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AIM：To investigate the synergistic anti-tumor effects of two novel inhibitors，zanubrutinib，a Bru? ton tyrosine kinase（BTK）inhibitor，and pracinostat，a histone deacetylase（HDAC）inhibitor，in diffuse large B-cell lym? phoma（DLBCL）cells. METHODS：（1）NU-DUL-1（ABC type DLBCL cell line）and SU-DHL-6（GCB type DLBCL cell line）were treated with single-agent zanubrutinib at concentrations of 0，2. 5，5，10，20，40，80 and 160 μmol/L and single-agent pracinostat at concentrations of 0，15. 63，31. 25，62. 5，125，250，500，1 000 and 2 000 nmol/L for 24，48，and 72 h. The CellTiter-Glo assay was used to detect the inhibition of cell proliferation，and the half-maximal inhibi? tory concentration（IC50）was calculated. （2）The NU-DUL-1 and SU-DHL-6 cells were treated with zanubrutinib at con? centrations of 0，5，7. 5，10，12. 5 and 15 μmol/L and pracinostat at concentrations of 0，15. 62，31. 25，62. 5，125 and 250 nmol/L，alone or in combination，for 48 h. The CellTiter-Glo assay was used to detect the inhibition of cell prolifera? tion，and the synergy index of the combination was calculated through zero interaction potency（ZIP）model on the Syner? gyFinder（https：//synergyfinder. fimm. fi）website. （3）The NU-DUL-1 cells were treated with zanubrutinib（0 and 20 μmol/L）and pracinostat（0 and 200 nmol/L），alone or in combination，for 48 h. The SU-DHL-6 cells were treated with zanubrutinib（0 and 20 μmol/L）and pracinostat（0 and 150 nmol/L），alone or in combination，for 48 h. Flow cytometry was used to detect the cell apoptosis. （4）The NU-DUL-1 cells were treated with zanubrutinib at concentrations of 0，10 and 20 μmol/L and pracinostat at concentrations of 0，100 and 200 nmol/L，alone or in combination，for 48 h. The SU-DHL-6 cells were treated with zanubrutinib at concentrations of 0，10 and 20 μmol/L and pracinostat at concentrations of 0，150 and 300 nmol/L，alone or in combination，for 48 h. Western blot analysis was used to detect the expression of apoptotic proteins，including poly（ADP-ribose）polymerase 1（PARP1），cleaved PARP1，caspase-3，cleaved caspase-3，caspase-8，and cleaved caspase-8. RESULTS：（1）Zanubrutinib and pracinostat can inhibit NU-DUL-1 and SU-DHL-6 cell proliferation in vitro，and exhibit time- and dose-dependent effects. （2）The combination of zanubrutinib and pracino? stat showed significant synergistic effects in NU-DUL-1 cells（synergy index，19. 553>10）and SU-DHL-6 cells（synergy index，19. 392>10）.（3）The combination of zanubrutinib and pracinostat can enhance cell apoptosis compared with the control group，the proportion of apoptotic cells in the combination group is significantly increased（P<0. 05）than that in the control and single-drug groups. （4）Western blot results showed that zanubrutinib combined with pracinostat increased the expression of apoptotic proteins cleaved caspase-3，cleaved caspase-8 and cleaved PARP1 in NU-DUL-1 and SU-DHL-6 cells. The relative expression of apoptotic protein in combination group was significantly increased compared with control group and monotherapy group（P<0. 05）. CONCLUSION：The combination of zanubrutinib and pracinostat can signifi? cantly inhibit the proliferation of NU-DUL-1 and SU-DHL-6 cells and promote cell apoptosis，exerting a synergistic effect by inducing cell apoptosis through increasing the cleavage of apoptotic proteins caspase-3，caspase-8 and PARP1. ? 2023 Editorial Board of Chinese Journal of Pathophysiology, Jinan University.
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