The Role And Mechanism Study Of Human Epidermal Growth Factor Receptor 2(HER2) In Arsenic-induced Bladder Epithelial Cell Malignant Transformation

Objective:Arsenic(As)is a metalloid element naturally present in the environment.Inorganic arsenic(i As)is a known high-exposure human carcinogen.Although long-term exposure to i As is considered a risk factor for the development of bladder cancer,its molecular mechanism remains unclear.Inorganic arsenic is one of the few human carcinogens that does not easily induce tumors in animals.Therefore,cell carcinogenesis models induced by i As are crucial for better understanding the potential carcinogenic process.Dimethylarsinic acid(DMA^?)is the most abundant arsenic in human and animal urine,and DMA^?as a bladder carcinogen in rats.The increased cell proliferation is the early phenotype of urothelial cells in the development of bladder cancer induced by DMA^?.Arsenic induce cancer by disturbing the epigenetic control of specific sites,resulting in abnormal gene expression.Human epidermal growth factor receptor 2(HER2)plays an important role in various oncogenic signaling pathways.HER2 is activated by binding to other members of the Erb B family,which induces and stimulates the phosphorylation of dimers and then activates downstream signaling pathways such as MAPK,PI3K/AKT and JAK2/STAT3,leading to malignant transformation of cells.Some reports show that the non-receptor protein tyrosine kinase Src is involved in the activation of the HER receptor.Binding of activated Src to the phosphorylated tyrosine residue of HER2 results in ligand-independent triggering of HER2.Epithelial-mesenchymal transition(EMT)is a key step in the development of cancer.The results show that chemokines and cytokines in the tumor microenvironment can affect EMT.Interleukin-8(IL-8)secreted by tumor cells can enhance tumor progression by inducing adjacent epithelial tumor cells to EMT.Recent studies have shown that inflammatory pathways and genes can be strongly up-regulated by oncogenes such as HER2,and are crucial for ability of transform.Therefore,in this study,we established the animal model of DMA^?exposure and HER2 inhibitor intervention in rats to study the role of HER2 in the proliferation of bladder epithelial cells induced by DMA^?.To investigate the role and mechanism of HER2 in the malignant transformation,EMT and migration of human bladder epithelial cells(SV-HUC-1)cells induced by arsenite,a model of malignant transformation of SV-HUC-1 cells was established.This study reveals the related molecular mechanism of i As-induced bladder epithelial cells malignant transformation,and provides an experimental basis for the study of i As carcinogenesis.Methods:1.Forty female Wistar rats were randomly divided into 4 groups.Group 1:Control group.Group 2:Drinking water exposed to 200 ppm(mg/L)DMA^?.Group 3:Drinking water exposed to 200 ppm DMA^?,and intraperitoneal injection of 8 mg/kg/body weight trastuzumab from the fifth week(three times a week).Group 4:Drinking water exposed to 200 ppm DMA^?,and intraperitoneal injection of 8 mg/kg/body weight saline from the fifth(three times a week)for 12 weeks.Immunohistochemical assays were used to detect HER2,p-HER2,Ki67,PCNA,Cyclin D1,COX-2,Raf1,ERK1/2,p-ERK1/2,AKT,p-AKT,JAK2,p-JAK2,STAT3 and p-STAT3 protein expression.The levels of VEGF were measured by ELISA assays.2.The SV-HUC-1 cells were treated with 0?M and 0.5?M Na As O₂ for 10,20,30,and 40 weeks to establish a malignant transformation model of SV-HUC-1 cells induced by arsenite.SV-HUC-1 cells were treated with 0,1,2,4,8,10?M Na As O₂ for24 h to establish a short-term arsenite exposure cell model.Cell viability was detected using the MTS assay.The degree of malignant