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Synergic prodegradative activity of Bicalutamide and trehalose on the mutant androgen receptor responsible for spinal and bulbar muscular atrophy.

Giorgetti E, Rusmini P, Crippa V, Cristofani R, Boncoraglio A, Cicardi ME, Galbiati M, Poletti A - Hum. Mol. Genet. (2014)

Bottom Line: Thus, (i) prevention of ARpolyQ nuclear localization, combined with (ii) an increased ARpolyQ cytoplasmic clearance, should reduce its detrimental activity.Using the antiandrogen Bicalutamide (Casodex(®)), which slows down AR activation and nuclear translocation, and the disaccharide trehalose, an autophagy activator, we found that, in motoneurons, the two compounds together reduced ARpolyQ insoluble forms with higher efficiency than that obtained with single treatments.Collectively, these data suggest that the combinatory use of Bicalutamide and trehalose is a novel approach to facilitate ARpolyQ clearance that has to be tested in other cell types target of SBMA (i.e. muscle cells) and in vivo in animal models of SBMA.

View Article: PubMed Central - PubMed

Affiliation: Sezione di Biomedicina ed Endocrinologia, Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Centro di Eccellenza sulle Malattie Neurodegenerative, Università degli Studi di Milano, Milano 20133, Italy Centro InterUniversitario sulle Malattie Neurodegenerative, Università degli Studi di Firenze, Genova e Roma Tor Vergata, Milano 20133, Italy Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA and.

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Effect of Bicalutamide or trehalose on testosterone-induced ARpolyQ accumulation. (A) NSC34 cells expressing AR.Q46 treated with ethanol (EtOH) as a vehicle control, 10 nm testosterone (T) in the absence of or in the presence of different doses of trehalose (10 mm/100 mm/1 m) for 48 h. FRA shows AR.Q46 insoluble species accumulation after T treatment, and the dose-dependent effects of trehalose (*P < 0.05 versus +T; **P < 0.001 versus +T). (B) NSC34 cells expressing AR.Q46 treated with ethanol (EtOH) as a vehicle control, 10 nm testosterone (T) and/or 100 nm Bicalutamide (Cas) for 48 h. (C) NSC34 cells expressing AR.Q46 treated with ethanol (EtOH) as a vehicle control or 10 nm testosterone (T), in the absence of or in the presence of 100 mm trehalose for 48 h. Western blot analysis (B and C, upper panels) shows soluble AR.Q46 protein levels following different treatments. Alpha-tubulin was used to normalize protein loading. FRA in B, lower panel, illustrates the different accumulation of insoluble species of AR.Q46 after T or/and Cas treatments (^P < 0.05 versus EtOH; °P < 0.05 versus +Cas; *P < 0.05 versus +T). FRA in C, lower panel, shows AR.Q46 insoluble species accumulation after T treatment, in the absence of or in the presence of trehalose (*P < 0.05 versus +T). (D) HRFM analysis (63× magnification) on NSC34 cells expressing GFP-AR.Q48 in the absence of (EtOH) or in the presence of 10 nm testosterone (T) and/or 100 nm Bicalutamide (Cas) for 48 h. Nuclei were stained with DAPI (blue). Scale bar = 10 μm. (E) HRFM analysis (63× magnification) on NSC34 cells expressing GFP-AR.Q48 in the absence of (EtOH) or in the presence of 10 nm testosterone (T), with or without 100 mm trehalose treatment for 48 h. Nuclei were stained with DAPI (blue). Scale bar = 10 μm.
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DDU419F1: Effect of Bicalutamide or trehalose on testosterone-induced ARpolyQ accumulation. (A) NSC34 cells expressing AR.Q46 treated with ethanol (EtOH) as a vehicle control, 10 nm testosterone (T) in the absence of or in the presence of different doses of trehalose (10 mm/100 mm/1 m) for 48 h. FRA shows AR.Q46 insoluble species accumulation after T treatment, and the dose-dependent effects of trehalose (*P < 0.05 versus +T; **P < 0.001 versus +T). (B) NSC34 cells expressing AR.Q46 treated with ethanol (EtOH) as a vehicle control, 10 nm testosterone (T) and/or 100 nm Bicalutamide (Cas) for 48 h. (C) NSC34 cells expressing AR.Q46 treated with ethanol (EtOH) as a vehicle control or 10 nm testosterone (T), in the absence of or in the presence of 100 mm trehalose for 48 h. Western blot analysis (B and C, upper panels) shows soluble AR.Q46 protein levels following different treatments. Alpha-tubulin was used to normalize protein loading. FRA in B, lower panel, illustrates the different accumulation of insoluble species of AR.Q46 after T or/and Cas treatments (^P < 0.05 versus EtOH; °P < 0.05 versus +Cas; *P < 0.05 versus +T). FRA in C, lower panel, shows AR.Q46 insoluble species accumulation after T treatment, in the absence of or in the presence of trehalose (*P < 0.05 versus +T). (D) HRFM analysis (63× magnification) on NSC34 cells expressing GFP-AR.Q48 in the absence of (EtOH) or in the presence of 10 nm testosterone (T) and/or 100 nm Bicalutamide (Cas) for 48 h. Nuclei were stained with DAPI (blue). Scale bar = 10 μm. (E) HRFM analysis (63× magnification) on NSC34 cells expressing GFP-AR.Q48 in the absence of (EtOH) or in the presence of 10 nm testosterone (T), with or without 100 mm trehalose treatment for 48 h. Nuclei were stained with DAPI (blue). Scale bar = 10 μm.

