Low-dose arsenite causes overexpression of EGF, TGFα, and HSP90 through Trx1-TXNIP-NLRP3 axis mediated signaling pathways in the human bladder epithelial cells.

Epidemiological studies have demonstrated an increased incidence of bladder cancer in arseniasis- endemic areas; however, the precise molecular mechanisms remain unknown. Our previous results have shown that the protein levels of EGF, TGFα, and HSP90 in arsenite-treated bladder uroepithelial cells increased markedly and contributed to hyperactivation of EGF receptors. The aim of this study was to further explore the regulatory ways underlying overexpression of EGF, TGFα, and HSP90 in these cells. The present results showed that both Trx and GSH systems were stimulated in arsenite-treated cells, and ROS levels in 2 µM arsenite-treated cells did not changed obviously; however, ROS levels in 4 µM arsenite-treated cells increased significantly. By using the antioxidant and specific inhibitors, we found that in 2 µM arsenite-treated cells, JNK/NF-κB signaling pathway was involved in overexpression of EGF and TGFα, and ERK/NF-κB signaling pathway contributed to HSP90 overexpression, however in 4 µM arsenite-treated cells, both ERK/ and JNK/NF-κB signaling pathways were involved in overexpression of EGF, TGFα, and HSP90, and PI3K/AKT/NF-κB signaling pathway contributed to overexpression of EGF and TGFα. Furthermore, our results also showed that the Trx1-TXNIP-NLRP3 axis was activated in arsenite-treated cells, and played a pivotal role in activation of the signaling pathways involved in overexpression of EGF, TGFα, and HSP90. In conclusion, the Trx1-TXNIP-NLRP3 axis might be activated by arsenite-induced redox imbalance in bladder uroepithelial cells, and mediate the activation of signaling pathways involved in overexpression of EGF, TGFα, and HSP90.

HER2 overexpression triggers the IL-8 to promote arsenic-induced EMT and stem cell-like phenotypes in human bladder epithelial cells.

Arsenic is a natural chemical element that is strongly associated with bladder cancer. Understanding the underlying mechanisms behind the association between arsenic and bladder cancer as well as identifying effective preventive interventions will help reduce the incidence and mortality of this disease. The epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties play key roles in cancer development and progression. Here, we reported that chronic exposure to arsenic resulted in EMT and increased levels of the CSC marker CD44 in human uroepithelial cells. Furthermore, IL-8 promoted a mesenchymal phenotype and upregulated CD44 by activating the ERK, AKT and STAT3 signaling. Phosphorylation of the human epidermal growth factor receptor 2 (HER2) was key for arsenic-induced IL-8 overexpression and depended on the simultaneous activation of the MAPK, JNK, PI3K/AKT and GSK3ß signaling pathways. We also found that genistein inhibited arsenic-induced HER2 phosphorylation and downregulated its downstream signaling pathways, thereby inhibiting progression of EMT, and reducing CD44 expression levels. These results demonstrate that the HER2/IL-8 axis is related to the acquisition of an EMT phenotype and CSCs in arsenic-treated cells. The inhibitory effects of genistein on EMT and CSCs provide a new perspective for the intervention and potential chemotherapy against arsenic-induced bladder cancer.

Arsenic-induced HER2 promotes proliferation, migration and angiogenesis of bladder epithelial cells via activation of multiple signaling pathways in vitro and in vivo.

Arsenic (As) is a known human carcinogen with a hitherto unknown mechanism of action. Dimethylarsinic acid (DMAV) is a methylated metabolite of arsenicals found in most mammals, and long-term exposure to DMAV can lead to bladder cancer in rats. Human epidermal growth factor receptor 2 (HER2) is an oncogenic factor that is overexpressed in bladder cancer, but its role in the initiation and progression of As-induced bladder cancer has not been elucidated. We found that HER2 was up-regulated in human uroepithelial cells treated with arsenite as well as in the bladder tissues of DMAV-exposed rats. HER2 overexpression correlated to increased cell proliferation, epithelial-to-mesenchymal transition (EMT), migration and angiogenesis in vitro. The anti-HER2 monoclonal antibody trastuzumab significantly decreased serum vascular endothelial-derived growth factor (VEGF) levels and that of proliferation-related proteins in the bladder tissues of DMAV-exposed rats. Furthermore, inhibition of HER2, as well as that of the MAPK, AKT and STAT3 pathways, attenuated arsenite-induced proliferation, migration and angiogenesis of human uroepithelial cells, and increased apoptosis rates in vitro. These findings indicate that HER2 mediates the oncogenic effects of As on bladder epithelial cells by activating the MAPK, PI3K/AKT and Src/STAT3 signaling pathways, and is therefore a promising biomarker.

Metal Biomonitoring and Comparative Assessment in Urine of Workers in Lead-Zinc and Steel-Iron Mining and Smelting.

