| 其他摘要 | Hedgehog (Hh)信号通路在发育和癌症中都发挥重要作用，异常激活Hh信号通路会导致很多癌症的形成和发展，尤其是基底细胞癌和髓母细胞瘤。基于 Hh信号通路的靶向抗癌药的研发在过去的十多年间取得了巨大的进展，尤其是最近有2个抑制剂Vismodegib和Sonidegib已上市用于治疗基底细胞癌。但是在临床治疗过程中和小鼠肿瘤模型中都很出现了广泛的抗药性，提示急需研发下一代新型的Hh信号通路抑制剂和研发针对多个靶点的联合用药疗法以克服抗药性的问题。我们筛选了约500个从药用植物中提取分离的化合物，经初筛和复筛验证发现了50个新型Hh抑制剂，根据其化学结构和对Hh信号通路的作用方式而分为8类。我们深入地研究了其中的两类：对映贝壳杉烷二萜和茜草科类型环肽。对映贝壳杉烷二萜抑制Smo激活剂SAG激活的Hh信号，和抑制Smo在纤毛的富集，但不结合Smo的环巴明位点。对映贝壳杉烷二萜不能抑制Sufu敲除后激活的Hh靶基因的表达，综合以上结果表明对映贝壳杉烷二萜可能作用于Smo除环巴明位点外的其它位点或Smo的下游，和Sufu的上游。此外对映贝壳杉烷二萜还可以延长初级纤毛，抑制有丝分裂纺锤体的组装形成，但不影响体外微管和Actin的聚合，表明对映贝壳杉烷二萜作用的靶蛋白可能同时参与了Smo在纤毛中的转运，纤毛的形成和纺锤体的组装，但不是通过直接作用于Tubulin和Actin。根据我们的实验结果和已发表的研究报道，我们推测对映贝壳杉烷二萜作用的靶蛋白很可能是Ran-Importin系统中的Ran结合蛋白。茜草科类型环肽是我们发现的所有新型Hh抑制剂中活性最强的，环肽分子对Hh信号通路的抑制具有多效性。环肽能抑制SAG激活的Hh信号，抑制SAG诱导的Smo在纤毛中的富集，不结合Smo的环巴明位点。环肽也能抑制下游Sufu敲除后激活的和Gli过表达激活的Hh靶基因的表达，但其抑制活性弱于在SAG激活的条件下。然而在Ptch-/- MEF细胞中，环肽却不能抑制由于Ptch敲除后激活的Hh靶基因的表达，和Smo向纤毛的转运富集，提示环肽分子作用的靶蛋白很可能是Ptch，可能通过增强Ptch对Smo的抑制。芯片分析结果显示环肽分子显著地下调了几乎所有胆固醇合成途径中的酶的表达，和胆固醇吸收途径中的关键调控蛋白低密度脂蛋白受体LDLR的表达。而这些酶和LDLR都是SREBP信号通路的靶基因，表明环肽还能抑制SREBP信号通路。环肽改变了胆固醇在细胞中的分布，导致胆固醇在细胞内的异常聚集，抑制LDLR介导的胆固醇吸收，以及长时间的处理能诱导脂滴的形成。基于以上结果和已有的研究报道，我们推测环肽分子很可能通过作用于Scap而抑制SREBP信号通路，而且Scap和Ptch都属于固醇感应结构域蛋白家族。环肽的这种同时能抑制胆固醇合成和吸收的作用方式，表明环肽还可能是一种潜在的降血脂药。此外，环肽分子抑制Hh和SREBP信号通路的活性是各自独立的，综上所述我们推测环肽分子可能分别通过作用于Ptch和Scap而抑制Hh和SREBP信号通路。最近我们成功地合成了放射性同位素氚标记的环肽[3H]-RA-VII，后续将进一步验证环肽分子是否能与Ptch和Scap直接结合。我们从天然植物化合物中发现了2类作用机制新颖的Hh信号通路抑制剂，这些活性分子能否成为潜在的抗癌药仍是未来值得深入研究和探讨的课题，同时也为研究Hh信号通路和纤毛形成的调控机制提供有用的工具分子。; The Hedgehog (Hh) signaling pathway plays critical roles in embryonic development and cancer, and aberrantlly activated Hh signaling can drive the formation of multiple cancers particularly basal cell carcinoma and medulloblastoma. In the past decade, major progress has been made in Hh pathway targeted cancer therapies with specific Hh pathway antagonists (HPAs). Especially, two inhibitors Vismodegib and Sonidegib were recently approved by USA FDA for treating basal cell carcinoma. However, acquired resistance to Vismodegib and Sonidegib were soon appeared in patients and mice with high percentage and resulted in the relapse of cancer. Thus, it is widely suggested to develop new types of HPAs and multiple-targeted combination therapies to conquer the problem of acquired resistance.To search for novel HPAs, we conducted a screen of natural products extracted from Chinese herbs, and identified 50 new HPAs that can be classified as eight types based on the distinct chemical structures and mechanism of actions on Hh pathway. We thoroughly investigated two types of them: ent-Kaurane diterpenoids (ent-Kauranoids) and Rubiaceae-type Cyclopeptides (RAs). After systematical analyses of Hh pathway, we found that ent-Kauranoids inhibited Smo agonist SAG activated Hh signaling and repressed the ciliary accumulation of Smo, but could not bind to the Cyclopamine (Cyc)-binding site of Smo and block the knockout of Sufu activated Hh signal. These evidence indicate that ent-Kauranoids may target to Smo other than the Cyc-binding site or downstream of Smo, and upstream of Sufu. ent-Kauranoids also can elongate the primary cilia, and