Chronic lactate exposure promotes cardiomyocyte cytoskeleton remodelling.

We investigated the effect of growing on lactate instead of glucose in human cardiomyocyte assessing their viability, cell cycle activity, oxidative stress and metabolism by a proteomic and metabolomic approach. In previous studies performed on elite players, we found that adaptation to exercise is characterized by a chronic high plasma level of lactate. Lactate is considered not only an energy source but also a signalling molecule and is referred as "lactormone"; heart is one of the major recipients of exogenous lactate. With this in mind, we used a cardiac cell line AC16 to characterize the lactate metabolic profile and investigate the metabolic flexibility of the heart. Interestingly, our data indicated that cardiomyocytes grown on lactate (72 h) show change in several proteins and metabolites linked to cell hypertrophy and cytoskeleton remodelling. The obtained results could help to understand the effect of this metabolite on heart of high-performance athletes.

Cytotoxic auranofin analogues bearing phosphine, arsine and stibine ligands: A study on the possible role of the ligand on the biological activity.

Three gold(I) linear compounds, sharing the general formula [AuI(LPh3)], have been synthesized and characterized. The nature of the ligand has been modified by moving down among some of the elements of group 15, i.e. phosphorus, arsenic and antimony. The structures of derived compounds have been solved through XRD and the reactivity behaviour towards selected biomolecules has been investigated through a multi-technique approach involving NMR, high-resolution mass spectrometry and IR. Moreover, the biological activity of the investigated compounds has been comparatively analyzed through classical methodologies and the disclosed differences are discussed in detail.

STAT3 Signalling Drives LDH Up-Regulation and Adiponectin Down-Regulation in Cachectic Adipocytes.

Cachexia is a devastating pathology that worsens the quality of life and antineoplastic treatment outcomes of oncologic patients. Herein, we report that the secretome from murine colon carcinoma CT26 induces cachectic features in both murine and human adipocytes that are associated with metabolic alterations such as enhanced lactate production and decreased oxygen consumption. The use of oxamate, which inhibits lactate dehydrogenase activity, hinders the effects induced by CT26 secretome. Interestingly, the CT26 secretome elicits an increased level of lactate dehydrogenase and decreased expression of adiponectin. These modifications are driven by the STAT3 signalling cascade since the inhibition of STAT3 with WP1066 impedes the formation of the cachectic condition and the alteration of lactate dehydrogenase and adiponectin levels. Collectively, these findings show that STAT3 is responsible for the altered lactate dehydrogenase and adiponectin levels that, in turn, could participate in the worsening of this pathology and highlight a step forward in the comprehension of the mechanisms underlying the onset of the cachectic condition in adipocytes.

The effects of two cytotoxic gold(i) carbene compounds on the metabolism of A2780 ovarian cancer cells: mechanistic inferences through NMR analysis.

NMR metabolomics is a powerful tool to characterise the changes in cancer cell metabolism elicited by anticancer drugs. Here, the large metabolic alterations produced by two cytotoxic gold carbene compounds in A2780 ovarian cancer cells are described and discussed in comparison to auranofin, in the frame of the available mechanistic knowledge.

A complex bearing TSPO PIGA ligand coordinated to the [Au(PEt3)]+ pharmacophore is highly cytotoxic against ovarian cancer cells.

Auranofin ([1-(thio-κS)-ß-D-glucopyranose-2,3,4,6-tetraacetato](triethylphosphine)-gold) is a leading gold-based drug clinically used to treat arthritis. In the last years, it entered various drug reprofiling programs, and it has been found promising against various forms of tumor, including ovarian cancer. Evidence showed as its antiproliferative profile mainly depends on the inhibition of thioredoxin reductase (TrxR), being this mitochondrial system its main target. In this context, we report here the synthesis and biological evaluation of a novel complex designed as auranofin analogue obtained through the conjugation of a phenylindolylglyoxylamide ligand (which belongs to the so-called PIGA TSPO ligand family) with the auranofin-derived cationic fragment [Au(PEt3)]+. This complex is characterized by two parts. The phenylindolylglyoxylamide moiety, owing to its high affinity for TSPO (in the low nM range) should drive the compound to target mitochondria, whereas the [Au(PEt3)]+ cation is the actual anticancer-active molecular fragment. Overall, we wanted to offer the proof-of-concept that by coupling PIGA ligands to anticancer gold active moieties, it is possible to preserve and even improve anticancer effects, opening the avenue to a reliable approach for targeted therapy.

