DataSheet_1_α-Hederin Inhibits the Proliferation of Hepatocellular Carcinoma Cells via Hippo-Yes-Associated Protein Signaling Pathway.pdf

AimsYes-associated protein (YAP), a downstream protein in the Hippo signaling pathway, plays an important role in tumor proliferation, including in hepatocellular carcinoma (HCC). α-hederin, a monodesmosidic triterpenoid saponin isolated from Fructus akebiae, displayed anti-cancer effects on several cancer cell lines but the precise mechanism has not been ascertained. In the present study, we explored the effects of α-hederin on cell proliferation and apoptosis in human HCC cell lines and the underlying mechanisms.
Main MethodCell proliferation and apoptosis were assessed using 5-ethynyl-2’-deoxyuridine staining, colony formation, flow cytometry. The expression patterns of components of Hippo signaling pathway and apoptotic genes were further examined via RT-qPCR and immunoblotting. A xenograft tumor model in nude mice was used to evaluate the anti-HCC effects of α-hederin in vivo.
Resultsα-hederin promoted the apoptosis and inhibited the proliferation of SMMC-7721 and HepG2 cells in vitro, and remarkably inhibited the tumor size and weight in the xenograft mouse model. Additionally, α-hederin increased the expression of pro-apoptosis proteins and suppressed the expression of anti-apoptosis proteins. Moreover, α-hederin treatment upregulated the expression of Hippo signaling pathway-related proteins and genes, while, effectively reduced the level of nuclear YAP, which resulted in the inhibition of proliferation and the induction of apoptosis of HCC cells. Finally, the effects of α-hederin on HCC cell proliferation and apoptosis were alleviated by XMU-MP-1, a Mst1/2 inhibitor in vitro.
SignificanceWe identified α-hederin is a novel agonist of Hippo signaling pathway and possesses an anti-HCC efficacy through inhibiting YAP activity.

研究目的：Yes相关蛋白（Yes-associated protein, YAP）是Hippo信号通路（Hippo signaling pathway）的下游效应蛋白，在包括肝细胞癌（hepatocellular carcinoma, HCC）在内的多种肿瘤增殖过程中发挥关键调控作用。α-常春藤皂苷（α-hederin）是从预知子（Fructus akebiae）中分离得到的单糖链三萜皂苷，已被证实对多种癌细胞系具有抗癌活性，但其具体分子机制尚未阐明。本研究旨在探讨α-常春藤皂苷对人肝癌细胞系增殖与凋亡的影响及其潜在作用机制。

主要方法：采用5-乙炔基-2’-脱氧尿苷（5-ethynyl-2’-deoxyuridine, EdU）染色、克隆形成实验及流式细胞术检测细胞增殖与凋亡水平；通过实时定量聚合酶链式反应（RT-qPCR）与免疫印迹实验分析Hippo信号通路组分及凋亡相关基因的表达模式；构建裸鼠异种移植瘤模型，体内评价α-常春藤皂苷的抗肝癌效应。

实验结果：体外实验显示，α-常春藤皂苷可显著促进SMMC-7721与HepG2细胞的凋亡并抑制其增殖；同时可明显抑制裸鼠异种移植瘤的体积与重量。进一步机制研究表明，α-常春藤皂苷可上调促凋亡蛋白的表达，下调抗凋亡蛋白的表达；此外，该化合物可上调Hippo信号通路相关蛋白及基因的表达水平，并有效降低细胞核内YAP的蛋白含量，进而抑制肝癌细胞增殖并诱导其凋亡。体外实验证实，Mst1/2抑制剂XMU-MP-1可逆转α-常春藤皂苷对肝癌细胞增殖与凋亡的调控作用。

研究意义：本研究证实α-常春藤皂苷是一种新型的Hippo信号通路激动剂，可通过抑制YAP活性发挥抗肝癌的治疗功效。