Dihydroartemisinin induces endoplasmic reticulum stress-mediated apoptosis in HepG2 human hepatoma cells.
OBJECTIVE
Previous studies showed that dihydroartemisinin (DHA) possessed antitumor activity in many human tumor cells through the induction of apoptosis. The aim of this study was to investigate the effects of DHA on apoptosis in the human hepatocellular carcinoma cell line HepG2 and the possible molecular mechanisms involved.


METHODS
The inhibitory effect of DHA on HepG2 cells was measured by MTT assay. The percentage of apoptotic cells was detected by flow cytometry with double staining of fluorescein isothiocyanate-annexin V/propidium iodide. The intracellular production of reactive oxygen species (ROS) and intracellular Ca2+ concentration ([Ca2+]i) were detected by fluorescence spectrophotometry. Protein expression of GADD153, Bcl-2 and Bax in HepG2 cells was examined by Western blot and immunocytochemistry.


RESULTS
DHA significantly inhibited proliferation of HepG2 cells in a dose- and time-dependent manner. The apoptosis rates in HepG2 cells treated with 0, 50, 100 and 200 μmol/L DHA for 24 hours were 2.53 ± 0.88%, 24.85 ± 3.63%, 35.27 ± 5.92% and 48.53 ± 7.76%, respectively. Compared with the control group, DHA significantly increased ROS generation and [Ca2+]i level (P <0.05), with the generation of ROS preceding the increase in [Ca2+]i. An increase in GADD153 and Bax expression and a decrease in Bcl-2 were observed in DHA-treated cells. Pretreatment with the antioxidant N-acetylcysteine could attenuate the effects of DHA in the experiments.


CONCLUSIONS
DHA could inhibit proliferation and induce apoptosis in HepG2 cell lines through increasing the intracellular production of ROS and [Ca2+]i. Endoplasmic reticulum stress-induced apoptosis may contribute to this effect by regulating the expression of GADD153, proapoptotic Bax, and antiapoptotic Bcl-2.
Institute of Life Science, Chongqing Medical University, Chongqing, PR China
Xiaoling
X
Gao
Ziguo
Z
Luo
Tingxiu
T
Xiang
Kejian
K
Wang
Jian
J
Li
Pilong
P
Wang
D017304:Annexin A5 / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D037621:Artemisinins / Q000494:pharmacology*; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D004305:Dose-Response Relationship, Drug; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D056945:Hep G2 Cells / Q000187:drug effects; D006801:Humans; D007150:Immunohistochemistry; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D051742:Transcription Factor CHOP / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D017304:Annexin A5; D000970:Antineoplastic Agents; D037621:Artemisinins; C494826:BAX protein, human; C497249:DDIT3 protein, human; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; D051742:Transcription Factor CHOP; C039060:artenimol