Doxorubicin-induced apoptosis in caspase-8-deficient neuroblastoma cells is mediated through direct action on mitochondria.
The induction of p53 expression and stimulation of the Fas/caspase-8 pathway represent major mechanisms by which cytotoxic drugs induce apoptosis, but in neuroblastomas, the caspase-8 gene is often not expressed.


OBJECTIVE
The aim of this study was to determine whether doxorubicin could induce apoptosis in caspase-8-deficient neuroblastoma cells and to define its mechanism of action.


METHODS
The caspase-8-deficient human neuroblastoma cell line, SKN-SH, was incubated with doxorubicin and the apoptotic response, as well as expression of apoptotic molecules in the p53/ Fas/caspase-8 pathway, were determined.


RESULTS
SKN-SH cells incubated with doxorubicin readily underwent apoptosis in a concentration-dependent manner. Western blot analyses with specific antibodies demonstrated that both p53 and Fas ligand were endogenously expressed in SKN-SH cells, but their expression was not stimulated by doxorubicin. Fas receptor was not detected in these cells and caspase-8 was totally absent. Electron microscopic analyses of SKN-SH cells treated with doxorubicin revealed pronounced alterations in mitochondrial structure. This treatment also induced the release of cytochrome c from mitochondria and activated the downstream apoptotic intermediate, caspase-3.


CONCLUSIONS
These results indicate that the p53/Fas/caspase-8 system does not play a role in mediating the apoptotic action of doxorubicin in the human neuroblastoma cell line SKN-SH. Thus, mitochondria and downstream apoptotic signaling intermediates may be considered as key targets for doxorubicin-induced apoptosis in neuroblastoma tumors having deficiencies in the Fas/caspase-8 system.
Rebbaa
A
A
Department of Pediatrics, Northwestern University Medical School, Children's Memorial Institute for Education and Research, Chicago, IL 60614, USA. arebbaa@childrensmemorial.org
Chou
P M
PM
Emran
M
M
Mirkin
B L
BL
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000096:biosynthesis / Q000172:deficiency / Q000378:metabolism*; D004317:Doxorubicin / Q000494:pharmacology*; D005786:Gene Expression Regulation; D016158:Genes, p53; D006801:Humans; D008928:Mitochondria / Q000502:physiology*; D009447:Neuroblastoma / Q000473:pathology; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D019014:fas Receptor
D000970:Antineoplastic Agents; D019014:fas Receptor; D004317:Doxorubicin; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases