Akiferidin suppresses cervical cancer growth and angiogenesis via the VEGF/DLL4-Notch pathway

Cervical cancer remains a major gynecological malignancy with a substantial global burden, particularly in low- and middle-income regions. Tumor angiogenesis driven by the VEGF/DLL4-Notch axis promotes cervical cancer progression, and the anti-VEGF antibody bevacizumab shows limited clinical benefit because of acquired resistance. Our prior work identified anti-tumor activity in the Xinjiang-native medicinal plant Ferula songorica Pall. ex Spreng, but the effects and mechanisms of its principal bioactive metabolite, akiferidin, in cervical cancer have not been defined. Here, we evaluated akiferidin's anti-tumor and anti-angiogenic activities in cervical cancer, examined its combinatorial potential with bevacizumab, and explored the underlying mechanisms. We developed and validated an HPLC method to quantify akiferidin in F. songorica ethanol extract. In vitro, we used MTT, wound-healing, Transwell, and Matrigel tube-formation assays to assess akiferidin's effects on U14 cell proliferation, migration, invasion, and HUVEC angiogenesis. In vivo, we evaluated the efficacy and safety of akiferidin alone and in combination with bevacizumab in U14 xenograft-bearing C57BL/6 mice. Modulation of the VEGF/DLL4-Notch pathway was examined by IHC and Western blot, and direct binding between akiferidin and VEGF-A was investigated by molecular docking followed by 100 ns molecular dynamics simulation. The HPLC method demonstrated excellent robustness (R ² = 0.999, all RSD<2%), yielding an akiferidin content of 57.45 ± 1.42 mg/g in the extract. In vitro, akiferidin inhibited U14 cell proliferation, migration, invasion, and HUVEC tube formation in a concentration-dependent manner (IC₅₀ = 8.06 μg/mL). In vivo, akiferidin suppressed tumor growth in a dose-dependent fashion without overt systemic toxicity, and its combination with bevacizumab markedly enhanced antitumor efficacy while reducing tumor microvessel density. Mechanistically, akiferidin significantly downregulated key proteins in the VEGF/DLL4-Notch pathway, with greater inhibition observed in the combination group. Molecular docking and dynamics confirmed stable binding between akiferidin and VEGF-A at five key sites. In conclusion, akiferidin exerts potent anti-cervical cancer activity by inhibiting angiogenesis through modulation of the VEGF/DLL4-Notch pathway, and its combination with bevacizumab enhances antitumor efficacy with favorable in vivo safety, representing a promising therapeutic strategy for cervical cancer.