Basic Study
Copyright ©The Author(s) 2018. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Sep 14, 2018; 24(34): 3861-3870
Published online Sep 14, 2018. doi: 10.3748/wjg.v24.i34.3861
Delta-like ligand 4 in hepatocellular carcinoma intrinsically promotes tumour growth and suppresses hepatitis B virus replication
Areerat Kunanopparat, Jiraphorn Issara-Amphorn, Asada Leelahavanichkul, Anapat Sanpavat, Suthiluk Patumraj, Pisit Tangkijvanich, Tanapat Palaga, Nattiya Hirankarn
Areerat Kunanopparat, Jiraphorn Issara-Amphorn, Asada Leelahavanichkul, Nattiya Hirankarn, Center of Excellence in Immunology and Immune Mediated Diseases, Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
Anapat Sanpavat, Department of Pathology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
Suthiluk Patumraj, Center of Excellence for Microcirculation, Department of Physiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
Pisit Tangkijvanich, Research Unit of Hepatitis and Liver Cancer, Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
Tanapat Palaga, Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
Author contributions: Kunanopparat A, Palaga T and Hirankarn N conceived and designed experiments; Kunanopparat A, Issara-Amphorn J, Leelahavanichkul A, Patumraj S and Tangkijvanich P conducted the experiments; Kunanopparat A and Sanpavat A analysed the data; Kunanopparat A, Palaga T and Hirankarn N wrote the manuscript.
Supported by National Research Council of Thailand 2013; the Ratchadaphiseksomphot Matching Fund from the Faculty of Medicine, Chulalongkorn University; the International Research Integration, Chula Research Scholar, Ratchadaphisek somphot Endowment Fund, Center of Excellence in Immunology and Immune-mediated Diseases; and the Rachadapisaek Sompote Post-Doctoral Fund, Chulalongkorn University.
Institutional review board statement: The study was approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University.
Institutional animal care and use committee statement: All protocols were carried out in accordance with relevant guidelines and regulations.
Conflict-of-interest statement: The authors declare no conflict of interest.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
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: Nattiya Hirankarn, MD, PhD, Lecturer, Professor, Center of Excellence in Immunology and Immune Mediated Diseases Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand. nattiya.H@chula.ac.th
Telephone: +66-2-2564132 Fax: +66-2-2525952
Received: May 25, 2018
Peer-review started: May 25, 2018
First decision: June 21, 2018
Revised: July 5, 2018
Accepted: July 16, 2018
Article in press: July 16, 2018
Published online: September 14, 2018
Abstract
AIM

To investigate the role of Delta-like ligand 4 (DLL4) on tumour growth in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) in vivo.

METHODS

We suppressed DLL4 expression in an HBV expressing HCC cell line, HepG2.2.15 and analysed the growth ability of cells as subcutaneous tumours in nude mice. The expression of tumour angiogenesis regulators, VEGF-A and VEGF-R2 in tumour xenografts were examined by western blotting. The tumour proliferation and neovasculature were examined by immunohistochemistry. The viral replication and viral protein expression were measured by quantitative PCR and western blotting, respectively.

RESULTS

Eighteen days after implantation, tumour volume in mice implanted with shDLL4 HepG2.2.15 was significantly smaller than in mice implanted with control HepG2.2.15 (P < 0.0001). The levels of angiogenesis regulators, VEGF-A and VEGF-R2 were significantly decreased in implanted tumours with suppressed DLL4 compared with the control group (P < 0.001 and P < 0.05, respectively). Furthermore, the suppression of DLL4 expression in tumour cells reduced cell proliferation and the formation of new blood vessels in tumours. Unexpectedly, increased viral replication was observed after suppression of DLL4 in the tumours.

CONCLUSION

This study demonstrates that DLL4 is important in regulating the tumour growth of HBV-associated HCC as well as the neovascularization and suppression of HBV replication.

Keywords: Hepatocellular carcinoma, Notch signalling, Delta-like ligand 4, HepG2.2.15

Core tip: We demonstrated that Delta-like ligand 4 (DLL4) is important for tumour growth of hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) in a xenograft model. We found that the level of angiogenesis regulators, VEGF-A and VEGF-R2 were significantly decreased in HCC xenograft tumours with suppressed DLL4 compared with the control group. Consistent with these findings, the suppression of DLL4 expression in the tumour cells reduced cell proliferation and the formation of new blood vessels in the tumour. Furthermore, this is the first report that DLL4 in an HBV expressing HCC cell line plays a key role in regulating tumour growth, angiogenesis, and viral replication in a mouse model of xenograft transplantation.