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H. Chung, K. Cho, J. Hwang, J. Koh, Y. Yoon; The Role of Chelatable Zinc in Tamoxifen-Induced Autophagic Retinal Cell Death. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5839. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Using optical coherence tomography (OCT), we recently observed intraretinal cyst, disruption of photoreceptor line, and/or macular atrophy in several patients who received tamoxifen. Whereas ocular toxicity of tamoxifen is well documented, its mechanism is not completely understood. Recently, tamoxifen was shown to induce autophagy and to inhibit the proliferation of MCF7 breast carcinoma cells. Here we investigated autophagic vacuole (AV) formation in tamoxifen-treated retinal cells in culture.
Retinal cell cultures were prepared from newborn rats as previously described. Immunocytochemistry to LC3 (microtubule-associated protein 1 light chain 3), LAMP-2 (Lysosomal membrane-associated glycoproteins-2) and immunoblotting for several autophagic markers and lysosomal enzymes were done. For staining of lysosomes and mitochondria, Lysotracker Red DND-99 and Mitotracker Red CM-H2XROS (Molecular Probes) were used, respectively. For zinc fluorescence, FluoZin-3 was used. After double staining, live cells were imaged using confocal microscope (UltraView, Perkin Elmer).
24 hr after exposure to 5 µM tamoxifen, severe vacuolar changes as well as retinal cell death were observed in retinal cell culture. Morphological changes under TEM (transmission electron microscopy), increased the expressions of Atg5/12, Beclin-1 and Atg5-dependent conversion of LC3-I to LC3-Π all indicated that these vacuoles were autophagic vacuoles (AV). A cell-permeant zinc chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) markedly attenuated AV formation and neuronal loss. FluoZin-3 staining showed that almost all AV contain high levels of labile zinc from the very early phase of AV formation. Double staining with lysotracker showed that increasing fraction of zinc-containing AV colocalizes with the acidic compartment such as lysosome as the time after exposure to tamoxifen increases. Immunocytochemistry of LC3 and LAMP-2 revealed the formation of autophagolysosome and immunoblotting of cathepsin D showed a subsequent increase of cytosolic lysosomal hydrolases.
The present results has demonstrated that endogenous zinc has a critical role in tamoxifen-induced AV formation in retinal cells. The possible causal link between the formation of zinc-containing AVs and cell death seems to warrant future investigation.
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