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BioMetals

, Volume 23, Issue 6, pp 997–1013 | Cite as

Zinc(II) ion mediates tamoxifen-induced autophagy and cell death in MCF-7 breast cancer cell line

  • Jung Jin Hwang
  • Ha Na Kim
  • Jean Kim
  • Dong-Hyung Cho
  • Mi Joung Kim
  • Yong-Sook Kim
  • Yunha Kim
  • Sung-Jin Park
  • Jae-Young KohEmail author
Article

Abstract

Treatment of MCF-7 cells with tamoxifen induced vacuole formation and cell death. Levels of the autophagy marker, microtubule-associated protein light chain 3 (LC3)-II also increased, and GFP-LC3 accumulated in and around vacuoles in MCF-7 cells exposed to tamoxifen, indicating that autophagy is involved in tamoxifen-induced changes. Live-cell confocal microscopy with FluoZin-3 staining and transmission electron microscopy with autometallographic staining revealed that labile zinc(II) ion (Zn2+) accumulated in most acidic LC3(+) autophagic vacuoles (AVs). Chelation of Zn2+ with N,N,N,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) blocked the increase in phospho-Erk and LC3-II levels, and attenuated AV formation and cell death. Conversely, the addition of ZnCl2 markedly potentiated tamoxifen-induced extracellular signal-regulated kinase (Erk) activation, autophagy and cell death, indicating that Zn2+ has an important role in these events. Tamoxifen-induced death was accompanied by increased oxidative stress and lysosomal membrane permeabilization (LMP) represented as release of lysosomal cathepsins into cytosol. Treatment with the antioxidant N-acetyl-l-cysteine (NAC) blunted the increase in Zn2+ levels and reduced LC3-II conversion, cathepsin D release and cell death induced by tamoxifen. And cathepsin inhibitors attenuated cell death, indicating that LMP contributes to tamoxifen-induced cell death. Moreover, TPEN blocked tamoxifen-induced cathepsin D release and increase in oxidative stress. The present results indicate that Zn2+ contributes to tamoxifen-induced autophagic cell death via increase in oxidative stress and induction of LMP.

Keywords

Cathepsin Extracellular signal-regulated kinase Lysosome Lysosomal membrane permeabilization Microtuble-associated protein light chain 3 Oxidative stress 

Abbreviations

3-MA

3-methyladenine

AMG

Autometallography

ATG

Autophagy-related gene

AV

Autophagic vacuole

ER

Estrogen receptor

Erk

Extracellular signal-regulated kinase

Lamp-2

Lysosomal-associated membrane protein-2

LC3-II

Microtuble-associated protein light chain 3-II

LDH

Lactate dehydrogenase

LMP

Lysosomal membrane permeabilization

MT

Metallothionein

NAC

N-acetyl-l-cysteine

ROI

Region of interest

ROS

Reactive oxygen species

SERM

Selective estrogen receptor modulator

TPEN

N,N,N’,N′-tetrakis (2-pyridylmethyl) ethylenediamine

Notes

Acknowledgments

We thank Drs. Noboru Mizushima and Maria Colombo for generous gifts of LC3 cDNA and RFP-LC3 plasmid respectively. This study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea [A084270].

Supplementary material

Movie 1

Live cell confocal microscopic images (2 minutes/frame) of MCF-7 cells transfected with LC3-GFP, 60-90 minutes after addition of 17 μM tamoxifen in HBSS. Images were obtained from z-series collections (35 z-section images of 1 μm thickness). (AVI 464 kb)

Movie 2

Live cell confocal microscopic images (2 minutes/frame) of a MCF-7 cell stained with FluoZin-3. Cells were treated with 17 μM tamoxifen and images were obtained from z-series collection (38 z-section images of 1 μm thickness) for 86 minutes. (AVI 170 kb)

Movie 3

Live cell confocal microscopic images (1 minutes/frame) of a MCF-7 cell stained with FluoZin-3. Cells were treated with 17 μM tamoxifen and images were obtained from z-series collection (33 z-section images of 1 μm thickness) for 90 minutes. (AVI 483 kb)

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Copyright information

© Springer Science+Business Media, LLC. 2010

Authors and Affiliations

  • Jung Jin Hwang
    • 1
  • Ha Na Kim
    • 2
  • Jean Kim
    • 2
  • Dong-Hyung Cho
    • 1
  • Mi Joung Kim
    • 1
  • Yong-Sook Kim
    • 1
  • Yunha Kim
    • 1
  • Sung-Jin Park
    • 1
    • 4
  • Jae-Young Koh
    • 2
    • 3
    Email author
  1. 1.Institute for Innovative Cancer ResearchCollege of Medicine, University of Ulsan, Asan Medical CenterSeoulKorea
  2. 2.Neural Injury Research Lab, Asan Institute for Life ScienceCollege of Medicine, University of Ulsan, Asan Medical CenterSeoulKorea
  3. 3.Department of NeurologyCollege of Medicine, University of UlsanSeoulKorea
  4. 4.Laboratory of Bioimaging Probe Development, Singapore Bioimaging ConsortiumAgency for Science, Technology and Research (A*STAR)SingaporeRepublic of Singapore

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