Hispidulin induces ER stress-mediated apoptosis in human hepatocellular carcinoma cells in vitro and in vivo by activating AMPK signaling pathway

Mei Han1, Hui Gao1, Jing Xie2, Yin-ping Yuan3,4, Quan Yuan5, Ming-quan Gao1, Kai-li Liu1, Xue-hong Chen2, Yan-tao Han2, Zhi-wu Han6
1 Department of Pharmacology, School of Pharmacy, Qingdao University, Qingdao 266021, China
2 Medical College, Qingdao University, Qingdao 266071, China
3 Shandong Cancer Hospital Affiliated to Shandong University, Jinan 250117, China
4 Shandong Academy of Medical Sciences, Jinan 250001, China
5 Qingdao Fifth People’s Hospital, Qingdao 266000, China
6 The Affiliated Hospital of Qingdao University, Qingdao 266003, China
Correspondence to: Hui Gao:,
DOI: 10.1038/s41401-018-0159-7
Received: 10 January 2018
Accepted: 4 July 2018
Advance online: 14 September 2018


Hispidulin (4’,5,7-trihydroxy-6-methoxyflavone) is a phenolic flavonoid isolated from the medicinal plant S. involucrata, which exhibits anti-neoplastic activity against several types of cancer. However, the mechanism underlying its anti-cancer activity against hepatocellular carcinoma (HCC) has not been fully elucidated. In this study, we investigated whether and how hispidulin-induced apoptosis of human HCC cells in vitro and in vivo. We showed that hispidulin (10, 20 μmol/L) dose-dependently inhibited cell growth and promoted apoptosis through mitochondrial apoptosis pathway in human HCC SMMC7721 cells and Huh7 cells. More importantly, we revealed that its pro-apoptotic effects depended on endoplasmic reticulum stress (ERS) and unfolded protein response (UPR), as pretreatment with salubrinal, a selective ERS inhibitor, or shRNA targeting a UPR protein CHOP effectively abrogated hispidulin-induced cell apoptosis. Furthermore, we showed that hispidulin-induced apoptosis was mediated by activation of AMPK/mTOR signaling pathway as pretreatment with Compound C, an AMPK inhibitor, or AMPK-targeting siRNA reversed the pro-apoptotic effect of hispidulin. In HCC xenograft nude mice, administration of hispidulin (25, 50 mg/kg every day, ip, for 27 days) dose-dependently suppressed the tumor growth, accompanied by inducing ERS and apoptosis in tumor tissue. Taken together, our results demonstrate that hispidulin induces ERS-mediated apoptosis in HCC cells via activating the AMPK/mTOR pathway. This study provides new insights into the anti-tumor activity of hispidulin in HCC.
Keywords: hispidulin; hepatocellular carcinoma; mitochondrial apoptosis; endoplasmic reticulum stress; CHOP; AMPK/mTOR; Z-LEHD-FMK; salubrinal; Compound C

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