神经前体细胞表达下调因子8类泛素修饰抑制因子MLN4924对单核巨噬细胞白血病细胞增殖和凋亡的影响

Effect of neddylation inhibitor MLN4924 on proliferation and apoptosis of mononuclear macrophage leukemia cells

目的观察神经前体细胞表达下调因子8（NEDD8）类泛素修饰抑制因子MLN4924对单核巨噬细胞白血病细胞增殖的影响,并探讨其作用机制。方法 MLN4924 0.25~4.0μmol·L-1与U937细胞、THP-1细胞、Mo7e细胞和RAW264.7细胞作用24 h,采用MTT法检测细胞存活。MLN4924 0.25~8.0μmol·L-1与RAW264.7细胞作用24 h,用Western蛋白质印迹法检测NEDD8类泛素化修饰的主要底物cullin蛋白表达水平。MLN4924 0.5μmol·L-1作用于RAW264.7细胞24 h,用流式细胞仪检测细胞周期和细胞凋亡率。结果 MLN4924 0.25~4.0μmol·L-1作用24 h对RAW264.7细胞、U937细胞、THP-1细胞和Mo7e细胞增殖具有抑制作用,IC50分别为0.52,3.11,2.89和0.76μmol·L-1;对正常小鼠骨髓巨噬细胞增殖无明显影响。MLN4924 0.25~8.0μmol·L-1作用24 h,NEDD8类泛素化修饰的cullin蛋白表达减少（P〈0.01）,表明RAW264.7细胞NEDD8修饰逐渐减少。MLN4924 0.5,1.0和2.0μmol·L-1作用于RAW264.7细胞24 h,G0/G1期细胞百分率由正常对照组的（61.3±1.3）%分别升高到（64.5±1.5）%（P〈0.05）,（69.5±2.3）%（P〈0.01）和（72.8±1.7）%（P〈0.01）,细胞凋亡率由正常对照组的（1.5±0.3）%分别升高到（2.3±0.5）%,（4.3±0.9）%（P〈0.05）和（7.5±0.8）%（P〈0.01）。结论 MLN4924能够明显抑制单核巨噬细胞白血病细胞增殖,并诱导其细胞周期阻滞和细胞凋亡。

OBJECTIVE To study the effect of MLN4924 on proliferation of mononuclear macrophage leukemia cells and the underlying mechanisms. METHODS U937, THP-1, Mo7e and RAW264.7 cells were treated with MLN4924 0.25-4.0 pmol.L-1 for 24 h, and cell viability was detected with MTT assay. Western blotting was used to determine the expression level of neddylated cullin after treatment with MLN4924 0.25-8.0 umol· L-1 for 24 h. Flow cytometry analysis was used to detect cell cycle of RAW264.7 ceils after treatment with MLN4924 0.5 pmol·L-1 for 24 h. Flow cytometry analysis was used to detect ap- optosis of RAW264.7 cells after treatment with MLN4924 0.5-2.0 iJmol·L-1 for 24 h. RESULTS MTT assay results showed that MLN4924 0.25-4.0 μmol· L-1 for 24 h had significant inhibitory effect on U937, THP-1, Mo7e and RAW264.7 cells. The ICs0 value was 0.52, 3.11, 2.89 and 0.76 μmol.L-1, respectively. Western blotting results showed that MLN4924 0.25-8.0 μmol· L-1 for 24 h on RAW264.7 cells was re- duced the expression of main substrate neddylated cullin（ P〈0.01 ）, suggesting that NEDD8 modification reduced gradually. Flow cytometry assay results showed that MLN4924 0.5-2.0 μmoI.L-1 for 24 h arres- ted RAW264.7 cells at Go/G1, the cell rate of which rose from （61.3±1.3）% to （64.5±1.5）%（ P〈0.05）, （69.5±2.3）%（P〈0.01） and （72.8±1.7）% （P〈0.01）, and increased the apoptosis rate of RAW264.7 cells significantly, from（1.5±0.3）% of cell control group to （2.3±0.5）%, （4.3±0.9）%（P〈0.05） and （7.5±0.8） % （ P〈0.01 ）, respectively. CONCLUSION MLN4924 can effectively inhibit the proliferation of mononuclear macrophage leukemia cells and induce cell cycle arrest and apoptosis.