Plasma metabolomics of oral squamous cell carcinomas based on NMR and MS approaches provides biomarker identification and survival prediction.

Metabolomics has proven to be an important omics approach to understand the molecular pathways underlying the tumour phenotype and to identify new clinically useful markers. The literature on cancer has illustrated the potential of this approach as a diagnostic and prognostic tool. The present study aimed to analyse the plasma metabolic profile of patients with oral squamous cell carcinoma (OSCC) and controls and to compare patients with metastatic and primary tumours at different stages and subsites using nuclear magnetic resonance and mass spectrometry. To our knowledge, this is the only report that compared patients at different stages and subsites and replicates collected in diverse institutions at different times using these methodologies. Our results showed a plasma metabolic OSCC profile suggestive of abnormal ketogenesis, lipogenesis and energy metabolism, which is already present in early phases but is more evident in advanced stages of the disease. Reduced levels of several metabolites were also associated with an unfavorable prognosis. The observed metabolomic alterations may contribute to inflammation, immune response inhibition and tumour growth, and may be explained by four nonexclusive views-differential synthesis, uptake, release, and degradation of metabolites. The interpretation that assimilates these views is the cross talk between neoplastic and normal cells in the tumour microenvironment or in more distant anatomical sites, connected by biofluids, signalling molecules and vesicles. Additional population samples to evaluate the details of these molecular processes may lead to the discovery of new biomarkers and novel strategies for OSCC prevention and treatment.

Glutathione levels are associated with methotrexate resistance in acute lymphoblastic leukemia cell lines.

Introduction: Methotrexate (MTX), a folic acid antagonist and nucleotide synthesis inhibitor, is a cornerstone drug used against acute lymphoblastic leukemia (ALL), but its mechanism of action and resistance continues to be unraveled even after decades of clinical use. Methods: To better understand the mechanisms of this drug, we accessed the intracellular metabolic content of 13 ALL cell lines treated with MTX by 1H-NMR, and correlated metabolome data with cell proliferation and gene expression. Further, we validated these findings by inhibiting the cellular antioxidant system of the cells in vitro and in vivo in the presence of MTX. Results: MTX altered the concentration of 31 out of 70 metabolites analyzed, suggesting inhibition of the glycine cleavage system, the pentose phosphate pathway, purine and pyrimidine synthesis, phospholipid metabolism, and bile acid uptake. We found that glutathione (GSH) levels were associated with MTX resistance in both treated and untreated cells, suggesting a new constitutive metabolic-based mechanism of resistance to the drug. Gene expression analyses showed that eight genes involved in GSH metabolism were correlated to GSH concentrations, 2 of which (gamma-glutamyltransferase 1 [GGT1] and thioredoxin reductase 3 [TXNRD3]) were also correlated to MTX resistance. Gene set enrichment analysis (GSEA) confirmed the association between GSH metabolism and MTX resistance. Pharmacological inhibition or stimulation of the main antioxidant systems of the cell, GSH and thioredoxin, confirmed their importance in MTX resistance. Arsenic trioxide (ATO), a thioredoxin inhibitor used against acute promyelocytic leukemia, potentiated MTX cytotoxicity in vitro in some of the ALL cell lines tested. Likewise, the ATO+MTX combination decreased tumor burden and extended the survival of NOD scid gamma (NSG) mice transplanted with patient-derived ALL xenograft, but only in one of four ALLs tested. Conclusion: Altogether, our results show that the cellular antioxidant defense systems contribute to leukemia resistance to MTX, and targeting these pathways, especially the thioredoxin antioxidant system, may be a promising strategy for resensitizing ALL to MTX.

Clinical Metabolomics Identifies Blood Serum Branched Chain Amino Acids as Potential Predictive Biomarkers for Chronic Graft vs. Host Disease.

