Anti-pancreatic cancer effects of luteolin and gemcitabine combination through network pharmacology, in silico, and in vitro studies

Pancreatic cancer remains a highly aggressive malignancy with poor survival rates due to late-stage diagnosis, rapid metastasis, and limited chemotherapy efficacy. Gemcitabine (GEM), a nucleoside analog, disrupts DNA synthesis but is often limited by rapid plasma degradation and chemoresistance. Luteolin (LUT), a naturally occurring flavonoid with anticancer properties, has been proposed as a chemosensitizing agent due to its ability to modulate multiple oncogenic pathways, including STAT3, VEGFA, and HIF-1α. This thesis investigates the therapeutic potential and synergistic effects of GEM and LUT combination therapy in pancreatic cancer using network pharmacology, molecular docking, and in vitro functional assays on the PANC-1 pancreatic cancer cell line. Network pharmacology analysis identified five key hub genes—STAT3, JUN, VEGFA, EGF, and HIF-1α—central to pancreatic cancer progression based on their high degree centrality in the protein-protein interaction (PPI) network constructed using the STRING database. Molecular docking revealed strong binding affinities between LUT and these oncogenic proteins, with binding energies of -7.8 kcal/mol for STAT3, -6.9 kcal/mol for VEGFA, and -6.5 kcal/mol for HIF-1α, suggesting a potential for direct interaction and pathway inhibition. In vitro cytotoxicity assays (CCK-8) demonstrated a time- and dose- dependent reduction in PANC-1 cell viability. GEM’s IC50 decreased from 494.2 μM at 24 hours to 57.04 μM at 72 hours, while LUT’s IC50 dropped from 209.7 μM to 91.10 μM over the same period, indicating enhanced cytotoxicity with extended exposure. Orthogonal experimental analysis further confirmed that LUT exerted a stronger inhibitory effect (F-value = 322.356, p < 0.001) compared to GEM (F-value = 116.085, p < 0.001). Response surface analysis identified significant synergy, with several combination treatments demonstrating a Combination Index (CI) below 0.9, such as GEM 60 μM + LUT 22.5 μM (CI = 0.99) and GEM 69.32 μM + LUT 56.25 μM (CI = 0.92), although some additive effects were also observed. Functional assays further validated the combination’s anti-migratory and anti-invasive effects. The wound healing assay revealed a significant reduction in wound closure by 50.7% (p < 0.001) at 24 hours for the highest GEM + LUT combination tested compared to control. Similarly, the transwell invasion assay showed a 68.2% reduction in invasive cells with combination treatment (p < 0.001). Western blot analysis confirmed significant downregulation of key oncogenic proteins, with reductions in STAT3 (64.3%, p < 0.001), JUN (55.7%, p < 0.001), VEGFA (47.8%, p < 0.01), EGF (52.1%, p < 0.01), and HIF-1α (45.7%, p < 0.01). These molecular findings correlated with the observed reduction in cellular proliferation, migration, and invasion, further supporting the disruption of multiple oncogenic pathways contributing to pancreatic cancer progression. While this study provides strong in vitro evidence of the synergistic anti-cancer effects of GEM and LUT, the absence of in vivo validation remains a limitation. Future studies using patient-derived xenograft models and pharmacokinetic optimization strategies, such as nanoparticle-based delivery systems, are recommended to further explore the clinical translational potential of this combination therapy for pancreatic cancer treatment.

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