The Crosstalk between GPR81/IGFBP6 Promotes Breast Cancer Progression by Modulating Lactate Metabolism and Oxidative Stress.

Breast cancer is the most frequent tumor and the leading cause of cancer deaths in women. In recent years, lactate metabolism and, in particular, its receptor GPR81 have been shown to play a vital role in cancer biology. GPR81 is upregulated in breast cancer and promotes tumor growth by tumor cell-derived lactate. Therefore, the search for possible crosstalk and the involvement of new molecules capable of generating this pathology is always in continuous development. In this study, the relationship between GPR81 and IGFBP6 protein in tumor growth and oxidative stress in the human breast cancer cell line MDA-MB-231 was studied. Cells were treated with lactate or the GPR81 receptor agonist and antagonist 3,5-DHBA and 3-OBA, respectively. In addition, oxidative stress and proliferation were also evaluated in cells challenged with the recombinant IGFBP6 protein. Our data showed that lactate induced cell proliferation and wound healing of the MDA-231 breast cancer cell through the overexpression of both the lactate receptor GPR81 and IGFBP6. The increase in IGFBP6 was able, in turn, to improve the mitochondrial fitness and redox state, as suggested by the reduced levels of mitochondrial ROS production after IGFBP6 treatment, presumably mediated by the increase in the ROS detoxifying genes HMOX1, GSTK1 and NQO1. In conclusion, our data highlight a novel axis between GPR81 and IGFBP6 in MDA-231 cells able to modulate lactate metabolism and oxidative stress. This complex signaling may represent a new therapeutic target for breast cancer.

Lung Surfactant Decreases Biochemical Alterations and Oxidative Stress Induced by a Sub-Toxic Concentration of Carbon Nanoparticles in Alveolar Epithelial and Microglial Cells.

Carbon-based nanomaterials are nowadays attracting lots of attention, in particular in the biomedical field, where they find a wide spectrum of applications, including, just to name a few, the drug delivery to specific tumor cells and the improvement of non-invasive imaging methods. Nanoparticles inhaled during breathing accumulate in the lung alveoli, where they interact and are covered with lung surfactants. We recently demonstrated that an apparently non-toxic concentration of engineered carbon nanodiamonds (ECNs) is able to induce oxidative/nitrosative stress, imbalance of energy metabolism, and mitochondrial dysfunction in microglial and alveolar basal epithelial cells. Therefore, the complete understanding of their "real" biosafety, along with their possible combination with other molecules mimicking the in vivo milieu, possibly allowing the modulation of their side effects becomes of utmost importance. Based on the above, the focus of the present work was to investigate whether the cellular alterations induced by an apparently non-toxic concentration of ECNs could be counteracted by their incorporation into a synthetic lung surfactant (DPPC:POPG in 7:3 molar ratio). By using two different cell lines (alveolar (A549) and microglial (BV-2)), we were able to show that the presence of lung surfactant decreased the production of ECNs-induced nitric oxide, total reactive oxygen species, and malondialdehyde, as well as counteracted reduced glutathione depletion (A549 cells only), ameliorated cell energy status (ATP and total pool of nicotinic coenzymes), and improved mitochondrial phosphorylating capacity. Overall, our results on alveolar basal epithelial and microglial cell lines clearly depict the benefits coming from the incorporation of carbon nanoparticles into a lung surfactant (mimicking its in vivo lipid composition), creating the basis for the investigation of this combination in vivo.

Modulation of Pro-Oxidant and Pro-Inflammatory Activities of M1 Macrophages by the Natural Dipeptide Carnosine.

