You K R, Shin M N, Park R K, Lee S O, Kim D G
Division of GI and Hepatology, Department of Internal Medicine, Research Institute of Clinical Medicine, Chonbuk National University Medical School and Hospital, Chonju, Republic of Korea.
Hepatology. 2001 Dec;34(6):1119-27. doi: 10.1053/jhep.2001.29199.
We observed that N-(4-hydroxyphenyl)retinamide (4HPR), a chemopreventive and chemotherapeutic agent, effectively induced apoptosis in hepatoma cells. Interestingly, Fas-negative (Hep 3B and PLC/PRF/5) hepatoma cells were shown to be more susceptible to apoptosis induced by 4HPR than were Fas-positive (Hep G2 and SK-HEP-1) hepatoma cells. Thus, we explored the mechanisms underlying 4HPR-induced apoptosis in Fas-defective hepatoma cells. Hep 3B cells stably expressing the dominant-negative Fas-associated death domain (dnFADD) showed no alteration in 4HPR drug susceptibility, but when stably expressing E1B19K, Crm A, or dominant-negative FLICE (dnFLICE), Hep 3B cells were resistant, suggesting that 4HPR-induced apoptosis was mediated by caspase-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-norleucinal (ALLN) (a calpain inhibitor I), or by Z-LEHD-FMK (a caspase-9 inhibitor). Time-sequence analysis of the induction of apoptosis by 4HPR revealed that an initial caspase-8 activation was followed by late mitochondrial cytochrome c release and minor caspase-9 activation, which suggested that caspase-8 activation is the primary upstream regulatory point. Activation of Bid or induction of proapoptotic Bax was not observed during apoptosis. In contrast, Bcl-xL expression was decreased during 4HPR-induced apoptosis. Taken together, these results indicate that 4HPR may be a potential chemotherapeutic drug, which is able to induce apoptosis in Fas-defective hepatoma cells through caspase-8 activation.
我们观察到，N-(4-羟基苯基)视黄酰胺（4HPR）作为一种化学预防和化疗药物，可有效诱导肝癌细胞凋亡。有趣的是，与Fas阳性（Hep G2和SK-HEP-1）肝癌细胞相比，Fas阴性（Hep 3B和PLC/PRF/5）肝癌细胞对4HPR诱导的凋亡更为敏感。因此，我们探究了4HPR在Fas缺陷型肝癌细胞中诱导凋亡的机制。稳定表达显性负性Fas相关死亡结构域（dnFADD）的Hep 3B细胞对4HPR的药物敏感性未发生改变，但当稳定表达E1B19K、Crm A或显性负性FLICE（dnFLICE）时，Hep 3B细胞具有抗性，这表明4HPR诱导的凋亡是由半胱天冬酶-8激活介导的。此外，凋亡可被Z-VAD-FMK（一种通用的半胱天冬酶抑制剂）或IETD-CHO（一种半胱天冬酶-8抑制剂）完全阻断，但仅被Ac-DEVD-CMK（一种半胱天冬酶-3抑制剂）、N-乙酰-L-半胱氨酸（NAC）（一种抗氧化剂）、N-乙酰-亮氨酰-亮氨酰-正亮氨酸（ALLN）（一种钙蛋白酶抑制剂I）或Z-LEHD-FMK（一种半胱天冬酶-9抑制剂）部分阻断。对4HPR诱导凋亡的时间序列分析显示，最初是半胱天冬酶-8激活，随后是晚期线粒体细胞色素c释放和轻微的半胱天冬酶-9激活，这表明半胱天冬酶-8激活是主要的上游调节点。在凋亡过程中未观察到Bid激活或促凋亡Bax的诱导。相反，在4HPR诱导的凋亡过程中Bcl-xL表达降低。综上所述，这些结果表明4HPR可能是一种潜在的化疗药物，能够通过半胱天冬酶-8激活在Fas缺陷型肝癌细胞中诱导凋亡。
