Chrysophanol Attenuates Breast Cancer Angiogenesis Through Blocking VEGFA/VEGFR2/ERK Activation via Inhibiting ACE2 Ubiquitination

Background Breast cancer progression depends on abnormal angiogenesis, driven by VEGF/VEGFR2/ERK and inhibited by Angiotensin-Converting Enzyme 2 (ACE2). Chrysophanol (CHR) shows anti-breast cancer efficacy, but its mechanism is unclear. Methods Anti-tumor effects were assessed by measuring tumor volume, H&E staining, Ki67 immunohistochemistry, MTT, scratch wound, and Transwell assays. Anti-angiogenic effects were evaluated via Platelet Endothelial Cell Adhesion Molecule-1 (CD31) immunohistochemistry, CAM assay and Western blot (WB) of VEGF-A, Hypoxia-Inducible Factor 1-α (HIF-1α), and VEGFA/VEGFR2/ERK pathway proteins. The mechanism was explored by WB analysis of ACE2 expression/ubiquitination, validated by siRNA knockdown and CETSA for binding site identification. Results Both in vivo and in vitro experiments demonstrated that chrysophanol reduces tumor cell viability, suppresses tumor cell growth, migration, and invasion, inhibiting the progression of breast cancer. Given the critical role of angiogenesis in tumor development, further studies revealed that chrysophanol downregulates the expression of proteins such as HIF-1α and Vascular Endothelial Growth Factor A (VEGFA), indicating that it inhibits tumor angiogenesis by suppressing the VEGFA/VEGFR2/ERK signaling pathway. Since prior evidence suggests that this pathway acts downstream of ACE2, we examined ACE2 protein levels and found that chrysophanol reduces ACE2 ubiquitination, and enhances its stability and expression. This upregulation of ACE2 further inhibits the downstream VEGFA/VEGFR2/ERK pathway. Moreover, molecular docking confirmed that chrysophanol binds specifically to the active pocket 888 of the ACE2 protein. All the observed inhibitory effects on breast cancer were dose-dependent. Conclusion All the data shows that chrysophanol can reduce tumor angiogenesis and alleviate breast cancer growth. It also confirms that chrysophanol can promote ACE2 expression to inhibit the VEGFa/VEGFR2/ERK signaling pathway, thereby suppressing breast cancer development.