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Combination of tert-butyl hydroperoxide with vorinostat induces cell death of Acanthamoeba through cell cycle arrest
Journal Pre-proof Combination of tert-butyl hydroperoxide with vorinostat induces cell death of Acanthamoeba through cell cycle arrest Hae-Ahm Lee, Eun-Kyung Moon, Fu-Shi Quan PII:
S0014-4894(19)30282-6
DOI:
https://doi.org/10.1016/j.exppara.2020.107833
Reference:
YEXPR 107833
To appear in:
Experimental Parasitology
Received Date: 24 June 2019 Revised Date:
6 December 2019
Accepted Date: 10 January 2020
Please cite this article as: Lee, H.-A., Moon, E.-K., Quan, F.-S., Combination of tert-butyl hydroperoxide with vorinostat induces cell death of Acanthamoeba through cell cycle arrest, Experimental Parasitology (2020), doi: https://doi.org/10.1016/j.exppara.2020.107833. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.
Author statement
Hae-Ahm Lee: Methodology, Formal analysis, Investigation Eun-Kyung Moon: Conceptualization, Validation, Writing – Original draft preparation Fu-Shi Quan: Writing – Reviewing and Editing
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Title: Combination of tert-butyl hydroperoxide with vorinostat induces cell death of Acanthamoeba through cell cycle arrest
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RT: Combination of tBHP with vorinostat
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Authors: Hae-Ahm Lee1, Eun-Kyung Moon2, and Fu-Shi Quan1, 2, *
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Address: 1Medical Research Center for Bioreaction to Reactive Oxygen Species and
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Biomedical Science Institute, School of Medicine, Graduate school, Kyung
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Hee University, Seoul, Republic of Korea, 2 Department of Medical Zoology,
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Kyung Hee University School of Medicine, Seoul, Republic of Korea
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* Corresponding author
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Fu-Shi Quan: Department of Medical Zoology, Kyung Hee University School of
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Medicine, Seoul, Republic of Korea
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Tel: +82-2-961-2302
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Fax: +82-2-961-0278
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E-mail: [email protected]
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ABSTRACT
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Safety precautions prior to contact lens usage is essential for preventing
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Acanthamoeba keratitis. Contact lens disinfecting solutions containing 3% hydrogen
28
peroxide (H2O2) are known to exert amoebicidal effect against Acanthamoeba. Yet,
29
these solutions need to be neutralized to prevent ocular irritation, which consequently
30
may result in incomplete disinfection. In this study, amoebicidal effect of tert-butyl
31
hydroperoxide (tBHP) was investigated and its efficacy was compared to those of
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hydrogen peroxide (H2O2). H2O2 and tBHP showed dose dependent amoebicidal effect,
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however high concentration of these compounds demonstrated cytotoxicity in human
34
corneal epithelial (HCE) cells. To reduce their cytotoxicity, the concentrations of both
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compounds were diluted to 50 µM and subsequently combined with 10 µM vorinostat to
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enhance amoebicidal effect. Addition of vorinostat induced high amoebicidal effect
37
against Acanthamoeba trophozoites, even at low concentrations of H2O2 or tBHP.
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Cellular damage induced by combined treatment of H2O2 or tBHP with vorinostat in
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Acanthamoeba were determined by assessing cell cycle arrest and apoptosis via FACS
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analysis. While 50 µM H2O2 combined with 10 µM vorinostat showed 36.26%
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cytotoxicity on HCE cells during 24 h exposure, 50 µM tBHP with 10 µM vorinostat did
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not show cytotoxicity on HCE cells. These findings suggest that the application of tBHP
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and vorinostat for Acanthamoeba keratitis treatment and contact lens disinfection
44
system is highly plausible.
