OBJECTIVE: To investigate the effect of VX-509 on CCSCs and elucidate the underlying mechanism.
METHODS: CCSCs were enriched from CRC cell lines by in conditioned serum-free medium. Western blot, Aldefluor, transwell and tumorigenesis assays were performed to verify the phenotypic characteristics of the CCSCs. The anticancer efficacy of VX-509 was assessed in HCT116 CCSCs and HT29 CCSCs by performing cell viability analysis, colony formation, sphere formation, flow cytometry, and western blotting assessments in vitro and tumor growth, immunohistochemistry and immunofluorescence assessments in vivo.
RESULTS: Compared with parental cells, sphere cells derived from HCT116 and HT29 cells presented increased expression of stem cell transcription factors and stem cell markers and were more potent at promoting migration and tumorigenesis, demonstrating that the CRC sphere cells displayed CSC features. VX-509 inhibited the tumor malignant biological behavior of CRC-stem-like cells, as indicated by their proliferation, migration and clonality in vitro, and suppressed the tumor of CCSC-derived xenograft tumors in vivo. Besides, VX-509 suppressed the CSC characteristics of CRC-stem-like cells and inhibited the progression of epithelial-mesenchymal transition (EMT) signaling in vitro. Nodal was identified as the regulatory factor of VX-509 on CRC stem-like cells through analyses of differentially expressed genes and CSC-related database information. VX-509 markedly downregulated the expression of Nodal and its downstream phosphorylated Smad2/3 to inhibit EMT progression. Moreover, VX-509 reversed the dedifferentiation of CCSCs and inhibited the progression of EMT induced by Nodal overexpression.
CONCLUSIONS: VX-509 prevents the EMT process in CCSCs by inhibiting the transcription and protein expression of Nodal, and inhibits the dedifferentiated self-renewal of CCSCs.
目的:探讨VX-509对CCSCs的作用及其机制。
方法:通过条件无血清培养基从CRC细胞系中富集CCSC。蛋白质印迹,Aldefluor,进行了transwell和肿瘤发生试验以验证CCSC的表型特征。通过进行细胞活力分析,在HCT116CCSCs和HT29CCSCs中评估了VX-509的抗癌功效。菌落形成,球体形成,流式细胞术,和蛋白质印迹评估体外和肿瘤生长,体内免疫组织化学和免疫荧光评估。
结果:与亲本细胞相比,来自HCT116和HT29细胞的球形细胞呈现干细胞转录因子和干细胞标志物的表达增加,并且在促进迁移和肿瘤发生方面更有效,证明CRC球体细胞显示CSC特征。VX-509抑制结直肠癌干细胞样细胞的肿瘤恶性生物学行为,正如它们的扩散所表明的那样,体外迁移和克隆性,并在体内抑制CCSC来源的异种移植肿瘤。此外,VX-509在体外抑制CRC干细胞样细胞的CSC特征并抑制上皮-间质转化(EMT)信号传导的进展。通过对差异表达基因和CSC相关数据库信息的分析,确定Nodal为VX-509对CRC干细胞样细胞的调节因子。VX-509显著下调Nodal及其下游磷酸化Smad2/3的表达以抑制EMT进展。此外,VX-509逆转了CCSCs的去分化,并抑制了Nodal过表达诱导的EMT的进展。
结论:VX-509通过抑制Nodal的转录和蛋白表达来阻止CCSCs中的EMT过程,抑制CCSCs的去分化自我更新。