Mechanism of Coumarin in the anti-liver cancer activity by promoting NK cell activation.

OBJECTIVE

This study aims to elucidate the molecular mechanisms by which Coumarin exerts anti-liver cancer effects via natural killer (NK) cell activation. We investigated whether Coumarin inhibits liver cancer progression by enhancing NK cell effector functions, providing novel strategies for immunotherapy.

MATERIALS AND METHOD: In vitro, Sophora tonkinensis extract were screened in NK-92 cells. Cytokines (e.g., IFN-γ) were measured by ELISA; granzyme B and perforin levels were assessed via western blot and qPCR. Flow cytometry analyzed surface receptors. Findings were verified in primary human NK cells. In vivo, an H22 hepatocellular carcinoma syngeneic mouse model evaluated tumor growth. Immune cell infiltration in spleen and tumor tissues was quantified by flow cytometry and qPCR. Transcriptome sequencing and Seahorse metabolic analysis explored underlying mechanisms.

RESULT: Coumarin significantly promoted NK cell activation. Synergizing with IL-12, it upregulated IFN-γ, granzyme B, and perforin transcription and enhanced their secretion and protein expression in both NK-92 and primary human NK cells. In vivo, Coumarin effectively inhibited H22 tumor growth, significantly reducing tumor volume. Flow cytometry revealed increased infiltration of NK cells and CD8⁺ T cells in tumor and spleen tissues. Transcriptomic analysis identified 194 upregulated and 67 downregulated genes, enriched in energy metabolism pathways. Seahorse analysis revealed that Cu significantly increased oxygen consumption rate (OCR), suggesting its ability to effectively restore energy supply in NK cells.

CONCLUSION

Cu potentiates IL‑12-induced NK cell activation, accompanied by increased IFN-γ secretion and upregulated granzyme B and perforin expression. Cu inhibits hepatocellular carcinoma growth by improving NK cell cytotoxic function, remodeling the tumor immune microenvironment, and enhancing mitochondrial bioenergetic activity in NK cells. The present study preliminarily reveals the immunological and metabolic phenotypic mechanisms underlying the anti-tumor activity of coumarin. Notably, the current data only demonstrate elevated mitochondrial respiratory capacity without confirming comprehensive metabolic reprogramming or the direct molecular targets of coumarin. Collectively, these findings support that coumarin serves as a promising preclinical natural product candidate for liver cancer immunotherapy.