Role of oxidative stress in the apoptosis of hepatocellular carcinoma induced by combination of arsenic trioxide and ascorbic acid

Aim:The present study was designed to determine the possible pathway under-lying the enhancement of apoptosis induced by the combined use of arsenictrioxide(As2O3)and ascorbic acid(AA).Methods:The level of intracellular reac-tive oxygen species(ROS)was detected by means of flow cytometry analysis withan oxidation-sensitive fuorescent probe(6-carboxy-2′,7′dichlorodihydrofluoresceindiacetate)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 analysiswith rhodamine 123 staining.Bcl-2,Bax,and p17 subunit of caspase-3 were ana-lyzed using the Western blot method.The apoptosis rate was determined by flowcytometry with annexin-V/propidium iodide staining.Results:Compared withAs2O3(2.0 μmol/L)treated alone,As2O3(2.0 μmol/L)in combination with AA(100μmol/L)decreased intracellular GSH content from 101.30+5.76 to 81.91+3.12 mg/gprotein,and increased ROS level from 127.61+5.12 to 152.60+5.88,which was rep-resented by the 2,7-dichlorofluorescein intensity.The loss of mitochondria mem-brane potential was increased from 1269.97±36.11 to 1540.52+52.63,which waspresented by fluorescence intensity.The p17 subunit of caspase-3 expressionwas increased approximately 2-fold.However,SOD and GPx depletion and theratio of Bcl-2 to Bax were equal to that of As2O3treated alone(P>0.05).When theROS scavenger,N-acetyl-L-cysteine,was added to As2O3and AA combined treat-ment 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.Conclusion:AApotentiated As2O3-induced apoptosis through the oxidative pathway by increas-ing the ROS level.This may be the result of depleting intracellular GSH.It mayinfluence the downstream cascade following ROS,including mitochondria depo-larization and caspase-3 activation.However,SOD and GPx depletion and theratio of Bcl-2 to Bax influenced by As2O3was not found to be potentiated by AA.