两种新型的BTK和HDAC抑制剂在弥漫性大B细胞淋巴瘤细胞中的协同抗肿瘤作用

Synergistic anti-tumor effect of two novel BTK and HDAC inhibitors in diffuse large B-cell lymphoma cells

目的:探索两种新型的布鲁顿激酶(BTK)抑制剂泽布替尼(zanubrutinib)和组蛋白脱乙酰酶(HDAC)抑制剂pracinostat在弥漫性大B细胞淋巴瘤(DLBCL)细胞中是否具有协同抗肿瘤作用。方法:(1)用浓度为0、2.5、5、10、20、40、80、160μmol/L的单药泽布替尼和浓度为0、15.63、31.25、62.5、125、250、500、1 000、2 000nmol/L的单药pracinostat分别处理NU-DUL-1(ABC型人弥漫性大B淋巴瘤细胞)和SU-DHL-6(GCB型人弥漫性大B淋巴瘤细胞)细胞24、48和72 h后,采用CellTiter-Glo法检测药物对细胞的增殖抑制率,并计算半数抑制浓度(IC_(50))。(2)用浓度为0、5、7.5、10、12.5、15μmol/L的泽布替尼和浓度为0、15.62、31.25、62.5、125、250 nmol/L的pracinostat单药或联合处理NU-DUL-1细胞和SU-DHL-6细胞48 h后,采用CellTiter-Glo法检测药物对细胞的增殖抑制率,在SynergyFinder(https://synergyfinder. fimm. fi)网站选用零相互作用效价模型计算联合用药的协同指数。(3)浓度为0、20μmol/L的泽布替尼和浓度为0、200 nmol/L的pracinostat单药或联合处理NU-DUL-1细胞,浓度为0、20μmol/L的泽布替尼和浓度为0、150 nmol/L的pracinostat单药或联合处理SU-DHL-6细胞,48 h后采用流式细胞术检测药物对细胞凋亡的影响。(4)浓度为0、10、20μmol/L的泽布替尼和浓度为0、100、200 nmol/L的pracinostat单药或联合处理NU-DUL-1细胞,浓度为0、10、20μmol/L的泽布替尼和浓度为0、150、300 nmol/L的pracinostat单药或联合处理SUDHL-6细胞,采用Western blot法检测凋亡相关蛋白多腺苷二磷酸核糖聚合酶1(PARP1)、cleaved PARP1、caspase-3、cleaved caspase-3、caspase-8和cleaved caspase-8的表达。结果:(1)泽布替尼和pracinostat可在体外抑制NU-DUL-1细胞和SU-DHL-6细胞增殖且具有时间和剂量依赖性。(2)泽布替尼和pracinostat联用在NU-DUL-1细胞中协同指数为19.553(>10),在SU-DHL-6细胞中协同指数为19.392(>10),两药联合具有明显的协同效应。(3)泽布替尼和pracinostat联合可增强细胞凋亡,联合组细胞凋亡细胞比例较对照组和单药组显著增加(P<0.05)。(4)Western blot结果表明,泽布替尼和pracinostat联合增加NU-DUL-1和SU-DHL-6细胞中凋亡蛋白cleaved caspase-3、cleaved caspase-8和cleaved PARP1的表达,诱导细胞凋亡,联合组凋亡蛋白相对表达量较对照组和单药组显著增加(P<0.05)。结论:泽布替尼和pracinostat联合可显著抑制NU-DUL-1和SU-DHL-6细胞增殖并促进其凋亡,通过增加凋亡蛋白caspase-3、caspase-8和PARP1的剪切诱导细胞凋亡,发挥协同效应。

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 160µmol/L and single-agent pracinostat at concentrations of 0,15.63,31.25,62.5,125,250,500,1000 and 2000 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 15µmol/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 20µmol/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 20µmol/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 20µmol/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 20µmol/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.