Carnosine is a natural endogenous dipeptide widely distributed in mammalian tissues, existing at particularly high concentrations in the muscles and brain and possesses well-characterized antioxidant and anti-inflammatory activities. In an in vitro model of macrophage activation, induced by lipopolysaccharide + interferon-gamma (LPS + IFN-γ), we here report the ability of carnosine to modulate pro-oxidant and pro-inflammatory activities of macrophages, representing the primary cell type that is activated as a part of the immune response. An ample set of parameters aimed to evaluate cytotoxicity (MTT assay), energy metabolism (HPLC), gene expressions (high-throughput real-time PCR (qRT-PCR)), protein expressions (western blot) and nitric oxide production (qRT-PCR and HPLC), was used to assess the effects of carnosine on activated macrophages challenged with a non cytotoxic LPS (100 ng/mL) + IFN-γ (600 U/mL) concentration. In our experimental model, main carnosine beneficial effects were: (1) the modulation of nitric oxide production and metabolism; (2) the amelioration of the macrophage energy state; (3) the decrease of the expressions of pro-oxidant enzymes (Nox-2, Cox-2) and of the lipid peroxidation product malondialdehyde; (4) the restoration and/or increase of the expressions of antioxidant enzymes (Gpx1, SOD-2 and Cat); (5) the increase of the transforming growth factor-ß1 (TGF-ß1) and the down-regulation of the expressions of interleukins 1ß and 6 (IL-1ß and IL-6) and 6) the increase of the expressions of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 (HO-1). According to these results carnosine is worth being tested in the treatment of diseases characterized by elevated levels of oxidative stress and inflammation (atherosclerosis, cancer, depression, metabolic syndrome, and neurodegenerative diseases).

Non-toxic engineered carbon nanodiamond concentrations induce oxidative/nitrosative stress, imbalance of energy metabolism, and mitochondrial dysfunction in microglial and alveolar basal epithelial cells.

Engineered nanoparticles are finding a wide spectrum of biomedical applications, including drug delivery and capacity to trigger cytotoxic phenomena, potentially useful against tumor cells. The full understanding of their biosafety and interactions with cell processes is mandatory. Using microglial (BV-2) and alveolar basal epithelial (A549) cells, in this study we determined the effects of engineered carbon nanodiamonds (ECNs) on cell viability, nitric oxide (NO) and reactive oxygen species (ROS) production, as well as on energy metabolism. Particularly, we initially measured decrease in cell viability as a function of increasing ECNs doses, finding similar cytotoxic ECN effects in the two cell lines. Subsequently, using apparently non-cytotoxic ECN concentrations (2 µg/mL causing decrease in cell number < 5%) we determined NO and ROS production, and measured the concentrations of compounds related to energy metabolism, mitochondrial functions, oxido-reductive reactions, and antioxidant defences. We found that in both cell lines non-cytotoxic ECN concentrations increased NO and ROS production with sustained oxidative/nitrosative stress, and caused energy metabolism imbalance (decrease in high energy phosphates and nicotinic coenzymes) and mitochondrial malfunctioning (decrease in ATP/ADP ratio).These results underline the importance to deeply investigate the molecular and biochemical changes occurring upon the interaction of ECNs (and nanoparticles in general) with living cells, even at apparently non-toxic concentration. Since the use of ECNs in biomedical field is attracting increasing attention the complete evaluation of their biosafety, toxicity and/or possible side effects both in vitro and in vivo is mandatory before these highly promising tools might find the correct application.

Potentially neuroprotective gene modulation in an in vitro model of mild traumatic brain injury.

In this study, we investigated the hypothesis that mild traumatic brain injury (mTBI) triggers a controlled gene program as an adaptive response finalized to neuroprotection, similar to that found in hibernators and in ischemic preconditioning. A stretch injury device was used to produce an equi-biaxial strain field in rat organotypic hippocampal slice cultures at a specified Lagrangian strain of 10 % and a constant strain rate of 20 s(-1). After 24 h from injury, propidium iodide staining, HPLC analysis of metabolites and microarray analysis of cDNA were performed to evaluate cell viability, cell energy state and gene expression, respectively. Compared to control cultures, 10 % stretch injured cultures showed no change in viability, but demonstrated a hypometabolic state (decreased ATP, ATP/ADP, and nicotinic coenzymes) and a peculiar pattern of gene modulation. The latter was characterized by downregulation of genes encoding for proteins of complexes I, III, and IV of the mitochondrial electron transport chain and of ATP synthase; downregulation of transcriptional and translational genes; downregulation and upregulation of genes controlling the synthesis of glutamate and GABA receptors, upregulation of calmodulin and calmodulin-binding proteins; proper modulation of genes encoding for proapoptotic and antiapoptotic proteins. These results support the hypothesis that, following mTBI, a hibernation-type response is activated in non-hibernating species. Unlike in hibernators and ischemic preconditioning, this adaptive gene programme, aimed at achieving maximal neuroprotection, is not triggered by decrease in oxygen availability. It seems rather activated to avoid increase in oxidative/nitrosative stress and apoptosis during a transient period of mitochondrial malfunctioning.

