5,7-dimethoxyflavone inhibits hepatocellular carcinoma progression via increasing intestinal Akkermansia muciniphila and hepatic CD8+ T cell infiltration

Background: Hepatocellular carcinoma (HCC) mainly develops in cases of fibrosis and cirrhosis and is accompanied by intestinal flora disorder. HCC also affects CD8⁺ T cell immune function. 5,7-Dimethoxyflavone (DMF), an active flavonoid with anti-tumor effect, is found in Kaempferia parviflora. However, whether DMF can treat HCC remains unclear. This study aims to investigate the effect of DMF on HCC and to explore its possible mechanism, focusing on the gut microbiota regulation and the effect of CD8⁺ T cells in a murine model.

Methods: The HCC mouse model was induced with diethylnitrosamine/carbon tetrachloride and orally administered DMF. DMF effects on HCC progression were assessed using hematoxylin and eosin staining, immunohistochemistry, and serum biochemical marker levels. The causal relationship between gut microbes and HCC was explored using 16S rRNA genome-derived taxonomic profiling, microbial transplantation, fecal high-throughput targeted metabolomics, and untargeted serum metabolomic analyses. Transcriptome analysis, molecular docking, quantitative real-time polymerase chain reaction, and Western blot were applied to study the genes targeted by DMF. CD8⁺ T cell infiltration and tumor-killing factors were studied using flow cytometry and immunofluorescence staining.

Results: DMF reduced the number of tumors, the largest tumor size, and the liver-to-body ratio while also improving liver function. An Antibiotic cocktail lowered the anti-tumor effect of DMF, indicating that DMF inhibition of HCC is partially dependent on the gut microbiota. DMF considerably upregulates Akkermansia muciniphila during chemical hepatocarcinogenesis in mice. DMF-upregulated A. muciniphila leading to intestinal barrier repair, which inhibited HCC progression by enhancing antioxidant capacity through glutathione regulation and 11,12-DIHETrE down-regulation. An untargeted serum metabolomic analysis showed that there existed additional mechanisms underlying DMF anti-tumor effect following its absorption into the bloodstream. DMF enhances the infiltration effect of CD8⁺ T cells and upregulates interferon-gamma expression in HCC tissue. Overall, 822 genes, including chemokine (C-C motif) ligand 2 (CCL2), were significantly downregulated by DMF treatment in HCC cells. Notably, DMF binds strongly with nuclear factor kappa-B (NF-κB) and inhibits NF-κB p65 phosphorylation, sequentially suppressing the expression of downstream protein CCL2, which mediate the crosstalk between tumor cells and CD8⁺ T cells.

Conclusion: DMF improves A. muciniphila-mediated intestinal barrier repair and inhibits the NF-κB/CCL2 pathway in HCC cells, enhancing the immunity of CD8⁺ T cells in the liver. Hence, it may serve as a potential candidate for HCC treatment.

5,7-Dimethoxyflavone; Antitumor immunity; Gut microbiota; Hepatocellular carcinoma; NF-κB/CCL2 pathway.