Reactive oxygen species-dependent nitric oxide production in reciprocal interactions of glioma and microglial cells

Shing Chuan Shen, Ming Shun Wu, Hui Yi Lin, Liang Yo Yang, Yi Hsuan Chen, Yen Chou Chen

Research output: Contribution to journalArticlepeer-review

25 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)2015-2026
Number of pages12
JournalJournal of Cellular Physiology
Issue number12
Publication statusPublished - Dec 2014

ASJC Scopus subject areas

  • Physiology
  • Clinical Biochemistry
  • Cell Biology


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