硼替佐米与高三尖杉酯碱在K562细胞中的联合作用及机制研究

Combined Effect of Bortezomib and Homoharringtonine on K562 Cells and their Mechanisms

目的:探讨硼替佐米(bortezomib,BTZ)与高三尖杉酯碱(homoharringonine,HHT)两药联合对K562细胞增殖和凋亡的影响及其联合作用机制与BCL-2、BAX、MCL-1蛋白的关系。方法:通过将K562细胞株按不同处理分为对照、BTZ 20 nmol/L、HHT 40 ng/ml、BTZ 20 nmol/L+HHT 40 ng/ml共4组。采用MTT方法检测各处理组细胞增殖抑制率。采用Annexin V-FITC/PI双染色法流式细胞术检测各处理组细胞早期凋亡率,采用Western blot法测定各处理组中BCL-2、BAX、MCL-1蛋白相对表达量。结果:BTZ+HHT联合组作用于K562细胞24、48和72h后的增殖抑制率(%)高于BTZ及HHT单药组(P<0.01)。联合组K562细胞的早期凋亡率(%)高于BTZ及HHT 2个单药组(P<0.05)。联合组K562细胞的BCL-2蛋白相对表达量明显低于BTZ及HHT单药组(P<0.05)。联合组BAX蛋白相对表达量明显高于BTZ及HHT单药组(P<0.05)。M CL-1蛋白相对表达量高低:BTZ组>对照组>BTZ+HHT联合组>HHT组(组间比较P<0.05)。结论:BTZ与HHT联合可起协同抑制细胞增殖、诱导凋亡效应,其机制与明显下调BCL-2蛋白、上调BAX蛋白有关。此外,HHT可通过下调MCL-1蛋白表达增加K562细胞对BTZ作用的敏感性。

Objective： To explore the effects of BTZ plus HHT on proliferation and apoptosis of K562 cells, and to clarify the relationship between the mechanism inderlying the effect of BTZ plus HI-IT on K562 cells and BCL-2, BAX, MCL-1 proteins. Methods： The K562 cells were divided into 4 groups by different treatment： BTZ（20 nmol/L）, HHT （40 ng/ml）, BTZ（20 nmol/L） ＋ HHT（40 ng/ml） and control. The proliferation inhibition rates of K562 cells in each group were detected by using MTT, and the early apoptosis rates of K562 cells in each group were assayed by using flow cytometry with Annexin V-FITC/PI staining. The proteins level of BCL-2, BAX and MCL-1 in each group were examined by using Western blot. Results： The inhibition rate of K562 cell proliferation in combined group was higher than that in BTZ, HHT alone group （P 〈 0.01 ）. The early apoptosis rate of K562 cells in combined group was increased significantly in comparison with BTZ and HHT alone group （ P 〈 0. 05 ）. The BCL-2 protein level of K562 cells in combined group was significantly lower than that in BTZ and HHT alone group（ P 〈 0.05 ）. BAX protein level of K562 cells in combined group was higher than that in BTZ and HHT alone group （ P 〈 0.05 ）. The Orders of the MCL-I protein level of K562 cells in 4 groups were BTZ 〉 Control 〉 BTZ plus HHT 〉 HHT （ P 〈 0. 05 ）. Conclusion： The combination of BTZ and HHT exerts the synergistic effect of anti-proliferative activity and induces apoptosis against K562 cells in vitro. The combination can induce apoptosis of K562 cells via suppression of BCL-2 protein and up- regulation of BAX protein. HHT can increase the sensitivity of K562 cells to BTZ by down-regulating the expression of MCL-1 protein.