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Key Words: Acanthamoeba, tBHP, vorinostat
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INTRODUCTION
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Acanthamoeba keratitis (AK) caused by the pathogenic Acanthamoeba is a rare
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disease, but its incidence is increasing (Auran et al., 1987). The risk factor of AK
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infection is associated with contact lens usage and poor lens hygiene (Jasim et al., 2012;
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Maycock and Jayaswal, 2016). Diagnosis of AK is difficult and often delayed. Limited
54
effective treatment options, as well as difficulties arising from highly drug-resistant cyst
55
stage of Acanthamoeba also remains a challenge that needs to be addressed. Most of the
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currently used agents for AK treatment are biguanides (0.02% polyhexamethylene
57
biguanide or 0.02% chlorhexidine) and diamidine (0.1% propamidine or 0.1%
58
hexamidine) (Lorenzo-Morales et al., 2015; Carrijo-Carvalho et al., 2017), whose potent
59
side effects are the results of drug toxicity and sensitive reaction of the corneal cell.
60
Approximately 90% of AK infections occur in contact lens wearers (Jasim et
61
al., 2012; Maycock and Jayaswal, 2016). Therefore, correct contact lens use and
62
disinfection can reduce AK incidence. Current trend in contact lens care system is the
63
use of multi-purpose solution (MPS) for cleansing and disinfection (Stevenson and Seal,
64
1998). Most of the MPSs contain polyhexamethylene biguanide, and they are effective
65
against microorganisms. However, they showed cytotoxicity in human corneal epithelial
66
(HCE) cells and its efficacy was insufficient to thwart Acanthamoeba infections,
67
particularly those arising from cysts (Beattie et al., 2003; Moon et al., 2016). Hydrogen
68
peroxide (H2O2) is known to be an effective antimicrobial agent, and 3% H2O2 solution
69
is used widely for contact lens disinfection (Gasset et al., 1975; Nichols et al., 2019).
70
H2O2-derived free radicals can serve as either essential components of cells or
71
detriments, which can destroy integral cell components (Lowe and Brennan, 1987).
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Because HCE cells are also susceptible to the oxidative effects of H2O2, lens care
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solution containing H2O2 require complete neutralization (Nichols et al., 2019). Varying
74
consequences may arise depending on the neutralization process, as extremely rapid
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H2O2 neutralization reaction can result in inadequate disinfection, whereas ocular
76
irritation may occur from incomplete neutralization (Kilvington and Winterton, 2017).
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For successful contact lens care, an investigation researching simple yet effective lens
78
disinfection system would be necessary.
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Oxidative stress caused by reactive oxygen species (ROS) induce cellular
80
damages to DNA, proteins, and lipids (Kehrer, 1993). Tert-butyl hydroperoxide (tBHP)
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is a representative oxidant that induces mitochondrial dysfunction and cell death
82
(Prakash and Kumar, 2014). Two stressors, H2O2 and tBHP enhanced cell damage and
83
ROS generation. Previous studies have documented apoptosis and necroptosis induction
84
by tBHP in endothelial cells (Zhao et al., 2017). The combination of tBHP plus
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cetyltrimethyl ammonium bromide and a tetra-amido macrocyclic ligand is effective in
86
killing Bacillus subtilis spores (Paul et al., 2007). While the research of amoebicdal
87
effects of H2O2 against Acanthamoeba is progressing well, the effect of tBHP in
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Acanthamoeba remains unknown.
89
Histone deacetylase (HDAC) inhibitors are chemical compounds that inhibit
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histone deacetylases, and they have shown to induce cell cycle arrest, differentiation, or
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apoptosis in tumor cells (Dokmanovic and Marks, 2005; Mei et al., 2004). Vorinostat is
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a one of the numerous types of HDAC inhibitor and their effects on cell growth,
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differentiation, and apoptosis have been reported (Bubna, 2015). Its application as a
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monotherapeutic agent or in combination with other drugs have been documented.
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In this study, we demonstrated the amoebicidal effect and its mechanism in
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tBHP compared to that of H2O2. Furthermore, we investigated the effect of tBHP
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combined with vorinostat to Acanthamoeba trophozoites and HCE cells.
