Differential Effects of 2-Hydroxypropyl-Cyclodextrins on Lipid Accumulation in Npc1-Null Cells

Abstract

Niemann-Pick disease type C (NPC) is an autosomal recessive disorder characterized by abnormal accumulation of free cholesterol and sphingolipids in lysosomes. The iminosugar miglustat, which inhibits hexosylceramide synthesis, is used for NPC treatment, and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), a cyclic oligosaccharide derivative, is being developed to treat NPC. Moreover, therapeutic potential of 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD) was shown in NPC models, although its mechanism of action remains unclear. Here, we investigated the effects of HP-β-CD, HP-γ-CD, and their homolog 2-hydroxypropyl-α-cyclodextrin (HP-α-CD) on lipid accumulation in Npc1-null Chinese hamster ovary (CHO) cells compared with those of miglustat. HP-β-CD and HP-γ-CD, unlike HP-α-CD, reduced intracellular free cholesterol levels and normalized the lysosome changes in Npc1-null cells but not in wild-type CHO cells. In contrast, miglustat did not normalize intracellular free cholesterol accumulation or lysosome changes in Npc1-null cells. However, miglustat decreased the levels of hexosylceramide and tended to increase those of sphingomyelins in line with its action as a glucosylceramide synthase inhibitor in both Npc1-null and wild-type CHO cells. Interestingly, HP-β-CD and HP-γ-CD, unlike HP-α-CD, reduced sphingomyelins in Npc1-null, but not wild-type, cells. In conclusion, HP-β-CD and HP-γ-CD reduce the accumulation of sphingolipids, mainly sphingomyelins, and free cholesterol as well as lysosome changes in Npc1-null, but not in wild-type, CHO cells.

Keywords: 2-hydroxypropyl-β-cyclodextrin; 2-hydroxypropyl-γ-cyclodextrin; Niemann–Pick disease type C; cholesterol; cyclodextrin; lysosome; miglustat; sphingolipid; sphingomyelin.

Figure 1

Concentration-dependent effects of cyclodextrins (HP-α-CD, HP-β-CD, and HP-γ-CD) and miglustat on impaired lysosomal compartments in Npc1-null CHO cells. The mean fluorescence intensity of LysoTracker^® was determined by flow cytometry at 24 h after exposure to cyclodextrins or miglustat. Each bar represents the mean ± S.E.M; (n = 3), ^## p < 0.01 compared with the wild-type (WT) group, ** p < 0.01 compared with the untreated Npc1-null group.

Figure 2

Effects of cyclodextrins (HP-α-CD, HP-β-CD, and HP-γ-CD) and miglustat on cellular free cholesterol levels in WT and Npc1-null CHO cells. Cellular free cholesterol levels were measured 24 h after exposure to cyclodextrins (1 mM) or miglustat (100 μM). Each bar represents the mean ± S.E.M. (n = 3); ^## p < 0.01 compared with the WT group, ** p < 0.01 compared with the untreated Npc1-null group.

Figure 3

Effects of cyclodextrins (HP-α-CD, HP-β-CD, and HP-γ-CD) and miglustat on total sphingolipid levels in WT and Npc1-null CHO cells. Total sphingolipid levels were measured 24 h after exposure to cyclodextrins (1 mM) or miglustat (100 μM). Each bar represents the mean ± S.E.M. (n = 3); ^## p < 0.01 compared with the WT group, ** p < 0.01 compared with the untreated Npc1-null group.

Figure 4

Effects of cyclodextrins (HP-α-CD, HP-β-CD, and HP-γ-CD) and miglustat on sphingolipid levels in WT (B) and Npc1-null (A) CHO cells. Sphingolipid levels were measured 24 h after treatment with 1 mM of each cyclodextrin and 100 µM of miglustat in Npc1-null and WT CHO cells. The x-axis shows the logarithm to base 2 of fold changes in each sphingolipid level induced by the treatments. The y-axis shows the negative logarithm to base 10 of t-test p-values. Open circle: sphingomyelins, closed triangle: hexosylceramides, open square: ceramides.