中药升麻环菠萝蜜烷三萜类成分基于内质网应激途径诱导肝癌细胞凋亡的活性和作用机制的研究

Primary hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Currently, there is no target specific drug developed for HCC. Thus, it is urgent to develop small molecular drug specifically targeting HCC. When carcinoma cells encounter endoplasmic reticulum stress (ER stress), an immediate response termed unfolded protein response (UPR) will be activated to protect cell from damage caused by ER stress and to make cell survival. Hence, UPR pathway has been regarded as a potential target for selective anticancer drug discovery, and has drawn extensive attention of researchers. In our previous studies, cycloartane triterpenoids of Cimicifugae Rhizoma, one of the commonly used traditional Chinese medicines, have shown a significant apoptosis induction against HCC cell line HepG2. Preliminary research on its apoptotic mechanism showed that some cycloartane triterpenoids can down-regulate expression of GRP78, a glucose-regulated protein, the key protein related to activation of UPR pathway; while up-regulate the expression of CHOP, the transcription factor C/EBP homologous protein which is known to promote apoptotic cell death. Above results indicate that apoptotic inducing effects against HepG2 of cycloartane tertripenoids may be associated with UPR pathway. On the basis of our preliminary work, a multi-disciplinary systematic study including phytochemistry and molecular biology techniques is proposed to isolated these compounds, to evaluate the apoptotic inducing activities and to further investigate the underlying mechanism for the inhibition of UPR pathway and to find related therapeutic molecular targets. The proposed study aims to establish a solid theoretical foundation for research and development of anti-HCC drugs based on UPR targets, as well as new anti-HCC leading compounds.

原发性肝癌是最常见的恶性肿瘤之一，目前尚无特效药物，发现靶标明确的抗肝癌药物具有重要的意义。肿瘤细胞通过启动非折叠蛋白反应 (UPR)来应对内质网应激，减少应激引起的损害，维持自身生存。UPR通路已成为筛选高度选择性抗肿瘤药物的一个很有潜力的靶标，已成为研究新热点。本课题组前期研究发现：中药升麻中的环菠萝蜜烷三萜类成分，能显著诱导肝癌细胞HepG2凋亡；初步机制研究表明，该类化合物能明显下调UPR通路启动关键蛋白GRP78，而上调凋亡促进因子CHOP的表达，提示着其诱导HepG2凋亡的机制可能与UPR通路有关。本项目拟在前期工作基础上，对3种升麻植物中环菠萝蜜烷三萜类成分进行系统分离；评价其诱导HepG2凋亡的生物活性；探索其基于UPR通路的抗癌分子机制；并阐明其作用的相关靶点以及通路。为阐明升麻抗肝癌活性的物质基础和作用机制提供理论依据，为靶向UPR抗肝癌新药研发提供先导化合物。

使肿瘤细胞发生过度内质网应激(ERS)，是诱导肿瘤细胞凋亡的重要途径之一,ERS已成为筛选高度选择性抗肿瘤药物的一个很有潜力的靶标。本课题拟从中药升麻中分离环菠萝蜜烷三萜类成分，探索其基于ERS的诱导肝癌细胞HepG2凋亡机制及相关靶点，为靶向ERS抗肝癌先导化合物的发现提供研究基础。.（一）研究结果如下：.(1)从3种升麻植物中分离了42个环菠萝蜜烷化合物，鉴定了31个，其中7为新化合物。.(2) MTT实验表明化合物（1-7）对HepG2 (肝癌)，HepG2/ADM (肝癌耐药株) ，MCF-7 (乳腺癌)生长具有一定抑制作用。其中25−O−乙酰基−7,8−去氢升麻醇−3−O−β−D−吡喃木糖苷 (1), 7,8−去氢升麻醇−3−O−β−D−吡喃木糖苷 (3), 25−O−乙酰基−7,8−去氢升麻醇−3−O−β−D−(2′−O−乙酰基) −吡喃木糖苷 (7)的活性较强。1和7处理MCF-7，HepG2和 HepG2/ADM 24小时，IC50值分别为17.7，22.0，24.6 μM和30.32, 40.73, 33.66 μM。而对正常乳腺细胞MCF-10A没有明显作用。.(3) 7能显著诱导MCF-7凋亡，下调细胞周期调节蛋白cyclinB1, CDK1和p-CDK1，阻滞细胞周期于G2/M期；显著抑制Raf/MEK/ERK通路和Akt蛋白。.(4) 1 诱导HepG2, HepG2/ADM凋亡,可能与三个途径有关：①线粒体途径；②内质网应激途径：抑制UPR通路，从而诱导肿瘤细胞的凋亡，显著上调与UPR通路相关的蛋白Calnexin, IRE1α, Chop的表达；③诱导细胞自噬流损伤有关。..（二）创新点：①首次发现升麻中三萜成分，通过ER stress途径诱导肝癌细胞凋亡的机制；②发现升麻中分离得到的三萜(1)是一个新的自噬流抑制剂，可导致耐药性肝癌细胞凋亡；③揭示1通过激活AKT而抑制降解酶cathepsin的成熟，导致溶酶体降解功能受损和自噬流抑制，最终导致细胞凋亡；④首次发现Akt在自噬降解过程中的调控作用。⑤对HepG2细胞PI3k/AKT信号通路的有一定的抑制作用。⑥化合物1可以作为靶向ERS和自噬的抗肝癌先导化合物，值得进一步研究。..（三）成果：已发表SCI论文6篇，中文核心期刊1篇，核心期刊已接收1篇，SCI论文投稿1篇。培养研究生3名