transformation of cells was measured by soft agar colony formation.The clone formation ability was measured using colony formation assay.Western blots were used to detect the expression levels of PCNA,Cyclin D1,HIF1-?,VEGF,HER2,Src,and the activation of Raf1/ERK1/2,PI3K/AKT and JAK2/STAT3 signaling pathways.The cells were treated with HER2 inhibitor and HER2 recombinant protein and the above indicators were detected.Cells were treated with HER2 inhibitor and AKT/STAT3 activator or HER2 recombinant protein and ERK inhibitor,and then the above indicators were detected.The cells were treated with Src inhibitor,and then the activation of Raf1/ERK,PI3K/AKT and JAK2/STAT3 signaling pathways was detected.The Co-IP experiment was used to detect the interaction between HER2 and Src.The cells were treated with HER2 inhibitor and Src inhibitor to detect cell viability and clonal formation ability.3.Based on the malignant transformation model of SV-HUC-1 cells induced by arsenite.The migratory capacities of cells were evaluated by wound healing assay and Transwell assay.Western blot and q RT-PCR were used to detect EMT protein and m RNA levels.The expression and secretion levels of IL-8 were determined by q RT-PCR and ELISA assay.Cells were treated with HER2 inhibitor and recombinant protein,then EMT,cell migration ability and IL-8 levels were measured.Cells were treated with ERK,p38,JNK,PI3K/AKT and GSK3?signaling pathway inhibitors,then IL-8expression and secretion levels were determined.Cells were treated with HER2recombinant protein and ERK,p38,JNK,PI3K/AKT,GSK3?signaling pathway inhibitors,then IL-8 expression and secretion levels were determined.The cells were treated with IL-8 inhibitor and IL-8 recombinant protein,then EMT,cell migration ability,and ERK,AKT,STAT3 and HER2 protein expression were detected.Cells were treated with IL-8 recombinant protein and ERK,PI3K/AKT,and STAT3 inhibitors,and then EMT and cell migration ability were detected.Cells were treated with HER2inhibitor and IL-8 recombinant protein,and then EMT,cell migration ability,and ERK,AKT,STAT3 protein expression were detected.Results:1.Effects of DMA^?on the expression of HER2 in rat bladder epithelial cellsThe expressions of HER2 and p-HER2 in bladder epithelial cells of DMA^?group were increased compared with the control group,and the levels of HER2 and p-HER2in trastuzumab group were significantly lower than that of DMA^?group(P<0.05).2.Effects of HER2 on the expression of proliferation-related proteins in bladder epithelial cells of rats exposed to DMA^?Compared with the control group,the number of Ki67,PCNA,Cyclin D1 positive cells and the expression level of COX-2 in the bladder tissues of the DMA^?group were increased,while the number of Ki67,PCNA,Cyclin D1 positive cells and the expression level of COX-2 in the trastuzumab group were decreased(P<0.05).3.Effects of HER2 on serum VEGF level in rats exposed to DMA^?Compared with the control group,the level of VEGF in the DMA^?group was significantly increased(P<0.05).Compared with the DMA^?group,the level of VEGF in the trastuzumab-treated group were significantly decreased(P<0.05).4.Effects of HER2 on activation of downstream signaling pathway in bladder epithelial cells of rats exposed to DMA^?Immunohistochemical analysis of Raf1,ERK1/2,p-ERK1/2,AKT,p-AKT,JAK2,p-JAK2,STAT3 and p-STAT3 found that the expression levels of these factors in bladder epithelial cells of DMA^?group were higher than that of the control group,while the expression levels of these factors in trastuzumab group were lower than that of DMA^?group(P<0.05).5.Long-term arsenite treatment induces malignant transformation of SV-HUC-1cellsThe SV-HUC-1 cells were treated with 0.5?M Na As O₂ for 