The exposure of heavy metals (lead (Pb), cadmium (Cd), copper (Cu), nickel (Ni), and metalloid arsenicals) and their effects on workers' health from a lead-zinc mine (145 workers) and a steel smelting plant (162 workers) was investigated. Information on subject characteristics was obtained through a questionnaire. We determined the urinary levels of Pb, Cd, Cu, Ni, and arsenicals (including inorganic arsenic (iAs), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA), as were 8-hydroxydeoxyguanosine (8-OHdG) and cystatin C. Lead-zinc mine foundry workers had significantly higher concentrations of urinary Pb, Cd, Cu, Ni, iAs, and MMA than did steel smelting plant workers. Individuals who had consumed seafood in the previous 3 days had higher concentrations of urinary Ni than did individuals who had not consumed seafood. The urinary Cd concentrations in the two groups of factory workers may have been affected by daily smoking. There was no significant difference in urinary 8-OHdG between workers from the lead-zinc mine foundry and the steel smelting plant. Urinary Pb and Cd had significant positive linear dose-dependent effects on 8-OHdG. Urinary cystatin C, a sensitive biological indicator reflecting early renal damage, was found at higher levels in lead-zinc mine workers than in steel smelting plant workers. Binary logistic regression analysis showed that age and urinary Cd were significantly associated with urinary cystatin C. These results indicated that workers from lead-zinc mines may be exposed to higher levels of heavy metals which could lead to greater risk of kidney damage.

HER2 and Src co-regulate proliferation, migration and transformation by downstream signaling pathways in arsenite-treated human uroepithelial cells.

Epidemiological studies have established a strong association between arsenic exposure in drinking water and an increased incidence of bladder cancer in arseniasis-endemic areas. Increased expression of HER2 has been observed in various types of human malignancies including bladder cancer. This study investigated the role of HER2 in arsenite-induced transformation of uroepithelial cells SV-HUC-1 and the role of Src family kinases in HER2 signaling. We found that the expression HER2 and Src were increased following chronic arsenite exposure in a time-dependent fashion. Chronic arsenite exposure also led to an upregulation of proliferation factors such as cyclin D1, COX2, PCNA, VEGF, and HIF-1α. Furthermore, Ras/Raf/MAPK, PI3K/AKT, and JAK2/STAT3 signaling pathways were activated by arsenite treatment. Importantly, these changes were inhibited by HER2 inhibitors and in HER2 knocked down cells. In addition, downregulation of HER2 inhibited cell growth and migration properties of arsenite-treated cells. Inhibition of Src also inhibits activation of signaling pathways and malignant transformation of cells. And we obtained evidence of an interaction between HER2 and Src in SV-HUC-1 cell lines. These results suggest that HER2 and Src may play an important role in arsenite-induced transformation by multiple downstream signals pathways.

HER2 Activation Factors in Arsenite-Exposed Bladder Epithelial Cells.

Chronic exposure to arsenic in drinking water is associated with an increased risk of bladder cancer in arseniasis-endemic areas throughout the world. Human epidermal growth factor receptor 2 (HER2) was recently reported to be involved in the development of bladder cancer. However, until this point, little is known about HER2 activation and its mechanism in arsenite-exposed urothelial cells. The aim of this study was to identify factors associated with HER2 activation in an arsenite-exposed human bladder epithelial cell line. Results of this study demonstrated that levels of phosphorylated HER2 increased significantly in cells treated with arsenite. Additionally, the protein levels of epidermal growth factor (EGF), transforming growth factor α (TGFα), soluble ectodomain fragment of E-cadherin (sE-cad), and neuregulin 1 (NRG1) were also increased significantly in these cells. Meanwhile, the protein levels of heat shock protein 90 (HSP90) and plasma membrane calcium ATPases 2 (PMCA2) increased, while those of Interleukin-6 (IL-6) and N-myc downstream regulated gene 1 (NDRG1) decreased significantly. Pretreatment of arsenite-exposed cells with exogenous EGF, TGFα, NRG1, and HSP90 could promote, whereas exogenous IL-6 and NDRG1 could suppress, the phosphorylation of HER2. Furthermore, reduction of EGF, TGFα, NRG1, PMCA2, or HSP90 via its neutralizing antibody, siRNA, or inhibitor suppressed, whereas knockdown of E-cadherin promoted, the phosphorylation of HER2. In conclusion, our results suggested that HER2 might be activated through promoting the dimerization of HER2 with other members of HER family, maintaining the stability of phosphorylated HER2, and attenuating the suppression of HER2 activation in arsenite-exposed cells.

Arsenite increases Cyclin D1 expression through coordinated regulation of the Ca2+/NFAT2 and NF-κB pathways via ERK/MAPK in a human uroepithelial cell line.

