Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Sep 28, 2015; 21(36): 10385-10399
Published online Sep 28, 2015. doi: 10.3748/wjg.v21.i36.10385
Equol inhibits proliferation of human gastric carcinoma cells via modulating Akt pathway
Zhi-Ping Yang, Yan Zhao, Fang Huang, Jie Chen, Ya-Hong Yao, Jun Li, Xiao-Nan Wu
Zhi-Ping Yang, Fang Huang, Jie Chen, Xiao-Nan Wu, Public Health College, Fujian Medical University, Fuzhou 350108, Fujian Province, China
Yan Zhao, Ya-Hong Yao, Jun Li, The First Affiliated Hospital of Xiamen University, Xiamen 361003, Fujian Province, China
Author contributions: Yang ZP and Wu XN designed the study; Yang ZP, Li J and Chen J performed the experiments; Zhao Y analyzed the data and prepared the figures; Huang F and Yao YH contributed to the interpretation of the data and preparation of the manuscript; Yang ZP and Zhao Y wrote the first version of the manuscript; all authors read, critically reviewed and approved the final manuscript.
Supported by Special Project on the Integration of Industry, Education and Research of Fujian Province, China, No. 2012N0014.
Institutional review board statement: The study was reviewed and approved by the Fujian Medical University Institutional Review Board.
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of Fujian Medical University (IACUC protocol number: 2014-022).
Conflict-of-interest statement: The authors have no conflict of interest to declare.
Data sharing statement: No additional data are available.
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: Xiao-Nan Wu, Professor, Public Health College, Fujian Medical University, No. 1 Xueyuan Road, Fuzhou 350108, Fujian Province, China. wuxiaonan728@163.com
Telephone: +86-591-22862865 Fax: +86-591-22862865
Received: April 23, 2015
Peer-review started: April 24, 2015
First decision: June 2, 2015
Revised: June 17, 2015
Accepted: July 18, 2015
Article in press: July 18, 2015
Published online: September 28, 2015
Processing time: 158 Days and 1.2 Hours
Abstract

AIM: To investigate the anti-tumor effects of equol in gastric cancer cells and the underlying molecular mechanisms.

METHODS: MGC-803 cells were employed for in vitro experiments in this study. Cells were treated with control (vehicle, 0.1% DMSO) or equol under specified dose titration or time courses. Cell viability was examined by MTS assay, and the levels of Ki67 were determined by qPCR and immunofluorescent assay. Changes in cell cycle distribution and apoptosis rate were detected by flow cytometry. The mRNA expression of cyclin E1 and P21WAF1 was determined by qPCR. The protein levels of cell cycle regulators, PARP and Caspase-3 cleavage, and the phosphorylation of Akt were examined by Western blot. In addition, to characterize the role of elevated Akt activation in the anti-tumor effect exerted by equol, Ly294002, a PI3K/AKT pathway inhibitor, was used to pretreat MGC-803 cells.

RESULTS: Equol (5, 10, 20, 40, or 80 μmol/L) inhibited viability of MGC-803 cells in a dose- and time-dependent manner after treatment for 24, 36, or 48 h (P < 0.05 for all). Equol also decreased the mRNA (P < 0.05 for 12 and 24 h treatment) and protein levels of Ki67. Equol treatment significantly induced G0/G1 cell cycle arrest (P < 0.05), with the percentages of G0/G1 cells of 32.23% ± 3.62%, 36.31% ± 0.24%, 45.58% ± 2.29%, and 65.10% ± 2.04% for equol (0, 10, 20, or 30 μmol/L) treatment, respectively, accompanied by a significant decrease of CDK2/4 (P < 0.05 for 24 and 48 h treatment) and Cyclin D1/Cyclin E1 (P < 0.05), and an increased level of P21WAF1 (P < 0.05). A marked increase of apoptosis was observed, with the percentages of apoptotic cells of 5.01% ± 0.91%, 14.57% ± 0.99%, 37.40% ± 0.58%, and 38.46% ± 2.01% for equol (0, 5, 10, or 20 μmol/L) treatment, respectively, accompanied by increased levels of cleaved PARP and caspase-3. In addition, we found that equol treatment increased P-Akt (Ser473 and Thr308) at 12 and 24 h compared to vehicle-treated control; longer treatment for 48 h decreased P-Akt (Ser473 and Thr308). P-Akt at Thr450, however, was decreased by equol treatment at all time points examined (P < 0.05 for all). Moreover, Akt inhibition by Ly294002 could not prevent but led to enhanced G0/G1 arrest and apoptosis.

CONCLUSION: Equol inhibits MGC-803 cells proliferation by induction of G0/G1 arrest and apoptosis. Its anti-cancer effects are likely mediated by dephosphorylation of Akt at Thr450.

Keywords: Equol; Apoptosis; Chemoprevention; Gastric cancer; G0/G1 arrest

Core tip: The results of our study demonstrated that equol could effectively inhibit the proliferation of human gastric cancer MGC-803 cells, which was associated with cell cycle arrest and apoptosis. Our data also suggested that the Akt signaling pathway may play a role in equol-mediated cell cycle arrest. To our knowledge, this is the first study demonstrating that equol exerts anticancer effect in human gastric cancer cells and that blockade of the Akt signaling pathway may be an antitumor mechanism of equol.