Staurosporine- and H-7-induced cell death in SH-SY5Y neuroblastoma cells is associated with caspase-2 and caspase-3 activation, but not with activation of the FAS/FAS-L-caspase-8 signaling pathway.
Apoptotic cell death is induced in SH-SY5Y neuroblastoma cells following exposure to the protein kinase inhibitors staurosporine (100 nM) and 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine: H-7 (100 microM). This is associated with reduced levels of PARP 117 kDa and with the concomitant formation of PARP-cleaved products of 89 kDa that result from caspase-3 activation. The process is inhibited with DEVD-fmk, a potent caspase-3 (and caspase-8) inhibitor, thus indicating that staurosporine- and H-7-induced cell death in SH-SY5Y is mediated by caspase activation. Increased caspase-2- and caspase-3-like activities, but not caspase-9-like activity, were demonstrated by monitoring proteolysis of the corresponding colorimetric substrates. Caspase-2 activity peaked at 6 h, whereas caspase-3 peaked at 12 h in parallel with the maximal loss of cell viability. No modifications in the expression levels of Fas and Fas-L were observed by Western blotting. Furthermore, no activation of caspase-8 was elicited by colorimetric assays through the process of apoptosis of neuroblastoma cells. These findings indicate that the Fas/Fas-L-caspase-8 pathway of cell death signaling is not involved in staurosporine- and H-7-induced apoptosis in SH-SY5Y neuroblastoma cells.
López
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E
Unitat de Neuropatologia, Department de Biologia Cel.lular i Anatomia Patològica, Universitat de Barcelona, Campus de Bellvitge, 08907 Hospitalet de Llobregat, Llobregat, Spain.
Ferrer
I
I
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D015153:Blotting, Western; D053143:Caspase 2; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D053222:Fas Ligand Protein; D006801:Humans; D008562:Membrane Glycoproteins / Q000032:analysis / Q000378:metabolism; D009447:Neuroblastoma; D009474:Neurons / Q000166:cytology / Q000201:enzymology; D011506:Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D019311:Staurosporine / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000032:analysis / Q000378:metabolism*
D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D011506:Proteins; D019014:fas Receptor; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053143:Caspase 2; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D019311:Staurosporine