block the assembly of mitosis spindle, but have no effect on the polymerization of tubulin and actin in vitro, suggesting that the ent-Kauranoids targeted protein might simultaneously participate in ciliary transport of Smo, ciliogenesis and spindle assembly. Based on the above evidence and previously reported researches, we speculate that the most possible target of ent-Kauranoids is the Ran binding protein of Ran-Importin system.Rubiaceae-type Cyclopeptides are the most potent HPAs among our discovery, and have multiple effects on different levels of Hh signaling pathway. With performing a variety of Hh pathway assays, we found that Cyclopeptides repressed SAG activated Hh signaling and ciliary enrichment of Smo, and were unable to bind to the Cyc-binding site of Smo. Cyclopeptides also can block the expression of Hh target genes actived by both the knockout of Sufu and overexpression of Gli. However, in the Ptch-/- MEF cells, Cyclopeptides can not inhibit the knockout of Ptch activated expression of Hh target gene and ciliary translocation of Smo, indicating that Cyclopeptides are most likely targeted to Ptch and enhance the Smo suppressing function of Ptch. The result of expression microarray analysis showed that Cyclopeptides strongly down-regulated the expressions of nearly all of enzymes in the cholesterol synthesis pathway, and also the key regulator LDLR in the cholesterol uptake pathway. Given that these enzymes of cholesterol synthesis pathway and LDLR are the target genes of SREBP pathway, Cyclopeptides turn out to also dramatically inhibit SREBP pathway. We also found that Cyclopeptides changed the distribution of cellular cholesterol with abnormal enriched in a still unknown cytoplasmic compartment, repressed the uptake of LDLR-mediated cholesterol endocytosis, and induced the formation of lipid droplets with 2 days’ treatment. Based on the above evidence and previously published studies, we speculate that Cyclopeptides are possibly targeted to Scap to inhibit SREBP pathway. Moreover, both Scap and Ptch are belonged to the sterol sensing domain protein family. Meanwhile, the strong effect of Cyclopeptides of repressing both cholesterol synthesis pathway and cholesterol uptake pathway suggest that they are also promising to be potential lipid-lowering drugs. Further investigation showed that Cyclopeptides independently repressed Hh and SREBP pathway, thus, we speculate that Cyclopeptides might target to Ptch and Scap to inhibit Hh and SREBP pathway, respectively. Currently, we successfully synthesized the radio-labeled [3H]-RA-VII, which would be a useful probe to determine whether Cyclopetides can directly to Ptch and Scap.Collectively, we identified two types of HPAs from natural chemical products, with novel mechanism of actions on Hh signaling pathway. Whether these compounds have the potential to be an-cancer drugs needs further investigation. They might provide new tools for revealing the mechanisms of cilia-dependent signal transduction of Hh pathway and ciliogenesis. |
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