The Role Of TNF-α In The Process Of Epithelial Mesenchymal Transition Induced By BleomycinA5 And The Related Molecular Mechanisms

Epithelial-mesenchymal transition(EMT)is a biological process in which polar epithelial cells are stimulated to change their characteristics and become mesenchymal phenotypes.In EMT,after being stimulated by different factors,the morphology,structure,adhesion and mobility of epithelial cells are altered,expression of the epithelial cell marker proteins(e.g.E-cadherin)are decreased;while that of the mesenchymal cell marker proteins(e.g.Fibronectin and Vimentin)are increased.These changes lead to the weakening of the intercellular junction and the enhancement of cell migration and invasion.EMT is very important for embryonic development,organogenesis and angiogenesis,but in recent years,it was also found that EMT participates in wound healing,fibrosis,invasion and metastasis of tumors,and is considered as an important step in the invasion and metastasis of tumors.Tumor Necrosis Factor alpha(TNF-a)is a bioactive cytokine that once was thought to be only produced by macrophages.It can induce a wide variety of reactions under different cell microenvironments.It has been reported that TNF-alpha can promote the metastasis and invasiveness of tumors and participate in the regulation of EMT.In different cell lines,TNF-alpha can induce EMT by regulating different signaling pathways,such as the nuclear factor kappa-B(NF-κB),phosphatidylinositol 3’-kinase/protein kinase B(PI3K/Akt),Wnt/β-catenin,and mitogen-activated protein kinases(MAPKK / ERK)pathways.Space radiation is one of the biggest obstacles to long-term manned space flight and deep space exploration.One of the major characteristics of space radiation is that it is composed of a large number of high-energy protons,particles,heavy ions,etc.These high linear energy transfer(LET)particles can cause complex cluster damage that is very difficult to repair,hence has strong lethal,mutagenic and carcinogenic effects,which will pose a serious threat to the health of the astronauts.Therefore,it is essential to thoroughly understand the biological effects of space radiation.BleomycinA5 is a glycopeptide antibiotic used clinically to treat a variety of cancers,including the squamous cell carcinoma,breast cancer,and liver cancer.Because of its special structure,it can produce biological effects such as DNA damage,mutagenic and carcinogenic effects that are similarto radiation.In the present study,we first confirmed that similar to alpha particle irradiation,BleomycinA5 could induce EMT in human bronchial epithelial cell BEAS-2B.Further,some preliminarily investigation was carried out for the molecular mechanisms involved in the phenomenon.The results obtained are as follows:1)The morphology of BEAS-2B cells was altered from epithelial cell-like to spindle-shaped mesenchymal cells after BleomycinA5 treatment.The cells were no longer tightly connected and arranged loosely.The results of cell wound healing assay and Transwell invasion assay also showed that the invasion and migration ability of BEAS-2B cells was enhanced after treated by BleomycinA5.The phenomenon is similar to the changes of BEAS-2B cells after alpha particle irradiation with a single dose of 0.5 Gy or 25 times of 0.02 Gy irradiation.It indicated that BEAS-2B cells treated with BleomycinA5 may also undergo epithelial-mesenchymal transition similar to that of irradiation.2)After BleomycinA5 treatment,marker proteins of epithelial-mesenchymal transition were analyzed.Compared with the control untreated group,the expression of E-cadherin,a marker protein of epithelial cells decreased;while the expression of Fibronectin and Vimentin,both of which are markers of mesenchymal cells,increased.The change of the protein expression is similar to that observed after alpha particle irradiation,suggesting that BleomycinA5 is an effective radiation simulator and can cause EMT similar to radiation.Hence,BleomycinA5 can be used for further study of the underlying mechanisms of the process.3)As an important cytokine,TNF-alpha has been found to be associated with tumor initiation and development.Studies have also found that TNF-alpha may be involved in the EMT process.In order to determine whether TNF-alpha also participated in the process of EMT induced by BleomycinA5,we first detected the expression level of TNF-alpha in BEAS-2B cells after BleomycinA5 treatment.It was found that BleomycinA5 significantly increased the expression of TNF-alpha.When BEAS-2B cells were treated with TNF-alpha along with BleomycinA5,the epithelial-mesenchymal transition phenomenon became stronger.On the other hand,when BEAS-2B cells were treated with both BleomycinA5 and TNF-alpha neutralizing antibodies,the epithelial-mesenchymal transition phenomenon was partially diminished.We also treated TNF-alpha knockdown cells with BleomycinA5,and found that when TNF-alpha was knocked down,the EMT process induced by BleomycinA5 was abolished.Similar EMT phenomena were observed when BEAS-2B cells were treated directly with TNF-alpha.These results suggested that TNF-alpha mayplay an important role in the induction of EMT in BEAS-2B cells by BleomycinA5.4)It has been reported that the main signaling pathways activated by TNF-alpha include the NF-κB,PI3K/AKT and MAPKK signaling pathways.In order to explore the molecular mechanism of the EMT process induced by BleomycinA5,we first treated BEAS-2B cells with TNF-alpha and inhibitor for different signaling pathways(NF-κB,PI3K/AKT and MAPKK).It was found that all three kinds of inhibitors exhibited a partial inhibitory effect on the EMT induced by TNF-alpha.When the key proteins in these signaling pathways were detected,it was found that the phosphorylation level of AKT and ERK were significantly increased,with no change observed for the total protein level.In addition,the nuclear level of NF-κB p65 increased as well,suggesting that multiple signaling pathways are involved in the TNF-alpha induced EMT process.Further analysis revealed that BAY11-7082,the inhibitor of NF-κB pathway,not only suppressed accumulation of p65 within the nucleus,but also inhibited phosphorylation of ERK.However,BAY11-7082 treatment did not affect the phosphorylation of Akt.These results implicated that the NF-κB pathway may be situated upstream of the MAPKK pathway.