Radicicol but not geldanamycin evokes oxidative stress response and efflux protein inhibition in ARPE-19 human retinal pigment epithelial cells Tuomas Ryhänen a , Eliisa Mannermaa c , Niku Oksala d , Johanna Viiri a , Tuomas Paimela a , Antero Salminen e , Mustafa Atalay f , Kai Kaarniranta a,b, ⁎ a Department of Ophthalmology, University of Kuopio, Kuopio, Finland b Department of Ophthalmology, Kuopio University Hospital, Kuopio, Finland c Department of Pharmaceutics, University of Kuopio, Kuopio, Finland d Division of Vascular Surgery, Department of Surgery, University of Tampere, Tampere, Finland e Department of Neurology and Neurosciences, University of Kuopio, Kuopio, Finland f Department of Physiology, University of Kuopio, Kuopio, Finland Received 9 May 2007; received in revised form 19 January 2008; accepted 6 February 2008 Available online 14 February 2008 Abstract Drug delivery to retinal cells has represented a major challenge for ophthalmologists for many decades. However, drug targeting to the retina is essential in therapies against retinal diseases such as age-related macular degeneration, the most common reason of blindness in the developed countries. Retinal cells are chronically exposed to oxidative stress that contributes to cellular senescence and may cause neovascularization in the most severe age-related macular degeneration cases. Various pre- and clinical studies have revealed that heat shock protein 90 (HSP90) inhibitors, such as geldanamycin and radicicol, are promising drugs in the treatment of different malignant processes. In this study, our goal was to compare the effects of 0.1 μM, 1 μM or 5 μM geldanamycin or radicicol on the oxidative stress response, cytotoxicity, and efflux protein activity (a protein pump which removes drugs from cells) in ARPE-19 (human retinal pigment epithelial, RPE) cells. Our findings indicate that geldanamycin and radicicol increased HSP70 and HSP27 expression analyzed by western blotting. Cellular levels of protein carbonyls were increased in response to 0.1 μM (P = 0.048 for 24 h, P =0.018 for 48 h) or 5 μM (P = 0.030 for 24 h, P = 0.046 for 48 h) radicicol but not to geldanamycin analyzed by ELISA assay. In addition, HNE-protein adducts were accumulated in the RPE cells exposed to 0.1 μM or 5 μM radicicol but not to geldanamycin analyzed by western blotting. However, MTT assay revealed that 5 μM geldanamycin reduced cellular viability 20–30% (P b 0.05 for 24 h, P b 0.01 for 48 h), but this was not observed at any radicicol concentration in RPE cells. Interestingly, the increased oxidative stress response was associated with efflux protein inhibition (20–30%) when the cells were exposed to 1 μM or 5 μM (P b 0.05) radicicol, but not in geldanamycin- treated RPE cells. These novel findings help in understanding the influence of HSP90 inhibition and regulatory mechanisms of drug delivery to retinal cells. © 2008 Elsevier B.V. All rights reserved. Keywords: Geldanamycin; Heat shock protein; Oxidative stress; Radicicol; Retinal pigment epithelium 1. Introduction Age-related macular degeneration (AMD) is the major cause of irreversible vision loss in the developed countries. AMD is characterized by progressive loss of central vision due to dys- function and cell death of photoreceptors in the macula. AMD pathology is attributable to degenerative and neovascular changes that occur at the interface between the neural retina, retinal pigment epithelial cells (RPE) and the underlying choroid vessels (Holz et al., 2004). RPE cells must endure a high level of oxidative stress because of their high oxygen con- sumption, their exposure to high levels of phagocytosed lipid derivatives from photoreceptors, as well as extensive exposure to light, and photosensitizing lipofuscin accumulation (Beatty et Available online at www.sciencedirect.com European Journal of Pharmacology 584 (2008) 229 – 236 www.elsevier.com/locate/ejphar ⁎ Corresponding author. Department of Ophthalmology, University of Kuopio, Kuopio, Finland. E-mail address: kaarnira@messi.uku.fi (K. Kaarniranta). 0014-2999/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.02.010