Chemical Modification of Auranofin Yields a New Family of Anticancer Drug Candidates: The Gold(I) Phosphite Analogues.

A panel of four novel gold(I) complexes, inspired by the clinically established gold drug auranofin (1-Thio-ß-D-glucopyranosatotriethylphosphine gold-2,3,4,6-tetraacetate), was prepared and characterized. All these compounds feature the replacement of the triethylphosphine ligand of the parent compound auranofin with a trimethylphosphite ligand. The linear coordination around the gold(I) center is completed by Cl-, Br-, I- or by the thioglucose tetraacetate ligand (SAtg). The in-solution behavior of these gold compounds as well as their interactions with some representative model proteins were comparatively analyzed through 31PNMR and ESI-MS measurements. Notably, all panel compounds turned out to be stable in aqueous media, but significant differences with respect to auranofin were disclosed in their interactions with a few leading proteins. In addition, the cytotoxic effects produced by the panel compounds toward A2780, A2780R and SKOV-3 ovarian cancer cells were quantitated and found to be in the low micromolar range, since the IC50 of all compounds was found to be between 1 µM and 10 µM. Notably, these novel gold complexes showed large and similar inhibition capabilities towards the key enzyme thioredoxin reductase, again comparable to those of auranofin. The implications of these results for the discovery of new and effective gold-based anticancer agents are discussed.

Chronic Training Induces Metabolic and Proteomic Response in Male and Female Basketball Players: Salivary Modifications during In-Season Training Programs.

The aim of this study was to characterize the salivary proteome and metabolome of highly trained female and male young basketball players, highlighting common and different traits. A total of 20 male and female basketball players (10 female and 10 male) and 20 sedentary control subjects (10 female and 10 male) were included in the study. The athletes exercised at least five times per week for 2 h per day. Saliva samples were collected mid-season, between 9:00 and 11:00 a.m. and away from sport competition. The proteome and metabolome were analyzed by using 2DE and GC-MS techniques, respectively. A computerized 2DE gel image analysis revealed 43 spots that varied in intensity among groups. Between these spots, 10 (23.2%) were differentially expressed among male athletes and controls, 22 (51.2%) between female basketball players and controls, 11 spots (25.6%) between male and female athletes, and 13 spots (30.2%) between male and female controls. Among the proteins identified were Immunoglobulin, Alpha-Amylase, and Dermcidin, which are inflammation-related proteins. In addition, several amino acids, such as glutamic acid, lysine, ornithine, glycine, tyrosine, threonine, and valine, were increased in trained athletes. In this study, we highlight that saliva is a useful biofluid to assess athlete performance and confirm that the adaptation of men and women to exercise has some common features, but also some different sex-specific behaviors, including differential amino acid utilization and expression of inflammation-related proteins, which need to be further investigated. Moreover, in the future, it will be interesting to examine the influence of sport-type on these differences.

Preliminary results indicate that regular training induces high protection against oxidative stress in basketball players compared to soccer.

In elite athlete several metabolic changes occur during regular training. These modifications are associated with changes in blood metabolic profile and can lead to adaptive mechanisms aimed at establish a new dynamic equilibrium, which guarantees better performance. The goal of this study was to characterize the plasma metabolic profile and redox homeostasis, in athletes practicing two different team sports such as soccer and basketball in order to identify potential metabolic pathways underlying the differences in training programs. A cohort of 30 male, 20 professional players (10 soccer and 10 basketballs) and 10 sedentary males as control were enrolled in the study. Plasma redox balance, metabolites and adiponectin were determined. The results show low levels of oxidative species (25.5%), with both high antioxidant capacity (17.6%) and adiponectin level (64.4%) in plasma from basketball players, in comparison to soccer players. Metabolic analysis indicates in basketball players a significant high plasma level of amino acids Valine and Ornithine both involved in redox homeostasis and anti-inflammatory metabolism.

Metal-Based Complexes in Cancer Treatment.

Metal-based complexes contribute a vital part to the available arsenal of cytotoxic agents today [...].

Pyruvate prevents the onset of the cachectic features and metabolic alterations in myotubes downregulating STAT3 signaling.

