| AIM: To study the influence of dopa mine (DA) on proliferation and survival of dopa minergic SH-SY5Y neuroblastoma cells; to deter mine whether Quinone oxidoreductase (NQO1) protects dopa minergic cells against dopa mine-induced cytotoxicity; to investigate the pathogenic mechanism of Parkinson's disease; to explore the potential therapeutic application of phase II inducers in the treatment of Parkinson's disease.METHODS:TH and VMAT2 expression in SH-SY5Y cells was deter mined by immunofluorescence staining. MTT assay (mono-nuclear cell direct cytotoxicity assay) was used to deter mine the DA-induced dose-dependant cytotoxicity in SH-SY5Y cells and the protective effects of various phaseâ…¡inducers. Western blot and Real Time PCR was applied to investigate the expression of endogenous phaseâ…¡enzymes after sulforaphane (SF) pre-incubation. The content of quinone protein was deter mined by the NBT/glycinate assay. Lipofection was applied to transfect NQO1 expression plasmid in to SH-SY5Y cells. The expression of NQ01 was detected by Real Time PCR, immunofluorescence staining and Western blot. MTT assay and Flow cytometry was used to estimate the influence of DA,6-hydroxy dopa (6-OHDA) or Rotenone on the survival of SH-SY5Y cells and the protective effect of NQO1. In transfected cells, the protective effect of NQO1 correlated to the amount of quinone protein. Adenovirus was used to infect various cell lines and NQO1 expression was detected by Western blot, In adenovirus infected SH-SY5Y cells that expressing high levels of NQO1, DA-induced reduction in cell viability was deter mined by MTT assay. In primary neurons, immunofluorescence was applied to deter mine the toxic effect of DA with or without SF pre-incubation or with or without NQO1 over-expression by adenovirus infection. Immunohistochemistry, Western blot and Real Time PCR was used to detect NQO1 expression in different brain regions after intraperitoneal injection of SF. RESULTS:Dopa mine reduces the viability of SH-SY5Y cells in a dose-dependant manner. SF was the most effective phase II inducer in protecting cells against DA-induced cytotoxicity. SF increases NQO1 expression and alleviates DA-induced toxicity in SH-SY5Y cells. Increased expression of NQO1 in SH-SY5Y cells either by transfecting an NQO1-expressing plasmid or by infection of an NQO1-expressing adeno virus alleviates DA-caused cytotoxicity, indicating that the SF protective effect was at least partly due to the higher levels of NQO1 expression. Moreover, high levels of NQO1 expression also protect cells against the toxicity caused by 6-OHDA and rotenone. In contrast, when NQO1 activity was reduced by NQO1 inhibitors, DA-induced toxicity was significantly increased in SH-SY5Y cells. The intracellular content of quinone protein correlated with cell viability, which was reduced by SF pre-incubation or NQOloverexpression. In primary neuronal cultures, DA-induced toxicity was also reduced by SF pre-incubation or by infecting cells with NQO1-expressing adenovirus. After intraperitoneal injection of SF, NQO1 expression was increased in the cortex, midbrain, caudate nucleus.CONCLUSIONS: Dopa mine reduces cell proliferation in SH-SY5Y cells in a dose-dependant manner. Expression of NQO1 can be increased via SF treatment, lipofection and adenovirus infection. Overexpression of NQO1 in SH-SY5Y cells protects cells against cytotoxicity caused by dopa mine and other related toxins. High levels of NQO1 expression also protect TH-positive neurons against DA-induced toxicity. The amount of intracellular quinone protein correlates with cell viability, indicating that the formation of quinone protein contributes to the toxicity in dopa minergic neurons. A pilot study indicated that an intraperitoneal SF injection enhances NQO1 expression in the cortex, midbrain, caudate nucleus, supporting the potentially application of phase II inducers like SF in the treatment of Parkinson's disease.
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