Betulonic acid, a bioactive triterpenoid from Celastrus orbiculatus extract, reverses chemoresistance in gastric cancer via CD44v6-mediated actin cytoskeleton remodeling.

Chemoresistance remains a major obstacle limiting the therapeutic efficacy of gastric cancer treatments, and cytoskeleton remodeling together with the abnormal membrane localization of drug efflux proteins may contribute to resistance phenotypes. This study aimed to investigate the role of CD44v6 in gastric cancer chemoresistance and to clarify whether betulonic acid, an active monomeric component of Celastrus orbiculatus extract, can reverse drug resistance via CD44v6. Analysis of public databases and validation in clinical tissue samples revealed that CD44v6 was highly expressed in gastric cancer cells and associated with poor prognosis, whereas CD44v6 expression was positively regulated by polypyrimidine tract binding protein 1. CD44v6 expression was markedly upregulated in MKN-45/DDP and HGC-27/Oxaliplatin resistant cells. CD44v6 knockdown inhibited GC cell proliferation, colony formation, migration/invasion, and tumor growth and increased the sensitivity of resistant cells to DDP or oxaliplatin. Mechanistically, CD44v6 knockdown suppressed F-actin remodeling, pseudopodia formation, cell motility, and the phosphorylation of VASP, Ezrin/Radixin/Moesin and Cofilin. Multiplex immunofluorescence analysis of tissue microarrays, molecular docking, and coimmunoprecipitation confirmed that CD44v6 was coexpressed and interacted with Ezrin/Radixin/Moesin family proteins in gastric cancer tissues. Betulonic acid was identified as a candidate active monomer of Celastrus orbiculatus extract that could potentially bind to CD44v6. Treatment with betulonic acid inhibited the malignant biological behaviors of gastric cancer cells, increased chemosensitivity, reduced membrane-associated P-glycoprotein expression, and weakened the interaction between CD44v6 and ERM proteins. In MKN-45/DDP xenograft models, betulonic acid combined with DDP significantly inhibited tumor growth and partially alleviated DDP-associated toxicity. In conclusion, CD44v6 promotes gastric cancer chemoresistance by interacting with Ezrin/Radixin/Moesin proteins to regulate cytoskeleton remodeling and membrane-associated P-glycoprotein expression, whereas betulonic acid reverses DDP resistance by through the CD44v6-ERM axis. This study provides mechanistic evidence supporting betulonic acid as a potential chemosensitizer for gastric cancer treatment.