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MATERIALS AND METHODS Cell cultures
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Acanthamoeba castellanii Castellani was obtained from the American Type
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Culture Collection (ATCC 30868). Acanthamoeba trophozoites were cultured axenically
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in PYG medium (20 g proteose peptone, 1 g yeast extract, 0.1M glucose, 4 mM MgSO4,
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0.4 mM CaCl2, 3.4 mM sodium citrate, 0.05 mM Fe(NH4)2(SO4)2, 2.5 mM Na2HPO4,
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and 2.5 mM K2HPO4 in 1 liter of distilled water with the final pH adjusted to 6.5) at
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25°C incubator. Human corneal epithelial (HCE) cells (ATCC PCS-700-010) were
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cultured in endothelial cell growth medium kits (KGM BulletKit; Lonza, Portsmouth,
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NH, USA) in a humidified incubator containing 5% CO2 at 37°C.
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Amoebicidal effects on Acanthamoeba
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Acanthamoeba trophozoites were seeded into 96-well culture plates at a density
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of 1 × 104 cells/well. To determine the effect of H2O2 (12.5, 25, 50, 100, and 200 µM)
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tBHP (12.5, 25, 50, 100, and 200 µM), vosinostat (0.5, 1, and 10 µM), combination of
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H2O2 and 10 µM vorinostat, and combination of tBHP and 10 µM vorinostat,
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monolayers of amoebae were incubated with aforementioned reagents for 24 h at 25°C.
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After incubation, 10 µl of cell counting kit-8 (CCK-8) was added to each well and the
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plate was incubated for 6 h at 25°C. The sample absorbance was measured at the 450
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nm wavelength against blank which contained medium only. The cell viability was
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calculated according to the following formula: (%) = (OD of sample - OD of
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blank)/(OD of control - OD of blank) × 100%. All assays were performed in triplicate.
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Cytopathic effects (CPE) on human corneal cells
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HCE cells were seeded into 96-well culture plates at a density of 1 × 104
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cells/well. The next day, HCE cell monolayers were incubated with H2O2 (50 and 500
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µM), tBHP (50 and 500 µM), vorinostat (10 µM), combination of 50 µM H2O2 and 10
126
µM vorinostat, or combination of 50 µM tBHP and 10 µM vorinostat at 37°C in 5%
127
CO2 for 24 h. After incubation, CPE on HCE cells were assessed using the
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aforementioned CCK-8. All assays were performed in triplicate.
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Detection of apoptosis and cell cycle arrest
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Apoptotic cell death was detected using FITC Annexin V Apoptosis Detection
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Kit (BD Bioscience, San Jose, California, United States). All experiments were
133
conducted following the manufacturer's protocol. Briefly, A. castellanii trophozoites
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were seeded into 6-well culture plates at a density of 5×105 cells per well. After
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overnight culture, monolayers of amoebae were incubated with 0.0002% PHMB, 50 µM
136
H2O2, combination of 50 µM H2O2 and 10 µM vorinostat, 50 µM tBHP, or combination
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of 50 µM tBHP and 10 µM vorinostat for 24 h at 25°C. Amoebae were washed with
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PBS to remove excess drug, and re-suspended in 1x binding buffer at a concentration of
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1×106 cells/ml. Amoebae (1×105 cells/100 µl) were transferred to a new tube and
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incubated with 5 µl of FITC annexin V and 5 µl of propidium iodide (PI) for 15 min at
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RT in the dark. Finally, 400 µl of 1x binding buffer was added, and cells were analyzed
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by a fluorescent microscope (Leica DMi8, Wetzlar, Germany) and flow cytometry using
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BD Accuri C6. To investigate the cell cycle arrest, amoebae were treated as above for
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apoptosis assessment. Samples were fixed with 70% ethanol for 30 min, and washed
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twice with PBS. Afterwards, cells were treated with ribonuclease (final conc. 10 µg/ml)
146
for 10 min at 37°C, and stained with PI for 30 min at RT in the dark. Stained cells were
147
analyzed by flow cytometry using BD Accuri C6.
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Statistical analysis
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Data are presented as mean ± SD from three independent experiments.
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Statistical analysis was performed using Student's t-test. A p value of < 0.05 was
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considered statistically significant.
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RESULTS
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H2O2 and tBHP showed amoebicidal effect on Acanthamoeba trophozoites
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Cell viability assay using CCK-8 showed that H2O2 decreased Acanthamoeba
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viability in a dose dependent manner (Fig. 1A). However, 50 µM of H2O2 that showed
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amoebicidal effect also demonstrated 34.98% of cytotoxicity against HCE cells (Fig.
