硼替佐米联合毛喉素诱导硼替佐米耐药骨髓瘤细胞凋亡的实验研究

Experimental study on forskolin combined with bortezomib inducing apoptosis in bortezomibresistant multiple myeloma cells

背景与目的:硼替佐米作为蛋白酶体抑制剂已成为新诊断和复发多发性骨髓瘤患者临床治疗的主要药物,但仍有部分患者对硼替佐米产生耐药而影响其长期生存。近年来研究发现,提高细胞内环腺苷酸浓度可以诱导骨髓瘤细胞凋亡并延缓其生长,成为骨髓瘤治疗新途径。该研究通过观察硼替佐米联合腺苷酸环化酶激动剂毛喉素(forskolin)对硼替佐米耐药骨髓瘤细胞的作用,探究克服骨髓瘤耐药的可能途径及其机制。方法:以硼替佐米耐药骨髓瘤细胞株H929-R和原代细胞为模型,通过观察细胞生长、形态、凋亡相关基因的改变来研究硼替佐米、毛喉素单独或联合处理对硼替佐米耐药细胞增殖及凋亡的影响;并应用Rh123/PI双染法检测药物处理前后细胞内线粒体跨膜电位的变化;同时采用实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)和蛋白[质]印迹法(Western blot)检测线粒体凋亡相关基因转录水平及抗凋亡基因Bcl-2和Mcl-1的蛋白水平的改变。结果:硼替佐米(20 nmol/L)与毛喉素(50 nmol/L)能够协同诱导硼替佐米耐药细胞凋亡。进一步研究发现,毛喉素可协同硼替佐米促使耐药细胞内线粒体跨膜电位下降并下调其Bcl-2和Mcl-1蛋白的表达。结论:毛喉素联合硼替佐米能够诱导硼替佐米耐药骨髓瘤细胞凋亡。

Background and purpose： Although bortezomib has become one of the major therapeutic agents against newly diagnosed or relapsed multiple myeloma （MM）, there are some patients who become resistant to bortezomib and then relapse, emerging as a major obstacle to long-term survival of MM patients. It has been found that elevation of intracellular cyclic adenosine monophosphate （cAMP） levels could induce cell cycle arrest and apoptosis in MM cells,which has become an interesting approach to MM therapy. This study aimed to investigate possible effects of forskolin combined with bortezomib on bortezomib-resistant myeloma cells and further explore its mechanisms. Methods： The bortezomib-resistant MM cell lines H929-R and primary cells from patients who do not respond to bortezomib were used as in vitro models. The influences of bortezomib and/or forskolin on MM cells were evaluated through cellular morphology, changes of cell distribution and apoptotic rate. Meanwhile, flow cytometry analysis was used to detect mitochondrial transmembrane potential （ΔΨm） and the expression levels of apoptosis regulators in these cells before and after the treatment were detected by Western blot. Results： Bortezomib （20 nmol/L） synergized with forskolin （50 nmol/L） to induce apoptosis of H929-R cells and bortezomib-resistant primary cells. In addition, bortezomib synergized with forskolin to induce collapse of mitochondrial transmembrane and facilitate the degradation of anti-apoptosis proteins including Bcl-2 and Mcl-1. Conclusion： Bortezomib could synergize with forskolin to induce apoptosis in bortezomib-resistant MM cells.