Human
hepatocellular carcinomas (HCCs) show resistance to apoptosis mediated by several
death receptors. Because cellular FLICE/
caspase-8-inhibitory
protein (cFLIP) is a recently identified intracellular inhibitor of
caspase-8 activation that potently inhibits death signaling mediated by all known
death receptors, including Fas,
TNF-receptor (TNF-R), and
TNF-related apoptosis-inducing ligand receptors (TRAIL-Rs), we investigated the expression and function of cFLIP in human HCCs. We found that cFLIP is constitutively expressed in all human HCC cell lines and is expressed more in human HCC tissues than in nontumor liver tissues. Metabolic inhibitors,
actinomycin D (ActD) or
cycloheximide (CHX), dramatically rendered HCC cells sensitive to Fas-mediated apoptosis. Neither
caspase-8 nor
caspase-3 was activated by agonistic anti-Fas antibody alone, but both
caspases were activated by Fas stimulation in the presence of ActD or CHX, indicating the importance of
caspase-8 inhibitors that are sensitive to metabolic inhibitors. Actually, cFLIP expression was decreased in ActD or CHX treatment. cFLIP down-regulation induced by cFLIP antisense
oligodeoxynucleotides sensitized HLE cells to Fas, TNF-R, and TRAIL-R-mediated apoptosis. Furthermore, cFLIP over-expression activated nuclear factor (
NF)-kappaB and cFLIP down-regulation attenuated
NF-kappaB activation induced by
TNF-alpha or TRAIL. Pretreatment with pan-
caspase-inhibitor,
benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl
ketone (
Z-VAD-fmk), restored
NF-kappaB activity attenuated by cFLIP down-regulation. cFLIP expression was increased by
TNF-alpha, TRAIL, or
vascular endothelial growth factor but decreased by
wortmannin, indicating that cFLIP expression is regulated by both the
NF-kappaB and phosphatidylinostiol-3
kinase (PI-3)/Akt pathways. These results suggest that cFLIP plays an important role in cell survival not simply by inhibiting
death-receptor-mediated apoptosis but also by regulating
NF-kappaB activation in human HCCs.