Mechanistic study of Clinacanthus nutans in treating triple-negative breast cancer based on network pharmacology, molecular docking, and experimental validation

• 34 bioactive compounds in Clinacanthus nutans identified via network pharmacology. • 128 key targets linked to TNBC, including PIK3R1, CDK1, and MAPK1. • PI3K-AKT and mitochondrial apoptosis pathways are core mechanisms. • Molecular docking confirmed strong binding (<-5 kcal/mol) of active compounds. • Extract inhibited MDA-MB-231 proliferation (IC50=800 μg/mL) and induced apoptosis. To investigate the effect of Clinacanthus nutans extract on the malignant biological behavior of triple-negative breast cancer MDA-MB-231 cells and its potential mechanism of action using network pharmacology and cellular experiments. Relevant targets of Clinacanthus nutans and triple-negative breast cancer were obtained from the SwissTargetPrediction and SuperPred drug databases, as well as the GeneCards and OMIM disease databases. A protein-protein interaction (PPI) network was constructed using the STRING database and Cytoscape 3.8.0 software, and core targets were screened. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on the common targets. Molecular docking validation between the core targets and Clinacanthus nutans was conducted using AutoDock Vina software. MDA-MB-231 cells were treated with different concentrations of Clinacanthus nutans extract. Cell proliferation was detected using the CCK-8 assay, cell migration was assessed by Scratch assay, and the expression levels of PIK3R1, Bcl-2, Bax, and Caspase-3 were measured using Western blot. Network pharmacology identified 970 targets of Clinacanthus nutans and 1,598 TNBC targets, with 128 overlapping targets. The PI3K-AKT and apoptosis signaling pathways were selected based on enrichment results. Cellular experiments showed that Clinacanthus nutans extract inhibited proliferation and migration of MDA-MB-231 cells concentration-dependently. Western blot indicated the extract inhibited PIK3R1/AKT signaling and promoted apoptosis by regulating Bax, Bcl-2, and Caspase-3. Clinacanthus nutans is a potential therapeutic agent for TNBC, inhibiting proliferation and migration by downregulating PIK3R1 and Bcl-2, and upregulating Bax and Caspase-3, thereby co-regulating PI3K-AKT signaling and mitochondrial apoptosis. Subsequent studies should focus on bioactive compound isolation and metabolomic profiling. This study employed an integrated approach combining network pharmacology, molecular docking, and experimental validation to elucidate the mechanisms of Clinacanthus nutans against triple-negative breast cancer (TNBC). Bioactive compounds were screened from C. nutans , and potential targets were predicted. Intersection with TNBC-related targets identified 128 common targets. Protein-protein interaction network analysis revealed core targets including TNF, STAT3, and PIK3R1. Enrichment analysis highlighted the PI3K-AKT and apoptosis pathways. Molecular docking validated strong binding between key compounds (e.g., maackiain) and core targets. Experimental assays demonstrated that C. nutans extract concentration-dependently inhibited MDA-MB-231 cell proliferation and migration, downregulated PIK3R1 and Bcl-2, and upregulated Bax and Caspase-3, confirming dual regulation of PI3K-AKT signaling and mitochondrial apoptosi (Figure 1).