Butein inhibits cancer cell growth by rescuing the wild-type thermal stability of mutant p53.

p53 is a transcription factor that activates the expression of various genes involved in the maintenance of genomic stability, and more than 50% of cancers harbor inactivating p53 mutations, which are indicative of highly aggressive cancer and poor prognosis. Pharmacological targeting of mutant p53 to restore the wild-type p53 tumor-suppressing function is a promising strategy for cancer therapy. In this study, we identified a small molecule, Butein, that reactivates mutant p53 activity in tumor cells harboring the R175H or R273H mutation. Butein restored wild-type-like conformation and DNA-binding ability in HT29 and SK-BR-3 cells harboring mutant p53-R175H and mutant p53-R273H, respectively. Moreover, Butein enabled the transactivation of p53 target genes and decreased the interactions of Hsp90 with mutant p53-R175H and mutant p53-R273H proteins, while Hsp90 overexpression reversed targeted p53 gene activation. In addition, Butein induced thermal stabilization of wild-type p53, mutant p53-R273H and mutant p53-R175H, as determined via CETSA. From docking study, we further proved that Butein binding to p53 stabilized the DNA-binding loop-sheet-helix motif of mutant p53-R175H and regulated its DNA-binding activity via an allosteric mechanism, conferring wild-type-like the DNA-binding activity of mutant p53. Collectively, the data suggest that Butein is a potential antitumor agent that restores p53 function in cancers harboring mutant p53-R273H or mutant p53-R175H. SIGNIFICANCE: Butein restores the ability of mutant p53 to bind DNA by reversing its transition to the Loop3 (L3) state, endows p53 mutants with thermal stability and re-establishes their transcriptional activity to induce cancer cell death.

Buxus alkaloid compound destabilizes mutant p53 through inhibition of the HSF1 chaperone axis.

BACKGROUND: P53 is the most frequently mutated gene in most tumour types, and the mutant p53 protein accumulates at high levels in tumours to promote tumour development and progression. Thus, targeting mutant p53 for degradation is one of the therapeutic strategies used to manage tumours that depend on mutant p53 for survival. Buxus alkaloids are traditionally used in the treatment of cardiovascular diseases. We found that triterpenoid alkaloids extracted from Buxus sinica found in the Yunnan Province exhibit anticancer activity by depleting mutant p53 levels in colon cancer cells. PURPOSE: To explore the anticancer mechanism of action of the triterpenoid alkaloid KBA01 compound by targeting mutant p53 degradation. STUDY DESIGN AND METHODS: Different mutant p53 cell lines were used to evaluate the anticancer activity of KBA01. MTT assay, colony formation assay and cell cycle analysis were performed to examine the effect of KBA01 on cancer cell proliferation. Western blotting and qPCR were used to investigate effects of depleting mutant p53, and a ubiquitination assay was used to determine mutant p53 ubiquitin levels after cells were treated with the compound. Co-IP and small interfering RNA assays were used to explore the effects of KBA01 on the interaction of Hsp90 with mutant p53. RESULTS: The triterpenoid alkaloid KBA01 can induce G2/M cell cycle arrest and the apoptosis of HT29 colon cancer cells. KBA01 decreases the stability of DNA contact mutant p53 proteins through the proteasomal pathway with minimal effects on p53 mutant protein conformation. Moreover, KBA01 enhances the interaction of mutant p53 with Hsp70, CHIP and MDM2, and knocking down CHIP and MDM2 stabilizes mutant p53 levels in KBA01-treated cells. In addition, KBA01 disrupts the HSF1-mutant p53-Hsp90 complex and releases mutant p53 to enable its MDM2- and CHIP-mediated degradation. CONCLUSION: Our study reveals that KBA01 depletes mutant p53 protein in a chaperone-assisted ubiquitin/proteasome degradation pathway in cancer cells, providing insights into potential strategies to target mutant p53 tumours.