Casticin-induced apoptosis involves death receptor 5 upregulation in hepatocellular carcinoma cells

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-dimethylthi- azol-2-yl]-2...

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Published in:World journal of gastroenterology : WJG 2011-10, Vol.17 (38), p.4298-4307
Main Authors: Yang, Jun, Yang, Yun, Tian, Li, Sheng, Xi-Feng, Liu, Fei, Cao, Jian-Guo
Format: Article
Language:English
Subjects:
Antimetabolites, Antineoplastic - pharmacology
Antimetabolites, Antineoplastic - therapeutic use
Antineoplastic Agents - pharmacology
Antineoplastic Agents - therapeutic use
Apoptosis - drug effects
Carcinoma, Hepatocellular - drug therapy
Carcinoma, Hepatocellular - metabolism
Carcinoma, Hepatocellular - pathology
Caspases - metabolism
Cell Line, Tumor
DNA Fragmentation
Flavonoids - chemistry
Flavonoids - pharmacology
Flavonoids - therapeutic use
Fluorouracil - pharmacology
Fluorouracil - therapeutic use
Glutathione - metabolism
Humans
Liver Neoplasms - drug therapy
Liver Neoplasms - metabolism
Liver Neoplasms - pathology
Molecular Structure
Original
Reactive Oxygen Species - metabolism
Receptors, TNF-Related Apoptosis-Inducing Ligand - metabolism
人肝癌细胞
死亡受体
流式细胞仪分析
牡荆
紫花
细胞凋亡
诱导凋亡
酶联免疫吸附试验
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Summary: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-dimethylthi- azol-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.
ISSN:1007-9327
2219-2840
DOI:10.3748/wjg.v17.i38.4298