NMR Metabolomics of Primary Ovarian Cancer Cells in Comparison to Established Cisplatin-Resistant and -Sensitive Cell Lines.

Cancer cell lines are frequently used in metabolomics, such as in vitro tumor models. In particular, A2780 cells are commonly used as a model for ovarian cancer to evaluate the effects of drug treatment. Here, we compare the NMR metabolomics profiles of A2780 and cisplatin-resistant A2780 cells with those of cells derived from 10 patients with high-grade serous ovarian carcinoma (collected during primary cytoreduction before any chemotherapeutic treatment). Our analysis reveals a substantial similarity among all primary cells but significant differences between them and both A2780 and cisplatin-resistant A2780 cells. Notably, the patient-derived cells are closer to the resistant A2780 cells when considering the exo-metabolome, whereas they are essentially equidistant from A2780 and A2780-resistant cells in terms of the endo-metabolome. This behavior results from dissimilarities in the levels of several metabolites attributable to the differential modulation of underlying biochemical pathways. The patient-derived cells are those with the most pronounced glycolytic phenotype, whereas A2780-resistant cells mainly diverge from the others due to alterations in a few specific metabolites already known as markers of resistance.

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.

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.

Therapy-Induced Stromal Senescence Promoting Aggressiveness of Prostate and Ovarian Cancer.

Cancer progression is supported by the cross-talk between tumor cells and the surrounding stroma. In this context, senescent cells in the tumor microenvironment contribute to the development of a pro-inflammatory milieu and the acquisition of aggressive traits by cancer cells. Anticancer treatments induce cellular senescence (therapy-induced senescence, TIS) in both tumor and non-cancerous cells, contributing to many detrimental side effects of therapies. Thus, we focused on the effects of chemotherapy on the stromal compartment of prostate and ovarian cancer. We demonstrated that anticancer chemotherapeutics, regardless of their specific mechanism of action, promote a senescent phenotype in stromal fibroblasts, resulting in metabolic alterations and secretion of paracrine factors, sustaining the invasive and clonogenic potential of both prostate and ovarian cancer cells. The clearance of senescent stromal cells, through senolytic drug treatment, reverts the malignant phenotype of tumor cells. The clinical relevance of TIS was validated in ovarian and prostate cancer patients, highlighting increased accumulation of lipofuscin aggregates, a marker of the senescent phenotype, in the stromal compartment of tissues from chemotherapy-treated patients. These data provide new insights into the potential efficacy of combining traditional anticancer strategies with innovative senotherapy to potentiate anticancer treatments and overcome the adverse effects of chemotherapy.

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.

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.

Comparative NMR metabolomics of the responses of A2780 human ovarian cancer cells to clinically established Pt-based drugs.

Pt-Based drugs play a very important role in current cancer treatments; yet, their cellular and mechanistic aspects are not fully understood. NMR metabolomics provides a powerful tool to investigate the metabolic perturbations induced by Pt drugs in cancer cells and decipher their meaning in relation to the presumed molecular mechanisms. We have carried out a systematic and comparative 1H NMR metabolomics study to analyze the responses of A2780 human ovarian cancer cells to the main clinically established Pt drugs, i.e., cisplatin, carboplatin and oxaliplatin. Notably, NMR analysis revealed some moderate and consistent changes in the metabolomic profiles of A2780 cells treated with the 3 Pt drugs with respect to controls, but only very small differences among them. Beyond alterations at the level of nucleic acid precursors, the observed changes highlight in all cases the induction of a significant endoplasmic reticulum stress. Owing to the clinical relevance of platinum resistance, the behavior of a cisplatin resistant A2780 cancer cell line upon cisplatin treatment was also evaluated.

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.

Modulation of Plasma Proteomic Profile by Regular Training in Male and Female Basketball Players: A Preliminary Study.

