Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Sep 7, 2015; 21(33): 9727-9735
Published online Sep 7, 2015. doi: 10.3748/wjg.v21.i33.9727
Notch1 downregulation combined with interleukin-24 inhibits invasion and migration of hepatocellular carcinoma cells
Bing Han, Shi-Hai Liu, Wei-Dong Guo, Bin Zhang, Jian-Ping Wang, Yu-Kun Cao, Jun Liu
Bing Han, Jian-Ping Wang, Yu-Kun Cao, Jun Liu, Department of Liver Transplantation and Hepatobiliary Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, Shandong Province, China
Shi-Hai Liu, Central Laboratory, The Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Province, China
Bing Han, Wei-Dong Guo, Bin Zhang, Department of Hepatobilliary and Pancreatic Surgery, The Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Province, China
Author contributions: Han B and Liu SH contributed equally to this work; Han B, Liu SH, Guo WD, Zhang B, Wang JP, Cao YK and Liu J designed the research; Han B, Liu SH, Wang JP and Cao YK performed the research; Han B, Liu SH, Guo WD and Zhang B analyzed the data; Han B and Liu SH wrote the paper; all authors read and approved the final manuscript.
Supported by Shandong Provincial Natural Science Foundation, China, No. ZR2012HQ039 and No. ZR2014HP065; National Natural Science Foundation of China, No. 81373172 and No.81402579.
Conflict-of-interest statement: There is no conflict of interest in this study.
Data sharing statement: No additional unpublished data are available.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: Jun Liu, MD, PhD, Professor, Department of Liver Transplantation and Hepatobiliary Surgery, Shandong Provincial Hospital Affiliated to Shandong University, No. 324 Jingwu Road, Jinan 250021, Shandong Province, China. dr_junliu@163.com
Telephone: +86-531-68776905 Fax: +86-531-68776905
Received: March 15, 2015
Peer-review started: March 15, 2015
First decision: April 13, 2015
Revised: June 6, 2015
Accepted: June 26, 2015
Article in press: June 26, 2015
Published online: September 7, 2015
Abstract

AIM: To confirm the anti-invasion and anti-migration effects of down-regulation of Notch1 combined with interleukin (IL)-24 in hepatocellular carcinoma (HCC) cells.

METHODS: γ-secretase inhibitors (GSIs) were used to down-regulate Notch1. HepG2 and SMMC7721 cells were seeded in 96-well plates and treated with GSI-I or/and IL-24 for 48 h. Cell viability was measured by MTT assay. The cellular and nuclear morphology was observed under a fluorescence microscope. To further verify the apoptotic phenotype, cell cultures were also analyzed by flow cytometry with Annexin V-FITC/propidium iodide staining. The expression of Notch1, SNAIL1, SNAIL2, E-cadherin, IL-24, XIAP and VEGF was detected by Western blot. The invasion and migration capacities of HCC cells were detected by wound healing assays. Notch1 and Snail were down-regulated by RNA interference, and the target proteins were analyzed by Western blot. To investigate the mechanism of apoptosis, we analyzed HepG2 cells treated with siNotch1 or siCON plus IL-24 or not for 48 h by caspase-3/7 activity luminescent assay.

RESULTS: GSI-I at a dose of 2.5 μmol/L for 24 h caused a reduction in cell viability of about 38% in HepG2 cells. The addition of 50 ng/mL IL-24 in combination with 1 or 2.5 μmol/L GSI-I reduced cell viability of about 30% and 15%, respectively. Treatment with IL-24 alone did not induce any cytotoxic effect. In SMMC7721 cells with the addition of IL-24 to GSI-I (2.5 μmol/L), the reduction of cell viability was only about 25%. Following GSI-I/IL-24 combined treatment for 6 h, the apoptotic rate of HepG2 cells was 47.2%, while no significant effect was observed in cells treated with the compounds employed separately. Decreased expression of Notch1 and its associated proteins SNAIL1 and SNAIL2 was detected in HepG2 cells. Increased E-cadherin protein expression was noted in the presence of IL-24 and GSI-I. Furthermore, the increased GSI-I and IL-24 in HepG2 cell was associated with downregulation of MMP-2, XIAP and VEGF. In the absence of treatment, HepG2 cells could migrate into the scratched space in 24 h. With IL-24 or GSI-I treatment, the wound was still open after 24 h. And the distance of the wound closure strongly correlated with the concentrations of IL-24 and GSI-I. Treatment of Notch-1 silenced HepG2 cells with 50 ng/mL IL-24 alone for 48 h induced cytotoxic effects very similar to those observed in non-silenced cells treated with GSI-I/IL-24 combination. Caspase-3/7 activity was increased in the presence of siNotch1 plus IL-24 treatment.

CONCLUSION: Down-regulation of Notch1 by GSI-I or siRNA combined with IL-24 can sensitize apoptosis and decrease the invasion and migration capabilities of HepG2 cells.

Keywords: Notch signaling pathway, Interleukin-24, γ-secretase inhibitor, Invasion, Migration, Hepatocellular carcinoma

Core tip: The down-regulation of Notch1 by γ-secretase inhibitor (GSI-I) or siRNA combined with interleukin (IL)-24, could sensitize apoptosis, increase expression of E-cadherin and decrease the invasion and migration capabilities of HepG2 cells. These results indicate for the first time that GSI-I/IL-24 combination might represent a novel and potentially effective tool for hepatocellular carcinoma treatment.