Mentions: In the first set of experiments performed using filter retardation assay (FRA), western blot (WB) and high-resolution fluorescence microscopy (HRFM) analysis, we compared the effects of single treatments with Bicalutamide or trehalose on ARpolyQ aggregation and clearance in the absence of or in the presence of testosterone (to trigger the formation of ARpolyQ toxic species) in NSC34 cells. The doses of trehalose have been selected by performing preliminary studies of dose-dependent response (Fig. 1A), while for Bicalutamide, which is a drug widely used in clinics against prostate cancer, and for which the pharmacokinetics is very well known, we used the doses classically adopted to counteract testosterone activity in all transcriptional and binding assays, as well as against ARpolyQ in SBMA (see, for example, 27,37,38). The results clearly demonstrate that Bicalutamide (Cas) reduces the accumulation of ARpolyQ insoluble species induced by testosterone (Fig. 1B, lower panel and relative quantification, FRA), and to a lesser extent the total levels of the monomeric ARpolyQ protein evaluated in western blot (Fig. 1B, upper panel, WB). This suggests that the misfolded ARpolyQ fraction, in particular, is targeted by the Bicalutamide prodegradative activity. Trehalose also significantly reduces the accumulation of mutant ARpolyQ insoluble species in FRA (Fig. 1C, lower panel and relative quantification), and reduces the monomeric ARpolyQ levels both in the absence of and in the presence of testosterone, as show in WB analysis (Fig. 1C, upper panel).Figure 1.


Synergic prodegradative activity of Bicalutamide and trehalose on the mutant androgen receptor responsible for spinal and bulbar muscular atrophy.

Giorgetti E, Rusmini P, Crippa V, Cristofani R, Boncoraglio A, Cicardi ME, Galbiati M, Poletti A - Hum. Mol. Genet. (2014)

