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. 2015 Nov 4;10(11):e0141946.
doi: 10.1371/journal.pone.0141946. eCollection 2015.

2-Hydroxypropyl-β-Cyclodextrin Acts as a Novel Anticancer Agent

Masako Yokoo  1 , Yasushi Kubota  1   2 , Keiichi Motoyama  3 , Taishi Higashi  3 , Masatoshi Taniyoshi  3 , Hiroko Tokumaru  3 , Rena Nishiyama  3 , Yoko Tabe  4 , Sakiko Mochinaga  5 , Akemi Sato  1 , Naoko Sueoka-Aragane  1 , Eisaburo Sueoka  2   6 , Hidetoshi Arima  3   7 , Tetsumi Irie  3   7 , Shinya Kimura  1
Affiliations

2-Hydroxypropyl-β-Cyclodextrin Acts as a Novel Anticancer Agent

Masako Yokoo et al. PLoS One. .

Abstract

2-Hydroxypropyl-β-cyclodextrin (HP-β-CyD) is a cyclic oligosaccharide that is widely used as an enabling excipient in pharmaceutical formulations, but also as a cholesterol modifier. HP-β-CyD has recently been approved for the treatment of Niemann-Pick Type C disease, a lysosomal lipid storage disorder, and is used in clinical practice. Since cholesterol accumulation and/or dysregulated cholesterol metabolism has been described in various malignancies, including leukemia, we hypothesized that HP-β-CyD itself might have anticancer effects. This study provides evidence that HP-β-CyD inhibits leukemic cell proliferation at physiologically available doses. First, we identified the potency of HP-β-CyD in vitro against various leukemic cell lines derived from acute myeloid leukemia (AML), acute lymphoblastic leukemia and chronic myeloid leukemia (CML). HP-β-CyD treatment reduced intracellular cholesterol resulting in significant leukemic cell growth inhibition through G2/M cell-cycle arrest and apoptosis. Intraperitoneal injection of HP-β-CyD significantly improved survival in leukemia mouse models. Importantly, HP-β-CyD also showed anticancer effects against CML cells expressing a T315I BCR-ABL mutation (that confers resistance to most ABL tyrosine kinase inhibitors), and hypoxia-adapted CML cells that have characteristics of leukemic stem cells. In addition, colony forming ability of human primary AML and CML cells was inhibited by HP-β-CyD. Systemic administration of HP-β-CyD to mice had no significant adverse effects. These data suggest that HP-β-CyD is a promising anticancer agent regardless of disease or cellular characteristics.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Function and structure of cyclodextrins, and effects of HP-β-CyD on leukemic cell growth.
(A) Cyclodextrins (CyDs) can form water-soluble complexes with lipophilic guests through encapsulation into the cavity of CyDs. (B and C) Chemical structure of methyl-β-cyclodextrin (M-β-CyD) (B), and 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD) (C). (D–F) Growth curves for each cell line were determined by trypan blue dye exclusion. Ba/F3 BCR-ABLWT cells (D), BV173 cells (E), and hepatocytes (F) were exposed to 0 mM (■), 5 mM (▲), 7.5 mM (●), 10 mM (□), 15 mM (△), 20 mM (○) HP-β-CyD. Data are the mean ± SD of at least three independent experiments. (G) Colony-forming capacity of murine bone marrow mononuclear cells treated with HP-β-CyD. Data represent the mean number of colonies ± SD (n = 3). *P < 0.05.
Fig 2
Fig 2. HP-β-CyD induces apoptosis in BV173 cells and K562 cells.
(A–D) BV173 cells and K562 cells were treated with 0, 5 mM, 10 mM, 15 mM HP-β-CyD, respectively. After 24 hours of culture, cells were collected and stained with Annexin V and 7-AAD. (A) FACS plots, representative of three independent experiments using BV173 cells. (B) Percentage of Annexin V-positive BV173 cells after culture with HP-β-CyD for 24 hours. Data are the mean ± SD of three independent experiments. **P < 0.01. (C) FACS plots, representative of three independent experiments using K562 cells. (D) Percentage of Annexin V-positive K562 cells after culture with HP-β-CyD for 24 hours. Data are the mean ± SD of three independent experiments. *P < 0.05. **P < 0.01.
Fig 3
Fig 3. HP-β-CyD causes cell-cycle arrest in leukemic cells.
(A–D) BV173 and K562 cells were treated with the indicated concentration of HP-β-CyD for 12 hours, then flow cytometric analysis of PI-stained nuclei was performed. (A) Representative flow cytometric histograms of PI-stained BV173 cells. (B) The percentage of cells in G0/G1, S, or G2/M phase was assessed in viable BV173 cells. White: G1-phase, gray: S-phase, black: G2/M-phase. (C) Representative flow cytometric histograms of PI-stained K562 cells. (D) The percentage of cells in G0/G1, S, or G2/M phase was assessed in viable K562 cells. White: G1-phase, gray: S-phase, black: G2/M-phase. (E and F) Effects of HP-β-CyD on the expression of G2/M cell-cycle-associated proteins. BV173 (E) and K562 cells (F) were treated with 10 mM HP-β-CyD for the indicated times. Cells were lysed and analyzed by Western blotting. Western blot images are representative results from at least two independent experiments. Detection of β-actin was used as a loading control. Intensity of the immunoblot signals after background subtraction was quantified using ImageJ software.
