[Proteasomal inhibitor lactacystin induces cell apoptosis and caspase 3 activation in PC12 cells].
OBJECTIVE
To explore whether proteasome dysfunction cam induce dopaminergic cell apoptosis and investigate the probable molecular mechanism.


METHODS
MTT assay was applied to measure the cell vitality of rat pheochrom-ocytoma cells of the Line PC12 exposed to highly specific proteasomal inhibitor lactacystin (0, 1 micromol/L, 5 micromol/L or 10 micromol/L) for 24 hours. After the PC12 cells were treated with 10 micromol/L lactacystin for 24 hours, apoptosis was estimated by Hoechst fluorescence staining and flow cytometry. When the PC12 cells were treated with 10 micromol/L lactacystin for 0, 24 or 48 hours, the level of caspase 3 cleaved fragments were analyzed by Western blotting.


RESULTS
The PC12 cells exposed to 5 micromol/L or 10 micromol/L lactacystin for 24 hours showed a significant decrease in cell vitality (P < 0.05). Following treated with 10 micromol/L lactacystin for 24 hours, PC12 cells were seen to be nuclear condensation and fragmentation consistent with an apoptotic nuclear morphology by were seen in the Hoechst fluorescence staining and confirmed to have a significant increase of apoptotic cells (about 31.4%) by flow cytometry. Western blotting showed that there was a very low level of caspase 3 cleaved fragments (17,000) in control cells. But, after PC12 cells were exposed to 10 micromol/L lactacystin for 48 hours, the protein level of caspase 3 cleaved fragments (17,000) increased obviously.


CONCLUSIONS
Proteasomal inhibitor lactacystin leads to dopaminergic cell apoptosis. The activation of caspase 3 protease may contribute to the mechanism of lactacystin-induced apoptosis in PC12 cells. Proteasome dysfunction may play an important role in the pathogenesis of Parkinson's disease.
Yang
Hui
H
Department of Neurology, Clinical & Research Center for Parkinson Disease, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China.
Chen
Sheng-di
SD
Lu
Guo-qiang
GQ
Li
Biao
B
Liang
Liang
L
Xu
Jie-yi
JY
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism*; D016716:PC12 Cells; D061988:Proteasome Inhibitors; D051381:Rats
D061988:Proteasome Inhibitors; C067713:lactacystin; D053148:Caspase 3; D000111:Acetylcysteine