关键词: 2-methoxyestradiol, 凋亡, 反应性氧基, 一氧化氮, 超氧化物离子

Abstract: Abstract: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 mol•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) (P<0.05). 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％) (P<0.05). There were no significant differences of apoptotic rate between 2-ME-treated group and control group at 1, 3, and 6 h time points（P>0.05）. 2.00 μmol•L^-1 2-ME also significantly increased the mean fluorescence of NO sensor dye and superoxide anions in U937 cells compared with control group (P<0.05). 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％, P>0.05). An markedly increase in apoptotic cells was detected after 2-ME treatment for 3 h(6.78％±1.01％ vs 1.59％±0.12％, P<0.05).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) (P<0.05). 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.

Key words: 2-methoxyestradiol, apoptosis, reactive oxygen species, nitric oxide, superoxide anion