Analysis of interferon related gene regulation as a proapoptotic function against hepatocellular carcinoma

干扰素(IFN)联合5-氟尿嘧啶(5-FU)治疗晚期肝细胞癌疗效显著。干扰素α2被广泛用于慢性肝病。但干扰素α8是最有效的诱导几种癌细胞凋亡的亚型。在本研究中，我们探讨了干扰素α2或α8联合5-FU诱导肝癌细胞凋亡的分子机制。检测了5种肝癌细胞株(Hep3B、HuH7、HLE、PLC/PRL/5和HepG2)在不加或不加5-FU的情况下，干扰素α诱导细胞凋亡的能力。对干扰素和5-FU联合作用最敏感的是Hep3B。Caspase-3、-9，尤其是caspase-8活性在干扰素和5-FU联合用药时明显高于单用干扰素或5-FU组。当5-FU加入两种干扰素α处理时，JAK-STAT途径转录因子ISRE被更协同地激活。对caspase-3，-8，9，c-jun氨基末端激酶(JNK)、磷脂酰肌醇3-激酶(PI3K)和p38丝裂原活化蛋白激酶(P38 MAPK)的抑制表明，抑制caspase-8是减少干扰素和/或5-FU诱导细胞凋亡的最有效的治疗方法。在JAK1和ISGF3g沉默的Hep3B细胞中，干扰素诱导的凋亡和caspase-8的激活水平被取消，甚至与5-FU联合使用也是如此。虽然干扰素α8比α2更有效地诱导肝癌细胞的凋亡，并且在芯片分析中呈现不同的簇，但JAK-STAT和caspase的激活状态是相似的，我们认为caspase-8是控制干扰素和5-FU诱导肝癌细胞凋亡的最重要的因素。

Interferon (IFN) combined with 5-Fluorouracil (5-FU) treatment is recently reported to show marked effects in patients with advanced hepatocellular carcinoma (HCC). IFN alpha 2 is widely provided for chronic liver diseases. But IFN alpha 8 is the most effective subtype to induce apoptosis in several cancer cell lines. In this study, we investigated the molecular mechanisms of apoptosis induction in hepatoma cell lines with IFN alpha 2 or alpha 8 in combination with 5-FU. Five hepatoma cell lines (Hep3B, Huh7, HLE, PLC/PRL/5, and HepG2) were tested for apoptosis inducibility by IFN alpha in the absence or presence of 5-FU. Hep3B was the most apoptosis sensitive to IFN plus 5-FU treatment. Caspases-3, -9, and especially caspase-8 activities were higher with IFN alpha plus 5-FU than IFN or 5-FU alone. The JAK-STAT pathway transcriptional factor ISRE was activated more synergistically when 5-FU was added to both IFN alpha treatments. Inhibition of caspase-3,-8,9,c-Jun N-terminal kinase (JNK), phosphatidylinositide 3-kinase (PI3K), and p38 mitogen-activated protein kinase (p38 MAPK) revealed that caspase-8 inhibition was the most effective treatment to decrease apoptotic effects of IFN and/or 5-FU. In JAK1, and ISGF3g-silenced Hep3B cells, the apoptosis induction and caspase-8 activation levels by IFN, even in combination with 5-FU, were abrogated. Although IFN alpha 8 induced apoptosis more effectively than alpha 2 and showed different cluster in microarray analysis, the JAK-STAT and caspase activation status were similar.We conclude that caspase-8 is the most important factor that controls IFN-and 5-FU- induced apoptosis in hepatoma cell lines.