Disulfiram联合铜对B淋巴细胞白血病细胞株增殖与凋亡影响机制探讨  

Disulfiram combination with copper inhibits proliferation and induces apoptosis of B lineage acute lymphoblastic leukemia cell line:nalm6 cells and its mechanism

作　　者：邓漫漫 蒋治武[2] 李洁[1] 陈凯[1] 李鹏[2] 周淑芸[1] 徐兵[1] 

机构地区：[1]南方医科大学南方医院血液科,广东广州510515 [2]中国科学院广州生物医院与健康研究院再生生物学重点实验室,广东广州510000

出　　处：《中华肿瘤防治杂志》2015年第11期840-845,共6页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(81070425;81400104);广东省科技计划(2009B050700028)

摘　　要：目的研究Disulfirum(DS)联合Cu(DS/Cu)对B淋巴细胞白血病(B-ALL)细胞株(nalm6细胞)增殖及凋亡的影响,并探讨其机制。方法 CCK8法检测不同浓度DS/Cu对Nalm6的增殖抑制作用,AnnexinⅤ/PI法检测不同浓度DS/Cu对Nalm6细胞的诱导凋亡作用,JC-1法检测不同浓度DS/Cu处理nalm6后线粒体膜电位的变化,蛋白质印迹法检测药物处理后线粒体通路相关蛋白(Bcl-2和Bcl-xl)的表达变化。结果 0.5μmol/L Cu作用24h后对nalm6细胞的增殖抑制率为(6.39±4.93)%,与对照组比较差异无统计学意义,t=-2.244,P=0.154。而不同浓度的(0.05、0.1、0.2、0.4、0.8、1.6、3.2和6.4μmol/L)DS联合0.5μmol/L Cu对nalm6细胞均具有显著的增殖抑制作用,抑制率分别为(22.29±6.69)%、(48.66±11.58)%、(50.83±12.61)%、(59.24±9.43)%、(62.74±9.17)%、(66.7±7.63)%、(72.13±7.94)%和(77.86±5.43)%,与对照组比较差异均有统计学意义,P值均<0.05;24h的IC50为(0.18±0.08)μmol/L。AnnexinⅤ/PI检测细胞凋亡结果显示,0.5μmol/L Cu作用24h后nalm6细胞的凋亡比例为(7.82±5.13)%,与对照组(8.34±6.23)%比较差异无统计学意义,t=0.112,P=0.916。而不同浓度的DS/Cu对nalm6细胞均具有显著的诱导凋亡作用,0.025、0.05、0.1、0.2和0.5μmol/L的DS联合Cu(0.5μmol/L)作用24h后的凋亡率分别为(17.84±7.68)%、(31.39±5.86)%、(60.41±13.87)%、(69.26±13.29)%和(81.37±12.72)%,与对照组比较差异有统计学意义,P值均<0.05。JC-1法检测线粒体膜电位变化结果显示,单药Cu(0.5μmol/L)组的JC-1多聚体/单体比值为1.12±0.09,与对照组(0.90±0.13)比较差异无统计学意义,t=-2.442,P=0.071。不同浓度的(0.025、0.05、0.1、0.2和0.5μmol/L)DS联合0.5μmol/L Cu组处理nalm6细胞12h后,线粒体膜电位水平明显降低,表现为JC-1多聚体/单体比值随浓度的增加而逐渐降低,分别为1.4±0.32、0.32±0.13、0.14±0.09、0.1±0.02和0.06±0.005,与对照组比较差异有统计学意义,P值均<0.001。蛋白质印迹法显示,DS/Cu�OBJECTIVE To investigate the effect of Disufiram combination with copper on cell proliferation and apoptosis and its molecule mechanism.METHODS The nalm6 cells were treated with various concentrations of DS/Cu for24 h,the inhibitory ratio was measured by CCK8 assay,the apoptosis percent was measured by flow cytometry.The JC-1assay was used to measure the change of the mitochondrial membrane potential.Western blot were used to analyze Bcl-2and Bcl-xl expression after treatment with CON,Cu,DS and DS/Cu.RESULTS The inhibition ratio of 0.5μmol/L Cu on nalm6 cells was（6.39±4.93）%,compared with CON group,there was no statistical difference between two groups（t=-2.244,P=0.154）.However,various concentrations（0.05,0.1,0.2,0.4,0.8,1.6,3.2and 6.4μmol/L）of DS with0.5μmol/L Cu had significantly inhibitory effect,and all the differences had statistical significance（P0.05）,and the specifical inhibition ratio were respectively（22.29±6.69）%,（48.66±11.58）%,（50.83±12.61）%,（59.24±9.43）%,（62.74±9.17）%,（66.7±7.63）%,（72.13±7.94）% and（77.86±5.43）%,with the 50% inhibitory concentration（IC50）values of（0.18±0.08）μmol/L.The results of Annexin Ⅴ/PI double staining showed that there was no significantly difference between CON（8.34±6.23）%and 0.5μmol/L Cu group（7.82±5.13）%（t=0.112,P=0.916）,while,after the treamtment of 0.025,0.05,0.1,0.2and 0.5μmol/L DS with 0.5μmol/L Cu for 24 hon nalm6cells,the percentage was significantly increased（P0.05）respectively（17.84±7.68）%,（31.39±5.86）%,（60.41±13.87）%,（69.26±13.29）%,（81.37±12.72）%.The results of mitochondrial membranes potential detecting by the JC-1staining indicated that JC-1polymer/monomer ratio of Cu（0.5μmol/L）on nalm6 cells was（1.12±0.09）,and CON group was（0.90±0.13）,with no statistical difference between two groups（t=-2.442,P=0.071）.However,treated with various concentrations DS/Cu could result in significant decrease of motichondrial membrane potential with P0

关 键 词：双硫仑 铜 nalm6 线粒体通路 急性B淋巴细胞白血病 

分 类 号：R733.71[医药卫生—肿瘤]