Effects of flavonoids on glycosaminoglycan synthesis: implications for substrate reduction therapy in Sanfilippo disease and other mucopolysaccharidoses

Abstract

Sanfilippo disease (mucopolysaccharidosis type III, MPS III) is a severe metabolic disorder caused by accumulation of heparan sulfate (HS), one of glycosaminoglycans (GAGs), due to a genetic defect resulting in a deficiency of GAG hydrolysis. This disorder is characterized as the most severe neurological form of MPS, revealing rapid deterioration of brain functions. Among therapeutic approaches for MPS III, one of the most promising appears to be the substrate reduction therapy (SRT). Genistein (5, 7-dihydroxy-3- (4-hydroxyphenyl)-4H-1-benzopyran-4-one) is an isoflavone that has been used in SRT for MPS III. In this report, we tested effects of other flavonoids (apigenin, daidzein, kaempferol and naringenin) on GAG synthesis. Their cytotoxicity and anti-proliferation features were also tested. We found that daidzein and kaempferol inhibited GAG synthesis significantly. Moreover, these compounds were able to reduce lysosomal storage in MPS IIIA fibroblasts. Interestingly, although genistein is believed to inhibit GAG synthesis by blocking the tyrosine kinase activity of the epidermal growth factor receptor, we found that effects of other flavonoids were not due to this mechanism. In fact, combinations of various flavonoids resulted in significantly more effective inhibition of GAG synthesis than the use of any of these compounds alone. These results, together with results published recently by others, suggest that combination of flavonoids can be considered as a method for improvement of efficiency of SRT for MPS III.

Fig. 1

Structural formulas of natural flavonoids used in this work: apigenin (a), daidzein (b), kaempferol (c), naringenin (d) and genistein (e)

Fig. 2

Dose-dependent effects of natural flavonoids on kinetics of glycosaminoglycan synthesis in fibroblasts presented as relative ³⁵S incorporation into GAGs after 3-day exposure to different concentrations of flavonoids [μΜ]. Labeling was conducted for 24 h with 20 μCi/ml H₂[³⁵S]O₄. Radioactivity of incorporated ³⁵S was measured in a scintillation counter, calculated per DNA amount [dpm/ng DNA], and expressed as the percentage of control. 100% (bold solid line, with SD indicated by bold dashed lines) corresponds to the relative ³⁵S incorporation into control cells (from the cell culture treated with 0.05% dimethylformamide). The results presented are average values obtained for five different cell lines with bars indicating standard deviation. Statistical analysis was performed by using the Tukey post-hoc test. Values of p < 0.05 (*) or p < 0.01 (**) are indicated

Fig. 3

Effects of mixtures of natural flavonoids (K—kaempferol, N—naringenin, D—daidzein, G—genistein, at 10 μM concentration each) on kinetics of glycosaminoglycan synthesis in fibroblasts. Relative ³⁵S incorporation into GAGs after 3-day exposure to mixtures of various flavonoids is presented. Labeling was conducted for 24 h with 20 μCi/ml H₂[³⁵S]O₄. Radioactivity of incorporated ³⁵S was measured in a scintillation counter, calculated per DNA amount [dpm/ng DNA], and expressed as the percentage of control (ctrl = cell culture treated with 0.05% dimethylformamide). The results presented are average values obtained for three different cell lines with bars indicating standard deviation. Statistical analysis was performed by using the t-Student two-tailed test. Values of p < 0.05 (*) or p < 0.01 (**) are indicated

Fig. 4

Different lysosomal structures observed in fibroblasts of the MPS IIIA patient. Electron microphotographs present: lysosome of lamellar and electron-dense structures (a), lysosome of amorphous, flocculent and electron-lucent structures (b), and complex lysosomal structure (autophagolysosome) with storage material of different electron density (c)

Fig. 5

Effects of flavonoids (at different concentrations) on tyrosine kinase activity of EGF receptor (EGFR). The results of phospho-EGFR fluorescence were normalized to the total EGFR fluorescence and presented as the percentage of the control cells (incubated without flavonoids). Abbreviations are as follows: K—kaempferol, D—daidzein, N—naringenin, G—genistein, I—a potent EGFR tyrosine kinase inhibitor, PD168390 (which was used as an additional positive control). The results presented are average values obtained from two different experiments with bars indicating standard deviation. Statistically significant differences (p < 0.05) are marked by asterisk