Abstract
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). CONCLUSION: 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.