Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Feb 7, 2015; 21(5): 1498-1509
Published online Feb 7, 2015. doi: 10.3748/wjg.v21.i5.1498
S-phase arrest after vincristine treatment may promote hepatitis B virus replication
Lei Xu, Zeng Tu, Ge Xu, Jie-Li Hu, Xue-Fei Cai, Xing-Xing Zhan, Yu-Wei Wang, Yuan Huang, Juan Chen, Ai-Long Huang
Lei Xu, Zeng Tu, Ge Xu, Jie-Li Hu, Xue-Fei Cai, Yu-Wei Wang, Yuan Huang, Juan Chen, Ai-Long Huang, Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing 400016, China
Lei Xu, Zeng Tu, Department of Microbiology, Chongqing Medical University, Chongqing 400016, China
Xing-Xing Zhan, Department of Pediatrics, Chongqing Medical University, Chongqing 400016, China
Author contributions: Xu L performed the majority of the experiments; Tu Z, Xu G, Hu JL and Cai XF made substantial contributions in conceiving and designing the study; Zhan XX, Wang YW and Huang Y contributed to the analysis and interpretation of the data; Chen J and Huang AL designed the study; Huang AL provided financial support for this work.
Supported by National Natural Science Foundation of China, No. 81201282 and No. 30901280; Grants from Chongqing Natural Science Foundation, No. cstc2012jjA10047; and the PhD Programs Foundation of the Ministry of Education of China, No. 20125503120004.
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: Ai-Long Huang, Professor, Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, No. 1 Yixueyuan Road, Chongqing 400016, China. ahuang@cqu.edu.cn
Telephone: +86-23-68486780 Fax: +86-23-68486780
Received: May 7, 2014
Peer-review started: May 8, 2014
First decision: June 27, 2014
Revised: July 4, 2014
Accepted: September 19, 2014
Article in press: September 19, 2014
Published online: February 7, 2015
Abstract

AIM: To observe the effect of vincristine on hepatitis B virus (HBV) replication in vitro and to study its possible mechanisms.

METHODS: Vincristine was added to the cultures of two cell lines stably expressing HBV. Then, the levels of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and hepatitis B core antigen (HBcAg) in the supernatants or cytoplasm were examined using by enzyme-linked immunosorbent assay and Western blot. The HBV pregenome RNA (pgRNA) was detected using reverse transcription-PCR and real-time fluorescent quantitative PCR (RT-qPCR), and viral DNA was detected using Southern blot and RT-qPCR. Cell proliferation after drug treatment was detected using the BrdU incorporation test and the trypan blue exclusion assay. Cell cycle and cell apoptosis were examined using flow cytometry and Western blot.

RESULTS: Vincristine up-regulated HBV replication directly in vitro in a dose-dependent manner, and 24-h exposure to 0.1 μmol/L vincristine induced more than 4-fold and 3-fold increases in intracellular HBV DNA and the secretion of viral DNA, respectively. The expression of HBV pgRNA, intracellular HBsAg and HBcAg, and the secretion of HBeAg were also increased significantly after drug treatment. Most importantly, vincristine promoted the cell excretion of HBV nucleocapsids instead of HBV Dane particles, and the nucleocapsids are closely related to the HBV pathogenesis. Furthermore, vincristine inhibited the proliferation of cells stably expressing HBV. The higher the concentration of the drug, the more significant the inhibition of the cell proliferation and the stronger the HBV replication ability in cells. Flow cytometry indicated that cell cycle arrest at S-phase was responsible for the cell proliferation inhibition.

CONCLUSION: Vincristine has a strong stimulatory effect on HBV replication and induces cell cycle arrest, and cell proliferation inhibition may be conducive to viral replication.

Keywords: Viral reactivation, Cytotoxic chemotherapy, Cell cycle arrest, Proliferation, Hepatitis B virus

Core tip: Hepatitis B virus (HBV) reactivation is an emerging clinical challenge for HBV carriers receiving anti-cancer chemotherapy. However, the mechanism for the sharp increase in viral replication in the early stages of chemotherapy is still unclear. In this research, we found that vincristine, which is a cytotoxic drug, had a direct stimulatory effect on HBV replication without the involvement of immune factors and that vincristine induced cell cycle arrest; cell proliferation inhibition may be conducive to viral replication, and this may be a novel mechanism of HBV reactivation following cytotoxic chemotherapy.