Warfarin and bevacizumab suppress tumor progression in pancreatic ductal adenocarcinoma by targeting EGFR-PI3K-Akt signaling: inhibition of proliferation/migration and apoptosis induction.

Background: Pancreatic ductal adenocarcinoma (PDAC) exhibits aggressive progression, dense stromal remodeling, and resistance to chemotherapy, resulting in extremely poor survival. Although bevacizumab-mediated vascular endothelial growth factor (VEGF) inhibition can suppress angiogenesis, clinical efficacy is limited by compensatory activation of alternative signaling pathways. Meanwhile, PDAC-associated hypercoagulability supports tumor progression, and warfarin has been reported to exert anti-tumor effects partly through inhibition of the growth arrest-specific protein 6 (Gas6)/Axl-phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) axis. Given that epidermal growth factor receptor (EGFR) also converges on the PI3K-Akt pathway, combining warfarin with bevacizumab may enhance therapeutic efficacy by co-targeting angiogenic and oncogenic signaling. This study aimed to evaluate the synergistic effects of warfarin and bevacizumab in PDAC and to elucidate the underlying molecular mechanisms. Methods: An integrated approach combining network pharmacology, molecular docking, and in vitro assays was used. Bioinformatics tools identified key targets and pathways, with docking simulations assessing warfarin-target binding. Functional assays, including the Cell Counting Kit-8 (CCK-8), wound healing, flow cytometry, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot, evaluated cell proliferation, migration, apoptosis, and gene/protein expression related to EGFR-PI3K-Akt pathway. Results: Integrated bioinformatics identified 70 overlapping targets between warfarin and pancreatic cancer, with EGFR, PI3K isoforms, and AKT1 as core hubs in the protein-protein interaction (PPI) network. Molecular docking demonstrated strong warfarin binding to EGFR, PI3K catalytic isoforms, and AKT1 (ΔG <-7.0 kcal/mol), while exhibiting moderate interaction with the PI3K regulatory subunit phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1, -6.1 kcal/mol). In vitro validation showed that 0.8 mmol/L warfarin combined with 500 mg/L bevacizumab exhibited optimal anti-proliferative synergy (24-h Bliss score: 0.335; 48-h inhibition: 50.5%), while reducing wound closure versus blank (P<0.001). The combination elevated apoptosis to 10.11% (P<0.001 vs. 1.19% blank) with B-cell lymphoma 2 (Bcl-2) suppression (0.41-fold), Bcl-2-associated X protein (Bax, 1.12-fold) and cysteine-aspartic acid protease-3 (caspase-3, 0.93-fold) upregulation. Combination therapy synergistically downregulated EGFR (0.41- vs. 0.74-fold warfarin, P<0.001) and PI3K (0.32- vs. 0.57-fold, P<0.001) at messenger RNA (mRNA)/protein levels, while AKT1 protein remained unchanged (P>0.05). Conclusions: The warfarin-bevacizumab combination synergistically impaired PDAC progression via multi-tiered EGFR-PI3K-Akt suppression and mitochondrial apoptosis activation, providing a rationale for clinical translation against oncogenic pathway plasticity.