Synergistic inhibitory effects of Trifolium pratense L. extract and doxorubicin on ۴T۱ tumor-bearing mice are mediated via targeting the Wnt/β-catenin pathway and reversal of epithelial-mesenchymal transition

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 ۴T۱ tumor-bearing BALB/c mice.Materials and Methods: Female BALB/c mice were divided into six (n=۱۰) groups: control, DOX (۵ mg/kg), and three treatment groups receiving ۱۰۰, ۲۰۰, or ۴۰۰ mg/kg T. pratense extract alongside DOX, and a single dose of ۴۰۰ 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, Cdh۱, Snai۱, Sfrp۲, Wif۱, Kremen۱, and ARHGAP۱۷. Immunohistochemical staining was performed to evaluate p۵۳, Ki-۶۷, β-catenin, Cdh۱, and vimentin expression.Results: Co-treatment of T. pratense (۴۰۰ mg/kg) with DOX (۵ mg/kg) synergistically reduced cell proliferation and increased apoptosis by increasing p۵۳ and decreasing Ki-۶۷ expression in a dose-dependent manner. This co-treatment effectively inhibited the Wnt/β-catenin pathway by upregulating antagonists (Wif۱ and Sfrp۲), modulating β-catenin accumulation, and reversing EMT through increased E-cadherin expression and decreased vimentin (protein level) and Snai۱ (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.