Archives of Toxicology

, Volume 93, Issue 12, pp 3523–3534 | Cite as

Chronic exposure to submicromolar arsenite promotes the migration of human esophageal Het1A cells induced by heparin-binding EGF-like growth factor

  • Daigo SumiEmail author
  • Yuri Yoshino
  • Riko Kameda
  • Seiichiro Himeno
Inorganic Compounds


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.


Arsenic Cyclin D1 HB-EGF Migration 



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).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

204_2019_2592_MOESM1_ESM.pptx (294 kb)
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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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Laboratory of Molecular Nutrition and Toxicology, Faculty of Pharmaceutical SciencesTokushima Bunri UniversityTokushimaJapan

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