Cholesterol-Lowering Effect of Octaarginine-Appended β-Cyclodextrin in Npc1-Trap-CHO Cells

Abstract

Niemann–Pick disease type C (NPC) is an autosomal recessive lysosomal storage disorder, which is an inherited disease characterized by the accumulation of unesterified cholesterol in endolysosomes. Recently, 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD) has been used for the treatment of NPC, and ameliorated a hepatosplenomegaly in the patients. However, to obtain the treatment efficacy, a high dose of HP-β-CyD was necessary. Therefore, the decrease in dose by using active intracellular delivery system of β-CyD to NPC cells is expected. In this study, to efficiently deliver β-CyD to NPC-like cells, we newly synthesized octaarginine (R8)-appended β-CyD with a spacer of γ-aminobutyric acid (R8-β-CyD) and evaluated its cytotoxicity, intracellular distribution, endocytosis pathway and cholesterol-lowering effect in Npc1-trap-Chinese hamster ovary (CHO) cells, cholesterol-accumulated cells through the impairment of NPC1 function. R8-β-CyD did not show cytotoxicity in the cells. In addition, Alexa568-labeled R8-β-CyD was actively internalized into Npc1-trap-CHO cells, possibly through micropinocytosis. Notably, R8-β-CyD significantly decreased intracellular cholesterol content compared with HP-β-CyD. These results suggest that R8-β-CyD may be a promising therapeutic agent for ameliorating cholesterol accumulation in NPC.

Niemann–Pick disease type C (NPC) is an atypical lysosomal storage disorder, which is an inherited disease characterized by the accumulation of unesterified cholesterol in endolysosomes. NPC is elicited by the mutations in either Npc1 or Npc2 gene, and elicits hepatosplenomegaly, neurodegeneration and failure to thrive childhood.^1–3) NPC1 protein in endolysosomes is dominantly associated with cholesterol trafficking in cells.^3,4) Therefore, in NPC patients with loss of function of NPC1 protein, an excessive accumulation of unesterified cholesterol in endolysosomes and a shortage of esterified cholesterol in other cellular compartments are observed. Therefore, decreasing the cholesterol level in endolysosomes was found to be crucial approach for the treatment of NPC.

To evaluate the cholesterol-decreasing ability of drug candidates against NPC, in vitro cell culture systems by utilizing NPC-like cells are necessary. Recently, Higaki et al. established the Npc1-deficient Chinese hamster ovary (CHO) cell mutants (Npc1-trap-CHO cells) by gene trap mutagenesis.⁵⁾ Npc1-trap-CHO cells exhibit the phenotype characteristics of Npc1-deficient cells; the accumulation of free cholesterol in endocytic vesicles and the upregulation of cholesterol synthesis through mevalonate pathway.^5,6) Therefore, Npc1-trap-CHO cells can be applicable for the model of NPC cells.

Cyclodextrins (CyDs) are cyclic oligosaccharides consisting of 6–8 glucose units and have been utilized for improvement of certain properties of drugs such as solubility, stability and bioavailability, etc., through the formation of inclusion complexes.^7,8) Recently, of various CyD derivatives, 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD) has utilized for the treatment of NPC,^4,9–11) based on the several reports demonstrating the gain of longevity of Npc1-deficient mice by a cholesterol decreasing ability of HP-β-CyD.^9–11) Unfortunately, HP-β-CyD was found to produce an increasing in hearing threshold.¹²⁾ Most recently, it is demonstrated that HP-γ-CyD, which is more biocompatible than HP-β-CyD, can also lower the accumulation of cholesterol in NPC-like cells.¹³⁾ However, further dosing as well as dose escalations are needed to ascertain sufficient efficacy of HP-β-CyD or HP-γ-CyD.

To deliver the various bioactive molecules into cells, arginine-rich cell-penetrating peptides (CPPs) such as the oligoarginine peptides and human immunodeficiency virus (HIV)-1 Tat peptide are promising carriers. So far, various cellular uptake mechanisms of arginine-rich CPPs were proposed, i.e., physiological cellular uptake (i.e., endocytosis) and direct permeation through the plasma membranes,^14,15) and recently macropinocytosis (accompanied by actin reorganization, plasma membrane ruffling, and the stimulated engulfment of large volumes of extracellular fluid) has been shown to be an important pathway.^16–20) Therefore, CPP-mediated intracellular delivery is likely to be promising approach to deliver CyDs to NPC disease cells. In this study, to efficiently deliver β-CyD to Npc1-trap-CHO cells, we newly fabricated octaarginine (R8)-appended β-CyD with a spacer of γ-aminobutyric acids (R8-β-CyD) and evaluated its cytotoxicity, cholesterol-lowering effect and intracellular distribution in Npc1-trap-CHO cells.

