Anti-Cancer Potential of Homemade Fresh Garlic Extract Is Related to Increased Endoplasmic Reticulum Stress.

The use of garlic and garlic-based extracts has been linked to decreased incidence of cancer in epidemiological studies. Here we examine the molecular and cellular activities of a simple homemade ethanol-based garlic extract (GE). We show that GE inhibits growth of several different cancer cells in vitro, as well as cancer growth in vivo in a syngeneic orthotopic breast cancer model. Multiple myeloma cells were found to be especially sensitive to GE. The GE was fractionated using solid-phase extractions, and we identified allicin in one GE fraction; however, growth inhibitory activities were found in several additional fractions. These activities were lost during freeze or vacuum drying, suggesting that the main anti-cancer compounds in GE are volatile. The anti-cancer activity was stable for more than six months in −20 °C. We found that GE enhanced the activities of chemotherapeutics, as well as MAPK and PI3K inhibitors. Furthermore, GE affected hundreds of proteins involved in cellular signalling, including changes in vital cell signalling cascades regulating proliferation, apoptosis, and the cellular redox balance. Our data indicate that the reduced proliferation of the cancer cells treated by GE is at least partly mediated by increased endoplasmic reticulum (ER) stress.

Altered neurochemical profile in the McGill-R-Thy1-APP rat model of Alzheimer's disease: a longitudinal in vivo 1 H MRS study.

We investigated metabolite levels during the progression of pathology in McGill-R-Thy1-APP rats, a transgenic animal model of Alzheimer's disease, and in healthy age-matched controls. Rats were subjected to in vivo (1) H magnetic resonance spectroscopy (MRS) of the dorsal hippocampus at age 3, 9 and 12 months and of frontal cortex at 9 and 12 months. At 3 months, a stage in which only Aß oligomers are present, lower glutamate, myo-inositol and total choline content were apparent in McGill-R-Thy1-APP rats. At age 9 months, lower levels of glutamate, GABA, N-acetylaspartate and total choline and elevated myo-inositol and taurine were found in dorsal hippocampus, whereas lower levels of glutamate, GABA, glutamine and N-acetylaspartate were found in frontal cortex. At age 12 months, only the taurine level was significantly different in dorsal hippocampus, whereas taurine, myo-inositol, N-acetylaspartate and total creatine levels were significantly higher in frontal cortex. McGill-R-Thy1-APP rats did not show the same changes in metabolite levels with age as displayed in the controls, and overall, prominent and complex metabolite differences were evident in this transgenic rat model of Alzheimer's disease. The findings also demonstrate that in vivo (1) H MRS is a powerful tool to investigate disease-related metabolite changes in the brain.

Tricarboxylic acid cycle activity measured by 13C magnetic resonance spectroscopy in rats subjected to the kaolin model of obstructed hydrocephalus.

Evaluating early changes in cerebral metabolism in hydrocephalus can help in the decision making and the timing of surgical intervention. This study was aimed at examining the tricarboxylic acid (TCA) cycle rate and (13)C label incorporation into neurotransmitter amino acids and other compounds 2 weeks after rats were subjected to kaolin-induced progressive hydrocephalus. In vivo and ex vivo magnetic resonance spectroscopy (MRS), combined with the infusion of [1,6-(13)C]glucose, was used to monitor the time courses of (13)C label incorporation into the different carbon positions of glutamate in the forebrains of rats with hydrocephalus as well as in those of controls. Metabolic rates were determined by fitting the measured data into a one-compartment metabolic model. The TCA cycle rate was 1.3 ± 0.2 µmoles/gram/minute in the controls and 0.8 ± 0.4 µmoles/gram/minute in the acute hydrocephalus group, the exchange rate between α-ketoglutarate and glutamate was 4.1 ± 2.5 µmoles/gram/minute in the controls and 2.7 ± 2.6 µmoles/gram/minute in the hydrocephalus group calculated from in vivo MRS. There were no statistically significant differences between these rates. Hydrocephalus caused a decrease in the amounts of glutamate, alanine and taurine. In addition, the concentration of the neuronal marker N-acetyl aspartate was decreased. (13)C Labelling of most amino acids derived from [1,6-(13)C]glucose was unchanged 2 weeks after hydrocephalus induction. The only indication of astrocyte impairment was the decreased (13)C enrichment in glutamine C-2. This study shows that hydrocephalus causes subtle but significant alterations in neuronal metabolism already early in the course of the disease. These sub-lethal changes, however, if maintained and if ongoing might explain the delayed and programmed neuronal damage as seen in chronic hydrocephalus.

