Rottlerin protected dopaminergic cell line from cytotoxicity of 6-hydroxydopamine by inhibiting PKCdelta phosphorylation.
OBJECTIVE
The present study aims to investigate the role of protein kinase C delta subtype (PKCdelta) phosphorylation in the process of 6-hydroxydopamine (6-OHDA)-induced dopaminergic cell death, and demonstrate the molecular basis of neurological disorders, such as Parkinson's disease.


METHODS
The pheochromocytoma (PC12) cell line was employed in the present study. Cells were treated with 2 mumol/L PKCdelta inhibitor Rottlerin, 10 nmol/L protein kinase C delta subtype (PKCdelta) inhibitor bisindolylmaleimide I, or 5 nmol/L Gö6976 that could specifically inhibit the calcium-dependent PKCdelta isoforms, respectively. PKC activator phorbol-12-myristate-13-acetate (PMA, 100 nmol/L) was also used in this study. All these agents were added to the medium before cells were incubated with 6-OHDA. Cells with no treatment served as control. The cytotoxicity of 6-OHDA was determined by methyl thiazolyl tetrazolium (MTT) reduction assay and PKCdelta phosphorylation levels in various groups were measured by western blotting.


RESULTS
Bisindolylmaleimide I and Gö6976 exerted no significant attenuation on the cytotoxicity of 6-OHDA, nor any effects on PKCdelta phosphorylation in PC12 cells. However, Rottlerin could inhibit the phosphorylation of PKCdelta and attenuate 6-OHDA-induced cell death, and the cell viability was raised to (69.6+/-2.63)% of that in control group (P<0.05). In contrast, PMA induced a significant increase in PKCdelta phosphorylation and also strengthened the cytotoxic effects of 6-OHDA. The cell viability of PMA-treated PC12 cells decreased to (49.8+/-5.06)% of that in control group (P<0.001).


CONCLUSIONS
Rottlerin can protect PC12 cells from cytotoxicity of 6-OHDA probably by inhibiting PKCdelta phosphorylation. The results suggest that PKCdelta may be a key regulator of neuron loss in Parkinson's disease.
Fan
Ying
Y
Department of Geriatrics, Second Affiliated Hospital, Harbin Medical University, Harbin 150080, China. fanyingyan@163.com
Zhang
Yan-Qiao
YQ
Sun
Dian-Jun
DJ
Zhang
Yi-Na
YN
Wu
Xiao-Wei
XW
Li
Jing
J
D000098:Acetophenones / Q000494:pharmacology*; D018663:Adrenergic Agents / Q000633:toxicity*; D000818:Animals; D001578:Benzopyrans / Q000494:pharmacology*; D002227:Carbazoles / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D004298:Dopamine / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D016627:Oxidopamine / Q000633:toxicity*; D016716:PC12 Cells; D010703:Phorbol Esters / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D013997:Time Factors
D000098:Acetophenones; D018663:Adrenergic Agents; D001578:Benzopyrans; D002227:Carbazoles; D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D010703:Phorbol Esters; C081021:Go 6976; C032974:12-O-retinoylphorbol-13-acetate; D016627:Oxidopamine; C085746:rottlerin; D011493:Protein Kinase C; C070515:bisindolylmaleimide I; D004298:Dopamine