Archives of Toxicology

, Volume 88, Issue 8, pp 1549–1559 | Cite as

Long-term low-dose exposure of human urothelial cells to sodium arsenite activates lipocalin-2 via promoter hypomethylation

  • Hsiu-Hua Wang
  • Meei-Maan Wu
  • Michael W. Y. Chan
  • Yeong-Shiau Pu
  • Chien-Jen Chen
  • Te-Chang Lee
Molecular Toxicology


We previously reported that the sustained exposure of human urothelial cells (HUCs) to low-dose sodium arsenite induces changes in the gene expression profile and neoplastic transformation. In this study, we used the HumanMethylation27 BeadChip to analyze genome-wide methylation profiles and 5-aza-2′-deoxycytidine to examine the involvement of promoter methylation in gene expression. Because the expression of lipocalin-2 (LCN2) was highly enhanced by promoter hypomethylation in inorganic arsenic (iAs)-HUCs cells as well as bladder cancer tissues, we further showed that mutations at the binding sequences for NF-κB and C/EBP-α significantly reduced LCN2 promoter activity. By chromatin immunoprecipitation assay, we demonstrated the significantly increased binding of RelA (p65) and NF-κB1 (p50) to the hypomethylated promoter of LCN2 in the iAs-HUCs. Furthermore, we also demonstrated that LCN2 overexpression was crucial for the neoplastic characteristics of the iAs-HUCs, such as enhanced anchorage-independent growth, resistance to serum deprivation and activation of NF-κB signaling. In addition, our results indicated that enhanced NF-κB activity in iAs-HUCs was via LCN2-mediated increase in intracellular iron and reactive oxygen species levels. Taken together, our results show that sustained low-dose arsenic exposure results in epigenetic changes and enhanced oncogenic potential via LCN2 overexpression.


Inorganic arsenic Bladder cancer DNA methylation Epigenetic alterations Lipocalin-2 Urothelial cells 



We thank the Institute of Biomedical Sciences, Academia Sinica (IBMS-CRC97-P01) and the National Science Council, Taiwan (NSC97-2314-B-001-003-MY3, NSC98-2320-B-001-002-MY3) for the grant supporting, the Sequencing Core Facility, Scientific Instrument Center at Academia Sinica for DNA sequencing, Dr. Jiunn-Liang Ko (Chung-Shan Medical University, Taichung) for providing the LCN2 promoter luciferase vector.

Conflict of interest

No potential conflicts of interest were disclosed.

Supplementary material

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Supplementary material 1 (DOCX 17 kb)
204_2014_1214_MOESM2_ESM.pdf (342 kb)
Supplementary material 2 (PDF 341 kb)
204_2014_1214_MOESM3_ESM.docx (46 kb)
Supplementary material 3 (DOCX 46 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Hsiu-Hua Wang
    • 1
    • 2
  • Meei-Maan Wu
    • 3
    • 7
  • Michael W. Y. Chan
    • 4
  • Yeong-Shiau Pu
    • 5
  • Chien-Jen Chen
    • 6
  • Te-Chang Lee
    • 1
    • 2
  1. 1.Graduate Institute of Life SciencesNational Defense Medical CenterTaipeiTaiwan
  2. 2.Institute of Biomedical SciencesAcademia SinicaTaipeiTaiwan
  3. 3.School of Public HealthTaipei Medical UniversityTaipeiTaiwan
  4. 4.Department of Life Science, Institute of Molecular BiologyNational Chung-Cheng UniversityChia-yiTaiwan
  5. 5.Department of UrologyNational Taiwan University HospitalTaipeiTaiwan
  6. 6.Genomics Research CenterAcademia SinicaTaipeiTaiwan
  7. 7.Graduate Institute of Oncology, College of MedicineNational Taiwan UniversityTaipeiTaiwan

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