Role of oxidative stress in the apoptosis of hepatocellular carcinoma induced by combination of arsenic trioxide and ascorbic acid.
OBJECTIVE
The present study was designed to determine the possible pathway underlying the enhancement of apoptosis induced by the combined use of arsenic trioxide (As(2)O(3)) and ascorbic acid (AA).


METHODS
The level of intracellular reactive oxygen species (ROS) was detected by means of flow cytometry analysis with an oxidation-sensitive fluorescent probe (6-carboxy-2',7' dichlorodihydrofluorescein diacetate) uploading. The activity of glutathione (GSH), glutathione peroxidase (GPx), and superoxide dismutase (SOD) were detected by biochemical methods. The mitochondrial membrane potential was measured by flow cytometry analysis with rhodamine 123 staining. Bcl-2, Bax, and p17 subunit of caspase-3 were analyzed using the Western blot method. The apoptosis rate was determined by flow cytometry with annexin-V/propidium iodide staining.


RESULTS
Compared with As(2)O(3) (2.0 micromol/L) treated alone, As(2)O(3) (2.0 micromol/L) in combination with AA (100 micromol/L) decreased intracellular GSH content from 101.30+/-5.76 to 81.91+/-3.12 mg/g protein, and increased ROS level from 127.61+/-5.12 to 152.60+/-5.88, which was represented by the 2, 7-dichlorofluorescein intensity. The loss of mitochondria membrane potential was increased from 1269.97+/-36.11 to 1540.52+/-52.63, which was presented by fluorescence intensity. The p17 subunit of caspase-3 expression was increased approximately 2-fold. However, SOD and GPx depletion and the ratio of Bcl-2 to Bax were equal to that of As2O3 treated alone (P>0.05). When the ROS scavenger, N-acetyl-L-cysteine, was added to As(2)O(3) and AA combined treatment group, the apoptosis rate decreased from 15.60 %+/-1.14% to 9.48%+/-0.67%, and the ROS level decreased from 152.60+/-5.88 to 102.77+/-10.25.


CONCLUSIONS
AA potentiated As(2)O(3)-induced apoptosis through the oxidative pathway by increasing the ROS level. This may be the result of depleting intracellular GSH. It may influence the downstream cascade following ROS, including mitochondria depolarization and caspase-3 activation. However, SOD and GPx depletion and the ratio of Bcl-2 to Bax influenced by As(2)O(3) was not found to be potentiated by AA.
Li
Jing-Jing
JJ
Department of Pharmacology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Tang
Qiang
Q
Li
Yuan
Y
Hu
Ben-Rong
BR
Ming
Zhang-Yin
ZY
Fu
Qin
Q
Qian
Jia-Qing
JQ
Xiang
Ji-Zhou
JZ
D000111:Acetylcysteine / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D000077237:Arsenic Trioxide; D001152:Arsenicals / Q000494:pharmacology*; D001205:Ascorbic Acid / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D004357:Drug Synergism; D016166:Free Radical Scavengers / Q000494:pharmacology; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology*; D018384:Oxidative Stress / Q000187:drug effects*; D010087:Oxides / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D000970:Antineoplastic Agents; D000975:Antioxidants; D001152:Arsenicals; D016166:Free Radical Scavengers; D010087:Oxides; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D001205:Ascorbic Acid; D000077237:Arsenic Trioxide; D000111:Acetylcysteine