Metabolic profile of amniotic fluid as a biochemical tool to screen for inborn errors of metabolism and fetal anomalies.

Physiologic concentration in amniotic fluid (AF) of several metabolites has not been established with certainty. In this study, we initially assayed purines, pyrimidines, and amino compounds in 1,257 AF withdrawn between the 15th and the 20th week of gestation from actually normal pregnancies (normal gestations, normal offspring). Results allowed to determine physiologic reference intervals for 45 compounds. In these AF, not all purines and pyrimidines were detectable and uric acid (238.35±76.31 µmol/l) had the highest concentration. All amino compounds were measurable, with alanine having the highest concentration (401.10±88.47 µmol/l). In the second part of the study, we performed a blind metabolic screening of AF to evaluate the utility of this biochemical analysis as an additional test in amniocenteses. In 1,295 additional AF from normal pregnancies, all metabolites fell within the confidence intervals determined in the first part of the study. In 24 additional AF from women carrying Down's syndrome-affected fetuses, glutamate, glutamine, glycine, taurine, valine, isoleucine, leucine, ornithine, and lysine were different from physiologic reference values. One AF sample showed phenylalanine level of 375.54 µmol/l (mean value in normal AF=65.07 µmol/l) and was from a woman with unreported phenylketonuria with mild hyperphenylalaninemia (serum phenylalanine=360.88 µmol/l), carrying the IVS 4+5 G-T and D394A mutations. The fetus was heterozygote for the maternal D394A mutation. An appropriate diet maintained the mother phenylalanine in the range of normality during pregnancy, avoiding serious damage in fetal and neonatal development. These results suggest that the metabolic screening of AF might be considered as an additional biochemical test in amniocenteses useful to highlight anomalies potentially related to IEM.

Brain energy depletion in a rodent model of diffuse traumatic brain injury is not prevented with administration of sodium lactate.

Lactate has been identified as an alternative fuel for the brain in situations of increased energy demand, as following a traumatic brain injury (TBI). This study investigates the effect of treatment with sodium lactate (NaLac) on the changes in brain energy state induced by a severe diffuse TBI. Rats were assigned to one of the eight groups (n=10 per group): 1-sham, normal saline; 2-TBI, normal saline; 3-TBI, hypertonic saline; 4-TBI, 100mM NaLac, 5-TBI, 500 mM NaLac; 6-TBI, 1280 mM NaLac; 7-TBI, 2000 mM NaLac and 8-TBI-500 mM NaLac+magnesium sulfate. Cerebrums were removed 6h after trauma. Metabolites representative of the energy state (ATP, ATP-catabolites), N-acetylaspartate (NAA), antioxidant defenses (ascorbic acid, glutathione), markers of oxidative stress (malondialdehyde, ADP-ribose) and nicotinic coenzymes (NAD(+)) were measured by HPLC. TBI induced a marked decrease in the cerebral levels of ATP, NAA, ascorbic acid, glutathione and NAD(+) and a significant rise in the content of ATP-catabolites, malondialdehyde and ADP-ribose. These alterations were not ameliorated with NaLac infusion. We observed a significant reduction in cerebral NAD(+), an essential co-enzyme for mitochondrial lactate-dehydrogenase that converts lactate into pyruvate and thus replenishes the tricarboxylic acid cycle. These results suggest that the metabolic pathway necessary to consume lactate may be compromised following a severe diffuse TBI in rats.

The protein kinase C activator phorbol myristate acetate decreases brain edema by aquaporin 4 downregulation after middle cerebral artery occlusion in the rat.

