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Aim:The present study was designed to determine the possible pathway under-lying the enhancement of apoptosis induced by the combined use of arsenictrioxide(As_2O_3)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 withAs_2O_3(2.0 μmol/L)treated alone,As_2O_3(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 As_2O_3 treated alone(P>0.05).When theROS scavenger,N-acetyl-L-cysteine,was added to As_2O_3 and 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 As_2O_3-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 As_2O_3 was not found to be potentiated by AA.
Aim: The present study was designed to determine the possible pathway under-lying the enhancement of apoptosis induced by the combined use of arsenictrioxide (As_2O_3) 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 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 ana-lyzed using the Western blot method. apoptosis rate was determined by flowcytometry with annexin -V / propidium iodide staining combined with AA (100μmol / L) decreased the intracellular GSH content from 101.30 + 5.76 to 81.91 + 3.12 mg / L compared with that of As_2O_3 (2.0 μmol / L) gprot ein, 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 increased increased from 1269.97 ± 36.11 to 1540.52 + 52.63, which waspresented by fluorescence intensity. The p17 subunit of caspase-3 expression was increased by approximately 2-fold. However, SOD and GPx depletion and the ratio of Bcl-2 to Bax were equal to that of As_2O_3 treated alone (P> 0.05) .When the ROS scavenger, N-acetyl-L-cysteine, was added to As_2O_3 and 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 As_2O_3-induced apoptosis through the oxidative pathway by increas-ing the ROS level. This may be the result of depleting intracellular GSH. At may may inhibit the downstream cascade following ROS, including mitochondria depo-larization and caspase-3 activation. , SOD and GPx depletion and theratio of Bcl-2 to Bax influenced by As_2O_3 was not found to be potentiated by AA.