五味子乙素调节非小细胞肺癌细胞死亡受体4表达的作用机制研究

Recombinant TRAIL is a novel tumor-targeted protein drug, however, non-small-cell lung cancer (NSCLC) is relatively resistant to TRAIL-mediated tumor surveillance，lack of DR4 expression is a major mechanism. In our previous investigation, we established drug screening system based on death receptor 4 expression and screened small molecules library of Chinese medicine. As a result, we found schisandrin lignans were able to significantly upregulate DR4 expression and schisandrin B showed the strongest effect. Microarray experiment showed that schisandrin B increased CHOP and Chac1 expressions, further experiments revealed that CHOP is involved in schisandrin B-induced DR4 mRNA expression, while Chac1 prolonged the half-life of DR4. Based on these results, we presume that schisandrin B is able to increase CHOP-dependent DR4 transcription and inhibit ubiquitin-mediated DR4 degradation by Chac1. By the combinatory effects of CHOP and Chac1, schisandrin B recovers DR4 expression in NSCLC, thus reversing the resistance of NSCLC towards TRAIL. To test this hypothesis, we will investigate the molecular mechanism underlying the effects of schisandrin B on CHOP and Chac1 expressions, effect of CHOP on DR4 transcription, and effect of Chac1 on DR4 protein degradation in this study. The results of this study will widen the therapeutic value of TRAIL, and is beneficial to identify more potent anti-NSCLC agents from Frutus Schisandrae that have high efficacy and low toxicity.

重组TRAIL是极具开发前景的靶向抗肿瘤药物，但非小细胞肺癌(NSCLC)细胞对其耐受，细胞死亡受体4(DR4)表达“沉默”是产生耐受重要机制。前期研究我们从中药小分子库中筛选、发现五味子乙素能重新激活NSCLC细胞中“沉默”的DR4表达，且该过程与CHOP与Chac1相关。进一步研究发现CHOP参与DR4 mRNA上调，而Chac1能延长DR4蛋白质半衰期，由此推测五味子乙素能通过CHOP参与DR4转录，通过Chac1抑制DR4泛素化降解，最终逆转NSCLC对TRAIL耐受。为验证该推测，本研究将探索五味子乙素调节CHOP与Chac1表达、CHOP调节DR4转录、Chac1抑制DR4泛素化降解的分子机制，从而拓宽TRAIL临床适应症，为从中药五味子中发现高效低毒的抗NSCLC增敏剂奠定基础。

五味子具有收敛固涩作用，在前期研究中，我们发现五味子能显著增加非小细胞肺腺癌NSCLC对TRAIL诱导细胞凋亡的敏感性，但有效成分与作用机制不详。本研究发现五味子木脂素类成分能显著增强NSCLC细胞对TRAIL诱导凋亡的敏感性，尤其以五味子乙素的作用最强，其作用机制在于五味子乙素能选择性增加NSCLC细胞膜死亡受体DR4的表达。基因芯片结果表明五味子乙素增加DR4表达可能与CHOP与Chac1（CYB35）表达相关。实验结果表明，五味子乙素促进CHOP的转录激活，且Chac1的启动子序列上存在CHOP的结合位点，因此Chac1（CYB35）是位于CHOP的下游基因。分子与细胞实验结果表明CYB35能在蛋白质翻译后水平调控DR4的蛋白表达，即CYB35能与DR4相互作用，并抑制DR4的E3连接酶结合DR4与诱导DR4的泛素化降解。TRAIL联合五味子乙素对NSCLC的联合增效作用能够被CYB35 siRNA所阻断，因此五味子乙素通过CHOP-CHAC1-DR4通路增加NSCLC细胞对TRAIL诱导细胞凋亡的敏感性。此外，通过对TCGA等NSCLC患者数据深度挖掘，本研究还发现基于CHAC1等17个基因组成的风险数学模型，能够很好地对临床NSCLC患者预后进行较为准确的预测，且该模型与肿瘤内质网应激、肿瘤免疫状态与外秘体信号通路有很好的相关性。