Mechanistic insights into 18β-glycyrrhetinic acid-induced apoptosis in SCC-9 cells revealed by TMT proteomics and network pharmacology.

18β-Glycyrrhetinic acid (18β-GRA), a major bioactive component of licorice, exhibits recognized biological activities in various tumor models. However, its cellular effects and underlying molecular mechanisms in tongue squamous cell carcinoma (TSCC) are not well characterized. This study aimed to investigate the molecular mechanisms of 18β-GRA against TSCC by integrating quantitative proteomics with network pharmacology. The CCK-8 assay was used to assess cell viability. Cell-cycle distribution and apoptosis were analyzed by flow cytometry. Intracellular Reactive Oxygen Species (ROS) levels were detected using DCFH-DA staining. Tandem Mass Tag (TMT)-based quantitative proteomics was employed to identify differentially expressed proteins, followed by functional enrichment and subcellular localization analyses. Western blotting was performed to examine the expression of proteins associated with apoptosis, autophagy, and the PI3K/AKT pathway. Network pharmacology was applied to predict potential targets of 18β-GRA. Common targets shared with TSCC were mapped onto a protein-protein interaction network, and molecular docking was used to evaluate the binding affinity of 18β-GRA to these overlapping proteins. 18β-GRA significantly inhibited SCC-9 cell viability in a dose- and time-dependent manner and induced G0/G1 phase arrest. The percentages of apoptotic cells and intracellular ROS levels increased with higher concentrations of 18β-GRA. Proteomic analysis revealed widespread alterations in pathways involved in metabolism, cellular structure, apoptosis, and autophagy. Western blotting confirmed upregulation of Bax, downregulation of Bcl-2, an increased LC3-II/LC3-I ratio, decreased p62 expression, and reduced phosphorylation of AKT1. Network pharmacology identified six intersection targets between 18β-GRA and TSCC, which were enriched in metabolic and cancer-related pathways including the PI3K-Akt pathway. Molecular docking suggested stable binding interactions between 18β-GRA and all six candidate proteins. 18β-GRA exerts multi-faceted effects on SCC-9 cells, including growth inhibition, cell-cycle arrest, apoptosis induction, oxidative stress enhancement, and modulation of autophagy markers. The integrated proteomic and computational approach implicates several key pathways and protein targets in the anti-TSCC activity of 18β-GRA, providing a broader molecular context for its mechanistic understanding.