MATERIALS AND METHODS

Materials

β-CyD was donated by Nihon Shokuhin Kako (Tokyo, Japan). LysoTracker^® was purchased from Life Technologies Japan (Tokyo, Japan). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Nichirei (Tokyo, Japan) and Nissui Pharmaceuticals (Tokyo, Japan), respectively.

Peptide Synthesis

A synthetic scheme of R8-β-CyD is shown in Fig. 1. The peptide chain GABA-[Arg(Pbf)]₈-resin (GABA: γ-aminobutyric acid; Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl) was constructed on a TGS-RAM resin (Shimadzu, Kyoto, Japan) by the Fmoc solid-phase peptide synthesis with the standard coupling system using N-hydroxybenzotriazole (HOBt)/O-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU)/N,N-diisopropylethylamine (DIEA). The peptide was cleaved from the resin and deprotected by treatment with trifluoroacetic acid (TFA)–ethanedithiol (EDT) (95 : 5), and purified by a reverse phase high-performance liquid chromatography (RP-HPLC). Introduction of β-CyD to the N-terminus of GABA-R8-amide was conducted using 6-deoxy-6-(N-hydroxysuccinimide)-β-CyD (NHS-CyD)²¹⁾ (5 equiv.) and N-methylmorpholine (2 equiv.) in 20 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) buffer (pH 6.8) at room temperature, followed by a RP-HPLC purification to yield CyD-GABA-R8 (R8-β-CyD). The product was confirmed by a matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (MALDI-TOFMS). MALDI-TOFMS: 2567.3 [calculated for (M+H)⁺: 2567.3]. Retention time in HPLC: 9.9 min [column: Cosmosil 5C₁₈-AR-II (4.6×150 mm); gradient: 5–55% B in A (A=H₂O containing 0.1% TFA, B=CH₃CN containing 0.1% TFA) over 50 min; flow: 1 mL/min; detection: 220 nm]. For a synthesis of Alexa568-R8-β-CyD, GABA-R8-Alexa568 was prepared by modifying GABA-(Arg)₈-Gly-Cys-amide with 1.1 eq of Alexa 568 C₅ maleimide sodium salt in dimethylformamide–methanol (1 : 1) for 1 h at 25°C, followed by a RP-HPLC purification. Introduction to the N-terminus of GABA-R8-Alexa568 of CyD was conducted as described above, followed by a RP-HPLC purification. The product was confirmed by a MALDI-TOFMS. MALDI-TOFMS: 3586.0 [calculated for (M+H)⁺: 3585.5]. Retention time in HPLC: 12.6 min [column: Cosmosil 5C₁₈-AR-II (4.6×150 mm); gradient: 12–62% B in A (A=H₂O containing 0.1% TFA, B=CH₃CN containing 0.1% TFA) over 50 min; flow: 1 mL/min; detection: 220 nm]. Actual sequences of the synthesized peptides: R8-β-CyD, CyD-GABA-(Arg)₈-amide; Alexa568-R8-β-CyD, CyD-GABA-(Arg)₈-Gly-Cys(Alexa568)-amide.

Fig. 1. Preparation of R8-β-CyD

Cell Culture

Npc1-trap-CHO cells were established by Higaki et al. (Tottori University Faculty of Medicine, Tottori, Japan) and kindly gifted. Npc1-trap-CHO cells were grown in DMEM with 10% FBS, containing penicillin (1×10⁵ mU/mL), streptomycin (0.1 mg/mL) at 37°C in a humidified 5% CO₂ incubator.

Cytotoxicity

Cytotoxicity was assayed by the WST-1 method, as reported previously.²²⁾ Briefly, Npc1-trap-CHO cells (5×10⁴ cells/96-well microplate) were incubated with 150 µL of DMEM containing R8-β-CyD (1, 10 or 100 µM) or Tween 20 for 24 h at 37°C. After washing twice with phosphate-buffered saline (PBS; pH 7.4) to remove R8-β-CyD, 100 µL of fresh Hanks’ balanced salt solution (HBSS; pH 7.4) and 10 µL of WST-1 reagent (Cell Counting Kit, Wako Pure Chemical Industries, Ltd., Osaka, Japan) were added. After incubation for 30 min at 37°C, the absorbance was measured with a microplate reader (Bio-Rad Model 550, Tokyo, Japan).