[2,4-(13)C]ß-hydroxybutyrate metabolism in astrocytes and C6 glioblastoma cells.

This study was undertaken to determine if the ketogenic diet could be useful for glioblastoma patients. The hypothesis tested was whether glioblastoma cells can metabolize ketone bodies. Cerebellar astrocytes and C6 glioblastoma cells were incubated in glutamine and serum free medium containing [2,4-(13)C]ß-hydroxybutyrate (BHB) with and without glucose. Furthermore, C6 cells were incubated with [1-(13)C]glucose in the presence and absence of BHB. Cell extracts were analyzed by mass spectrometry and media by (1)H magnetic resonance spectroscopy and HPLC. Using [2,4-(13)C]BHB and [1-(13)C]glucose it could be shown that C6 cells, in analogy to astrocytes, had efficient mitochondrial activity, evidenced by (13)C labeling of glutamate, glutamine and aspartate. However, in the presence of glucose, astrocytes were able to produce and release glutamine, whereas this was not accomplished by the C6 cells, suggesting lack of anaplerosis in the latter. We hypothesize that glioblastoma cells kill neurons by not supplying the necessary glutamine, and by releasing glutamate.

Detoxification of ammonia in mouse cortical GABAergic cell cultures increases neuronal oxidative metabolism and reveals an emerging role for release of glucose-derived alanine.

Cerebral hyperammonemia is believed to play a pivotal role in the development of hepatic encephalopathy (HE), a debilitating condition arising due to acute or chronic liver disease. In the brain, ammonia is thought to be detoxified via the activity of glutamine synthetase, an astrocytic enzyme. Moreover, it has been suggested that cerebral tricarboxylic acid (TCA) cycle metabolism is inhibited and glycolysis enhanced during hyperammonemia. The aim of this study was to characterize the ammonia-detoxifying mechanisms as well as the effects of ammonia on energy-generating metabolic pathways in a mouse neuronal-astrocytic co-culture model of the GABAergic system. We found that 5 mM ammonium chloride affected energy metabolism by increasing the neuronal TCA cycle activity and switching the astrocytic TCA cycle toward synthesis of substrate for glutamine synthesis. Furthermore, ammonia exposure enhanced the synthesis and release of alanine. Collectively, our results demonstrate that (1) formation of glutamine is seminal for detoxification of ammonia; (2) neuronal oxidative metabolism is increased in the presence of ammonia; and (3) synthesis and release of alanine is likely to be important for ammonia detoxification as a supplement to formation of glutamine.

Complex glutamate labeling from [U-13C]glucose or [U-13C]lactate in co-cultures of cerebellar neurons and astrocytes.

Glutamate metabolism was studied in co-cultures of mouse cerebellar neurons (predominantly glutamatergic) and astrocytes. One set of cultures was superfused (90 min) in the presence of either [U-(13)C]glucose (2.5 mM) and lactate (1 mM) or [U-(13)C]lactate (1 mM) and glucose (2.5 mM). Other sets of cultures were incubated in medium containing [U-(13)C]lactate (1 mM) and glucose (2.5 mM) for 4 h. Regardless of the experimental conditions cell extracts were analyzed using mass spectrometry and nuclear magnetic resonance spectroscopy. (13)C labeling of glutamate was much higher than that of glutamine under all experimental conditions indicating that acetyl-CoA from both lactate and glucose was preferentially metabolized in the neurons. Aspartate labeling was similar to that of glutamate, especially when [U-(13)C]glucose was the substrate. Labeling of glutamate, aspartate and glutamine was lower in the cells incubated with [U-(13)C]lactate. The first part of the pyruvate recycling pathway, pyruvate formation, was detected in singlet and doublet labeling of alanine under all experimental conditions. However, full recycling, detectable in singlet labeling of glutamate in the C-4 position was only quantifiable in the superfused cells both from [U-(13)C]glucose and [U-(13)C]lactate. Lactate and alanine were mostly uniformly labeled and labeling of alanine was the same regardless of the labeled substrate present and higher than that of lactate when superfused in the presence of [U-(13)C]glucose. These results show that metabolism of pyruvate, the precursor for lactate, alanine and acetyl-CoA is highly compartmentalized.

Cortical glutamate metabolism is enhanced in a genetic model of absence epilepsy.

