| Objective:A large number of studies have shown that TNF-?,an important pro-inflammatory factor in chronic lung inflammation,plays a critical role in the development of lung adenocarcinoma.Manganese superoxide dismutase(SOD-2),as an antioxidant in mitochondria,is closely related to the progress of malignant tumors.Our recent in vitro study found that TNF-?promoted EMT and migration in A549 cells and induces proliferation and chemotherapeutic resistance in esophageal cancer cells by upregulateing SOD-2 expression.These results suggest that TNF-?-dependent inflammatiory responses may up-regulation of SOD-2 expression in tumor cells.However,these studies all simulate inflammation by administering TNF-?in vitro.Whether TNF-?mediates lung tissue inflammation and regulates SOD-2 to promote the progression of lung adenocarcinoma is not clear at the overall level.In this study,we established a model of TNF-?-dependent inflammation-driven lung adenocarcinoma in mice induced by urethane and collected human lung adenocarcinoma specimens to investigate the effect of chronic inflammation mediated by TNF-?on SOD-2 expression at the overall level.Methods:1.We used urethane to induce lung inflammation-driven lung adenocarcinoma(IDLA)in mice.After mice were subjected to urethane-treatment for 8 weeks,chronic lung inflammatory responses were induced in their lung tissues;When the mice were treated with urethane for 8weeks,and then followed for a further 4 months after the end of the urethane treatment period,the mice developed lung adenocarcinoma.In some experiments,sTNFR:Fc was given to neutralize TNF-?in urethane-induced lung inflammation and lung adenocarcinoma.The expression of TNF-?,SOD-2,as well as CD68 macrophage infiltrations were measured by immunohistochemistry and immunofluorescence staining.2.Human paraffin-embedded cancer samples were obtained from the Department of Pathology,Hebei Chest Hospital,from 2010 to 2015,with the Institutional Internal Review Board's approval.A total of 44 cases of lung adenocarcinoma(LA)and 20 cases of normal lung tissue(NLT)were collected in the study.The expression of SOD-2 and TNF-?as well as CD68~+macrophages infiltrations in human lung adenocarcinoma was measured by immunohistochemical staining and immunofluorescence analysis.3.The expression of SOD-2 in A549 and H1299 cells was inhibited by siRNA technique,and then treated with TNF-?.The expression of SOD-2,CyclinD1 and Survivin was detected by Western Blot.The proliferation of A549 and H1299 cells was detected by CCK8 test and clone formation test.Results:1 In urethane-induced IDLA,SOD-2 was highly expressed in inflammatory tissues and lung adenocarcinoma tissues1.1 SOD-2 expression increased in urethane-induced IDLAIDLA was induced by urethane in abdominal cavity.Tissue samples of control group,inflammatory group and lung adenocarcinoma group were collected.It was found that the expression of SOD-2 in inflammatory lung tissue and lung adenocarcinoma tissue was significantly increased,and the expression of SOD-2 in lung adenocarcinoma group was significantly higher than that in inflammatory group.It is suggested that SOD-2 may be involved in the development of lung adenocarcinoma in the IDLA model from inflammation to tumorigenesis.1.2 Increased SOD-2 expression in lung adenocarcinoma cells arising from AT-IIIF staining was used to observe the co-expression of SP-C and SOD-2.A large number of co-expression cells of SP-C and SOD-2 were found in inflammatory tissue and lung adenocarcinoma tissues,and that in lung adenocarcinoma tissues was higher than that in inflammatory tissue.It is suggested that SOD-2 expression in AT-II increases gradually from inflammation to tumorigenesis.1.3 TNF-?secreted by macrophages in IDLAIn inflammatory lung tissues,the expression of TNF-?in CD68~+macrophages was significantly increased by IF.In lung adenocarcinoma tissues,TNF-?was expressed not only in CD68~+macrophages,but