背景:PD-1/PD-L1抑制剂BMS-1166,抑制PD-L1与PD-1的结合,恢复T细胞功能,增强肿瘤免疫反应。然而,肿瘤抑制基因的突变或受损的细胞信号通路也可能导致细胞转化。在这项研究中,使用SW480和SW480R细胞系作为模型,阐明BMS-1166,BEZ235及其组合的治疗.方法:采用MTT法和集落形成法评价细胞增殖。伤口愈合测定用于评估细胞迁移。通过流式细胞术分析细胞周期和细胞凋亡。PI3K/Akt/mTOR和MAPK通路中关键激酶的磷酸化水平,PD-L1和与增殖相关的蛋白质水平,迁移,使用蛋白质印迹法评估细胞凋亡。结果:BEZ235增强BMS-1166介导的SW480和SW480R细胞增殖和迁移抑制作用,促进细胞凋亡。有趣的是,负调节剂PTEN的下调提高了PD-L1水平,它被Akt的抑制所废除。BMS-1166推广了PI3K,Akt,mTOR,和Erk磷酸化。然而,BEZ235与BMS-1166的组合抑制PI3K的表达,p-Akt,p-mTOR,通过抑制PD1与PD-L1的结合,与BMS-1166或BEZ235单次处理相比,SW480和SW480R细胞中的p-Erk。结论:PD-1与PD-L1结合并激活PI3K/mTOR和MAPK通路,这可能是CRC对BMS-1166获得性耐药的分子机制。两种药物联合抑制PI3K的磷酸化,Akt,和Erk在PI3K/mTOR和MAPK通路中,即,BEZ235通过阻断PI3K/mTOR通路和干扰MAPK通路的串扰增强BMS-1166治疗效果。因此,本研究结果为BMS-1166联合BEZ235治疗结直肠癌提供了理论依据。
Background: BMS-1166, a PD-1/PD-L1 inhibitor, inhibits the binding of PD-L1 to PD-1, restores T cell function, and enhances tumor immune response. However, mutations in the tumor suppressor or impaired cellular signaling pathways may also lead to cellular transformation. In this study, the SW480 and SW480R cell lines were used as the model to elucidate the treatment with BMS-1166, BEZ235, and their combination. Methods: MTT and colony-formation assays were used to evaluate cell proliferation. Wound-healing assay was used to assess cell migration. Cell cycle and apoptosis were analyzed by flow cytometry. The phosphorylation level of the key kinases in the PI3K/Akt/mTOR and MAPK pathways, PD-L1, and the protein levels related to the proliferation, migration, and apoptosis were assessed using western blotting. Results: BEZ235 enhanced BMS-1166-mediated cell proliferation and migration inhibition in SW480 and SW480R cells and promoted apoptosis. Interestingly, the downregulation of the negative regulator PTEN raised the PD-L1 level, which was abolished by the inhibition of Akt. BMS-1166 promoted PI3K, Akt, mTOR, and Erk phosphorylation. However, the combination of BEZ235 with BMS-1166 suppressed the expression of PI3K, p-Akt, p-mTOR, and p-Erk in SW480 and SW480R cells compared to BMS-1166 or BEZ235 single treatment by inhibiting the binding of PD1 to PD-L1. Conclusions: PD-1 binds to PD-L1 and activates the PI3K/mTOR and MAPK pathways, which might be the molecular mechanism of acquired resistance of CRC to BMS-1166. The combination of the two drugs inhibited the phosphorylation of PI3K, Akt, and Erk in the PI3K/mTOR and MAPK pathway, i.e., BEZ235 enhanced the BMS-1166 treatment effect by blocking the PI3K/mTOR pathway and interfering with the crosstalk of the MAPK pathway. Therefore, these findings provide a theoretical basis for BMS-1166 combined with BEZ235 in the trial treatment of colorectal cancer.