Inflammation-mediated SOD-2 Upregulation Contributes To Epithelial-Mesenchymal Transition,Migration And Proliferation Of Tumor Cells In Aflatoxin G₁-induced Lung Adenocarcinoma

Aflatoxins（AF）,including AFB₁,AFG₁ and AFM₁,are a type of mycotoxins,which are hepatotoxic and strongly carcinogenic,produced by certain molds of dietary staples in Asia and Africa.Recently,we found that oral administration of AFG₁ for 6 months induced chronic lung inflammation in BALB/c mice.Before the initiation of AFG₁-induced lung adenocarcinoma,we also reported that AFG₁-induced chronic inflammation might cause alveolar epithelial oxidative stress evidenced by the increase of manganese superoxide dismutase（SOD-2）expression.SOD-2 is one of the primary cellular antioxidant enzymes,which is vital in the regulation of oxidative stress by catalyzing the conversion of superoxide to hydrogen peroxide.Up-regulation of SOD-2 was shown to contribute to the migration and invasion in several carcinomas.However,the role of SOD-2 in tumorigenesis has been widely investigated and is still being explored.Whether SOD-2contributes to the migration and invasion of AFG₁-induced lung adenocar-cinoma is unknown.Several studies have shown that TNF-α/NF-κB pathway in tumor-associated inflammatory microenvironment is important to trigger epithelial-mesenchyme transition（EMT）to facilitate cancer cell motility,invasiveness,and metastatic potential.Kinugasa et al.reported that SOD-2 is upregulated through NF-κB pathway in well-defined transformed oral and esophageal human epithelial cell lines undergoing EMT.If AFG₁ induces tumor-associated inflammatory responses,there is a critical need to identify whether the tumor-associated inflammation mediates SOD-2 upregulation to contribute to EMT and migration in AFG₁-induced lung adenocarcinoma.In this study,we aim to explore the contribution of SOD-2 involved in tumor cell EMT,migration,and proliferation in AFG₁-induced lung adenocarcinoma,as well as whether tumor-associated inflammation mediates SOD-2 upregulation in the lung tumorigenesis.Part one The expression of inflammatory cytokines,SOD-2 and the relevant indicators of EMT,migration and proliferation in AFG₁ induced lung adenocarcinomaObjective:The primary objective to explore the expression of inflammatory cytokines,SOD-2 as well as the relevant indicators of EMT,migration and proliferation in AFG₁-induced lung adenocarcinoma.Methods:We collected AFG₁-induced lung adenocarcinoma samples from our previous study.The expression of IL-6 and TNF-αwas measured by IHC,Western Blot and Real time PCR.The expression of E-cadherin,vimentin,α-SMA,Twist1,CyclinD1,Survivin,PCNA,BCL-XL and SOD-2was detected by IHC and Western Blot.Results:1.Tumor-associated inflammation microenvironment is induced in AFG₁induced lung adenocarcinoma.The expression of TNF-αand IL-6 at the m RNA and protein levels were significantly upregulated in AFG₁-induced lung adenocarcinoma tissues.Immunohistochemical staining showed there were more TNF-αand IL-6positive cells and CD68+macrophages infiltration in tumor-adjacent tissues.The results suggest that AFG₁-induced tumor-associated inflammatory microenvironment consists of cytokines production by cancer cells and the surrounding tumor-adjacent cells as well as infiltrating macrophages.2.Enhanced SOD-2 expression in AFG₁-induced lung adenocarcinomaSOD-2 expression was significantly upregulated in AFG₁-induced lung adenocarcinoma compared to that in control lung tissues.3.EMT is induced in AFG₁-induced lung adenocarcinomaTo further investigate the relation between SOD-2 overexpression and EMT,we examined the well-defined markers of EMT.Increased expression of vimentin,α-SMA,and Twist1 and lower expression of E-cadherin were observed in AFG₁-induced lung adenocarcinoma.Those findings suggest that EMT of tumor cell is induced in AFG₁-induced lung adenocarcinoma.Since EMT is also associated with the acquisition of migratory and invasive properties of tumor cells,we also examined the expression of MMP-2and MMP-9.We found elevated expression of MMP-2 and MMP-9 in AFG₁-induced lung adenocarcinoma.4.Increased cell proliferation expression in AFG₁-induced lung adenoc-arcinomaCompared to control,increased expression of cyclinD1,survivin,PCNA and BCL-xL was observed in AFG₁-induced adenocarcinomas.These results indicate enhanced tumor cell proliferation was induced in AFG₁-induced lung adenocarcinoma.Part two Inflammation-mediated SOD-2 upregulation contributes to epithelial-mesenchymal transition and migration in A549 cellsObjective:In this study,we aim to explore whether inflammatory cytokines activate NF-κB pathway to upregulate SOD-2 expression,which may contribute to EMT and migration in A549 cells.Methods:A549 cells and MΦ-THP-1 cells were treated with AFG₁、IL-6and TNF-α,and SOD-2 were measured.The A549 cells transfected with STAT3 si RNA or NF-κB siRNA,were treated with IL-6 and TNF-α,STAT3/NF-κB signaling pathway related proteins were measured.Finally,A549 cells were transfected with SOD-2 siRNA,and then treated with TNF-α.The EMT and cell migration as well as E-cadherin,vimentin,MMP-2and MMP-9 expression was detected.Results:1.Upregulation of SOD-2 in cancer cells is dependent on the inflam-mation-activated NF-κB pathwayTo establish the mechanism of SOD-2 upregulation in AFG₁-induced lung tumor inflammatory microenvironment,A549 cells and MΦ-THP-1 cells were treated with different concentrations of AFG₁,IL-6 and TNF-α.IL-6 and AFG₁ did not induce SOD-2 expression in A549 cells,while TNF-αcould