Timosaponin AⅢ and Gefitinib Synergistically Inhibit NSCLC by Targeting RAGE/AKT/NF-κB/VEGF Pathway.

INTRODUCTION: EGFR tyrosine kinase inhibitors (EGFR-TKIs) are the standard therapy for EGFR-mutated advanced Non-Small Cell Lung Cancer (NSCLC). Gefitinib (GEF), as a first-generation EGFR-TKI, has made significant progress in the treatment of advanced lung cancer. However, acquired resistance leading to treatment failure severely limits its clinical benefits. Therefore, there is an urgent need for a new and effective strategy to improve the therapeutic effect of GEF. METHODS: In this study, Timosaponin AⅢ (Tim-AⅢ) was combined with GEF for administration. The in vitro and in vivo efficacy of the combination in NSCLC cells was analyzed through cell viability assays, cell clone experiments, cell migration and invasion assays, flow cytometry, and animal experiments. The mechanism of the combination was analyzed and verified through RNA-seq and Western blotting experiments. RESULTS: In this study, Tim-AⅢ and GEF showed a synergistic effect (CI<1). Compared with the single-drug groups, the combination group could inhibit cell proliferation, migration, and invasion, induce apoptosis, and arrest cells in the G0/G1 phase. The combination also significantly inhibited the growth of lung cancer cell xenografts in vivo without obvious toxicity. Mechanistically, this regimen could increase E-cadherin and downregulate Vimentin, thereby inhibiting the Epithelial-Mesenchymal Transition (EMT) process. It also downregulated the RAGE-mediated AKT/NF-κB/VEGF signaling pathway, thereby inhibiting tumor angiogenesis. DISCUSSION: Tim-AⅢ combined with GEF exerts synergistic anti-tumor effects on NSCLC by regulating EMT and angiogenesis, with good biosafety. This combination provides a promising strategy to enhance the efficacy of EGFR-TKIs and supports further clinical translation. CONCLUSION: This study not only provides sufficient preclinical in vitro and in vivo pharmacological evidence for the synergistic anti-tumor effect of Tim-AⅢ and GEF, but also clarifies its new mechanism of action through two pathways-EMT and angiogenesis. This provides theoretical support for the development of new combined drug regimens in clinical practice.