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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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The autophagy activation is mediated by trehalose and not influenced by Bicalutamide. (A and B) Real-time PCR on SQSTM1/p62 (A) and on LC3 (B) mRNA expression levels on NSC34 cells expressing AR.Q46, in the absence of (ethanol as a vehicle control) or in the presence of 10 nm testosterone (T) and/or 100 nm Bicalutamide (Cas) and/or 100 mm trehalose for 48 h (°°P < 0.01 versus EtOH; §§P < 0.01 versus +T; ^^P < 0.01 versus +Cas; **P < 0.01 versus +T/+Cas). Not transfected cells (NT) show that AR.Q46 transfection does not alter SQSTM1/p62 and LC3 mRNA expression levels. (C) Western blot analysis on NSC34 cells expressing AR.Q46 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. Two well-known markers of the autophagic pathway (SQSTM1/p62 and LC3-II) were used to show that the activation of autophagy is mediated by trehalose and not influenced by Bicalutamide. NT demonstrates that AR.Q46 transfection does not influence SQSTM1/p62 and LC3-II protein expression levels. Alpha-tubulin was used to normalize protein loading. (D and E) The two histograms, relative to western blot analysis in (C), represent a quantitative evaluation of SQSTM1/p62 protein level normalized on alpha-tubulin (D) (°P < 0.05 versus EtOH; §P < 0.05 versus +T; ^P < 0.05 versus +Cas; *P < 0.05 versus +T/+Cas) and a quantitative evaluation of LC3-II/LC3-I ratio protein level (E) (°P < 0.05 versus EtOH; §P < 0.05 versus +T; ^P < 0.05 versus +Cas; *P < 0.05 versus +T/+Cas), carried out by densitometric scanning of the blots.
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DDU419F4: The autophagy activation is mediated by trehalose and not influenced by Bicalutamide. (A and B) Real-time PCR on SQSTM1/p62 (A) and on LC3 (B) mRNA expression levels on NSC34 cells expressing AR.Q46, in the absence of (ethanol as a vehicle control) or in the presence of 10 nm testosterone (T) and/or 100 nm Bicalutamide (Cas) and/or 100 mm trehalose for 48 h (°°P < 0.01 versus EtOH; §§P < 0.01 versus +T; ^^P < 0.01 versus +Cas; **P < 0.01 versus +T/+Cas). Not transfected cells (NT) show that AR.Q46 transfection does not alter SQSTM1/p62 and LC3 mRNA expression levels. (C) Western blot analysis on NSC34 cells expressing AR.Q46 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. Two well-known markers of the autophagic pathway (SQSTM1/p62 and LC3-II) were used to show that the activation of autophagy is mediated by trehalose and not influenced by Bicalutamide. NT demonstrates that AR.Q46 transfection does not influence SQSTM1/p62 and LC3-II protein expression levels. Alpha-tubulin was used to normalize protein loading. (D and E) The two histograms, relative to western blot analysis in (C), represent a quantitative evaluation of SQSTM1/p62 protein level normalized on alpha-tubulin (D) (°P < 0.05 versus EtOH; §P < 0.05 versus +T; ^P < 0.05 versus +Cas; *P < 0.05 versus +T/+Cas) and a quantitative evaluation of LC3-II/LC3-I ratio protein level (E) (°P < 0.05 versus EtOH; §P < 0.05 versus +T; ^P < 0.05 versus +Cas; *P < 0.05 versus +T/+Cas), carried out by densitometric scanning of the blots.

Mentions: Based on these data we wished to evaluate the involvement of the autophagic process in the combined activity of Bicalutamide (Cas) and trehalose. Thus, we analyzed SQSTM1/p62 and LC3 expression and processing. As shown in Figure 4A, trehalose, alone or in combination with Bicalutamide (Cas), significantly increases SQSTM1/p62 mRNA expression levels in our cells expressing ARpolyQ; Bicalutamide (Cas) is unable to enhance SQSTM1/p62 mRNA expression levels and does not amplify the trehalose-stimulated SQSTM1/p62 expression, both in the absence and presence of testosterone. Overlapping results were obtained when, in the same conditions, we analyzed the LC3 mRNA expression levels (Fig. 4B). In addition, the levels of the SQSTM1/p62 protein and the conversion of LC3-I to its lipidated and autophagosome-associated form, LC3-II, were increased after trehalose treatment but not influenced by the co-treatment with Bicalutamide (Fig. 4C and relative quantifications for SQSTM1/p62 in D and for LC3-II/LC3-I ratio in E).Figure 4.


