Etoposide induces protein kinase Cdelta- and caspase-3-dependent apoptosis in neuroblastoma cancer cells.
In this report, we reveal that etoposide inhibits the proliferation of SK-N-AS neuroblastoma cancer cells and promotes protein kinase Cdelta (PKCdelta)- and caspase-dependent apoptosis. Etoposide induces the caspase-3-dependent cleavage of PKCdelta to its active p40 fragment, and active PKCdelta triggers the processing of caspase-3 by a positive-feedback mechanism. Treatment of cells with the caspase-3-specific inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone or caspase-3-specific small interacting RNA (siRNA) prevented the etoposide-induced activation of caspase-8 and inhibited apoptosis. The silencing of the caspase-2 or caspase-8 genes using siRNAs did not affect the etoposide-induced processing of caspase-3, indicating that these caspases lie downstream of caspase-3 in this signaling pathway. Furthermore, the etoposide-induced processing of caspase-2 required the expression of caspase-8, and the etoposide-mediated processing of caspase-8 required the expression of caspase-2, indicating that these two caspases activate each other after etoposide treatment. We also observed that etoposide-mediated apoptosis was decreased by treating the cells with the caspase-6-specific inhibitor benzyloxycarbonyl-Val-Glu(OMe)-Ile-Asp-(OMe)-fluoromethyl ketone and that caspase-6 was activated by a caspase-8-dependent mechanism. Finally, we show that rottlerin blocks etoposide-induced apoptosis by inhibiting the PKCdelta-mediated activation of caspase-3 and by degrading caspase-2, which prevents caspase-8 activation. Our results add important insights into how etoposide mediates apoptotic signaling and how targeting these pathways may lead to the development of novel therapeutics for the treatment of neuroblastomas.
Day
Travis W
TW
Department of Pharmacology and Toxicology and Indiana University Simon Cancer Center, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Wu
Ching-Huang
CH
Safa
Ahmad R
AR
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D005047:Etoposide / Q000494:pharmacology*; D006801:Humans; D009447:Neuroblastoma / Q000201:enzymology*; D051745:Protein Kinase C-delta / Q000378:metabolism*
D000972:Antineoplastic Agents, Phytogenic; D005047:Etoposide; D051745:Protein Kinase C-delta; D053148:Caspase 3