Formononetin suppresses colitis-associated colon cancer by targeting lipid synthesis and mTORC2/Akt signaling

BACKGROUND: Colitis-associated colon cancer (CAC) is a serious gastrointestinal malignancy, with a significantly increased incidence among patients with inflammatory bowel disease, posing major challenges to patients' quality of life and prognosis. Modern research highlights Formononetin (FN) for its significant anti-inflammatory and extensively studied anti-cancer properties; however, its precise mechanisms, particularly in CAC, remain unclear and warrant further investigation. OBJECTIVE: To investigate the anti-inflammatory and anti-tumor activity of FN and its effect on CAC, using biological fluid metabolomics to identify potential diagnostic markers for CAC. METHODS: ) of FN on murine CT-26 and human HCT116 cells IL-6, IL-1β, and TNF-α levels were detected by ELISA. Western blotting was used to analyze autophagic and apoptotic pathways. Utilizing an in vivo mouse model of colitis, the anti-inflammatory activity of FN was assessed by monitoring relevant indicators such as body weight, DAI score, and colon index. And an in vitro mouse colon cancer model was used to observe FN's anti-tumor activity by measuring tumor volume, size, and inhibition rate. Metabolomics analyzed differential serum metabolites and lipid metabolic pathways. Intestinal flora in mice was also analyzed. RESULTS: FN can inhibit the activation of the NF-κB/MAPK signaling pathways in LPS-induced RAW264.7 cells, thereby exerting its anti-inflammatory effects. Moreover, FN significantly enhanced the colon length and DAI score in mice, notably suppressed the production of inflammatory cytokines, and inhibited the NF-κB/MAPK signaling pathway, leading to an improvement in DSS-induced colitis. FN significantly inhibited CT-26 and HCT116 cell growth, reduced tumor growth rate, improved pathological damage in CAC mice, and inhibited inflammatory factor production, enhancing intestinal mucosa protection. FN promoted apoptosis of colon cancer cells by increasing autophagy proteins (LC3, Beclin-1) and apoptosis proteins (CL-Caspase3, Bax), while reducing Bcl-2 expression. Metabolomics identified 34 differential metabolites, including glycerophospholipids and fatty acids, showing anti-tumor effects by regulating lipid metabolism. FN reduced IGF-1, ACLY, A-CoA, FAS, HSL, ATGL, and FFA levels, and increased GSK-3 levels (p < 0.01). FN also inhibited the expression of P-mTOR, Rictor, P-Akt, ACLY, PDE3B, P-PKA, and P-HSL (p < 0.01). CONCLUSIONS: FN significantly inhibits colon cancer cell growth and exerts anti-CAC effects by activating autophagy and apoptosis pathways and regulating lipid metabolism. This study is the first to comprehensively integrate metabolomics, intestinal flora analysis, and molecular mechanisms to unveil FN's multifaceted role in CAC treatment, offering novel insights into its therapeutic application.