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

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.

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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

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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].

Author information

Correspondence to Jae-Young Koh.

Electronic supplementary material

Below is the link to the electronic supplementary material.

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)

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)

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)

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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Hwang, J.J., Kim, H.N., Kim, J. et al. Zinc(II) ion mediates tamoxifen-induced autophagy and cell death in MCF-7 breast cancer cell line. Biometals 23, 997–1013 (2010) doi:10.1007/s10534-010-9346-9

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Keywords

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