The Role Of Autophagy In Epithelial Mesenchymal Transition Of Human Bronchial Cells Induced With Chronic Arsenic

Objective:Long-term exposure to arsenic could induce the occurrence of a wide variety of tumor. Epithelial-mesenchymal Transition (EMT) is an important step of tumorigenesis. Autophagy is a cell self-protective mechanism against cell transformation. The aim of this paper is to reveal the role of autophagy on epithelial mesenchymal transition of human bronchial cells induced with chronic sodium arsenite exposure.Methods:1. Manipulation of BEAS-2B cellsThe cells were acutely exposed with2.5ummol/L sodium arsenic for48hours and chronicly induced with0.25ummol/L sodium arsenic for66days, BEAS-2B cells were cultured for66days without sodium arsenite as control in the same conditions. The BEAS-2B cells were transfected by Beclin-1shRNA plasmid to restrain activity of autophagy; The BEAS-2B cells were treated by10nM rapamycin for24hours to enhance activity of autophagy and by10nM Bafilomycin A1for8hours to inhibit activity of autophagy.2. Soft agar assayBEAS-2B cells were maintained in culture medium with0.25uM sodium arsenite for66days and colony formation assay in soft agar were performed. The colony was picked up after14days and cultured as transformed BEAS-2B cells.3. Activity of autophagy detectionAutophagic protein beclin-1, LC3, P62expression were detected by western blot, autophagosome was detected with Monoda-nsylcadaverine (MDC) staining and checked by fluorescence microscope.4. Detection of Epithelial marker E-cadherin, mesenehymal marker Vimentin and inflammatory related proteinE-cadherin, Vimentin, ASC were detected by western blot after artificial regulation of autophagy.Results:1. The rate of clony formation in BEAS-2B cells with chronic-arsenite exposure is22.23%with significant difference compared to the control (3.06%)(p<0.01).2. The morphology of BEAS-2B cells induced with chronic-arsenite exposure transformed from epithelial cells to mesenehymal-like cells with long spindle-shaped. The BEAS-2B cell successfully transfected by pDNA-Bclin-1shRNA plasmid.3. The expression level of beclin-1and LC3Ⅱin arsenic acute exposed group were highest, while its expression level were declined in arsenic chronic exposed group but were still higher than control group. The P62in arsenic acute exposed group and chronic exposed group was down regulated compare with control group.4. MDC staining showed stronger green fluorescence signal in arsenic acute exposed and chronic exposed group than control group. The strongest signal was in arsenic acute exposed group.5. The expression level of epithelial cells marker E-Cadherin was increased and mesenehymal cells marker protein Vimentin was decreased in BEAS-2B cells with Rapamycin treatment, while the expression level were reversed with Bafilomycin A1treatment.6. IL-1β and ASC protein expression were down regulated by rapamycin treatment while up regulated by bafilomycin A1treatment.Conclusion:1. The morphology of BEAS-2B cells was transformed induced with chronic arsenic exposure and its capacity was improved for clony formation.2. The activation of autophagy in BEAS-2B cells was enhanced by sodium arsenic induction.3. Aautophagy regulate EMT negatively and inflammatory related proteins ASC and IL-1βin BEAS-2B cells.