Activation of caspase-8 triggers anoikis in human neuroblastoma cells.
Cells require appropriate interaction with extracellular matrix proteins mediated by integrins to grow, differentiate and survive. Many cell types including nervous cells undergo anoikis, a substrate-dependent apoptosis, when adhesion is impaired. Resistance of tumors to cytotoxic drugs is probably due to disturbed apoptosis programs. The proteolytic enzymes caspases are the main executioners of apoptosis. It was reported that caspase-8 expression is deficient in some neuroblastoma cells. We demonstrated that human neuroblastoma cell line SK-B-BE, differentiated with retinoic acid, expressed caspases 3, 8 and 9. Caspases 8 and 3, but not caspase-9 were activated in SK-N-BE cells cultured in suspension or on aspecific adhesive substrate. Cell positive to caspase-8 were classified into four stages, by morphometric and densitometric parameters. The use of the specific caspase-8 inhibitor Z-IETD-FMK dramatically reduced apoptosis, demonstrating that caspase-8 is the upstream initiator caspase during SK-N-BE cells anoikis. Among matrix proteins, type I collagen is the most effective and fibronectin the least in delaying anoikis. The activation of caspases 8 and 3 by unligated integrins was dependent on the state of neuronal differentiation, since the most differentiated cell was the most vulnerable to anoikis. These data show that activation of caspase-8 is specifically required to promote anoikis in SK-N-BE neuroblastoma cells.
Bozzo
Chiarella
C
Department of Clinical and Experimental Medicine, University of Eastern Piedmont A. Avogadro, Novara, Italy. bozzo@med.unipmn.it
Sabbatini
Maurizio
M
Tiberio
Rossana
R
Piffanelli
Valentina
V
Santoro
Claudio
C
Cannas
Mario
M
D000704:Analysis of Variance; D023102:Anoikis / Q000187:drug effects / Q000502:physiology*; D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western / Q000379:methods; D053148:Caspase 3 / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D016326:Extracellular Matrix Proteins / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods; D009447:Neuroblastoma; D009842:Oligopeptides / Q000494:pharmacology; D013997:Time Factors; D014212:Tretinoin / Q000494:pharmacology
D000970:Antineoplastic Agents; D015815:Cell Adhesion Molecules; D015853:Cysteine Proteinase Inhibitors; D016326:Extracellular Matrix Proteins; D009842:Oligopeptides; C403753:benzyloxycarbonyl-isoleucyl-glutamyl-threonyl-aspartic acid fluoromethyl ketone; D014212:Tretinoin; D053148:Caspase 3; D053181:Caspase 8