蟾毒内酯化合物14α-artebufogenin靶向LRP6抑制结肠癌上皮间质转化作用机制研究

Epithelial-Mesenchymal transition (EMT) is one of the most important reasons that leads to metastasis of colorectal cancer. Wnt/β-catenin pathway is known for regulating EMT of colorectal cancer. Thus, inhibition of EMT through blocking Wnt/β-catenin pathway has been a new strategy to inhibit metastasis of colorectal cancer. LRP6 is a vital receptor of Wnt/β-catenin pathway, which binds to Wnt3a and Frizzled to activate Wnt/β-catenin pathway. Small molecule compounds directly binding to LRP6 to block the interaction of Wnt3a and LRP6 haven’t been found yet. In our previously study, a natural bufadienolide 14α-artebufogenin could inhibit EMT of high metastatic colorectal cancer cell in vitro and in vivo. Preliminary mechanistic study also showed that 14α-artebufogenin, as a novel ligand for LRP6, could directly bind to LRP6 and block the interaction of Wnt3a and LRP6, thereby leading to the inhibition of Wnt/β-catenin pathway. In the present study, we will focus on the binding effect of 14α-artebufogenin to LRP6 for its selectivity, action model and interaction position, and declare the target (LRP6) and signaling pathway (Wnt/β-catenin) through which 14α-artebufogenin regulates the EMT of colorectal cancer cell, as well as evaluate the anti-metastasis effect of 14α-artebufogenin on colorectal cancer cell in vivo. Our research will provide evidence for the development of 14α-artebufogenin to be an anti-metastasis drug with specific target and action mechanisms for treatment of colorectal cancer.

上皮间质转化（EMT）是结肠癌转移的主要原因，Wnt/ß-catenin是调控结肠癌EMT的重要信号通路，阻断该通路抑制EMT已成为抗结肠癌转移药物研究的新策略。LRP6是该通路的关键受体，与Wnt3a结合后激活该通路，但未见LRP6的小分子配体能阻断Wnt3a和LRP6相互作用。我们前期研究发现蟾毒内酯化合物14α-artebufogenin (14α-art)是LRP6的小分子配体，能抑制Wnt3a和LRP6结合，进而抑制Wnt/ß-catenin通路；且14α-art在体内外均能显著抑制结肠癌EMT。本项目拟进一步研究14α-art与LRP6相互作用的选择性、结合特征、作用方式和位点，阐明14α-art调控结肠癌EMT的作用靶标（LRP6）和信号通路（Wnt/ß-catenin），体内评价14α-art抑制结肠癌转移活性，为其发展成为靶点清楚、机制明确的抗结肠癌转移新药奠定科学基础。

上皮间质转化（EMT）是肿瘤转移的主要原因，Wnt/ß-catenin是调控肿瘤EMT的重要信号通路，阻断该通路抑制EMT已成为抗肿瘤转移药物研究的新策略。LRP6是该通路的关键受体，与Wnt3a结合后激活该通路，但未见LRP6的小分子配体能阻断Wnt3a和LRP6相互作用。我们的研究发现蟾毒内酯化合物14α-artebufogenin和arenobufagin能抑制肿瘤EMT，且发现它们是LRP6的小分子配体，揭示了蟾毒内酯化合物14α-artebufogenin和arenobufagin与LRP6相互作用的选择性、结合特征，初步探索了作用方式和位点，明确了蟾毒内酯化合物14α-artebufogenin和arenobufagin对LRP6的调控作用，并证明了14α-artebufogenin和arenobufagin诱导肿瘤细胞EMT是依赖于LRP6。发现蟾毒内酯化合物14α-artebufogenin和arenobufagin能抑制Wnt3a和LRP6结合进而抑制Wnt/ß-catenin通路，包括抑制细胞浆ß-catenin 降解复合物形成，抑制ß-catenin的入核和 ß-catenin/TCF介导的 EMT相关基因（E-cadherin、N-cadherin、Vimentin、Slug、Snail 和 Twist）的转录和表达，体内研究也表明在裸鼠移植瘤模型上蟾毒内酯化合物14α-artebufogenin和arenobufagin能显著抑制肿瘤生长和EMT。本项目的研究揭示了蟾毒内酯化合物14α-artebufogenin和arenobufagin调控肿瘤EMT的作用靶标LRP6和信号通路Wnt/ß-catenin，体内证明了14α-artebufogenin和arenobufagin抑制肿瘤EMT活性，为其发展成为靶点清楚、机制明确的抗肿瘤转移新药奠定科学基础。

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