目标:最近,喹唑啉衍生物由于其独特的抗肿瘤作用而引起了人们的极大兴趣。在这项研究中,我们旨在研究活性三氟甲基化喹唑啉衍生物KZL204的作用,在人类多形性胶质母细胞瘤(GBM)细胞系U251MG上。此外,我们试图确定KZL204治疗GBM的潜在靶点。
方法:细胞计数试剂盒-8(CCK-8)检测细胞毒性,5-乙炔基-2-脱氧尿苷(EdU)染色细胞增殖,流式细胞术检测细胞凋亡和细胞周期,细胞迁移的伤口划痕试验,将U251MG细胞暴露于不同浓度的KZL204后,进行细胞侵袭的transwell测定。此外,蛋白质印迹分析,基于网络药理学的分析,分子对接测定,细胞热转移测定(CETSA),进行环己酰亚胺追踪测定。
结果:我们的结果显示KZL204浓度依赖性地抑制U251MG细胞增殖,诱导细胞凋亡,细胞周期停滞在G2/M期,并抑制细胞的侵袭和迁移能力。基于网络药理学的进一步分析显示,表皮生长因子受体(EGFR),FYN,YES1LYN,ephrinA型受体2(EPHA2),EPHA4是抑制细胞生长的前6个核心靶标,凋亡,细胞周期,和GBM细胞的转移。分子对接和CETSA显示KZL204与EPHA2具有强的靶向结合亲和力。环己酰亚胺追踪实验和蛋白质印迹结果表明,KZL204可以下调EPHA2的蛋白水平。
结论:KZL204对多形性胶质母细胞瘤细胞具有有效的抑制活性,这可能与其促进EPHA2降解的作用有关。
OBJECTIVE: Recently, there has been much interest in quinazoline derivatives due to their unique anti-tumor effects. In this study, we aimed to investigate the effects of KZL204, an active trifluoromethylated quinazoline derivative, on a human glioblastoma multiforme (GBM) cell line U251MG. Additionally, we tried to identify the potential target of KZL204 for treating GBM.
METHODS: Cell counting kit-8 (CCK-8) assay for cytotoxicity, 5-ethynyl-2-deoxyuridine (EdU) staining for cell proliferation, flow cytometry for cell apoptosis and cell cycle, wound scratch test for cell migration, and transwell assay for cell invasion were carried out on U251MG cells after exposing them to different concentrations of KZL204. In addition, western blot analysis, network pharmacology-based analysis, molecular docking assay, cellular thermal shift assay (CETSA), and
cycloheximide chase assay were performed.
RESULTS: Our results showed that KZL204 concentration-dependently inhibited U251MG cell proliferation, induced apoptosis, arrested cell cycle in the G2/M phase, and inhibited cell invasion and migration capacity. Further network pharmacology-based analysis revealed that epidermal growth factor receptor (EGFR), FYN, YES1, LYN, ephrin type-A receptor 2 (EPHA2), and EPHA4 are the top 6 core targets for inhibiting cell growth, apoptosis, cell cycle, and metastasis of the GBM cells. Molecular docking and CETSA showed that KZL204 had a strong targeting binding affinity with EPHA2.
Cycloheximide chase assay and western blot results demonstrated that KZL204 could down-regulate the protein level of EPHA2.
CONCLUSIONS: KZL204 exhibits potent inhibitory activity for glioblastoma multiforme cells, which may be related to its role in promoting the degradation of EPHA2.