Pro-oxidative Effect Of Xanthohumol In HL-60 Cells

Objective:To explore the reactive oxygen species (ROS) related mechanisms of xanthohumol (XN) bioactivity.Methods:The human promyelocytic leukemia cells HL-60 cell line was used as a study model. The cytotoxicity of xanthohumol on HL-60 cells were detected by Trypan Blue Test. Flow cytomitry techniques were used to detect apoptosis (cells were stained by Annexin-V FITC/PI), cell cycle (stained by PI) and ROS generation (stained by 2',7'-dichlorofluore-seein-diacetate, DCFH-DA). Furthermore, the lipid peroxidation and the intracellular levels of total antioxidant capacity after 24 hours were measured with kits.Results:The cell viability was 96.0% to control when treated with XN 10μM for 24 h, and which decreased rapidly to 76.0% and 14.1% when the concentration of xanthohumol up to 15 and 20μM respectively. This indicated that xanthohumol exhibited a strong cytotoxicity effect on HL-60 cells. xanthohumol induced apoptosis at 10μM and 15μM, the apoptosis rates were 9.8% and 12.5% respectively. xanthohumol induced cell cycle arrested at G1 phase. ROS were induced by all the xanthohumol treatments (0.1,1 and 10μM) at 30 min or 2 h. while the ROS levels restored to the same as control group under the lower concentrations (0.1,1μM) treated after 24 h except 10μM group, which kept on exhibiting higher ROS level. The NADPH oxidase inhibitor, Diphenyleneiodonium (DPI), was used to treat cells with different concentrations of xanthohumol together, and the ROS levels were reduced in all the tested group (0.1,1, and 10μM), which indicated that NADPH xoidase is involved in the ROS generation of xanthohumol treated HL-60 cells. After treated with 10μM for 24 h, the lipid peroxidation of cell lysate was significantly higher than the control group, while the results of 0.1 and 1μM treatment groups displayed no difference with the control. This is consistant with the ROS data. However, there no significantly changes were found in the cellular total antioxidant capacity.Conclusions:Xanthohumol significantly influenced the ROS levels in HL-60 cells, induced apoptosis and blocked the cell cycle at G1 phase. A short term treatment with xanthohumol can cause ROS generation in HL-60 cells, while the ROS level was restored after prolonged treatment in lower concentrations (0.1 and 1μM), and the high concentration group maintain high levels of ROS. Further more, we found that NADPH oxidase is one of the intracellular sources of ROS induced by xanthohumol.