Lactoferrin Hydrolysate Mitigates Azoxymethane/Dextran Sulfate Sodium–Induced Colon Cancer by Modulating Gut Microbiota and Microbial Metabolites

Abstract Colon cancer is one of the most common malignant tumors worldwide and is associated with high morbidity and mortality. Conventional treatments are typically administered at advanced stages and often lead to severe adverse effects. Therefore, developing safe and effective nutritional interventions from natural food sources is of great importance for the prevention and management of colon cancer. Dairy-derived protein hydrolysates have received increasing attention as functional food components due to their biological activities and favorable safety profiles. In this study, lactoferrin (LF) was hydrolyzed using alkaline protease to obtain lactoferrin hydrolysate (LFH). LFH was administered to mice with azoxymethane/dextran sulfate sodium-induced colon cancer to evaluate its protective effects. LFH markedly alleviated colonic tissue injury, reduced serum levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, and decreased the expression of tumor markers CEA, CA19-9, as well as the proliferation marker Ki-67, thereby inhibiting tumor progression. At the molecular level, LFH downregulated key genes in the Wnt/β-catenin pathway, including c-Myc, β-catenin, and Cyclin D1. Furthermore, LFH upregulated pro-apoptotic genes Bax, caspase-9, caspase-3, and p53, while downregulating anti-apoptotic genes Bcl-2 and Bcl-xl, suggesting enhanced apoptotic responses in tumor cells. LFH also significantly improved gut microbiota composition by reducing harmful bacteria such as Bacillus, Enterorhabdus, and Odoribacter, while increasing beneficial genera including Ruminococcus and Eubacterium_siraeum_group. Correspondingly, LFH elevated several gut microbial metabolites such as Chrysogine, Agmatine, 5′-Deoxy-5-Fluorocytidine, and Glutamyl-γ-Glutamate. Overall, this study provides new insights into the biological effects of LFH on colon cancer and supports its potential application in the development of LFH-based functional foods.