Protective Effect of Nebivolol against Oxidative Stress Induced by Aristolochic Acids in Endothelial Cells.

Aristolochic acids (AAs) are powerful nephrotoxins that cause severe tubulointerstitial fibrosis. The biopsy-proven peritubular capillary rarefaction may worsen the progression of renal lesions via tissue hypoxia. As we previously observed the overproduction of reactive oxygen species (ROS) by cultured endothelial cells exposed to AA, we here investigated in vitro AA-induced metabolic changes by 1H-NMR spectroscopy on intracellular medium and cell extracts. We also tested the effects of nebivolol (NEB), a ß-blocker agent exhibiting antioxidant properties. After 24 h of AA exposure, significantly reduced cell viability and intracellular ROS overproduction were observed in EAhy926 cells; both effects were counteracted by NEB pretreatment. After 48 h of exposure to AA, the most prominent metabolite changes were significant decreases in arginine, glutamate, glutamine and glutathione levels, along with a significant increase in the aspartate, glycerophosphocholine and UDP-N-acetylglucosamine contents. NEB pretreatment slightly inhibited the changes in glutathione and glycerophosphocholine. In the supernatants from exposed cells, a decrease in lactate and glutamate levels, together with an increase in glucose concentration, was found. The AA-induced reduction in glutamate was significantly inhibited by NEB. These findings confirm the involvement of oxidative stress in AA toxicity for endothelial cells and the potential benefit of NEB in preventing endothelial injury.

Targeting Metabolic Reprogramming to Improve Breast Cancer Treatment: An In Vitro Evaluation of Selected Metabolic Inhibitors Using a Metabolomic Approach.

Characteristic metabolic adaptations are recognized as a cancer hallmark. Breast cancer, like other cancer types, displays cellular respiratory switches-in particular, the Warburg effect-and important fluctuations in the glutamine and choline metabolisms. This cancer remains a world health issue mainly due to the side effects associated with chemotherapy, which force a reduction in the administered dose or even a complete discontinuation of the treatment. For example, Doxorubicin is efficient to treat breast cancer but unfortunately induces severe cardiotoxicity. In the present in vitro study, selected metabolic inhibitors were evaluated alone or in combination as potential treatments against breast cancer. In addition, the same inhibitors were used to possibly potentiate the effects of Doxorubicin. As a result, the combination of CB-839 (glutaminase inhibitor) and Oxamate (lactate dehydrogenase inhibitor) and the combination of CB-839/Oxamate/D609 (a phosphatidylcholine-specific phospholipase C inhibitor) caused significant cell mortality in both MDA-MB-231 and MCF-7, two breast cancer cell lines. Furthermore, all inhibitors were able to improve the efficacy of Doxorubicin on the same cell lines. Those findings are quite encouraging with respect to the clinical goal of reducing the exposure of patients to Doxorubicin and, subsequently, the severity of the associated cardiotoxicity, while keeping the same treatment efficacy.

BNIP3 promotes HIF-1α-driven melanoma growth by curbing intracellular iron homeostasis.

BNIP3 is a mitophagy receptor with context-dependent roles in cancer, but whether and how it modulates melanoma growth in vivo remains unknown. Here, we found that elevated BNIP3 levels correlated with poorer melanoma patient's survival and depletion of BNIP3 in B16-F10 melanoma cells compromised tumor growth in vivo. BNIP3 depletion halted mitophagy and enforced a PHD2-mediated downregulation of HIF-1α and its glycolytic program both in vitro and in vivo. Mechanistically, we found that BNIP3-deprived melanoma cells displayed increased intracellular iron levels caused by heightened NCOA4-mediated ferritinophagy, which fostered PHD2-mediated HIF-1α destabilization. These effects were not phenocopied by ATG5 or NIX silencing. Restoring HIF-1α levels in BNIP3-depleted melanoma cells rescued their metabolic phenotype and tumor growth in vivo, but did not affect NCOA4 turnover, underscoring that these BNIP3 effects are not secondary to HIF-1α. These results unravel an unexpected role of BNIP3 as upstream regulator of the pro-tumorigenic HIF-1α glycolytic program in melanoma cells.

