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Leo Kim, Dhanesh Amarnani, Wen Tseng, Demetrios Vavvas, Patricia D'Amore; Receptor-Interacting Protein Kinases and the NLRP3 Inflammasome Mediate Tamoxifen Toxicity of the Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1810.
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© ARVO (1962-2015); The Authors (2016-present)
We investigated the mechanism of tamoxifen-induced cell death in human cultured retinal pigment epithelial cells (ARPE-19) with a focus on alternative mechanisms of cell death including pyroptosis and necroptosis.
ARPE-19 cells were cultured to confluence and treated with tamoxifen (20 μM) for two hours. Cell death was evaluated by measuring lactate dehydrogenase (LDH) release. Lysosomal membrane permeabilization was assessed using acridine orange staining of lysosomes. Fluorescent detection of active caspase-1 was performed with the fluorochrome-labeled inhibitor of caspases (FLICA) probe specific for caspase-1, FAM-YVAD-FMK. Caspase involvement was evaluated using the pan-caspase inhibitor Z-VAD-FMK and the caspase-1-specific inhibitor Z-YVAD-FMK. Necrostatin-1 was used to inhibit receptor-interacting protein kinase 1 (RIP1). IL-1β production was quantified by ELISA.
Tamoxifen treatment of confluent ARPE-19 cells induced lysosomal membrane permeabilization and cell death. Neither inhibition of caspase-1 with Z-YVAD-FMK (82%, p > 0.05) nor blockage of RIP1 kinase with necrostatin-1 (87.8%, p > 0.05) significantly decreased LDH release compared against tamoxifen alone (91.2%). Pan-caspase inhibition with Z-VAD-FMK did lead to a significant reduction in LDH release (68.9%, p < 0.05) compared to tamoxifen alone and simultaneous addition of Z-YVAD-FMK and necrostatin resulted in near-total inhibition of cell death (12.6%, p < 0.05). Similarly, the combination of Z-VAD-FMK and necrostatin completely inhibited LDH release (1.1 %, p < 0.05). Visualization of caspase-1 using the FLICA probe revealed caspase-1 activation in all cells treated with tamoxifen. Tamoxifen treatment of IL-1α-primed ARPE-19 cells resulted in release of 17.6 pg/ml of mature IL-1β, which was reduced to 6.4 pg/ml mature IL-1β in the presence of Z-YVAD-FMK.
Tamoxifen induces cell death through concurrent mechanisms of pyroptosis and necroptosis and is associated with inflammasome and caspase-1 activation, as well as the expected release of mature IL-1β. This mechanism may explain the maculopathy that is associated with chronic tamoxifen treatment.
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