The Mechanism Of UT-B Overexpression Inducing B16 Melanoma Cell Death By Activating P53 And Mitochondrial Apoptosis Pathway

Background: Malignant melanoma occurs mostly in the skin.Although the incidence rate accounts for only 4% of all skin cancers,its mortality rate accounts for 80% of skin cancer mortality.Only 14% of patients with metastatic melanoma survived for five years,and the incidence of cutaneous melanoma has increased year by year.P53 is a common tumor suppressor and has been extensively studied for its transcriptional regulation of cell cycle and apoptosis genes.P53 limits glycolysis and drives transcription of genes required for ETC assembly and maintenance.In addition to transcriptional regulation of mitochondrial activity,p53 also acts directly on mitochondria to induce apoptosis in response to stress by interacting with members of the Bcl-2 family.P53 is a transcriptional inducer of Bax,which is a link between DNA damage and apoptosis.At the same time,p53 also restricts glycolysis and inhibits excessive nucleotide,amino acid and fatty acid synthesis in many ways,and malignant melanoma metabolism has attracted attention.Metabolic reprogramming is the energy basis for cancer cell proliferation and metastasis.UT-B is a membrane transporter protein involved in urea transmembrane transport.It has been reported in the literature that the expression of urea transporter B(UT-B)in bladder cancer is decreased,and the tumor malignancy increases.UT-B is a membrane transporter protein that mediates rapid transmembrane transport of urea.It is mainly involved in the expression of urine in the kidney,and is also widely distributed in extrarenal tissues such as skin,brain,heart and liver.It has been shown that changing the expression of UT-B on the membrane affects the instantaneous concentration of urea inside and outside the cell,which may cause changes in the amino acids,sugar,fat,etc.of the tumor cells,thereby affecting the energy metabolism of the cells.Therefore,it is speculated that UT-B may be related to the development of melanoma.P53 plays an important role in initiating cell cycle delay after exposure to high salt and urea,and thus may provide sufficient time to repair DNA damage or initiate apoptosis,which may prevent the proliferation of damaged DNA.It is reported that mitochondrial dysfunction and energy metabolism remodeling occur in cardiomyocytes of UT-B knockout mice.It is suggested that overexpression of UT-B-induced apoptosis may be related to the mitochondrial apoptosis pathway.Objective:To up-regulated the expression of UT-B in B16 cells by transfecting UT-B over-expression plasmid.The effect of UT-B on the growth of melanoma cells was studied in vitro and in vivo,and its possible mechanism was preliminary studied.Methods and results:(1)The expression of UT-B in human melanoma tissue decreased.We used HE immunohistochemical staining method to detect the expression level of UT-B in each tissue of human skin cancer chip(Shanghai Core Super).UT-B positive expression can be seen in normal skin tissue,and there were a large number of brown patches and particles mainly in the basal layer cells.Only a few brown granules and patches were present in the 8 skin melanoma tissues on the chip,indicating that the UT-B protein was less expressed in the melanoma tumor than in the normal skin.We also collected4 cases human melanoma tissue and extracted RNA for RT-PCR experiments to detect the expression level of UT-B mRNA.The results showed that there were few UT-B mRNA expressions in melanoma tissues of 3 patients,which further confirmed that the UT-B mRNA and protein levels in melanoma were significantly decreased.(2)Overexpression of UT-B in melanoma B16 cells inhibited cell growth and increased B16 cells apoptosis.RT-PCR showed no expression of UT-B mRNA in melanin B16 cell line.The expression level of UT-B in B16 cells was up-regulated by transfecting UT-B expression plasmid,and the control group was transfected with p CDNA3.1 empty plasmid.Western Blotting and immunofluorescence showed that B16 cells transfected with pUT-B plasmid had higher UT-B protein expression levels after 48 hours.Flow cytometry experiments revealed that after transfectedwith the UT-B expression plasmid,B16 cells exhibited G2/M arrest compared to the control group.MTT assay demonstrated that after transfected with pUT-B plasmid48 hours,the viability of B16 cells decreased(decreased to 32.4% of the control group).Scratch tests showed that the migration ability of B16 cells after over-expressing UT-B also significantly decreased by about 4 times.And colony formation experiments showed that the colony forming ability of B16 cells decreased.The above results indicate that overexpression of UT-B inhibits the growth and proliferation of B16 cells.Flow cytometry was used to detect apoptosis after Annexin V/PI staining.It was found that after transfection of UT-B expression plasmid,compared with the control group(6.85%±1.24%),the apoptosis of B16 cells was significantly increased(11.63%±1.67%).Hoechst staining showed nuclear condensation and nuclear debris in B16 cells over-expressing UT-B increased and apoptotic bodies increased(p<0.05),which also demonstrated apoptosis was increased.In addition,Western Blotting experiments showed that the expression levels of the pro-apoptotic proteins Bax and Cleaved-Caspase3 were increased(p<0.05)and the protein level of the anti-apoptotic protein Bcl2 was decreased(p<0.05),indicating that over-expression of UT-B promoted B16 cell apoptosis.