Buxus alkaloid KBA01 targets Valosin-containing protein to inhibit colorectal cancer progression via the p53-Wnt/β-catenin axis

ABPP identifies valine-containing protein (VCP) as a KBA01 direct action target. • KBA01 specifically degrades p53 R273 by targeting VCP. • Distinct from p53 degraders, KBA01 has a unique role in disrupting the dominant negative effect of p53 R273H Background Mutant p53 is closely associated with tumor proliferation and metastasis, making an attractive therapeutic target in cancer treatment. Buxus alkaloids exhibit antibacterial and antiviral properties, however, research into their antitumor potential remains limited. KBA01, a triterpenoid alkaloid isolated from the leaves and stems of Buxus bodinieri Levl, activates the p53 signaling pathway in colorectal cancer cells; however, its drug targets and mechanism of action remain unclear. Purpose To identify the molecular target of KBA01 and elucidate how it suppresses tumor growth through activation of the p53 signaling pathway. Methods Activity-based protein profiling (ABPP) assay was performed to identify the potential target of KBA01. Wound healing, cell colony formation and transwell assays were conducted to elucidate the effects of KBA01 on cell proliferation, migration, and invasion. Western blot, RT-qPCR, Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), Electrophoretic mobility shift assay (EMSA), Chromatin immunoprecipitation (ChIP), and Luciferase reporter assays were applied to investigate the effects of KBA01 on p53 function. The colorectal cancer xenograft experiment was performed to evaluate the in vivo anticancer activity. Results KBA01 targets Valosin-containing protein (VCP), reducing its interaction with p53 R273H and thereby promoting the ubiquitination and degradation of mutant p53. Consequently, KBA01 disrupts the dominant negative effect of mutant p53. KBA01 elevated the p53 downstream targeted genes p21 and PUMA mRNA and protein levels, thereby activating the p53 signaling pathway and ultimately inhibiting tumor growth and metastasis in vitro . In addition, KBA01 inhibits the Wnt/β-catenin pathway in tumor cells harboring mutant p53. Further in vivo studies demonstrated that KBA01 significantly suppressed tumor growth in colorectal cancer xenograft models by inducing mutant p53 depletion and reactivating p53 downstream target genes, achieving a tumor inhibition rate of 61.39%. Conclusion These findings identify VCP as the molecular target of KBA01 and suggest that KBA01 selectively promotes the degradation of mutant p53 R273H, thereby alleviating its dominant-negative effect and restoring p53 signaling. Collectively, this study broadens the potential pharmacological applications of Buxus alkaloids and supports the further investigation of KBA01 as a lead compound for mutant p53-targeted therapy. Download: Download high-res image (255KB) Download: Download full-size image