The Research Center of Chiral Drugs, Innovation Research Institute of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; Shanghai Frontiers Science Center for Traditional Chinese Medicine Chemical Biology, Innovation Research Institute of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201203, China.
The Research Center of Chiral Drugs, Innovation Research Institute of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; Shanghai Frontiers Science Center for Traditional Chinese Medicine Chemical Biology, Innovation Research Institute of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201203, China.
Biochem Pharmacol. 2024 Sep;227:116458. doi: 10.1016/j.bcp.2024.116458. Epub 2024 Aug 3.
Vasculogenic mimicry (VM) serves as a vascular-like channel that provides important substances for tumor growth and is a primary factor in glioblastoma (GBM) drug resistance. Human Antigen R (HuR)-an mRNA-binding protein-is highly expressed in GBM, closely related to tumor progression, and deemed a potential drug target. Although some small-molecule compounds have been identified to disrupt HuR binding to target mRNA, they remain in the preclinical research stage, suggesting the need for further validation and development of HuR inhibitors. In our study, we aim to screen for potential HuR inhibitors and investigate their efficacy and molecular mechanisms in GBM. We employed the fluorescence polarization method to identify HuR inhibitors from a natural compound library, confirming the efficacy of juglone in effectively inhibiting the binding of HuR to ARE. Further validation of the binding of juglone to HuR at the protein level was conducted through electrophoretic mobility shift analysis, surface plasmon resonance, and molecular docking. Furthermore, juglone demonstrated inhibitory effects on glioma growth and VM formation in vitro and in vivo. Moreover, it was observed that juglone reversed epithelial-mesenchymal transition by inhibiting the VEGF-A/VEGFR2/AKT/SNAIL signaling pathway. Finally, we established the capability of juglone to target HuR in U251 cells through HuR knockdown, mRNA stability, and cell thermal shift assays. Therefore, this study identifies juglone as a novel HuR inhibitor, potentially offering promise as a lead compound for anti-VM therapy in GBM by targeting HuR. Abbreviations: AKT, protein kinase B; ARE, adenine-and uridine-rich elements; CETSA, cellular thermal shift assay; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme linked immune sorbent assay; EMSA, electrophoretic mobility shift assay; EMT, epithelial mesenchymal transition; FP, fluorescence polarization; GBM, glioblastoma; HTS, high-throughput screening; HuR, human antigen R; IF, Immunofluorescence; PAS, periodic acid-Schiff; PI3K, phosphoinositide-3 kinase; qRT-PCR, quantitative real-time PCR; RRMs, RNA recognition motifs; SPR, surface plasmon resonance. TMZ, temozolomide; VM, vasculogenic mimicry; VEGF-A, Vascular endothelial growth factor-A; VEGFR2, Vascular endothelial growth factor receptor-2.
血管生成拟态 (VM) 作为一种类似血管的通道，为肿瘤生长提供重要物质，是胶质母细胞瘤 (GBM) 耐药的主要因素。人类抗原 R (HuR)-一种 mRNA 结合蛋白-在 GBM 中高度表达，与肿瘤进展密切相关，被认为是潜在的药物靶点。虽然已经鉴定出一些小分子化合物可以破坏 HuR 与靶 mRNA 的结合，但它们仍处于临床前研究阶段，这表明需要进一步验证和开发 HuR 抑制剂。在我们的研究中，我们旨在筛选潜在的 HuR 抑制剂，并研究其在 GBM 中的疗效和分子机制。我们采用荧光偏振法从天然化合物库中鉴定 HuR 抑制剂，证实胡桃醌能有效抑制 HuR 与 ARE 的结合。通过电泳迁移率变动分析、表面等离子体共振和分子对接进一步验证了胡桃醌与 HuR 蛋白的结合。此外，胡桃醌在体外和体内均显示出抑制神经胶质瘤生长和 VM 形成的作用。此外，观察到胡桃醌通过抑制 VEGF-A/VEGFR2/AKT/SNAIL 信号通路逆转上皮间质转化。最后，我们通过 HuR 敲低、mRNA 稳定性和细胞热转移测定，在 U251 细胞中证实了胡桃醌靶向 HuR 的能力。因此，本研究鉴定胡桃醌为一种新型 HuR 抑制剂，通过靶向 HuR，可能为 GBM 的抗 VM 治疗提供一种有前途的先导化合物。缩写：AKT，蛋白激酶 B；ARE，腺嘌呤和尿嘧啶丰富元件；CETSA，细胞热转移测定；DMEM，杜尔贝科改良伊格尔培养基；ELISA，酶联免疫吸附测定；EMSA，电泳迁移率变动分析；EMT，上皮间质转化；FP，荧光偏振；GBM，胶质母细胞瘤；HTS，高通量筛选；HuR，人类抗原 R；IF，免疫荧光；PAS，过碘酸希夫染色；PI3K，磷酸肌醇 3-激酶；qRT-PCR，实时定量 PCR；RRMs，RNA 识别基序；SPR，表面等离子体共振；TMZ，替莫唑胺；VM，血管生成拟态；VEGF-A，血管内皮生长因子 A；VEGFR2，血管内皮生长因子受体 2。
