Combining repurposed drugs with tamoxifen (Tam) provides a powerful targeted strategy to mitigate chemotherapy-induced toxicities in breast cancer. This investigation aimed to evaluate the combinatorial effects of Tam, teriflunomide (Ter), a repurposed immunomodulatory drug, and Vitamin D3 (Vit. D3) in both dual and triple regimens against MCF-7 cells. Utilizing the MTT assay, flow cytometry, and Annexin V/PI staining, we assessed cytotoxicity, cell cycle progression, and apoptotic activity, respectively, with synergy quantified via the Chou-Talalay method. Our results revealed that IC₅₀ value for Tam+ Ter combination was 12.999± 0.071 μM, with combination index CI= 1.093, indicating an additive pharmacological activity, accompanied by an increased total apoptotic ratio (48.39%) compared to Tam monotherapy (38.44%), and a strong S-phase accumulation. Conversely, the Tam+ Vit. D3 regimen yielded an IC50= 171.816± 11.937 μM, with CI= 11.72, demonstrating a very strong antagonistic relationship, with a total apoptotic ratio of 41%, accumulating the majority of cells in the G1 phase. Notably, the triple combination Tam+ Ter+ Vit. D3 resulted in IC50= 24.044± 1.453 μM and a CI= 1.35, showing a moderate antagonistic effect, alongside a massive S-phase arrest and near-elimination of the G2 phase. In summary, our findings reveal that while teriflunomide significantly potentiates the anti-cancer activity of tamoxifen through enhanced apoptosis, inhibition of proliferation, and cell cycle arrest, the dual Tam+ Vit. D3 regimen paradoxically yielded antagonistic efficacy. Most importantly, adding teriflunomide to the Tam+ Vit. D3 combination mitigates this negative interaction, shifting it from strong to slight antagonism.

Keywords:

Tamoxifen; Teriflunomide; Vitamin D3; Combination therapy; Breast cancer