DS/Cu对急性髓系白血病干细胞增殖与凋亡影响及其机制的探讨  

Effects of disulfiram and Disulfiram/Cu on the proliferation and apopsis of leukemia stem cells and their mechanism

作　　者：王诗韵[1] 查洁[1] 董慧娟[1] 余乐[2] 李蓉蔚[1] 张妍琰[1] 史鹏程[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]南方医科大学药学院免疫与生化药理学科,广东广州510515

出　　处：《中华肿瘤防治杂志》2013年第6期411-415,共5页Chinese Journal of Cancer Prevention and Treatment

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

摘　　要：目的:探讨双硫仑(disulfiram,DS)单药及DS联合Cu(DS/Cu)在体外对白血病干细胞的影响及其分子机制。方法:应用流式细胞仪分选KG1a细胞中具有LSCs特性的CD34+CD38-亚群;MTT检测DS和DS/Cu对CD34+CD38-KG1a细胞的抑制增殖作用;Annexin-Ⅴ/PI法检测DS及DS/Cu对CD34+CD38-KG1a细胞的诱导凋亡作用;蛋白质印迹法检测药物处理后NF-кb通路和JNK通路蛋白表达的变化。结果:CD34+CD38-亚群占KG1a细胞的(59.4±6.2)%,流式细胞分选后的CD34+CD38-细胞可达(93.16±2.7)%,其中(65.3±6.4)%处于G0/G1期。DS和DS/Cu能抑制CD34+CD38-KG1a细胞增殖能力,24h的IC50分别为(0.54±0.18)μmol/L和(0.21±0.03)μmol/L,DS/Cu的作用显著强于DS,P=0.036。DS和DS/Cu对CD34+CD38-KG1a细胞均有诱导凋亡的作用,0.05、0.5和5μmol/L的DS和DS/Cu作用细胞24h后凋亡率分别为〔(7.69±3.44)%、(15.06±4.58)%、(32.67±6.06)%〕和〔(28.83±6.34)%、(42.33±3.21)%、(58.93±1.53)%〕,DS/Cu诱导CD34+CD38-KG1a细胞凋亡的比例显著高于DS单药,P<0.01;DS及DS/Cu均能抑制p65蛋白及下游蛋白c-myc、Survivin的表达、并上调磷酸化JNK及下游转录因子p-c-jun的表达,DS/Cu的作用更加显著。结论:DS在体外能抑制LSCs增殖及诱导其凋亡,Cu可以显著增强这种作用,其作用机制与抑制NF-κb通路及激活JNK通路相关。OBJECTIVE：To investigate the effect of Disulfiram （DS） and DS/Cu on LSCs and the relationship with NF-κb and JNK pathway. METHODS： CD34＋ CD38-KGla cells was sorted from KGla cell lines by fluorence-activated cell sorting （FACS） analysis and were treated with various concentrations of DS and DS/Cu for 24 h,and the inhibitory ratio was measured by MTT assay. The effects of DS and DS/Cu on apoptosis of CD34＋ CD38- KGla cells were deter- mined by flow cytometry Annexin- V/PI double staining; the expression of JNK, p-JNK, p-c-jun,p65 and its downstream protein in CD34＋ CD38- KG1a cells after treatment with DS and DS/Cu are tested by western bloting. RESULTS： The percentage of CD34＋ CD38- was （59.4±6.2） % ,in KGla cells. After separated by using flow-cytometry,the percentage was （93.16±2.7） % ,and （65.3±6.4） % was in G0/G1 stage. Different concentrations of DS could inhibit the prolifera- tion of CD34＋ CD38-KGla cells with the 50% inhibitory concentration （IC50） values of （0. 54±0.18） μmol/L,in combi- ning with low concentration of Cu （ CuC12 1 μmol/L）,the IC50 was significantly reduced to （0.21±0.03） μmol/L （P= 0. 036） ; With the concentration of 0.05,0.5,5 μmol/L,DS could respectively induce apoptosis with the apoptotic rate of（7.69±.44） %, （15.06±4.58） %, （32.67±6.06） % after 24 h treatment, while DS/Cu （Cu= 1 μmol/L） could induce （28.83±6.34）%, （42. 33 ±3.21）%, （58.93 ± 1.53）%. DS and DS/Cu could induced the apoptosis of CD34＋ CD38- KGla cells in a dose-dependent manner,while DS/Cu showed significantly higher effect in apoptosis than DS alone （P〈0.01）. Western blot results showed markedly increase the expression of p-JNK,c-Jun and decrease the expression of p65 in CD34＋ CD38-KGla cells treated with DS and DS/Cu. CONCLUSION： DS and DS/Cu may interfere in the growth of CD34＋ CD38- KGla cells by simultaneous activation of JNK pathway and inhibition of NF-κB pathway.

关 键 词：白血病干细胞 双硫仑 铜 CD34+ CD38+ KGla NF-κB JNK 

分 类 号：R733.7[医药卫生—肿瘤]