BTK抑制剂BGB-3111联合硼替佐米对人多发性骨髓瘤细胞凋亡的影响及其机制

doi:10.13481/j.1671-587X.20250305

摘要/Abstract

摘要：

目的探讨泽布替尼（BGB-3111）联合硼替佐米（Btz）对人多发性骨髓瘤（MM）细胞凋亡的影响，并阐明其可能的机制。方法体外培养人MM U266、PS-R、RPMI8226、KMS28-PE、KMS28-BM和H929细胞，Western blotting法检测不同细胞中布鲁顿酪氨酸激酶（BTK）蛋白表达水平。采用细胞计数试剂盒8（CCK-8）法检测0、10、20、30、40和50 μmol·L^-1 BGB-3111处理的RPMI8226、U266及KMS28-BM细胞存活率。取对数生长期RPMI8226、U266和KMS28-BM细胞，分为对照组、BGB-3111组、Btz组和BGB-3111+Btz组，采用流式细胞术检测各组细胞凋亡率，Western blotting法检测各组细胞中髓细胞白血病因子1（MCL-1）、B细胞淋巴瘤2（Bcl-2）、Bcl-2相互作用细胞死亡介质蛋白（Bim）、磷酸化Bim（p-Bim）、P65、磷酸化P65（p-P65）、肿瘤坏死因子受体相关因子（TRAF）2和肿瘤坏死因子α（TNF-α）诱导蛋白3（A20）蛋白表达水平。U266细胞分为A20过表达组（A20-OE组）和空载对照组（EV组），2组细胞各分为对照组、BGB-311组、BTZ组和BGB-311+BTZ组，转染相应质粒，采用Western blotting法检测各组细胞转染效率，流式细胞术检测过表达A20后各组细胞凋亡率。结果 Western blotting法检测，与KMS28-BM细胞比较，U266、RPMI8226和H929细胞中BTK蛋白表达水平均明显升高（P<0.05或P<0.01）。CCK-8法检测，与0 μmol·L^－1 BGB-3111组比较，10、20、30、40和50 μmol·L^-1 BGB-3111组RPMI8226细胞及U266细胞存活率均明显降低（P<0.05或P<0.01），20、30、40和50 μmol·L^-1 BGB-3111组KMS28-BM细胞存活率均明显降低（P<0.01）。与RPMI8226和U266细胞比较，20、30和40 μmol·L^-1 BGB-3111组KMS28-BM细胞存活率均明显升高（P<0.05），选取10 μmol·L^-1 BGB-3111用于后续实验。流式细胞术检测，与对照组比较，BGB-3111组、Btz组和BGB-3111+Btz组RPMI8226及U266细胞凋亡率均明显升高（P<0.05或P<0.01）；与BGB-3111组和Btz组比较，BGB-3111+Btz组RPMI8226及U266细胞凋亡率均明显升高（P<0.01）；与对照组比较，Btz组和BGB-3111+Btz组KMS28-BM细胞凋亡率均明显升高（P<0.01）；与BGB-3111组比较，BGB-3111+Btz组KMS28-BM细胞凋亡率明显升高（P<0.01）；与EV组细胞比较，Btz组和BGB-3111+Btz组中A20-OE细胞凋亡率均明显升高（P<0.01）。Western blotting法检测，与对照组比较，BGB-3111组、Btz组和BGB-3111+Btz组RPMI8226细胞及U266细胞中Bim蛋白表达水平均明显升高（P<0.05），Btz组和BGB-3111+Btz组RPMI8226细胞及U266细胞中MCL-1、p-Bim和Bcl-2蛋白表达水平均明显降低（P<0.05）；与BGB-3111组和Btz组比较，BGB-3111+Btz组RPMI8226细胞和U266细胞中Bim蛋白表达水平均明显升高（P<0.05），MCL-1、p-Bim和Bcl-2蛋白表达水平均明显降低（P<0.05）。与对照组比较，Btz组和BGB-3111+Btz组RPMI8226及U266细胞中p-P65蛋白表达水平均明显升高（P<0.05），TRAF2和A20蛋白表达水平均明显降低（P<0.05）；与BGB-3111组和Btz组比较，BGB-3111+Btz组RPMI8226和U266细胞中p-P65蛋白表达水平均明显升高（P<0.05），TRAF2和A20蛋白表达水平均明显降低（P<0.05）。与EV组比较，A20-OE组细胞中A20蛋白表达水平明显升高（P<0.01）。结论 BGB-3111通过抑制BTK活性诱导MM细胞发生凋亡，并增强Btz的促凋亡作用，过表达A20增加MM细胞对联合用药的敏感性，其抗肿瘤作用可能与核因子κB（NF-κB）信号通路抑制有关。

