Berberine and celecoxib synergistically suppress non-small cell lung cancer with EGFR/AKT/COX-2-related signaling: network pharmacology, molecular docking and experimental validation

Abstract

Non-small cell lung cancer (NSCLC) remains a major cause of cancer-related mortality, and safer combination strategies with improved antitumor efficacy are still needed. Berberine (BBR), a natural isoquinoline alkaloid with multi-target pharmacological activity, and celecoxib (CXB), a selective cyclooxygenase-2 (COX-2) inhibitor, have shown antitumor potential. However, their combined efficacy and underlying mechanisms in NSCLC remain unclear. Network pharmacology, molecular docking, Chou–Talalay combination index analysis, complementary synergy analysis using SynergyFinder+, in vitro functional assays, molecular validation, and a PC9 xenograft model were integrated to evaluate the antitumor effects and associated molecular changes of BBR combined with CXB. Candidate targets and pathways were screened using protein–protein interaction analysis, GO/KEGG enrichment, and Target–Pathway network analysis. Molecular docking was performed to assess the predicted binding affinity of BBR and CXB with key targets, and native-ligand re-docking was performed to validate the docking protocol. Cell viability, proliferation, colony formation, migration, invasion, apoptosis, and molecular changes were examined using CCK-8, EdU, colony formation, wound-healing, Transwell invasion, flow cytometry, Western blotting, qRT-PCR, immunohistochemistry, and in vivo experiments. Network analysis identified EGFR, AKT1, and PTGS2 (encoding COX-2) as candidate nodes, and molecular docking suggested predicted binding of BBR and CXB with EGFR, AKT1, and COX-2. Native-ligand re-docking showed acceptable reproduction of the crystallographic ligand poses. BBR and CXB synergistically reduced A549 and PC9 cell viability, with ZIP, HSA, Loewe, and Bliss analyses further supporting the multidose synergy, and a representative working combination dose was used for validation. The combination significantly inhibited proliferation, colony formation, migration, and invasion while promoting apoptosis. These effects were accompanied by suppression of EGFR and AKT phosphorylation, reduced COX-2 expression, downregulation of TNF-α, IL-1β, and CXCL8, decreased the Bcl-2/Bax ratio, and increased cleaved-Caspase-3. In vivo , BBR + CXB more effectively inhibited PC9 xenograft growth than either monotherapy without obvious body weight loss, accompanied by reduced Ki-67, p-EGFR, p-AKT, and COX-2 expression and increased cleaved-Caspase-3 staining. BBR combined with CXB exhibits synergistic antitumor activity in NSCLC cells and enhanced antitumor efficacy in vivo , associated with suppression of EGFR/AKT/COX-2-related signaling, attenuation of inflammatory responses, and activation of apoptosis, providing support for this potential combination strategy.