Intracellular Distribution of R8-β-CyD

Npc1-trap-CHO cells (1×10⁴ cells/96 well microplate) were incubated with 150 µL of DMEM containing 10 µM Alexa568-R8-β-CyD for 24 h. After washing with PBS, the cells were incubated with 100 nM LysoTracker^® for 1 h. After washing with PBS, the fluorescence derived from Alexa568 and LysoTracker^® in Npc1-trap-CHO cells was detected by an IN Cell Analyzer 6000 (GE Healthcare Life Sciences, U.K.).

Effect of Endocytosis Inhibition on Cellular Uptake of R8-β-CyD

Npc1-trap-CHO cells (1×10⁵ cells in 35 mm dish with glass bottom) were incubated with 1 mL of culture medium containing 10 µM Alexa568-R8-β-CyD at 37 or 4°C for 1 h. After washing with PBS, the fluorescence derived from Alexa568 in Npc1-trap-CHO cells was detected by an IN Cell Analyzer 6000 (GE Healthcare Life Sciences). A BZ-II analyzer (Keyence, Osaka, Japan) was used for determination of the fluorescence intensities.

Effect of a Macropinocytosis Inhibitor on Cellular Uptake of R8-β-CyD

Npc1-trap-CHO cells (5×10⁴ cells/35 mm dish with glass bottom) were pretreated with 1 mg/mL of amiloride, a macropinocytosis inhibitor, for 30 min. Then, the cells were incubated with 10 µM Alexa568-R8-β-CyD in medium (FBS (−)) for 1 h at 37°C. After washing with PBS, the fluorescence derived from Alexa568 in Npc1-trap-CHO cells was detected by a fluorescence microscope (Keyence Biozero BZ-8000, Osaka, Japan).

Intracellular Distribution of Cholesterol

Npc1-trap-CHO cells (5×10⁴ cells in 35 mm dish with glass bottom) were incubated in the presence of 150 µL of R8-β-CyD (0.1, 1, 5 or 10 µM) in DMEM for 24 h. After washing with PBS, the intracellular cholesterol was visualized by the cholesterol cell-based detection assay kit (Ann Arbor, MI, U.S.A.). Then, the cells were fixed by cell-based assay fixative solution (4% formaldehyde) and added 150 µL of cholesterol detection assay buffer containing 50 µg/mL of Filipin III. After incubation for 1 h, the fluorescence derived from Filipin III in Npc1-trap-CHO cells was detected by a fluorescence microscope (Keyence Biozero BZ-8000). A BZ-II analyzer (Keyence) was used for determination of the fluorescence intensities.

Data Analysis

Data are presented as the mean±standard error of the mean (S.E.M.) for each group. Statistical significance of mean coefficients for the studies was performed by ANOVA followed by Scheffe’s test. p-Values for significance were set at 0.05.

RESULTS AND DISCUSSION

Cytotoxicity of R8-β-CyD

To evaluate cytotoxicity of R8-β-CyD in Npc1-trap-CHO cells as NPC-like cells,^5,6) we examined the WST-1 method. As shown in Fig. 2, no cytotoxicity of R8-β-CyD was observed in Npc1-trap-CHO cells up to 100 µM for 24 h. The following studies were performed under the present experimental conditions.

Fig. 2. Cytotoxic Activity of R8-β-CyD in Npc1-Trap-CHO Cells after Treatment for 24 h

Npc1-trap-CHO cells (5×10⁴ cells/well) were incubated with 150 µL of medium (FBS (−)) containing R8-β-CyD for 24 h at 37°C. After washing once with PBS, the cells were incubated with 100 µL of fresh HBSS and 10 µL of WST-1 reagent for 30 min at 37°C. The absorbance at 450 nm against a reference wavelength of 655 nm was measured. Each value represents the mean±S.E.M. of 6–8 experiments

Intracellular Distribution of R8-β-CyD

To reveal whether R8-β-CyD enters Npc1-trap-CHO cells, we evaluated the intracellular distribution of Alexa568-labeled R8-β-CyD. As shown in Fig. 3, the cellular uptake of Alexa568-R8-β-CyD in Npc1-trap-CHO cells was observed at 24 h after incubation. Additionally, Alexa568-R8-β-CyD was co-localized with endolysosomes stained by LysoTracker^®. Meanwhile, the intracellular levels of β-CyD and HP-β-CyD labeled with tetramethylrhodamine isothiocyanate (TRITC-β-CyD and TRITC-HP-β-CyD) in Npc1-trap-CHO cells were not observed (Supplementary Fig. 1). These results indicate that R8-β-CyD distributed in endolysosomes after the cellular uptake into Npc1-trap-CHO cells. Meanwhile, it is possible to affect the intracellular distribution of R8-β-CyD by the introduction of Alexa568 due to changes of molecular weight and/or hydrophobicity of the fluorescent probe. Therefore, to reveal the intracellular distribution of R8-β-CyD precisely, a mass imaging system should be necessary to detect R8-β-CyD directly.

