解毒三根汤多靶点干预EGFR/Hippo/YAP通路抑制上皮间质转化抗结肠癌侵袭转移的研究

The invasion and metastasis of tumor is the cause of the death of more than 90% cancer patients. It is demonstrated that the epithelial mesenchymal transition of epithelial cell in infiltrating area plays an important role. K-ras, the downstream effector genes of EGFR, is regarded as the key that starts canceration. The overexpression of YAP genes, which is the downstream effector of Hippo, can lead to proliferation and epithelial mesenchymal transition process of tumor. So we guess there is relation between the downstream pathway of EGFR and Hippo-YAP pathway, or even they interact to regulate epithelial mesenchymal transition. Therefore, finding some effective drugs to restrain EGFR/Hippo/YAP axis so as to inhibit invasion and metastasis of cancer becomes a new thinking. We choose empirical formula Detoxifying three root soup(Radix Actinidiae chinensis,Polygonum Cuspidatum Root and Geum japonicum Thumnb) ,which is manufactured in the 1970s according to the colon cancer therapeutic principle "Clearing Heat and Detoxicating" .We apply the vitro EMT colon cancer model to detect the ability of invasion and metastasis and the phosphorylation expression of some signal proteins and metastasis-associated proteins which are related to EGFR/Hippo/YAP axis. At the same time, we build up colonic liver metastases model in nude mice and analyze expression of metastasis-associated proteins related to EGFR/Hippo/YAP axis. With that, we verify the effectiveness of Detoxifying Three Root Soup on inhibition of metastasis of colon cancer through multi-target interference with EGFR/Hippo/YAP pathway.

超过90%实体瘤患者死亡直接原因是肿瘤的侵袭转移，肿瘤浸润区上皮细胞发生上皮间质转化（EMT）发挥重要作用。EGFR下游的K-ras基因被认为是启动癌变的关键，Hippo-YAP信号通路中YAP基因的过表达可促使肿瘤增殖及EMT过程。我们猜测EGFR下游通路与Hippo-YAP通路有关，甚至发生互作而调控EMT的发生，寻找有效药物抑制EGFR/Hippo/YAP轴从而抑制肿瘤的侵袭转移成为新的思路。选用本单位肿瘤科上世纪70 年代根据"清热解毒法"治疗结肠癌研制的验方解毒三根汤（藤梨根、虎杖根和水杨梅根），应用体外结肠癌EMT模型，检测细胞侵袭转移能力及EGFR/Hippo/YAP轴信号蛋白、转移相关蛋白等磷酸化及表达水平，验证解毒三根汤多靶点干预EGFR/Hippo/YAP通路抗结肠癌转移的有效性。

目的：阐述解毒三根汤对EGFR/Hippo/YAP轴为核心的上皮间质转化（EMT）相关信号通路及调控蛋白的干预靶点及机制，为进一步临床应用推广奠定理论基础。.方法：应用结肠癌SW480细胞EGF诱导的EMT模型，构建Lats1 shRNA SW480细胞（3477细胞），再通过划痕实验、Transwell及侵袭实验、免疫荧光及激光共聚焦实验、Western Blot 实验及qRT-PCR实验对比沉默前后解毒三根汤对SW480细胞干预影响的区别。.结果：划痕结果：3477细胞10%ZYXQ及SGT组的迁移距离均大于SW480对应组(P <0.05)。Transwell结果：3477细胞10%ZYXQ组过膜细胞数较SW480、NC细胞组多(P<0.05)。Transwell侵袭结果：SGT组比EGF组过膜细胞数明显减少(P <0.01)。Wb结果：SGT组较EGF组EGFR表达下调； ZYXQ组能上调E-cad、Occludin，下调N-cad、Vimentin的表达；SGT能下调Fibronectin、Vimentin，上调Occludin表达；ZYXQ组较NSXQ组能上调NF2及p-Mst1的表达；SGT组较EGF组TAZ和YAP的表达明显减弱。Lats1沉默后，3477细胞10%ZYXQ组能明显抑制E-cadherin，上调N-cadherin的表达；3477细胞SGT组的N-cadherin，较SW480及NC细胞组均上调表达。qRT-PCR结果：SGT组较EGF组Ki67、c-myc、Sox4、BIRC5/survivin的表达均降低(P <0.001)。3477细胞10%ZYXQ及SGT组较SW480及NC细胞，Ki67、c-myc、Sox4基因表达均降低(P <0.05)。.结论：(1)、解毒三根汤是通过EGFR/Hippo/YAP轴发挥抑制SW480细胞的迁移、侵袭以及逆转SW480细胞EMT的作用；(2)、解毒三根汤可通过抑制Ki67、c-myc、Sox4及BIRC5/survivin基因多靶点作用抑制肿瘤的发生发展。