23-hydroxybetulinic acid induces cell cycle arrest in esophageal cancer cells via the BUB1/STAT3 signaling pathway

Background23-Hydroxybetulinic acid (23-HBA), a key bioactive compound in the traditional Chinese herb Pulsatilla chinensis, has garnered substantial scientific interest due to its potent antitumor activity. However, the inhibitory effects of 23-HBA on esophageal cancer growth have not been fully elucidated.MethodsIn this study, EC9706 and KYSE150 cells were treated with 23-hydroxybetulinic acid (23-HBA). Cell viability was assessed using the CCK-8 assay. Cell cycle distribution and apoptosis were analyzed by flow cytometry. Cell migration and invasion abilities were evaluated using wound healing and Transwell invasion assays, respectively. Protein expression levels of BUB1, STAT3, p-STAT3, CCNB1, CDK1, Bcl-2 and Caspase-3 were determined by Western blot analysis. For the in vivo study, a nude mouse xenograft model of esophageal carcinoma was established. Tumor-bearing mice were randomly assigned to the following groups: model group, low-dose (15 mg/kg) and high-dose (30 mg/kg) 23-HBA groups, and cisplatin group (4 mg/kg). Body weight and tumor volume were monitored regularly. At the end of the experiment, tumor tissues were collected, weighed, and subjected to histopathological examination via hematoxylin and eosin (HE) staining. Protein expression of BUB1, STAT3, and p-STAT3 in tumor tissues was further analyzed by Western blot and immunohistochemistry (IHC). Meanwhile, the protein expressions of Bcl-2 and Caspase-3 were detected.Results23-HBA inhibited the proliferation, migration, and invasion of EC cells, while inducing apoptosis and G2/M phase cell cycle arrest. Treatment with 23-HBA significantly downregulated the expression of p-STAT3, BUB1, CCNB1, CDK1, and Bcl-2 in EC cells, while significantly upregulating the expression of Caspase-3, although STAT3 protein levels remained unchanged. In the xenograft mouse model, 23-HBA treatment led to a significant reduction in tumor volume and weight compared with the model group, accompanied by extensive tumor necrosis. Western blot and immunohistochemical analyses further confirmed that p-STAT3 and BUB1 expression were markedly downregulated in tumor tissues from the 23-HBA-treated group, whereas STAT3 expression did not show significant alterations. The results showed that 23-HBA downregulated Bcl-2 and upregulated Caspase-3 protein expression.Conclusion23-HBA inhibits mitosis in esophageal cancer by blocking the BUB1/STAT3 signaling pathway, suggesting its potential as a therapeutic agent for esophageal cancer treatment.