Epileptic seizures are a common and poorly understood comorbidity for individuals with primary brain tumors. To investigate peritumoral seizure etiology, we implanted human-derived glioma cells into ...severe combined immunodeficient mice. Within 14-18 d, glioma-bearing mice developed spontaneous and recurring abnormal electroencephalogram events consistent with progressive epileptic activity. Acute brain slices from these mice showed marked glutamate release from the tumor mediated by the system x(c)(-) cystine-glutamate transporter (encoded by Slc7a11). Biophysical and optical recordings showed glutamatergic epileptiform hyperexcitability that spread into adjacent brain tissue. We inhibited glutamate release from the tumor and the ensuing hyperexcitability by sulfasalazine (SAS), a US Food and Drug Administration-approved drug that blocks system x(c)(-). We found that acute administration of SAS at concentrations equivalent to those used to treat Crohn's disease in humans reduced epileptic event frequency in tumor-bearing mice compared with untreated controls. SAS should be considered as an adjuvant treatment to ameliorate peritumoral seizures associated with glioma in humans.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Glutathione (GSH) is an essential antioxidant responsible for the maintenance of intracellular redox homeostasis. As tumors outgrow their blood supply and become hypoxic, their redox homeostasis is ...challenged by the production of nitric oxide and reactive oxygen species (ROS). In gliomas, the sustained import of l-cystine via the l-cystine/l-glutamate exchanger, system xc−, is rate-limiting for the synthesis of GSH. We show that hypoxia causes a significant increase in NO and ROS but without affecting glioma cell growth. This is explained by a concomitant increase in the utilization of GSH, which is accompanied by an increase in the cell-surface expression of xCT, the catalytic subunit of system xc−, and l-cystine uptake. Growth was inhibited when GSH synthesis was blocked by buthionine sulfoximine (BSO), an inhibitor of the enzyme required for GSH synthesis, or when cells were deprived of l-cystine. These findings suggest that glioma cells show an increased requirement for GSH to maintain growth under hypoxic conditions. Therefore, approaches that limit GSH synthesis such as blocking system xc− may be considered as an adjuvant to radiation or chemotherapy.
Malignant gliomas have been shown to release glutamate, which kills surrounding brain cells, creating room for tumor expansion. This glutamate release occurs primarily via system xC, a ...Na+-independent cystine-glutamate exchanger. We show here, in addition, that the released glutamate acts as an essential autocrine/paracrine signal that promotes cell invasion. Specifically, chemotactic invasion and scrape motility assays each show dose-dependent inhibition of cell migration when glutamate release was inhibited using either S-(4)-CPG or sulfasalazine, both potent blockers of system xC. This inhibition could be overcome by the addition of exogenous glutamate (100 micromol/L) in the continued presence of the inhibitors. Migration/invasion was also inhibited when Ca2+-permeable alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPA-R) were blocked using GYKI or Joro spider toxin, whereas CNQX was ineffective. Ca2+ imaging experiments show that the released glutamate activates Ca2+-permeable AMPA-R and induces intracellular Ca2+ oscillations that are essential for cell migration. Importantly, glioma cells release glutamate in sufficient quantities to activate AMPA-Rs on themselves or neighboring cells, thus acting in an autocrine and/or paracrine fashion. System xC and the appropriate AMPA-R subunits are expressed in all glioma cell lines, patient-derived glioma cells, and acute patient biopsies investigated. Furthermore, animal studies in which human gliomas were xenographed into scid mice show that chronic inhibition of system xC-mediated glutamate release leads to smaller and less invasive tumors compared with saline-treated controls. These data suggest that glioma invasion is effectively disrupted by inhibiting an autocrine glutamate signaling loop with a clinically approved candidate drug, sulfasalazine, already in hand.
