Bcl-2 Family and Caspases are Involved in CoCl2-Induced Apoptosis of PC12 Cells

Abstract

Hypoxic/ischemic condition induces the neuronal apoptotic events, consequently resulting in neuronal damages. Cobalt chloride (CoCl₂) could mimic the hypoxic condition including the production of reactive oxygen species (ROS). This study aimed to investigate the roles of Bcl-2 family and caspases as central regulators of apoptosis, in CoCl₂-induced apoptosis of PC12 cells. Cell viability was determined by MTT assay and DNA fragmentation was detected by DNA laddering. The expression levels of Bcl-2, Bax, Bid, cytochrome c and Fas/APO-1 were determined by RT-PCR or Western blotting analysis in CoCl₂-treated PC12 cells. Caspase-9 and caspase-3 activities were assessed using spectrophotometry and caspase-8 activity was measured with fluorospectrocytometry. Administration of CoCl₂ decreased viability of cells in a dose- and time-dependent manner. Furthermore, fragmentation of the genomic DNA and apoptotic bodies were induced in CoCl₂-treated PC12 cells. Bcl-2, an anti-apoptotic Bcl-2 family, was downregulated, whereas Bax, pro-apoptotic molecule, was upregulated in CoCl₂-treated cells. Treatment of CoCl₂ augumented the release of cytochrome c into the cytoplasm and increase of caspase-8, -9, and -3 activities. In addition, CoCl₂ upregulated Fas and downregulated pro-Bid, which are known to be correlated with death receptor-mediated apoptotic signaling pathway. Therefore, these results suggest that Bcl-2 family and caspase play crucial roles in CoCl₂-induced apoptosis through mitochondria- and death receptor-dependent pathways in PC12 cells.