Role of oxidative stress in the apoptosis of hepatocellular carcinoma induced by combination of arsenic trioxide and ascorbic acid
Role of oxidative stress in the apoptosis of hepatocellular carcinoma induced by combination of arsenic trioxide and ascorbic acid
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DOI：
10.1111/j.1745-7254.2006.00345.x
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发表时间：
2006-08-01
影响因子：
8.2
通讯作者：
Xiang, Ji-zhou
中科院分区：
文献类型：
--
作者：
Li, Jing-jing;Tang, Qiang;Xiang, Ji-zhou
目的：为探讨三氧化二砷（As 2 O3）和抗坏血酸（AA）联合应用诱导细胞凋亡的可能途径，采用流式细胞术检测细胞内活性氧（ROS）水平，以6-羧基-2 '，7'二氯二氢荧光素二乙酸酯（6-carboxy-2 '，7' dichlorodihydrofluorescein diacetate，AA）为氧化敏感荧光探针。生化法检测谷胱甘肽（GSH）、谷胱甘肽过氧化物酶（GPx）、超氧化物歧化酶（SOD）活性。采用流式细胞仪罗丹明123染色法测定线粒体膜电位。Western blot法检测Bcl-2、Bax和caspase-3的p17亚基. Annexin-V/propidium iodide染色流式细胞仪检测细胞凋亡率。与As 2 O3相比（2.0 μ mol/L）单独处理，As 2 O3（2.0 μ mol/L）与AA联合（100 μ mol/L）使细胞内GSH含量从101.30 +/- 5.76降至81.91 +/- 3.12 mg/g蛋白，并将ROS水平从127.61 +/-5.12增加到152.60 +/-5.88，其由2，7-二氯荧光素强度表示。线粒体膜电位的损失由1269.97 ± 36.11增加到1540.52 ± 52.63，以荧光强度表示。caspase-3表达的p17亚基增加约2倍。As_2O_3处理后，SOD、GPx下降，Bcl-2/Bax比值下降，但与As_2O_3单独处理组相比，差异无显著性（P > 0.05）。当As 2 O3和AA联合处理组加入ROS清除剂N-乙酰-L-半胱氨酸时，细胞凋亡率从15.60% ± 1.14%下降到9.48% ± 0.67%，ROS水平从152.60 ± 5.88下降到102.77 ± 10.25。AA通过增加ROS水平，增强As 2 O3诱导的细胞凋亡。这可能是细胞内GSH耗尽的结果。它可能影响ROS后的下游级联反应，包括线粒体去极化和caspase-3激活。AA对As（2）O（3）引起的SOD和GPx的耗竭及Bcl-2/Bax比值的影响无明显增强作用。
Aim: The present study was designed to determine the possible pathway underlying the enhancement of apoptosis induced by the combined use of arsenic trioxide (As2O3) 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 p 17 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 As2O3 (2.0 mu mol/L) treated alone, As2O3 (2.0 mu mol/L) in combination with AA (100 mu mol/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 As2O3 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.Conclusion: AA potentiated As2O3-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.