吉西他滨增强ABT-199对费城染色体阳性急性淋巴细胞白血病细胞杀伤作用的机制研究

Mechanism of gemcitabine enhances ABT-199 induced apoptosis in Philadelphia chromosome-positive acute lymphoblastic leukemia： a preliminary study

目的 研究吉西他滨联合ABT-199对费城染色体阳性（Ph＋）急性淋巴细胞白血病（ALL）细胞株SUP-B15的增殖抑制及凋亡诱导作用,并探讨其协同作用机制.方法 取对数生长期SUP-B15细胞,分别接受吉西他滨（0.025、0.050μmol/L）、ABT-199（0、0.5、1.0、2.0、4.0、8.0μmol/L）及两药联合处理24 h后,采用CCK-8法检测细胞增殖情况,流式细胞术（FCM）检测细胞凋亡情况,JC-1法检测线粒体膜电位的变化,蛋白质印迹法分析线粒体凋亡通路相关蛋白的表达变化.结果 ABT-199作用SUP-B15细胞24 h的半数抑制浓度（IC50）值为（4.13±0.89）μmol/L,吉西他滨（0.025、0.050μmol/L）能增强ABT-199对SUP-B15细胞的增殖抑制作用,其IC50值分别为（2.23±0.73）、（1.15±0.45）μmol/L,差异均有统计学意义（t=28.65,P〈0.01;t=35.12,P〈0.01）.FCM检测细胞凋亡结果显示,与0.025μmol/L吉西他滨单药组[（7.33±1.54）％]相比,0.025μmol/L吉西他滨联合ABT-199（1.0、2.0μmol/L）作用SUP-B15细胞24 h后,凋亡细胞比例分别为（32.42±1.45）％和（44.33±1.86）％,差异具有统计学意义（F=70.78,P〈0.001）;与0.050μmol/L吉西他滨单药组[（9.60±2.76）％]相比,0.050μmol/L吉西他滨联合ABT-199（1.0、2.0μmol/L）后,凋亡细胞比例增加,分别为（47.63±3.81）％和（58.73±4.33）％,差异有统计学意义（F=79.21,P〈0.001）.吉西他滨联合ABT-199作用SUP-B15细胞12 h后,去极化细胞的比例高于单药组,差异有统计学意义（P〈0.001）.吉西他滨联合ABT-199作用SUP-B15细胞12 h后,抗凋亡蛋白bcl-2、bcl-xL、Mcl-1水平下降.结论 吉西他滨可增强ABT-199对Ph＋ALL细胞的增殖抑制及凋亡诱导作用,机制可能与下调抗凋亡相关蛋白相关.

Objective To investigate the effects of gemcitabine and ABT-199 on proliferation inhibition and apoptosis induction of Philadelphia chromosome-positive （Ph＋） acute lymphoblastic leukemia （ALL） cell line SUP-B15, and to explore its synergistic mechanism. Methods SUP-B15 cells in logarithmic growth phase were treated with gemcitabine （0.025 and 0.050 μmol/L）, ABT-199 （0, 0.5, 1.0, 2.0, 4.0, 8.0 μmol/L） or two drugs for 24 h. Cell proliferation was detected by CCK-8 method, apoptosis was detected by flow cytometry （FCM）, mitochondrial membrane potential was detected by JC-1 method, and expression of mitochondrial apoptosis pathway-related protein was analyzed by Western blot. Results The 50 % inhibitory concentration （IC50） of SBT-B15 cells treated with ABT-199 for 24 h was （4.13±0.89） μmol/L. However, gemcitabine （0.025, 0.050 μmol/L） significantly enhanced the inhibitory effect of ABT-199 on proliferation of SUP-B15 cells, the IC50 values were （2.23 ±0.73） and （1.15 ±0.45） μmol/L, respectively. The results of FCM assay showed that compared with the monotherapy group [（7.33±1.54）%], 0.025 umol/L gemcitabine combined with ABT-199 （1.0 and 2.0 μmol/L） acted on SUP-B15 cells for 24 h, the proportions of apoptotic cells were （32.42±1.45） %and （44.33±1.86） %, the difference was statistically significant （F=70.78, P〈0.001）;compared with the monotherapy group [（9.60 ±2.76） %], 0.05 μmol/L gemcitabine combined with ABT-199 （1.0 and 2.0 μmol/L） acted on SUP-B15 cells for 24 h, the proportion of apoptotic cells increased to （47.63 ± 3.81） % and （58.73 ±4.33） %, respectively, and the difference was statistically significant （F= 79.21, P〈0.001）. The JC-1 experiment showed that treated with ABT-199 and gemcitabine for 12 h, the percentage of depolarizing cell was significantly higher than that in single agent group, and the difference was statistical significant （P〈0.001）. Western blot showed that the anti-apoptotic proteins bcl-2, bcl-xL and Mcl-1 decreased after treated by gemcitabine combined with ABT-199 for 12 h. Conclusion Gemcitabine could enhance the proliferation inhibition and induce apoptosis of Ph＋ALL cells by ABT-199, and its mechanism may be related to down-regulation of anti-apoptosis-related proteins.