(3)Overexpression of UT-B in melanoma B16 cells may induce apoptosis through activation of p53 and mitochondrial damage.It has been reported that mitochondrial dysfunction occurs in cardiomyocytes of UT-B knockout mice,suggesting that changes in UT-B expression levels are likely to affect mitochondrial function.Flow cytometry was used to detect ROS level after staining with DCFH-DA fluorescent probe.The results showed that after 48 hours of UT-B expression,the ROS production in B16 cells increased(18.7% compared to the control group,p<0.05),and the mitochondrial membrane potential decreased(p<0.05),while the oxygen consumption of the cells decreased significantly(decreased to 54.8% of the control group,p<0.05).At the same time,the expression levels of NDUFV1,CYC1,COX7 C,and ATP5F1,which are important subunits of complexes I,III,IV,and V in the mitochondrial electron transport chain,,were significantly lower(p<0.05).The expression of SOD2,an enzyme that scavenges oxygen free radicals,also decreased significantly(p<0.05),and CYC1 released from the mitochondria to the cytoplasm increased significantly.The above shows that the mitochondrial function of B16 cells is decreased after UT-B overexpression,thereby activating the mitochondrial apoptosis pathway to induce apoptosis.Mitochondrial damage in bladder epithelial cells is p53-dependent in UT-B knockout mice,and this study also investigated whether the effect of overexpressing UT-B in melanoma is also related to p53.Overexpression of UT-B by Western Blotting was found to up-regulate the expression of p53 and its downstream p21 in B16 cells.The activation of AKT promotes nuclear entry of MDM2 and reduces the cellular level of p53.In this study,it was found that the expression level of p-AKT was down-regulated after transfection of UT-B expression plasmid,and the expression of MDM2 was down-regulated(p<0.05).The above shows that transfection of UT-B expression plasmid inhibits the proliferation of B16 cells and may induce apoptosis through activation of p53 and mitochondrial pathways.(4)Lipid metabolism related enzyme expression and glucose uptakeIn B16 cells,Western Blotting assay revealed that overexpression of UT-B reduced the expression of the glucose transporter GLUT1,and spectrophotometric detection revealed that overexpression of UT-B reduced glucose uptake.RT-PCR assay showed that FASN,a key enzyme for fat synthesis,and mRNA expression of SREBP1 were decreased.The above results indicate that overexpression of UT-B in B16 cells may affect cellular glucose metabolism and lipid metabolism.(5)UT-B overexpression in mouse melanoma xenograft tumors also significantly inhibits tumor growth,which may be related to the p53 pathway.We constructed a mouse melanoma xenograft model and divided the mice into PQ group,PQ-P(PQ-p CDNA3.1)group and PQ-U(PQ-UT-B)group.A tumor-targeting attenuated Salmonella was used as a carrier to carry p CDNA3.1 and UT-B plasmids and administered to mice by tail vein injection.After that,tumor growth and growth status were observed.The results showed that the expression of UT-B in tumors of PQ-U group was higher than that of PQ group and PQ-P group.UT-B was overexpressed in tumors for treatment(16 days).The tumor volume growth rate and weight were significantly lower than those in the control group,indicating that overexpression of UT-B inhibited melanoma growth.At the same time,Western Blotting detected the up-regulation of Bax in tumor cells and the down-regulation of Bcl2.Overexpression of UT-B increased the expression of p53 and p21 and decreased the expression of Mmd2 and p-AKT.Conclusions:1.Compared with normal human skin,human melanoma tissue has a lower expression level of UT-B.It is speculated that changing the expression of UT-B may affect the proliferation and growth of melanoma cells.2.UT-B can induce the expression of proapoptotic proteins BAX and Cleaved-Caspase3 in B16 cells,and also low expression of Bcl2.It indicates that UT-B can promote the apoptosis of B16 cells and inhibit its growth and proliferation.3.UT-B can induce the decrease of mitochondrial membrane potential in B16 cells,decrease the expression of cytochrome C in mitochondria,and increase the expression of cytochrome C in cells,suggesting that UT-B may promote apoptosis through mitochondrial pathway.4.Lipid metabolism related enzyme expression and sugar uptakeIn B16 cells,after overexpression of UT-B,Western Blotting assay showed a decrease in the expression of the glucose transporter GLUT1,and a decrease in glucose uptake was detected by spectrophotometry.RT-PCR assay showed that the mRNA expression of FASN and SREBP1,the key enzymes of fat synthesis,was decreased.The above results indicate that overexpression of UT-B in B16 cells may affect cellular glucose metabolism and lipid metabolism.5.UT-B can inhibit the decrease of phosphorylated AKT expression,and activate p53 through p53 negative regulator MDM2 to inhibit the growth of melanoma B16 cells.In summary,after up-regulating UT-B expression levels in B16 cells,inhibition of glucose metabolism and lipid metabolism and induction of mitochondrial pathway apoptosis inhibits the growth and proliferation of melanoma B16 cells and promotes their death by activating p53.We have demonstrated the role of UT-B in the development of cutaneous melanoma and provide new strategies for the treatment of melanoma.