The allogeneic hematopoietic stem cell transplantation procedure-the only curative therapy for many types of hematological cancers-is increasing, and graft vs. host disease (GVHD) is the main cause of morbidity and mortality after transplantation. Currently, GVHD diagnosis is clinically performed. Whereas, biomarker panels have been developed for acute GVHD (aGVHD), there is a lack of information about the chronic form (cGVHD). Using nuclear magnetic resonance (NMR) and gas chromatography coupled to time-of-flight (GC-TOF) mass spectrometry, this study prospectively evaluated the serum metabolome of 18 Brazilian patients who had undergone allogeneic hematopoietic stem cell transplantation (HSCT). We identified and quantified 63 metabolites and performed the metabolomic profile on day -10, day 0, day +10 and day +100, in reference to day of transplantation. Patients did not present aGVHD or cGVHD clinical symptoms at sampling times. From 18 patients analyzed, 6 developed cGVHD. The branched-chain amino acids (BCAAs) leucine and isoleucine were reduced and the sulfur-containing metabolite (cystine) was increased at day +10 and day +100. The area under receiver operating characteristics (ROC) curves was higher than 0.79. BCAA findings were validated by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in 49 North American patients at day +100; however, cystine findings were not statistically significant in this patient set. Our results highlight the importance of multi-temporal and multivariate biomarker panels for predicting and understanding cGVHD.

Leucine-rich diet alters the 1H-NMR based metabolomic profile without changing the Walker-256 tumour mass in rats.

BACKGROUND: Cachexia is one of the most important causes of cancer-related death. Supplementation with branched-chain amino acids, particularly leucine, has been used to minimise loss of muscle tissue, although few studies have examined the effect of this type of nutritional supplementation on the metabolism of the tumour-bearing host. Therefore, the present study evaluated whether a leucine-rich diet affects metabolomic derangements in serum and tumour tissues in tumour-bearing Walker-256 rats (providing an experimental model of cachexia). METHODS: After 21 days feeding Wistar female rats a leucine-rich diet, distributed in L-leucine and LW-leucine Walker-256 tumour-bearing groups, we examined the metabolomic profile of serum and tumour tissue samples and compared them with samples from tumour-bearing rats fed a normal protein diet (C - control; W - tumour-bearing groups). We utilised 1H-NMR as a means to study the serum and tumour metabolomic profile, tumour proliferation and tumour protein synthesis pathway. RESULTS: Among the 58 serum metabolites examined, we found that 12 were altered in the tumour-bearing group, reflecting an increase in activity of some metabolic pathways related to energy production, which diverted many nutrients toward tumour growth. Despite displaying increased tumour cell activity (i.e., higher Ki-67 and mTOR expression), there were no differences in tumour mass associated with changes in 23 metabolites (resulting from valine, leucine and isoleucine synthesis and degradation, and from the synthesis and degradation of ketone bodies) in the leucine-tumour group. This result suggests that the majority of nutrients were used for host maintenance. CONCLUSION: A leucine rich-diet, largely used to prevent skeletal muscle loss, did not affect Walker 256 tumour growth and led to metabolomic alterations that may partially explain the positive effects of leucine for the whole tumour-bearing host.

A redox 2-Cys mechanism regulates the catalytic activity of divergent cyclophilins.

The citrus (Citrus sinensis) cyclophilin CsCyp is a target of the Xanthomonas citri transcription activator-like effector PthA, required to elicit cankers on citrus. CsCyp binds the citrus thioredoxin CsTdx and the carboxyl-terminal domain of RNA polymerase II and is a divergent cyclophilin that carries the additional loop KSGKPLH, invariable cysteine (Cys) residues Cys-40 and Cys-168, and the conserved glutamate (Glu) Glu-83. Despite the suggested roles in ATP and metal binding, the functions of these unique structural elements remain unknown. Here, we show that the conserved Cys residues form a disulfide bond that inactivates the enzyme, whereas Glu-83, which belongs to the catalytic loop and is also critical for enzyme activity, is anchored to the divergent loop to maintain the active site open. In addition, we demonstrate that Cys-40 and Cys-168 are required for the interaction with CsTdx and that CsCyp binds the citrus carboxyl-terminal domain of RNA polymerase II YSPSAP repeat. Our data support a model where formation of the Cys-40-Cys-168 disulfide bond induces a conformational change that disrupts the interaction of the divergent and catalytic loops, via Glu-83, causing the active site to close. This suggests a new type of allosteric regulation in divergent cyclophilins, involving disulfide bond formation and a loop-displacement mechanism.