Glioma is the most common malignant primary brain tumor. Its rapid growth is aided by tumor-mediated glutamate release, creating peritumoral excitotoxic cell death and vacating space for tumor ...expansion. Glioma glutamate release may also be responsible for seizures, which complicate the clinical course for many patients and are often the presenting symptom. A hypothesized glutamate release pathway is the cystine/glutamate transporter System xc (-) (SXC), responsible for the cellular synthesis of glutathione (GSH). However, the relationship of SXC-mediated glutamate release, seizures, and tumor growth remains unclear. Probing expression of SLC7A11/xCT, the catalytic subunit of SXC, in patient and mouse-propagated tissues, we found that ~50% of patient tumors have elevated SLC7A11 expression. Compared with tumors lacking this transporter, in vivo propagated and intracranially implanted SLC7A11-expressing tumors grew faster, produced pronounced peritumoral glutamate excitotoxicity, induced seizures, and shortened overall survival. In agreement with animal data, increased SLC7A11 expression predicted shorter patient survival according to genomic data in the REMBRANDT (National Institutes of Health Repository for Molecular Brain Neoplasia Data) database. In a clinical pilot study, we used magnetic resonance spectroscopy to determine SXC-mediated glutamate release by measuring acute changes in glutamate after administration of the U.S. Food and Drug Administration-approved SXC inhibitor, sulfasalazine (SAS). In nine glioma patients with biopsy-confirmed SXC expression, we found that expression positively correlates with glutamate release, which is acutely inhibited with oral SAS. These data suggest that SXC is the major pathway for glutamate release from gliomas and that SLC7A11 expression predicts accelerated growth and tumor-associated seizures.
•Redox homeostasis is important in the brain due to high O2 consumption and ROS production.•Glutathione is an important mediator of redox homeostasis in the brain.•System xc- and EAAT provide ...intracellular substrates for glutathione synthesis.•Gliomas cells with increased system xc- have a growth and survival and advantage.•System xc- and glutathione may be important players in tumor treatment resistance.
Redox homeostasis is especially important in the brain where high oxygen consumption produces an abundance of harmful oxidative by-products. Glutathione (GSH) is a tripeptide non-protein thiol. It is the central nervous system’s most abundant antioxidant and the master controller of brain redox homeostasis. The glutamate transporters, System xc- (SXC) and the Excitatory Amino Acid Transporters (EAAT), play important, synergistic roles in the synthesis of GSH. In glial cells, SXC mediates the uptake of cystine, which after intracellular reduction to cysteine, reacts with glutamate during the rate-limiting step of GSH synthesis. EAAT3 mediates direct cysteine uptake for neuronal GSH synthesis. SXC and EAAT work in concert in glial cells to provide two intracellular substrates for GSH synthesis, cystine and glutamate. Their cyclical basal function also prevents a buildup of extracellular glutamate, which SXC releases extracellularly in exchange for cystine uptake. Maintaining extracellular glutamate homeostasis is critical to prevent neuronal toxicity, as well as glutamate-mediated SXC inhibition, which could lead to a depletion of intracellular GSH and loss of cellular redox control. Many neurological diseases show evidence of GSH dysfunction, and increased GSH has been widely associated with chemotherapy and radiotherapy resistance of gliomas. We present evidence suggesting that gliomas expressing elevated levels of SXC are more reliant on GSH for growth and survival. They have an increased inherent radiation resistance, however, inhibition of SXC can increase tumor sensitivity at low radiation doses. GSH depletion through SXC inhibition may be a viable mechanism to enhance current glioma treatment strategies and make tumors more sensitive to radiation and chemotherapy protocols.
Changes in the glioma microenvironment including oxygen (O2) levels, supply of amino acid such as L-glutamate and L-cystine and glutathione (GSH) concentrations play a critical role in glioma ...biology. Previous data from our laboratory and others have implicated the L-cystine/L-glutamate exchanger, system xc - in the invasion and proliferation of cancers including glioma. The central aim of this dissertation was to characterize the contribution of L-cystine uptake, GSH synthesis and L-glutamate release to migration and proliferation of glioma cells. In my first study, I examined the role of system xc- mediated L-glutamate release on glioma migration. I show that activation of Ca2+ permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA-Rs) induces intracellular Ca2+ oscillations which promote migration. These findings suggest that a primary role of L-glutamate release in glioma cells is to promote migration through the autocrine and paracrine activation of Ca2+ permeable AMPA-Rs. In my second study, I examined how hypoxia affects the dependency of glioma growth on L-cystine and GSH. I show that glioma cells respond to hypoxia with increases in free radical production. When D54-MG cells were propagated under hypoxic conditions, we observed an increase in surface expression of xCT alone. I also show that hypoxia increases system xc- sensitive L-cystine uptake and consumption rates of GSH. In my last study, I examined the expression profile of xCT across high grade human glioma cell lines and in biopsied glioma samples from over 30 patients. The results show a heterogeneous expression of xCT ranging from no expression to abundant expression. Interestingly, glioma cells with minimal xCT expression no longer depend on L-cystine or GSH for growth. These studies show multiple important roles for the system x c- transporter in glioma biology.
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