Basic Study
Copyright ©The Author(s) 2018. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. May 7, 2018; 24(17): 1901-1910
Published online May 7, 2018. doi: 10.3748/wjg.v24.i17.1901
Mitochondrial pathway mediated by reactive oxygen species involvement in α-hederin-induced apoptosis in hepatocellular carcinoma cells
Jiao Li, Dan-Dan Wu, Ji-Xiang Zhang, Jing Wang, Jing-Jing Ma, Xue Hu, Wei-Guo Dong
Jiao Li, Dan-Dan Wu, Ji-Xiang Zhang, Jing-Jing Ma, Xue Hu, Department of Gastroenterology, Renmin Hospital of Wuhan University, Central Laboratory of Renmin Hospital, Wuhan 430060, Hubei Province, China
Jing Wang, Department of Gastroenterology, Beijing Shijitan Hospital of Capital Medical University, Beijing 100038, China
Wei-Guo Dong, Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China
Author contributions: Li J, Wu DD, Zhang JX, Wang J and Dong WG designed the research; Li J, Wu DD and Ma JJ performed the research; Zhang JX and Wang J contributed new reagents/analytical tools; Li J, Hu X and Ma JJ analyzed the data; Li J wrote the manuscript.
Supported by the National Natural Science Foundation of China, No. 81572426; and the Natural Science Foundation of Hubei Province, No. 2015CKB755.
Institutional animal care and use committee statement: All animal experiments were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Wuhan University.
Conflict-of-interest statement: The authors declare that there are no conflicts-of-interest regarding the publication of this paper.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The ARRIVE guidelines have been adopted.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Wei-Guo Dong, MD, PhD, Department of Gastroenterology, Renmin Hospital of Wuhan University, 238 Jiefang Road, Wuhan 430060, Hubei Province, China. dongweiguo@whu.edu.cn
Telephone: +86-13986167388
Received: March 1, 2018
Peer-review started: March 2, 2018
First decision: March 30, 2018
Revised: April 4, 2018
Accepted: April 9, 2018
Article in press: April 9, 2018
Published online: May 7, 2018
Processing time: 66 Days and 3 Hours
Abstract
AIM

To investigate the antitumor activity of α-hederin in hepatocellular carcinoma (HCC) cells and its underlying mechanisms in vitro and in vivo.

METHODS

SMMC-7721, HepG-2 and Huh-7 HCC cells were cultured in vitro and treated with α-hederin (0, 5 μmol/L, 10 μmol/L, 15 μmol/L, 20 μmol/L, 25 μmol/L, 30 μmol/L, 35 μmol/L, 40 μmol/L, 45 μmol/L, 50 μmol/L, 55 μmol/L, or 60 μmol/L) for 12 h, 24 h, or 36 h, and cell viability was then detected by the Cell Counting Kit-8. SMMC-7721 cells were treated with 0, 5 μmol/L, 10 μmol/L, or 20 μmol/L α-hederin for 24 h with or without DL-buthionine-S,R-sulfoximine (2 mmol/L) or N-acetylcysteine (5 mmol/L) pretreatment for 2 h, and additional assays were subsequently performed. Apoptosis was observed after Hoechst staining. Glutathione (GSH) and adenosine triphosphate (ATP) levels were measured using GSH and ATP Assay Kits. Intracellular reactive oxygen species (ROS) levels were determined by measuring the oxidative conversion of 2’,7’-dichlorofluorescin diacetate. Disruption of the mitochondrial membrane potential was evaluated using JC-1 staining. The protein levels of Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9, apoptosis-inducing factor and cytochrome C were detected by western blotting. The antitumor efficacy of α-hederin in vivo was evaluated in a xenograft tumor model.

RESULTS

The α-hederin treatment induced apoptosis of HCC cells. The apoptosis rates in the control, low-dose α-hederin (5 μmol/L), mid-dose α-hederin (10 μmol/L) and high-dose α-hederin (20 μmol/L) groups were 0.90% ± 0.26%, 12% ± 2.0%, 21% ± 2.1% and 37% ± 3.8%, respectively (P < 0.05). The α-hederin treatment reduced intracellular GSH and ATP levels, induced ROS, disrupted the mitochondrial membrane potential, increased the protein levels of Bax, cleaved caspase-3, cleaved caspase-9, apoptosis-inducing factor and cytochrome C, and decreased Bcl-2 expression. The α-hederin treatment also inhibited xenograft tumor growth in vivo.

CONCLUSION

The α-hederin saponin induces apoptosis of HCC cells via the mitochondrial pathway mediated by increased intracellular ROS and may be an effective treatment for human HCC.

Keywords: Hepatic carcinoma; α-hederin; Apoptosis; Reactive oxygen species; Mitochondria

Core tip: The α-hederin saponin induces apoptosis of hepatocellular carcinoma cells in vitro and in vivo. We found that reactive oxygen species and the mitochondrial pathway play a vital role in α-hederin-induced apoptosis.