摘要
Objective： Yes Associated Protein （YAP） is a downstream effector that negatively regulated by Hippo kinase LATS1/2. As a transcriptional coactivator, YAP controls the transactivation of variety target genes to promote cell proliferation which is a critical survival input for cancer cells, thus the inhibition of YAP function is a promising strategy to treat cancer patients. The aim of this study was to explore YAP inhibitors derived from natural products using a cell-based YAP-TEADs luciferase reporter assay and investigate the functional activities of the novel inhibitor. Methods： natural compounds were used by 8×GTIIC luciferase reporter assay to screen YAP inhibitor. Phosphorylation of YAP and AMPK were detected by Western Blotting. The target genes of YAP were determined through RT-PCR. Inhibition on HepG2 cells of screened compounds were assessed by the Sulforhodamine B （SRB） assay. Results： we found that Shikonin （derived from the traditional Chinese medical herb Zicao （Lithospermum erythrorhizon）） exerted significant suppression against the transcriptional activity of YAP （inhibition ratio=74.3%）, accompanied with increased phosphorylation of YAP protein upon within short-exposure to cancer cells. Shikonin treated on HepG2 induced phosphorylation of AMPK. In HepG2 cell lines, Shikonin exhibited a profound cytotoxicity in a concentration manner. Conclusion： our results indicated that the inhibition activity of Shikonin on YAP function was probably due to the activation of AMPK by phosphorylation. Moreover, Shikonin exhibited potent cytotoxicity on cancer cells. In summary, the present study identifies Shikonin as a novel natural inhibitor of YAP function and could be an anti-cancer drug candidate for cancer treatment.
目的：YAP是Hippo信号通路的关键下游蛋白,受到Hippo激酶LATS1/2的负性调控.作为转录共激活子,YAP可转录激活下游靶基因促进细胞增殖,对调控肿瘤细胞的存活发挥关键的作用,因而阻断其活性被视为肿瘤治疗的全新策略.本研究的主要目的是在细胞水平上利用YAP-TEADs荧光报告系统筛选来源于天然化合物的新型YAP抑制剂,进-步探究该抑制剂的抗肿瘤活性.方法：利用8×GTIICluciferase系统对70个天然化合物进行筛选抑制YAP活性的抑制剂.WesternBlotting实验考察筛选到的化合物对p-YAP、p-AMPK的作用.通过RT-PCR实验考察筛选到的化合物对YAP下游靶基因的影响.利用SRB实验考察紫草素对HepG2增殖抑制的作用.结果：通过上述系统,我们筛选得到YAP天然抑制剂紫草素（来源于中草药紫草）,可显著抑制YAP的活性（抑制率为74.3%）,并且发现该天然活性化合物可在短时间内迅速上调肿瘤细胞内YAP的磷酸化水平,抑制YAP的转录活性.进-步实验发现紫草素可显著上调AMPK的磷酸化水平.紫草素可抑制肝癌细胞HepG2的增殖.结论：本研究在机制上发现紫草素主要通过上调AMPK的磷酸化水平抑制YAP的活性.进-步研究发现,紫草素作用后对肿瘤细胞有显著的细胞毒性.综上,本研究发现了YAP的新型抑制剂-紫草素,该天然化合物可作为肿瘤治疗的候选药物.
基金
This work was supported National Key R＆D Program of China （No. 2017YFE0102200）, National Natural Science Foundation for Distinguished Young Scholar of China （81625024） and National Natural Science Foundation of China （81773753） to B. Yang.
