Overexpression of NQO1 protects human SK-N-MC neuroblastoma cells against dopamine-induced cell death.
NAD(P)H quinone oxidoreductase 1 (NQO1) can metabolize dopamine-derived quinones (DAQ) and absence of NQO1 due to the NQO1*2 polymorphism has been suggested to be a risk factor for Parkinson's disease. In order to define whether NQO1 plays a protective role in dopamine toxicity, we have examined the potential role of NQO1 in the SK-N-MC human neuroblastoma cell line. SK-N-MC cells were stably transfected with NQO1 to generate stable clones with NQO1 enzymatic activity of 245 nmol/mgmin while vector control and parental cells had NQO1 activities of less than 12 nmol/mgmin. Incubation of dopamine for 24 h in both parental and vector control SK-N-MC cells resulted in 85% and 72% cell death as assessed by annexin-V/propidium iodide analysis. In agreement, 88% and 84% of parental and vector control cells, respectively underwent loss of mitochondrial membrane potential (MMP) assessed by tetramethylrhodamine ethyl ester. In contrast, NQO1-transfected cells were resistant to dopamine toxicity and both cell death and loss of MMP were markedly abrogated in NQO1-transfected SK-N-MC cells. When dopamine was added to medium, oxygen uptake could be detected indicating autoxidation with concomitant formation of oxygen radicals and quinones. However, dopamine-induced cell death was not affected by the inclusion of either superoxide dismutase or catalase suggesting that superoxide and hydrogen peroxide were not involved in toxicity. Quinones formed in medium may exert toxicity extracellularly or intracellularly but the protective role of NQO1 argues for an intracellular mechanism. In summary, transfection of SK-N-MC cells with NQO1 protects against dopamine-induced toxicity.
Zafar
K S
KS
Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado at Denver and Health Sciences Center, Denver, CO 80262, USA.
Inayat-Hussain
S H
SH
Siegel
D
D
Bao
A
A
Shieh
B
B
Ross
D
D
D017304:Annexin A5; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000473:pathology*; D002374:Catalase / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D004298:Dopamine / Q000633:toxicity*; D005434:Flow Cytometry; D005609:Free Radicals; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D051336:Mitochondrial Membranes / Q000187:drug effects; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000096:biosynthesis* / Q000502:physiology; D009447:Neuroblastoma / Q000473:pathology*; D011809:Quinones / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism; D014162:Transfection
D017304:Annexin A5; D005609:Free Radicals; D011809:Quinones; D002374:Catalase; D013482:Superoxide Dismutase; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human; D004298:Dopamine