Chk1抑制剂逆转TP53突变型乳腺癌细胞的顺铂耐药

Chk1 Inhibitor Reverses Cisplatin Resistance of TP53 Mutant Breast Cancer Cells

目的研究细胞周期检查点激酶1抑制剂(cell cycle checkpoint kinase 1 inhibitor,Chk1 i)AZD7762对三阴性乳腺癌(TNBC)细胞的作用,以及AZD7762逆转TNBC细胞对顺铂的耐药性及其机制。方法将AZD7762孵育BT-549、HCC70、MDA-MB-231、T47D和MCF-7细胞,利用CCK8法检测细胞存活率。构建MCF-7-shp53细胞系,利用CCK8和集落形成实验检测细胞MCF-7-shp53与野生型细胞的增殖能力,利用流式细胞术与膜联蛋白-Ⅴ染色法检测两组细胞凋亡与细胞周期情况。将AZD7762和顺铂加入两组细胞联合孵育72 h,利用Chou-Talalay方程计算两种化合物的协同效应,并利用免疫印迹法检测相关蛋白表达。结果通过评估AZD7762对5种TNBC细胞活力的影响,发现MCF-7是最具AZD7762耐药性的细胞系。TNBC对AZD7762的敏感性依赖于TP53状态。探讨AZD7762对TNBC细胞毒性作用的潜在机制:AZD7762终止TP53突变细胞S期和G2/M期阻滞,通过促进DNA损伤和凋亡导致细胞死亡。AZD7762联合顺铂作用可以逆转TP53突变型乳腺癌细胞对顺铂的耐药性,并可诱导TP53突变型乳腺癌细胞有丝分裂改变。结论AZD7762通过促进DNA损伤、凋亡、终止G2/M期阻滞和诱导有丝分裂改变而导致细胞死亡。对AZD7762的敏感性取决于TNBC细胞中的TP53状态。此外,AZD7762逆转TNBC细胞对顺铂的耐药性,Chk1 i是治疗TNBC患者的潜在药物。

Objective To investigate the sensitivity of triple-negative breast cancer cells(TNBCs)to cell cycle checkpoint kinase 1(Chk1)inhibitor AZD7762 and its mechanism of reversing cisplatin resistance of TNBCs.Methods BT-549,HCC70,MDA-MB-231,T47 D and MCF-7 cells were incubated with AZD7762,and the cell survival rates were detected by CCK8,which resulted in the construction of MCF-7-shp53 cell line.CCK8 and colony formation assay were used to detect the proliferation of MCF-7-shp53 and wild-type cells.Flow cytometry and annexin-Ⅴstaining were used to detect the cell apoptosis and cell cycle.Afterwards,the two groups of cells were added with AZD7762 and cisplatin,and then incubated for 72 h.The synergistic effects of these two drugs were calculated by Chou-Talalay equation,and the expression of related proteins was detected by Western blotting.Results The evaluation of the effect of AZD7762 on cell viability of five TNBCs cell lines revealed that MCF-7 was the most resistant type.The sensitivity of TNBCs to AZD7762 depends on TP53 status.The potential mechanism of the cytotoxicity of AZD7762 on TNBCs was explored.AZD7762 blocked the S phase and G2/M phase arrest of TP53 mutant cells,and induced cell death by promoting DNA damage and apoptosis.The cisplatin resistance of TP53 mutant breast cancer cells was reversed by the synergistic effects of AZD7762 and cisplatin.And the mitotic mutation was also induced.Conclusion AZD7762 induces cell death by promoting DNA damage and apoptosis,blocking G2/M cell cycle arrest,and inducing mitotic mutation.The sensitivity of TNBCs to AZD7762 depends on TP53 status.Moreover,AZD7762 reverses the cisplatin resistance of TNBCs cells,indicating Chk1 inhibitor could serve as a potential therapeutic drug for TNBC patients.