10,20,30,and 40weeks.The results showed that with the prolongation of Na As O₂ exposure time,cell viability was increased(P<0.05).SV-HUC-1 cells treated with 0.5?M arsenite for 40weeks formed colonies on soft agar.6.Arsenite induces HER2 overexpression in SV-HUC-1 cellsThe phosphorylation level of HER2 increased after the cells were treated with 2?M Na As O₂ for 24 h.After the cells were treated with 0.5?M Na As O₂ for 20 weeks,the expression level of p-HER2 protein increased gradually(P<0.05).As the treatment time of Na As O₂ prolonged,the level of p-HER2 increased significantly.7.HER2 participates in the proliferation of SV-HUC-1 cells induced by arseniteCompared with the control group,PCNA and Cyclin D1 levels increased after 24h of Na As O₂ treatment(P<0.05).After long-term treatment of cells with Na As O₂,the protein expression levels of PCNA and Cyclin D1 increased significantly(P<0.05),promoted cell cycle progression.HER2 inhibitor significantly reduced Na As O₂-induced cell viability and the expression of PCNA and Cyclin D1,and inhibited cell cycle progression(P<0.05).Overexpression of HER2 further increased the cell viability induced by arsenite and the expression of PCNA and Cyclin D1(P<0.05).8.HER2 participates in the expression of HIF1-?and VEGF induced by arseniteCompared with the control group,the protein expression levels of VEGF and HIF1-?increased after short-term and long-term arsenite treatment.HER2 inhibitor can significantly reduce the expression of arsenite-induced HIF1-?and VEGF,and overexpression of HER2 further increases the expression of HIF1-?and VEGF induced by arsenite.9.The role of HER2 in activation of Raf1/ERK1/2,PI3K/AKT and JAK2/STAT3signaling pathways in SV-HUC-1 cellsAfter treatment of SV-HUC-1 cells with different concentrations of Na As O₂ for24 h,ERK1/2,Raf1,AKT,STAT3 and JAK2 phosphorylation levels were increased(P<0.05).After 20 weeks of 0.5?M Na As O₂ treatment,the phosphorylation levels of ERK1/2,Raf1,AKT,STAT3 and JAK2 increased significantly(P<0.05).HER2inhibitor and HER2 recombinant protein significantly inhibited and enhanced activation of Raf1/ERK,PI3K/AKT and STAT3 signaling pathways induced by arsenite,respectively(P<0.05).10.HER2 participates in the proliferation and angiogenesis of SV-HUC-1 cells induced by arsenite through Raf1/ERK1/2,PI3K/AKT and STAT3 signaling pathwaysERK inhibitors reversed the increase in cell viability and the expression levels of PCNA,Cyclin D1,HIF-1?,and VEGF induced by HER2 overexpression.AKT and STAT3 activator reversed the decrease of cell viability and expression levels of PCNA,Cyclin D1,HIF-1?and VEGF caused by HER2 inhibitor.11.Effects of HER2 and Src positive feedback loop on the proliferation and clone formation of SV-HUC-1 cells induced by arseniteShort-term and long-term arsenite treatment can activate the Src.Co-IP results showed that HER2 and p-HER2 were detected in cell lysates with Src antibody.Src inhibitor inhibits the level of p-HER2 induced by Na As O₂.HER2 inhibitor inhibits Na As O₂-induced the level of p-Src.The activation of Raf1/ERK,PI3K/AKT and STAT3 signaling pathways induced by arsenite was significantly inhibited by Src inhibitor(P<0.05).Compared with single inhibitor,Src inhibitor with HER2 inhibitor co-treated significantly inhibited the cell viability and clone formation ability caused by arsenite(P<0.05).12.Effects of arsenite on cell migration,EMT and IL-8 expressionAfter treating SV-HUC-1 cells with 0.5?M Na As O₂ for 10,20,30,and 40 weeks,with the prolongation of arsenite treatment time,cell migratory capacities and the number of cell migration were increased.Compared with the control group,long-term arsenite treatment reduced E-cadherin levels,increased vimentin,Snail,Slug,and