Southern Brazilian autumnal propolis shows anti-angiogenic activity: an in vitro and in vivo study.

The present study focuses on the effects of a hydro-alcoholic propolis extract collected in autumn (2010) in Santa Catarina State (Southern Brazil), on the angiogenesis, using in vitro and in vivo models. Cultures of human umbilical vein endothelial cells were used to assess the effects of propolis on viability, proliferation, and cell migration, as well as capillary tube formation. The propolis autumnal extracts significantly decreased the cell viability, based on CC50 values, which decreased (56%) from 297 to 130 µg/ml in 24 h and 72 h of treatment, respectively (cytotoxicity assay). The process of cell proliferation was decreased by 81.7 to 48.4% due to exposure (72 h) to 130-180 µg/ml of propolis extract, as compared with control (vehicle). In these same concentrations, the cell migration was also reduced by 39.6 to 12.6%, respectively (versus control). Furthermore, autumnal propolis extract (100-200 µg/ml) inhibited the tube-like structure formation (tubulogenesis) of endothelial cells on Matrigel™ (16.2-69.9% inhibition). The treatments performed in vivo with administration of 450 mg propolis.kg(-1) inhibited both angiogenesis and vasculogenesis by 82.3 and 66.5% in the chorioallantoic and yolk-sac membranes of chick embryos. Furthermore, by means of UV-vis-spectrophotometry, reverse phase-high performance liquid chromatography analysis and 1D and 2D-nuclear magnetic resonance experiments reveal higher contents of flavonoids and total phenolic compounds with predominance of the flavonol quercetin and the phenolic acids, e.g., gallic acid, protocatechuic acid and chlorogenic acid in the propolis hydro-alcoholic extract. Our findings related to the anti-proliferative, anti-migration, and anti-tubulogenic actions on human umbilical vein endothelial cell line agree with the inhibitory effects in the in vivo vessel formation exerted by propolis extract under study. The results also suggest that autumnal propolis extract might be potentially instrumental in providing alternative tools for angiogenic disease therapeutics.

Identification of novel interaction between ADAM17 (a disintegrin and metalloprotease 17) and thioredoxin-1.

ADAM17, which is also known as TNFα-converting enzyme, is the major sheddase for the EGF receptor ligands and is considered to be one of the main proteases responsible for the ectodomain shedding of surface proteins. How a membrane-anchored proteinase with an extracellular catalytic domain can be activated by inside-out regulation is not completely understood. We characterized thioredoxin-1 (Trx-1) as a partner of the ADAM17 cytoplasmic domain that could be involved in the regulation of ADAM17 activity. We induced the overexpression of the ADAM17 cytoplasmic domain in HEK293 cells, and ligands able to bind this domain were identified by MS after protein immunoprecipitation. Trx-1 was also validated as a ligand of the ADAM17 cytoplasmic domain and full-length ADAM17 recombinant proteins by immunoblotting, immunolocalization, and solid phase binding assay. In addition, using nuclear magnetic resonance, it was shown in vitro that the titration of the ADAM17 cytoplasmic domain promotes changes in the conformation of Trx-1. The MS analysis of the cross-linked complexes showed cross-linking between the two proteins by lysine residues. To further evaluate the functional role of Trx-1, we used a heparin-binding EGF shedding cell model and observed that the overexpression of Trx-1 in HEK293 cells could decrease the activity of ADAM17, activated by either phorbol 12-myristate 13-acetate or EGF. This study identifies Trx-1 as a novel interaction partner of the ADAM17 cytoplasmic domain and suggests that Trx-1 is a potential candidate that could be involved in ADAM17 activity regulation.

The repeat domain of the type III effector protein PthA shows a TPR-like structure and undergoes conformational changes upon DNA interaction.