Cachexia is a systemic disease associated with several pathologies, including cancer, that leads to excessive weight loss due to enhanced protein degradation. Previously, we showed that cachectic features in myotubes are provoked by a metabolic shift toward lactic fermentation. Our previous results led us to hyphotesise that increasing pyruvate concentration could impede the metabolic modifications responsible for induction of cachexia in myotubes. Here, we demonstrated that the addition of sodium pyruvate in conditioned media from CT26 colon cancer cells (CM CT26) prevents the onset of either phenotypic and metabolic cachectic features. Myotubes treated with CM CT26 containing sodium pyruvate show a phenotype similar to the healthy counterpart and display lactate production, oxygen consumption, and pyruvate dehydrogenase activity as control myotubes. The use of the Mitochondrial Pyruvate Carrier inhibitor UK5099, highlights the importance of mitochondrial pyruvate amount in the prevention of cachexia. Indeed, UK5099-treated myotubes show cachectic features as those observed in myotubes treated with CM CT26. Finally, we found that sodium pyruvate is able to decrease STAT3 phosphorylation level, a signaling pathway involved in the induction of cachexia in myotubes. Collectively, our results show that cachexia in myotubes could be prevented by the utilization of sodium pyruvate which impedes the metabolic modifications responsible for the acquisition of the cachectic features.

The effects of two gold-N-heterocyclic carbene (NHC) complexes in ovarian cancer cells: a redox proteomic study.

PURPOSE: Ovarian cancer is the fifth leading cause of cancer-related deaths in women. Standard treatment consists of tumor debulking surgery followed by platinum and paclitaxel chemotherapy; yet, despite the initial response, about 70-75% of patients develop resistance to chemotherapy. Gold compounds represent a family of very promising anticancer drugs. Among them, we previously investigated the cytotoxic and pro-apoptotic properties of Au(NHC) and Au(NHC)2PF6, i.e., a monocarbene gold(I) complex and the corresponding bis(carbene) complex. Gold compounds are known to alter the redox state of cells interacting with free cysteine and selenocysteine residues of several proteins. Herein, a redox proteomic study has been carried out to elucidate the mechanisms of cytotoxicity in A2780 human ovarian cancer cells. METHODS: A biotinylated iodoacetamide labeling method coupled with mass spectrometry was used to identify oxidation-sensitive protein cysteines. RESULTS: Gold carbene complexes cause extensive oxidation of several cellular proteins; many affected proteins belong to two major functional classes: carbohydrate metabolism, and cytoskeleton organization/cell adhesion. Among the affected proteins, Glyceraldehyde-3-phosphate dehydrogenase inhibition was proved by enzymatic assays and by ESI-MS studies. We also found that Au(NHC)2PF6 inhibits mitochondrial respiration impairing complex I function. Concerning the oxidized cytoskeletal proteins, gold binding to the free cysteines of actin was demonstrated by ESI-MS analysis. Notably, both gold compounds affected cell migration and invasion. CONCLUSIONS: In this study, we deepened the mode of action of Au(NHC) and Au(NHC)2PF6, identifying common cellular targets but confirming their different influence on the mitochondrial function.

Redox proteome analysis of auranofin exposed ovarian cancer cells (A2780).

The effects of Auranofin (AF) on protein expression and protein oxidation in A2780 cancer cells were investigated through a strategy based on simultaneous expression proteomics and redox proteomics determinations. Bioinformatics analysis of the proteomics data supports the view that the most critical cellular changes elicited by AF treatment consist of thioredoxin reductase inhibition, alteration of the cell redox state, impairment of the mitochondrial functions, metabolic changes associated with conversion to a glycolytic phenotype, induction of ER stress. The occurrence of the above cellular changes was extensively validated by performing direct biochemical assays. Our data are consistent with the concept that AF produces its effects through a multitarget mechanism that mainly affects the redox metabolism and the mitochondrial functions and results into severe ER stress. Results are discussed in the context of the current mechanistic knowledge existing on AF.

S1P Signalling Axis Is Necessary for Adiponectin-Directed Regulation of Electrophysiological Properties and Oxidative Metabolism in C2C12 Myotubes.

BACKGROUND: Adiponectin (Adn), released by adipocytes and other cell types such as skeletal muscle, has insulin-sensitizing and anti-inflammatory properties. Sphingosine 1-phosphate (S1P) is reported to act as effector of diverse biological actions of Adn in different tissues. S1P is a bioactive sphingolipid synthesized by the phosphorylation of sphingosine catalyzed by sphingosine kinase (SK) 1 and 2. Consolidated findings support the key role of S1P in the biology of skeletal muscle. METHODS AND RESULTS: Here we provide experimental evidence that S1P signalling is modulated by globular Adn treatment being able to increase the phosphorylation of SK1/2 as well as the mRNA expression levels of S1P4 in C2C12 myotubes. These findings were confirmed by LC-MS/MS that showed an increase of S1P levels after Adn treatment. Notably, the involvement of S1P axis in Adn action was highlighted since, when SK1 and 2 were inhibited by PF543 and ABC294640 inhibitors, respectively, not only the electrophysiological changes but also the increase of oxygen consumption and of aminoacid levels induced by the hormone, were significantly inhibited. CONCLUSION: Altogether, these findings show that S1P biosynthesis is necessary for the electrophysiological properties and oxidative metabolism of Adn in skeletal muscle cells.

