Ibrutinib抑制弥漫大B细胞淋巴瘤细胞生存的作用研究

Ibrutinib inhibits diffuse large B-cell lymphoma cell survival

目的:探讨Bruton酪氨酸激酶(Bruton's tyrosine kinase,BTK)抑制剂ibrutinib抑制弥漫大B细胞淋巴瘤(diffuse large B cell lymphoma,DLBCL)细胞生存的作用及其相关机制。方法:用不同浓度的ibrutinib处理DLBCL细胞系SUDHL-10、HBL-1和患者原代细胞,以MTT法检测细胞的增殖抑制情况;以Annexin V/PI流式细胞术和DAPI染色法检测细胞的凋亡情况;应用Western blot法检测细胞表达磷酸化BTK、AKT、ERK的变化。DLBCL细胞与MSC共培养后,体外克隆形成实验和NOD/SCID肿瘤模型小鼠检测ibrutinib在肿瘤微环境里对DLBCL细胞的抑制作用。结果:2.5μmol/L及更高浓度的ibrutinib对DLBCL细胞的增殖有明显的抑制作用,且呈剂量依赖性。1.0、2.5μmol/L ibrutinib作用于SUDHL-10细胞24 h,细胞凋亡率分别为(21.73±3.64)%和(34.71±2.36)%,高于对照组(3.55±1.89)%(P<0.05)。5、10μmol/L ibrutinib处理24 h后,DLBCL细胞系均出现核皱缩(5μmol/L)、碎裂(10μmol/L)。ibrutinib处理细胞后磷酸化BTK、AKT、ERK的表达均明显降低。ibrutinib抑制共培养时DLBCL细胞的体外克隆形成(P<0.01)及DLBCL细胞在体内的增殖生长,差异均具有统计学意义(P<0.05)。结论:ibrutinib可抑制细胞系SUDHL-10和HBL-1的增殖,诱导凋亡,其机制可能通过阻断AKT、ERK信号途径而实现;在肿瘤微环境中ibrutinib同样对DLBCL细胞具有较强的抑制生存的作用,该药物有望为DLBCL的治疗带来希望。

Objective： To illustrate the effect and mechanism of ibrutinib, a Bruton＇s tyrosine kinase（BTK） inhibitor that inhibits diffuse large B-cell lymphoma（DLBCL） cell survival. Methods： DLBCL cell lines SUDHL-10 and HBL-1 were treated with ibrutinib at different concentrations. A MTT assay was used to detect the inhibition of cell proliferation. Cell apoptosis was analyzed by Annexin V-binding assay, as well as flow cytometry and DAPI staining. The expression of phosphorylated BTK, AKT and ERK was detected by Western blot.DLBCL cells were co-cultured with MSC. The inhibitory effect of ibrutinib on DLBCL cells in tumor microenvironment was assessed in clonogenicity in vitro and in a tumor-bearing non-obese diabetic/severe combined immunodeficient mice in vivo. Results： Up to 2.5μmol/L and high concentrations of ibrutinib significantly inhibited the proliferation of DLBCL cells in a dose-dependent manner. Approximately 1 and 2.5 μmol/L ibrutinib was added on SUDHL-10 cells for 24 h, and the cell apoptotic rates were（21.73±3.64）% and（34.71±2.36）%, respectively. Both were superior to that of the control group（3.55±1.89）%（P0.05）. Both two DLBCL cell lines pretreated with5 and 10 μmol/L ibrutinib for 24 h and exhibited nuclear shrinkage at 5 μmol/L and nuclear fragmentation at 10 μmol/L. The expression of phosphorylated BTK, AKT, and ERK decreased significantly after ibrutinib treatment. Ibrutinib inhibited clonogenicity in vitro（P0.01） and cell proliferation and growth in vivo of DLBCL cells in co-culture system. The differences were statistically significant. Conclusion： Ibrutinib can inhibit the proliferation and induce apoptosis of SUDHL-10 and HBL-1 cell lines through a mechanism of blocking the AKT and ERK signaling pathways, as well as the proliferation of DLBCL cells in tumor microenvironment. This finding can significantly benefit DLBCL treatment.