Sulfasalazine decreases mouse cortical hyperexcitability.

OBJECTIVE: Currently prescribed antiepileptic drugs (AEDs) are ineffective in treating approximately 30% of epilepsy patients. Sulfasalazine (SAS) is an US Food and Drug Administration (FDA)-approved drug for the treatment of Crohn disease that has been shown to inhibit the cystine/glutamate antiporter system xc- (SXC) and decrease tumor-associated seizures. This study evaluates the effect of SAS on distinct pharmacologically induced network excitability and determines whether it can further decrease hyperexcitability when administered with currently prescribed AEDs. METHODS: Using in vitro cortical mouse brain slices, whole-cell patch-clamp recordings were made from layer 2/3 pyramidal neurons. Epileptiform activity was induced with bicuculline (bic), 4-aminopyridine (4-AP) and magnesium-free (Mg2+ -free) solution to determine the effect of SAS on epileptiform events. In addition, voltage-sensitive dye (VSD) recordings were performed to characterize the effect of SAS on the spatiotemporal spread of hyperexcitable network activity and compared to currently prescribed AEDs. RESULTS: SAS decreased evoked excitatory postsynaptic currents (eEPSCs) and increased the decay kinetics of evoked inhibitory postsynaptic currents (eIPSCs) in layer 2/3 pyramidal neurons. Although application of SAS to bic and Mg2+ -free-induced epileptiform activity caused a decrease in the duration of epileptiform events, SAS completely blocked 4-AP-induced epileptiform events. In VSD recordings, SAS decreased VSD optical signals induced by 4-AP. Co-application of SAS with the AED topiramate (TPM) caused a significantly further decrease in the spatiotemporal spread of VSD optical signals. SIGNIFICANCE: Taken together this study provides evidence that inhibition of SXC by SAS can decrease network hyperexcitability induced by three distinct pharmacologic agents in the superficial layers of the cortex. Furthermore, SAS provided additional suppression of 4-AP-induced network activity when administered with the currently prescribed AED TPM. These findings may serve as a foundation to assess the potential for SAS or other compounds that selectively target SXC as an adjuvant treatment for epilepsy.

Glutamate release by primary brain tumors induces epileptic activity.

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.

Inhibition of the Sodium-Potassium-Chloride Cotransporter Isoform-1 reduces glioma invasion.

Malignant gliomas metastasize throughout the brain by infiltrative cell migration into peritumoral areas. Invading cells undergo profound changes in cell shape and volume as they navigate extracellular spaces along blood vessels and white matter tracts. Volume changes are aided by the concerted release of osmotically active ions, most notably K(+) and Cl(-). Their efflux through ion channels along with obligated water causes rapid cell shrinkage. Suitable ionic gradients must be established and maintained through the activity of ion transport systems. Here, we show that the Sodium-Potassium-Chloride Cotransporter Isoform-1 (NKCC1) provides the major pathway for Cl(-) accumulation in glioma cells. NKCC1 localizes to the leading edge of invading processes, and pharmacologic inhibition using the loop diuretic bumetanide inhibits in vitro Transwell migration by 25% to 50%. Short hairpin RNA knockdowns of NKCC1 yielded a similar inhibition and a loss of bumetanide-sensitive cell volume regulation. A loss of NKCC1 function did not affect cell motility in two-dimensional assays lacking spatial constraints but manifested only when cells had to undergo volume changes during migration. Intracranial implantation of human gliomas into severe combined immunodeficient mice showed a marked reduction in cell invasion when NKCC1 function was disrupted genetically or by twice daily injection of the Food and Drug Administration-approved NKCC1 inhibitor Bumex. These data support the consideration of Bumex as adjuvant therapy for patients with high-grade gliomas.

Cloning and characterization of glioma BK, a novel BK channel isoform highly expressed in human glioma cells.

Voltage-dependent large-conductance Ca2+-activated K+ channels (BK channels) are widely expressed in excitable and nonexcitable cells. BK channels exhibit diverse electrophysiological properties, which are attributable in part to alternative splicing of their alpha-subunits. BK currents have been implicated in the growth control of glial cells, and BK channels with novel biophysical properties have recently been characterized in human glioma cells. Here we report the isolation, cloning, and functional characterization of glioma BK (gBK), a novel splice isoform of hSlo, the gene that encodes the alpha-subunits of human BK channels. The primary sequence of gBK is 97% identical to its closest homolog hbr5, but it contains an additional 34-amino-acid exon at splice site 2 in the C-terminal tail of BK channels. hSlo transcripts containing this novel exon are expressed ubiquitously in various normal tissues as well as in neoplasmic samples, suggesting that the novel exon may modulate important physiological functions of BK channels. Expression of gBK in Xenopus oocytes gives rise to iberiotoxin-sensitive (IbTX) currents, with an IC(50) for IbTX of 5.7 nm and a Hill coefficient of 0.76. Single gBK channels have a unitary conductance of similar250 pS, and the currents show significantly slower activation and higher Ca2+ sensitivity than hbr5. Ca2+ sensitivity was enhanced specifically at physiologically relevant [Ca2+]i (100-500 nm). Examination of biopsies from patients with malignant gliomas has revealed specific overexpression of BK channels in gliomas compared with nonmalignant human cortical tissues. Importantly, tumor malignancy grades have correlated positively with BK channel expression, suggesting an important role for the gBK channel in glioma biology.