Fig. 3. Intracellular Distribution of R8-β-CyD in Npc1-Trap-CHO Cells

Npc1-trap-CHO cells (5×10⁴ cells/well) were incubated in DMEM (FBS (−)) with or without 10 µM Alexa568-R8-β-CyD for 24 h. After washing once with PBS, the cells were incubated with 100 nM LysoTracker^® for 15 min. The cells were washed once with PBS, fixed with 4% paraformaldehyde, and observed by an In Cell Analyzer 6000. The images reflect similar results from three separate experiments.

To investigate whether R8-β-CyD is endocytosed in Npc1-trap-CHO cells, we next examined the cellular uptake of Alexa568-R8-β-CyD under the treatment at 4°C, in which endocytosis is inhibited. As shown in Fig. 4, the fluorescence derived from Alexa568 in Npc1-trap-CHO cells was significantly inhibited at 4°C, but not at 37°C. In cellular uptake of arginine-rich CPPs, macropinocytosis is found to be an important pathway.^16–20) In addition, to induce the macropinocytosis by arginine-rich CPPs, the formation of divalent hydrogen bonds and electrostatic interactions between the arginines and sulfates in glycosaminoglycans (GAGs) are important to accumulate CPPs on plasma membranes.¹⁴⁾ Recently, Takechi-Haraya et al. reported that R8 interacted with GAGs and highly internalized into CHO cells.²³⁾ Indeed, the cellular uptake of Alexa568-R8-β-CyD was inhibited by the presence of amiloride, an inhibitor of macropinocytosis in Npc1-trap-CHO cells (Fig. 5). Therefore, the R8 moieties of R8-β-CyD are likely to interact with GAGs and endocytosed via the macropinocytosis pathway. Collectively, these findings indicate that R8-β-CyD was endocytosed in Npc1-trap-CHO cells, possibly via macropinocytosis.

Fig. 4. Effects of Endocytosis Inhibition to R8-β-CyD in Npc1-Trap-CHO Cells

Npc1-trap-CHO cells (1×10⁵ cells/35 mm dish) were treated with 10 µM R8-β-CyD in medium (FBS (−)) for 1 h at 37°C (A) or at 4°C (B). The cells were washed once with PBS, fixed, and observed by an In Cell Analyzer 6000. The images reflect similar results from three separate experiments.

Fig. 5. Effect of Amiloride, a Macropinocytosis Inhibitor, on Cellular Uptake of R8-β-CyD in Npc1-Trap-CHO Cells

Npc1-trap-CHO cells (5×10⁴ cells/35 mm dish) were pretreated with 1 mg/mL of amiloride for 30 min. Then, the cells were incubated with 10 µM Alexa568-R8-β-CyD in medium (FBS (−)) for 1 h at 37°C. The experiments were performed independently three times.

Effects of R8-β-CyD on Intracellular Cholesterol Levels

To evaluate the cholesterol-decreasing ability of R8-β-CyD, the effects of R8-β-CyD on cholesterol levels in Npc1-trap-CHO cells were examined. Here, to detect the cholesterol levels in the cells, we used Filipin III, as a specific binder to unesterified cholesterol. The fluorescence intensity derived from Filipin III was detected by a fluorescence microscope after treatment with β-CyDs for 24 h (Fig. 6). As shown in Fig. 6A, the treatment with 10 µM HP-β-CyD and R8-β-CyD for 24 h lowered the fluorescent intensity derived from Filipin III in Npc1-trap-CHO cells. In the results of quantification of the fluorescence intensity, the significant lowering effect of R8-β-CyD was shown, compared to that of HP-β-CyD (Fig. 6B). In addition, this cholesterol- lowering effect of R8-β-CyD was in a concentration and time dependent manner (Figs. 7, 8). Therefore, these results indicate that R8-β-CyD lowered the cholesterol levels in Npc1-trap-CHO cells through R8-mediated endocytosis.

