Pinusolide and 15-methoxypinusolidic acid attenuate the neurotoxic effect of staurosporine in primary cultures of rat cortical cells.
OBJECTIVE
Apoptosis is a fundamental process required for neuronal development but also occurs in most of the common neurodegenerative disorders. In an attempt to obtain an anti-apoptotic neuroprotective compound from natural products, we isolated the diterpenoids, pinusolide and 15-MPA, from B. orientalis and investigated their neuroprotective activity against staurosporine (STS) -induced neuronal apoptosis. In addition, we determined the anti-apoptotic mechanism of these compounds in rat cortical cells.


METHODS
Primary cultures of rat cortical cells injured by STS were used as an in vitro assay system. Cells were pretreated with pinusolide or 15-MPA before exposure to STS. Anti-apoptotic activities were evaluated by the measurement of cytoplasmic condensation and nuclear fragmentation. The levels of cellular peroxide, malondialdehyde (MDA) and [Ca(2+)]i, as well as the activities of superoxide dismutase (SOD) and caspase-3/7, were measured.


RESULTS
Pinusolide and 15-MPA, at a concentration of 5.0 ìM, reduced the condensed nuclei and rise in [Ca(2+)]i that accompanies apoptosis induced by 100 nM STS. Pinusolide and 15-MPA also protected the cellular activity of SOD, an antioxidative enzyme reduced by STS insult. Furthermore, the overproduction of reactive oxygen species and lipid peroxidation induced by STS was significantly reduced in pinusolide and 15-MPA treated cells. In addition, pinusolide and 15-MPA inhibited STS-induced caspase-3/7 activation.


CONCLUSIONS
These results show that pinusolide and 15-MPA protect neuronal cells from STS-induced apoptosis, probably by preventing the increase in [Ca(2+)]i and cellular oxidation caused by STS, and indicate that they could be used to treat neurodegenerative diseases.
Koo
K A
KA
College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University, San 56-1, Shillim-Dong, Gwanak-Gu, Seoul 151-742, Korea.
Lee
M K
MK
Kim
S H
SH
Jeong
E J
EJ
Kim
S Y
SY
Oh
T H
TH
Kim
Y C
YC
D000818:Animals; D002118:Calcium / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D004224:Diterpenes / Q000494:pharmacology*; D004789:Enzyme Activation; D008315:Malondialdehyde / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D019311:Staurosporine / Q000633:toxicity*; D013482:Superoxide Dismutase / Q000378:metabolism
C464724:15-methoxypinusolidic acid; D004224:Diterpenes; D017382:Reactive Oxygen Species; C101973:pinusolide; D008315:Malondialdehyde; D013482:Superoxide Dismutase; D020169:Caspases; D019311:Staurosporine; D002118:Calcium