Steppogenin suppresses tumor growth and sprouting angiogenesis through inhibition of HIF-1α in tumors and DLL4 activity in the endothelium

Background: Hypoxia is a characteristic feature of many solid tumors. As an adaptive response to hypoxia, tumor cells activate hypoxia-inducible factor-1 alpha (HIF-1 alpha). Under hypoxic conditions, angiogenesis mediated by HIF-1 alpha 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-1 alpha or DLL4 have been widely studied Purpose: We tried to find inhibitors targeting both HIF-1 alpha and DLL4 in tumor which have not yet been developed. Study design: In this study, we examined a natural compound that inhibits sprouting angiogenesis and tumor growth by targeting both HIF-1 alpha and DLL4 under hypoxic conditions. Methods: After examining cell viability of 70 selected natural compounds, we assessed the effects of compounds on HIF-1 alpha 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-1 alpha and DLL4, as well as HIF-1 alpha 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. Results: Among these compounds, steppogenin, which is extracted from the root bark of Morus alba l, respectively inhibited the transcriptional activity of HIF-1 alpha under hypoxic conditions in HEK293T cells and vascular endo-thelial 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-1 alpha 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. Conclusion: 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-1 alpha and DLL4.