Glial cells play an important role in sequestering neuronally released glutamate via Naϩ -dependent transporters. Surprisingly, these transporters are not operational in glial-derived tumors (gliomas). Instead, gliomas release glutamate, causing excitotoxic death of neurons in the vicinity of the tumor. We now show that glutamate release from glioma cells is an obligatory by-product of cellular cystine uptake via system x c Ϫ , an electroneutral cystine-glutamate exchanger. Cystine is an essential precursor for the biosynthesis of glutathione, a major redox regulatory molecule that protects cells from endogenously produced reactive oxygen species (ROS). Glioma cells, but not neurons or astrocytes, rely primarily on cystine uptake via system x c Ϫ for their glutathione synthesis. Inhibition of system x c Ϫ causes a rapid depletion of glutathione, and the resulting loss of ROS defense causes caspase-mediated apoptosis. Glioma cells can be rescued if glutathione status is experimentally restored or if glutathione is substituted by alternate cellular antioxidants, confirming that ROS are indeed mediators of cell death. We describe two potent drugs that permit pharmacological inhibition of system x c Ϫ . One of these drugs, sulfasalazine, is clinically used to treat inflammatory bowel disease and rheumatoid arthritis. Sulfasalazine was able to reduce glutathione levels in tumor tissue and slow tumor growth in vivo in a commonly used intracranial xenograft animal model for human gliomas when administered by intraperitoneal injection. These data suggest that inhibition of cystine uptake into glioma cells through the pharmacological inhibition of system x c Ϫ may be a viable therapeutic strategy with a Food and Drug Administration-approved drug already in hand.

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 x C À , 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 x C À . This inhibition could be overcome by the addition of exogenous glutamate (100 Mmol/L) in the continued presence of the inhibitors. Migration/invasion was also inhibited when Ca 2+ -permeable A-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPA-R) were blocked using GYKI or Joro spider toxin, whereas CNQX was ineffective. Ca 2+ imaging experiments show that the released glutamate activates Ca 2+ -permeable AMPA-R and induces intracellular Ca 2+ 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 x C À 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 x C À -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.

Highly migratory neuroectodermal cells share a common embryonic origin with cells of the central nervous system (CNS). They include enteric, parasympathetic, sympathoadrenal, and sensory neurons of the peripheral nervous system, Schwann cells, melanocytes, endocrine cells, and cells forming connective tissue of the face and neck. Because of their common embryologic origin, these cells and the tumors that derive from them can share genetic and antigenic phenotypes with gliomas, tumors derived from CNS glia. We recently discovered that chlorotoxin (ClTx), a 4-kD peptide purified from Leiurus quinquestriatus scorpion, is a highly specific marker for glioma cells in biopsy tissues (Soroceanu et al. Cancer Res 58:4871-4879, 1998) that can target tumors in animal models. We report on the specificity of ClTx as a marker for tumors of neuroectodermal origin that include peripheral neuroectodermal tumors (PNET) and gliomas. Specifically, we histochemically stained frozen and paraffin tissue sections of human biopsy tissues from 262 patients with a synthetically manufactured and biologically active ClTx bearing an N-terminal biotin. The vast majority (74 of 79) of primary human brain tumors investigated showed abundant binding of ClTx with greater than 90% ClTx-positive cells in each section. By comparison, 32 biopsies of uninvolved brain used for comparison were largely ClTx-negative, with only a few isolated reactive astrocytes showing some ClTx binding. However, as with gliomas, the vast majority of PNETs examined showed specific ClTx binding (31 of 34). These include medulloblastomas (4 of 4), neuroblastomas (6 of 7), ganglioneuromas (4 of 4), melanomas (7 of 7), adrenal pheochromocytomas (5 of 6), primitive PNET (1), small cell lung carcinoma (2 of 3), and Ewing's sarcoma (2 of 2). Under identical staining conditions, normal tissues from brain, skin, kidney, and lung were consistently negative for ClTx. These results suggest that chlorotoxin is a reliable and specific histopathological marker for tumors of neuroectodermal origin and that chlorotoxin derivatives with cytolytic activity may have therapeutic potential for these cancers.

Voltage-gated chloride channels have recently been implicated as being important for cell proliferation and invasive cell migration of primary brain tumors cells. In the present study we provide several lines of evidence that glioma Cl- currents are primarily mediated by ClC-2 and ClC-3, two genes that belong to the ClC superfamily. Transcripts for ClC-2 thru ClC-7 were detected in a human glioma cell line by PCR, whereas only ClC-2, ClC-3, and ClC-5 protein could be identified by Western blot. Prominent ClC-2, -3, and -5 channel expression was also detected in acute patient biopsies from low- and high-grade malignant gliomas. Immunogold electron microscopic studies as well as digital confocal imaging localized a portion of these ClC channels to the plasma membrane. Whole-cell patch-clamp recordings show the presence of two pharmacologically and biophysically distinct Cl- currents that could be specifically reduced by 48 hr exposure of cells to channel-specific antisense oligonucleotides. ClC-3 antisense selectively and significantly reduced the expression of outwardly rectifying current with pronounced voltage-dependent inactivation. Such currents were sensitive to DIDS (200-500 microm) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (165 microm). ClC-2 antisense significantly reduced expression of inwardly rectifying currents, which were potentiated by hyperpolarizing prepulses and inhibited by Cd2+ (200-500 microm). Currents that were mediated by ClC-5 could not be demonstrated. We suggest that ClC-2 and ClC-3 channels are specifically upregulated in glioma membranes and endow glioma cells with an enhanced ability to transport Cl-. This may in turn facilitate rapid changes in cell size and shape as cells divide or invade through tortuous extracellular brain spaces.