Despite these results, the pharmacological inhibition (using BAY 11-7082 and parthenolide) or stimulation (using exogenous TNF-α supplementation) of the NF-κB pathway did not alter the MTX resistance of the cell lines significantly, evidencing a complex interplay between MTX and NF-κB in ALL

Xanthan Gum Removal for 1H-NMR Analysis of the Intracellular Metabolome of the Bacteria Xanthomonas axonopodis pv. citri 306

Xanthomonas is a genus of phytopathogenic bacteria, which produces a slimy, polysaccharide matrix known as xanthan gum, which involves, protects and helps the bacteria during host colonization. Although broadly used as a stabilizer and thickener in the cosmetic and food industries, xanthan gum can be a troubling artifact in molecular investigations due to its rheological properties. In particular, a cross-reaction between reference compounds and the xanthan gum could compromise metabolic quantification by NMR spectroscopy. Aiming at an efficient gum extraction protocol, for a 1H-NMR-based metabolic profiling study of Xanthomonas, we tested four different interventions on the broadly used methanol-chloroform extraction protocol for the intracellular metabolic contents observation. Lower limits for bacterial pellet volumes for extraction were also probed, and a strategy is illustrated with an initial analysis of X. citri’s metabolism by 1H-NMR spectroscopy

Early metabolic response after resistance exercise with blood flow restriction in well-trained men: a metabolomics approach

The present study aimed to compare the early metabolic response between high-load resistance exercise (HL-RE) and low-load resistance exercise with blood flow restriction (LL-BFR). Nine young well-trained men participated in a randomized crossover design in which each subject completed LL-BFR, HL-RE or condition control (no exercise) with a one-week interval between them. Blood samples were taken immediately before and five minutes after the exercise sessions. Nuclear magnetic resonance (NMR) spectroscopy identified and quantified 48 metabolites, six of which presented significant changes among the exercise protocols. The HL-RE promoted a higher increase in pyruvate, lactate and alanine compared to the LL-BFR and the control. HL-RE and LL-BFR promoted a higher increase in succinate compared to the control, however, there was no difference between HL-RE and LL-BFR. Also, while there was no difference in acetoacetate between HL-RE and LL-BFR, a greater decrease was observed in both compared to the control. Finally, LL-BFR promoted a greater decrease in choline compared to the control. In conclusion, this study provides by metabolomics a new insight in metabolic response between LL-BFR and HL-RE by demonstrating a distinct response to some metabolites that are not commonly analyzed.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author