Network pharmacology reveals functional components and molecular mechanisms of andrographis combined with lactoferrin in pancreatic cancer treatment

Background: Pancreatic cancer (PC) remains the most lethal malignancies, underscoring the need for novel therapeutic strategies. Natural plant-derived bioactive compounds have garnered increasing attention as potential anticancer agents due to their safety, affordability, and ability to target multiple pathways regulating cellular growth. Network pharmacology has emerged as a powerful approach to elucidate the multi-target and multi-pathway mechanisms of various bioactive compounds, offering theoretical insights into their synergistic therapeutic potential in complex diseases. Among natural agents, Andrographis (Andro) and Lactoferrin (Lf) have shown potent anticarcinogenic properties. However, their potential synergistic interactions and the underlying mechanisms remain largely unexplored. Methods: In this study, we investigated the synergistic anticancer effects of Andro and Lf on PC cell lines (MIA-PaCa-2 and BxPC-3), and explored growth-regulatory pathways underlying their combined activities. Cell viability, migration, and invasion assays were performed to evaluate the antiproliferative and anti-invasive activities. Using Network pharmacology and pathway enrichment analyses, we further identified key targets and signaling pathways associated with the synergistic effects of these two compounds in PC. Subsequently, the findings from cell-culture experiments were validated using patient-derived 3D tumor organoids. Results: The combined treatment with Andro and Lf demonstrated marked synergistic anti-tumor activity, significantly suppressing cell viability, proliferation, migration, and invasion in PC cells. Network pharmacology identified 13,778 disease-related targets and 164 putative drug targets, with 148 overlapping targets between PC and the combination treatment were deciphered. Notably, AKT1 emerged as a central node, indicating strong associations between PC progression and the pharmacological activity of Andro and Lf. Pathway enrichment analyses further indicated that the synergistic effects of the combination were predominantly linked to the PI3K-AKT signaling pathway ( P <0.05) and accompanied by a significant reduction in intracellular calcium levels. Consistently, AKT1 expression was significantly higher in PC tissues compared with normal tissues in the TCGA dataset (FC = 1.24, P <0.05). Finally, in 3D organoid models, the combined treatment showed stronger anticancer activity, significantly reducing organoid number and size ( P <0.05), highlighting its therapeutic potential. Conclusion: Our study provides evidence that the synergistic anticancer effects of Andro and Lf in PC are mediated via the PI3K-AKT signaling pathway and modulation of intracellular calcium levels, highlighting their promise as a combinatorial therapeutic strategy.