title: The p53/HSP70 inhibitor, 2-phenylethynesulfonamide, causes oxidative stress, unfolded protein response and apoptosis in rainbow trout cells.
abstract: The effect of 2-phenylethynesulfonamide (PES), which is a p53 and HSP70 inhibitor in mammalian cells, was studied on the rainbow trout (Oncorhynchus mykiss) gill epithelial cell line, RTgill-W1, in order to evaluate PES as a tool for understanding the cellular survival pathways operating in fish. As judged by three viability assays, fish cells were killed by 24h exposures to PES, but cell death was blocked by the anti-oxidant N-acetylcysteine (NAC). Cell death had several hallmarks of apoptosis: DNA laddering, nuclear fragmentation, Annexin V staining, mitochondrial membrane potential decline, and caspases activation. Reactive oxygen species (ROS) production peaked in several hours after the addition of PES and before cell death. HSP70 and BiP levels were higher in cultures treated with PES for 24h, but this was blocked by NAC. As well, PES treatment caused HSP70, BiP and p53 to accumulate in the detergent-insoluble fraction, and this too was prevented by NAC. Of several possible scenarios to explain the results, the following one is the simplest. PES enhances the generation of ROS, possibly by inhibiting the anti-oxidant actions of p53 and HSP70. ER stress arises from the ROS and from PES inhibiting the chaperone activities of HSP70. The ER stress in turn initiates the unfolded protein response (UPR), but this fails to restore ER homeostasis so proteins aggregate and cells die. Despite these multiple actions, PES should be useful for studying fish cellular survival pathways.
authors.affiliation: Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
authors.forename: Fanxing
authors.initials: F
authors.lastname: Zeng
authors.affiliation: Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
authors.forename: Catherine
authors.initials: C
authors.lastname: Tee
authors.affiliation: Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
authors.forename: Michelle
authors.initials: M
authors.lastname: Liu
authors.affiliation: Aquatic Contaminants Research Division, Environment Canada, Burlington, Ontario L7R 4A6, Canada.
authors.forename: James P
authors.initials: JP
authors.lastname: Sherry
authors.affiliation: Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
authors.forename: Brian
authors.initials: B
authors.lastname: Dixon
authors.affiliation: Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
authors.forename: Bernard P
authors.initials: BP
authors.lastname: Duncker
authors.affiliation: Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada. Electronic address: ncbols@uwaterloo.ca.
authors.forename: Niels C
authors.initials: NC
authors.lastname: Bols
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002460:Cell Line; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D017686:Oncorhynchus mykiss / Q000502:physiology*; D018384:Oxidative Stress / Q000187:drug effects*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013449:Sulfonamides / Q000633:toxicity*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D056811:Unfolded Protein Response / Q000187:drug effects*; D014874:Water Pollutants, Chemical / Q000633:toxicity*
chemical_list: C545662:2-phenylethylenesulfonamide; D018840:HSP70 Heat-Shock Proteins; D017382:Reactive Oxygen Species; D013449:Sulfonamides; D016159:Tumor Suppressor Protein p53; D014874:Water Pollutants, Chemical; D000111:Acetylcysteine
keywords: 2-Phenylethynesulfonamide; Apoptosis; BiP; ER; ER stress; HSP70; Pifithrin-μ; ROS; Unfolded protein response; binding immunoglobulin protein; endoplasmic reticulum; heat shock protein70