Effect of fraxetin on antioxidant defense and stress proteins in human neuroblastoma cell model of rotenone neurotoxicity. Comparative study with myricetin and N-acetylcysteine.
Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production. Recently, it has been shown that fraxetin (coumarin) and myricetin (flavonoid) have significant neuroprotective effects against apoptosis induced by rotenone, increase the total glutathione levels in vitro, and inhibit lipid peroxidation. Thus, these considerations prompted us to investigate the way in which fraxetin and myricetin affect the endogenous antioxidant defense system, such as Mn and CuZn superoxide dismutase (MnSOD, CuZnSOD), catalase, glutathione reductase (GR), and glutathione peroxidase (GPx) on rotenone neurotoxicity in neuroblastoma cells. N-acetylcysteine (NAC), a potent antioxidant, was employed as a comparative agent. Also, the expression and protein levels of HSP70 by Northern and Western blot analysis were assayed in SH-SY5Y cells. After incubation for 16 h, rotenone significantly increased the expression and activity of MnSOD, GPx, and catalase. When cells were preincubated with fraxetin, there was a decrease in the protein levels and activity of both MnSOD and catalase, in comparison with the rotenone treatment. The myricetin effect was less pronounced. Activity and expression of GPx were increased by rotenone and pre-treatment with fraxetin did not modify significantly these levels. The significant enhancement in HSP70 expression at mRNA and protein levels induced by fraxetin was observed by pre-treatment of cells 0.5 h before rotenone insult. These data suggest that major features of rotenone-induced neurotoxicity are partially mediated by free radical formation and oxidative stress, and that fraxetin partially protects against rotenone toxicity affecting the main protection system of the cells against oxidative injury.
Molina-Jiménez
María Francisca
MF
Departamento de Farmacología, Facultad de Farmacia, Universidad Complutense, Plaza de Ramón y Cajal s/n, 28040 Madrid, Spain.
Sánchez-Reus
María Isabel
MI
Cascales
María
M
Andrés
David
D
Benedí
Juana
J
D000111:Acetylcysteine / Q000378:metabolism / Q000494:pharmacology*; D000975:Antioxidants / Q000378:metabolism*; D015152:Blotting, Northern; D015153:Blotting, Western; D002374:Catalase / Q000037:antagonists & inhibitors / Q000378:metabolism; D003374:Coumarins / Q000378:metabolism / Q000494:pharmacology*; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D005419:Flavonoids / Q000378:metabolism / Q000494:pharmacology*; D005434:Flow Cytometry; D005453:Fluorescence; D005979:Glutathione Peroxidase / Q000235:genetics / Q000378:metabolism; D005980:Glutathione Reductase / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D008163:Luminescent Measurements; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology; D017382:Reactive Oxygen Species / Q000378:metabolism; D012402:Rotenone / Q000494:pharmacology*; D013482:Superoxide Dismutase / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000975:Antioxidants; D003374:Coumarins; D005419:Flavonoids; D018840:HSP70 Heat-Shock Proteins; D017382:Reactive Oxygen Species; D012402:Rotenone; C040015:myricetin; C105671:fraxetin; D002374:Catalase; D005979:Glutathione Peroxidase; D013482:Superoxide Dismutase; D005980:Glutathione Reductase; D000111:Acetylcysteine