Fig. 6. Effects of R8-β-CyD on Cholesterol Levels in Npc1-Trap-CHO Cells

Npc1-trap-CHO cells (5×10⁴ cells/well) were treated with 10 µM HP-β-CyD or 10 µM R8-β-CyD for 24 h. (A) The cells were washed once with PBS, fixed, and observed by an In Cell Analyzer 6000. (B) The fluorescence intensities were determined by a BZ-II analyzer. The experiments were performed independently three times. Each value represents the mean±S.E.M. of 3 experiments. * p<0.05, compared with Npc1-trap-CHO. ^† p<0.05, compared with HP-β-CyD.

Fig. 7. Effects of R8-β-CyD Concentration on Cholesterol Levels in Npc1-Trap-CHO Cells

Npc1-trap-CHO cells (5×10⁴ cells/well) were treated with R8-β-CyD (0.1, 1 and 10 µM) for 24 h. (A) The cells were washed once with PBS, fixed, and observed by an In Cell Analyzer 6000. (B) Determination of fluorescence intensities by a BZ-II analyzer. Each value represents the mean±S.E.M. of 3 experiments. * p<0.05, compared with Npc1-trap-CHO.

Fig. 8. Effects of Incubation Time of R8-β-CyD on Cholesterol Levels in Npc1-Trap-CHO Cells

Npc1-trap-CHO cells (5×10⁴ cells/well) were treated with 10 µM R8-β-CyD for 1, 6, 12 and 24 h. (A) The cells were washed once with PBS, fixed, and observed by an In Cell Analyzer 6000. (B) Determination of fluorescence intensities by a BZ-II analyzer. Each value represents the mean±S.E.M. of 3 experiments. * p<0.05, compared with Npc1-trap-CHO.

Recently, Rosenbaum et al. demonstrated that endocytosis of β-CyDs was responsible for cholesterol reduction in NPC mutant cells.^24,25) Therefore, a macropinocytosis of R8-β-CyD through cell-penetrating peptides of R8 was found to be crucial to lower the cholesterol accumulation in Npc1-trap-CHO cells. Actually, the cholesterol-lowering effect of R8-β-CyD was much higher than that of HP-β-CyD, which cannot enter cells due to lacking of CPPs, in Npc1-trap-CHO cells (Fig. 6). However, the role of R8-β-CyD endocytosed in cholesterol-lowering effect still remains unclear. Therefore, to reveal the detail mechanism of cholesterol-lowering effect of R8-β-CyD, further elaborate studies on not only cholesterol trafficking system but also an interaction with endolysosomal membranes are necessary.

Autophagy is one of the bulk degradation systems of cytoplasmic protein aggregates and subcellular organelles, and is crucial for the regulation of various diseases. In addition, a basal autophagy plays a pivotal role in the constitutive turnover of cytoplasmic components for maintaining cellular function.^26–29) Indeed, the impairment of basal autophagy has been reported in various neurodegenerative diseases and lysosomal storage disorders including NPC.^30,31) Therefore, an amelioration of impaired autophagy is still a challenging issue for the treatment of NPC. Recently, Tamura and Yui reported that HP-β-CyD markedly increased the number of LC3-positive puncta and the p62 levels in NPC1 patient-derived fibroblasts, indicating that autophagic flux was further perturbed.³²⁾ In sharp contrast, the intracellular β-CyD, which was delivered by the biocleavable polyrotaxane system, significantly reduced the number of LC3-positive puncta and the p62 levels in NPC1 patient-derived fibroblasts, indicating the amelioration of impaired autophagy.³²⁾ Therefore, the intracellular R8-β-CyD delivered by CPPs may have the potential to ameliorate autophagy impaired in Npc1-trap-CHO cells. Thereafter, further elaborate studies of not only autophagic function after treatment with R8-β-CyD in Npc1-trap-CHO cells, but also the in-vivo studies in NPC mice are necessary.

CONCLUSION

In the present study, we successfully prepared R8-β-CyD and examined its cholesterol-lowering effect in Npc1-trap-CHO cells. As the results, R8-β-CyD was endocytosed via a cell-penetrating peptide of R8 and reduced the cholesterol accumulation in Npc1-trap-CHO cells. Therefore, R8-β-CyD may be a promising therapeutic for ameliorating cholesterol accumulation in NPC.

Acknowledgments

This work was funded by the Japan Agency of Medical Research and Development (AMED). We thank Dr. K. Higaki and Dr. K. Ohno for the donation of the Npc1-trap-CHO cells.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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