Quantitative comparison of the efficacy of various compounds in lowering intracellular cholesterol levels in Niemann-Pick type C fibroblasts.

Niemann-Pick Type C disease (NPC) is a lethal, autosomal recessive disorder caused by mutations in the NPC1 and NPC2 cholesterol transport proteins. NPC's hallmark symptoms include an accumulation of unesterified cholesterol and other lipids in the late endosomal and lysosomal cellular compartments, causing progressive neurodegeneration and death. Although the age of onset may vary in those affected, NPC most often manifests in juveniles, and is usually fatal before adolescence. In this study, we investigated the effects of various drugs, many of which modify the epigenetic control of NPC1/NPC2 gene expression, in lowering the otherwise harmful elevated intracellular cholesterol levels in NPC cells. Our studies utilized a previously described image analysis technique, which allowed us to make quantitative comparisons of the efficacy of these drugs in lowering cholesterol levels in a common NPC1 mutant model. Of the drugs analyzed, several that have been previously studied (vorinostat, panobinostat, and β-cyclodextrin) significantly lowered the relative amount of unesterified cellular cholesterol, consistent with earlier observations. In addition, a novel potential treatment, rapamycin, likewise alleviated the NPC phenotype. We also studied combinations of effective compounds with β-cyclodextrin; the addition of β-cyclodextrin significantly enhanced the cholesterol-lowering activity of vorinostat and panobinostat, but had mixed effects with rapamycin. Collectively, these results may provide a basis for the eventual development of improved NPC therapies

Dose Dependence Summary Statistical Data for Treatments in Combination with 200 µM β-Cyclodextrin.

***<p> = significantly reduced from solvent control at p<0.001.</p><p>1–LSO Ratio = 0.321; 67.9% cholesterol reduction.</p><p>2–See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048561#pone-0048561-t001" target="_blank">Table 1</a> for “Drug-Only” Results.</p><p>3–200 µM β-cyclodextrin significantly enhanced the cholesterol-lowering activity of all four compounds at p<0.001.</p

Dose Response Curves for Cholesterol-Lowering Efficacy of Tested Drugs in Combination with β-Cyclodextrin.

<p>NPC1 mutant fibroblasts were treated with vorinostat, panobinostat, rapamycin, and decitabine, both with and without 200 µM β-cyclodextrin, for 48 h, after which they were fixed, filipin stained, imaged, and processed to give an LSO ratio value as described in Materials and Methods. β-Cyclodextrin increased the impact vorinostat, panobinostat, rapamycin, and decitabine had on late endosomal and lysosomal cholesterol levels at all concentrations. The red lines indicate vehicle controls (LSO ratio = 1), while green lines indicate the average result of treatment with only 200 µM β-cyclodextrin (LSO ratio = 0.321). Each point represents the average of at least three independent experiments totaling 36 images (three experiments×twelve images/experiment) and error bars are +/− SE.</p

Dose Dependence Summary Statistical Data.

*<p> = significantly reduced from solvent control at p<0.05.</p>**<p> = significant at p<0.01.</p>***<p> = significant at p<0.001.</p

Representative Images of NPC1 Mutant Cells After Treatment with Various Compounds.

<p>GM03123 NPC1 mutant fibroblasts were treated with various drugs and combinations of drugs with 200 µM β-cyclodextrin for 48 hours, after which they were fixed, filipin stained, and imaged under UV excitation as described in Materials and Methods. Images are shown after contrast adjustment but prior to threshold application. The concentrations used in each displayed image for vorinostat, panobinostat, β-cyclodextrin, rapamycin, decitabine, chloroquine, and chlorpromazine were 10 µM, 75 nM, 200 µM, 7.5 nM, 0.625 µM, 50 µM, and 20 µM, respectively. Reduced cholesterol levels, as evidenced through decreased filipin fluorescence relative to solvent controls, are clearly observed in the vorinostat, panobinostat, β-cyclodextrin, and rapamycin treated cells. Further diminished fluorescence is observed in cells treated with both the drug and β-cyclodextrin (Drug + β-CD). No visibly obvious decrease in fluorescence is observed between cells treated with decitabine, chloroquine, or chlorpromazine, and their respective controls.</p