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)-α and monocyte chemoattractant protein (MCP)-1 messenger (m)RNA in U87 and C6 cells were detected by an RT-PCR, and TNF-α and MCP-1 induced iNOS protein expression in time- and concentration-dependent manners. Neutralization of TNF-α or MCP-1 in U87-CM and C6-CM using a TNF-α or MCP-1 antibody inhibited iNOS protein expression, and increased intracellular peroxide by TNF-α 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-α and MCP-1 is elucidated.
Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan.
Shing-Chuan
SC
Shen
Ming-Shun
MS
Wu
Hui-Yi
HY
Lin
Liang-Yo
LY
Yang
Yi-Hsuan
YH
Chen
Yen-Chou
YC
Chen
D000873:Anthracenes / Q000008:administration & dosage; D002070:Butadienes / Q000008:administration & dosage; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D018932:Chemokine CCL2 / Q000378:metabolism; D018920:Coculture Techniques; D017077:Culture Media, Conditioned; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005910:Glioma / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D017628:Microglia / Q000378:metabolism* / Q000473:pathology; D016328:NF-kappa B / Q000096:biosynthesis / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D009570:Nitriles / Q000008:administration & dosage; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D000873:Anthracenes; D002070:Butadienes; C111835:CCL2 protein, human; D018932:Chemokine CCL2; D017077:Culture Media, Conditioned; D016328:NF-kappa B; D009570:Nitriles; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; C113580:U 0126; C432165:pyrazolanthrone; D009569:Nitric Oxide; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; D048031:JNK Mitogen-Activated Protein Kinases