Original Article

Hispidulin mediates apoptosis in human renal cell carcinoma by inducing ceramide accumulation

Authors: GAO Hui1,2, GAO Ming-quan3, PENG Jian-jun4, HAN Mei3, LIU Kai-li3, HAN Yan-tao5
1 Department of Pharmacology, School of Pharmacy, Qingdao University, Qingdao 266021, China
2 College of Life Sciences, Chongqing Normal University, Chongqing 401331, China;
3 Department of Pharmacology, School of Pharmacy, Qingdao University, Qingdao 266021, China
4 College of Life Sciences, Chongqing Normal University, Chongqing 401331, China
5 Medical College, Qingdao University, Qingdao 266071, China
Corresponding to: PEG Jian-jun: jianjunpeng@126.com, HA Yan-tao: hanyt19@126.com,
DOI: 10.1038/aps.2017.154
Received: 15 May 2017
Accepted: 9 July 2017
Advance online: 9 November 2017

Abstract

Abstract
Hispidulin, a polyphenolic flavonoid extracted from the traditional Chinese medicinal plant S involucrata, exhibits anti-tumor effects in a wide array of human cancer cells, mainly through growth inhibition, apoptosis induction and cell cycle arrest. However, its precise anticancer mechanisms remain unclear. In this study, we investigated the molecular mechanisms that contribute to hispidulininduced apoptosis of human clear-cell renal cell carcinoma (ccRCC) lines Caki-2 and ACHN. Hispidulin (10, 20 μmol/L) decreased the viability of ccRCC cells in dose- and time-dependent manners without affecting that of normal tubular epithelial cells. Moreover, hispidulin treatment dose-dependently increased the levels of cleaved caspase-8 and caspase-9, but the inhibitors of caspase-8 and caspase-9 only partly abrogated hispidulin-induced apoptosis, suggesting that hispidulin triggered apoptosis via both extrinsic and intrinsic pathways. Moreover, hispidulin treatment significantly inhibited the activity of sphingosine kinase 1 (SphK1) and consequently promoted ceramide accumulation, thus leading to apoptosis of the cancer cells, whereas pretreatment with K6PC-5, an activator of SphK1, or overexpression of SphK1 significantly attenuated the anti-proliferative and pro-apoptotic effects of hispidulin. In addition, hispidulin treatment dose-dependently activated ROS/JNK signaling and led to cell apoptosis. We further demonstrated in Caki-2 xenograft nude mice that injection of hispidulin (20, 40 mg·kg-1·d-1, ip) dose-dependently suppressed tumor growth accompanied by decreased SphK1 activity and increased ceramide accumulation in tumor tissues. Our findings reveal a new explanation for the antitumor mechanisms of hispidulin, and suggest that SphK1 and ceramide may serve as potential therapeutic targets for the treatment of ccRCC.
Keywords: hispidulin; SphK1; ceramide; ROS/JNK signaling; apoptosis; clear-cell renal cell carcinoma