Upgrade of an old drug: Auranofin in innovative cancer therapies to overcome drug resistance and to increase drug effectiveness.

Auranofin is an oral gold(I) compound, initially developed for the treatment of rheumatoid arthritis. Currently, Auranofin is under investigation for oncological application within a drug repurposing plan due to the relevant antineoplastic activity observed both in vitro and in vivo tumor models. In this review, we analysed studies in which Auranofin was used as a single drug or in combination with other molecules to enhance their anticancer activity or to overcome chemoresistance. The analysis of different targets/pathways affected by this drug in different cancer types has allowed us to highlight several interesting targets and effects of Auranofin besides the already well-known inhibition of thioredoxin reductase. Among these targets, inhibitory-κB kinase, deubiquitinates, protein kinase C iota have been frequently suggested. To rationalize the effects of Auranofin by a system biology-like approach, we exploited transcriptomic data obtained from a wide range of cell models, extrapolating the data deposited in the Connectivity Maps website and we attempted to provide a general conclusion and discussed the major points that need further investigation.

Cell instructive Liquid Crystalline Networks for myotube formation.

Development of biological tissues in vitro is not a trivial task and requires the correct maturation of the selected cell line. To this aim, many attempts were done mainly by mimicking the biological environment using micro/nanopatterned or stimulated scaffolds. However, the obtainment of functional tissues in vitro is still far from being achieved. In contrast with the standard methods, we here present an easy approach for the maturation of myotubes toward the reproduction of muscular tissue. By using liquid crystalline networks with different stiffness and molecular alignment, we demonstrate how the material itself can give favorable interactions with myoblasts helping a correct differentiation. Electrophysiological studies demonstrate that myotubes obtained on these polymers have more adult-like morphology and better functional features with respect to those cultured on standard supports. The study opens to a platform for the differentiation of other cell lines in a simple and scalable way.

Au2phen and Auoxo6, Two Dinuclear Oxo-Bridged Gold(III) Compounds, Induce Apoptotic Signaling in Human Ovarian A2780 Cancer Cells.

Au2phen ((2,9-dimethyl-1,10-phenanthroline)2Au2(µ-O)2)(PF6)2 and Auoxo6 ((6,6'-dimethyl-2,2'-bipyridine)2Au2(µ-O)2)(PF6)2 are two structurally related gold(III) complexes that were previously reported to display relevant and promising anticancer properties in vitro toward a large number of human cancer cell lines. To expand the knowledge on the molecular mechanisms through which these gold(III) complexes trigger apoptosis in cancer cells, further studies have been performed using A2780 ovarian cancer cells as reference models. For comparative purposes, parallel studies were carried out on the gold(III) complex AuL12 (dibromo(ethylsarcosinedithiocarbamate)gold(III)), whose proapoptotic profile had been earlier characterized in several cancer cell lines. Our results pointed out that all these gold(III) compounds manifest a significant degree of similarity in their cellular and proapoptotic effects; the main observed perturbations consist of potent thioredoxin reductase inhibition, disruption of the cell redox balance, impairment of the mitochondrial membrane potential, and induction of associated metabolic changes. In addition, evidence was gained of the remarkable contribution of ASK1 (apoptosis-signal-regulating kinase-1) and AKT pathways to gold(III)-induced apoptotic signaling. Overall, the observed effects may be traced back to gold(III) reduction and subsequent formation and release of gold(I) species that are able to bind and inhibit several enzymes responsible for the intracellular redox homeostasis, in particular the selenoenzyme thioredoxin reductase.

A Metabolic Change towards Fermentation Drives Cancer Cachexia in Myotubes.