The protein kinase C activator phorbol 12-myristate 13-acetate (PMA) is known to interact with aquaporin 4 (AQP 4), a water-selective transporting protein that is abundant in astrocytes, and has experimentally been found to decrease osmotically-induced cell swelling. The purpose of this study was to examine whether PMA reduces brain edema following focal ischemia induced by middle cerebral artery (MCA) occlusion by modulation of AQP4 expression. Male Sprague-Dawley rats were randomly assigned to either sham surgery (n = 6), or a continuous intravenous infusion of vehicle (1% dimethylsulfoxide), followed by MCA occlusion (n = 18), and administration of PMA at 50 microg/kg (n = 6) or at 200 microg/kg (n = 6) starting 60 min before or 30 min (200 microg/kg; n = 6) or 60 min (200 microg/kg; n = 6) after MCA occlusion. Cerebral blood flow was monitored with laser Doppler over the MCA territory, and confirmed a 70% reduction during occlusion. After a 2-h period of ischemia and 2 h of reperfusion, the animals were sacrificed for assessment of brain water content and sodium and potassium concentration. AQP4 expression was assessed by immunoblotting and quantified by densitometry (n = 24). Statistical analysis was performed by ANOVA followed by Tukey's post-hoc test. PMA treatment at 200 microg/kg significantly reduced brain water concentration in the infarcted area when started 60 min before or 30 min after occlusion (p < 0.001 and p = 0.022, respectively), and prevented the subsequent sodium shift (p < 0.05). PMA normalized the AQP4 upregulation in ischemia (p = 0.021). A downregulation of AQP4 in the ischemic area paralleling the reduction in brain edema formation following PMA treatment suggests that the effect was mediated by AQP4 modulation.

Transient alterations of creatine, creatine phosphate, N-acetylaspartate and high-energy phosphates after mild traumatic brain injury in the rat.

In this study, the concentrations of creatine (Cr), creatine phosphate (CrP), N-acetylaspartate (NAA), ATP, ADP and phosphatidylcholine (PC) were measured at different time intervals after mild traumatic brain injury (mTBI) in whole brain homogenates of rats. Anaesthetized animals underwent to the closed-head impact acceleration "weight-drop" model (450 g delivered from 1 m height = mild traumatic brain injury) and were killed at 2, 6, 24, 48 and 120 h after the insult (n = 6 for each time point). Sham-operated rats (n = 6) were used as controls. Compounds of interest were synchronously measured by HPLC in organic solvent deproteinized whole brain homogenates. A reversible decrease of all metabolites but PC was observed, with minimal values recorded at 24 h post-injury (minimum of CrP = 48 h after impact). In particular, Cr and NAA showed a decrease of 44.5 and 29.5%, respectively, at this time point. When measuring NAA in relation to other metabolites, as it is commonly carried out in "in vivo" (1)H-magnetic resonance spectroscopy ((1)H-MRS), an increase in the NAA/Cr ratio and a decrease in the NAA/PC ratio was observed. Besides confirming a transient alteration of NAA homeostasis and ATP imbalance, our results clearly show significant changes in the cerebral concentration of Cr and CrP after mTBI. This suggests a careful use of the NAA/Cr ratio to measure NAA by (1)H-MRS in conditions of altered cerebral energy metabolism. Viceversa, the NAA/PC ratio appears to be a better indicator of actual NAA levels during energy metabolism impairment. Furthermore, our data suggest that, under pathological conditions affecting the brain energetic, the Cr-CrP system is not a suitable tool to buffer possible ATP depletion in the brain, thus supporting the growing indications for alternative roles of cerebral Cr.

Increase of uric acid and purine compounds in biological fluids of multiple sclerosis patients.

OBJECTIVES: In this study, the concentrations of uric acid, purine profile and creatinine in samples of cerebrospinal fluid and serum of multiple sclerosis (MS) patients were measured by HPLC and compared with corresponding values recorded in patients without MS (cerebrospinal fluid) and healthy subjects (serum). DESIGN AND METHODS: All samples were deproteinized with ultrafiltration (which ensures minimal sample manipulation and efficient protein removal) and then assayed for the synchronous HPLC separation of uric acid, hypoxanthine, xanthine, inosine, adenosine, guanosine and creatinine. RESULTS: The values of all compounds assayed were significantly higher in both biological fluids of MS patients with respect to values measured in controls. In particular, serum hypoxanthine, xanthine, uric acid and sum of oxypurines were, respectively, 3.17, 3.11, 1.23 and 1.27-fold higher in these patients than corresponding values recorded in controls (p<0.001). CONCLUSIONS: Differently from what previously reported, we here demonstrate that all purine compounds, including uric acid, are elevated in biological fluids of MS patients. Reinforced by the trend observed for creatinine, this corroborates the notion of sustained purine catabolism, possibly due to imbalance in ATP homeostasis, under these pathological conditions. These results cast doubt on the hypothesis that uric acid is depleted in MS because of increased oxidative stress, rather suggesting that this disease causes a generalized increase in purine catabolism. As observed in other pathological states, uric acid, purine compounds and creatinine, can be considered markers of metabolic energy imbalance rather than of reactive oxygen species, even in MS.