Many plant pathogenic bacteria rely on effector proteins to suppress defense and manipulate host cell mechanisms to cause disease. The effector protein PthA modulates the host transcriptome to promote citrus canker. PthA possesses unusual protein architecture with an internal region encompassing variable numbers of near-identical tandem repeats of 34 amino acids termed the repeat domain. This domain mediates protein-protein and protein-DNA interactions, and two polymorphic residues in each repeat unit determine DNA specificity. To gain insights into how the repeat domain promotes protein-protein and protein-DNA contacts, we have solved the structure of a peptide corresponding to 1.5 units of the PthA repeat domain by nuclear magnetic resonance (NMR) and carried out small-angle X-ray scattering (SAXS) and spectroscopic studies on the entire 15.5-repeat domain of PthA2 (RD2). Consistent with secondary structure predictions and circular dichroism data, the NMR structure of the 1.5-repeat peptide reveals three α-helices connected by two turns that fold into a tetratricopeptide repeat (TPR)-like domain. The NMR structure corroborates the theoretical TPR superhelix predicted for RD2, which is also in agreement with the elongated shape of RD2 determined by SAXS. Furthermore, RD2 undergoes conformational changes in a pH-dependent manner and upon DNA interaction, and shows sequence similarities to pentatricopeptide repeat (PPR), a nucleic acid-binding motif structurally related to TPR. The results point to a model in which the RD2 structure changes its compactness as it embraces the DNA with the polymorphic diresidues facing the interior of the superhelix oriented toward the nucleotide bases.

Structure, dynamics, and RNA interaction analysis of the human SBDS protein.

Shwachman-Bodian-Diamond syndrome is an autosomal recessive genetic syndrome with pleiotropic phenotypes, including pancreatic deficiencies, bone marrow dysfunctions with increased risk of myelodysplasia or leukemia, and skeletal abnormalities. This syndrome has been associated with mutations in the SBDS gene, which encodes a conserved protein showing orthologs in Archaea and eukaryotes. The Shwachman-Bodian-Diamond syndrome pleiotropic phenotypes may be an indication of different cell type requirements for a fully functional SBDS protein. RNA-binding activity has been predicted for archaeal and yeast SBDS orthologs, with the latter also being implicated in ribosome biogenesis. However, full-length SBDS orthologs function in a species-specific manner, indicating that the knowledge obtained from model systems may be of limited use in understanding major unresolved issues regarding SBDS function, namely, the effect of mutations in human SBDS on its biochemical function and the specificity of RNA interaction. We determined the solution structure and backbone dynamics of the human SBDS protein and describe its RNA binding site using NMR spectroscopy. Similarly to the crystal structures of Archaea, the overall structure of human SBDS comprises three well-folded domains. However, significant conformational exchange was observed in NMR dynamics experiments for the flexible linker between the N-terminal domain and the central domain, and these experiments also reflect the relative motions of the domains. RNA titrations monitored by heteronuclear correlation experiments and chemical shift mapping analysis identified a classic RNA binding site at the N-terminal FYSH (fungal, Yhr087wp, Shwachman) domain that concentrates most of the mutations described for the human SBDS.

Deuterium/hydrogen exchange factors measured by solution nuclear magnetic resonance spectroscopy as indicators of the structure and topology of membrane proteins.

Deuterium/hydrogen exchange factors (chi) were measured for the backbone amide sites of the membrane-bound forms of the 50-residue fd coat protein and the 23-residue magainin2 peptide in lipid micelles by solution nuclear magnetic resonance spectroscopy. By combining kinetic and thermodynamic effects, deuterium/hydrogen exchange factors overcome the principal limitations encountered in the measurements of kinetic protection factors and thermodynamic fractionation factors for membrane proteins. The magnitudes of the exchange factors can be correlated with the structure and topology of membrane-associated polypeptides. In fd coat protein, residues in the transmembrane helix have exchange factors that are substantially smaller than those in the amphipathic surface helix or the loop connecting the two helices. For the amphipathic helical peptide, magainin2, the exchange factors of residues exposed to the solvent are appreciably larger than those that face the hydrocarbon portion of membrane bilayers. These examples demonstrate that deuterium/hydrogen exchange factors can be measured by solution NMR spectroscopy and used to identify residues in transmembrane helices as well as to determine the polarity of amphipathic helices in membrane proteins.