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Calcium and reactive oxygen species mediate staurosporine-induced mitochondrial dysfunction and apoptosis in PC12
1Sanders-Brown Research Center on Aging and Department of Anatomy and Neurobiology, University of Kentucky, Lexington 40536-0230, USA.
Abstract:
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.
Insights
Staurosporine induces apoptosis by increasing intracellular calcium and reactive oxygen species (ROS). Early interventions can prevent these changes and rescue cells, revealing critical early events in programmed cell death.
Area of Science:
- Cell Biology
- Biochemistry
- Neuroscience
Background:
- Staurosporine is a bacterial alkaloid used to induce apoptosis in various cell types, including neurons.
- The precise intracellular mechanisms driving staurosporine-induced apoptosis remain largely unelucidated.
Purpose of the Study:
- To investigate the early intracellular events and signaling cascades involved in staurosporine-induced apoptosis in PC12 cells.
- To determine the roles of intracellular calcium and reactive oxygen species (ROS) in this process.
Main Methods:
- PC12 cells were exposed to staurosporine and monitored for changes in intracellular free calcium levels ([Ca2+]i), mitochondrial ROS, and MTT reduction.
- Cells were treated with protective agents like serum, nerve growth factor, BAPTA, uric acid, zVAD-fmk, or Bcl-2 to assess their impact on apoptosis.
Main Results:
- Staurosporine rapidly increased [Ca2+]i and mitochondrial ROS, followed by lipid peroxidation, loss of mitochondrial potential, and nuclear apoptosis.
- Serum or nerve growth factor treatment rescued cells by normalizing [Ca2+]i and ROS levels.
- Inhibition of [Ca2+]i and ROS accumulation with BAPTA or uric acid protected cells, indicating their necessity for apoptosis.
- Caspase inhibition and Bcl-2 expression blocked sustained [Ca2+]i and ROS increases, suggesting early caspase involvement.
Conclusions:
- An increase in intracellular calcium ([Ca2+]i) is an early and critical trigger for staurosporine-induced apoptosis.
- The apoptotic pathway involves ROS production, oxidative stress, and mitochondrial dysfunction, initiated by calcium influx.
- Caspases play a crucial early role in mediating the sustained calcium and ROS increases that lead to apoptosis.