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TMR Modern Herbal Medicine  2018, Vol. 1 Issue (3): 136-142    DOI: 10.12032/TMRmhm2017A26
Orginal Articles     
A novel natural compound Shikonin inhibits YAP function by activating AMPK
Yan Fang-Jie1, Qian Mei-Jia1, Luo Hong2, Zeng Chen-Ming1, Yuan Tao1, He Qiao-Jun1, Zhu Hong1,*(), Yang Bo1,*()
1Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Science, Zhejiang University, Hangzhou, China
2Zhejiang cancer hospital, Hangzhou, China.
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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.

Highlights

Aberrant activation of YAP, a transcriptional co-activator, would result in the transactivation of target genes, and ultimately leading to the tumor development and malignance. Thus YAP has been regarded as a promising target for cancer therapy; however it has been challenged by the lack of effective inhibitors against YAP activity, particularly those origins from the natural resources. The current study identifies Shikonin, a natural compound derived from the traditional Chinese medical herb Zicao (Lithospermum erythrorhizon), as a potent inhibitor against YAP pathway so as to exert anti-cancer activities. And the YAP inhibitory effect was probably due to the activation of AMPK pathway.



Key wordsYAP      Shikonin      anti-cancer      AMPK     
Received: 04 July 2018      Published: 25 July 2018
Fund:  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.
Corresponding Authors: Zhu Hong,Yang Bo     E-mail: hongzhu@zju.edu.cn;yang924@zju.edu.cn
Cite this article:

Yan Fang-Jie, Qian Mei-Jia, Luo Hong, Zeng Chen-Ming, Yuan Tao, He Qiao-Jun, Zhu Hong, Yang Bo. A novel natural compound Shikonin inhibits YAP function by activating AMPK. TMR Modern Herbal Medicine, 2018, 1(3): 136-142. doi: 10.12032/TMRmhm2017A26

URL:

https://www.tmrjournals.com/mhm/EN/10.12032/TMRmhm2017A26     OR     https://www.tmrjournals.com/mhm/EN/Y2018/V1/I3/136

Figure 1. Identification of Shikonin as a natural inhibitor of YAP.
(A) 8×GTIIC luciferase reporter assay showed that Shikonin was a potential YAP inhibitor. (B) The chemical structure of Shikonin. (C) Shikonin prohibited the YAP-TEADs Luciferase activity. **p < 0.01; vs. control.
Figure 2. Shikonin suppressed YAP signaling by induced the phosphorylation of YAP.
(A) Immunoblotting analyses revealed that Shikonin increased YAP phosphorylation in a concentration-dependent manner, as Atorvastatin is a positive control. (B&C) Western Blotting showed that the phosphorylation of YAP upon short-exposure of Shikonin (2 μM) in cell lines of HepG2 and Hela. (D) Shikonin abrogated the mRNA levels of YAP target genes.
Figure 3. AMPK activation by Shikonin may contribute to the YAP-inhibition.
(A)Typical Western blots showed that glucose starvation enhanced the expression level of p-YAP and p-AMPK in HepG2 cells in a time-dependent manner. (B)Shikonin increased the p-AMPK levels in HepG2 cells in a concentration-dependent manner. (C) Shikonin increased both p-YAP and p-AMPK levels in HepG2 cells in a concentration-dependent manner, using Atorvastatin as a positive control.
Figure 4. The anti-cancer activities of Shikonin in liver cancer cells.
(A) The cytotoxicity of Shikonin was determined by the SRB assay in HepG2 cell line treated with Shikonin at 0, 2.5, 5, 7.5, 10 and 20 μM for 24 h. The results were expressed as the mean ± SD (n = 3). (B) The cell morphologies were photographed under an electron microscope.
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