GPR91 Receptor Mediates Protection against Doxorubicin-Induced Cardiotoxicity without Altering Its Anticancer Efficacy. An In Vitro Study on H9C2 Cardiomyoblasts and Breast Cancer-Derived MCF-7 Cells.

Doxorubicin (DOX) is an anticancer drug widely used in oncology, especially for breast cancer. The main limitation of DOX treatment is its cardiotoxicity due to the cumulative dose. Clinically, DOX-induced cardiomyopathy develops as a progressive heart failure caused by a progressive cardiomyocyte's death. For long, the oxidative stress induced by DOX was considered as the main toxic mechanism responsible for heart damage, but it is now controverted, and other processes are investigated to develop cardioprotective strategies. Previously, we studied DOX-induced cardiotoxicity and dexrazoxane (DEX), the only cardioprotective compound authorized by the FDA, by 1H-NMR metabonomics in H9C2 cells. We observed an increased succinate secretion in the extracellular fluid of DEX-exposed cardiomyocytes, a finding that led us to the hypothesis of a possible protective role of this agonist of the GPR91 receptor. The objective of the present work was to study the effect of succinate (SUC) and cis-epoxysuccinate (cis-ES), two agonists of the GPR91 receptor, on DOX-induced cardiotoxicity to H9C2 cells. To this purpose, several toxicity parameters, including cell viability, oxidative stress and apoptosis, as well as the GPR91 expression, were measured to assess the effects of DEX, SUC and cis-ES either alone or in combination with DOX in H9C2 cells. A 1H-NMR-based metabonomic study was carried out on cellular fluids collected after 24 h to highlight the metabolic changes induced by those protective compounds. Moreover, the effects of each agonist given either alone or in combination with DOX were evaluated on MCF-7 breast cancer cells. GPR91 expression was confirmed in H9C2 cells, while no expression was found in MCF-7 cells. Under such experimental conditions, both SUC and cis-ES decreased partially the cellular mortality, the oxidative stress and the apoptosis induced by DOX. The SUC protective effect was similar to the DEX effect, but the protective effect of cis-ES was higher on oxidative stress and apoptosis. In addition, the metabonomics findings pointed out several metabolic pathways involved in the cardioprotective effects of both GPR91 agonists: the stimulation of aerobic metabolism with glucose as the main fuel, redox balance and phospholipids synthesis. Finally, none of the GPR91 agonists jeopardized the pharmacological effects of DOX on MCF-7 breast cancer cells.

Metabolic Reprogramming in Metastatic Melanoma with Acquired Resistance to Targeted Therapies: Integrative Metabolomic and Proteomic Analysis.

Treatments of metastatic melanoma underwent an impressive development over the past few years, with the emergence of small molecule inhibitors targeting mutated proteins, such as BRAF, NRAS, or cKIT. However, since a significant proportion of patients acquire resistance to these therapies, new strategies are currently being considered to overcome this issue. For this purpose, melanoma cell lines with mutant BRAF, NRAS, or cKIT and with acquired resistances to BRAF, MEK, or cKIT inhibitors, respectively, were investigated using both 1H-NMR-based metabonomic and protein microarrays. The 1H-NMR profiles highlighted a similar go and return pattern in the metabolism of the BRAF, NRAS, and cKIT mutated cell lines. Indeed, melanoma cells exposed to mutation-specific inhibitors underwent metabolic disruptions following acute exposure but partially recovered their basal metabolism in long-term exposure, most likely acquiring resistance skills. The protein microarrays inquired about the potential cellular mechanisms used by the resistant cells to escape drug treatment, by showing decreased levels of proteins linked to the drug efficacy, especially in the downstream part of the MAPK signaling pathway. Integrating metabonomic and proteomic findings revealed some metabolic pathways (i.e., glutaminolysis, choline metabolism, glutathione production, glycolysis, oxidative phosphorylation) and key proteins (i.e., EPHA2, DUSP4, and HIF-1A) as potential targets to discard drug resistance.

New Insights About Doxorubicin-Induced Toxicity to Cardiomyoblast-Derived H9C2 Cells and Dexrazoxane Cytoprotective Effect: Contribution of In Vitro 1H-NMR Metabonomics.