upregulate SOD-2 as well as IL-6 may enhance TNF-α-upregulated SOD-2expression.Then,we found AFG₁ could activate NF-κB pathway and upregulate SOD-2 expression in MΦ-THP-1 cells.Furthermore,NF-κB and STAT3 signaling pathways were activated by TNF-αplus IL-6 treatment in A549 cells.NF-κB knockdown significantly inhibited SOD-2 upregulation in the cells treated with TNF-αplus IL-6,while blocking STAT3 did not affect SOD-2 upregulation.This suggests that the NF-κB pathway in AFG₁-induced lung tumor inflammatory microenvironment may play a key role in upregulating SOD-2 expression.2.TNF-αworking with IL-6,not AFG₁,induced EMT and migration in lung cancer cellsWe treated A549 with AFG₁ and/or TNF-αwith IL-6 to investigate to the key contributor of EMT and migration in AFG₁-induced lung adenocarcinoma.The combined treatment of TNF-αand IL-6 could induce morphological changes in A549 cells,characterized by fibroblast-like cells,while AFG₁ did not cause EMT in the tumor cells.Combination of TNF-αplus IL-6 treatment induced higher level expression of vimentin,Twist1,MMP-2 and MMP-9 as well as lower level expression of E-cadherin in A549 cells compared with TNF-αalone.The wound healing migration assay and transwell migration assay demonstrated that TNF-αworking together with IL-6 significantly enhanced A549 cell migration compared to TNF-αor IL-6 alone.Taken together,the results suggest that TNF-αis the key contributor of EMT and migration in AFG₁-induced lung adenocarcinoma.3.SOD-2 contributes to TNF-α-mediated EMT and migration in A549cellsTo determine whether SOD-2 is involved in inflammation-mediated EMT and migration in cancer cells,SOD-2 was knockdown by si RNA in A549 cells,and then the cells were treated with TNF-α.Blocking SOD-2 expression partly inhibited E-cadherin downregulation and vimentin upregulation induced by TNF-α.The EMT-like phenotypic changes were confirmed by detecting the expression of characteristic molecular markers using immunofluorescence.Thus,the suppression of SOD-2 partly reversed TNF-α-mediated EMT in A549 cells.The blocking of SOD-2 expression also inhibited the upregulation of MMP-2 and MMP-9 in A549 cells treated with TNF-α,which suggest that SOD-2 upregulation may be responsible for cell migration.The wound healing assay showed that blocking SOD-2 expression partly inhibited cells migration in TNF-α-treated cells.Transwell assay results showed that cell numbers in bottom chamber of SOD-2 knockdown cells treated with TNF-αwere lower than that of control cells.Those results support that blocking SOD-2expression partly inhibited TNF-α-induced migration in A549 cells.All above results suggest that SOD-2 upregulation upon inflammation responses may contribute to TNF-α-mediated EMT and migration in tumor cells in AFG₁-induced lung adenocarcinoma.Part three The expression of SOD-2 associated with Survivin and CyclinD1 as well as clinical outcomes in human lung adenocarcinomaObjective:To further explore the relationship between the expression of SOD-2 and TNF-α,Survivin and CyclinD1 as well as clinical outcomes in human lung adenocarcinoma.Method:The specimens of lung adenocarcinoma after surgery were collected,and the expression of TNF-α,SOD-2,Survivin and CyclinD1,were detected by IHC staining.Statistical methods were used to determine the correlation between SOD-2 and TNF-α,Survivin and CyclinD1,as well as the clinicopathological significance of SOD-2 and its relationship with the poor prognosis.Results:1.The expression of TNF-α,SOD-2,Survivin and CyclinD1 in human lung adenocarcinoma.IHC staining showed that the positive rates of TNF-α,SOD-2,Survivin and CyclinD1 were 82.4%,48.5%,50%and 61.8%,respectively,which were higher than those in normal lung tissues（P<0.05）.2.Correlation between TNF-αand SOD-2,as well as SOD-2 and Survivin and CyclinD1 in lung adenocarcinomaThere is a positive correlation between TNF-αand SOD-2（P<0.05）.There was a positive correlation between SOD-2 and Survivin and CyclinD1（P<0.05）.The results suggest that TNF-αmay promotes SOD-2 expression,which is associated with Survivin and CyclinD1 expression in human lung adenocarcinoma.3.Clinicopathological significance of SOD-2 protein expression and its relationship with poor prognosisSOD-2 is associated with lymph node metastasis in lung adenocarcinoma,but there is no correlation between its expression and sex,age and differen-tiation.Kaplan-Meier survival analysis showed that the increased expression of SOD-2 was related to a poor prognosis（P<0.05）.Conclusions:1.AFG₁-induced tumor-associated inflammatory microenvironment consists of cytokines production by cancer cells and the surrounding tumor-adjacent cells as well as infiltrating macrophages.2.Enhanced SOD-2 expression in AFG₁-induced lung adenocarcinoma.3.EMT and increased expression of proliferation-related proteins were induced in AFG₁-induced lung adenocarcinoma.4.Upregulation of SOD-2 in cancer cells is dependent on the inflam-mation-activated NF-κB pathway.5.TNF-αworking with IL-6,not AFG₁,induced EMT and migration in lung cancer cells.6.SOD-2 contributes to TNF-α-mediated EMT and migration in A549cells7.In human lung adenocarcinoma specimens,the expressions of TNF-α,SOD-2,Survivin and CyclinD1 were increased,and there was a positive correlation between TNF-αand SOD-2,as well as SOD-2 and Survivin and CyclinD1.8.SOD-2 is associated with lymph node metastasis in lung adenoca-rcinoma,but not with sex,age and differentiation.Increased expression of SOD-2 is associated with poor prognosis in the patients.