ABSTRACT We previously reported that hydrangenol has potent antitumor activity against human bladder cancer EJ cells. Here, we investigated the antiangiogenic activity of hydrangenol using in vitro and ex vivo models. Treatment with hydrangenol significantly inhibited the proliferation of vascular endothelial growth factor (VEGF)-induced HUVECs in a concentration-dependent manner (EC50 = 10 μM). Flow cytometry analysis revealed that hydrangenol suppressed the VEGF-induced inhibition of G1-cell cycle phase and also decreased cyclin D1, cyclin E, CDK2, and CDK4 levels. Hydrangenol-mediated arrest in the G1-cell cycle phase was associated with p27KIP1 level, but not p21WAF1 or p53 level. Hydrangenol also significantly inhibited VEGFR-2-mediated signaling pathways including ERK1/2, AKT, and endothelial nitric oxide synthase. Interestingly, immunoprecipitation assay demonstrated that the inhibition of VEGFR-2 activation was independent of VEGF binding, thereby suggesting an allosteric regulation of hydrangenol against VEGFR-2. Additionally, hydrangenol inhibited migration, invasion, and capillary-like tubular formation in VEGF-stimulated HUVECs. Zymography and immunoblot analyses revealed that these inhibitory activities were partially owing to the VEGF-induced inhibition of matrix metalloproteinase-2 activity. Finally, VEGF-mediated microvessel sprouting was inhibited in the presence of hydrangenol in ex vivo aortic ring assay. Taken together, hydrangenol possesses a potent antiangiogenesis potential; thus we believe that hydrangenol may be developed as a therapeutic reagent to treat angiogenesis-mediated diseases.

中文翻译：

---

Hydrangenol 通过靶向 p27KIP1 依赖性 G1 细胞周期停滞、VEGFR-2 介导的信号传导和 MMP-2 表达来抑制 VEGF 刺激的血管生成

摘要 我们之前报道过，Hydrangenol 对人膀胱癌 EJ 细胞具有有效的抗肿瘤活性。在这里，我们使用体外和离体模型研究了绣球醇的抗血管生成活性。用绣球花醇处理以浓度依赖性方式显着抑制血管内皮生长因子 (VEGF) 诱导的 HUVEC 的增殖 (EC50 = 10 μM)。流式细胞术分析显示，水螅醇抑制了 VEGF 诱导的 G1 细胞周期期抑制，并降低了细胞周期蛋白 D1、细胞周期蛋白 E、CDK2 和 CDK4 的水平。在 G1 细胞周期阶段，Hydrangenol 介导的停滞与 p27KIP1 水平相关，但与 p21WAF1 或 p53 水平无关。Hydrangenol 还显着抑制 VEGFR-2 介导的信号通路，包括 ERK1/2、AKT 和内皮一氧化氮合酶。有趣的是，免疫沉淀试验表明，VEGFR-2 激活的抑制作用与 VEGF 结合无关，从而表明对 VEGFR-2 的 hydrangenol 的变构调节。此外，在 VEGF 刺激的 HUVEC 中，绣球醇抑制迁移、侵袭和毛细血管样小管形成。酶谱和免疫印迹分析表明，这些抑制活性部分是由于 VEGF 诱导的基质金属蛋白酶 2 活性的抑制。最后，在离体主动脉环试验中，在存在绣球花醇的情况下，VEGF 介导的微血管发芽受到抑制。综上所述，Hydrangenol 具有强大的抗血管生成潜力。因此，我们相信可以将 hydrangenol 开发为治疗血管生成介导的疾病的治疗剂。