RETRACTED ARTICLE: Hispidulin mediates apoptosis in human renal cell carcinoma by inducing ceramide accumulation

Hui Gao,Mei Han,Kai-Li Liu,Ming-Quan Gao,Jian-Jun Peng,Yan-Tao Han

doi:10.1038/aps.2017.154

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 hispidulin-induced 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 anti-tumor mechanisms of hispidulin, and suggest that SphK1 and ceramide may serve as potential therapeutic targets for the treatment of ccRCC.

Highlights

T induced apoptosis of human clear-cell renal cell carcinoma lines Caki-2 and ACHN
Our findings reveal a new explanation for the antitumor mechanisms of hispidulin, and suggest that sphingosine kinase 1 (SphK1) and ceramide may serve as potential therapeutic targets for the treatment
Our findings collectively Reactive oxygen species (ROS) (Figure 7A), and this effect was markedly abrogated by indicated that hispidulin inhibits SphK1 activity and subseK6PC-5, suggesting that the increases in the ROS level quently induces the accumulation of ceramide, which depended on SphK1 inhibition and subsequent ceramide accu- activates ROS/JNK signaling and leads to apoptosis. mulation

Summary

E Materials and methods T Cell culture

The human ccRCC cell lines Caki-2 and ACHN were purchased from the ATCC. C purchased from the Cell Bank of the Shanghai Institute of Biological Science (Shanghai, China). A in a humidified atmosphere with 5% CO2 in DMEM medium (HyClone, Logan, UT, USA) with 10% (v/v) heat-inactivated.

E Primary culture of human RCC cells

E Western blotting

D Analysis of sphingosine kinase 1 activity

Findings

A Hispidulin induces apoptosis in ccRCC cells