Akt-dependent heme oxygenase-1 induction by NS-398 in C6 glial cells: a potential role for CO in prevention of oxidative damage from hypoxia.
We investigated whether increased heme oxygenase (HO)-1 activity by NS-398 is responsible for protection against hypoxia-induced damage in C6 cells. The expression of HO-1 was analyzed by Western blot and cell viability was analyzed by lactate dehydroxygease (LDH) activity. NS-398 increased HO-1 expression in a concentration- and time-dependent manner during both normoxia and hypoxia (95% N(2)/5% CO(2)), but the latter was much more sensitive. Because induction of HO-1 occurred due to hypoxia itself, NS-398 seemed to potentiate the expression of HO-1. The reduced cell viability due to hypoxia was significantly reversed by either NS-398 or [Ru(CO)(3)(Cl)(2)](2), a CO-donor. Zinc protophorphrin (ZnPPIX), a HO-1 inhibitor, inhibited the protective effect of NS-398 against hypoxia. Treatment with glucose oxidase (GOX, 20 mU/ml) increased ROS production and caused apoptotic death, as assayed by DCFH-DA and TUNEL, respectively. NS-398 significantly reduced GOX-induced cell death and ROS production; these effects were reversed by pre-treatment with oxyhemoglobin (HbO(2)), a CO/NO scavenger, or ZnPPIX. Finally, NS-398 increased PPAR-gamma luciferase activity in transiently PPAR-gamma transfected C6 cells, which was antagonized by ZnPPIX. NS-398 increased phosphorylation of Akt, and LY-294002, a specific PI(3) kinase inhibitor, inhibited NS-398-induced HO-1 expression. Taken together, we conclude that therapeutic use of NS-398 in the treatment of oxidative stress-oriented neuronal disorders would be beneficial through dual actions: HO-1 induction and COX-2 inhibition.
Department of Pharmacology, School of Medicine, and Institute of Health Sciences, Gyeongsang National University, Jinju 660-751, Republic of Korea.
Min Kyu
MK
Park
Chang Hee
CH
Kim
Young Min
YM
Kim
Young Jin
YJ
Kang
Hyo Jung
HJ
Kim
Hye Jung
HJ
Kim
Han Geuk
HG
Seo
Jae Heun
JH
Lee
Ki Churl
KC
Chang
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002248:Carbon Monoxide / Q000494:pharmacology; D002460:Cell Line; D004790:Enzyme Induction / Q000187:drug effects; D051547:Heme Oxygenase-1 / Q000096:biosynthesis*; D000860:Hypoxia / Q000503:physiopathology / Q000517:prevention & control; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D009457:Neuroglia / Q000187:drug effects / Q000502:physiology*; D009578:Nitrobenzenes / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt / Q000502:physiology*; D013449:Sulfonamides / Q000494:pharmacology*; D014162:Transfection
D000894:Anti-Inflammatory Agents, Non-Steroidal; D009578:Nitrobenzenes; D013449:Sulfonamides; C080955:N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; D002248:Carbon Monoxide; D007770:L-Lactate Dehydrogenase; D051547:Heme Oxygenase-1; D051057:Proto-Oncogene Proteins c-akt