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)

The autophagy activation is mediated by trehalose and not influenced by Bicalutamide. (A and B) Real-time PCR on SQSTM1/p62 (A) and on LC3 (B) mRNA expression levels on NSC34 cells expressing AR.Q46, in the absence of (ethanol as a vehicle control) or in the presence of 10 nm testosterone (T) and/or 100 nm Bicalutamide (Cas) and/or 100 mm trehalose for 48 h (°°P < 0.01 versus EtOH; §§P < 0.01 versus +T; ^^P < 0.01 versus +Cas; **P < 0.01 versus +T/+Cas). Not transfected cells (NT) show that AR.Q46 transfection does not alter SQSTM1/p62 and LC3 mRNA expression levels. (C) Western blot analysis on NSC34 cells expressing AR.Q46 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. Two well-known markers of the autophagic pathway (SQSTM1/p62 and LC3-II) were used to show that the activation of autophagy is mediated by trehalose and not influenced by Bicalutamide. NT demonstrates that AR.Q46 transfection does not influence SQSTM1/p62 and LC3-II protein expression levels. Alpha-tubulin was used to normalize protein loading. (D and E) The two histograms, relative to western blot analysis in (C), represent a quantitative evaluation of SQSTM1/p62 protein level normalized on alpha-tubulin (D) (°P < 0.05 versus EtOH; §P < 0.05 versus +T; ^P < 0.05 versus +Cas; *P < 0.05 versus +T/+Cas) and a quantitative evaluation of LC3-II/LC3-I ratio protein level (E) (°P < 0.05 versus EtOH; §P < 0.05 versus +T; ^P < 0.05 versus +Cas; *P < 0.05 versus +T/+Cas), carried out by densitometric scanning of the blots.
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DDU419F4: The autophagy activation is mediated by trehalose and not influenced by Bicalutamide. (A and B) Real-time PCR on SQSTM1/p62 (A) and on LC3 (B) mRNA expression levels on NSC34 cells expressing AR.Q46, in the absence of (ethanol as a vehicle control) or in the presence of 10 nm testosterone (T) and/or 100 nm Bicalutamide (Cas) and/or 100 mm trehalose for 48 h (°°P < 0.01 versus EtOH; §§P < 0.01 versus +T; ^^P < 0.01 versus +Cas; **P < 0.01 versus +T/+Cas). Not transfected cells (NT) show that AR.Q46 transfection does not alter SQSTM1/p62 and LC3 mRNA expression levels. (C) Western blot analysis on NSC34 cells expressing AR.Q46 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. Two well-known markers of the autophagic pathway (SQSTM1/p62 and LC3-II) were used to show that the activation of autophagy is mediated by trehalose and not influenced by Bicalutamide. NT demonstrates that AR.Q46 transfection does not influence SQSTM1/p62 and LC3-II protein expression levels. Alpha-tubulin was used to normalize protein loading. (D and E) The two histograms, relative to western blot analysis in (C), represent a quantitative evaluation of SQSTM1/p62 protein level normalized on alpha-tubulin (D) (°P < 0.05 versus EtOH; §P < 0.05 versus +T; ^P < 0.05 versus +Cas; *P < 0.05 versus +T/+Cas) and a quantitative evaluation of LC3-II/LC3-I ratio protein level (E) (°P < 0.05 versus EtOH; §P < 0.05 versus +T; ^P < 0.05 versus +Cas; *P < 0.05 versus +T/+Cas), carried out by densitometric scanning of the blots.
Mentions: Based on these data we wished to evaluate the involvement of the autophagic process in the combined activity of Bicalutamide (Cas) and trehalose. Thus, we analyzed SQSTM1/p62 and LC3 expression and processing. As shown in Figure 4A, trehalose, alone or in combination with Bicalutamide (Cas), significantly increases SQSTM1/p62 mRNA expression levels in our cells expressing ARpolyQ; Bicalutamide (Cas) is unable to enhance SQSTM1/p62 mRNA expression levels and does not amplify the trehalose-stimulated SQSTM1/p62 expression, both in the absence and presence of testosterone. Overlapping results were obtained when, in the same conditions, we analyzed the LC3 mRNA expression levels (Fig. 4B). In addition, the levels of the SQSTM1/p62 protein and the conversion of LC3-I to its lipidated and autophagosome-associated form, LC3-II, were increased after trehalose treatment but not influenced by the co-treatment with Bicalutamide (Fig. 4C and relative quantifications for SQSTM1/p62 in D and for LC3-II/LC3-I ratio in E).Figure 4.

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