Effects of plumbagin on proliferation and apoptosis of hepatocellular carcinoma HepG2R cells resistant to sorafenib and its mechanism
10.13481/j.1671-587x.20180620
- Author:
Deqiang ZHU
1
Author Information
1. Department of Pathology and Pathophysiology, School of Basic Medical Sciences, Jinzhou Medical University
- Publication Type:Journal Article
- Keywords:
Apoptosis;
Cell proliferation;
HepG2;
Liver neoplasms;
Plumbagin;
Sorafenib
- From:
Journal of Jilin University(Medicine Edition)
2018;44(6):1223-1230
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To explore the effects of plumbagin (PLB) on the proliferation and apoptosis of hepatocellular carcinoma hepG2R cells resistant to sorafenib, and to clarify its mechanism. Methods: The hepatocellular carcinoma cells hepG2 were cultured in vitro and the models of sorafenib-resistant HepG2R cells were set up. MTT assay was used to identify the resistance factor and screen the concentration and time of drug. According to the results, the concentrations of sorafenib and PLB were confirmed as 5 μmol · L-1 and 2 μmol · L-1. The action time in various groups was 48 h. The HepG2R cells were randomly divided into control group, sorafenib (5 μmol · L-1) group, PLB (2 μmol · L-1) group, sorafenib (5 μmol · L-1) combined PLB (2 μmol · L-1) group (combination group). MTT assay was used to detect the cell vitality in various groups. The clone formation rates of cells in various groups were detected by clone formation assay. The apoptotic rates of cells in various groups were determined with Hoechst33342 assay and flow cytometry (FCM). The expression levels of cleaved Caspase-3, Bax and Bel-2 proteins in the cells in various groups were examined by Western blotting method, and the Bax/Bcl-2 ratio was calculated. The reactive oxygen (ROS) levels in the cells in various groups were detected by ROS detector kit. Results: As the increasing of concentrations of sorafenib, the cell vitalities of HepG2 and HepG2R cells were gradually decreased; the IC5o of sorafenib-resistant HepG2R cells was 4. 5 times as much as HepG2 cells (P<0. 05). The sensitivities of sorafenib resistant HepG2R cells were increased with the increasing of PLB concentrations and prolongation of time. Compared with control group, sorafenib group and PLB group, the clone formation rate of the cells in combination group was decreased (P<0. 05 or P<0. 01). The Hoechst 33342 assay results showed the nuclei were lightly stained; a few of the nuclei in sorafenib group and PLB group were strongly stained and bright; while in combination group, the nuclei were mostly stained and chromatin was condensed and bright. The FCM results showed that compared with control group, sorafenib group and PLB group, the apoptotic rate of cells in combination group was significantly increased (P< 0. 05 or P< 0. 01). The Western blotting results showed that the expression level of cleaved Caspase-3 in the cells and the Bax/Bcl -2 ratio in combination group were increased significantly (P<0. 05 or P<0. 01). The ROS level in the cells in combination group was higher than those in the other groups (P< 0.05 or P<0. 01). Conclusion: PLB can improve the resistance of hepartocellular carcinoma to sorafenib and the mechanism may be related to increasing the ROS level.
