TNF-? Secreted By Macrophages Upregulates SOD-2 To Contribute To Tumor Cells Migration And Invasion In Glioblastoma

Objective: Glioblastoma is the most common malignant tumor of central nervous system in adults with high malignant degree and poor prognosis.In recent years,great attention has been paid to the relationship between inflammatory microenvironment and the development of tumor.Studies have shown that TNF-?-related inflammation plays a critical role in the progression of different type of cancer,such as lung cancer,colorectal cancer,and glioblastoma.In our previous study,we found that TNF-? upregulated SOD-2 expression in A549 cells as well as Eca109 cells,which may be related to tumor promotion.Manganese superoxide dismutase?SOD-2?plays an important role in oxidative stress,which can function as both oncogene and tumor suppressor gene in different tumors.The effect and mechanism of SOD-2 on migration and invasion in glioblastoma have not been shown,and it is not clear whether TNF-? in tumor microenvironment is involved in the regulation of SOD-2 in glioblastoma.In this study,we collected human paraffin-embedded glioblastoma samples to explore the expression of SOD-2 in glioblastoma progression.We also measured the role of TNF-? in the migration and invasion in glioblastoma cells,U87 and U251 cells.We isolated supernatant from human monocyte-macrophage THP-1 cells to stimulate U87 and U251 cells,and explore whether macrophages induce SOD-2 upregulation through TNF-? secretion.The purpose of this study is to reveal the effect of oxidative stress-related proteins on glioma progression,which may provide a new theoretical basis for biological targeted therapy of GBM.Methods:1. The expression of SOD-2 at m RNA level in GBM and its clinical outcome were analyzed in TCGA?TCGA The Cancer Genome Atlas?.2.We collected 45 cases of human paraffin-embedded glioblastoma samples from the Department of pathology in second Hospital of Hebei Medical University.Immunohistochemical and immunofluorescence staining were processed for detecting the expression of SOD-2 and TNF-?in tumor tissues,as well as number expression of SOD-2 and TNF-?,CD68⁺macrophage infiltration,as well as the overall survival status of the patients were analyzed.3.U87 and U251 cells were transfected with or without SOD-2si RNA,and then treated with TNF-?.The expression of SOD-2,cell proliferation and cell migration were measured.4.Human monocyte-macrophage THP-1 cells were cultured and induced to differentiate into M1 macrophages in vitro.The supernatant blocked with anti-TNF-?neutralizing antibody was collected to stimulate U87/U251 cells,and the expression of SOD-2was detected by Western blot.Results:1 The expression of SOD-2 and TNF-?and the infiltration of CD68⁺macrophages in human glioblastoma tissues.1.1 SOD-2 was highly expressed in glioblastoma from TCGA database.High expression of SOD-2 was related to the patients with lower survival rate.1.2 The relationship between SOD-2 and TNF-?expression and the infiltration of CD68⁺macrophages in human glioblastoma tissues.45 cases of human glioblastoma tissues and 27 cases of normal brain tissue adjacent to GBM were collected.Immunohistochemical staining showed that high expression of SOD-2 and TNF-?,and increased CD68⁺macrophages infiltration was observed in tumor.Kaplan-Meier survival curve showed that the survival rate of patients with high expression of SOD-2 tend to lower than those with low expression of SOD-2.The expression of TNF-?was closely related SOD-2 in tumor tissues.Immunofluorescence staining showed that TNF-?and CD68were co-expressed in glioblastoma,indicating that both tumor cells and CD68⁺macrophages in glioblastoma could secrete TNF-?.2 Effect of SOD-2 on migration and invasion in U87 and U251 cells.U87 and U251 cells were cultured in vitro,and transfected with si RNA to inhibit the expression of SOD-2.Wound healing and transwell assay showed that inhibition of SOD-2 decreased the migration and invasion ability of glioblastoma cells.3 Effects of SOD-2 on mitochondrial function,glycolysis level,SOD-2 activity and H₂O₂ content in glioblastoma.Blocking SOD-2 inhibited the expression of mitochondrial transcription factor-a?TFAM?in U87/U251 cells.RT-PCR results showed that blocking SOD-2 could down-regulate the expression of PKM2 and HK2 at m RNA level.SOD-2 activity and H₂O₂ content were decreased significantly by SOD-2 inhibition.4 TNF-?regulates migration and invasion in U87 and U251cell through SOD-2 expression.TNF-?significantly up-regulated the expression of SOD-2 and increased the ability of cell migration and invasion in U87 and U251cells.Blocking SOD-2 by si RNA could inhibit TNF-?-induced cell migration and invasion,which suggests that TNF-?enhances the ability of tumor cell migration and invasion by up-regulating SOD-2.5 TNF-?secreted by THP-1-M1 macrophages upregulated the expression of SOD-2 in U87 and U251 cells.To further explore whether macrophages contribute to SOD-2upregulation by secreting TNF-a,we collected the supernatant from LPS-activated PMA-induced THP1?M1?to stimulate U87 and U251 cells.The expression of SOD-2 was measured.We found that M1 supernatant significantly upregulated SOD-2 expression in the cells.Then neutralization of TNF-?secreted by M1 macrophages using anti-TNF-?antibody induced a significant decrease in SOD-2 in U87 and U251 cells,compared to the supernatant of M1.Thus,the findings indicate that THP1-M1 can induce SOD-2 upregulation in U87 and U251 cells by secreting TNF-?.Conclusions:1.The high expression of SOD-2 is closely related to the poor macrophage infiltration in tumor tissues are positively correlated with the high expression of SOD-2.2.High expression of SOD-2 can promote the migration and invasion of tumor cells,and up-regulate mitochondrial biogenesis,the level of glycolysis,the activity of SOD-2 and the content of H₂O₂.3.TNF-?enhanced the migration and invasion in glioblastoma cells by up-regulating SOD-2,and TNF-?secreted by M1macrophages up-regulated the expression of SOD-2 in tumor cells.