The p53/HSP70 inhibitor, 2-phenylethynesulfonamide, causes oxidative stress, unfolded protein response and apoptosis in rainbow trout cells

• Published in: Aquatic toxicology 2014-01, Vol.146, p.45-51
• Main Authors: Zeng, Fanxing, Tee, Catherine, Liu, Michelle, Sherry, James P., Dixon, Brian, Duncker, Bernard P., Bols, Niels C.
• Format: Article
• Language: English
• Subjects: 2-Phenylethynesulfonamide; Acetylcysteine - pharmacology; Agnatha. Pisces; Animal, plant and microbial ecology; Animals; Apoptosis; Apoptosis - drug effects; Applied ecology; Biological and medical sciences; Cell Line; Ecotoxicology, biological effects of pollution; Effects of pollution and side effects of pesticides on vertebrates; ER stress; Fundamental and applied biological sciences. Psychology; HSP70 Heat-Shock Proteins - metabolism; Oncorhynchus mykiss; Oncorhynchus mykiss - physiology; Oxidative Stress - drug effects; Pifithrin-μ; Reactive Oxygen Species - metabolism; ROS; Sulfonamides - toxicity; Tumor Suppressor Protein p53 - metabolism; Unfolded protein response; Unfolded Protein Response - drug effects; Water Pollutants, Chemical - toxicity
• ISSN: 0166-445X; 1879-1514
• DOI: 10.1016/j.aquatox.2013.10.026

•2-Phenylethynesulfonamide (PES) is an inhibitor of p53 and HSP 70 in mammals.•In the fish epithelial cell line, RTgill-W1, PES enhanced ROS generation and was cytotoxic.•RTgill-W1 death was by apoptosis and blocked by the anti-oxidant N-acetylcysteine.•This is the first report linking PES-induced cell death to ROS.•With this background PES should be useful for studying fish cell survival pathways. 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.