小檗碱抑制肝癌细胞的新生蛋白合成

We and others have demonstrated that berberine, the major active compound in Coptidis Rhizoma, has a potent effect of inhibiting human hepatocellular carcinoma (HCC). Berberine is able to suppress the angiogenesis, progression and metastasis of HCC as previously reported, which may indicate a global mechanism may be involved. Our preliminary data has shown that berberine could suppress the nascent protein synthesis in HCC cells. Inhibition of eukaryotic translation elongation factor-2 (eEF2) by berberine was observed in HCC cells, while berberine has no significant effect on the translational initiation process of proteins in HCC cells. This may indicate that berberine may target on the translation elongation process during the HCC cell protein synthesis through regulating eEF2. And we further observed that berberine could regulate the eEF2 kinase (eEF2K), whose activation could block the eEF2 activity by inducing its phosphoryaltion at Th56 site. Since eEF2K has been found to be the major cellular regulator of eEF2, we proposed that eEF2K play a key role in mediating protein synthesis inhibition by berberine in HCC. As activation of eEF2K in HCC is a novel property of berberine, we will determine if eEF2K play a dominant role in the inhibitory effect of berberine against angiogenesis, progression and metastasis of HCC using a new orthotopical HCC-implantation model with luciferase expression. Liposome-based shRNA against eEF2K will be intravascularly injected to block the activation of eEF2K by berberine. The effect of berberine on nascent protein translation will be profoundly determined, and we will determine if eEF2 is the executive regulator in mediating the inhibitory effect of berberine on nascent protein synthesis. Mutation on Th56 site of eEF2 will be performed to block the effect berberine on eEF2 and synthesis of nascent protein will be determined. And we will move to determine if the inhibition of eEF2K phospholrylation at Ser366 is dominant in mediating the activation of eEF2K by berberine. Furthermore, our preliminary data also observed that berberine could up-regulate the activity of AMPKα, one of the key up-streamed activator of eEF2K. Suppression of AMPKα by either pharmacological inhibitor Compound C or RNA interference blocks the induction of eEF2 phosphorylation by berberine. Berberine was also found to suppress mTOR signaling, which inactivates eEF2K by inducing its Ser366 phosphorylation. We will examine if AMPKα/mTOR pathway is involved in the regulation of eEF2K activity by berberine in HCC cells. Successful elucidation on the role and regulation of eEF2K in mediating the inhibitory effect of berberine against HCC will shed light on the potential of berberine as a novel therapeutic agent against HCC.

肿瘤细胞高效率的新生蛋白合成与肿瘤发生, 恶变及转移密切相关。我们及其他研究者揭示小檗碱可抑制人类肝细胞肝癌(HCC)的血管生成、恶变、迁移，但其抗肿瘤作用是否与其新生蛋白合成相关尚未明确。我们的预实验发现小檗碱可抑制HCC的新生蛋白合成并抑制了肝癌细胞真核翻译延伸因子2（eEF2）活性。小檗碱能够调节eEF2激酶eEF2K活性，激活eEF2K上游通路AMPKα。因此我们假设eEF2K在小檗碱抑制HCC新生蛋白生物合成中发挥重要作用。我们将应用HCC原位移植的荧光素酶表达荷瘤小鼠为模型，观察小檗碱对HCC血管生成、恶变、迁移的抑制作用是否依赖于抑制新生蛋白合成；小檗碱抑制HCC新生蛋白合成是否取决于eEF2K的磷酸化；检测AMPKα/mTOR是否参与了小檗碱对HCC细胞中eEF2K的激活。本研究将为eEF2K参与小檗碱对HCC细胞的抑制提供理论依据，为小檗碱抗肿瘤作用提供新靶点。

肝癌是影响人类生命健康的一种重大危险疾病。中国是肝癌的重灾区.本项目从以下几个方面进行了研究，阐明了化合物小檗碱对于肝癌细胞新生蛋白合成的分子作用机制：.a. 我们首次采用表达荧光素酶的肝癌原位移植动物模型，发现了腹腔注射和口服给药小檗碱均能够显著抑制肝癌的发生；腹腔注射小檗碱效果优于口服给药；.b. 我们通过基因沉默技术，阐释了eEF2K通路在小檗碱对于肝癌体内生长、血管新生和转移的抑制作用中的影响；证明了eEF2K通路的缺失会导致小檗碱抑制肝癌的作用失效；.c. 我们采用Click-IT的新技术检测肝癌细胞的新生蛋白合成，首次发现了小檗碱对于不同的肝癌细胞株的新生蛋白合成具有抑制作用；.d. 我们采用位点突变的办法，阐明了eEF2的Th56位点磷酸化对于小檗碱抑制新生蛋白合成具有决定性作用；.e. 我们采用位点突变的办法，证明了eEF2K的Ser366去磷酸化对于小檗碱调节eEF2活性及肝癌细胞新生蛋白合成具有显著影响；.f. 我们证明了小檗碱是通过AMPKα/mTOR通路影响肝癌细胞的新生蛋白合成；.g. 我们首次发现了黄连中类小檗碱化合物都具有调节eEF2活性作用，并且可能具有协同作用；.h. 我们阐明了中药复方黄连解毒汤中的其他化合物，对于小檗碱调节eEF2活性的作用具有协同功能；. 通过本课题的完成，我们先后发表5篇文章（其中SCI文章5篇），部分数据仍待发表，培养了2名博士研究生；部分成果在1次国际性会议上进行了口头汇报。