Vessel-Organ Interaction Research Center, VOICE (MRC), College of Pharmacy, Kyungpook National University, Daegu, 41566, Republic of Korea; BK21 FOUR Community-Based Intelligent Novel Drug Discovery Education Unit, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 41566, Republic of Korea.
Graduate School of Biotechnology and Department of Oriental Medicinal Biotechnology, Kyung Hee University, Yongin 17104, Korea.
Phytomedicine. 2023 Jan;108:154513. doi: 10.1016/j.phymed.2022.154513. Epub 2022 Oct 27.
Hypoxia is a characteristic feature of many solid tumors. As an adaptive response to hypoxia, tumor cells activate hypoxia-inducible factor-1α (HIF-1α). Under hypoxic conditions, angiogenesis mediated by HIF-1α is involved in the growth and metastasis of tumor cells. During the angiogenic process, differentiated tip endothelial cells (ECs) characterized by high expression of DLL4 promote angiogenic germination through filopodia. Inhibitors of HIF-1α or DLL4 have been widely studied PURPOSE: We tried to find inhibitors targeting both HIF-1α and DLL4 in tumor which have not yet been developed.
In this study, we examined a natural compound that inhibits sprouting angiogenesis and tumor growth by targeting both HIF-1α and DLL4 under hypoxic conditions.
After examining cell viability of 70 selected natural compounds, we assessed the effects of compounds on HIF-1α and DLL4 transcriptional activity using a dual-luciferase reporter assay. Western blot analysis, immunofluoresecnt assay and real-time qPCR were performed to identify expression of proteins, such as HIF-1α and DLL4, as well as HIF-1α target genes under hypoxic conditions. In vitro angiogenesis assay and in vivo allograft tumor experiment were performed to investigate inhibition of tumor growth through anti-angiogenic activity.
Among these compounds, steppogenin, which is extracted from the root bark of Morus alba l, respectively inhibited the transcriptional activity of HIF-1α under hypoxic conditions in HEK293T cells and vascular endothelial growth factor (VEGF)-induced DLL4 expression in vascular ECs in a dose-dependent manner. In tumor cells and retinal pigment epithelial cells, steppogenin significantly suppressed HIF-1α protein levels under hypoxic conditions as well as VEGF-induced DLL4 expression in ECs. Furthermore, steppogenin suppressed hypoxia-induced vascular EC proliferation and migration as well as VEGF-induced sprouting of EC spheroids.
These results suggest that the natural compound steppogenin could potentially be used to treat angiogenic diseases, such as those involving solid tumors, because of its dual inhibition of HIF-1α and DLL4.
缺氧是许多实体瘤的特征。肿瘤细胞作为对缺氧的适应性反应，激活缺氧诱导因子-1α（HIF-1α）。在缺氧条件下，由 HIF-1α介导的血管生成参与肿瘤细胞的生长和转移。在血管生成过程中，高表达 DLL4 的分化尖端内皮细胞（ECs）通过丝状伪足促进血管生成发芽。HIF-1α 或 DLL4 的抑制剂已被广泛研究。
我们试图寻找尚未开发的针对肿瘤中 HIF-1α 和 DLL4 的双重抑制剂。
在这项研究中，我们研究了一种天然化合物，该化合物通过靶向缺氧条件下的 HIF-1α 和 DLL4 来抑制发芽血管生成和肿瘤生长。
在检查了 70 种选定的天然化合物的细胞活力后，我们使用双荧光素酶报告基因检测法评估了化合物对 HIF-1α 和 DLL4 转录活性的影响。Western blot 分析、免疫荧光测定和实时 qPCR 用于鉴定蛋白质的表达，例如 HIF-1α 和 DLL4 以及 HIF-1α 靶基因在缺氧条件下。进行体外血管生成实验和体内同种异体肿瘤实验，以通过抗血管生成活性研究抑制肿瘤生长。
在这些化合物中，从桑树根皮中提取的 steppogenin 分别以剂量依赖性方式抑制了 HEK293T 细胞中缺氧条件下的 HIF-1α 转录活性和血管内皮生长因子（VEGF）诱导的血管 EC 中 DLL4 的表达。在肿瘤细胞和视网膜色素上皮细胞中，steppogenin 在缺氧条件下显著抑制 HIF-1α 蛋白水平以及 VEGF 诱导的 EC 中 DLL4 的表达。此外，steppogenin 抑制了缺氧诱导的血管 EC 增殖和迁移以及 VEGF 诱导的 EC 球体发芽。
这些结果表明，天然化合物 steppogenin 由于其对 HIF-1α 和 DLL4 的双重抑制作用，可能潜在地用于治疗血管生成性疾病，例如涉及实体瘤的疾病。
