北大博士再发封面文章 解析甲胎蛋白

　　

生物通报道：北京大学和海南医学院联合对肝癌细胞特异性标志物甲胎蛋白（AFP）进行长时间的研究，发现AFP具有抑制PTEN的生物学功能，导致肝癌细胞耐受全反式维甲酸诱导的凋亡，这是AFP新功能的发现。近日以题为“Alpha-fetoprotein: a new member of intracellular signal molecules in regulation of the PI3K/AKT signaling in human hepatoma cell lines”在国际著名癌症杂志《International Journal of Cancer》再次发表封面论文。

甲胎蛋白（α-fetoprotein, AFP）是肝癌细胞表达的高特异性蛋白质，很多肝癌病人（70-80%）在发病期间都有AFP基因高表达的特征，AFP是一种胚原蛋白，其基因在胎儿发育过程开放并表达，而在人出生两年后基本处于关闭状态，但是成人发生肝癌或肝脏良性再生时，AFP的基因重新被激活而大量表达，在临床上被认为是肝癌的经典肿瘤标记物，因而被当作诊断肝癌的金标准。北京大学博士李孟森课题组成员在国家自然科学基金的连续资助下，经过多年的潜心研究，逐步揭示了AFP所隐藏的生物学功能。该课题组研究发现AFP与肿瘤细胞的恶性生长、转移和侵袭密切相关，并且认为AFP是肝癌细胞耐药的关键性细胞因子，研究结果显示AFP具有潜在的抗凋亡诱导作用的生物学性质。

全反式维甲酸（ATRA）在临床上是用于治疗白血病的一线药物，其主要通过与胞内受体结合诱导恶性细胞“改邪归正”，由于ATRA与受体结合后改变后者的空间结构，导致受体进入细胞核内调节靶基因的转录，发挥其抑癌作用。尽管ATRA能诱导恶性细胞分化和抑制癌细胞生长，但是肝癌细胞对ATRA失去敏感性。ATRA能诱导PTEN （Phosphatase and tension homolog deleted on chromosome ten）的表达，而PTEN是水解3-磷酸肌醇（PIP3）为2-磷酸肌醇（PIP2）的磷酸酶，其能阻止3-磷酸肌醇激酶（PI3K）磷酸化蛋白激酶B（Protein kinase B, PKB/AKT），阻断PI3K/AKT的信号传递，因而被认为是一个重要的抑癌基因，其表达下降或功能的丧失是引起生长信号错误传递的分子基础，后果是导致癌细胞的过度分裂。约有12.5%的肝癌病人的癌细胞里PTEN基因关闭，而70%的病人PTEN基因开放并高表达，PTEN蛋白合成量与正常组织没有显著性差别，因而PTEN在肝癌内丧失发挥抑癌作用可能是由于其功能失活导致的。由于PTEN表达缺失或下降与AFP高表达密切相关，AFP是肝癌细胞高表达的特异性蛋白质，课题组基于AFP与PTEN在肝癌细胞内表达相关性的设想，深入研究肝癌细胞内高表达的AFP是否存在曾未发现的生物学功能。

北京大学博士李孟森研究员课题组联合北京大学李刚教授实验室，采用RNA干扰、免疫共沉淀（Co-IP）、荧光共振能量转移（FRET）和染色体免疫共沉淀（ChIP）等技术研究肝癌细胞内AFP新功能，发现在肝癌细胞内的AFP能与PTEN结合，并抑制PTEN 的生物学活性，促进PI3K/AKT信号的传递，而且发现AFP也能特异性与caspase-3结合，抑制caspase-3的活性，阻断 caspase信号的级联反应，导致肝癌细胞耐受肿瘤坏死因子相关凋亡诱导配体（TRAIL）的作用。传统的医学理论认为AFP基因的高表达是肝癌发生过程中的一个伴随现象，是肝癌发生的特异性肿瘤标志物。李孟森博士的研究结果表明细胞外的AFP能与其受体结合，介导生长信息的传递；细胞内的AFP不仅能抑制凋亡信号的转导，而且也能促进生长信号的传递，这是AFP具有抗凋亡和促进增殖功能的新发现，该研究结果赋予AFP新功能的认识和寻找到治疗肝癌的新靶点。研究结果在2009年6月15日出版的《International Journal of Cancer》上以封面介绍论文发表，2011年2月1日出版的《International Journal of Cancer》再次以封面介绍形式公布了AFP新功能的研究成果。