Effect of Bicalutamide or trehalose on testosterone-induced ARpolyQ accumulation. (A) NSC34 cells expressing AR.Q46 treated with ethanol (EtOH) as a vehicle control, 10 nm testosterone (T) in the absence of or in the presence of different doses of trehalose (10 mm/100 mm/1 m) for 48 h. FRA shows AR.Q46 insoluble species accumulation after T treatment, and the dose-dependent effects of trehalose (*P < 0.05 versus +T; **P < 0.001 versus +T). (B) NSC34 cells expressing AR.Q46 treated with ethanol (EtOH) as a vehicle control, 10 nm testosterone (T) and/or 100 nm Bicalutamide (Cas) for 48 h. (C) NSC34 cells expressing AR.Q46 treated with ethanol (EtOH) as a vehicle control or 10 nm testosterone (T), in the absence of or in the presence of 100 mm trehalose for 48 h. Western blot analysis (B and C, upper panels) shows soluble AR.Q46 protein levels following different treatments. Alpha-tubulin was used to normalize protein loading. FRA in B, lower panel, illustrates the different accumulation of insoluble species of AR.Q46 after T or/and Cas treatments (^P < 0.05 versus EtOH; °P < 0.05 versus +Cas; *P < 0.05 versus +T). FRA in C, lower panel, shows AR.Q46 insoluble species accumulation after T treatment, in the absence of or in the presence of trehalose (*P < 0.05 versus +T). (D) HRFM analysis (63× magnification) on NSC34 cells expressing GFP-AR.Q48 in the absence of (EtOH) or in the presence of 10 nm testosterone (T) and/or 100 nm Bicalutamide (Cas) for 48 h. Nuclei were stained with DAPI (blue). Scale bar = 10 μm. (E) HRFM analysis (63× magnification) on NSC34 cells expressing GFP-AR.Q48 in the absence of (EtOH) or in the presence of 10 nm testosterone (T), with or without 100 mm trehalose treatment for 48 h. Nuclei were stained with DAPI (blue). Scale bar = 10 μm.
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DDU419F1: Effect of Bicalutamide or trehalose on testosterone-induced ARpolyQ accumulation. (A) NSC34 cells expressing AR.Q46 treated with ethanol (EtOH) as a vehicle control, 10 nm testosterone (T) in the absence of or in the presence of different doses of trehalose (10 mm/100 mm/1 m) for 48 h. FRA shows AR.Q46 insoluble species accumulation after T treatment, and the dose-dependent effects of trehalose (*P < 0.05 versus +T; **P < 0.001 versus +T). (B) NSC34 cells expressing AR.Q46 treated with ethanol (EtOH) as a vehicle control, 10 nm testosterone (T) and/or 100 nm Bicalutamide (Cas) for 48 h. (C) NSC34 cells expressing AR.Q46 treated with ethanol (EtOH) as a vehicle control or 10 nm testosterone (T), in the absence of or in the presence of 100 mm trehalose for 48 h. Western blot analysis (B and C, upper panels) shows soluble AR.Q46 protein levels following different treatments. Alpha-tubulin was used to normalize protein loading. FRA in B, lower panel, illustrates the different accumulation of insoluble species of AR.Q46 after T or/and Cas treatments (^P < 0.05 versus EtOH; °P < 0.05 versus +Cas; *P < 0.05 versus +T). FRA in C, lower panel, shows AR.Q46 insoluble species accumulation after T treatment, in the absence of or in the presence of trehalose (*P < 0.05 versus +T). (D) HRFM analysis (63× magnification) on NSC34 cells expressing GFP-AR.Q48 in the absence of (EtOH) or in the presence of 10 nm testosterone (T) and/or 100 nm Bicalutamide (Cas) for 48 h. Nuclei were stained with DAPI (blue). Scale bar = 10 μm. (E) HRFM analysis (63× magnification) on NSC34 cells expressing GFP-AR.Q48 in the absence of (EtOH) or in the presence of 10 nm testosterone (T), with or without 100 mm trehalose treatment for 48 h. Nuclei were stained with DAPI (blue). Scale bar = 10 μm.
Mentions: In the first set of experiments performed using filter retardation assay (FRA), western blot (WB) and high-resolution fluorescence microscopy (HRFM) analysis, we compared the effects of single treatments with Bicalutamide or trehalose on ARpolyQ aggregation and clearance in the absence of or in the presence of testosterone (to trigger the formation of ARpolyQ toxic species) in NSC34 cells. The doses of trehalose have been selected by performing preliminary studies of dose-dependent response (Fig. 1A), while for Bicalutamide, which is a drug widely used in clinics against prostate cancer, and for which the pharmacokinetics is very well known, we used the doses classically adopted to counteract testosterone activity in all transcriptional and binding assays, as well as against ARpolyQ in SBMA (see, for example, 27,37,38). The results clearly demonstrate that Bicalutamide (Cas) reduces the accumulation of ARpolyQ insoluble species induced by testosterone (Fig. 1B, lower panel and relative quantification, FRA), and to a lesser extent the total levels of the monomeric ARpolyQ protein evaluated in western blot (Fig. 1B, upper panel, WB). This suggests that the misfolded ARpolyQ fraction, in particular, is targeted by the Bicalutamide prodegradative activity. Trehalose also significantly reduces the accumulation of mutant ARpolyQ insoluble species in FRA (Fig. 1C, lower panel and relative quantification), and reduces the monomeric ARpolyQ levels both in the absence of and in the presence of testosterone, as show in WB analysis (Fig. 1C, upper panel).Figure 1.

Bottom Line: Thus, (i) prevention of ARpolyQ nuclear localization, combined with (ii) an increased ARpolyQ cytoplasmic clearance, should reduce its detrimental activity.Using the antiandrogen Bicalutamide (Casodex(®)), which slows down AR activation and nuclear translocation, and the disaccharide trehalose, an autophagy activator, we found that, in motoneurons, the two compounds together reduced ARpolyQ insoluble forms with higher efficiency than that obtained with single treatments.Collectively, these data suggest that the combinatory use of Bicalutamide and trehalose is a novel approach to facilitate ARpolyQ clearance that has to be tested in other cell types target of SBMA (i.e. muscle cells) and in vivo in animal models of SBMA.

View Article: PubMed Central - PubMed

Affiliation: Sezione di Biomedicina ed Endocrinologia, Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Centro di Eccellenza sulle Malattie Neurodegenerative, Università degli Studi di Milano, Milano 20133, Italy Centro InterUniversitario sulle Malattie Neurodegenerative, Università degli Studi di Firenze, Genova e Roma Tor Vergata, Milano 20133, Italy Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA and.

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Related in: MedlinePlus