Systems biology based elucidation and experimental validation of anticancer effects of warfarin in vulvar cancer

Abstract Objectives To evaluate the efficacy of warfarin in inhibiting vulvar cancer cell growth and explore its molecular mechanisms, including effects on immune-infiltration and pathways utilizing both computational and experimental approaches. Methods The pharmacokinetic and toxicity profiles of warfarin were computationally evaluated to confirm drug-likeness and safety. Biological targets were then predicted, and the resulting genes were analyzed using a systems biology framework to construct a protein–protein interaction network, identifying key hub-genes. These genes underwent survival analysis to assess prognostic significance and were further analyzed for immune cell infiltration, indicating possible immunomodulatory effects. Molecular docking simulations examined warfarin’s binding affinity to the PD-1/PD-L1 immune checkpoint complex, suggesting immune regulatory potential. Experimental validation using SW-962 (vulvar-cancer) and HaCaT (normal) cell lines assessed cytotoxicity, oxidative stress, nuclear morphology, and cell cycle dynamics. Results Warfarin has promising physicochemical properties: high GI-absorption, BBB-permeability, and drug-likeness. Its predicted targets overlap with 20-vulvar cancer-related genes and form a PPI-network of key cancer-related pathways. GO and KEGG analyses reveal that the genes’ involvement in stress response, structural components, and cancer pathways. The top hub genes include AKT1 and CASP3, both of which have poor survival, and studies on immune infiltration show that warfarin interacts with immune cells. The docking analysis shows a strong binding affinity to the PD-1/PD-L1-complex, whereas cytotoxicity tests show selective action against cancer cells through oxidative stress, apoptosis, and G0/G1-cell cycle arrest. Conclusions Warfarin demonstrates promising anti-cancer effects by promoting apoptosis, cell cycle arrest, ROS-generation, and immune modulation, therefore warranting further clinical studies.