Abstract
Chronic arsenic exposure causes cancers in multiple organs in humans. However, the mechanisms underlying arsenic-induced carcinogenesis remain obscure. Here, we examined whether chronic arsenite (As(III)) exposure promotes cell migration induced by heparin-binding EGF-like growth factor (HB-EGF) in human esophageal immortalized Het1A cells. When Het1A cells were exposed to 0.5 μM As(III) for 4 months, HB-EGF-induced migration was enhanced in As(III)-exposed Het1A cells compared to controls. To elucidate the mechanisms underlying the promotion of HB-EGF-induced migration by chronic exposure to As(III), we compared ERK phosphorylation between As(III)-exposed and control Het1A cells and found that HB-EGF-induced ERK phosphorylation was enhanced in the As(III)-exposed cells. We next measured mRNA levels of 88 genes related to cell cycle regulation. The results showed elevated cyclin D1 mRNA levels in As(III)-exposed Het1A cells. The inhibitors of ERK and cyclin D/Cdk4 markedly suppressed HB-EGF-induced upregulation of cyclin D1 and the migration of Het1A cells, respectively, suggesting that cyclin D1 is located downstream of ERK and is required for HB-EGF-induced migration of Het1A cells. Collectively, these findings indicate that the promotion of HB-EGF-induced migration of Het1A cells chronically exposed to submicromolar As(III) might be caused by increased expression of cyclin D1 mediated by enhanced activation of the ERK pathway.
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This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Culture, and Sports of Japan (No. 24310048).
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Fig. S1. Effects of acute exposure to As(III) on HB-EGF-induced HaCaT cell migration.
(A) HaCaT cells were seeded onto 12-well plates (3 × 105 cells per well) and exposed to As(III) for 24 h. The medium was replaced with serum-free medium at 24 h after the cells were grown to confluence for 1 day, and then scratch-wound migration assays were conducted. After being wounded, the cells were maintained in fresh serum-free medium with 1 ng/mL of HB-EGF. Photos were taken at 18 h, and both the area occupied by migration and the scratched area were quantified by Image J software. The recovered surface area was calculated as (occupied area ̶ scratched area) × 100/scratched area (B). Each value represents the mean ± SEM of three individual determinations. *p < 0.05, **p < 0.01 vs. control HaCaT cells stimulated with HB-EGF. Fig. S2. Chronic exposure to As(III) did not affect Het1A cell proliferation. Het1A cells exposed to 0.5 μM As(III) for 4 months were seeded onto 48-well plates (1.25 × 105 cells per well). The medium was replaced with serum-free medium at 12 h after the cells were grown to confluence for 1 day. After being wounded, the cells were maintained in fresh serum-free medium without or with 50 ng/mL of HB-EGF for 24 h. alamarBlue solution (1/50 volume) was added to the cells, and the mixture was cultured for another 3 h in a CO2 incubator. Absorbance at 540 nm was measured. Each value represents the mean ± SD of three individual determinations. Data are presented as values relative to those of the control cells. Fig. S3. Effects of chronic exposure to As(III) on HB-EGF-induced EGFR phosphorylation. Het1A cells exposed to 0.5 μM As(III) for 4 months were seeded onto 6-cm dishes (1.2 × 106 cells per well). The cells were treated with 50 ng/mL HB-EGF for 5, 15, 30, and 60 min. Western blot analyses were performed for phospho-EGFR (Tyr1068) and EGFR. Representative data from three individual determinations are shown. (PPTX 294 kb)
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Sumi, D., Yoshino, Y., Kameda, R. et al. Chronic exposure to submicromolar arsenite promotes the migration of human esophageal Het1A cells induced by heparin-binding EGF-like growth factor. Arch Toxicol 93, 3523–3534 (2019). https://doi.org/10.1007/s00204-019-02592-6
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DOI: https://doi.org/10.1007/s00204-019-02592-6