2-3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline and Glioma

The neurosteroid pregnenolone sulfate (PREGS), which is synthesized in glial cells, plays a significant role in learning and memory performance. The aim of this study was to investigate the regulation of expression of the steroid sulfotransferase SULT2B1a, which catalyzes the conversion of pregnenolone to PREGS, using the rat C6 glioma cell line. Rat C6 glioma cells expressed the SULT2B1a isoform, which sulfonates pregnenolone, but, neither the SULT2B1b isoform, which catalyzes cholesterol, nor the prototypical steroid sulfotransferase SULT2A1 were expressed in these cells. Increasing concentrations of l-glutamic acid in the presence of cyclothiazide, which prevents AMPA receptor desensitization, attenuated SULT2B1a mRNA expression; however, neither NMDA nor kainic acid had a significant effect. Exposure to the synthetic glutamate analogue alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) in the presence of cyclothiazide also inhibited SULT2B1a expression. Attenuation of SULT2B1a expression by L-glutamic acid was reversed by the selective AMPA/kainate receptor antagonist 2,3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), and partially reversed by the specific neuronal nitric oxide synthase (NOS) inhibitor 7-nitroindazole (7-NI). Induction of inducible NOS by TNF-alpha in combination with lipopolysaccharide (LPS) dramatically attenuated SULT2B1a expression; this was partially reversed by the specific inducible NOS inhibitor N(6)-(1-iminoethyl)-L-lysine hydrochloride (L-NIL). Furthermore, exposure to exogenous NO donors inhibited SULT2B1a mRNA expression, and exposure to sodium nitroprusside, LPS/TNF-alpha and L-glutamic acid in combination with cyclothiazide increased the production of nitrite, a stable degradation product of NO. These findings suggest that expression of SULT2B1a, which catalyzes PREGS production, is inhibited by activation of excitatory amino acid receptors of the AMPA subtype, via facilitation of intracellular NO signaling.

Topics: Animals; Benzothiadiazines; Cell Line, Tumor; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glioma; Glutamic Acid; Isoenzymes; Neuroglia; Nitric Oxide; Pregnenolone; Quinoxalines; Rats; Receptors, AMPA; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Sulfotransferases

Glutamate may cause Ca(2+) entry through Ca(2+)-permeable glutamate receptors, which in turn stimulates the anti-apoptotic signaling cascade in glioma cells. It was found that a human glioma cell line, U-87 MG, expressed subunits of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate acid-type glutamate receptors (AMPAR). Ca(2+) entry through AMPAR was detected in approximately 40% of U-87 MG cells. AMPAR agonists facilitated cell proliferation in low-serum medium containing 0.5% fetal calf serum (FCS). Unexpectedly, cell proliferation by the activation of AMPAR was not detected in serum-rich medium containing 10% FCS. Overexpression of Ca(2+)-permeable AMPAR facilitated proliferation of U-87 MG cells in the low-serum condition, whereas it had again no effect in the serum-rich condition. Cell proliferation of U-87 MG cells is likely to be under the regulation of both growth factors contained in the serum and Ca(2+) entry through AMPAR, and that the latter regulation becomes evident only when serum factors are deprived of culture medium.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Astrocytes; Calcium; Cattle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Drug Combinations; Gene Expression; Glioma; Glutamic Acid; Humans; Quinoxalines; Rats; Receptors, AMPA; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Serum