基于PI3K/AKT/mTOR探索硼替佐米对多发性骨髓瘤侧群细胞的作用机制

目的 探讨26S蛋白酶体抑制剂硼替佐米对多发性骨髓瘤细胞系RPMI8226和骨髓瘤侧群(side population,SP)细胞的作用机制及其耐药性。 方法 在RPMI8226和SP细胞增殖以及生长周期和凋亡差异性研究基础上,采用Western blotting方法检测硼替佐米对2种细胞内信号通路PI3K/AKT/mTOR磷酸化水平的影响;通过硼替佐米耐药性研究,开展RPMI8226和SP细胞对硼替佐米耐药机制的探索。 结果 SP细胞48 h相对细胞活力(3.62±0.28)显著高于RPMI8226细胞(2.81±0.24,P=0.028);克隆形成能力结果显示SP细胞显著强于RPMI8226细胞(P<0.05)。流式细胞术检测显示硼替佐米增加了2种细胞S期、降低G₂/M期,对RPMI8226的促凋亡率(51.23±5.35)显著高于SP细胞凋亡率(29.62±2.61,P=0.008)。Western blotting结果显示硼替佐米显著降低RPMI8226和SP细胞AKT、mTOR及4E-RP1磷酸化水平;耐药性结果显示SP细胞耐药相关蛋白P-gp、Caveolin-1(Cav-1)、Fatty acid synthase(FASN)及CYP3A4表达显著高于RPMI8226细胞。 结论 硼替佐米主要通过细胞凋亡和降低PI3K/AKT/mTOR信号蛋白磷酸化水平,发挥对RPMI8226和SP细胞的抑制作用;通过P-gp、Cav-1、FASN以及CYP3A4耐药蛋白发挥对多发性骨髓瘤的耐药性。 

Exploration of mechanism of bortezomib on side population cell from multiple myeloma based on PI3K/AKT/mTOR

Objective To investigate the mechanism and drug resistance of 26 S proteasome inhibitor bortezomib on multiple myeloma cell line RPMI8226 and myeloma side population(SP) cells. Methods The effect of bortezomib on the phosphorylation of PI3 K/AKT/mTOR signaling pathways was detected by Western blotting. The mechanism of resistance to bortezomib in RPMI8226 and SP cells was explored. Results The relative cell viability of SP cells at 48 h(3.62±0.28) was significantly higher than that of RPMI8226 cells(2.81±0.24, P=0.028). The clone formation ability results showed that SP cells were significantly stronger than RPMI8226 cells(P<0.05). Flow cytometry showed that bortezomib increased the S phase of both cells and decreased the G₂/M phase. The pro-apoptotic rate of RPMI8226(51.23±5.35) was significantly higher than that of SP cells(29.62±2.61, P=0.008). Western blotting results showed that bortezomib significantly reduced the phosphorylation levels of ART, mTOR and 4 E-RP1 in RPMI8226 and SP cells. The drug resistance results showed that SP cell resistance-related proteins P-gp, Caveolin-1(Cav-1), Fatty acid synthase(FASN) and CYP3 A4 expression were higher than those in RPMI8226 cells. Conclusion The inhibitory activity of bortezomib on RPMI8226 and SP cells is exhibited by cell apoptosis and phosphorylation levels of AKT and mTOR signaling proteins. The resistance is developed by the high expression of P-gp, CYP3 A4, Cav-1 and FASN.