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.

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.