OBJECTIVE  To elucidate the mechanisms responsible for the inhibitory effects of limonene on the proliferation of non-small cell lung cancer(NSCLC) by non-targeted metabolomics and additional approaches.

METHODS  The CCK-8 assay was utilized to evaluate the inhibitory effects of limonene on NSCLC A549 cell viability and to ascertain the IC₅₀. In vitro experiments, encompassing colony formation, flow cytometry, iron content assessment, and mitochondrial staining, were conducted to assess the anti-lung cancer and iron-induced cell death effects of limonene. Metabolomic analysis was employed to identify potential pathways influenced by limonene, and Western blotting was carried out to validate pivotal proteins within these pathways.

RESULTS  In comparison to the control group, the limonene-treated group demonstrated a significant, dose-dependent reduction in A549 cell proliferation and colony formation. Optical microscopy revealed cellular detachment and pronounced changes in cellular morphology following exposure to limonene. Limonene induced apoptosis in A549 cells and arrested them in the G₀-G₁ phase of the cell cycle. Confocal microscopy unveiled diminished mitochondrial fluorescence and an augmented intracellular iron content, indicative of the classical phenomenon of ferroptosis. Metabolomic investigations unveiled divergent metabolic pathways, including glutathione(GSH) metabolism, arginine biosynthesis, D-glutamine and D-glutamate metabolism, as well as cysteine and methionine metabolism, with many of them intricately linked to intracellular GSH synthesis. Western blotting experiments underscored a marked reduction in the levels of SLC40A1, SLC7A11(xCT), and GPX4 proteins within the cells post-limonene treatment.

CONCLUSION  Limonene may induce ferroptosis in lung cancer cells by reducing GSH synthesis and increasing Fe²⁺ levels.