Baicalein Sensitizes Radioresistant BT549 and MCF7-R Breast Cancer Cells by Modulating Rac1 Signaling

Radioresistance remains a key barrier in breast cancer (BC) treatment. Baicalein, a flavonoid from Scutellaria baicalensis, has shown potential to enhance radiosensitivity. This study investigates its efficacy and mechanism in radioresistant BC models. Radioresistant BC cell models were established via fractionated irradiation and validated by clonogenic assays. Baicalein's radiosensitizing effects were assessed using clonogenic survival and DNA damage assays (γ-H2AX foci and neutral comet). KEGG and protein-protein interaction analyses identified Rac1 as a key mediator. Molecular docking and 100 ns molecular dynamics (MD) simulations evaluated the binding stability of the Rac1-baicalein complex, with binding free energy calculated via gmx_MMPBSA. Rac1 activity, immunoblotting, immunofluorescence, and comet assays demonstrated that baicalein inhibits Rac1 activity and delays DNA repair. In vivo xenograft studies (n = 5 mice/group) with immunohistochemistry validated Rac1 pathway inhibition. Baicalein enhanced radiosensitivity in BT549 (radiation enhancement ratio, ER = 2.20 ± 0.12) and MCF7-R cells (ER = 1.38 ± 0.10). Molecular docking revealed a strong binding affinity (-6.87 kcal/mol) between baicalein and Rac1, with MD simulations indicating a relatively stable interaction (binding free energy: -73.42 kJ/mol). Surface plasmon resonance (SPR) analysis revealed a direct interaction between baicalein and Rac1 protein with moderate affinity (K_D = 2.91 × 10^-6 M). High Rac1 expression correlated with poorer clinical outcomes in overall survival (OS), disease specific survival (DSS), and progress free interval (PFI). Baicalein delayed DNA repair after radiation by inhibiting Rac1 activity via the NHEJ pathway, an effect reversed by a constitutively active Rac1-Q61L plasmid. In vivo, baicalein inhibited Rac1, slowing tumor growth and enhancing radiosensitivity. Baicalein is a potential radiosensitizer, offering a novel strategy to overcome radioresistance in breast cancer therapy.