title: Tamoxifen toxicity in cultured retinal pigment epithelial cells is mediated by concurrent regulated cell death mechanisms.
abstract: OBJECTIVE
  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.
authors.affiliation: Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States Schepens Eye Research Institute/Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, United States.
authors.forename: Leo A
authors.initials: LA
authors.lastname: Kim
authors.affiliation: Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States Schepens Eye Research Institute/Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, United States.
authors.forename: Dhanesh
authors.initials: D
authors.lastname: Amarnani
authors.affiliation: Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States Schepens Eye Research Institute/Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, United States.
authors.forename: Gopalan
authors.initials: G
authors.lastname: Gnanaguru
authors.affiliation: Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States Schepens Eye Research Institute/Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, United States.
authors.forename: Wen Allen
authors.initials: WA
authors.lastname: Tseng
authors.affiliation: Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.
authors.forename: Demetrios G
authors.initials: DG
authors.lastname: Vavvas
authors.affiliation: Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.
authors.forename: Patricia A
authors.initials: PA
authors.lastname: D'Amore
mesh_terms: D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival; D002478:Cells, Cultured; D004965:Estrogen Antagonists / Q000633:toxicity; D006801:Humans; D053583:Interleukin-1beta / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism / Q000473:pathology; D012164:Retinal Diseases / Q000139:chemically induced* / Q000378:metabolism / Q000473:pathology; D055213:Retinal Pigment Epithelium / Q000187:drug effects / Q000473:pathology*; D013629:Tamoxifen / Q000633:toxicity*
chemical_list: D004965:Estrogen Antagonists; D053583:Interleukin-1beta; D013629:Tamoxifen; D020169:Caspases
keywords: RPE; apoptosis; necroptosis; pyroptosis; tamoxifen