原文摘要：
1. Alpha fetoprotein is a novel protein-binding partner for caspase-3 and blocks the apoptotic signaling pathway in human hepatoma cells. International Journal of Cancer, 2009; 124(12): 2845-2854.
Although there is increasing evidence that alpha fetoprotein (AFP) may function as regulatory factor in the growth of tumor cells, the precise mechanism is still unclear. In the current study, we investigated the role of the cytoplasmic AFP in caspase-3-mediated signaling of apoptosis. Our results showed that low doses of TNF-related apoptosis-inducing ligand (TRAIL) elevated the activity of caspase-8, but not caspase-3. Caspase-3 colocalized and interacted with AFP in the cytoplasm of Bel 7402 cells, and translocated into nuclei in association with the occurrence of apoptosis while cells were under cotreatment with all-trans retinoic acid (ATRA) or TRAIL. AFP was able to form complexes with caspase-3 and block onward transmission of signaling from caspase-8. Knockdown of AFP increased the sensitivity of Bel 7402 cells to TRAIL, and thereby, triggered caspase-3 signaling. No intermolecule interaction occurred between AFP and caspase-8, nor was caspase-8 activity altered after AFP knockdown, demonstrating the selectivity of AFP in interfering with the apoptotic signaling pathway. The effect of AFP on caspase-3 was further confirmed by transfection of the AFP gene into HLE cells (AFP negative). We conclude that ATRA or TRAIL resistance in AFP producing hepatoma is at least, in part, attributable to the high level of the cytoplasmic AFP. Therefore, it is possible that the combination of AFP gene silencing together with ATRA/TRAIL cotreatment will benefit the enhancement of the chemotherapeutic efficiency of these agents on tumors.
2. Alpha-fetoprotein: a new member of intracellular signal molecules in regulation of the PI3K/AKT signaling in human hepatoma cell lines. International Journal of Cancer, 2011; 128(3): 524-532.
Despite its well-defined role as a serum growth factor during fetal liver development and hepatic oncogenesis, the biological significance of cytoplasmic alpha-fetoprotein (AFP) remains incompletely understood. Here, we provide evidence to illustrate that cytoplasmic AFP may function as a regulator in the phosphatidylinositol 3-kinase (PI3K)/AKT pathway in human hepatocellular carcinoma cells. The results demonstrated colocalization and interaction of AFP and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in the cytoplasm of AFP-producing Bel 7402 and HepG2 cells, with an interaction distance of 12.62.7 A˚ as determined with the fluorescence resonance energy transfer technique. Knockdown of AFP mRNA or inhibition of AFP expression by all trans-retinoic acid resulted in enhancement of the PTEN level with a synchronous decrease in phosphorylated AKT. Transfection of the afp gene into HLE cells (originally AFP negative) led to a significant activation of AKT signaling. The inhibition of PI3K signaling by LY294002 was simultaneously reversed by transfection, accompanied by diminution of all trans-retinoic acid-induced upregulation of PTEN and enhancement of cell growth. In conclusion, these results demonstrate that cytoplasmic AFP is involved in regulation of hepatocellular growth and tumorigenesis.

作者简介
李孟森；北京大学理学博士，研究员。海南医学院分子生物学重点实验室主任，2010年当选为海南省有突出贡献优秀专家，并进入海南省“515人才工程”第一层次人才。1988.9-1993.7在北京医科大学（现为北京大学医学部）基础医学系读书，获得医学学士学位，1997.8-2000.7北京大学基础医学院生物化学与分子生物学系硕士研究生，获得理学硕士学位，2003.12-2005.7在海南医学院生物化学教研室工作，2003年12月晋升副教授/副研究员；2005.8-2008.7北京大学基础医学院生物化学与分子生物学系博士研究生，获得北京大学理学博士学位；2008.12-现在 在海南医学院分子生物学重点实验室工作，任实验室主任，2009年12月晋升研究员，以第一完成人身份分别于2009年和2002年获得海南省科技进步一等奖和二等奖。

发表与AFP生物学功能相关的论文
[1] Li M, Li H, Li C, et al. Alpha-fetoprotein, a new member of intracellular signal molecules in regulation of the PTEN/AKT signaling in human hepatoma cells lines. International Journal of Cancer, 2011; 128(3): 524-532.
[2] Li M, Li H, Li C, et al. Alpha fetoprotein is a novel protein-binding partner for caspase-3 and blocks the apoptotic signaling pathway in human hepatoma cells. International Journal of Cancer, 2009; 124(12): 2845-2854.
[3] Li M, Li H, Li C, et al. Cytoplasmic alpha-fetoprotein functions as a co-repressor in RA-RAR signaling to promote the growth of human hepatoma Bel7402 cells. Cancer Letters, 2009; 285(2): 190-199.
[4] Li MS, Zhou S, Liu X, et al. Alpha fetoprotein shields hepatocellular carcinoma cells from apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand. Cancer Letters, 2007; 249(2): 227-234.
[5] Li MS, Liu X, Zhou S, et al. Effects of alpha fetoprotein on escape of Bel 7402 cells from attack of lymphocytes. BMC Cancer, 2005; 5: 96.
[6] Li MS, Li PF, Yang FY, et al. Molecular mechanism of alpha-fetoprotein on the growth of NIH3T3 cells. Cell Research, 2002; 12(2): 151-156.
[7] Li MS, Li PF, He SP, et al. Promoting molecular mechanism of alpha-fetoprotein on the growth of human hepatoma Bel7402 line cells. World Journal Gastroenterology, 2002; 8(3): 467-472.