**Background:** Cancer, particularly breast cancer, remains a leading cause of death globally, with challenges including drug resistance and side effects from conventional therapies. Free fatty acids (FFAs) derived from diet and gut microbiota have been suggested to play protective roles against chronic diseases, including cancer, by acting as cytostatic and apoptosis-inducing agents. Histone deacetylase (HDAC) is an epigenetic enzyme overexpressed in many cancers, and its inhibition is a promising therapeutic strategy. Notably, cows and other ruminants have a rare occurrence of mammary cancer compared to humans, prompting investigation into whether metabolites in cow urine possess anticancer properties. This study aimed to extract and identify FFAs from cow urine, evaluate their effects on breast cancer cells and normal cells, and explore their molecular interactions with HDAC.
**Methods:** Cow urine was dried, reconstituted in DMSO, and centrifuged and filtered to obtain the cow urine DMSO fraction (CUDF). MCF-7 and ZR-75-1 breast cancer cells, as well as normal human gingival mesenchymal stem cells (hGMSCs), were treated with CUDF at concentrations of 15, 25, and 50 µg/mL for 72 hours. Cell viability was assessed using trypan blue exclusion and MTT assays. Apoptosis was evaluated using acridine orange/ethidium bromide (AO/EB) dual fluorescent staining and flow cytometry with annexin V/PI staining. Intracellular metabolites from CUDF-treated MCF-7 cells were purified using a novel vertical tube gel electrophoresis (VTGE) system and identified by LC-HRMS. Molecular docking (AutoDock Vina) and 10 ns molecular dynamics (MD) simulations (Desmond) were performed to study the binding of identified FFAs to HDAC (PDB ID: 1C3R), with trichostatin A (TSA) as a positive control.
**Key Results:** CUDF treatment reduced MCF-7 cell viability by up to 41.63% (p ≤ 0.001) at 50 µg/mL in the trypan blue assay and by 62.69% (p ≤ 0.01) in the MTT assay. Flow cytometry confirmed 58.27% apoptotic cells in CUDF-treated MCF-7 cells relative to DMSO control. In ZR-75-1 cells, CUDF reduced viable cells by 34.08% (p ≤ 0.01) and induced 35.15% apoptosis (p ≤ 0.01). The IC50 values were 39.64 µg/mL for MCF-7 and 38.88 µg/mL for ZR-75-1 cells. Importantly, CUDF showed no significant toxicity or apoptosis in normal hGMSCs. LC-HRMS analysis identified five FFAs in the intracellular compartment of CUDF-treated MCF-7 cells that were not detectable in controls: tetracosanedioic acid, 13Z-docosenoic acid (erucic acid), nervonic acid, 3-hydroxy-tridecanoic acid, and 8-hydroxycaprylic acid. Molecular docking revealed that tetracosanedioic acid had the highest binding affinity to HDAC (−7.5 kcal/mol), comparable to TSA (−7.9 kcal/mol). MD simulations over 10 ns showed stable binding of tetracosanedioic acid to HDAC with RMSD values between 0.8–1.8 Å, similar to TSA (1–2 Å), and both ligands interacted with key inhibitory domain residues including Tyr297, Phe198, and His132. ADMET predictions indicated tetracosanedioic acid is non-carcinogenic and not a P-gp substrate, with a maximum recommended therapeutic dose of 15,601 mg/day, compared to TSA's 150 mg/day.
**Clinical Implications:** This study identifies a novel, non-toxic source of anticancer FFAs from cow urine that selectively induce apoptosis in breast cancer cells without affecting normal stem cells. The FFAs, particularly tetracosanedioic acid, demonstrate HDAC inhibitory potential comparable to known inhibitors like TSA but with a superior safety profile. These findings support the concept of xeno-tumor heterogeneity—the rare occurrence of cancer in ruminants may be linked to their unique FFA profiles. The results warrant further preclinical and clinical evaluation of these FFA compositions (COFFAs) as potential dietary or therapeutic agents for breast cancer, especially considering the caspase 3 and p53 status of target cells. The novel VTGE-assisted metabolite profiling method also provides a valuable tool for intracellular drug metabolite analysis.