Change of Reactive Oxygen Species and Antioxidative Capacity on Nitric Oxide Induced Apoptosis in HL-60 Cells

This study was aimed to investigate the changes of reactive oxygen species and antioxidative capacity on nitric oxide induced apoptosis in HL-60 cells. By means of in vitro incubation of HL-60 cells with sodium nitroprusside (SNP), the growth inhibition was detected by MTT assay. Cell morphology was observed by transmission electronmicroscopy and light microscopy. The apoptosis was analyzed by DNA agarose gel electrophoresis, DNA content and Annexin-Ⅴ/PI labeling method. Reactive oxygen species (ROS) labeled with dihydrorhodamin 123 in cells was determinated by flow cytometry. The SNP-treated cells were examined for glutathione (GSH) level and activity of catalase (CAT), glutathione S-transferase (GST) and glutathione peroxidase (GPX). The results indicated that SNP could inhibit HL-60 cell growth. Cell apoposis was confirmed by typical cell morphology, DNA fragment, sub-G_1 phase and Annexin-Ⅴ/PI labeling method. HL-60 cell apoptosis was induced by SNP in a dosage- and time- dependent manner.After exposing to SNP at the concentration of 0.5-3.0 mmol/L for 48 hours, the mean fluorescence intensity of ROS in cells was significantly higher than those in groups control and potassium ferricyanide (PFC). During the apoptosis process, level of ROS in cells increased, levels of GSH, CAT,GPTand GPX decreased. The significant dose-effect relationship existed between the levels of ROS,CAT,GST,GPX and SNP dose. It is concluded that change of intracellular reactive oxygen species and antioxidative capacity are an important factors during the process of SNP-induced apoptosis in HL-60 cell.