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

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2.00 μmol?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 (2.00 μmol?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 (2.00 μmol?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 (2.00 μmol?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。

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)(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·L1 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.