Mcl-1在阿霉素诱导骨肉瘤细胞凋亡中的作用

摘要

Objective To explore the expression level and functional role of Mcl -1 under doxorubicin induced ap- optosis. Methods The mRNA levels of Mcl4, Bcl-2 and β-actin were detected by semi-quantitative RT-PCR. The protein levels of Mcl-1, Bcl-2 and β-actin were detected by Western blot. Transfections of pcDNA-f1ag-Mcl-1 and pcDNA-f1ag -Bcl-2 were used to overexpress Mcl-1 and Bcl-2 in cells. Caspase-3/7 activities were measured by Caspase-3/7 assay kit. Results Although the expression of Mcl -1 and Bcl-2 mRNA stayed the same level, Mcl4 protein level decreased in osteosarcoma under doxorubicin induced apoptosis. Overexpression of Mcl-1 , not Bcl-2, significantly inhibited cleavage of PARP , which was marker for apoptosis. Meanwhile , overexpression of Mcl -1 also inhibited activity of caspase-3/7 by doxorubicin. Conclusion The downregulation of Mcl-1 protein level leads to an increase of apoptosis in osteosarcoma cells under doxorubicin treatment . In addition, this process is related to Caspase.%目的 研究阿霉素诱导骨肉瘤细胞凋亡过程中Mcl-1含量的变化以及Mcl-1对骨肉瘤细胞凋亡的调控作用.方法 采用半定量PCR法检测Mcl-1、Bcl-2、β-actin的mRNA水平变化;Western blot法检测Mcl-1、Bcl-2、β-actin蛋白变化;转染pcDNA3-Mcl-1质粒使细胞高表达Mcl-1、Bcl-2;采用Caspase3/7活性试剂盒检测Caspase3/7活性.结果 骨肉瘤细胞在阿霉素作用下,Mcl-1和Bcl-2的mRNA水平无变化,而蛋白水平下调;过表达Mcl-1可以明显抑制凋亡标志物PARP的剪切,而Bcl-2不具有抑制效应;同样,过表达Mcl-1可以抑制阿霉素作用下Caspase3/7的活化.结论 阿霉素作用下,Mcl-1蛋白水平下降是骨肉瘤细胞发生凋亡的重要原因,并且该凋亡过程与Caspase的活化有关.