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1B). Amoebicidal effects of tBHP were noticeable at 50, 100, and 200 µM (Fig 1C).
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Although miniscule levels of amoebicidal effect were induced by 50 µM tBHP, its
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cytotoxicity to HCE cells were significantly low (Fig 1D). Neither Acanthamoeba nor
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HCE cell viabilities were affected by vorinostat concentrations of 0.1 ~ 10 µM (Fig. 1E
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and F).
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Vorinostat increased the amoebicidal effects of H2O2 and tBHP
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To increase the amoebidical effects of H2O2 and tBHP, 10 µM vorinostat was
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combined with them. Upon addition of vorinostat, morphological changes were
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observed in Acanthamoeba treated with 50 ~ 200 µM H2O2 or tBHP (Fig. 2B and D).
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Cell viability assay confirmed increased amoebicidal effects at low concentrations of
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H2O2 and tBHP when vorinostat was combined (Fig. 2E and F). Cyst formation was not
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observed from these samples. Compared to single treatment of H2O2 and tBHP, the
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combination of 50 µM H2O2 with 10 µM vorinostat showed increased amoebicidal
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effect from 25.12% to 34.89%, and the combination of 50 µM tBHP with 10 µM
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vorinostat showed drastically increased amoebicidal effects from 2.63% to 34.58% (Fig.
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2G and H). These results suggest that vorinostat enhanced the amoebicidal effects of
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low concentration of H2O2 and tBHP.
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H2O2 induced apoptosis in Acanthamoeba
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To investigate the effects of two combinations on Acanthamoeba, apoptosis
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analysis was performed (Fig. 3). Acanthamoeba treated with 50 µM H2O2 induced
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20.7% apoptosis (Fig. 3B), and the combination of 50 µM H2O2 with 10 µM vorinostat
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induced 18.3% apoptosis (Fig. 3C), while untreated control cells showed 13.2%
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apoptosis (Fig. 3A). The 50 µM tBHP showed 8.1% apoptotic cell death, and the
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combination of 50 µM tBHP with 10 µM vorinostat showed 9.0% apoptosis in
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Acanthamoeba (Fig. 3D and E). The addition of vorinostat did not affect apoptosis by
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H2O2 and tBHP. These results suggest that 50 µM H2O2 induced apoptosis in
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Acanthamoeba, whereas 50 µM tBHP did not induce apoptotic cell death in
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Acanthamoeba.
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tBHP combined with vorinostat arrested the cell cycle of Acanthamoeba
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To determine whether these combinations arrest the cell cycle of
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Acanthamoeba, flow cytometry was performed (Fig. 4). We divided the DNA
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replication cycle of Acanthamoeba into three segments: M1, M2, and M3 (Fig. 4).
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Because Acanthamoeba trophozoites in this study were examined in asynchronous
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culture, majority of the amoebae were in a single phase of the cycle. We supposed that
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M2 segment corresponded to G2 phase, which were set to be approximately 90%.
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Population changes and cell distribution among the three segments were investigated
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after treatment with the four aforementioned combinations. Acanthamoeba exposed to
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50 µM H2O2 showed reduced M2, and increased M1 and M3 population compared to
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control (Fig. 4B). The addition of 10 µM vorinostat to H2O2 increased M3 population
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(Fig. 4C). Acanthamoeba treated with 50 µM tBHP alone revealed reduced M2 and M3,
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but increased M1 populations compared to control cells (Fig. 4D). Combining 50 µM
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tBHP with 10 µM vorinostat significantly increased M1 population, whereas M3
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population was reduced (Fig. 4E). These findings suggested that H2O2 combined with
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vorinostat induced cell cycle arrest at G2/M phase (Fig. 4C, M3 segment), and tBHP
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combined with vorinostat induced cell cycle arrest at G1 phase (Fig. 4E, M1 segment).