Twist levels,and increased IL-8 levels(P<0.05).13.HER2 participates in migration,EMT and IL-8 expression caused by arseniteHER2 inhibitor resulted in decreased cell migration ability,decreased levels of vimentin,Snail,Slug,and Twist,increased levels of E-cadherin and decreased levels of IL-8 in arsenite-treated cells(P<0.05).The overexpression of HER2 significantly increased cell migration ability,the levels of vimentin,Snail,Slug,and Twist,and caused the decrease of E-cadherin level and the increase of IL-8 level(P<0.05).14.HER2 regulates the expression of IL-8 through MAPK,PI3K/AKT and GSK3?signaling pathwaysThe specific inhibition of ERK,p38,JNK,PI3K/AKT and GSK3?significantly reduced the level of IL-8 caused by long-term Na As O₂ treatment.And these pathway inhibitors significantly reduced the level of IL-8 in arsenite-treated cells induced by HER2 overexpression(P<0.05).15.Effects of IL-8 on the migration and EMT of SV-HUC-1 cells induced by arseniteIL-8 treatment significantly increased the levels of vimentin,Snail,Slug,and Twist,decreased the level of E-cadherin,and increased cell migration capacity in arsenite-treated cells(P<0.05).IL-8 inhibitor decreased cell migration capacity,the levels of vimentin,Snail,Slug,and Twist,and increased E-cadherin levels.16.IL-8 participates in the migration of SV-HUC-1 cells caused by arsenite through ERK,PI3K/AKT and STAT3 signaling pathwaysERK,STAT3,or AKT inhibitors significantly reduced vimentin,Snail,Slug,and Twist levels,and increased E-cadherin level(P<0.05).Overexpression of IL-8 further increased the phosphorylation levels of ERK,AKT and STAT3 expression level induced by arsenite.IL-8 inhibitor caused a significant decrease in the phosphorylation levels of ERK,AKT and STAT3(P<0.05).Inhibition of ERK,AKT and STAT3eliminated IL-8-induced increase in vimentin,Snail,Slug and Twist levels,decrease in E-cadherin level and increase in cell migration capacity.17.HER2 regulates IL-8 to affect the activation of ERK,AKT and STAT3signaling pathways and migration,EMT in SV-HUC-1 cells induced by arseniteHER2 inhibitor significantly reduced IL-8-induced vimentin,Snail,Slug,and Twist levels,increased E-cadherin levels,and reduced cell migration and ERK,AKT,and STAT3 phosphorylation levels(P<0.05).18.The feedback regulation of IL-8 on HER2The phosphorylation level of HER2 significantly increased after treatment with IL-8(P<0.05),while level of p-HER2 decreased significantly after treatment with IL-8 inhibitor(P<0.05).Conclusion:1.HER2 was overexpression in bladder tissue of rats exposed to DMA^?.DMA^?activated Raf/ERK,AKT and JAK2/STAT3 signaling pathways and increased cell proliferation factors expression and serum VEGF level.HER2 participated in the increase of proliferation factors,VEGF levels and activation of Raf1/ERK1/2,AKT and JAK2/STAT3 signal pathways induced by DMA^?in rat bladder epithelial cells.2.Both short-term and long-term Na As O₂ treatment increased the level of HER2in SV-HUC-1 cells.The interaction between HER2 and Src further activated Raf1/ERK,PI3K/AKT and STAT3 signaling pathways,and participated in the proliferation,angiogenesis and clone formation of SV-HUC-1 cells induced by arsenite.3.HER2 overexpression induced by long-term Na As O₂ treatment induces IL-8expression and secretion through ERK,p38/MAPK,JNK,PI3K/AKT and GSK3?pathways.IL-8 triggered ERK,PI3K/AKT and STAT3 pathways leading to EMT and migration.It was found that arsenic could activate HER2,and the positive feedback interactions between HER2 and Src and IL-8,respectively,further the activation of downstream Raf1/ERK1/2,PI3K/AKT and STAT3 signaling pathways,and finally promote bladder epithelial cell proliferation and angiogenesis,induce cell EMT and migration.