Induction of autophagy and cell death by tamoxifen in cultured retinal pigment epithelial and photoreceptor cells.
OBJECTIVE
We investigated the mechanism of tamoxifen (TAM) retinotoxicity using human retinal pigment epithelial (RPE)-derived (ARPE-19) and photoreceptor-derived (661W) cells.


METHODS
Cultured ARPE-19 and 661W cells were treated with 5 to 10 μM TAM, and the resultant cell death was quantified using lactate dehydrogenase (LDH) release assay. Cellular oxidative stress was determined by measuring 5-(and-6)-carboxy-2',7'-dichlorohydrofluorescein diacetate (H(2)-DCFDA) fluorescence. Changes in intracellular free zinc levels were monitored using the zinc-specific fluorescent dye, FluoZin-3 AM. Autophagic vacuole formation was assessed morphologically in ARPE-19 and 661W cells transfected with the fluorescent protein-conjugated markers, RFP-LC3 or GFP-LC3.


RESULTS
Following exposure to TAM, both ARPE-19 and 661W cells had cytosolic vacuoles within 1 hour and underwent cell death within 18 hours. In both cell types, TAM-induced cell death was accompanied by increased oxidative stress and elevated zinc levels, and was attenuated by the antioxidant N-acetyl-L-cysteine (NAC) or the zinc chelator N,N,N'N'-tetrakis(-)(2-pyridylmethyl)-ethylenediamine (TPEN). The levels of LC3-II as well as the number of autophagic vacuoles (AVs) increased after TAM treatment. Double staining for lysosomes and AVs showed that autolysosome formation proceeded normally. Consistent with this, autophagy flux was increased. Finally, as shown in other cases of autophagic cell death, lysosomal membrane permeabilization (LMP) as well as caspase-dependent apoptosis contributed to TAM-induced cell death.


CONCLUSIONS
ARPE-19 and 661W cells were vulnerable similarly to TAM-induced cytotoxicity. Increases in zinc levels and oxidative stress, excessive activation of autophagy flux, and ultimately the occurrence of LMP and consequent caspase activation may contribute to the well-established retinal cytotoxicity of TAM.
Department of Ophthalmology, NRL Neural Injury Research Center, University of Ulsan, College of Medicine, Seoul, Korea.
Kyung Sook
KS
Cho
Young Hee
YH
Yoon
Jeong A
JA
Choi
Sook-Jeong
SJ
Lee
Jae-Young
JY
Koh
D001343:Autophagy / Q000187:drug effects*; D016923:Cell Death / Q000187:drug effects*; D002478:Cells, Cultured; D003600:Cytosol / Q000473:pathology; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D020419:Photoreceptor Cells, Vertebrate / Q000473:pathology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000473:pathology*; D020845:Selective Estrogen Receptor Modulators / Q000494:pharmacology*; D013629:Tamoxifen / Q000494:pharmacology*; D014617:Vacuoles / Q000473:pathology; D015032:Zinc / Q000378:metabolism
C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D017382:Reactive Oxygen Species; D020845:Selective Estrogen Receptor Modulators; D013629:Tamoxifen; D015032:Zinc