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

Abstract

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

CLC Number:

R733.3

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^**

Effect of BTK inhibitor BGB-3111 combined with bortezomib on apoptosis of human multiple myeloma cells and its mechanism

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