Calcium and reactive oxygen species mediate staurosporine-induced mitochondrial dysfunction and apoptosis in PC12 cells.
The bacterial alkaloid staurosporine is widely employed as an inducer of apoptosis in many cell types including neurons. The intracellular cascades that mediate staurosporine-induced apoptosis are largely unknown. Exposure of cultured PC12 cells to staurosporine resulted in a rapid (min) and prolonged (1-6 hr) elevation of intracellular free calcium levels [Ca2+]i, accumulation of mitochondrial reactive oxygen species (ROS), and decreased mitochondrial 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction (1-4 hr). These early events were followed by membrane lipid peroxidation, loss of mitochondrial transmembrane potential, and nuclear apoptotic changes. Treatment of cells with serum or nerve growth factor within 1-2 hr of staurosporine exposure resulted in recovery of [Ca2+]i and ROS levels, and rescued the cells from apoptosis. The increased [Ca2+]i and ROS production were required for staurosporine-induced apoptosis because the intracellular calcium chelator BAPTA and uric acid (an agent that scavenges peroxynitrite) each protected cells against apoptosis. The caspase inhibitor zVAD-fmk and the anti-apoptotic gene product Bcl-2 prevented the sustained [Ca2+]i increase and ROS accumulation induced by staurosporine indicating that caspases act very early in the apoptotic process. Our data indicate that a [Ca2+]i increase is an early and critical event in staurosporine-induced apoptosis that engages a cell death pathway involving ROS production, oxidative stress, and mitochondrial dysfunction.
Kruman
I
I
Sanders-Brown Research Center on Aging and Department of Anatomy and Neurobiology, University of Kentucky, Lexington 40536-0230, USA.
Guo
Q
Q
Mattson
M P
MP
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D002614:Chelating Agents / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D016716:PC12 Cells; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology*
D002614:Chelating Agents; D004791:Enzyme Inhibitors; D017382:Reactive Oxygen Species; D019311:Staurosporine; D002118:Calcium