Abstract:

Objective To investigate the anti-tumoral effect of ACT001, a novel antitumor compound, on the nuclear factor-κB (NF-κB) pathway and programmed cell death protein ligand 1 (PDL1) expression in U87 glioblastoma cell line. Methods We analyzed the correlation between the expression of P65 and PDL1, pathological grade, and prognosis by introducing The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) databases. CCK-8 was used to observe the effect of ACT001 on the proliferation of U87 cells, and calculate the median inhibition concentration (IC50). Immunofluorescence staining was used to detect the effect of ACT001 on nuclear translocation of the transcription factor P65. U87 cells were transfected with small interference RNA (siRNA), and P65 knockdown efficiency was determined by quantitative real-time polymerase chain reaction (qRT-PCR). The relative expression of P65, phospho-P65 (p-P65) and PDL1 in different transfected groups and drug treatment groups were determined by Western blotting. Results 1) Bioinformatics analysis showed that gliomas with higher pathological grade hourbored higher P65 and PDL1 expression (P<0.05, for all). Moreover, patients with high expression of P65 and PDL1 mRNA predicted poor prognosis (P<0.05, for all). 2) CCK-8 assay showed that treatment with different concentrations of ACT001 (5, 10, 20, 40, 80, 160 and 320 μmol/L), the inhibition rates of U87 cells were (9.01 ±4.75)%, (17.03 ±2.91)%, (28.50 ±4.85)%, (45.50 ±5.15)%, (67.67 ±8.46)%, (83.02 ±5.79)% and (94.33 ±1.59)%, respectively, and the IC50 was about 42.98 μmol/L. 3) Immunofluorescence staining showed that ACT001 inhibited nuclear translocation of P65. 4) qRT-PCR showed that the P65 mRNA expression of U87 cells in different siRNA transfected groups was statistically significant (F=164.200, P=0.000), and the mRNA expression of P65 in si-P65 -1 group (t=13.290, P=0.000) and si-P65-2 group (t=12.730, P=0.000) was lower than that of si-NC group. 5) Western blotting showed that the relative expression of P65 (F=681.300, P=0.000), p-P65 (F=128.800, P=0.000) and PDL1 (F=20.470, P=0.002) in U87 cells of different siRNA transfection groups were statistically significant different. Among them, P65 (t=59.630, P=0.000; t=29.380, P=0.000), p-P65 (t=24.370, P=0.000; t=13.460, P=0.000) and PDL1 (t=6.025, P=0.004; t=6.728, P=0.003) in si-P65-1 group and si-P65-2 group were lower than those in si-NC group. 6) There were statistically significant differences in the relative expression of p-P65 (F=269.700, P=0.000) and PDL1 (F=128.800, P=0.000) in U87 cells treated with different concentrations of drugs. P-P65 (t=19.750, P=0.000; t=24.640, P=0.000) and PDL1 (t=12.690, P=0.000; t=19.230, P=0.000) in ACT001 25 μmol/L group and 50 μmol/L group were lower than those in normal control group, ACT001 50 μmol/L group were also lower than those of 25 μmol/L group (t=5.288, P=0.006; t=2.868, P=0.046). Conclusions ACT001 can inhibit the proliferation of U87 glioma cells. By inhibiting the phosphorylation and nuclear translocation of P65 in U87 cells, ACT001 decreased the expression of PDL1 to improve the immunosuppressive environment of glioblastoma and thus inhibit the progression of glioblastoma.

Key words: Glioblastoma, Sesquiterpenes, NF-kappa B, Programmed cell death 1 receptor, Ligands, Cell proliferation, Transfection, Microscopy, fluorescence, Immunoblotting, Tumor cells, cultured

摘要：

目的 探讨新型抗肿瘤药物ACT001对胶质母细胞瘤细胞核因子-κB（NF-κB）信号转导通路活性和细胞程序性死亡蛋白配体1（PDL1）表达的影响。方法 采用生物信息学分析肿瘤基因组学图谱计划（TCGA）和中国脑胶质瘤基因组学图谱计划（CGGA）数据库中胶质瘤患者病理分级和预后与P65和PDL1 mRNA表达量的关系。CCK-8法观察ACT001对U87细胞增殖活性的影响并计算细胞抑制率和ACT001半数抑制浓度，免疫荧光法检测P65蛋白在U87细胞中的定位变化，小干扰RNA（siRNA）转染U87细胞，实时定量聚合酶链反应（qRT-PCR）检测P65蛋白敲低效率，Western blotting法检测不同转染组和不同浓度药物处理组P65、磷酸化P65（p-P65）和PDL1相对表达量。结果 （1）生物信息学分析显示，胶质瘤患者P65和PDL1 mRNA表达量随肿瘤病理分级的升高而呈上升趋势（均P<0.05），且P65和PDL1高表达组生存率和总生存期均低于低表达组（均P<0.05）。（2）细胞增殖活性检测显示，经不同药物浓度ACT001（5、10、20、40、80、160和320 μmol/L）处理后，U87细胞抑制率分别为（9.01±4.75）%、（17.03±2.91）%、（28.50±4.85）%、（45.50±5.15）%、（67.67±8.46）%、（83.02±5.79）%和（94.33±1.59）%，ACT001对U87细胞的半数抑制浓度为42.98 μmol/L。（3）免疫荧光法显示，经50 μmol/L ACT001处理的U87细胞P65蛋白入核减少。（4）qRT-PCR法显示，不同siRNA转染组U87细胞P65 mRNA表达量差异有统计学意义（F=164.200，P=0.000），其中si-P65-1组（t=13.290，P=0.000）和si-P65-2组（t=12.730，P=0.000）P65 mRNA表达量均低于si-NC组。（5）Western blotting法显示，不同siRNA转染组U87细胞P65（F=681.300，P=0.000）、p-P65（F=128.800，P=0.000）和PDL1（F=20.470，P=0.002）相对表达量差异有统计学意义，其中si-P65-1组和si-P65-2组P65（t=59.630，P=0.000；t=29.380，P=0.000）、p-P65（t=24.370，P=0.000；t=13.460，P=0.000）和PDL1（t=6.025，P=0.004；t=6.728，P=0.003）相对表达量均低于si-NC组。（6）不同浓度药物处理组U87细胞p-P65（F=269.700，P=0.000）和PDL1（F=128.800，P=0.000）相对表达量差异有统计学意义，其中ACT001 25 μmol/L组和50 μmol/L组p-P65（t=19.750，P=0.000；t=24.640，P=0.000）和PDL1（t=12.690，P=0.000；t=19.230，P=0.000）相对表达量均低于正常对照组，ACT001 50 μmol/L组亦低于25 μmol/L组（t=5.288，P=0.006；t=2.868，P=0.046）。结论 ACT001不仅可以抑制U87胶质瘤细胞的增殖，还可以通过抑制P65的磷酸化及核转位，从而抑制PDL1的表达，以改善胶质瘤的免疫抑制微环境，进而抑制胶质母细胞瘤进展。

关键词: 胶质母细胞瘤, 倍半萜类, NF-&kappa, B, 程序性细胞死亡受体1, 配体, 细胞增殖, 转染, 显微镜检查, 荧光, 免疫印迹法, 肿瘤细胞, 培养的