Doxorubicin (DOX) is an anticancer drug widely used in oncology. The main limitation to DOX treatments though is due to the cumulative dose that may lead to cardiotoxicity. Clinically, DOX-induced cardiomyopathy develops as a progressive heart failure consecutive to a progressive loss in cardiomyocytes due to cell necrosis and apoptosis induced by DOX. For many years, the cardiac oxidative stress caused by DOX was considered as its main toxic mechanism. Therefore, several clinical trials were carried out to assess the efficacy of various antioxidants as a cardioprotective strategy. Only dexrazoxane (DEX), did significantly reduce DOX cardiotoxicity. However, since other antioxidants used later on to counteract DOX cardiotoxicity were not as successful as DEX, DOX-induced oxidative stress and DEX antioxidant activity are not considered as the main feature anymore and this led the scientific world to suspect other involved mechanisms which are still unknown. The objective of the present work was to study from a metabolic point of view the side effects of DOX and the protective properties of DEX. In vitro 1H-NMR metabonomics was applied to the rat cardiomyoblastic H9C2 cell line. This strategy was used with the hope of unveiling possible new targets to cope with DOX cardiotoxicity. Another underlying goal was the validation of H9C2 in vitro model for metabolic investigations of DOX and DEX effects. For this purpose, several parameters, including oxidative stress, cell mortality, and apoptosis, were measured to assess the effects of DOX and DEX alone or in combination. The metabonomic study was carried out on cellular fluids collected after either 4 or 24 hours of DOX-exposure. Under such experimental conditions, both the major adverse effects reported in patients exposed to DOX and the protective effect of DEX were demonstrated in vitro, suggesting that the H9C2 in vitro model is relevant to investigate both DOX cardiotoxicity and putative cardioprotective strategies. In addition, the metabonomics findings highlighted several metabolic pathways involved in DOX cardiotoxicity and DEX cardioprotective effects as potential metabolic targets for cardioprotection: energy metabolism, redox balance, as well as phospholipids and proteins metabolism.

Short halt in vaping modifies cardiorespiratory parameters and urine metabolome: a randomized trial.

Propylene glycol and glycerol are e-cigarette constituents that facilitate liquid vaporization and nicotine transport. As these small hydrophilic molecules quickly cross the lung epithelium, we hypothesized that short-term cessation of vaping in regular users would completely clear aerosol deposit from the lungs and reverse vaping-induced cardiorespiratory toxicity. We aimed to assess the acute effects of vaping and their reversibility on biological/clinical cardiorespiratory parameters [serum/urine pneumoproteins, hemodynamic parameters, lung-function test and diffusing capacities, transcutaneous gas tensions (primary outcome), and skin microcirculatory blood flow]. Regular e-cigarette users were enrolled in this randomized, investigator-blinded, three-period crossover study. The periods consisted of nicotine-vaping (nicotine-session), nicotine-free vaping (nicotine-free-session), and complete cessation of vaping (stop-session), all maintained for 5 days before the session began. Multiparametric metabolomic analyses were used to verify subjects' protocol compliance. Biological/clinical cardiorespiratory parameters were assessed at the beginning of each session (baseline) and after acute vaping exposure. Compared with the nicotine- and nicotine-free-sessions, a specific metabolomic signature characterized the stop-session. Baseline serum club cell protein-16 was higher during the stop-session than the other sessions (P < 0.01), and heart rate was higher in the nicotine-session (P < 0.001). Compared with acute sham-vaping in the stop-session, acute nicotine-vaping (nicotine-session) and acute nicotine-free vaping (nicotine-free-session) slightly decreased skin oxygen tension (P < 0.05). In regular e-cigarette-users, short-term vaping cessation seemed to shift baseline urine metabolome and increased serum club cell protein-16 concentration, suggesting a decrease in lung inflammation. Additionally, acute vaping with and without nicotine decreased slightly transcutaneous oxygen tension, likely as a result of lung gas exchanges disturbances.

A New Classification Method of Metastatic Cancers Using a 1H-NMR-Based Approach: A Study Case of Melanoma, Breast, and Prostate Cancer Cell Lines.

