Robustaflavone induces G0/G1 cell cycle arrest and apoptosis in human umbilical vein endothelial cells and exhibits anti-angiogenic effects in vivo

• Published in: Scientific reports 2020-07, Vol.10 (1), p.11070-11070, Article 11070
• Main Authors: Sim, Woo Kyung, Park, Jong-Hwa, Kim, Ki-Young, Chung, In Sik
• Format: Article
• Language: English
• Subjects: 631/154/433; 631/67/1059/99; Angiogenesis; Angiogenesis Inhibitors - pharmacology; Animal models; Animals; Apoptosis; Apoptosis - drug effects; Apoptosis-inducing factor; Biflavonoids - pharmacology; Caspase-3; Cell cycle; Cell Cycle Checkpoints - drug effects; Cell Proliferation; Colonic Neoplasms - blood supply; Colonic Neoplasms - drug therapy; Colonic Neoplasms - metabolism; Colonic Neoplasms - pathology; Cyclin D1; Cyclin-dependent kinase 4; Cytochrome c; Cytotoxicity; DNA nucleotidylexotransferase; Endothelial cells; Female; Flow cytometry; G1 Phase; Human Umbilical Vein Endothelial Cells - drug effects; Humanities and Social Sciences; Humans; Membrane potential; Metastases; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; multidisciplinary; Neovascularization, Pathologic - drug therapy; p53 Protein; Resting Phase, Cell Cycle; Ribose; Science; Science (multidisciplinary); Tumor Cells, Cultured; Tumors; Umbilical vein; Vascular endothelial growth factor; Xenograft Model Antitumor Assays
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-020-67993-5

We investigated the anti-angiogenic and pro-apoptotic effects of robustaflavone (RF), a naturally occurring biflavonoid, on human umbilical vein endothelial cells (HUVECs). RF inhibited HUVEC proliferation and showed cytotoxicity that inhibited HUVEC viability. RF-induced apoptosis was characterized by flow cytometry and caspase 3 analysis. We found that RF increased the number of sub-G1 cells and terminal deoxynucleotidyl transferase dUTP nick end-labeled cells. Additionally, RF induced caspase 3 and poly (ADP-ribose) polymerase activation. Potential molecular targets were identified using a human apoptosis antibody array. RF upregulated Bax, Bad, cleaved caspase 3, p21, and phosphorylated p53 levels. RF induced mitochondrial membrane potential loss and the release of cytochrome c and apoptosis-inducing factor. Cell cycle arrest at G0/G1 phase and the downregulation of Cdk4, Cdk6, and cyclin D1 expression were induced by RF. In vivo anti-angiogenic effects were investigated using a tumor allograft animal model and a Matrigel plug assay. RF reduced the volumes and weights of CT-26 cell-derived tumors. The blood vessel density was significantly decreased in RF-treated tumors. RF also inhibited VEGF-A-stimulated blood vessel formation in vivo in Matrigel plugs. These results suggest that RF can potentially inhibit angiogenesis-dependent tumor growth and metastasis.