Anti-breast cancer effects of the Xingxiao Pill are associated with inhibition of the ErbB3/PI3K/AKT/mTOR pathway, modulation of amino acid metabolism, and promotion of apoptosis.

Authors

Jingyue Zhang, Jianbang Cheng, Fengchuan Cao, Qiangli Qi, Sihan Zhang, Yujia Wang, Ruoyun Wu, Heng Li, Chen Li, Nu Zhang, Wen Li

Published in

Journal of ethnopharmacology. Pages 122321. Aug 26, 2026. Epub Aug 26, 2026.

Abstract

The Xingxiao Pill (XXP), a representative traditional Chinese medicine (TCM) formula used to promote blood circulation and remove blood stasis in tumor therapy, has shown promising clinical potential. According to the book Famous Traditional Chinese Medicine Formulas and Case Studies for Cancer Treatment, XXP has demonstrated favorable efficacy in the treatment of breast cancer.
The therapeutic effects and mechanisms of action of XXP in breast cancer have not been reported. The aim of the study was to evaluate the therapeutic efficacy of XXP in breast cancer and to investigate its potential mechanisms of action and active components.
First, the proliferation-inhibiting activity of XXP against eight types of breast cancer cell lines was evaluated using the CCK-8 assay. Subsequently, the effects of XXP on the biological behavior of 4T1 cells were further investigated using cloning assays, EDU assays, and flow cytometric apoptosis assays. In addition, the mechanism of apoptosis was examined by western blotting (WB). A 4T1 subcutaneous tumor model was established to evaluate the in vivo antitumor activity of XXP. Concurrently, the safety of XXP was assessed through H&E staining of tissue specimens from major organs and by determining serum biochemical markers of liver and kidney function. Furthermore, weighted gene co-expression network analysis (WGCNA) was used to integrate metabolomics and transcriptomics data, to elucidate the mechanisms responsible for the anti-breast cancer activity of XXP. These findings were validated through WB experiments. Finally, bioactive components of XXP were identified and screened using UPLC-Q-TOF-MS/MS (UPLC-MS) and the SwissADME database, and their activity was validated using CCK-8 assays. Bioactive components with potential binding to mTOR protein were screened and identified using Biolayer Interferometry (BLI) combined with UPLC-MS technology and further confirmed through affinity experiments.
XXP exhibited proliferation-inhibiting activity against various subtypes of breast cancer cells. In further studies, XXP demonstrated both proliferation-inhibiting and apoptosis-promoting activities against 4T1 breast cancer cells in vitro and in vivo. XXP induced mitochondrial apoptosis by activating the cytochrome C/caspase-9/caspase-3/PARP signaling cascade. Subsequently, integrated metabolomic and transcriptomic analyses revealed that XXP reshaped amino acid metabolism. WGCNA also identified a gene module highly correlated with differential changes in amino acid metabolites, which was enriched in the ErbB3/PI3K/AKT/mTOR signaling pathway. WB analysis revealed reduced levels of ErbB3 and PI3K proteins, as well as decreased phosphorylation levels of AKT/mTOR, although total AKT/mTOR levels remained unchanged. Consequently, we hypothesized that XXP regulates amino acid metabolism and promotes apoptosis by inhibiting the ErbB3/PI3K/AKT/mTOR signaling pathway. Finally, seven bioactive components of XXP were identified by UPLC-MS analysis and screening against the SwissADME database. Among these, the inhibitory activity of cinnamic acid, palmitic acid, eugenol, 11-keto-β-boswellic acid, and asiatic acid against 4T1 breast cancer cell proliferation was validated using CCK-8 assays. Furthermore, cinnamic and palmitic acid were identified as active components that may bind to mTOR.
XXP exhibits pharmacological activity that inhibits 4T1 breast cancer cell proliferation and promotes apoptosis in vitro and in vivo. XXP may exert these effects by inhibiting the ErbB3/PI3K/AKT/mTOR signaling pathway, regulating amino acid metabolism, and promoting caspase-mediated endogenous apoptosis. Cinnamic acid, palmitic acid, eugenol, 11-keto-β-boswellic acid, and asiatic acid were identified and validated as active compounds in XXP. Among these, cinnamic acid and palmitic acid are active compounds that potentially bind to mTOR. This study provides experimental data supporting the use of XXP for the treatment of breast cancer.

PMID:
42648410
Bibliographic data and abstract were imported from PubMed on 27 Aug 2026.

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