伊布替尼联合BH3拟似物ABT737协同抗肿瘤作用及机制

Synergistic Anti-tumor Activity and Mechanism of Ibrutinib Combined with BH3 Analogue ABT737

关键词:  伊布替尼  ABT737  肝细胞  Sox2  CD44

Key words:  ibrutinib  ABT737  stem cell  Sox2  CD44

摘要:

目的 研究伊布替尼联合BH3拟似物ABT737的协同抗实体肿瘤作用及机制。方法 以2种类型实体肿瘤细胞，人非小细胞肺癌细胞株A549、H1299和人脑胶质瘤细胞U251、U87为对象，采用SRB法检测不同浓度伊布替尼(20，15，10，7.5，5 μmol·L^-1)和BH3拟似物ABT737(20，15，10，7.5，5 μmol·L^-1)单独或共同作用24 h后的细胞增殖情况，计算细胞存活率和合用指数；采用克隆形成试验检验10 μmol·L^-1伊布替尼与10 μmol·L^-1ABT737 联合作用120 h后的细胞克隆形成情况；成球试验检测两药联合作用7 d对细胞成球能力的影响；采用PI染色结合流式细胞术检测10 μmol·L^-1伊布替尼与10 μmol·L^-1ABT737联用对U87和U251细胞凋亡的影响；RT-PCR检测两药联合作用24 h后对U87和U251细胞中干细胞标记物Sox2、Nanog和Oct4 mRNA水平影响；Western blotting检测脑胶质瘤干细胞标记物CD44蛋白水平的表达。结果 不同浓度伊布替尼与BH3拟似物ABT737在非小细胞肺癌和脑胶质瘤中均有协同作用，合用组增殖抑制率均高于单用组(P<0.05)；两药合用可抑制细胞的克隆形成能力，协同诱导细胞凋亡；同时两者合用可抑制肿瘤干细胞标记物Sox2的mRNA表达水平，使CD44蛋白表达水平降低。结论 伊布替尼联合ABT737可协同抑制实体肿瘤细胞的增殖，促进凋亡发生，其机制可能是通过抑制肿瘤干细胞蛋白的表达。

Abstract:

OBJECTIVE To investigate the synergistic anti-tumor effect and mechanism of ibrutinib combined with BH3 analogue ABT737. METHODS Human non-small cell lung cancer cell lines A549 and H1299, and glioma cells U251 and U87 were used as subjects. SRB method was used to detect cell proliferation after 24 h of single or combined action of different concentrations of ibutinib(20, 15, 10, 7.5, 5 μmol·L^-1) and BH3 analogues ABT737(20, 15, 10, 7.5, 5 μmol·L^-1), cell survival rate and combined index was calculated. The clone formation assay was used to detect the characteristic of stemness after the combination treatment of 10 μmol·L^-1 ibrutinib and 10 μmol·L^-1 ABT737 for 120 h. Cell spheroidization test was used to investigate the effect of the combination treatment of two drugs on cell spheroidization ability for 7 d. PI staining together with flow cytometry was used to detected effect of the combination treatment of 10 μmol·L^-1ibrutinib and 10 μmol·L^-1ABT737 on apoptosis of U87 and U251 cells. RT-PCR was used to detect the effect of the combination treatment of the two drugs on the mRNA levels of stem cell markers Sox2, Nanog, and Oct4 in U87 and U251 cells for 24 h. Western blotting was used to detect the expression of brain glioma stem cell marker CD44 protein. RESULTS Different concentrations of ibrutinib and BH3 analogue ABT737 had synergistic effects in non-small cell lung cancer and glioma, and the combined group had a higher proliferation inhibition rate than the single group(P<0.05). The combination of two drugs could inhibit the ability of cell clone formation and synergistically induce cell apoptosis. At the same time, the combination of the two drugs could inhibit the mRNA expression level of tumor stem cell marker Sox2 and reduce the expression level of CD44 protein. CONCLUSION Ibrutinib combined with ABT737 can synergistically inhibit the proliferation of solid tumor cells and promote apoptosis, the mechanism is possibly by inhibiting the expression of tumor stem cell proteins.