Synergistic inhibitory effects of Trifolium pratense L. extract and doxorubicin on 4T1 tumor-bearing mice are mediated via targeting the Wnt/β-catenin pathway and reversal of epithelial-mesenchymal transition

Khazayel, Saeed; Faraji, Mohammad Hossein; Akbari Bazm, Mohsen; Khazaei, Mozafar; Niromand, Elham; Khazaei, Mohammad Rasool

doi:10.22038/ajp.2025.25940

Synergistic inhibitory effects of Trifolium pratense L. extract and doxorubicin on 4T1 tumor-bearing mice are mediated via targeting the Wnt/β-catenin pathway and reversal of epithelial-mesenchymal transition

Document Type : Original Research Article

Authors

Saeed khazayel ¹

Mohammad Hossein Faraji ²

mohsen akbari bazm ³

Mozafar khazaei ⁴

Elham Niromand ¹

Mohammad Rasool khazaei ¹

¹ Fertility and Infertility Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran.

² Physiology Division, Department of Basic Science, School of Veterinary Medicine, Shiraz University, Shiraz, Iran.

³ Department of Anatomical Sciences, Kermanshah University of Medical Sciences

⁴ Fertility and Infertility Research Center, Health Technology Institute School of Medicine, University Ave., Shahid Shiroudi Blvd, Kermanshah, Iran

10.22038/ajp.2025.25940

Abstract

Objective: Triple-negative breast cancer (TNBC) presents significant therapeutic challenges. This study investigates the combination effects of Trifolium pratense L. (red clover) and doxorubicin (DOX) on the Wnt/β-catenin signaling pathway, epithelial-mesenchymal transition (EMT) and apoptosis in 4T1 tumor-bearing BALB/c mice.
Materials and Methods: Female BALB/c mice were divided into six (n=10) groups: control, DOX (5 mg/kg), and three treatment groups receiving 100, 200, or 400 mg/kg T. pratense extract alongside DOX, and a single dose of 400 mg/kg T. pratense. Tumor size was measured using Vernier calipers, and survival rates were analyzed through Kaplan-Meier curves. Tumors were removed to analyze histological examinations and gene expression of Ccnd, Myc, Cdh1, Snai1, Sfrp2, Wif1, Kremen1, and ARHGAP17. Immunohistochemical staining was performed to evaluate p53, Ki-67, β-catenin, Cdh1, and vimentin expression.
Results: Co-treatment of T. pratense (400 mg/kg) with DOX (5 mg/kg) synergistically reduced cell proliferation and increased apoptosis by increasing p53 and decreasing Ki-67 expression in a dose-dependent manner. This co-treatment effectively inhibited the Wnt/β-catenin pathway by upregulating antagonists (Wif1 and Sfrp2), modulating β-catenin accumulation, and reversing EMT through increased E-cadherin expression and decreased vimentin (protein level) and Snai1 (gene expression) levels.
Conclusion: T. pratense extract shows potential as an adjuvant therapy against TNBC by targeting the Wnt/β-catenin pathway and reversing EMT while enhancing DOX efficacy. Further research is warranted to explore additional anticancer mechanisms of T. pratense extract.

Keywords

Trifolium pratense L. Doxorubicin Epithelial

mesenchymal transition Wnt signaling pathway beta catenin

Subjects