@article {313171,
	title = {Tamoxifen toxicity in cultured retinal pigment epithelial cells is mediated by concurrent regulated cell death mechanisms.},
	journal = {Invest Ophthalmol Vis Sci},
	volume = {55},
	number = {8},
	year = {2014},
	month = {2014 Aug},
	pages = {4747-58},
	abstract = {PURPOSE: To evaluate the mechanism of tamoxifen-induced cell death in human cultured RPE cells, and to investigate concurrent cell death mechanisms including pyroptosis, apoptosis, and necroptosis. METHODS: Human RPE cells were cultured until confluence and treated with tamoxifen; cell death was measured by detecting LDH release. Tamoxifen-induced cell death was further confirmed by 7-aminoactinomycin D (7-AAD) and annexin V staining. Lysosomal destabilization was assessed using lysosomal-associated membrane protein-1 (LAMP-1) and acridine orange staining. The roles of lysosomal enzymes cathepsin B and L were examined by blocking their activity. Caspase activity was evaluated by caspase-1, -3, -8, and -9 specific inhibition. Cells were primed with IL-1α and treated with tamoxifen; mature IL-1β production was quantified via ELISA. Caspase activity was verified with the fluorochrome-labeled inhibitor of caspases (FLICA) probe specific for each caspase. Regulated cell necrosis or necroptosis was examined with 7-AAD and inhibition of receptor-interacting protein 1 (RIP1) kinase using necrostatin-1 (Nec-1). RESULTS: Cell death occurred within 2 hours of tamoxifen treatment of confluent RPE cells and was accompanied by lysosomal membrane permeabilization. Blockade of cathepsin B and L activity led to a significant decrease in cell death, indicating that lysosomal destabilization and cathepsin release occur prior to regulated cell death. Tamoxifen-induced toxicity was shown to occur through both caspase-dependent and caspase-independent cell death pathways. Treatment of RPE cells with caspase inhibitors and Nec-1 resulted in a near complete rescue from cell death. CONCLUSIONS: Tamoxifen-induced cell death occurs through concurrent regulated cell death mechanisms. Simultaneous inhibition of caspase-dependent and caspase-independent cell death pathways is required to protect cells from tamoxifen. Inhibition of upstream activators, such as the cathepsins, may represent a novel approach to block multiple cell death pathways.},
	keywords = {Blotting, Western, Caspases, Cell Death, Cell Survival, Cells, Cultured, Estrogen Antagonists, Humans, Interleukin-1beta, Lysosomes, Retinal Diseases, Retinal Pigment Epithelium, Tamoxifen},
	issn = {1552-5783},
	doi = {10.1167/iovs.13-13662},
	author = {Kim, Leo A and Amarnani, Dhanesh and Gnanaguru, Gopalan and Tseng, Wen Allen and Vavvas, Demetrios G and D{\textquoteright}Amore, Patricia A}
}
