Urea Transporter B Down-regulates The Levels Of Polyamines In Melanoma B16 Cells By Activating P53

Background:Melanoma is a malignant tumor derived from melanocytes,which is characterized by a high degree of malignancy,high mortality and easy metastasis.Cancer cells typically increase their metabolic processes to meet their high biological demands,and the urea cycle can be used to eliminate the excess nitrogen and ammonia that accompanies the breakdown of proteins in the body or the synthesis of nitrogencontaining compounds.Urea transporter B(UT-B)is a membrane channel protein involved in urea transmembrane transport.The expression of UT-B in most tumors,especially melanoma,is significantly decreased compared with normal tissues,and UTB may be involved in the development of tumors.Previous studies have found that UTB in melanoma B16 cells significantly reduced the proliferation ability of B16 cells after overexpression of UT-B compared with normal tissues,and the molecular mechanism remains unclear.Ornithine,another product of the urea formation process,is a substrate for de novo synthesis of polyamines.The most common polyamines found in human cells are putrescine,spermidine,and spermine.Dysregulation of polyamines metabolism is associated with various pathological conditions,including cancer,and plays an important role in regulating tumor cell proliferation,transformation,and stabilizing cell membrane and chromatin structure.The effect of UT-B on polyamine metabolism in melanoma cells and its mechanism have not been reported.This study will provide new clues for further revealing the role of UT-B in melanoma.Aims:This study aims to observe the changes of polyamine metabolism and urea cycle in melanoma B16 cells with UT-B overexpression,which is helpful to explore the roles of mitochondrial dysfunction and p53 activation in the cell growth and polyamines metabolism.Methods:1.MTT assay was used to detect the cell viability of B16 cells,and flow cytometry was used to detect the apoptosis,ROS level and changes of mitochondrial membrane potential of B16 cells.2.Enzyme-linked immunosorbent assay and liquid chromatography were used to detect the contents of intracellular polyamines in B16 cells with overexpression of UTB.Biochemical kits were used to detect the contents of intracellular urea and nitric oxide.3.The m RNA and protein expression levels of ODC,SSAT,and Arg1,key enzymes of polyamine metabolism and urea generation,in B16 cells overexpressing UT-B,were detected by q PCR and Western blot.4.Exogenous polyamines,p53 inhibitor pifithrin-α(PFT-α),ROS scavenging agent N-acetyl-L-cysteine(N-acetyl-L-cysteine,NAC)and antioxidant coenzyme Q10(Co Q10)were added into the B16 cells with overexpression of UT-B to observe the effects of these drugs on the B16 cells with overexpression of UT-B.Results:1.In B16 cells with the UT-B overexpression,cell viability decreased,apoptosis increased,ROS level increased,and mitochondrial membrane potential decreased.2.In B16 cells with the UT-B overexpression,the spermine content decreased,the expression of ornithine decarboxylase(ODC)was down-regulated,and the expression of spermidine/spermidine-N-acetyltransferase(SSAT)was up-regulated.At the same time,the expression of the transcription factor c-Myc regulating ODC was inhibited,and the transcription factor p53 regulating SSAT was activated.3.In B16 cells with the UT-B overexpression,urea content was decreased,arginaseⅠ(ARG1)expression were inhibited;At the same time,nitric oxide(NO)content increased,nitric oxide synthase expression increased.4.Supplementation of exogenous putroamine can increase the viability,proliferation ability,apoptosis and ROS level of B16 cells with overexpression of UTB,but exogenous spermine has no significant effect.5.The p53 inhibitor PFT-α can reduce the apoptosis,increase the production of urea and polyamine,decrease the production of nitric oxide,and slightly increase the mitochondrial membrane potential of B16 cells with overexpression of UT-B.6.ROS scavenging agent NAC and antioxidant Co Q10 can increase the cell viability,mitochondrial membrane potential and polyamine production of B16 cells with overexpression of UT-B.Moreover,NAC can inhibit the expression of p53.Conclusion:1.In B16 cells with the UT-B overexpression,the growth ability was decreased,apoptosis was increased,and intracellular metabolic pathways related to the urea cycle were significantly changed,which were manifested as decreased production of urea and polyamines,and increased production of nitric oxide.2.Maintaining appropriate levels of polyamines in B16 cells contributes to the viability and proliferation,as well as reduction of apoptosis.3.UT-B overexpression causes mitochondrial dysfunction and oxidative stress in B16 cells,thus activating p53 expression,which may be one of the main mechanisms leading to the decrease of intracellular polyamines.