雷帕霉素联合2-DG对多发性骨髓瘤细胞增殖和凋亡的影响

Effects of rapamycin combined with 2-DG on cell proliferation and apoptosis of multiple myeloma

背景多发性骨髓瘤(multiple myeloma,MM)是血液系统第二常见的恶性肿瘤,新药时代使骨髓瘤患者的生存得到极大改善,但仍不可治愈,需要探索新的治疗方法以进一步提高骨髓瘤的治疗效果。目的研究哺乳动物雷帕霉素靶蛋白抑制剂雷帕霉素(rapamycin,Rapa)联合糖酵解抑制剂2-脱氧-D-葡萄糖(2-deoxy-D-glucose,2-DG)对体外培养人多发性骨髓瘤RPMI8226细胞周期和凋亡的影响。方法CCK-8法分析两药单独应用及联用对人多发性骨髓瘤RPMI8226细胞增殖和毒性的影响,并通过CompuSyn软件计算合用指数(combination index,CI);利用流式细胞术检测Rapa和2-DG单药及联合作用下对骨髓瘤细胞周期和凋亡的影响;Western blot检测Rapa和2-DG单药及联合作用下骨髓瘤细胞周期调控分子CyclinD1、凋亡相关指标P53、Bax、Bcl-2的表达。结果CCK-8法显示多种不同浓度组合下Rapa联合2-DG作用48 h有显著的抗骨髓瘤协同作用,其CI值介于0.6~0.8,其中Rapa(50 nmol/L)和2-DG(0.5 mmol/L)合用的CI值为0.642,显示出很好的协同作用;流式细胞术显示联合应用Rapa(50 nmol/L)和2-DG(0.5 mmol/L)较空白对照组和单药组使细胞周期阻滞在G0/G1期、S期的比例下降,凋亡率明显升高(P均<0.05);Western blot结果显示Rapa(50 nmol/L)和2-DG(0.5 mmol/L)联用组促凋亡指标P53、Bax等表达增加,凋亡抑制指标Bcl-2表达下降(P均<0.05)。结论Rapa和2-DG联合应用可显著抑制RPMI8226细胞增殖,并显著增加其细胞凋亡,显示出较好的协同作用,对于进一步探索Rapa和2-DG在多发性骨髓瘤中的应用有一定的启示作用。

Background Multiple myeloma(MM)is the second most common hematological malignancy.Introduction of several new classes of drugs for MM treatment has increased response rates and survival substantially,but MM also remains incurable and new therapies are needed to induce more profound clinical outcomes.Objective To investigate the synergistic anti-tumor effect between the mammalian target of Rapamycin inhibitor Rapamycin(Rapa)and glycolysis inhibitor 2-deoxy-D-glucose(2-DG)on the human MM RPMI8226 cells.Methods CCK-8 was used to analyze the effects of the two drugs alone and in combination on the proliferation and toxicity of human myeloma RPMI8226 cells,and the combination index(CI)was calculated by CompuSyn software;Cell cycle and apoptosis were detected by the flow cytometry;Western blot was used to detect the expression of cell cycle regulation molecules CyclinD1,apoptosis-related indicators p53,Bax and Bcl-2.Results The CCK-8 method showed that Rapa combining with 2-DG had significant anti-myeloma synergistic effect under various combinations of different concentrations after48 h treatment,with the CI value of 0.6-0.8,among which the CI value of Rapa(50 nmol/L)and 2-DG(0.5 mmol/L)was 0.642,showing a good synergistic effect.Flow cytometry showed that cell cycle was arrested in the G0/G1 phrase,S phase decreased,while the apoptosis rate significantly increased compared with the control group and single drug group after combined application of Rapa(50 nmol/L)and 2-DG(0.5 nmol/L)(all P<0.05).Western blot showed that the expression of pro-apoptotic indicators p53 and Bax increased,and the anti-apoptotic protein Bcl-2 decreased in the combination group(all P<0.05).Conclusion The combined application of Rapa and 2-DG shows a good synergistic effect by inhibiting the proliferation of RPMI8226 cells,blocking cell cycle and increasing apoptosis,which brings enlightenment for further exploration of the usage of Rapa and 2-DG in multiple myeloma.