Effect of Bruton’s tyrosine kinase inhibitors combined with bortezomib on human multiple myeloma cell lines and its mechanism / 第二军医大学学报

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.