Original Article
Copyright ©2011 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Oct 14, 2011; 17(38): 4298-4307
Published online Oct 14, 2011. doi: 10.3748/wjg.v17.i38.4298
Casticin-induced apoptosis involves death receptor 5 upregulation in hepatocellular carcinoma cells
Jun Yang, Yun Yang, Li Tian, Xi-Feng Sheng, Fei Liu, Jian-Guo Cao
Jun Yang, Department of Pathology, The Third Xiangya Hospital of Central South University, Changsha 410013, Hunan Province, China
Yun Yang, Li Tian, Xi-Feng Sheng, Fei Liu, Jian-Guo Cao, Laboratory of Medical Engineering, Medical College, Hunan Normal University, Changsha 410013, Hunan Province, China
Author contributions: Yang J, Yang Y, Tian L and Liu F performed the majority of experiments; Sheng XF provided the vital reagents and analytical tools and was involved in editing the manuscript; Cao JG designed the study and wrote the manuscript.
Supported by The Scientific Research Project of Hunan Provincial Administration Bureau of Traditional Chinese Medicine, No. 2010081; Scientific Research Project of Hunan Provincial Health Department, No. B2010-030; Major Projects of Scientific Research of Hunan Provincial Department of Education, No. 09A054
Correspondence to: Jian-Guo Cao, Professor, Laboratory of Medical Engineering, Medical College, Hunan Normal University, Changsha 410013, Hunan Province, China. caojianguo2005@yahoo.com.cn
Telephone: +86-731-8912434 Fax: +86-731-8912417
Received: January 26, 2011
Revised: June 9, 2011
Accepted: June 16, 2011
Published online: October 14, 2011
Abstract

AIM: To investigate the apoptotic activities of casticin in hepatocellular carcinoma (HCC) cells and its molecular mechanisms.

METHODS: PLC/PRF/5 and Hep G2 cell lines were cultured in vitro and the inhibitory effect of casticin on the growth of cells was detected by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolim bromide (MTT) assay. The apoptotic cell death was examined using the cell apoptosis enzyme linked immunosorbent assay (ELISA) detection kit, flow cytometry (FCM) after propidium iodide (PI) staining and DNA agarose gel electrophoresis. The caspase activities were measured using ELISA. Reactive oxygen species (ROS) production was evaluated by FCM after dichlorodihydrofluorescein diacetate (DCFH-DA) probe labeling. Intracellular glutathione (GSH) content was measured using a glutathione assay kit. The expression of death receptor (DR)4 and DR5 proteins was analyzed by Western blotting and FCM.

RESULTS: Casticin significantly inhibited the growth of human HCC (PLC/PRF/5 and Hep G2) cells in a dose-dependent manner (P < 0.05). Casticin increased the percentage of the sub-G1 population in HCC cells in a concentration-dependent manner. The potency of casticin to PLC/PRF/5 cells was higher than that of 5-flurouracil (26.8% ± 4.8% vs 17.4% ± 5.1%) at 10 μmol/L for 24 h. Casticin increased the levels of Histone/DNA fragmentation and the levels of active caspase-3, -8 and -9 in a concentration-dependent manner (P < 0.05). Treatment with 30 μmol/L casticin for 24 h resulted in the formation of a DNA ladder. Casticin reduced the GSH content (P < 0.05), but did not affect the level of intracellular ROS in PLC/PRF/5 and Hep G2 cells. The thiol antioxidants, acetylcysteine (NAC) and GSH restored GSH content and attenuated casticin-induced apoptosis. In contrast, the nonthiol antioxidants, butylated hydroxyanisole and mannitol failed to do so. In the HCC cells treated with casticin for 24 h, DR5 protein level was increased. The expression of DR5 protein induced by casticin was inhibited by NAC. Pretreatment with DR5/Fc chimera protein, a blocking antibody, effectively attenuated the induction of apoptosis by casticin.

CONCLUSION: Casticin-induced apoptosis of HCC cells is involved in GSH depletion and DR5 upregulation.

Keywords: Hepatocellular carcinoma, Casticin, Glutathione, Death receptor 5