Fig 4
Fig 4. Disruption of cellular cholesterol homeostasis by β-CyDs.
(A and B) Effect of β-CyDs on cholesterol efflux from Ba/F3 BCR-ABLWT and BV173 cells. Ba/F3 BCR-ABLWT cells (A) and BV173 cells (B) were incubated with β-CyDs (5 mM, 10 mM) for 1, 2, or 3 hours. Then the cholesterol concentration of the culture supernatants was determined. (●), control; (△), 5 mM HP-β-CyD; (▲), 10 mM HP-β-CyD; (□), 5 mM M-β-CyD; (■), 10 mM M-β-CyD. Data are the mean ± SD of three experiments. (C–E) Measurement of the intracellular cholesterol content of β-CyD-treated cells. Ba/F3 BCR-ABLWT (C), BV173 (D), and hepatocytes (E) were incubated with vehicle, HP-β-CyD (5 mM, 10 mM), or M-β-CyD (5 mM, 10 mM), respectively. After 1 hour, cellular lipids were extracted, and cholesterol contents were determined. HP, HP-β-CyD; M, M-β-CyD; TC, total cholesterol; FC, free cholesterol; EC, esterified cholesterol. (F and G) Images of filipin staining for Ba/F3 BCR-ABLWT (F) or BV173 cells (G) treated with 10 mM β-CyDs for 1 hour are shown. Scale bar: 10 μm.
Fig 5
Fig 5. Protein expression and phosphorylation of BCR-ABL-associated kinases in leukemic cell lines treated with HP-β-CyD.
BV173 and K562 cells were treated with 10 mM HP-β-CyD for the indicated times, after which STAT5, AKT, LYN, and ERK1/2 protein levels, and the phosphorylation status of each protein were determined by western blot analysis. Detection of β-actin was used as a loading control. Data are representative of three independent experiments. Intensity of the immunoblot signals after background subtraction was quantified using ImageJ software, and the relative intensities of p-STAT5, p-AKT, p-LYN, and p-ERK1/2 compared to those of STAT5, AKT, LYN, and ERK1/2 were respectively calculated.
Fig 6
Fig 6. Effect of HP-β-CyD on survival in leukemia mouse models.
(A) Survival of mice that received EGFP+ Ba/F3 BCR-ABLWT cells. EGFP+ Ba/F3 BCR-ABLWT cells (1×106) were transplanted into 6-week-old nude mice. Three days after transplantation, 200 μL vehicle, 50 mM (695.5 mg/kg) HP-β-CyD, or 150 mM (2086.5 mg/kg) HP-β-CyD were intraperitoneally injected twice a day for 20 days, and survival was monitored daily. Gray lines, dotted lines, and black lines indicate the survival curves of vehicle-, 50 mM HP-β-CyD- and 150 mM HP-β-CyD-treated mice, respectively. Survival data were analyzed using a log-rank nonparametric test and are shown as Kaplan-Meier survival curves (n = 10). **P < 0.01. (B) Survival of human leukemia xenografted mice transplanted with BV173 cells. BV173 cells (1×106) were intravenously injected into 2 Gy irradiated NOD/SCID mice. Three days after transplantation, 200 μL vehicle, 50 mM HP-β-CyD (695.5 mg/kg HP-β-CyD), or 150 mM HP-β-CyD (2086.5 mg/kg HP-β-CyD) were administered intraperitoneally for 5 consecutive days every week for 13 weeks, and survival was monitored daily. Gray lines, dotted lines and black lines indicate the survival curves of vehicle-, 50 mM HP-β-CyD-, and 150 mM HP-β-CyD-treated mice, respectively. Survival data were analyzed using a log-rank nonparametric test and are shown as Kaplan-Meier survival curves (n = 10). *P < 0.05. (C–F) Histological examination of lungs from age-matched controls, and HP-β-CyD-treated NOD/SCID mice. (C and D) hematoxylin and eosin (H&E) stained control lung. (C) Original magnification, ×100; (D), ×400. (E and F) H&E stained 150 mM HP-β-CyD-treated lung. (E) Original magnification, ×100; (F), ×400. Representative images from at least two samples are shown.
Fig 7
Fig 7. HP-β-CyD inhibits the in vitro colony-forming ability of human primary leukemic cells.
Mononuclear cells (MNCs) obtained from two patients with AML and from one patient with accelerated phase CML (CML-AP) were plated in methylcellulose containing HP-β-CyD and cultured for 8–13 days. Colonies (>50 cells) were counted. Results are expressed as the percentage of colonies relative to the untreated control ± SD of three replicates. (A and B) Colony formation assays of MNCs from AML patients (FAB: M0) in the presence of HP-β-CyD. (C) Colony formation assays of MNCs from the CML-AP patient in the presence of HP-β-CyD. *P < 0.05. **P < 0.01.
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This work was supported in part by a Grant-in-Aid for Challenging Exploratory Research 25670452 (YK) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan.