Exposure treated with 100 ?M of 4
Exposure of phosphatidylserine (PS) on the surface of the cell is a general and early marker of apoptosis. AnnexinV-FITC /PI double staining method confirmed apoptosis of KG1a treated-cells. The cells in the lower left quarter (Annx? /PI?) show viable cells, in the lower right quarter (Annx+ /PI-) show early apoptotic cells, in the upper left quarter (Annx- /PI-) show necrotic cells and in the upper right quarter (Annx+ /PI+) show late apoptotic cells. Flow cytometric analysis revealed that the apoptotic cell percentage in the control cells from 0.17% increased to 69.31% after 72 h of treatment (Fig. 4C). These results detected the occurrence of apoptosis in KG1a treated-cells.
Recent studies showed that chemotherapy agents can induce apoptosis in malignant cells using the increase in ROS level or decrease in ROS scavenging potency20. To assay ROS generation by treated KG1a myeloid cells with the 4 -HBTC, we evaluated intracellular peroxide levels using DCFH-DA staining. The KG1a cells were treated with 100 ?M of 4 -HBTC for 24-72 h. Then, cells incubated with DCFH-DA and the level of cellular ROS was measured by flow cytometry. As shown in Figure 4B, 4 -HBTC (30 ?M) enhanced ROS generation 23.82% and 36.03% after 48 and 72 h treatment, respectively, in compared to control cells.
Evaluation of stress oxidative parameters
In order to detect the effect of 4 -HBTC on the cellular redox status in the Leukemia stem cells, antioxidant defense system capabilities were evaluated according to appropriate methods reported in materials and methods. SOD and CAT play the essential role in the cellular antioxidant defense system. The activities of these enzymes (IU/mg of protein) in KG1a cells line were enhanced notably in 4 -HBTC treated cells for 24 h compared to controls (P< 0.001). However, it was observed a significant decrease in the activity of both enzymes after 48 and 72 h (Fig 2). Moreover, our conclusion showed that the formation of TBARS levels as the index of lipid peroxidation enhanced in the cells treated with 100 ?M 4 -HBTC after 24, 48 and 72 h compared to the control group. Diminution in total thiol levels in cells is a basic indicator of oxidative stress. There was an elevation in the total thiol (SH) level after 24 h and notably reduced after 48 and 72 h of treatment, compared with untreated groups.