Activation of caspase-8 during N-(4-hydroxyphenyl)retinamide-induced apoptosis in Fas-defective hepatoma cells

We observed that N-(4-hydroxyphenyl)retinamide (4HPR), a chemopreventive an d chemotherapeutic agent, effectively induced apoptosis in hepatoma cells. Interestingly, Fas-negative (Hep 3B and PLC/PRF/5) hepatoma cells were show n to be more susceptible to apoptosis induced by 4HPR than were Fas-positiv e (Hep G2 and SK-HEP-1) hepatoma cells. Thus, we explored the mechanisms un derlying 4HPR-induced apoptosis in Fas-defective hepatoma cells. Hep 3B cel ls stably expressing the dominant-negative Fas-associated death domain (dnF ADD) showed no alteration in 4HPR drug susceptibility, but when stably expr essing E1B19K, Crm A, or dominant-negative FLICE (dnFLICE), Hep 3B cells we re resistant, suggesting that 4HPR-induced apoptosis was mediated by caspas e-8 activation. Furthermore, apoptosis could be completely blocked by Z-VAD -FMK (a general caspase inhibitor) or by IETD-CHO (a caspase-8 inhibitor), but was only partially blocked by Ac-DEVD-CMK (a caspase-3 inhibitor), by N -acetyl-L-cysteine (NAC) (an antioxidant), by N-acetyl-leucyl-leucyl-norleu cinal (ALLN) (a calpain inhibitor I), or by Z-LEHD-FMK (a caspase-9 inhibit or). Time-sequence analysis of the induction of apoptosis by 4HPR revealed that an initial caspase-8 activation was followed by late mitochondrial cyt ochrome c release and minor caspase-9 activation, which suggested that casp ase-8 activation is the primary upstream regulatory point. Activation of Bi d or induction of proapoptotic Bax was not observed during apoptosis. In co ntrast, Bcl-xL expression was decreased during 4HPR-induced apoptosis. Take n together, these results indicate that 4HPR may be a potential chemotherap eutic drug, which is able to induce apoptosis in Fas-defective hepatoma cel ls through caspase-8 activation.