Cachexia is a disorder associated with several pathologies, including cancer. In this paper, we describe how cachexia is induced in myotubes by a metabolic shift towards fermentation, and the block of this metabolic modification prevents the onset of the cachectic phenotype. Cachectic myotubes, obtained by the treatment with conditioned medium from murine colon carcinoma cells CT26, show increased glucose uptake, decreased oxygen consumption, altered mitochondria, and increased lactate production. Interestingly, the block of glycolysis by 2-deoxy-glucose or lactate dehydrogenase inhibition by oxamate prevents the induction of cachexia, thus suggesting that this metabolic change is greatly involved in cachexia activation. The treatment with 2-deoxy-glucose or oxamate induces positive effects also in mitochondria, where mitochondrial membrane potential and pyruvate dehydrogenase activity became similar to control myotubes. Moreover, in myotubes treated with interleukin-6, cachectic phenotype is associated with a fermentative metabolism, and the inhibition of lactate dehydrogenase by oxamate prevents cachectic features. The same results have been achieved by treating myotubes with conditioned media from human colon HCT116 and human pancreatic MIAPaCa-2 cancer cell lines, thus showing that what has been observed with murine-conditioned media is a wide phenomenon. These findings demonstrate that cachexia induction in myotubes is linked with a metabolic shift towards fermentation, and inhibition of lactate formation impedes cachexia and highlights lactate dehydrogenase as a possible new tool for counteracting the onset of this pathology.

Proteomic Profiling of BRAFV600E Mutant Colon Cancer Cells Reveals the Involvement of Nucleophosmin/c-Myc Axis in Modulating the Response and Resistance to BRAF Inhibition by Vemurafenib.

BRAFV600E mutations are found in approximately 10% of colorectal cancer patients and are associated with worse prognosis and poor outcomes with systemic therapies. The aim of this study was to identify novel druggable features of BRAFV600E-mutated colon cancer (CC) cells associated with the response and resistance to BRAFV600E inhibitor vemurafenib. Towards this aim, we carried out global proteomic profiling of BRAFV600E mutant vs. KRAS mutant/BRAF wild-type and double wild-type KRAS/BRAF CC cells followed by bioinformatics analyses. Validation of selected proteomic features was performed by immunohistochemistry and in silico using the TCGA database. We reveal an increased abundance and activity of nucleophosmin (NPM1) in BRAFV600E-mutated CC in vitro, in silico and in tumor tissues from colon adenocarcinoma patients and demonstrate the roles of NPM1 and its interaction partner c-Myc in conveying the resistance to vemurafenib. Pharmacological inhibition of NPM1 effectively restored the sensitivity of vemurafenib-resistant BRAF-mutated CC cells by down-regulating c-Myc expression and activity and consequently suppressing its transcriptional targets RanBP1 and phosphoserine phosphatase that regulate centrosome duplication and serine biosynthesis, respectively. Altogether, findings from this study suggest that the NPM1/c-Myc axis could represent a promising therapeutic target to thwart resistance to vemurafenib in BRAF-mutated CC.

NMR reveals the metabolic changes induced by auranofin in A2780 cancer cells: evidence for glutathione dysregulation.

NMR metabolomics represents a powerful tool to characterize the cellular effects of drugs and gain detailed insight into their mode of action. Here, we have exploited NMR metabolomics to illustrate the changes in the metabolic profile of A2780 ovarian cancer cells elicited by auranofin (AF), a clinically approved gold drug now repurposed as an anticancer agent. An early and large increase in intracellular glutathione is highlighted as the main effect of the treatment accompanied by small but significant changes in the levels of a few additional metabolites; the general implications of these findings are discussed in the frame of the current mechanistic knowledge of AF.

Follicular microenvironment: Oxidative stress and adiponectin correlated with steroids hormones in women undergoing in vitro fertilization.

Research has been focused on determining the follicular microenviroment produced by the theca and granulosa cells since the molecular characterisation of this body fluid could lead to the understanding of several fertility problems. Oxidative stress may be one of the factors involved in female infertility since it plays a key role in the modulation of oocyte maturation and finally pregnancy. An increase in oxidative stress is correlated with inflammation and intense research was developed to understand the interaction between inflammation and adiponectin, based on the fact that many adipokines are inflammation related proteins linked to reactive oxygen species production. The aim of this study is to investigate the correlation between total adiponectin levels and oxidative stress amount in the serum and follicular fluid (FF) of women who undergone in vitro fertilization. Moreover we verified the expression of adiponectin in granulosa and cumulus cells. To clarify the predictive value of steroid hormones in human assisted reproduction, twelve steroid hormones in FF and serum, were quantified in a single run liquid chromatography/mass spectrometry, by using a multiple reaction monitoring mode and we related the serum and follicular fluids adiponectin levels with the concentration of the investigated steroid hormones.