Role And Mechanism Of Sodium Thiodicarbonate In The Inhibition Of Sodium Arsenite Induced EMT And Acquisition Of Tumor Stem Cell-like Features In Human Lung Epithelial Cells

BackgroundArsenic contamination has long been a global public health problem,with exposure occurring mainly through drinking water,groundwater and food,and is a Class I carcinogen identified by the International Agency for Research on Cancer（IARC）.Epidemiological studies have demonstrated that arsenic exposure leads to an increased risk of bladder,skin and lung cancer in susceptible people,with lung cancer being the leading cause of cancer-related deaths worldwide.Studies have shown that arsenic contamination in drinking water affects at least 140 million people in 50 countries and is considered to be one of the major challenges to public health.Hydrogen sulphide（H₂S）has been considered a toxic gas for decades and has only recently been recognised as a gaseous transmitter with a variety of physiological and pathophysiological effects including vasodilatory,antioxidant and anti-inflammatory.Several studies have demonstrated that exogenous hydrogen sulphide donors,such as sodium hydrosulphide（Na HS）,have anti-inflammatory and anti-fibrotic properties and are therapeutic and protective against organ fibroproliferative diseases However,it is unclear whether sodium hydrosulphide plays a role in the carcinogenic effects of sodium arsenite（Na As O₂）or the mechanism.ObjectiveIn order to investigate the specific role and potential molecular mechanism of sodium thioredoxin in trivalent arsenic-induced lung cancer,human lung bronchial epithelial cell line BEAS-2B cells were exposed to sodium arsenite for a long period of time,and thus a trivalent arsenic-induced malignant transformation BEAS-2B cell line（Trivalent arsenic-transformed cells,Tas-B-2B）model was established to further investigate the role and molecular mechanism of sodium thioredoxin in trivalent arsenic-induced lung cancer.Methods（1）To confirm the exposure time and dose of sodium arsenite,the effect of sodium arsenite exposure at different time and concentration on the survival rate of BEAS-2B cells was examined by MTT（tetramethylazole salt）method.（2）To investigate the promotion effect of Na As O₂on tumor stem cell formation,BEAS-2B cells were treated with long-term low dose Na As O₂（0.5μM,5months）,and the self-renewal and growth ability of the cells were detected by suspension into balls and colony assay;the cell morphology was also observed and recorded by inverted microscope.（3）The contents of epithelial-mesenchymal marker and tumor stem cell marker molecules were determined by Western Blot method at different concentrations of sodium arsenite（0,0.25,0.5,1.0,2.0μM-24h）,different times（0,6,12,24,36h-1.0μM）and long-term（0.5μM,5 months）.（4）To investigate the role of hydrogen sulfide in sodium arsenite-induced lung epithelial-mesenchymal transition and acquisition of tumor stem cell-like features,BEAS-2B cells were treated with different concentrations（0,0.25,0.5,1.0,2.0μM-24h）and times（0,6,12,24,36h-1.0μM）,and then intracellular H₂S content was detected by chemofluorescence assay;the intracellular H₂S content was detected by Western Blot assay to detect the protein expression levels of three H₂S synthases CTH,CBS and MPST and observe their trends.（5）To investigate the role of HIF-1αgene and protein in the induction of epithelial mesenchymal transition and malignant transformation in BEAS-2B cells by sodium arsenite,the protein and m RNA expression levels of HIF-1αin sodium arsenite-exposed BEAS-2B cells were detected by Western Blot and q RT-PCR.To investigate the relationship between HIF-1αand the Wnt/β-catenin signaling pathway,cells were pretreated with the HIF-1αinhibitor YC-1,and changes in the expression of Wnt3a protein and genes were detected by Western Blot and q RT-PCR.The distribution and content ofβ-catenin protein in the cells were detected by cellular immunofluorescence.（6）To investigate the role of hydrogen sulfide in sodium arsenite-induced increase in HIF-1αexpression and activation of Wnt/β-catenin signaling pathway,which in turn leads to epithelial-mesenchymal transition of cells and acquisition of tumor stem cell-like features,pretreatment with Na HS was performed,and the expression levels of HIF-1α,Wnt3a protein and genes were measured by Western Blot and q RT-PCR,and the distribution and content ofβ-catenin protein were detected by cellular immunofluorescence assay.Suspension sphere-forming assay and colony assay were used to assess the changes in the ability of Tas-B-2B cells to migrate and proliferate on their own after Na HS.Result1.Long-term exposure to Na As O₂increases the self-renewal capacity and growth of BEAS-2B cells,while altering cell morphology.2.Sodium arsenite exposure induced epithelial-mesenchymal transition in BEAS-2B cells and regulated the expression levels of tumor stem cell marker molecules.The expression levels of epithelial marker E-cadherin,mesenchymal marker Vimentin and transcriptional repressor protein Slug were decreased,and the expression levels of tumor stem cell markers CD44,OCT4 and NANOG were increased after sodium arsenite exposure,showing a significant time-dose-dependent effect.3.Sodium arsenite exposure reduced the ambient H₂S content in BEAS-2B cells,while downregulating the expression level of H₂S synthase protein CBS,showing a significant time-dose-dependent effect relationship,upregulating the expression level of hypoxia-inducible factor HIF-1αand activating the Wnt/β-catenin signaling pathway.4.Pretreatment with sodium sulfide at a concentration of 100μM reversed the reduction of endogenous H₂S induced by sodium arsenite in BEAS-2B cells,and supplementation with exogenous hydrogen sulfide reduced the level of hypoxia-inducible factor HIF-1αexpression,inhibited sodium arsenite-induced epithelial-mesenchymal transition in BEAS-2B cells and reversed tumor stem cell characteristics.5.Sodium arsenite further led to the accumulation of hypoxia-inducible factor HIF-1αby reducing the endogenous H₂S content,which further activated the Wnt/β-catenin signaling pathway and led to the epithelial-mesenchymal transition and the acquisition of tumor stem cell-like features in BEAS-2B cells.Conclusion1.Decreased endogenous H₂S content in BEAS-2B cells was associated with the occurrence of epithelial-mesenchymal transition and the acquisition of tumor stem cell-like characteristics of the cells.2.Sodium arsenite exposure induced a decrease in endogenous H₂S content in BEAS-2B cells,inhibited the protein expression level of hydrogen sulfide synthase,contributed to the accumulation of hypoxia-inducible factor HIF-1α,and activated the Wnt/β-catenin signaling pathway.3.Exogenous hydrogen sulfide donor（Na HS）reduces the expression level of hypoxia-inducible factor HIF-1αby elevating the intracellular hydrogen sulfide level,which in turn reverses the epithelial-mesenchymal transition occurring in the cells and the acquisition of tumor stem cell-like characteristics.