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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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Combined effect of Bicalutamide and trehalose on PC12 cells expressing AR.Q112. (A and B) AR.Q112 protein expression in PC12 stable transfected cell line was induced by 1 μg/ml of doxycycline; after 12 h the cells were treated with ethanol (EtOH) as a vehicle control, 10 nm testosterone (T) and/or 100 nm Bicalutamide (Cas) and/or 100 mm trehalose for 48 h. The cells were re-suspended in RIPA buffer and centrifuged to separate the pellet and the supernatant fractions. The supernatant fraction was loaded in FRA (A). Both the supernatant and the pellet fractions were analyzed by western blot (B). GAPDH was used to normalize protein loading.
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DDU419F6: Combined effect of Bicalutamide and trehalose on PC12 cells expressing AR.Q112. (A and B) AR.Q112 protein expression in PC12 stable transfected cell line was induced by 1 μg/ml of doxycycline; after 12 h the cells were treated with ethanol (EtOH) as a vehicle control, 10 nm testosterone (T) and/or 100 nm Bicalutamide (Cas) and/or 100 mm trehalose for 48 h. The cells were re-suspended in RIPA buffer and centrifuged to separate the pellet and the supernatant fractions. The supernatant fraction was loaded in FRA (A). Both the supernatant and the pellet fractions were analyzed by western blot (B). GAPDH was used to normalize protein loading.

Mentions: To further characterize the effects of the combinatory use of Bicalutamide and trehalose, their activity was also tested on a form of AR containing a very long polyQ (Q112) tract, which has a different kinetic of aggregation and generates nuclear inclusions. To accomplish this, we used the PC12/AR.Q112 TET-On inducible cell model of SBMA. We demonstrated in FRA (Fig. 6A) that both Bicalutamide (Cas) and trehalose decrease the ARpolyQ(112) aggregation, but the combinatory use of the two compounds is far more efficient than the two compounds used singularly. A similar effect was observed on the insoluble fraction (pellet) recovered after high-speed centrifugation of cell lysates of PC12/AR.Q112 cells (Fig. 6B). These results demonstrate a reduction in the amount of ARpolyQ(112) present in the pellet fraction from cells treated simultaneously with Bicalutamide and trehalose (in the presence of testosterone). We also observed a trend toward a reduction in the levels of the monomeric form of ARpolyQ in the supernatant fractions, but these data did not reach statistical significance (Fig. 6B). This may reflect the possibility that the insoluble aggregated forms of ARpolyQ (more than the soluble monomeric forms of ARpolyQ) are preferentially degraded after the combinatory use of Bicalutamide and trehalose. Moreover, it is noteworthy that there is a reduction in the amount of ARpolyQ oligomers, observed in the stacking gel, due to the combined treatment.Figure 6.


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)

Combined effect of Bicalutamide and trehalose on PC12 cells expressing AR.Q112. (A and B) AR.Q112 protein expression in PC12 stable transfected cell line was induced by 1 μg/ml of doxycycline; after 12 h the cells were treated with ethanol (EtOH) as a vehicle control, 10 nm testosterone (T) and/or 100 nm Bicalutamide (Cas) and/or 100 mm trehalose for 48 h. The cells were re-suspended in RIPA buffer and centrifuged to separate the pellet and the supernatant fractions. The supernatant fraction was loaded in FRA (A). Both the supernatant and the pellet fractions were analyzed by western blot (B). GAPDH was used to normalize protein loading.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4262493&req=5

DDU419F6: Combined effect of Bicalutamide and trehalose on PC12 cells expressing AR.Q112. (A and B) AR.Q112 protein expression in PC12 stable transfected cell line was induced by 1 μg/ml of doxycycline; after 12 h the cells were treated with ethanol (EtOH) as a vehicle control, 10 nm testosterone (T) and/or 100 nm Bicalutamide (Cas) and/or 100 mm trehalose for 48 h. The cells were re-suspended in RIPA buffer and centrifuged to separate the pellet and the supernatant fractions. The supernatant fraction was loaded in FRA (A). Both the supernatant and the pellet fractions were analyzed by western blot (B). GAPDH was used to normalize protein loading.
Mentions: To further characterize the effects of the combinatory use of Bicalutamide and trehalose, their activity was also tested on a form of AR containing a very long polyQ (Q112) tract, which has a different kinetic of aggregation and generates nuclear inclusions. To accomplish this, we used the PC12/AR.Q112 TET-On inducible cell model of SBMA. We demonstrated in FRA (Fig. 6A) that both Bicalutamide (Cas) and trehalose decrease the ARpolyQ(112) aggregation, but the combinatory use of the two compounds is far more efficient than the two compounds used singularly. A similar effect was observed on the insoluble fraction (pellet) recovered after high-speed centrifugation of cell lysates of PC12/AR.Q112 cells (Fig. 6B). These results demonstrate a reduction in the amount of ARpolyQ(112) present in the pellet fraction from cells treated simultaneously with Bicalutamide and trehalose (in the presence of testosterone). We also observed a trend toward a reduction in the levels of the monomeric form of ARpolyQ in the supernatant fractions, but these data did not reach statistical significance (Fig. 6B). This may reflect the possibility that the insoluble aggregated forms of ARpolyQ (more than the soluble monomeric forms of ARpolyQ) are preferentially degraded after the combinatory use of Bicalutamide and trehalose. Moreover, it is noteworthy that there is a reduction in the amount of ARpolyQ oligomers, observed in the stacking gel, due to the combined treatment.Figure 6.

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