Hypoxia induces apoptosis in human neuroblastoma SK-N-MC cells by caspase activation accompanying cytochrome c release from mitochondria.
We have attempted to elucidate the mechanism of apoptotic cell death induced by hypoxia (very low oxygen conditions) in neuronal cells. Human neuroblastoma SK-N-MC cells under hypoxic conditions resulted in apoptosis in a time-dependent manner estimated by DNA fragmentation assay and nuclear morphology stained with fluorescent chromatin dye. Pretreatment with Z-Asp-CH2-DCB, a caspase inhibitor, suppressed the DNA ladder in response to hypoxia in a concentration-dependent manner. An increase in caspase-3-like protease (DEVDase) activity was observed during apoptosis, but no caspase-1 activity (YVADase) was detected. To confirm the involvement of caspase-3 during apoptosis, Western blot analysis was performed using anti-caspase-3 antibody. The 20- and 17-kDa proteins, corresponding to the active products of caspase-3, were generated in hypoxia-challenged lysates in which processing of the full length form of caspase-3 was evident. With a time course similar to this caspase-3 activation, hypoxic stress caused the cleavage of PARP, yielding an 85-kDa fragment typical of caspase activity. In addition, caspase-2 was also activated by hypoxia, and the stress elicited the release of cytochrome c into the cytosol during apoptosis. These results suggest that caspase activation and cytochrome c release play roles in hypoxia-induced neuronal apoptosis.
Araya
R
R
Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Uehara
T
T
Nomura
Y
Y
D017209:Apoptosis; D020170:Caspase 1 / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D015687:Cell Hypoxia; D003574:Cytochrome c Group / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D008928:Mitochondria / Q000201:enzymology*; D009447:Neuroblastoma / Q000201:enzymology* / Q000473:pathology; D010100:Oxygen / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D003574:Cytochrome c Group; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D020170:Caspase 1; D010100:Oxygen