Berberine Chloride Induces Apoptosis and Inhibits Adhesion, Migration, and Invasion in MDA‐MB‐231 and 4T1 Breast Cancer Cells: An Integrative In Vitro and In Silico Study

Berberine chloride (BRB), an isoquinoline alkaloid isolated from Berberis vulgaris, demonstrated significant anticancer activity against breast cancer. This study assessed the therapeutic effects of BRB on cell proliferation, cell-cycle progression, apoptosis, and metastasis in human (MDA-MB-231) and murine (4T1) triple-negative breast cancer cells. Cytotoxicity testing using a crystal violet assay showed IC₅₀ values of 40 µM for MDA-MB-231 and 10 µM for 4T1 after 48 h of treatment. BRB induced S-phase arrest in MDA-MB-231 and G2/M-phase arrest in 4T1 cells. It also induced late apoptosis in both cell lines, along with increased reactive oxygen species production and loss of mitochondrial membrane potential. Furthermore, BRB exhibited marked anti-metastatic potential by inhibiting cell adhesion, migration, and invasion. Scratch-wound and Transwell assays demonstrated a significant, concentration-dependent reduction in migratory capacity, while 3D spheroid assays confirmed strong suppression of invasive behavior. BRB also reduced adhesion to extracellular matrix components, particularly collagen IV. At the molecular level, BRB significantly downregulated MMP2 and MMP9 mRNA expression in both cell lines, supporting its role in inhibiting extracellular matrix remodeling and metastatic progression. Molecular docking simulations indicated that BRB has a favorable binding energy with multiple cancer-related targets, including MDM2-P53, BCL2, Caspases (3, 8, and 9), MCL1 complexes, and matrix metalloproteinases (MMP-2 and MMP-9). The compound maintained stable hydrogen bonds, ππ-stacking interactions, and hydrophobic contacts, often achieving higher docking scores than the co-crystallized ligand. Overall, the results suggest that BRB exerts multi-targeted anticancer effects by regulating processes such as proliferation, apoptosis, migration, and adhesion, indicating that BRB could be a promising candidate for breast cancer therapy.