Chung Yih-Lin, Sheu Meei-Ling, Yen Sang-Hue
Department of Radiation Oncology, Koo Foundation Sun Yat-Sen Cancer Center, Taipei, Taiwan, Republic of China.
Int J Cancer. 2003 Oct 20;107(1):65-73. doi: 10.1002/ijc.11303.
Treatment of hepatocellular carcinoma (HCC) cells with butyrate can induce apoptosis irrespective of hepatitis B virus integration. No information is available, however, regarding the effect of butyrate on HCC in the presence of hepatitis C virus (HCV) because some HCV proteins can regulate cell survival. By gene transfer, we found that HCV core enhances but HCV NS5A antagonizes sodium phenylbutyrate (NaPB)-induced apoptosis in HCC cells, which is independent of p53. We then chose the p53-negative Hep3B HCC cell to investigate the mechanism of anti-apoptosis mediated by NS5A. In the NaPB-treated Hep3B cells without NS5A expression, induction of apoptosis was associated with Bax redistribution from the cytosol to the nucleus interior and subsequently, to a nuclear membrane-bound form. In the NS5A expressing Hep3B cells, NaPB treatment also triggered relocalization of both Bax and NS5A from the cytosol to the nucleus interior but Bax retained inside the nucleus and did not finally move to the nuclear membrane. Using double immunofluorescence and coimmunoprecipitation, we demonstrated that NS5A co-localizes and interacts with Bax in the nucleus. The HCV NS5A protein was further found to contain Bcl-2 homology domains (BH3, BH1 and BH2). Additional studies using deleted NS5A constructs were carried out to determine whether the BH2 domain or nuclear localization signal (NLS) in NS5A is required for interaction with Bax in the nucleus or inhibition of apoptosis. NS5A with deletion of both BH2 domain and NLS localized in the cytoplasm, dissociated with Bax, and lost anti-apoptosis activity during NaPB treatment. In contrast, NS5A with intact BH domains except NLS still bound directly to Bax in the perinuclear region or the nucleus, but showed less association with Bax in the nucleus and lower effect in apoptosis inhibition than full-length NS5A. These results suggest that HCV NS5A as a Bcl-2 homologue interacts with Bax to protect p53-negative HCC cells from NaPB-induced apoptosis.
用丁酸盐处理肝细胞癌（HCC）细胞可诱导细胞凋亡，而与乙型肝炎病毒整合无关。然而，关于在丙型肝炎病毒（HCV）存在的情况下丁酸盐对HCC的影响尚无相关信息，因为一些HCV蛋白可调节细胞存活。通过基因转移，我们发现HCV核心蛋白增强但HCV NS5A拮抗苯丁酸钠（NaPB）诱导的HCC细胞凋亡，这与p5３无关。然后我们选择p5３阴性的Hep3B HCC细胞来研究NS5A介导的抗凋亡机制。在未表达NS5A的经NaPB处理的Hep3B细胞中，细胞凋亡的诱导与Bax从细胞质重新分布到细胞核内部，随后再到核膜结合形式有关。在表达NS5A的Hep3B细胞中，NaPB处理也引发了Bax和NS5A从细胞质重新定位到细胞核内部，但Bax保留在细胞核内，最终未移动到核膜。通过双重免疫荧光和免疫共沉淀，我们证明NS5A在细胞核中与Bax共定位并相互作用。进一步发现HCV NS5A蛋白含有Bcl-2同源结构域（BH3、BH1和BH2）。使用缺失NS5A构建体进行了额外的研究，以确定NS5A中的BH2结构域或核定位信号（NLS）是否是与细胞核中的Bax相互作用或抑制细胞凋亡所必需的。在NaPB处理期间，缺失BH2结构域和NLS的NS5A定位于细胞质，与Bax解离，并失去抗凋亡活性。相比之下，除NLS外具有完整BH结构域( BH3、BH1和BH2)的NS5A仍直接与核周区域或细胞核中的Bax结合，但与细胞核中Bax的结合较少，在抑制细胞凋亡方面的作用低于全长NS5A。这些结果表明，作为Bcl-2同源物的HCV NS5A与Bax相互作用，保护p5３阴性的HCC细胞免受NaPB诱导的细胞凋亡。
