衰老细胞清除剂ABT-263联合替莫唑胺治疗胶质母细胞瘤增敏作用及机制

Effect and mechanism of senescent cell scavenger ABT-263 combined with temozolomide in the treatment of glioblastoma

目的探究衰老细胞清除剂ABT-263联合替莫唑胺(TMZ)治疗胶质母细胞瘤的效果及作用机制。方法体外培养U87MG细胞系,分为对照组(不进行干预)、TMZ组和TMZ+ABT组。应用SA-β-gal染色检测细胞的衰老;应用MTT法检测细胞活力;应用EdU染色检测细胞增殖能力;应用划痕试验检测细胞迁移能力;应用蛋白印迹法检测不同药物处理组U87MG细胞中P53、P21、Bcl-2、Bax、PARP1和Caspase3等蛋白的表达量。应用转录组测序分析不同药物治疗组中U87MG细胞基因的表达差异。结果与TMZ(200μmol/L)组相比,TMZ(200μmol/L)+ABT(2μmol/L)组细胞衰老率显著下降(P<0.05),EdU阳性细胞比例显著降低(P<0.05),细胞迁移速率显著减慢(P<0.05)。与相同TMZ剂量单药给药组相比,TMZ(10μmol/L)+ABT(2μmol/L)低剂量组、TMZ(50μmol/L)+ABT(2μmol/L)中剂量组、TMZ(200μmol/L)+ABT(2μmol/L)高剂量组细胞P21蛋白表达水平均显著降低(P<0.05),细胞Bcl-2和Caspase3蛋白的表达水平均显著升高(P<0.05);低剂量联合给药组细胞P53蛋白表达水平显著降低(P<0.05),中、高剂量联合给药组细胞PARP1剪切体蛋白的表达水平显著升高(P<0.05),中、低剂量联合给药组细胞Bax蛋白的表达水平显著升高(P<0.05)。此外,TMZ(100μmol/L)+ABT(1μmol/L或2μmol/L)组和TMZ(100μmol/L)组相比,显著改变的差异表达基因共有312个,其中上调基因141个,下调基因171个,富集分析涉及多条炎症反应以及调控细胞过程相关的通路。结论ABT-263联合TMZ可以抑制U87MG细胞的增殖和迁移,在一定程度上清除由TMZ诱导的U87MG衰老细胞,干扰生长停滞肿瘤细胞的恢复能力,防止肿瘤复发。

Objective To investigate the effect and mechanism of senescent cell scavenger ABT-263 combined with temozolomide(TMZ)in the treatment of glioblastoma.Methods U87MG cells were cultured in vitro and were divided into the control group(without treatment),the TMZ group and the TMZ combined with ABT group.The senescence of U87MG cells was detected by SA-β-gal staining.MTT method was used to detect the cell viability.The proliferation ability of the cells was detected by EdU staining.The migration ability of the cells was measured by scratch test.The expression levels of P53,P21,Bcl-2,Bax,PARP1 and Caspase3 protein in the cells was detected by Western blot.RNA-seq was used to analyze the gene expression differences of U87MG cells in different drug treatment groups.Results Compared with the TMZ(200μmol/L)group,the cell senescence rate,the proportion of EdU-positve cells and migration rate were significantly decreased(P<0.05)in TMZ(200μmol/L)combined with ABT(2μmol/L)group.Compared with the single-drug administration group of the same TMZ dose,not only was the expression level of P21 protein in cells notably reduced(P<0.05),but also the expression level of Bcl-2 and Caspase3 protein were markedly increased in cells(P<0.05),in TMZ(10μmol/L)combined with ABT(2μmol/L)low-dose group,TMZ(50μmol/L)combined with ABT(2μmol/L)medium-dose group and TMZ(200μmol/L)combined with ABT(2μmol/L)high-dose group.In addition,the expression level of P53 protein was significantly decreased in the low-dose combination group(P<0.05),the expression level of PARP1 spliceosome protein was significantly increased in medium-dose and high-dose combination groups(P<0.05),and the expression level of Bax protein was significantly increased in low-does and medium-dose groups(P<0.05),in comparison with the same dose of TMZ group.Furthermore,312 differentially expressed genes were significantly changed in TMZ(100μmol/L)and ABT(1μmol/L or 2μmol/L)combination group including 141 up-regulated genes and 171 down-regulated genes,compared with the TMZ(100μmol/L)group.Enrichment analysis involved multiple pathways related to inflammatory response and regulation of cellular processes.Conclusion ABT-263 combined with TMZ can inhibit the proliferation and migration of U87MG cells,clear U87MG senescent cells induced by TMZ to some extent,interfere with the recovery ability of growth-arrested tumor cells,and prevent tumor recurrence.