Temporal window of metabolic brain vulnerability to concussions: oxidative and nitrosative stresses--part II.

OBJECTIVE: In the present study, we investigated the occurrence of oxidative and nitrosative stresses in rats undergoing repeat mild traumatic brain injury (mTBI) delivered with increasing time intervals. METHODS: Rats were subjected to two diffuse mTBIs (450 g/1 m height), with the second mTBI delivered after 1 (n = 6), 2 (n = 6), 3 (n = 6), 4 (n = 6), or 5 days (n = 6). The rats were sacrificed 48 hours after the last mTBI. Sham-operated animals were used as controls (n = 6). Concentrations of biochemical indices of oxidative stress (malondialdehyde, ascorbic acid, reduced and oxidized glutathione) and nitrosative stress (nitrite, nitrate) were synchronously measured by high-performance liquid chromatography in deproteinized tissue extracts (three right + three left hemispheres for each group of animals). RESULTS: Increase of malondialdehyde, reduced/oxidized glutathione ratio, nitrite, nitrate, and decrease of ascorbic acid and glutathione were dependent on the interval between impacts with maximal changes recorded when mTBIs were spaced by 3 days. Biochemical markers of oxidative and nitrosative stresses were near control levels only in animals receiving mTBIs 5 days apart. CONCLUSION: This study shows the remarkable negative contribution of reactive oxygen species overproduction and activation of inducible nitric oxide synthase in repeat mTBI. Because these effects were maximal when mTBIs were spaced by 3 days, it can be inferred that occurrence of a second mTBI within the temporal window of brain vulnerability not only causes profound derangement of mitochondrial functions, but also induces sustained oxidative and nitrosative stresses. Both phenomena certainly play a major role in the overall brain tissue damage occurring under these pathological conditions.

Temporal window of metabolic brain vulnerability to concussions: mitochondrial-related impairment--part I.

OBJECTIVE: In the present study, we investigate the existence of a temporal window of brain vulnerability in rats undergoing repeat mild traumatic brain injury (mTBI) delivered at increasing time intervals. METHODS: Rats were subjected to two diffuse mTBIs (450 g/1 m height) with the second mTBI delivered after 1 (n = 6), 2 (n = 6), 3 (n = 6), 4 (n = 6), and 5 days (n = 6) and sacrificed 48 hours after the last impact. Sham-operated animals were used as controls (n = 6). Two further groups of six rats each received a second mTBI after 3 days and were sacrificed at 120 and 168 hours postinjury. Concentrations of adenine nucleotides, N-acetylated amino acids, oxypurines, nucleosides, free coenzyme A, acetyl CoA, and oxidized and reduced nicotinamide adenine dinucleotides, oxidized nicotinamide adenine dinucleotide phosphate, and reduced nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide phosphate nicotinic coenzymes were measured in deproteinized cerebral tissue extracts (three right and three left hemispheres), whereas the gene expression of N-acetylaspartate acylase, the enzyme responsible for N-acetylaspartate (NAA) degradation, was evaluated in extracts of three left and three right hemispheres. RESULTS: A decrease of adenosine triphosphate, adenosine triphosphate/adenosine diphosphate ratio, NAA, N-acetylaspartylglutamate, oxidized and reduced nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide, and acetyl CoA and increase of N-acetylaspartate acylase expression were related to the interval between impacts with maximal changes recorded when mTBIs were spaced by 3 days. In these animals, protracting the time of sacrifice after the second mTBI up to 1 week failed to show cerebral metabolic recovery, indicating that this type of damage is difficult to reverse. A metabolic pattern similar to controls was observed only in animals receiving mTBIs 5 days apart. CONCLUSION: This study shows the existence of a temporal window of brain vulnerability after mTBI. A second concussive event falling within this time range had profound consequences on mitochondrial-related metabolism. Furthermore, because NAA recovery coincided with normalization of all other metabolites, it is conceivable to hypothesize that NAA measurement by 1H-NMR spectroscopy might be a valid tool in assessing full cerebral metabolic recovery in the clinical setting and with particular reference to sports medicine in establishing when to return mTBI-affected athletes to play. This study also shows, for the first time, the influence of TBI on acetyl-CoA, N-acetylaspartate acylase gene expression, and N-acetylaspartylglutamate, thus providing novel data on cerebral biochemical changes occurring in head injury.