Monitoring fatigue and recovery during training periods contributes to identifying the best training methods to achieve sports performance. To date, little is known about sex-related differences in sports adaptations. The aim of the present study is to identify sex-related sports adaptation proteins in female basketball players and male basketball players using proteomics approach on plasma samples withdrawn from athletes during in-season training period but far from a competition. A cohort of 20 professional basketball players, 10 female (BF) and 10 male (BM), and 20 sedentary male (10 CM) and female (10 CF) as control, of comparable age and BMI, were involved in this study. Protein profiles of plasma samples obtained from BM, BF, CM, and CF were analyzed by two-dimensional electrophoresis (2-DE). Differentially expressed proteins were identified by mass spectrometry. The computational 2-DE gel image analysis pointed out 33 differentially expressed protein spots (ANOVA p-value < 0.05) and differences between male and female basketball players are more evident among the players than controls. The expression profile of 54.5% of the total proteins is affected by sports activity. Furthermore, 14 proteins are differentially expressed in basket female players in comparison with their relative controls while seven are differentially expressed in basket male players in comparison with their controls. In conclusion, we identify in female athletes a reduction in proteins related to transcription regulation, most of these modulate chronic inflammation confirming the anti-inflammatory effect of regular training in female muscle metabolism. In male and female athletes, we found a decrease in Transthyretin involved in muscle homeostasis and regeneration and Dermcidin a stress-induced myokine linked to inflammatory and it will be interesting to fully understand the role of its different isoforms in male and female skeletal muscle contraction.

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.

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.

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.

Relationship between the metabolic and lipid profile in follicular fluid of women undergoing in vitro fertilization.

Among the follicular fluid (FF) components promoting the development of the oocyte are included glycoproteins, several fatty acids, and steroid hormones synthesized by the dominant follicle. For this, the analysis of the metabolites present in FF can determine the quality of the oocyte. FF composition is in part determined by local follicular metabolic processes and in part a plasma transudate. Since the causes of impaired fertility may be due to a metabolic imbalance, metabolomics is useful to identify low molecular weight metabolites. Oxidative stress is involved in human infertility and the use of metabolomics can be crucial to identify which other metabolites besides reactive oxygen species are involved in oxidative stress correlated to infertility. To obtain new information on the study of signaling molecules in FF, the knowledge of the lipid content will be important to improve information on the understanding of follicular development. The objective of this study is to identify (a) a metabolic profile and a lipid profile of FF in women undergoing in vitro fertilization and (b) to correlate the previous information obtained regarding adiponectin and oxidative stress with the metabolic and lipid profile obtained in the present study. As result, we found an increase in oxidative stress due to both an increase of androgens and an accumulation of lipids in the follicular environment and we suggest that this might be one of the causes of reduced fertility.

The Adipokines in Cancer Cachexia.

Cachexia is a devastating pathology induced by several kinds of diseases, including cancer. The hallmark of cancer cachexia is an extended weight loss mainly due to skeletal muscle wasting and fat storage depletion from adipose tissue. The latter exerts key functions for the health of the whole organism, also through the secretion of several adipokines. These hormones induce a plethora of effects in target tissues, ranging from metabolic to differentiating ones. Conversely, the decrease of the circulating level of several adipokines positively correlates with insulin resistance, metabolic syndrome, diabetes, and cardiovascular disease. A lot of findings suggest that cancer cachexia is associated with changed secretion of adipokines by adipose tissue. In agreement, cachectic patients show often altered circulating levels of adipokines. This review reported the findings of adipokines (leptin, adiponectin, resistin, apelin, and visfatin) in cancer cachexia, highlighting that to study in-depth the involvement of these hormones in this pathology could lead to the development of new therapeutic strategies.

Oxidative stress in exercise training: the involvement of inflammation and peripheral signals.

The evidence about the health benefits of regular physical activity is well established. Exercise intensity is a significant variable and structured high-intensity interval training (HIIT) has been demonstrated to improve both whole-body and skeletal muscle metabolic health in different populations. Conversely, fatigue accumulation, if not resolved, leads to overwork, chronic fatigue syndrome (CFS), overtraining syndrome up to alterations of endocrine function, immune, systemic inflammation, and organic diseases with health threat. In response to temporary increases in stress during training, some athletes are unable to maintain sufficient caloric intake, thus suffering a negative energy balance that causes further stress. The regulation of the energy balance is controlled by the central nervous system through an elaborate interaction of the signalling that involves different tissues such as leptin, adiponectin and ghrelin whose provide important feedback to the hypothalamus to regulate the energy balance. Although exercise-induced reactive oxygen species are required for normal force production in muscle, high levels of ROS appear to promote contractile dysfunction. However, a high level of oxidative stress in may induce a rise in inflammatory markers and a disregulation in expression of adiponectin, leptin and grelin.

miR-210-3p mediates metabolic adaptation and sustains DNA damage repair of resistant colon cancer cells to treatment with 5-fluorouracil.

