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©The Author(s) 2016. Published by Baishideng Publishing Group Inc. All rights reserved.
Increased ATG5-ATG12 in hepatitis B virus-associated hepatocellular carcinoma and their role in apoptosis
Areerat Kunanopparat, Ingorn Kimkong, Tanapat Palaga, Pisit Tangkijvanich, Boonchoo Sirichindakul, Nattiya Hirankarn
Areerat Kunanopparat, Nattiya Hirankarn, Center of Excellence in Immunology and Immune-mediated Diseases, Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
Ingorn Kimkong, Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
Ingorn Kimkong, Center for Advanced Studies in Tropical Natural Resources, National Research University - Kasetsart University, Bangkok 10900, Thailand
Tanapat Palaga, Department of Microbiology, Faculty of Science, 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
Boonchoo Sirichindakul, Department of Surgery, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
Author contributions: Kunanopparat A performed the majority of experiments and analyzed the data; Tangkijvanich P and Sirichindakul B participated in tissue biopsy collection, clinical data and data analysis; Palaga T was involved in data analysis and editing the manuscript; Kimkong I and Hirankarn N designed the research; Kunanopparat A, Kimkong I and Hirankarn N wrote the manuscript.
Supported by National Research Council of Thailand 2013 and the Ratchadaphiseksomphot Matching Fund from the Faculty of Medicine, Chulalongkorn University; International Research Integration, Chula Research Scholar, Ratchadaphiseksomphot 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 reviewed and approved by the Institutional Review Board (IRB No. 396/55), Faculty of Medicine, Chulalongkorn University.
Conflict-of-interest statement: The authors have no conflicts 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: Dr. Ingorn Kimkong, Assistant Professor, Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
fsciiok@ku.ac.th
Telephone: +66-25625555 Fax: +66-25792081
Received: May 21, 2016
Peer-review started: May 23, 2016
First decision: June 20, 2016
Revised: July 4, 2016
Accepted: August 1, 2016
Article in press: August 1, 2016
Published online: October 7, 2016
Processing time: 132 Days and 15 Hours
AIM
To investigate autophagy-related genes, particularly ATG12, in apoptosis and cell cycle in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) and non-HBV-HCC cell lines.
METHODS
The expression of autophagy-related genes in HBV-associated hepatocellular carcinoma and non-HBV-HCC cell lines and human liver tissues was examined by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blotting. The silencing of target genes was used to examine the function of various genes in apoptosis and cell cycle progression.
RESULTS
The expression of autophagy related genes ATG5, ATG12, ATG9A and ATG4B expression was analyzed in HepG2.2.15 cells and compared with HepG2 and THLE cells. We found that ATG5 and ATG12 mRNA expression was significantly increased in HepG2.2.15 cells compared to HepG2 cells (P < 0.005). Moreover, ATG5-ATG12 protein levels were increased in tumor liver tissues compared to adjacent non-tumor tissues mainly from HCC patients with HBV infection. We also analyzed the function of ATG12 in cell apoptosis and cell cycle progression. The percentage of apoptotic cells increased by 11.4% in ATG12-silenced HepG2.2.15 cells (P < 0.005) but did not change in ATG12-silenced HepG2 cells under starvation with Earle’s balanced salt solution. However, the combination blockade of Notch signaling and ATG12 decreased the apoptotic rate of HepG2.2.15 cells from 55.6% to 50.4% (P < 0.05).
CONCLUSION
ATG12 is important for HBV-associated apoptosis and a potential drug target for HBV-HCC. Combination inhibition of ATG12/Notch signaling had no additional effect on HepG2.2.15 apoptosis.
Core tip: ATG5-ATG12 protein expression was increased in hepatitis B virus (HBV)-transfected HepG2.2.15 cells when they were compared with HepG2 cells and was increased in tumor liver tissues compared to adjacent non-tumor tissues from hepatocellular carcinoma (HCC) patients with HBV infection. We showed that silencing of ATG12 increased cell apoptosis of HepG2.2.15 cells but not HepG2 cells under starvation conditions. These results suggest that ATG5-ATG12 proteins are important for the survival of HBV-associated HCC during states of limited tumor nutrients. The inhibition of ATG12 might be a good target for HBV-associated HCC treatment.