also in lung adenocarcinoma cells.It is suggested that macrophages can secrete TNF-?in the inflammatory stage,and after the occurrence of lung adenocarcinoma,tumor cells themselves can also secrete TNF-?.1.4 Increased SOD-2 expression in IDLA is associated with macrophage-related chronic inflammationImmunofluorescence showed that the number of CD68~+cells and the expression of SOD-2 were significantly increased in inflammatory lung tissue and lung adenocarcinoma tissue,and were significantly higher in lung adenocarcinoma tissue than in inflammatory lung tissue.It is suggested that macrophage infiltration is closely related to SOD-2 expression and may play an important role in the development of lung adenocarcinoma.2 TNF-?-dependent lung inflammatory responses regulates SOD-2 at the pro-tumor stageIn the pro-tumor stage,TNF-?neutralizing antibody sTNFR:Fc was given to inhibit the inflammatory response of lung tissue.Inflammatory cell infiltrations,the expression of TNF-?,SOD-2,SP-C and the number of CD68~+cells in inflamed lung tissues were significantly decreased by TNF-?neutralizing.SOD-2 expression in AT-II is decreased,and TNF-?secretion by macrophages is also reduced.These results suggest that TNF-?-dependent chronic inflammation may up-regulate SOD-2 expression associated with macrophages in lung tissues at pro-tumor stage.3 Effect of inhibiting TNF-?-dependent lung inflammatory response on SOD-2 expression in lung adenocarcinomaInhibitation of TNF-?-dependent lung inflammation by sTNFR:Fc in pro-tumor stage did downregulate the expression of SOD-2,CyclinD1 and Survivin in lung adenocarcinoma.CD68~+macrophage cells inflitation was also decreased after TNF-?inhibitation.These results suggest that blocking TNF-?can down-regulate SOD-2 expression in lung adenocarcinoma cells and affect the proliferation of lung adenocarcinoma cells.Macrophage infiltration may be closely related to SOD-2 expression.4 Macrophage-related inflammatory environment is associated with SOD-2and TNF-?expression in human lung adenocarcinoma4.1 CD68 is associated with SOD-2 expression in human lung adenocarcinomaIn 44 human lung adenocarcinoma specimens,the expression of SOD-2was significantly higher than that in adjacent control tissues by IHC assay;the infiltration of CD68~+macrophages and the expression of SOD-2,CyclinD1and Survivin was also increased in specimens;Spearman correlation analysis showed that the number of CD68~+cells was positively correlated with SOD-2(P<0.05,r=0.8117).suggesting that Macrophage infiltration in the inflammatory environment is positively correlated with the expression of SOD-2 in human lung adenocarcinoma tissues.4.2 TNF-?is associated with SOD-2 expression in human lung adenocarcinoma.IHC results showed the expression of TNF-?and SOD-2 was increased in lung adenocarcinoma.Spearman correlation analysis showed that TNF-?was positively correlated with SOD-2 expression(P<0.05,r=0.3768).4.2 The relationship between clinicopathological features and SOD-2expression in lung adenocarcinomaSpearman correlation analysis showed that SOD-2 expression in lung adenocarcinoma was significantly higher than that in normal control group.The expression of SOD-2 in human lung adenocarcinoma was positively correlated with lymph node metastasis and TNM stage(P<0.05),but not with age,sex and differentiation of tumors(P>0.05).These results further confirm that SOD-2 plays an oncogene role in lung adenocarcinoma.5.Upregulation of SOD-2 by TNF-?promotes proliferation in A549 and H1299 cellsTNF-?-induced cell proliferation was significantly inhibited by SOD-2blocking in A549 and H1299 cells(P<0.05),suggesting that low dose of TNF-?could promote the proliferation of lung adenocarcinoma cells by up-regulation of SOD-2.Conclusion:TNF-?-dependent lung inflammation up-regulates SOD-2 expression,which may promote cells proliferation in lung adenocarcinoma. |
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