| 摘要: |
| 目的 探讨布鲁顿酪氨酸激酶（Bruton’s tyrosine kinase，BTK）抑制剂依鲁替尼（ibrutinib）和AVL-292单药及联合蛋白酶体抑制剂硼替佐米对人多发性骨髓瘤细胞系H929和RPMI8226的作用及其机制。方法 用不同浓度的依鲁替尼、AVL-292单药以及联合硼替佐米处理H929、RPMI8226细胞。采用CCK-8法检测细胞增殖情况，流式细胞术检测细胞凋亡情况，蛋白质印迹法检测药物处理前后细胞内BTK信号通路蛋白及凋亡相关蛋白的表达水平。结果 依鲁替尼和AVL-292均可抑制H929、RPMI8226细胞增殖，其抑制作用呈浓度依赖性，依鲁替尼对H929、RPMI8226细胞48 h的半数抑制浓度（median inhibitory concentration，IC50）分别为（10.41±3.29）μmol/L和（51.65±13.58）μmol/L，AVL-292对H929、RPMI8226细胞48 h的IC50分别为（7.77±2.99）μmol/L和（6.44±1.06）μmol/L。不同浓度的依鲁替尼（5、10 μmol/L）和AVL-292（5、10 μmol/L）分别与不同浓度的硼替佐米（5、10、20、50 nmol/L）联合应用对H929、RPMI8226细胞增殖的抑制率均高于相应浓度单药组（P<0.05，P<0.01），不同组合的协同系数R均>1.0。10 μmol/L依鲁替尼、10 μmol/L AVL-292和20 nmol/L硼替佐米单独作用48 h后，H929细胞的凋亡率分别为（15.12±1.59）%、（18.23±6.38）%和（10.71±1.62）%，均高于对照组[（6.46±1.18）%；P<0.05，P<0.01]；RPMI8226细胞的凋亡率分别为（9.29±1.44）%、（15.01±4.99）%和（7.58±1.13）%，10 μmol/L依鲁替尼和10 μmol/L AVL-292单药组与对照组[（5.54±1.61）%]比较差异均有统计学意义（P<0.05）；10 μmol/L依鲁替尼和10 μmol/L AVL-292分别与20 nmol/L硼替佐米联合后，H929细胞凋亡率分别为（40.31±3.94）%和（51.55±6.39）%，RPMI8226细胞凋亡率分别为（31.86±1.93）%和（43.23±4.03）%，均高于相应单药组（P<0.01）。10 μmol/L依鲁替尼单药和10 μmol/L AVL-292单药作用24 h后，H929细胞内BTK、NF-κB p65、Akt和ERK的磷酸化水平及Bcl-xL蛋白表达水平均较对照组降低（P<0.05），cleaved caspase-3表达水平均较对照组升高（P<0.01）；两药分别联合20 nmol/L硼替佐米后，对上述蛋白的调节作用均较相应单药组增强（P<0.05，P<0.01）。结论 BTK抑制剂依鲁替尼和AVL-292对多发性骨髓瘤细胞系H929、RPMI8226有增殖抑制和凋亡诱导作用，并与蛋白酶体抑制剂硼替佐米有协同作用，其机制可能与抑制细胞内BTK活性及下游NF-κB、Akt、ERK信号通路活性，下调抗凋亡蛋白Bcl-xL表达、激活caspase-3依赖的凋亡途径有关。 |
| 关键词: 多发性骨髓瘤 布鲁顿酪氨酸激酶 依鲁替尼 AVL-292 硼替佐米 药物协同作用 |
| DOI：10.16781/j.0258-879x.2016.11.1325 |
| 投稿时间：2016-08-17修订日期：2016-10-28 |
| 基金项目:国家自然科学基金（30873042，81100361），上海市自然科学基金（07ZR14146）. |
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| Effect of Bruton's tyrosine kinase inhibitors combined with bortezomib on human multiple myeloma cell lines and its mechanism |
| ZHANG Wen,CHENG Hui,TANG Gu-sheng,DING Jing,HU Xiao-xia,YANG Jian-min,WANG Jian-min,LÜ Shu-qing* |
(Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
*Corresponding author.) |
| Abstract: |
| Objective To explore the effect of Bruton's tyrosine kinase (BTK) inhibitors, ibrutinib and AVL-292 alone or in combination with proteasome inhibitor bortezomib on human multiple myeloma (MM) cell lines H929 and RPMI8226 and the related mechanism. Methods H929 and RPMI8226 cells were treated with ibrutinib or AVL-292 alone or in combination with bortezomib in vitro. The cell viability was detected by CCK-8 assay after treatment, the apoptosis levels were analyzed by flow cytometry, the expression and phosphorylation levels of BTK pathway proteins and apoptosis-related proteins were measured by Western blotting analysis. Results The proliferation of H929 and RPMI8226 cells was inhibited by ibrutinib and AVL-292 in a dose-dependent manner. The 48 h median inhibitory concentration (IC50) values of ibrutinib in the two cell lines were (10.41±3.29) μmol/L and (51.65±13.58) μmol/L, respectively, and the 48 h IC50 values of AVL-292 were (7.77±2.99) μmol/L and (6.44±1.06) μmol/L, respectively. The inhibition effects of different concentrations of ibrutinib (5 μmol/L, 10 μmol/L) and AVL-292 (5 μmol/L, 10 μmol/L) combined with different concentrations of bortezomib (5 nmol/L, 10 nmol/L, 20 nmol/L, and 50 nmol/L) were significantly higher than those of the corresponding single agents (P<0.05, P<0.01); the coefficients of concordance (R) of different combinations were all above 1.0. After treatment with 10 μmol/L ibrutinib, 10 μmol/L AVL-292 and 20 nmol/L bortezomib alone for 48 h, the apoptosis levels of H929 cells were (15.12±1.59)%, (18.23±6.38)% and (10.71±1.62)%, respectively, which were all significantly higher than that of the control group ([6.46±1.18]%; P<0.05, P<0.01); the apoptosis levels of RPMI8226 cells were (9.29±1.44)%, (15.01±4.99)% and (7.58±1.13)%, respectively, and those of treated with 10 μmol/L ibrutinib and 10 μmol/L AVL-292 were significantly higher than that of the control group ([5.54±1.61]%, P<0.05). The apoptosis levels of H929 cells in the 20 nmol/L bortezomib+10 μmol/L ibrutinib and 20 nmol/L bortezomib+10 μmol/L AVL-292 groups were (40.31±3.94)% and (51.55±6.39)%, respectively, and those of RPMI8226 cells were (31.86±1.93)% and (43.23±4.03)%, respectively, and they were all significantly higher than those in their corresponding single agent groups (P<0.01). Compared with control group, the phosphorylation levels of BTK, NF-κB p65, Akt and ERK and expression of Bcl-xL protein were significantly decreased in H929 cells treated with 10 μmol/L ibrutinib or 10 μmol/L AVL-292 for 24 h (P<0.05), and the expression of cleaved caspase-3 was significantly increased (P<0.01). The regulation effects on the above indices were significantly more evident when the two agents were combined with 20 nmol/L bortezomib (P<0.05, P<0.01). Conclusion Both ibrutinib and AVL-292 can inhibit proliferation and induce apoptosis of human MM cell lines H929 and RPMI8226; there are significant synergistic effects between them and proteasome inhibitor bortezomib, which may be related to inhibition of BTK activity and the downstream pathways (NF-κB, Akt and ERK), down-regulation of Bcl-xL and activation of caspase-3 apoptotic pathway. |
| Key words: multiple myeloma Bruton's tyrosine kinase ibrutinib AVL-292 bortezomib drug synergism |
