Department of Ophthalmology, NRL Neural Injury Research Center, University of Ulsan, College of Medicine, Seoul, Korea.
Invest Ophthalmol Vis Sci. 2012 Aug 9;53(9):5344-53. doi: 10.1167/iovs.12-9827.
We investigated the mechanism of tamoxifen (TAM) retinotoxicity using human retinal pigment epithelial (RPE)-derived (ARPE-19) and photoreceptor-derived (661W) cells.
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.
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.
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.
我们使用人视网膜色素上皮（ARPE-19）和光感受器衍生（661W）细胞研究了他莫昔芬（TAM）视网膜毒性的机制。
用 5 至 10μM 的 TAM 处理培养的 ARPE-19 和 661W 细胞，通过测定乳酸脱氢酶（LDH）释放试验来定量细胞死亡。通过测量 5-（和-6）-羧基-2'，7'-二氯荧光素二乙酸酯（H（2）-DCFDA）荧光来确定细胞氧化应激。使用锌特异性荧光染料 FluoZin-3 AM 监测细胞内游离锌水平的变化。用荧光蛋白缀合标记物 RFP-LC3 或 GFP-LC3 转染 ARPE-19 和 661W 细胞，评估自噬空泡的形成。
TAM 暴露后，ARPE-19 和 661W 细胞在 1 小时内出现细胞质空泡，并在 18 小时内发生细胞死亡。在这两种细胞类型中，TAM 诱导的细胞死亡伴随着氧化应激和锌水平升高的增加，并且抗氧化剂 N-乙酰-L-半胱氨酸（NAC）或锌螯合剂 N，N，N'，N'-四（-）（2-吡啶基甲基）-乙二胺（TPEN）可减轻其损伤。TAM 处理后 LC3-II 的水平和自噬小体（AV）的数量增加。溶酶体和 AV 的双重染色表明自溶酶体的形成正常进行。与此一致，自噬通量增加。最后，与其他自噬细胞死亡情况一样，溶酶体膜通透性（LMP）和 caspase 依赖性凋亡导致 TAM 诱导的细胞死亡。
ARPE-19 和 661W 细胞对 TAM 诱导的细胞毒性同样敏感。锌水平和氧化应激的增加，自噬通量的过度激活，以及最终发生 LMP 和随后的 caspase 激活，可能导致 TAM 确立的视网膜细胞毒性。
