The effect of rage ligands on a β25-35 induced cytotoxicity in rat pc-12 cells

David Sykes, Carol Austin, Mark Shaxted, Robert Hull, Iain McDonald, David Dunstone & Barret Kalindjian (Peter Hill). James Black Foundation, Dulwich, London, UK.

The receptor for advanced glycation endproducts (RAGE) has been implicated in the pathogenesis of Alzheimer’s disease. Rat PC-12 phaeochromocytoma cells provide a neuronal-related cell culture model for examining RAGE activation (Yan et al., 1996). PC-12 cells exposed to Aβ 25-35 exhibit a concentration-dependent inhibition of the MTT response, indicative of cellular toxicity, which is partially prevented by blockade of RAGE signalling. A low molecular weight non-peptide RAGE antagonist belonging to TransTech Pharma, and described in their patent (WO-00192210 06) was synthesised, namely (2-(4-Benzyloxy-phenyl)-1-(2,4- bis -(3-diethylamino-propoxy)-phenylcarbamoyl)-ethyl)-carbamic acid tert-butyl ester, herein referred to as Compound 1, to test against Aβ 25-35-induced neurotoxicity using rat PC-12 cells as the model.

PC-12 cells were seeded in RPMI containing 10% horse serum and 5% foetal bovine serum into collagen-coated 96-multiwell plates maintained at 37oC in an atmosphere of 5% CO2. PC-12 cells were preincubated with antagonists for 60min prior to the addition of Aβ 25-35. Cells were incubated for 46hr, and then treated with 15μl of MTT (Promega) for 4hr at 37oC. MTT/formazan was extracted by addition of 100μl of lysis buffer. Optical densities were determined at 570 nm.

Both Aβ25-35 and Aβ1-40 produced concentration-dependent attenuation of MTT reduction. A p[A]50 of value of 7.09±0.08 (mean ± s.e. mean, n=9) was estimated for Aβ25-35. In contrast, the other reported RAGE ligands S100B (1μM), S100P (1μM) and HMGB-1 (3μg/ml) had no effect on MTT reduction in PC-12 cells. Compound 1 (2μM) inhibited the effect of Aβ25-35 producing a parallel rightward shift of the Aβ25-35 concentration response curve (Figure 1). A pA2 value of 6.12±0.20 (mean ± s.e. mean, n=3) was estimated for Compound 1, this value is similar to the pIC50 value of 5.91 estimated by TransTech Pharma when tested in a RAGE binding assay.

Figure 1. Concentration response curves for Aβ25-35 in the absence and presence of Compound 1 in rat PC-12 cells. The values shown are mean ± s.e. mean, n=3. Cells were treated with Compound 1 or vehicle for 60min before addition of Aβ25-35.

S100B (10μM) also produced rightward shift of the Aβ25-35 concentration response curve and a pA2 value of 5.42 was estimated.

We describe in a rat in-vitro model of neuronal cell death the protective action of a non-peptide RAGE antagonist against Aβ25-35 induced cell toxicity.

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