神经生长因子受体TrkA特异性诱导剂藤黄酰胺通过双重作用抗神经母细胞瘤的研究

Neuroblastoma (NB) is a common solid tumor of childhood and originates from primitive cells of the sympathetic nervous system. Major of NBs have rapid metastasis and high mortality, while some NBs can differentiate and regress spontaneously. It is with intense interests in the research field to induce NB cells to differentiate and regress artificially. Many evidences have shown that overexpression of tropomyosin-related kinase A (TrkA)， a member of neurotrophin receptors of the tyrosine kinase (Trk) family, has been identified as a major prognostic and biological factor for this disease. Gambogic amide, a derivative of gambogic acid, has been reported to selectively activate TrkA. Our primary work in vitro and in vivo showed that gambogic amide does not only activate TrkA protein but also promotes TrkA transcription and enhances TrkA protein level. Moreover，gambogic amide possesses the cytotoxicity on cancer cells. So we premise that gambogic amide has double actions on neuroblastoma. In current study, we will explore the anti-NB potency of gambogic amide, using cultured human neuroblastoma SH-SY5Y cells， which express TrkA , and murine neuroblastoma Neuro-2a cells, which express no TrkA , as well as their tumor-bearing mouse models. We will explore the molecular mechanisms of gambogic amide on neuroblasoma, to provide basic research data from further development of a novel NB drug.

神经母细胞瘤（Neuroblastoma, NB）是儿童常见的一种交感神经系统的恶性肿瘤，大多转移早，死亡率高，但是部分NB有自行分化消退的特性。如何人为地诱导NB细胞分化消退，成为NB研究的热点之一。很多临床和实验室研究证明高表达的 TrkA 参与了NB细胞的分化消退。藤黄酰胺是藤黄酸的衍生物，最近发现它能特异性活化TrkA。我们的前期工作表明藤黄酰胺不仅能活化TrkA受体，而且能促进TrkA的转录，增加总TrkA的蛋白水平；而且藤黄酰胺还表现出对肿瘤细胞的细胞毒性，因此我们推测藤黄酰胺对NB细胞既有诱导分化的活性， 又有直接的细胞毒性。本项目采用表达TrkA的人NB细胞SH-SY5Y和不表达TrkA的小鼠NB细胞Neuro-2a体外培养以及荷瘤小鼠体内实验相结合的研究手段，开展藤黄酰胺抗NB的研究，在确定药效的基础上研究其作用机制，为开发一种针对NB的新型药物奠定基础。

神经母细胞瘤（Neuroblastoma, NB）是儿童常见的一种神经系统恶性肿瘤，TrkA高表达的NB细胞可自行分化消退，预后良好。藤黄酰胺是藤黄酸的衍生物，被报道可特异性活化TrkA。我们在工作中发现藤黄酰胺可促进PC12, P23A和K562细胞中TrkA转录和TrkA的总体水平，活化TrkA下游信号通路。本课题我们采用两种神经母细胞瘤（鼠源的Neuro-2a和人源的SH-SY5Y），研究藤黄酰胺对NB细胞的细胞毒性和诱导分化作用。研究内容主要包括两部分：1）确定藤黄酰胺对NB细胞促分化的药效；2）主要从TrkA信号通路探讨藤黄酰胺促NB细胞分化的分子机制。我们发现：1）高浓度的藤黄酸和藤黄酰胺（>500 nM）对两种NB细胞和正常的神经元均有细胞毒性，但是NB细胞较正常神经元对药物更加敏感；2）低浓度的藤黄酰胺 (40 nM)可促进两种NB细胞的分化，但是对Neuro-2a促分化作用更明显；3）低浓度的藤黄酰胺 (40 nM)可促进Neuro-2a细胞中分化相关基因tubulinIII, PAX6和SLUG2的表达，而对PHOX2A,PHOX2B,RARg,Tau等基因的表达影响不大；4）低浓度的藤黄酰胺 (40 nM)可诱导Neuro-2a细胞中TrkA的表达以及下游信号通路的活化，而藤黄酸没有该作用；5）低浓度的藤黄酰胺 (40 nM)对SH-SY5Y细胞虽然也有促分化作用，但是对其TrkA表达没有促进作用，提示了TrkA不依赖的促分化通路被活化；6）动物实验显示大剂量（8 mg/kg,腹腔注射，每两天一次，连续5次）藤黄酸和藤黄酰胺均有很强的毒性，但是藤黄酸毒性较藤黄酰胺更强。.本课题工作证明，藤黄酰胺在高浓度可引起NB细胞毒性，低浓度可促进NB细胞的分化，且毒性较藤黄酸小，有开发成为抗NB药物的潜能。另一个有趣的出乎意料的现象是，相同条件下藤黄酰胺对Neuro-2a 和SH-SY5Y中TrkA表达的差异，提示Trk信号通路在不同神经母细胞瘤中有细胞特异性，值得进一步研究。