关键词: 多发性骨髓瘤, 布鲁顿酪氨酸激酶抑制剂, 硼替佐米, 肿瘤坏死因子α诱导蛋白3, 核因子κB

Abstract:

Objective To discuss the effect of zanubrutinib （BGB-3111） combined with bortezomib （Btz） on the apoptosis of the human multiple myeloma （MM） cells， and to clarify its possible mechanism. Methods The human MM cell lines U266， PS-R， RPMI8226， KMS28-PE， KMS28-BM， and H929 were cultured in vitro. Western blotting method was used to detect the expression level of Bruton’s tyrosine kinase （BTK） protein in various cells； cell counting kit-8（CCK-8） method was used to detect the survival rates of the RPMI8226， U266， and KMS28-BM cells after treated with 0， 10， 20， 30， 40， and 50 μmol·L?1 BGB-3111. The RPMI8226， U266， and KMS28-BM cells at the logarithmic growth phase were selected and divided into control group， BGB-3111 group， Btz group， and BGB-3111+Btz group. Flow cytometry was used to detect the apoptotic rates of the cells in various groups； Western blotting method was used to detect the expression levels of myeloid cell leukemia 1 （MCL-1）， B-cell lymphoma-2（Bcl-2）， Bcl-2-interacting mediator of cell death （Bim）， phosphorylated Bim （p-Bim）， P65， phosphorylated P65 （p-P65）， tumor necrosis factor receptor-associated factor （TRAF） 2， and tumor necrosis factor alpha-induced protein 3 （A20） in different kinds of cells. The U266 cells were divided into A20 overexpression group （A20-OE group） and empty vector control group （EV group）. Each group was further divided into control group， BGB-3111 group， Btz group， and BGB-3111+Btz group. The corresponding plasmids were transfected； Western blotting method was used to detect the transfection efficiency of the cells in various groups； flow cytometry was used to detect the apoptotic rates of the cells in various groups after over-expression of A20. Results The Western blotting results showed that compared with KMS28-BM cells， the expression levels of BTK protein in the U266， RPMI8226， and H929 cells were significantly increased （P<0.05 or P<0.01）. The CCK-8 results showed that compared with 0 μmol·L?1 BGB-3111 group， the survival rates of the RPMI8226 and U266 cells in 10， 20， 30， 40， and 50 μmol·L?1 BGB-3111 groups were significantly decreased （P<0.05 or P<0.01）， and the survival rates of the KMS28-BM cells in 20， 30， 40， and 50 μmol·L?1 BGB-3111 groups were significantly decreased （P<0.05）. Compared with RPMI8226 and U266 cells， the survival rates of the KMS28-BM cells in 20， 30， and 40 μmol·L?1 BGB-3111 groups were significantly increased （P<0.05）. Therefore， 10 μmol·L?1 BGB-3111 was selected for subsequent experiments. The flow cytometry results showed that compared with control group， the apoptotic rates of the RPMI8226 and U266 cells in BGB-3111 group， Btz group， and BGB-3111+Btz group were significantly increased （P<0.05 or P<0.01）； compared with BGB-3111 group and Btz group， the apoptotic rates of the RPMI8226 and U266 cells in BGB-3111+Btz group were significantly increased （P<0.01）； compared with control group， the apoptotic rates of the KMS28-BM cells in Btz group and BGB-3111+Btz group were significantly increased （P<0.01）； compared with BGB-3111 group， the apoptotic rate of the KMS28-BM cells in BGB-3111+Btz group was significantly increased （P<0.01）； compared with EV group， the apoptotic rates of the cells in A20-OE group in Btz group and BGB-3111+Btz group were significantly increased （P<0.05）. The Western blotting results showed that compared with control group， the expression levels of Bim protein in the RPMI8226 and U266 cells in BGB-3111 group， Btz group， and BGB-3111+Btz group were significantly increased （P<0.05）， while the expression levels of MCL-1， p-Bim， and Bcl-2 proteins in the RPMI8226 and U266 cells in Btz group and BGB-3111+Btz group were significantly decreased （P<0.05）； compared with BGB-3111 group and Btz group， the expression levels of Bim protein in the RPMI8226 and U266 cells in BGB-3111+Btz group were significantly increased （P<0.05）， while the expression levels of MCL-1， p-Bim， and Bcl-2 proteins were significantly decreased （P<0.05）. Compared with control group， the expression levels of p-P65 protein in the RPMI8226 and U266 cells in Btz group and BGB-3111+Btz group were significantly increased （P<0.05）， while the expression levels of TRAF2 and A20 proteins were significantly decreased （P<0.05）； compared with BGB-3111 group and Btz group， the expression levels of p-P65 protein in the RPMI8226 and U266 cells in BGB-3111+Btz group were significantly increased （P<0.05）， while the expression levels of TRAF2 and A20 proteins were significantly decreased （P<0.05）. The flow cytometry results showed that compared with EV group， the expression level of A20 protein in A20-OE group cells was significantly increased （P<0.01）. Conclusion BGB-3111 induces apoptosis in the MM cells by inhibiting BTK activity and enhances the pro-apoptotic effect of Btz. Over-expression of A20 increases the sensitivity of the MM cells to the combined treatment. The antitumor effect may be related to the inhibition of the nuclear factor kappa B （NF-κB） signaling pathway.

