Basic Study
Copyright ©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Feb 21, 2017; 23(7): 1171-1179
Published online Feb 21, 2017. doi: 10.3748/wjg.v23.i7.1171
Thymoquinone suppresses migration of LoVo human colon cancer cells by reducing prostaglandin E2 induced COX-2 activation
Hsi-Hsien Hsu, Ming-Cheng Chen, Cecilia Hsuan Day, Yueh-Min Lin, Shin-Yi Li, Chuan-Chou Tu, Viswanadha Vijaya Padma, Hui-Nung Shih, Wei-Wen Kuo, Chih-Yang Huang
Hsi-Hsien Hsu, Division of Colorectal Surgery, Mackay Memorial Hospital, Taipei 10449, Taiwan
Hsi-Hsien Hsu, Mackay Medicine, Nursing and Management College, Taipei 10449, Taiwan
Ming-Cheng Chen, Division of Colorectal Surgery, Taichung Veterans General Hospital, Taichung 40705, Taiwan
Cecilia Hsuan Day, Department of Nursing, MeiHo University, Pingtung 912, Taiwan
Yueh-Min Lin, Department of Pathology, Changhua Christian Hospital, Changhua 500, Taiwan
Yueh-Min Lin, Jen-Teh Junior College of Medicine, Nursing and Management, Miaoli 35664, Taiwan
Shin-Yi Li, Graduate Institute of Basic Medical Science, China Medical University, Taichung 40402, Taiwan
Chuan-Chou Tu, Division of Chest Medicine, Department of Internal Medicine, Armed Force Taichung General Hospital, Taichung 41152, Taiwan
Viswanadha Vijaya Padma, Department of Biotechnology, Bharathiar University, Coimbatore 641046, India
Hui-Nung Shih, Chih-Yang Huang, Graduate Institute of Chinese Medical Science, China Medical University, Taichung 41352, Taiwan
Wei-Wen Kuo, Department of Biological Science and Technology, China Medical University, Taichung 40402, Taiwan
Chih-Yang Huang, Department of Health and Nutrition Biotechnology, Asia University, Taichung 41352, Taiwan
Author contributions: Hsu HH, Kuo WW and Huang CY contributed equally to this work; Hsu HH, Chen MC, Day CH, Lin YM, Li SY, Tu CC, Padma VV, Shih HN, Kuo WW and Huang CY designed the research; Hsu HH, Chen MC, Day CH, Lin YM, Li SY, Tu CC, Padma VV, Kuo WW and Huang CY performed the research; Hsu HH and Shih HN wrote the paper.
Supported by (in part) the Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence, No. MOHW105-TDU-B-212-133019.
Institutional review board statement: The study was reviewed and approved by the Institutional Review Board of China Medical University, Taiwan.
Institutional animal care and use committee statement: All protocols were reviewed and approved by the Institutional Review Board (IRB, ethical clearance number 104-223-N), Animal Care and Use Committee of China Medical University, Taichung, China, and the study was conducted in accordance with the principles of laboratory animal care.
Conflict-of-interest statement: We declare that there are no conflicts of interest to disclose.
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: Chih-Yang Huang, PhD, Graduate Institute of Chinese Medical Science, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan. cyhuang@mail.cmu.edu.tw
Telephone: +886-4-22053366-3313 Fax: +886-4-22032295
Received: September 6, 2016
Peer-review started: September 8, 2016
First decision: October 20, 2016
Revised: November 9, 2016
Accepted: December 16, 2016
Article in press: December 19, 2016
Published online: February 21, 2017
Processing time: 167 Days and 7.2 Hours
Abstract
AIM

To identify potential anti-cancer constituents in natural extracts that inhibit cancer cell growth and migration.

METHODS

Our experiments used high dose thymoquinone (TQ) as an inhibitor to arrest LoVo (a human colon adenocarcinoma cell line) cancer cell growth, which was detected by cell proliferation assay and immunoblotting assay. Low dose TQ did not significantly reduce LoVo cancer cell growth. Cyclooxygenase 2 (COX-2) is an enzyme that is involved in the conversion of arachidonic acid into prostaglandin E2 (PGE2) in humans. PGE2 can promote COX-2 protein expression and tumor cell proliferation and was used as a control.

RESULTS

Our results showed that 20 μmol/L TQ significantly reduced human LoVo colon cancer cell proliferation. TQ treatment reduced the levels of p-PI3K, p-Akt, p-GSK3β, and β-catenin and thereby inhibited the downstream COX-2 expression. Results also showed that the reduction in COX-2 expression resulted in a reduction in PGE2 levels and the suppression of EP2 and EP4 activation. Further analysis showed that TG treatment inhibited the nuclear translocation of β-catenin in LoVo cancer cells. The levels of the cofactors LEF-1 and TCF-4 were also decreased in the nucleus following TQ treatment in a dose-dependent manner. Treatment with low dose TQ inhibited the COX-2 expression at the transcriptional level and the regulation of COX-2 expression efficiently reduced LoVo cell migration. The results were further verified in vivo by confirming the effects of TQ and/or PGE2 using tumor xenografts in nude mice.

CONCLUSION

TQ inhibits LoVo cancer cell growth and migration, and this result highlights the therapeutic advantage of using TQ in combination therapy against colorectal cancer.

Keywords: Thymoquinone; LoVo cell; Cyclooxygenase 2; Prostaglandin E2; Migration

Core tip: Prostaglandin E2 (PGE2) induces migration of human LoVo colon cancer cells, and the major mechanism involves the activation of the p-Akt/p-PI3K/p-GSK3β/β-catenin/LEF-1/TCF-4 pathway that ultimately up-regulates cyclooxygenase 2 (COX-2) expression. Thymoquinone (TQ) suppresses cancer cell migration and represents a potential therapeutic target for colon adenocarcinoma metastasis. PGE2 activation of COX-2 and β-catenin to induce human LoVo colon cancer cell migration was blocked by TQ. Our study used cell proliferation assay, immunoblotting assay, immunofluorescence assay, nuclear extraction and in vivo experiments to examine the COX2 protein, which affects the metastasis of highly metastatic LoVo cancer cells treated with TQ.