2-methoxyestradiol通过反应性氧基对U937白血病细胞的凋亡诱导作用

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

目的:探讨2-ME诱导U937白血病细胞凋亡的作用和机制。方法:实验分为白血病细胞株U937细胞含有等量DMSO RPMI 1640培养液的对照组、2-ME处理组、NAC处理组和2-ME+NAC处理组。MTT测定评价对U937细胞毒作用;采用Annexin V和NO染料标记细胞,流式细胞术检测细胞内NO生成和细胞凋亡;DHE测定细胞内超氧阴离子。结果:不同浓度2-ME(0.25、0.50、1.00、和2.00μmol.L-1)处理U937细胞48 h后,U937细胞活性均较对照组明显减少(P<0.05),随着2-ME浓度的增高,U937细胞的生存率逐渐减低,呈剂量依赖性。2-ME(2.00μmol.L-1)处理U937细胞8、12、18和24 h后的细胞凋亡率均高于对照组(P<0.05),随着处理时间的延长,细胞凋亡率逐渐升高;而2-ME处理U937细胞1、3及6 h后的细胞凋亡率与对照组比较差异均无显著性(P>0.05)。2-ME(2.00μmol.L-1)处理U937细胞产生NO荧光值显著增加,超氧阴离子为1.21±0.02,与对照组比较差异具有显著性(P<0.05)。2-ME处理U937细胞1 h时,细胞NO阳性率是46.74%±0.15%,与对照组(0.52%±0.21%)比较差异具有显著性(P<0.05);而细胞凋亡率(1.28%±0.07%)与对照组(1.59%±0.12%)比较差异无显著性(P>0.05);2-ME处理U937细胞3 h后,细胞凋亡率高于对照组(P<0.05),提示U937细胞NO的产生早于细胞凋亡。用NAC抑制反应性氧基(ROS)可保护U937细胞逃避2-ME的细胞毒作用和避免2-ME诱导的细胞凋亡。2-ME(2.00μmol.L-1)处理组U937细胞活性和细胞凋亡率分别是0.47%±0.02%和13.87%±0.69%,与对照组和2-ME+NAC处理组(0.82%±0.08%及2.98%±0.19%)比较差异均具有显著性(P<0.05)。结论:2-ME通过ROS诱导U937细胞凋亡,提示产生ROS的物质可能增强抗白血病作用。

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） （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 ofapoptotic 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 2.00μmol · 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.