To understand the direct link between Cyclin D1, and nuclear factor of activated T cells 2 (NFAT2) and nuclear factor (NF)-κB in arsenic-treated bladder cells, as well as the association between MAPK and NFAT signaling, we determined whether or not the Ca2+/NFAT pathway is activated in an arsenic-treated normal urothelial cell line and determined the roles of NFAT and NF-κB signals in the regulation of Cyclin D1 expression. The SV-40 immortalized human uroepithelial cell line, SV-HUC-1, was treated with NaAsO2 for 24 h (0, 1, 2, 4, 8, and 10 µM) and 10, 20, 30, and 40 weeks (0 and 0.5 µM). We found that arsenite increased the intracellular Ca2+ levels and induced NFAT2 nuclear translocation after treatment for 24 h. The level of NFAT2 mRNA and expression of total protein and nuclear protein were increased after long-term treatment with 0.5 µM arsenite for 30 and 40 weeks compared to the cells treated for 24 h. In addition, NF-κB p50 and p65 nuclear protein expression increased significantly in cells treated with 2-8 µM arsenite for 24 h, which was consistent with NFAT2 nuclear expression. Furthermore, an ERK inhibitor (U0126) significantly reduced the expression of NFAT2 nuclear protein, and an ERK and JNK inhibitor decreased the levels of p65 and p50 nuclear protein. Cyclin D1 is known as a proto-oncogene and the level of this protein was increased in SV-HUC-1 cells treated with arsenite for 24 h and long-term. An NFAT inhibitor (CsA) and NF-κB inhibitor (PDTC) all markedly reduced Cyclin D1 protein expression. Treatment with U0126 also significantly decreased Cyclin D1 protein expression while JNK and p38 inhibitors did not attenuate the arsenite-associated increase in Cyclin D1 protein expression. The results suggest that regulation of Cyclin D1 protein expression by arsenite in SV-HUC-1 cells is dependent on ERK/NFAT2 and ERK/NF-κB, but is not dependent on JNK or p38.

sEcad and EGF Levels Increased in Urine of Non-ferrous Metal Workers and Medium of Uroepithelial Cell Line Treated by Arsenic.

Inorganic arsenic (iAs) is a carcinogen and could increase the risks of bladder, lung, and skin cancer. Mining and smelting of non-ferrous metals are common occupational arsenic exposures. In this study, 125 individuals working in non-ferrous metal smelting plants were separated into two groups according to urinary total arsenic (TAs) levels: group 1, TAs < 100 µg/g Cr; group 2, TAs ≥ 100 µg/g Cr. Demographic characteristics of participants were obtained by questionnaire interview. Levels of E-cadherin soluble ectodomain fragment (sEcad) and epidermal growth factor (EGF) in workers urine were determined by ELISA test. We found that concentrations of sEcad and EGF present in urine were significantly elevated in the high urinary arsenic group 2 compared with the low urinary arsenic group 1. Urinary levels of the shedding of E-cadherin soluble ectodomain fragment (sEcad) and epidermal growth factor (EGF) were positively related to the concentrations of iAs in urine after adjusting for the confounding effects. A positive correlation between sEcad and EGF concentrations in urine was also observed. In order to verify the effects of iAs on sEcad and EGF, the human uroepithelial cell line (SV-HUC-1) was treated with NaAsO2 for 24 h in vitro. sEcad and EGF levels in the 4 µM NaAsO2-treated SV-HUC-1 cell medium significantly increased compared to the control group. In conclusion, urinary levels of sEcad and EGF increased in higher urinary arsenic workers of non-ferrous metal plants and are closely associated with urinary iAs concentration. The results suggested that sEcad and EGF may potentially be preclinical prognostic factors of bladder injury and early detection in arsenic exposure individuals.

ATF2 partly mediated the expressions of proliferative factors and inhibited pro-inflammatory factors' secretion in arsenite-treated human uroepithelial cells.

Inorganic arsenic (iAs) could induce the expression of activating transcription factor-2 (ATF2) in the human urinary bladder epithelial cell line (SV-HUC-1 cells). ATF2, as a member of the bZIP transcription factor family, has been implicated in a transcriptional response leading to cell growth, migration and malignant tumor progression. However, little is known about the effects of ATF2 on proliferative factors in iAs treated human urothelial cells. In this study, ATF2 siRNA was employed to investigate the relationship between ATF2 activation and the expressions of proliferative factors, such as BCL2, cyclin D1, COX-2, MMP1 and PCNA, and pro-inflammatory factors (TNFα, TGFα and IL-8) in SV-HUC-1 cells. The results showed that low concentration arsenite increased the expressions of proliferative factors BCL2, cyclin D1, COX-2, MMP1 and PCNA in SV-HUC-1 cells, and ATF2 siRNA partly decreased the expressions of BCL2, cyclin D1, and COX-2. A neutralizing antibody of IL-8 was used for attenuating the levels of IL-8 and neutralizing antibody of IL-8 did not relieve the expressions of ATF2 and proliferative factors induced by arsenite in SV-HUC-1 cells. In addition, ATF2 knockdown did not decrease the expressions of pro-inflammatory cytokines induced by arsenite in SV-HUC-1 cells, but dramatically increased mRNA expressions of TNFα, TGFα and IL-8 under arsenite and non-arsenite conditions. In conclusion, our present study indicated that ATF2, but not IL-8, played a partial role in the expressions of proliferative factors induced by arsenite in human uroepithelial cells.