Disturbances in GABAergic and glutamatergic neurotransmission in the thalamocortical loop are involved in absence seizures. Here, we examined potential disturbances in metabolism and interactions between neurons and glia in 5-month-old genetic absence epilepsy rats from Strasbourg (GAERS) and nonepileptic rats (NER). Animals received [1-(13)C]glucose and [1,2-(13)C]acetate, the preferential substrates of neurons and astrocytes, respectively. Extracts from cerebral cortex, thalamus, and hippocampus were analyzed by (13)C nuclear magnetic resonance spectroscopy. Most changes were detected in the cortex. Pyruvate metabolism was enhanced as evidenced by increases of lactate, and labeled and unlabeled alanine. Neuronal mitochondrial metabolism was also enhanced as detected by elevated amounts of N-acetylaspartate and nicotinamide adenine dinucleotide as well as increased incorporation of label from [2-(13)C]acetyl CoA into glutamate, glutamine, and aspartate. Likewise, mitochondrial metabolism in astrocytes was increased. Changes in thalamus were restricted to increased concentration and labeling of glutamine. Changes in the hippocampus were similar to those in the cortex. This increase in glutamate-glutamine metabolism in cortical neurons and astrocytes accompanied by a decreased gamma aminobyturic acid level may lead to impaired thalamic filter function. Hence, reduced sensory input to cortex could allow the occurrence of spike-and-wave discharges in the thalamocortical loop. Increased glutamatergic output from the cortex to hippocampus may be the underlying cause of improved learning in GAERS.

Metabolism is normal in astrocytes in chronically epileptic rats: a (13)C NMR study of neuronal-glial interactions in a model of temporal lobe epilepsy.

The aim of the present work was to study potential disturbances in metabolism and interactions between neurons and glia in the lithium-pilocarpine model of temporal lobe epilepsy. Rats chronically epileptic for 1 month received [1-(13)C]glucose, a substrate for neurons and astrocytes, and [1,2-(13)C]acetate, a substrate for astrocytes only. Analyses of extracts from cerebral cortex, cerebellum, and hippocampal formation (hippocampus, amygdala, entorhinal, and piriform cortices) were performed using (13)C and (1)H nuclear magnetic resonance spectroscopy and HPLC. In the hippocampal formation of epileptic rats, levels of glutamate, aspartate, N-acetyl aspartate, adenosine triphosphate plus adenosine diphosphate and glutathione were decreased. In all regions studied, labeling from [1,2-(13)C]acetate was similar in control and epileptic rats, indicating normal astrocytic metabolism. However, labeling of glutamate, GABA, aspartate, and alanine from [1-(13)C]glucose was decreased in all areas possibly reflecting neuronal loss. The labeling of glutamine from [1-(13)C]glucose was decreased in cerebral cortex and cerebellum and unchanged in hippocampal formation. In conclusion, no changes were detected in glial-neuronal interactions in the hippocampal formation while in cortex and cerebellum the flow of glutamate to astrocytes was decreased, indicating a disturbed glutamate-glutamine cycle. This is, to our knowledge, the first study showing that metabolic disturbances are confined to neurons inside the epileptic circuit.

Manganese, copper, and zinc in cerebrospinal fluid from patients with multiple sclerosis.

The concentrations of manganese, copper, and zinc in cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) and patients with no known neurological disease (control group) were measured. Manganese and copper levels were determined by two different analytical methods: atomic absorption spectrometry (AAS) and high-resolution inductively coupled plasma-mass spectrometry (HR-ICP-MS), whereas zinc levels were determined by HR-ICP-MS only. Manganese levels (mean+/-SEM) were significantly decreased in the CSF of MS patients (1.07+/-0.13 microg/L, ICP-MS; 1.08+/-0.11 microg/L, AAS) compared to the levels in the control group (1.78+/-0.26 microg/L, ICP-MS; 1.51+/-0.17 microg/L, AAS). Copper levels were significantly elevated in the CSF of MS patients (10.90+/-1.11 microg/L; ICP-MS, 11.53+/-0.83 microg/L, AAS) compared to the levels in the control group (8.67+/-0.49 microg/L, ICP-MS; 9.10+/-0.62 microg/L, AAS). There were no significant differences between the CSF zinc levels of MS and control patients. The physiological basis for the differences in manganese and copper concentrations between MS patients and controls is unknown, but could be related to alterations in the manganese- containing enzyme glutamine synthetase and the copper-containing enzyme cytochrome oxidase.