Integrative In Vitro and In Silico Analysis Reveals Multi- Targeted Anticancer Effects of Berberine Chloride on Breast Cancer

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 with a crystal violet assay showed an IC₅₀ of 40 µM for MDA-MB-231 and 10 µM for 4T1 after 48 hours of treatment. BRB induced S-phase cell cycle 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 anti-angiogenic effects by inhibiting cell migration, as demonstrated by scratch wound and Transwell assays. Additionally, BRB reduced adhesion to various extracellular matrix components, with the most noticeable effect seen with collagen IV in both cell lines. 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.