三氧化二砷诱导HL-60细胞凋亡过程中ROS、NF-κB和C-IAP2的变化

Alteration of Reactive Oxygen Species,NF-kB and C-IAP2 in Apoptosis of HL-60 Cell Induced by Arsenic Trioxide

背景与目的:探讨As2O3(arsenic trioxide,ATO)对HL-60细胞凋亡过程中细胞内活性氧(reactive oxygen species,ROS)水平、NF-κB(nuclear factor kappa B),及C-IAP2(cellular inhibitor of apoptosis proteins 2)的影响。材料与方法:以7.5μmol/L As2O3单独应用及与500μmol/L N-乙酰半胱氨酸(N-acetyl-L-cysteine,NAC)联合作用于HL-60细胞12、24 h后,流式细胞术检测HL-60细胞ROS的产生量;Western blot法检测NF-κB P65核蛋白的变化情况;半定量RT-PCR法检测C-IAP2的相对表达量。结果:7.5μmol/L的As2O3作用HL-60细胞12、24 h后细胞内ROS的生成增加,与阴性对照比较,差异具有统计学意义(P<0.05)。NF-κBP65核蛋白的相对含量分别为49.3%±4.4%和23.1%±2.1%,C-IAP2的相对表达分别为72.9%±5.8%和59.3%±4.4%,较对照组均明显降低(P<0.05)。500μmol/L NAC和7.5μmol/L As2O3共同作用HL-60细胞12、24 h后细胞ROS生成量相对减少,与AS2O3单独作用组比较,差异具有统计学意义(P<0.05);NF-κB P65核蛋白的相对含量分别为65.4%±4.9%和37.1%±3.4%,C-IAP2的相对表达量分别为81.1%±5.8%和73.7%±4.9%。较AS2O3单独作用组均明显增加(P<0.05)。结论:As2O3诱导HL-60细胞凋亡过程中,细胞内ROS生成增加,抑制NF-κB活性,同时下调其靶基因C-IAP2等的表达;NAC能阻断As2O3诱导HL-60细胞凋亡过程中ROS的生成,部分阻断了NF-κB活性的抑制。

BACKGROUND AND AIM: To investigate the alteration of reactive oxygen species(ROS) level,the activity of nuclear factor kappa B(NF-κB)and the expression of C-IAP2 in apoptosis of HL-60 cells induced by As2O3(arsenic trioxide,ATO).MATERIALS AND METHODS: HL-60 cells were treated with 7.5 μmol/L As2O3 alone or together with 500 μmol/L N-acetyl-L-cysteine(NAC) for 12 and 24 h.Intracellular ROS level was measured by flow cytometry(FCM),the activity of NF-κB p65 was determined by Western blot and the expression of C-IAP2 mRNA was determined by semi-quantitative RT-PCR.RESULTS: After treatment with 7.5 μmol/L As2O3 for 12 and 24 h,the level of ROS increased obviously,and the relative amount of NF-κB p65 were 49.3%±4.4% and 23.1%±2.1%,and the relative expressions of C-IAP2 mRNA were 72.9%±5.8% and 59.3%±4.4%.After co-treatment of 7.5 μmol/L As2O3 and 500 μmol/L NAC for 12 and 24 h in HL-60 cells,the level of ROS was decreased,the relative amount of NF-κB p65 were 65.4%±4.9% and 37.1%±3.4%,and the relative expressions of C-IAP2 mRNA were 81.1%±5.8% and 73.7%±4.9%.CONCLUSION: As2O3 could increase the level of ROS in HL-60,inhibited the activity of NF-κB and down-regulated the expression of C-IAP2 mRNA.Moreover,co-treatment of As2O3 and NAC could protect HL-60 cells from apoptosis through decreasing reactive oxygen species(ROS),prohibiting partly the suppression of the activity ofNF-κB and the expression of C-IAP2 mRNA.