In this study, metastatic melanoma, breast, and prostate cancer cell lines were analyzed using a 1H-NMR-based approach in order to investigate common features and differences of aggressive cancers metabolomes. For that purpose, 1H-NMR spectra of both cellular extracts and culture media were combined with multivariate data analysis, bringing to light no less than 20 discriminant metabolites able to separate the metastatic metabolomes. The supervised approach succeeded in classifying the metastatic cell lines depending on their glucose metabolism, more glycolysis-oriented in the BRAF proto-oncogene mutated cell lines compared to the others. Other adaptive metabolic features also contributed to the classification, such as the increased total choline content (tCho), UDP-GlcNAc detection, and various changes in the glucose-related metabolites tree, giving additional information about the metastatic metabolome status and direction. Finally, common metabolic features detected via 1H-NMR in the studied cancer cell lines are discussed, identifying the glycolytic pathway, Kennedy's pathway, and the glutaminolysis as potential and common targets in metastasis, opening up new avenues to cure cancer.

Balkan Endemic Nephropathy and the Causative Role of Aristolochic Acid.

Balkan endemic nephropathy is a chronic tubulointerstitial disease with insidious onset, slowly progressing to end-stage renal disease and frequently associated with urothelial carcinoma of the upper urinary tract (UTUC). It was described in South-East Europe at the Balkan peninsula in rural areas around tributaries of the Danube River. After decades of intensive investigation, the causative factor was identified as the environmental phytotoxin aristolochic acid (AA) contained in Aristolochia clematitis, a common plant growing in wheat fields that was ingested through home-baked bread. AA initially was involved in the outbreak of cases of rapidly progressive renal fibrosis reported in Belgium after intake of root extracts of Aristolochia fangchi imported from China. A high prevalence of UTUC was found in these patients. The common molecular link between Balkan and Belgian nephropathy cases was the detection of aristolactam-DNA adducts in renal tissue and UTUC. These adducts are not only biomarkers of prior exposure to AA, but they also trigger urothelial malignancy by inducing specific mutations (A:T to T:A transversion) in critical genes of carcinogenesis, including the tumor-suppressor TP53. Such mutational signatures are found in other cases worldwide, particularly in Taiwan, highlighting the general public health issue of AA exposure by traditional phytotherapies.

Involvement of HPV Infection in the Release of Macrophage Migration Inhibitory Factor in Head and Neck Squamous Cell Carcinoma.

Human papilloma virus (HPV) infection has been well-established as a risk factor in head and neck squamous cell carcinoma (HNSCC). The carcinogenic effect of HPV is mainly due to the E6 and E7 oncoproteins, which inhibit the functions of p53 and pRB, respectively. These oncoproteins could also play a role in the Warburg effect, thus favoring tumor immune escape. Here, we demonstrated that the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) is expressed at higher levels in HPV-negative patients than in HPV-positive patients. However, the secretion of MIF is higher in HPV-positive human HNSCC cell lines, than in HPV-negative cell lines. In-HPV positive cells, the half inhibitory concentration (IC50) of MIF inhibitor (4-iodo-6-phenylpyrimidine (4-IPP)) is higher than that in HPV-negative cells. This result was confirmed in vitro and in vivo by the use of murine SCCVII cell lines expressing either E6 or E7, or both E6 and E7. Finally, to examine the mechanism of MIF secretion, we conducted proton nuclear magnetic resonance (¹H-NMR) experiments, and observed that lactate production is increased in both the intracellular and conditioned media of HPV-positive cells. In conclusion, our data suggest that the stimulation of enzymes participating in the Warburg effect by E6 and E7 oncoproteins increases lactate production and hypoxia inducible factor 1α (HIF-1α) expression, and finally induces MIF secretion.

Does the 1H-NMR plasma metabolome reflect the host-tumor interactions in human breast cancer?

Breast cancer (BC) is the most common diagnosed cancer and the leading cause of cancer death in women worldwide. There is an obvious need for a better understanding of BC biology. Alterations in the serum metabolome of BC patients have been identified but their clinical significance remains elusive. We evaluated by 1H-Nuclear Magnetic Resonance (1H-NMR) spectroscopy, filtered plasma metabolome of 50 early (EBC) and 15 metastatic BC (MBC) patients. Using Principal Component Analysis, Partial Least-Squares Discriminant Analysis and Hierarchical Clustering we show that plasma levels of glucose, lactate, pyruvate, alanine, leucine, isoleucine, glutamate, glutamine, valine, lysine, glycine, threonine, tyrosine, phenylalanine, acetate, acetoacetate, ß-hydroxy-butyrate, urea, creatine and creatinine are modulated across patients clusters. In particular lactate levels are inversely correlated with the tumor size in the EBC cohort (Pearson correlation r = -0.309; p = 0.044). We suggest that, in BC patients, tumor cells could induce modulation of the whole patient's metabolism even at early stages. If confirmed in a lager study these observations could be of clinical importance.

