Metabolic inhibitors sensitize for CD95 (APO-1/Fas)-induced apoptosis by down-regulating Fas-associated death domain-like interleukin 1-converting enzyme inhibitory protein expression.

Protein or RNA synthesis inhibitors are known to sensitize some resistant cells for death receptor-induced apoptosis. However, the molecular mechanism(s) involved in sensitization have not yet been defined exactly. Here, we report that metabolic inhibitors such as cycloheximide (CHX) or actinomycin D (ActD) sensitize for CD95-induced apoptosis by strongly down-regulating FLIP and RIP expression. Metabolic labeling studies revealed that CHX or ActD inhibited protein or RNA synthesis at concentrations required for sensitization. In contrast to Fas-associated death domain (FADD) or caspase-8, FADD-like interleukin 1-converting enzyme-inhibitory protein (FLIP) and RIP protein levels rapidly decreased upon treatment with CHX or ActD, indicating that both molecules have a high turnover rate. Selective down-regulation of FLIP expression by FLIP antisense oligonucleotides sensitized for CD95-induced apoptosis. Reduction of FLIP levels resulted in undetectable amounts of FLIP at the CD95 death-inducing signaling complex (DISC) upon CD95 stimulation, thereby enhancing the recruitment of caspase-8 to the DISC and caspase-8 activation. CHX- or ActD-mediated sensitization to CD95-induced apoptosis was predominantly found in type I cells in which FADD and caspase-8 are recruited to CD95 upon stimulation but not in type II cells in which no DISC formation is detected. Pretreatment with CHX or ActD sensitized for subsequent CD95 stimulation compared with cells without pretreatment. CHX or ActD also reduced XIAP expression and similarly sensitized for tumor necrosis factor-related apoptosis-inducing ligand- or tumor necrosis factor-alpha-induced apoptosis. Because blockade of death receptor triggering by FLIP overexpression has recently been implicated in tumorigenesis and treatment resistance in vivo, strategies to inhibit FLIP expression, e.g., by metabolic inhibitors, may prove to be a useful complementary tool for the treatment of cancer.