Reactive Oxygen Species-Dependent Nitric Oxide Production in Reciprocal Interactions of Glioma and Microglial Cells

Conditioned mediums (CMs) from glioma cells U87, GBM-8401, and C6 significantly induced iNOS protein and NO production by microglial cells BV-2 but without altering the cell viability or cell-cycle progression of BV2 microglia. Significant increases in intracellular peroxide by U87-CM and C6-CM were detected by a DCHF-DA assay, and vitamin (Vit) C and N-acetyl cysteine (NAC)-reduced intracellular peroxide levels elicited by CMs lead to inhibition of iNOS/NO production The extracellular signal-regulated kinase (ERK) inhibitor, U0126, and c-Jun N-terminal kinase (JNK) inhibitor, SP600125, suppressed U87-CM- and C6-CM-induced iNOS/NO production by respectively blocking phosphorylated ERK (pERK) and JNK (pJNK) protein expressions stimulated by U87-CM and C6-CM. Increased migration of U87 and C6 glioma cells by a co-culture with BV-2 microglial cells or adding the nitric oxide (NO) donor, sodium nitroprusside (SNP) was observed, and that was blocked by adding an NO synthase (NOS) inhibitor, N-nitro L-arginine methyl ester (NAME). Contributions of ROS, pERK, and pJNK to the migration of glioma cells was further demonstrated in a transwell coculture system of U87 and C6 gliomas with BV-2 microglial cells. Furthermore, expressions of tumor necrosis factor (TNF)- and monocyte chemoattractant protein (MCP)-1 messenger (m)RNA in U87 and C6 cells were detected by an RT-PCR, and TNF- and MCP-1 induced iNOS protein expression in time- and concentration-dependent manners. Neutralization of TNF- or MCP-1 in U87-CM and C6-CM using a TNF- or MCP-1 antibody inhibited iNOS protein expression, and increased intracellular peroxide by TNF- or MCP-1 was identified in BV-2 cells. The reciprocal activation of glioma cells and microglia via ROS-dependent iNOS/NO elevation at least partially mediated by TNF- and MCP-1 is elucidated. J. Cell. Physiol. 229: 2015-2026, 2014. (c) 2014 Wiley Periodicals, Inc.