甲氧胺联合手霉素对白血病U937细胞凋亡的影响  

Effects of Manumycin Combined with Methoxyamine on Apoptosis in Myeloid Leukemia U937 Cells

作　　者：佘妙容[1] 郭坤元[1] 牛新清[1] 卢晓珣[1] 

机构地区：[1]南方医科大学珠江医院血液内科,广东广州510282

出　　处：《癌症》2008年第8期835-839,共5页Chinese Journal of Cancer

基　　金：广东省自然科学基金项目(No.7001196);广东省医学科研基金项目(No.A2006020)~~

摘　　要：背景与目的:DNA损伤修复对细胞生存很重要。我们既往的研究发现手霉素可诱导肿瘤细胞出现DNA损害反应。因此,碱基切除修复抑制剂甲氧胺(methoxyamine)有可能增强手霉素(manumycin)的抗肿瘤作用。为此,本实验研究甲氧胺对手霉素诱导白血病细胞凋亡的影响,探讨联合甲氧胺和手霉素诱导白血病细胞凋亡过程中线粒体凋亡途径的变化。方法:不同浓度手霉素和甲氧胺处理U937细胞48h后,MTT细胞毒性测定U937细胞存活率,软琼脂法检测细胞的克隆形成。应用流式细胞术检测细胞凋亡。免疫印迹技术检测细胞色素C、Caspase-9、聚腺苷核糖聚合酶(poly-adenosyl ribose polymerase,PARP)的表达。中位效应方法评价两药的相互作用。结果:甲氧胺使手霉素的剂量反应曲线向左边移动,结合指数(CI)<1(P<0.05)。1μmol/L手霉素、5mmol/L甲氧胺和联合两药处理U937细胞,相对于对照组的克隆形成分别是0.3641±0.0463,0.7541±0.0379,0.0473±0.0024(联合用药组与对照组、手霉素组或甲氧胺组相比,P<0.05);进一步发现,联合两药协同诱导细胞凋亡,引起Annexin V荧光值增加,阳性细胞增加,分别是(2.34±0.30)%、(8.80±0.95)%、(2.21±0.19)%、(13.37±0.91)%,联合用药组与对照组、手霉素组或甲氧胺组相比,差异有统计学意义(P<0.05)。联合甲氧胺和手霉素增强细胞色素C从线粒体释放到细胞浆,Caspase-9激活,PARP特异性裂解。结论:甲氧胺增强手霉素通过线粒体凋亡途径诱导U937细胞凋亡,提示甲氧胺联合FTIs和DNA修复抑制剂治疗白血病的可能性。BACKGROUND ＆ OBJECTIVE： Repair of DNA damage is important to cell survival. Our previous study showed DNA damage response induced by manumycin in cancer cells. We hypothesized that methoxyamine, an inhibitor of base-excision repair, can enhance the antineoplastic effect of manumycin. This study was to investigate apoptosis induced by manumycin combined with methoxyamine in myeloid leukemia cell line U937, and to explore the role of mitochondrial apoptotic pathway in apoptosis induction of the two drugs. METHODS： U937 cells were treated with various concentrations of manumycin and/or methoxyamine for 48 h. The celt viability was analyzed by MTT assay. Colony formation was evaluated by soft agar clonogenic assay. Cell apoptosis was investigated by flow cytometry. Protein expressions of cytochrome c, caspase-9, and poly ADP-ribose polymerase （PARP） were determined by Western blott. RESULTS： The dose-response curve of manumycin was shifted to the left after addition of methoxyamine. The combination index （CI） was less than 1 （P〈0.05） in U937 cells （P〈 0.05）, indicating a synergistic effect of manumycin and methoxyamine. Rates of colony formation of U937 cells treated with 1umol/L manumycin, or 5 mmol/L methoxyamine, or the combination of the two were 0.3641±0.0463, 0.7541 ±0.0379, and 0.0473±0.0024, respectively compared with that of control cells （P〈0.05）. Moreover, the drug combination resulted in enhanced apoptosis in U937 cells. The apoptotic rates of the control, manumycin, methoxyamine and combination group were （2.34±0.30）%, （8.80±0.95）%, （2.21 ±0.19）%, and （13.37±0.91）%, respectively. The combination of manumycin with methoxyamine also promoted the release of cytochrome c from mitochondria into the cytosol, activated caspase-9, and ted appearance of specific cleavage of PARP in U937 cells. CONCLUSION： Methoxyamine enhances manumycin-induced apoptosis in U937 myeloid leukemia cells.

关 键 词：白血病 法尼基转移酶抑制剂 甲氧胺 手霉素 碱基切除修复抑制剂 U937细胞 凋亡 

分 类 号：R733.7[医药卫生—肿瘤]