Effect of fraxetin and myricetin on rotenone-induced cytotoxicity in SH-SY5Y cells: comparison with N-acetylcysteine.
The purpose of this study was to investigate the potential neuroprotective effects of myricetin (flavonoid) and fraxetin (coumarin) on rotenone-induced apoptosis in SH-SY5Y cells, and the possible signal pathway involved in a neuronal cell model of Parkinson's disease. These two compounds were compared to N-acetylcysteine. The viability of cells was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and cytotoxicity was assayed by lactate dehydrogenase (LDH) released into the culture medium. Parameters related to apoptosis, such as caspase-3 activity, the cleavage of poly(ADP-ribose) polymerase and the levels of reactive oxygen species were also determined. Rotenone caused a time- and dose-dependent decrease in cell viability and the degree of LDH release was proportionally to the effects on cell viability. Cells were pretreated with fraxetin, myricetin and N-acetylcysteine at different concentrations for 30 min before exposure to rotenone. Cytotoxicity of rotenone (5 microM) for 16 h was significantly diminished as well as the release of LDH into the medium, by the effect of fraxetin, myricetin and N-acetylcysteine, with fraxetin (100 microM) and N-acetylcysteine (100 microM) being more effective than myricetin (50 microM). Rotenone-induced apoptosis in SH-SY5Y cells was detected by an increase in caspase-3 activity and in the cleavage of poly(ADP-ribose) polymerase. After exposing these cells to rotenone, a significant increase in reactive oxygen species preceded apoptotic events. Fraxetin (100 microM) and N-acetylcysteine (100 microM) not only reduced rotenone-induced reactive oxygen species formation, but also attenuated caspase-3 activity and poly(ADP-ribose) polymerase cleavage at 16 h against rotenone-induced apoptosis. The effect of fraxetin in both experiments was similar to that of N-acetylcysteine. These results demonstrated the protective action of fraxetin and suggest that it can reduce apoptosis, possibly by decreasing free radical generation in SH-SY5Y cells. Myricetin at 100 microM was without any preventive effect.
Molina-Jimenez
M Francisca
MF
Departamento de Farmacologia, Facultad de Farmacia, Universidad Complutense, Plaza de Ramon y Cajal s/n 28040, Madrid, Spain.
Sanchez-Reus
M Isabel
MI
Benedi
Juana
J
D000111:Acetylcysteine / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003374:Coumarins / Q000494:pharmacology*; D005419:Flavonoids / Q000494:pharmacology*; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009447:Neuroblastoma / Q000201:enzymology / Q000473:pathology; D018696:Neuroprotective Agents / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D012402:Rotenone / Q000633:toxicity*
D003374:Coumarins; D005419:Flavonoids; D018696:Neuroprotective Agents; D017382:Reactive Oxygen Species; D012402:Rotenone; C040015:myricetin; C105671:fraxetin; D007770:L-Lactate Dehydrogenase; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D000111:Acetylcysteine