Network Pharmacology Prediction and Experimental Validation of Apocynin Against Colon Adenocarcinoma: Identification of CCND1 and CXCL8 as Key Targets

Background Colon adenocarcinoma (COAD) remains a leading cause of cancer-related mortality, necessitating novel therapeutic strategies. Apocynin, a natural NADPH oxidase inhibitor, exhibits anti-tumor potential, yet its precise mechanisms against COAD remain poorly characterized. Methods An integrative approach combining, bioinformatics, molecular docking, and in vitro experimental validation was employed. Apocynin targets and COAD differentially expressed genes (DEGs) were retrieved from GeneCards and TCGA databases, respectively. Protein-protein interaction (PPI) networks, functional enrichment, molecular docking, and survival analyses were performed. Phenotypic effects—including apoptosis, cell cycle progression, invasion, and mitotic activity—were evaluated in colon cancer cells treated with Apocynin (APO), hyperthermia (HT), or their combination (HT + APO). Results 58 intersecting targets were identified between Apocynin and COAD DEGs, from which seven hub genes (ALB, CCND1, CXCL12, CXCL8, SERPINE1, SPP1, TIMP1) were extracted. KEGG enrichment predominantly highlighted the PI3K/Akt signaling pathway. Molecular docking confirmed robust binding affinities, with CCND1 exhibiting the strongest interaction (− 6.8 kcal/mol). Survival analysis identified CXCL8 as a significant adverse prognostic biomarker (P = 0.00048). In vitro, Apocynin induced apoptosis, triggered G0/G1 cell cycle arrest, suppressed invasion, and reduced the mitotic index. Furthermore, the HT + APO combination demonstrated pronounced synergistic anti-tumor effects across all phenotypic assays. Conclusions Apocynin exerts its anti-COAD effects primarily through the regulation of CCND1 and CXCL8. The synergistic efficacy of Apocynin combined with hyperthermia provides a compelling rationale for its clinical development as a combinatorial therapeutic strategy for colon adenocarcinoma.