Background IFN-γ has been traditionally recognized as an inflammatory cytokine that involves in inflammation and autoimmune diseases. Previously we have shown that sustained IFN-γ induced malignant transformation of bovine mammary epithelial cells (BMECs) via arginine depletion. However, the molecular mechanism underlying this is still unknown. Methods In this study, the amino acids contents in BMECs were quantified by a targeted metabolomics method. The acquisition of differentially expressed genes was mined from RNA-seq dataset and analyzed bioinformatically. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), western blotting, and immunohistochemistry (IHC) assay were performed to detect gene mRNA and protein expression levels. CCK-8 and would healing assays were used to detect cell proliferation and migration abilities, respectively. Cell cycle phase alternations were analyzed by flow cytometry. Results The targeted metabolomics analysis specifically discovered IFN-γ induced arginine depletion through accelerating arginine catabolism and inhibiting arginine anabolism in BMECs. Transcriptome analysis identified leucine aminopeptidase 3 (LAP3), which was regulated by p38 and ERK MAPKs, to downregulate arginine level through interfering with argininosuccinate synthetase (ASS1) as IFN-γ stimulated. Moreover, LAP3 also contributed to IFN-γ-induced malignant transformation of BMECs by upregulation of HDAC2 (histone deacetylase 2) expression and promotion of cell cycle proteins cyclin A1 and D1 expressions. Arginine supplementation did not affect LAP3 and HDAC2 expressions, but slowed down cell cycle process of malignant BMECs. In clinical samples of patients with breast cancer, LAP3 was confirmed to be upregulated, while ASS1 was downregulated compared with healthy control. Conclusions These results demonstrated that LAP3 mediated IFN-γ-induced arginine depletion to malignant transformation of BMECs. Our findings provide a potential therapeutic target for breast cancer both in humans and dairy cows.

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Background IFN-γ is a pleiotropic cytokine that has been shown to affect multiple cellular functions of bovine mammary epithelial cells (BMECs) including impaired milk fat synthesis and induction of malignant transformation via depletion of arginine, one of host conditionally essential amino acids. But the molecular mechanisms of these IFN-γ induced phenotypes are still unknown. Methods BMECs were treated with IFN-γ for 6 h, 12 h, and 24 h. The metabolomic profiling in BMECs upon IFN-γ induction were assessed using untargeted ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) metabolomic analysis. Key differentially expressed metabolites (DEMs) were quantified by targeted metabolomics. Results IFN-γ induction resulted in significant differences in the contents of metabolites. Untargeted analysis identified 221 significantly DEMs, most of which are lipids and lipid-like molecules, organic acids and derivatives, organ heterocyclic compounds and benzenoids. According to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, DEMs were enriched in fructose and mannose metabolism, phosphotransferase system (PTS), β-alanine metabolism, arginine and proline metabolism, methane metabolism, phenylalanine metabolism, and glycolysis/gluconeogenesis. Quantification of selected key DEMs by targeted metabolomics showed significantly decreased levels of D-(-)-mannitol, argininosuccinate, and phenylacetylglycine (PAG), while increased levels in S-hydroxymethylglutathione (S-HMG) and 2,3-bisphospho-D-glyceric acid (2,3-BPG). Conclusions These results provide insights into the metabolic alterations in BMECs upon IFN-γ induction and indicate potential theoretical basis for clarifying IFN-γ-induced diseases in mammary gland.

Accumulating evidence suggests that amino acids are important indicators of nutritional and metabolic status. A high‐resolution mass spectrometry method based on sequential window acquisition of all theoretical mass spectra acquisition was developed for the simultaneous determination of 16 amino acids in human plasma. Sample preparation by protein precipitation using a mixture of acetonitrile and formic acid was followed by a BEH Amide column. The superiority of this method was investigated by comparing it to time‐of‐flight scan and multiple reaction monitoring modes. The limit of detection in sequential window acquisition of all theoretical mass spectra mode for threonine, methionine, histidine, citrulline, and tryptophan is 0.1 ng on the column; for lysine and asparagine is 0.2 ng; for alanine, pyroglutamic acid, leucine, ornithine, and aspartate is 0.5 ng, for arginine is 1.0 ng; for glutamate and serine is 2.0 ng; for glutamine is 10.0 ng. This method was linear in the range 0.8–40 μg/mL for arginine, citrulline, glutamate, histidine, leucine, methionine, pyroglutamic acid, threonine, tryptophan; 2–100 μg/mL for asparagine, aspartate, lysine, ornithine, serine; and 4–200 μg/mL for alanine, glutamine with good accuracy and precision. Significantly different levels in 11 amino acids were observed between childhood and adulthood, representing the growth and development of individuals relating to the level of amino acids.