南蛇藤多萜通过调控内质网-线粒体偶联抑制肺腺癌侵袭转移的机制研究

In view of the clinical dilemma that lung adenocarcinoma has prone to invasion and metastasis, we found that the total terpenoids of Celastrus orbiculatus Thunb (TTC) could effectively inhibit the migration and invasion in lung adenocarcinoma cells. Endoplasmic reticulum - mitochondrial couple (MAMs ) and mitochondria related protein DJ-1 are closely related to tumor invasion and metastasis. It has been reported that DJ-1 is positioned at MAMs and DJ-1 could regulate MAMs. Preliminary studies have shown that TTC could regulate MAMs and inhibit the expression of DJ-1 protein in lung adenocarcinoma cells. TTC may inhibit the invasion and metastasis of lung adenocarcinoma by regulating the MAMs through the DJ-1/PTEN axis. In this study, we will establish a small clinical biological sample bank of lung adenocarcinoma and analyze the correlation between DJ-1 and MAMs in lung adenocarcinoma based on pathological data. In vitro, we will investigate whether DJ-1 could be used as the target to regulate the invasion and metastasis of lung adenocarcinoma by the MAMs. In vivo, we will investigate the mechanism of DJ-1/PTEN axis in anti-metastasis effects of TTC in lung adenocarcinoma using subcutaneous tumor transplantation nude model and hematogenous metastatic tumor model with DJ-1 knockout mice. Consequently, these studies will provide the initial underpinnings of TTC in treating invasiveness and metastasis of lung adenocarcinoma as an effective agent with novel mechanisms in future clinical applications, and this will lay the foundation for the development of new drugs with high bioavailability.

针对肺腺癌易侵袭转移的临床困境，课题组从中药南蛇藤中提取了具有抗肿瘤活性的组分南蛇藤多萜（TTC），发现其显著抑制肺腺癌细胞迁移及侵袭。内质网-线粒体偶联（MAMs）与线粒体相关蛋白DJ-1均与肿瘤的侵袭转移密切相关，且DJ-1定位于MAMs并可调控MAMs。初步研究显示，TTC可调控MAMs，并抑制肺腺癌细胞中DJ-1的蛋白水平表达。TTC是否通过DJ-1/PTEN轴调控MAMs抑制肺腺癌侵袭转移，有待研究。本课题拟通过临床样本分析DJ-1与肺腺癌MAMs的相关性，体外研究TTC能否以DJ-1/PTEN为靶点调控MAMs进而抑制肺腺癌侵袭转移；并利用裸鼠皮下移植瘤模型及DJ-1敲基因小鼠构建肺腺癌血行转移动物模型进一步探明DJ-1/PTEN在TTC抑制肺腺癌转移的作用。本项目将为TTC未来能够抑制肺腺癌转移并提高临床疗效开展有价值有意义的科学探索，并为开发生物利用度高的抗癌新药奠定基础。

本研究围绕南蛇藤多萜（TTC）通过DJ-1调控内质网线粒体结构偶联（MAMs）抑制肺腺癌细胞侵袭转移为科研假说开展相关工作。高内涵成像动态追踪细胞轨迹结果表明TTC能够有效抑制肺腺癌细胞的侵袭和迁移。同时，Western Blot验证了TTC阻碍了肺腺癌细胞的EMT进程。通过外源性表达DJ-1蛋白，我们挽救了被TTC抑制的侵袭和迁移表型，因此TTC通过DJ-1抑制肺腺癌细胞的侵袭和迁移假说成立。. 通过透射电镜和线粒体荧光标记三维重建发现，TTC作用后的肺腺癌细胞内线粒体大量破坏和降解。同时，线粒体的功能显著受损，具体表现在线粒体膜电位的崩溃，线粒体内部超氧化合物（ROS）的堆积和ATP生成的减少。给予谷胱甘肽的前体—乙酰半胱氨酸后，TTC对肺腺癌细胞的侵袭和迁移抑制作用显著降低，因此可以确认TTC抑制肺腺癌细胞侵袭和迁移作用是通过ROS生效的。与这些数据一致的是，TTC对裸鼠皮下移植瘤模型同样起效，并极大程度地抑制了肺腺癌细胞的EMT进程。. 除了上述机制外，我们发现TTC介导的DJ-1降解显著上调了PTEN的表达，这与我们之前的假设一致，DJ-1/PTEN/FAK轴是TTC抑制肺腺癌侵袭和迁移的一条重要通路。本次研究还偶然发现高浓度TTC（≥80mg/ml）导致了Hippo通路的激活，并以此促进了YAP的磷酸化和抑制了YAP的核转位，为首次发现。TTC介导的Hippo通路激活参与调控了肺腺癌细胞的增殖、干性、衰老。同时，裸鼠皮下移植瘤中的YAP蛋白被磷酸化并显著降解，肿瘤生长速度大幅度减缓。深入研究发现，这是基于DJ-1降解引发的下游效应之一。我们发现乙酰半胱氨酸能够显著抑制TTC对MOB1的磷酸化作用。因此可以认为TTC通过DJ-1引发的活性氧堆积能够导致Hippo通路激活。. 综上，本项目通过体外细胞实验及体内裸鼠皮下移植瘤模型，探明TTC通过DJ-1/PTEN/FAK以及Hippo通路调控MAMs抑制肺腺癌侵袭转移，为后期评估TTC的临床运用价值提供科学依据，并为开发生物利用度高的抗癌新药奠定基础。同时，为肺腺癌的靶向治疗提供新的方向，为寻找DJ-1抑制剂提供了可能的先导化合物。