Curcumin Has Beneficial Effects on Lysosomal Alpha-Galactosidase: Potential Implications for the Cure of Fabry Disease

Abstract

Fabry disease is a lysosomal storage disease caused by mutations in the GLA gene that encodes alpha-galactosidase (AGAL). The disease causes abnormal globotriaosylceramide (Gb3) storage in the lysosomes. Variants responsible for the genotypic spectrum of Fabry disease include mutations that abolish enzymatic activity and those that cause protein instability. The latter can be successfully treated with small molecules that either bind and stabilize AGAL or indirectly improve its cellular activity. This paper describes the first attempt to reposition curcumin, a nutraceutical, to treat Fabry disease. We tested the efficacy of curcumin in a cell model and found an improvement in AGAL activity for 80% of the tested mutant genotypes (four out of five tested). The fold-increase was dependent on the mutant and ranged from 1.4 to 2.2. We produced evidence that supports a co-chaperone role for curcumin when administered with AGAL pharmacological chaperones (1-deoxygalactonojirimycin and galactose). The combined treatment with curcumin and either pharmacological chaperone was beneficial for four out of five tested mutants and showed fold-increases ranging from 1.1 to 2.3 for DGJ and from 1.1 to 2.8 for galactose. Finally, we tested a long-term treatment on one mutant (L300F) and detected an improvement in Gb3 clearance and lysosomal markers (LAMP-1 and GAA). Altogether, our findings confirmed the necessity of personalized therapies for Fabry patients and paved the way to further studies and trials of treatments for Fabry disease.

Keywords: AGAL; Fabry disease; curcumin; drug repositioning; lysosomal storage diseases; nutraceutical; pharmacological chaperone.

Figure 1

Curcumin treatment enhanced AGAL activity. IF-GLA and IF-GLA-MUTs were treated with 20 µM curcumin for 48 h. The AGAL-specific activity was then tested on cell protein extracts. Wild-type cells and four mutants showed a significant AGAL improvement upon curcumin treatment (two-tailed unpaired t-test, *** = p < 1 × 10⁻³, **** = p < 1 × 10⁻⁴). (A) IF-GLA-A37T: n = 9, p. 1.85 × 10⁻⁴; (B) IF-GLA-D244H: n = 12, p. 1.02 × 10⁻⁴ (C) IF-GLA-L300F: n = 10, p. 1.43 × 10⁻⁴; (D) IF-GLA-Q280K: n = 6, p. 9.68 × 10⁻⁵; (E) IF-GLA-V269M: n = 6, p. 1.08 × 10⁻¹ (F) IF-GLA: n = 7, p. 5.72 × 10⁻⁴. IF-NULL cells were used as a negative control (G).

Figure 2

Curcumin treatment increased AGAL. IF-GLA and IF-GLA-MUTs were treated with 20 µM curcumin for 48 h. AGAL was visualized via immunoblotting on cell protein extracts. The figure shows IF-GLA-MUTs treated with (+) or without (−) 20 µM curcumin.

Figure 3

Combined treatment with DGJ and curcumin enhanced AGAL activity. IF-GLA and IF-GLA-MUTs were treated for 48 h with 10 µM DGJ in the presence or absence of 20 µM curcumin. The AGAL-specific activity was then tested on cell protein extracts. The presence of curcumin improved AGAL stabilization induced by DGJ in four out of the five tested mutants (two-tailed unpaired t-test, * = p < 5 × 10⁻², ** = p < 1 × 10⁻², **** = p < 1 × 10⁻⁴). (A) IF-GLA-A37T: n = 5, p. 8.01 × 10⁻¹; (B) IF-GLA-D244H: n = 9, p. 2.64 × 10⁻³; (C) IF-GLA-L300F: n = 6, p. 5.03 × 10⁻⁵; (D) IF-GLA-Q280K: n = 2, p. 1.22 × 10⁻²; (E) IF-GLA-V269M: n = 3, p. 2.66 × 10⁻²; (F) IF-GLA: n = 4, p. 1.89 × 10⁻¹. IF-NULL cells were used as a negative control (G).

Figure 4

Combined treatment with DGJ and curcumin treatment increased AGAL. IF-GLA and IF-GLA-MUTs were treated for 48 h with 10 µM DGJ in the presence or absence of 20 µM curcumin. AGAL was visualized via immunoblotting on cell protein extracts. The figure shows IF-GLA-MUTs treated with 10 µM DGJ in the presence (+) or the absence (−) of 20 µM curcumin.

Figure 5

AGAL activity increased upon combined treatment with galactose and curcumin. IF-GLA and IF-GLA-MUTs were treated for 48 h with 100 mM galactose in the presence or the absence of 20 µM curcumin. The AGAL-specific activity was then tested on cell protein extracts. The effect of galactose potentiation was appreciated in four out of the five tested mutants (two-tailed unpaired t-test, * = p < 5 × 10⁻², *** = p < 1 × 10⁻³). (A) IF-GLA-A37T: n = 3, p. 2.06 × 10⁻²; (B) IF-GLA-D244H: n = 3, p. 8.61 × 10⁻²; (C) IF-GLA-L300F: n = 2, p. 3.41 × 10⁻²; (D) IF-GLA-Q280K: n = 3, p. 6.31 × 10⁻⁴; (E) IF-GLA-V269M: n = 3, p. 3.1 × 10⁻²; (F) IF-GLA: n = 3, p. 7.36 × 10⁻². IF-NULL cells were used as a negative control (G).

Figure 6

Combined treatment with galactose and curcumin treatment increased AGAL. IF-GLA and IF-GLA-MUTs were treated for 48 h with 100 mM galactose in the presence or absence of 20 µM curcumin. AGAL was visualized via immunoblotting on cell protein extracts. The figure shows IF-GLA-MUTs treated with 100 mM galactose in the presence (+) or the absence (−) of 20 µM curcumin.

Figure 7

Curcumin treatment improved Gb3 clearance and lysosomal markers. (A,B) IF-GLA-L300F cells were treated for 50 days with drug administration once a week. At the end of the treatment (day 50), cells were collected and lipids were extracted through a methanol:chloroform:water protocol. Lipid content was measured via LC-MS/MS. (A) IF-GLA-L300F treated with 20 µM curcumin or with no drug showed improved Gb3 clearance upon curcumin treatment (two-tailed unpaired t-test, * = p < 5 × 10⁻², n = 3, p. 3.79 × 10⁻²). (B) IF-GLA-L300F treated with 10 µM DGJ and 20 µM curcumin showed Gb3 clearance improved with respect to DGJ monotherapy (two-tailed unpaired t-test; ** = p < 1 × 10⁻², n = 3, p. 8.96 × 10⁻³). (C–E) IF-GLA-L300F cells were treated for 15 days with 10 µM DGJ, 20 µM curcumin, or with both drugs. At the end of the treatment, the immunoblot showed a reduction in the levels of lysosome-associated membrane glycoprotein 1 (LAMP-1) upon curcumin treatment with or without DGJ (C). In addition, a reduction in GAA activity was determined via an enzyme activity assay both in monotherapy (D) (two-tailed unpaired t-test, **** = p < 1 × 10⁻⁴, n = 6, p. 3.95 × 10⁻⁶) or in combined therapy with DGJ (E) (two-tailed unpaired t-test * = p < 5 × 10⁻², n = 6, p. 3.3 × 10⁻²).