Chemoresistance is the primary cause of chemotherapy failure. Compelling evidence shows that micro RNAs (miRNAs) contribute to reprogram cancer cells toward a resistant phenotype. We investigate the role of miRNAs in the response to acute treatment with 5-FU in colon cancer-resistant cells. We performed a global gene expression profile for the entire miRNA genome and found a change in the expression of four miRNAs following acute treatment with 5-FU. Among them, we focused on miR-210-3p, previously described as a key regulator of DNA damage repair mechanisms and mitochondrial metabolism. We show that miR-210-3p downregulation enables resistant cells to counteract the toxic effect of the drug increasing the expression of RAD-52 protein, responsible for DNA damage repair. Moreover, miR-210-3p downregulation enhances oxidative phosphorylation (OXPHOS), increasing the expression levels of succinate dehydrogenase subunits D, decreasing intracellular succinate levels and inhibiting HIF-1α expression. Altogether, these adaptations lead to increased cells survival following drug exposure. These evidence suggest that miR-210-3p downregulation following 5-FU sustains DNA damage repair and metabolic adaptation to counteract drug treatment.

Adiponectin in Myopathies.

In skeletal muscle, adiponectin has varied and pleiotropic functions, ranging from metabolic, anti-inflammatory, insulin-sensitizing to regenerative roles. Despite the important functions exerted by adiponectin, the study of the hormone in myopathies is still marginal. Myopathies include inherited and non-inherited/acquired neuromuscular pathologies characterized by muscular degeneration and weakness. This review reports current knowledge about adiponectin in myopathies, regarding in particular the role of adiponectin in some hereditary myopathies (as Duchenne muscular dystrophy) and non-inherited/acquired myopathies (such as idiopathic inflammatory myopathies and fibromyalgia). These studies show that some myopathies are characterized by decreased concentration of plasma adiponectin and that hormone replenishment induces beneficial effects in the diseased muscles. Overall, these findings suggest that adiponectin could constitute a future new therapeutic approach for the improvement of the abnormalities caused by myopathies.

Cancer-associated fibroblasts promote prostate cancer malignancy via metabolic rewiring and mitochondrial transfer.

Cancer-associated fibroblasts (CAFs) are the major cellular stromal component of many solid tumors. In prostate cancer (PCa), CAFs establish a metabolic symbiosis with PCa cells, contributing to cancer aggressiveness through lactate shuttle. In this study, we report that lactate uptake alters the NAD+/NADH ratio in the cancer cells, which culminates with SIRT1-dependent PGC-1α activation and subsequent enhancement of mitochondrial mass and activity. The high exploitation of mitochondria results in tricarboxylic acid cycle deregulation, accumulation of oncometabolites and in the altered expression of mitochondrial complexes, responsible for superoxide generation. Additionally, cancer cells hijack CAF-derived functional mitochondria through the formation of cellular bridges, a phenomenon that we observed in both in vitro and in vivo PCa models. Our work reveals a crucial function of tumor mitochondria as the energy sensors and transducers of CAF-dependent metabolic reprogramming and underscores the reliance of PCa cells on CAF catabolic activity and mitochondria trading.

Replacement of the Thiosugar of Auranofin with Iodide Enhances the Anticancer Potency in a Mouse Model of Ovarian Cancer.

In recent years, a few successful attempts were made to repurpose the clinically approved antiarthritic gold drug, Auranofin (AF), as an anticancer agent. The present study shows that the iodido(triethylphosphine)gold(I) complex, (Et 3 PAuI hereafter)-an AF analogue where the thiosugar ligand is simply replaced by one iodide ligand-manifests a solution chemistry resembling that of AF and exerts similar cytotoxic and proapoptotic effects on A2780 human ovarian cancer cells in vitro. However, when evaluated in a preclinical orthotopic model of ovarian cancer, Et 3 PAuI produces a far superior anticancer action than AF inducing a nearly complete tumor remission. The highly promising in vivo performances here documented for Et 3 PAuI warrant its further evaluation as a drug candidate for ovarian cancer treatment.