Prospective evaluation of potential toxicity of repeated doses of Thymus vulgaris L. extracts in rats by means of clinical chemistry, histopathology and NMR-based metabonomic approach.

In the field of natural extracts, research generally focuses on the study of their biological activities for food, cosmetic, or pharmacological purposes. The evaluation of their adverse effects is often overlooked. In this study, the extracts of Thymus vulgaris L. were obtained by two different extraction methods. Intraperitoneal injections of both extracts were given daily for four days to male Wistar Han rats, at two different doses for each extract. The evaluation of the potential toxic effects included histopathological examination of liver, kidney, and lung tissues, as well as serum biochemistry of liver and kidney parameters, and (1)H-NMR-based metabonomic profiles of urine. The results showed that no histopathological changes were observed in the liver and kidney in rats treated with both extracts of thyme. Serum biochemical investigations revealed significant increases in blood urea nitrogen, creatinine, and uric acid in animals treated with polyphenolic extract at both doses. In these latter groups, metabonomic analysis revealed alterations in a number of urine metabolites involved in the energy metabolism in liver mitochondria. Indeed, the results showed alterations of glycolysis, Krebs cycle, and ß-oxidative pathways as evidenced by increases in lactate and ketone bodies, and decreases in citrate, α-ketoglutarate, creatinine, hippurate, dimethylglycine, and dimethyalanine. In conclusion, this work showed that i.p. injection of repeated doses of thyme extracts causes some disturbances of intermediary metabolism in rats. The metabonomic study revealed interesting data which could be further used to determine the cellular pathways affected by such treatments.

PPARs: Interference with Warburg' Effect and Clinical Anticancer Trials.

The metabolic/cell signaling basis of Warburg's effect ("aerobic glycolysis") and the general metabolic phenotype adopted by cancer cells are first reviewed. Several bypasses are adopted to provide a panoramic integrated view of tumoral metabolism, by attributing a central signaling role to hypoxia-induced factor (HIF-1) in the expression of aerobic glycolysis. The cancer metabolic phenotype also results from alterations of other routes involving ras, myc, p53, and Akt signaling and the propensity of cancer cells to develop signaling aberrances (notably aberrant surface receptor expression) which, when present, offer unique opportunities for therapeutic interventions. The rationale for various emerging strategies for cancer treatment is presented along with mechanisms by which PPAR ligands might interfere directly with tumoral metabolism and promote anticancer activity. Clinical trials using PPAR ligands are reviewed and followed by concluding remarks and perspectives for future studies. A therapeutic need to associate PPAR ligands with other anticancer agents is perhaps an important lesson to be learned from the results of the clinical trials conducted to date.

Cesium adsorption in hydrated iron oxide particles suspensions: an NMR study.

137Cs is an important component of nuclear waste which may pollute water. Its migration in natural environments is slowed down by adsorption on minerals. Cesium adsorption on akaganeite (beta-FeOOH) particles, dextran-coated ferrihydrite (5 Fe(2)O(3)-9H(2)O) particles, and ferritin in aqueous solutions is studied with (133)Cs nuclear magnetic resonance measurements. The longitudinal relaxation time (T(1)) of (133)Cs in the presence of such magnetic particles depends on whether the ions bind to the particle or not. T(1) of (133)Cs ions in aqueous solutions containing the same amount of magnetized particles will not depend on cesium concentration if relaxation is governed by diffusion (when cesium is not able to bind), but it will depend on cesium concentration if exchange governs relaxation (when cesium is able to bind). The method is successfully tested using TEMPO, a nitroxide stable free radical whose relaxation is due to diffusion. (133)Cs relaxation in solutions of ferritin, akaganeite, and dextran-coated ferrihydrite particles is found to result from a cationic exchange of cesium ions between particles surface and bulk ions, owing to adsorption. The effect of pH on (133)Cs relaxation in solutions of the particles is consistent with the adsorption properties of cations on hydrated iron oxides.