五味子乙素抑制肿瘤转移的关键靶分子鉴定

Cancer metastasis is the major cause of cancer-related death, but there is no clinical available drug against cancer metastasis. It is proposed by a group of well-established scientists 'given the fact that primary tumors can often be controlled using conventional therapies, could agents that act specifically on the process of metastasis be more likely to increase long term patient survival'. Hence, it is urgent to develop safe and efficacious agents to disturb the process of metastasis. Schisandrin B (Sch B), a naturally occurring compound, appears to be a safe and efficacious agent targeting the process of metastasis. We recently found that Sch B significantly suppressed the spontaneous lung metastasis of 4T1 cells inoculated s.c. without significant effect on primary tumor growth and significantly extend the survival time of these mice. Sch B did not inhibit lung metastasis of 4T1 cells that were injected via tail vein, suggesting that Sch B acted at the step of local invasion. Histopathological evidence demonstrated that the primary tumor in Sch B group were significantly less locally invasive than control tumor. In vitro assays demonstrated that Sch B could inhibit TGF-β induced EMT of 4T1 cells and of primary human breast cancer cells. Schisandrin B is the most abundant dibenzocyclooctadiene lignan present in the traditional Chinese medicinal herb Schisandra chinensis (Turcz.) Baill. The herb has been used in our country for several thousand years without reports of severe adverse effect, suggesting the safety of Schisandrin B. Previous animal studies from many laboratories also proved that the systematic toxicity of this compound was very low. These lines of evidence prompted us to further study the molecular target by Sch B, i.e., the mechanism by which Sch B inhibits cancer metastasis. In this proposal, we would use solid-phase Sch B to extract cellular proteins, whose identities are then defined by mass spectroscopy. These extracted proteins represent potential targets by Sch B. We will determine the dissociation constant between these potential targets with Sch B. The dissociation constants will tell the affinity between these potential targets with Sch B. The protein with highest affinity with Sch B is rationally the most important target by Sch B. We will observe if the potential targets indeed interact with Sch B in cells, and if the interaction will affect cancer invasion and metastasis. Finally, we will examine the clinical relevance of these targets by Sch B.

肿瘤转移是导致肿瘤病人死亡的主要原因。目前，临床上没有特异性针对肿瘤转移的药物。因此，寻找低毒并特异性抗肿瘤转移的药物有重要的临床意义。传统中药五味子在我国已有数千年的医药史，五味子乙素是其主要的药效成分之一。该化合物毒性极低，对人体是安全的。我们前期的体内外实验证明：五味子乙素不抑制原位肿瘤，但能显著降低肿瘤的转移；其细胞毒性极低，但能抑制肿瘤细胞系和原代人乳腺癌细胞的EMT降低后者的迁移和侵袭能力。鉴此，五味子乙素可考虑为一种安全和特异性针对肿瘤转移的候选药物。然而，五味子乙素抑制肿瘤转移的分子靶不清。我们拟用固相五味子亲和技术提取肿瘤细胞中与之特异结合的蛋白质；用质谱鉴定这些蛋白质的身份；测定五味子乙素与这些蛋白质的解离常数；观察这些蛋白质与五味子乙素在细胞内的相互作用；鉴定这些蛋白质与肿瘤细胞侵袭转移的关系。最终明确五味子乙素抑制肿瘤转移的关键靶分子。

肿瘤转移是导致肿瘤病人死亡的主要原因，因而抑制肿瘤转移应是治疗肿瘤疾病的一个重要策略。目前，临床上没有特异性针对肿瘤转移的药物。因此，寻找低毒并特异性针对肿瘤转移的药物有重要的临床意义。传统中药五味子在我国已有数千年的医药史，五味子乙素是其主要的药效成分之一。该化合物毒性极低，对人体是安全的。体外实验证明：五味子乙素细胞毒性很低，但能抑制肿瘤细胞系和原代人乳腺癌细胞的EMT降低后者的迁移和侵袭能力；体内实验证明，五味子乙素不抑制原位肿瘤，能显著降低了4T1远处肺转移和骨转移。进一步研究发现，五味子乙素是NADPH氧化酶的抑制剂，对NADPH氧化酶4抑制作用的IC50值是9.3 µM；过去有文献报道：TGF-β可调控NADPH氧化酶的活性。NADPH氧化酶产生的活性氧族，对TGF-β信号通路又具有重要的调控作用。NADPH氧化酶家族包括NADPH氧化酶1-5，双重氧化酶1-2（小鼠缺乏NADPH氧化酶5基因）。我们得到的结果表明：在4T1乳腺癌细胞系中，TGF-β促进的ROS产生及细胞运动能力增加依赖于NADPH氧化酶4的表达，RNA干扰敲低NADPH氧化酶4显著降低了4T1细胞本身及TGF-β刺激增加的细胞运动能力，而且4T1细胞的远处肺转移及骨转移也显著降低。这些证据表明，NADPH氧化酶4是五味子乙素治疗肿瘤转移的一个靶点。本研究揭示了五味子乙素在体外能抑制肿瘤细胞EMT，在体内能抑制肿瘤细胞的侵袭和远端转移，其作用机理是通过抑制NOX4。结合我们前期对五味子乙素的研究，该天然化合物具有多种药理作用，抑制P-糖蛋白和多药耐药蛋白MRP逆转肿瘤多要耐药性，增强化疗药诱导肿瘤细胞的凋亡，降低蒽环类药物的心机毒性，加上本研究揭示的抑制肿瘤的转移。五味子乙素是中药五味子的主要药理成分。五味子在我国已有千年的用药史，被证明是安全的。这些事实都支持五味子乙素有可能成为一个抗肿瘤的潜在药物。