2-methoxyestradiol-induced apoptosis in U937 cells through generation of reactive oxygen species

Objective To investigate the effect of 2-methoxyestradiol(2-ME) on U937 myeloid leukemia cell line and its mechanism.Methods The experiment was divided into control group(myeloid leukemia U937 cell in RPMI 1640 culture medium with equal DMSO),2-ME-treated group,NAC-treated group,and 2-ME+NAC-treated group.The cytotoxicity was analyzed by MTT assay.Apoptosis and cellular nitric oxide(NO) were detected by flow cytometry using annexin V and NO sensor dye.Superoxide anion was measured with a fluorescent plate reader by DHE.Results Viabilities of U937 cells treated with 2-ME(0.25,0.50,1.00,and 2.00 μmol·L-1) for 48 h were gradually reduced to 0.68±0.05,0.28±0.07,0.18±0.07,and 0.11±0.04,respectively.The differences were significant compared with control group(1.00±0.05)(P0.05).2-ME resulted in viability decrease in a dose-dependent manner.The apoptotic rates in 2-ME(2.00 μmol·L-1)-treated group were 4.64%±0.21%,9.86%±0.9%,14.62%±0.67%,and 19.49%±0.90% at 8,12,18,and 24 h time points,respectively,and significantly higher than that in control group(1.74%±0.08%)(P0.05).There were no significant differences of apoptotic rate between 2-ME-treated group and control group at 1,3,and 6 h time points(P0.05).2.00 μmol·L1 2-ME also significantly increased the mean fluorescence of NO sensor dye and superoxide anions in U937 cells compared with control group(P0.05).The percentage of NO positive cells increased from 0.52%±0.21% to 46.74%±0.15% after treatment for 1 h with 2.00 μmol·L-1 2-ME(P 0.05),whereas,there was no significant difference of apoptotic cells at this point between 2-ME group and control group(1.28%±0.07% vs 1.59%±0.12%,P0.05).An markedly increase in apoptotic cells was detected after 2-ME treatment for 3 h(6.78%±1.01% vs 1.59%±0.12%,P0.05).These results indicated that the generation of NO was earlier than apoptosis underwent.Furthermore,quenching of ROS with NAC protected leukemia cells from the cytotoxicity of 2-ME and prevented apoptosis induced by 2-ME.The viabilities and apoptotic rate of 2.00 μmol·L-1 2-ME-treated group were 0.47±0.02 and 13.87%±0.69%,respectively.The differences were significant compared with control group or 2-ME+NAC group(0.82±0.08 and 2.98%±0.19%,respectively)(P0.05).Conclusion 2-ME can induce apoptosis in U937 cells through generation of ROS.It is possible to use ROS-generation agents to enhance the antileukemic effect.