Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jan 21, 2015; 21(3): 854-861
Published online Jan 21, 2015. doi: 10.3748/wjg.v21.i3.854
Antiproliferative effects of cinobufacini on human hepatocellular carcinoma HepG2 cells detected by atomic force microscopy
Qing Wu, Wei-Dong Lin, Guan-Qun Liao, Li-Guo Zhang, Shun-Qian Wen, Jia-Ying Lin
Qing Wu, Wei-Dong Lin, Guan-Qun Liao, Li-Guo Zhang, Shun-Qian Wen, Jia-Ying Lin, Department of General Surgery, the Affiliated Foshan Hospital of Southern Medical University, Foshan 528000, Guangdong Province, China
Author contributions: Wu Q and Lin WD designed the research and wrote the paper; Wu Q and Liao GQ performed the research; Zhang LG, Wen SQ and Lin JY analyzed the data.
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: Wei-Dong Lin, Chief, Professor, Department of General Surgery, the Affiliated Foshan Hospital of Southern Medical University, No. 78 Weiguo Road, Foshan 528000, Guangdong Province, China. 13433123361@163.com
Telephone: +86-757-88032005 Fax: +86-757-88032005
Received: May 13, 2014
Peer-review started: May 13, 2014
First decision: July 9, 2014
Revised: July 30, 2014
Accepted: September 18, 2014
Article in press: September 19, 2014
Published online: January 21, 2015
Processing time: 252 Days and 8.6 Hours
Abstract

AIM: To investigate the antiproliferative activity of cinobufacini on human hepatocellular carcinoma HepG2 cells and the possible mechanism of its action.

METHODS: HepG2 cells were treated with different concentrations of cinobufacini. Cell viability was measured by methylthiazolyl tetrazolium (MTT) assay. Cell cycle distribution was analyzed by flow cytometry (FCM). Cytoskeletal and nuclear alterations were observed by fluorescein isothiocyanate-phalloidin and DAPI staining under a laser scanning confocal microscope. Changes in morphology and ultrastructure of cells were detected by atomic force microscopy (AFM) at the nanoscale level.

RESULTS: MTT assay indicated that cinobufacini significantly inhibited the viability of HepG2 cells in a dose-dependent manner. With the concentration of cinobufacini increasing from 0 to 0.10 mg/mL, the cell viability decreased from 74.9% ± 2.7% to 49.41% ± 2.2% and 39.24% ± 2.1% (P < 0.05). FCM analysis demonstrated cell cycle arrest at S phase induced by cinobufacini. The immunofluorescence studies of cytoskeletal and nuclear morphology showed that after cinobufacini treatment, the regular reorganization of actin filaments in HepG2 cells become chaotic, while the nuclei were not damaged seriously. Additionally, high-resolution AFM imaging revealed that cell morphology and ultrastructure changed a lot after treatment with cinobufacini. It appeared as significant shrinkage and deep pores in the cell membrane, with larger particles and a rougher cell surface.

CONCLUSION: Cinobufacini inhibits the viability of HepG2 cells via cytoskeletal destruction and cell membrane toxicity.

Keywords: Cinobufacini; Cell viability; Atomic force microscopy; HepG2 cells; Hepatocarcinoma

Core tip: Cinobufacini is effective against hepatocarcinoma. However, its mechanism of action has not been determined. The present study investigated the effect of cinobufacini on HepG2 cells and the alterations in cell morphology and membrane ultrastructure. We used atomic force microscopy (AFM) to study the changes in cell membrane ultrastructure induced by cinobufacini. We demonstrated that AFM is a useful tool in verifying cell response to cinobufacini treatment. We observed nuclear morphology and actin filaments in the cytoskeleton by laser scanning confocal microscopy. The cellular changes allowed us to understand better the biophysical functions of HepG2 cells induced by cinobufacini.