The Mechanism Of Arginine Antagonist Malignant Transformation Of Mammary Epithelial Cells Induced By IFN-γ And Its Validation By Animal Model

Objective:Breast cancer is one of the most common malignant tumors in women worldwide,it seriously affects women’s physical and mental health and is life threatening.And the incidence of cancer in China’s residents has been on the rise for the past decade or so.IFN-γ is a pleiotropic cytokine that plays a dual role in tumorigenesis and progression.Our previous study demonstrated that IFN-γ promoted the malignant transformation of primary bovine mammary epithelial cells by accelerating arginine depletion.Arginine,a non-essential amino acid,is closely related to the biological behavior of tumor.It’s supposed that arginine addition can antagonize malignant transformation of mammary epithelial cells induced by IFN-γ.The study aimed to investigate the mechanism of arginine on the malignant transformation of mammary epithelial cells under the action of IFN-γ.Methods:In the present study,malignant transformed mammary epithelial cells induced by IFN-γ were selected as experimental subjects.Malignant transformation of cells was observed by adding different concentrations of arginine,including cell proliferation(MTT assay),cell migration(would healing assay)and cell clone formation assay.The molecular mechanisms of its occurrence further were explored by Screening cancer-related genes from transcriptomics.Western blotting was used to verify the pathway proteins.Secondly,the effects of arginine on the development of breast cancer and its molecular mechanism were verified by animal tumorigenesis experiments.Finally,ELISA was used to detect human breast cancer specimens and immunohistochemistry was used to verify the protein molecules.Results:(1)IFN-γ can promote the malignant transformation of BMECs through arginine depletion mediated by GCN2/e IF2α signaling pathway.However,arginine addition can antagonize the malignant transformation of mammary epithelial cells induced by IFN-γ through NF-κB/GCN2/eIF2α signaling pathway,performing as cell proliferation weaken,cell migration slower,and plate colony formation reduced.(2)The vivo animal experiments confirmed that arginine supplementation could significantly inhibit tumor growth in tumorbearing mice,and the expression of p-p65,p-GCN2,p-EIF2S1 in arginine addition group was much lower than that of IFN-γ alone treatment group.(3)Clinical studies further verify that the plasma or tissue from human breast cancer patients showed lower arginine level and higher IFN-γ level than that from patients with benign breast disease,and the expression levels of arginine and IFN-γ are correlated with TNM staging of cancer,ER(-/+),PR(-/+)and other related.And the expression of p-p65,p-GCN2,p-EIF2S1 in breast cancer specimens was much higher than that of benign breast disease.Conclusion:Arginine supplement could antagonize the malignant transformation of mammary epithelial cells induced by IFN-γ through NF-κB/GCN2/eIF2α signaling pathway in vitro and in vivo,which is provided a new idea for the prevention and treatment of breast cancer.Inhibition of p65,GCN2,and eIF2α activity can be used as a target for breast cancer treatment.IFN-γ reduction or arginine addition may be one of the methods for breast cancer prevention.