From silico to benchtop: cosmosiin as a PD-1/PDL-1 immune checkpoint inhibitor revealed through DFT, network pharmacology analysis, and molecular docking integrated experimental verification

Abstract

), confirmed by molecular dynamics simulations. GEPIA analysis linked the expression of these genes to survival outcomes and disease stage in breast cancer. Experimentally, cosmosiin was tested on MCF-7 (breast cancer) and MCF-10 (non-tumorigenic) cells. Cytotoxicity was evaluated using the MTT assay, showing dose-dependent viability reduction in MCF-7 cells with minimal impact on MCF-10 cells, thus exhibiting selective cytotoxicity. Phase-contrast microscopy revealed altered morphology in treated MCF-7 cells. Annexin V/PI flow cytometry confirmed increased early and late apoptosis, while EdU incorporation assays indicated decreased DNA synthesis and reduced proliferation. Transwell assays demonstrated up to 81% inhibition of cell migration at higher concentrations. Western blotting validated downregulation of CDK5, NFKB1, and PTGS2, aligning with computational predictions. These findings highlight selective, multi-targeted anticancer activity of cosmosiin, particularly through PTGS2, NFKB1, and CDK5, supporting its therapeutic potency for breast cancer with favorable effects on apoptosis, proliferation, and cell migration, while correlating with survival and immune infiltration outcomes.