Overexpression of 14-3-3 protein protects pheochromocytoma cells against 1-methyl-4-phenylpyridinium toxicity.
Objective To investigate the effects of 14-3-3 protein overexpression on the 1-methyl-4-phenylpyridinium (MPP(+)) induced pheochromocytoma (PC12) cell death and the potential mechanisms. Methods pcDNA3.1(+)-14-3-3 plasmids, which could be expressed in mammalian cell, were constructed and transfected into PC12 cells with Lipofectamine 2000. The expression of 14-3-3 protein, Bcl-2 protein, and BAD protein were determined by western blot. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, microplate reader, and flow cytometric analysis were used to measure cell viability, the caspase activity, and apoptotic ratio respectively. Results (1) The expression of 14-3-3 protein increased significantly three weeks after pcDNA3.1 (+)-14-3-3 plasmids transfected into PC12 cells. (2) MPP(+) caused a decrease of cell viability in a dose-dependent manner. At 100 mu mol/L MPP(+), cell viability reduced approximately 50%. (3) The caspase activity increased along with the MPP(+) concentrations rising and reached its maximum value (0.34 mu mol/mg protein) at 100 mu mol/L MPP(+). However caspase activity decreased significantly when the MPP(+) concentration exceeded 100 mu mol/L. (4) Overexpression of 14-3-3 protein decreased the apoptosis ratio of PC12 cells treated with 100 mu mol/L MPP(+) from 26.5% to 8.6%. (5) Bcl-2 protein tended to decrease but BAD protein tended to increase after treatment of PC12 cells with 100 mu mol/L MPP(+). Overexpression of 14-3-3 protein significantly increased the cellular level of Bcl-2 protein and decreased that of BAD protein. Conclusion Overexpression of 14-3-3 protein may reduce MPP(+)-induced apoptotic cell death in PC12 cells by up-regulating the Bcl-2 expression and down-regulating the BAD expression. These results may provide a promising target for treatment of Parkinson' s disease.
Chen
Xiao-Wu
XW
Department of Neurology, the Affiliated Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China; E-mail: sunshenggang@126.com.
Sun
Sheng-Gang
SG
Cheng
Dao-Bin
DB
Tian
You-Yong
YY