Key words: Multiple myeloma, Bruton’s tyrosine kinase inhibitor, Bortezomib, Tumor necrosis factor alpha-induced protein 3, Nuclear factor kappa B

中图分类号:

R733.3

李洪杰,兰茂卓,王潇,冯冉冉,陶燕燕,刘佳庆,孙海柏. BTK抑制剂BGB-3111联合硼替佐米对人多发性骨髓瘤细胞凋亡的影响及其机制[J]. 吉林大学学报(医学版), 2025, 51(3): 599-609.

Hongjie LI,Maozhuo LAN,Xiao WANG,Ranran FENG,Yanyan TAO,Jiaqing LIU,Haibai SUN. Effect of BTK inhibitor BGB-3111 combined with bortezomib on apoptosis of human multiple myeloma cells and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2025, 51(3): 599-609.

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Group	Surival rate
Group	0 μmol·L^－1 BGB-311	10 μmol·L^－1 BGB-311	20 μmol·L^－1 BGB-311	30 μmol·L^－1 BGB-311	40 μmol·L^－1 BGB-311	50 μmol·L^－1 BGB-311
RPMI8226	91.18±1.16	83.97±1.55^*	65.57±2.44^**	47.30±1.63^**	32.27±1.39^**	21.51±6.43^**
U266	90.21±2.49	82.67±2.86^*	69.63±2.39^**	53.67±0.62^**	40.53±0.55^**	27.28±2.13^**
KMS28-BM	89.87±2.45	85.83±0.91	72.63±2.95^**△#	61.17±3.20^**△#	45.00±4.22^**△#	24.61±4.61^**