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Combining tBHP with vorinostat did not show cytotoxicity to HCE cells
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To determine the cytotoxicity of two combinations in HCE cells, HCE cells
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were treated with 50 µM H2O2, 50 µM H2O2 combined with 10 µM vorinostat, 50 µM
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tBHP, and 50 µM tBHP combined with 10 µM vorinostat (Fig. 5). The combination of
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50 µM H2O2 and 10 µM vorinostat showed 36.26% cytotoxicity to HCE cells, which
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was similar level to that of 50 µM H2O2 (Fig. 5A). The combination of 50 µM tBHP and
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10 µM vorinostat showed slight cytotoxicity (6.98%) to HCE cells (Fig. 5B). The
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addition of 10 µM vorinostat did not increase the cytotoxicity by 50 µM tBHP, and this
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combination was considered to be non-toxic to HCE cells.
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DISCUSSION
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In this study, we report that oxidant tBHP has amoebicidal effect on
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Acanthamoeba trophozoite. The combination of low concentration of tBHP (50 µM) and
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vorinostat (10 µM) showed high amoebicidal effect on Acanthamoeba, and low
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cytotoxicity on HCE cells (Fig. 2 and 5).
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Although
vorinostat
alone have
not
shown
amoebicidal
effect
on
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Acanthamoeba, it showed strong amoebicidal effect when combined with tBHP or H2O2.
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However, there were no significant differences among different concentrations of H2O2
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or tBHP (Fig. 2E and F). A 3% H2O2 solution can effectively disinfect soft contact
227
lenses (Gasset et al., 1975), but H2O2 is widely regarded as a cytotoxic agent whose
228
levels must be minimized (Halliwell et al., 2000). In this study, to reduce the
229
cytotoxicity of H2O2, the concentration was diluted to 50 µM. One mole solution of
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H2O2 is equivalent to 3.4% H2O2. By addition of 10 µM of vorinostat to 50 µM H2O2,
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the amoebicidal effect was increased (Fig. 2).
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The actual mechanism of action for the combined treatments remains unclear.
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H2O2 induced apoptosis in Acanthamoeba, while tBHP did not induce apoptotic cell
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death (Fig. 3). We speculate that the concentration of tBHP (50 µM) was too low to
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induce apoptosis of Acanthamoeba (Fig. 1C and 3D). Spector et al. found that H2O2-
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induced cell death was related to the extensive DNA damage caused by peroxide, and
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tBHP under similar conditions caused little DNA damage (Spector et al., 2002). In line
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with this notion, results acquired from the present study also showed a pattern similar to
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this. The addition of vorinostat did not affect induction of apoptosis.
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The DNA replication cycle of Acanthamoeba has been examined. In synchrony
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cultured Acanthamoeba, trophozoites appeared to consist of 37-47% G1 (presynthetic
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gap), 13% S (synthesis), and 40-50% G2 (postsynthetic gap) plus M (mitosis) (Byers et
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al., 1991). In asynchronous cultures of Acanthamoeba, the cycle appeared to consist of
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at least 90% G2, essentially no G1, and 8-10% M plus S (Byers et al., 1991).
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Acanthamoeba treated with 50 µM H2O2 showed increased G1/S and M (Fig. 4B -
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segments M1 and M3) compared to untreated control cells. After addition of 10 µM
247
vorinostat, M was increased (Fig. 4C - segment M3). Acanthamoeba treated with 50 µM
248
tBHP showed increased G1/S (Fig. 4D - segment M1). After addition of 10 µM
249
vorinostat, G1/S was increased further and M was decreased (Fig. 4E - segments M1
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and M3). Vorinostat induces cell-cycle arrest at the G1 or the G2/M boundary in a cell-
251
type dependent and/or dose-related fashion (Richon et al., 2009). In Acanthamoeba,
252
vorinostat with H2O2 induced cell cycle arrest at the G2M boundary, and vorinostat with
253
tBHP induced cell cycle arrest at the G1 phase. These results suggest that vorinostat
254
combined with H2O2 or tBHP increased amoebicidal effect on Acanthamoeba by cell
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cycle arrest. Prospective studies investigating the effects of these combinations on
256
Acanthamoeba cyst and various clinical strains would be of great value.
257
The low concentration of tBHP (50 µM) combined with 10 µM vorinostat did
258
not show cytotoxicity, while 50 µM H2O2 combined with 10 µM vorinostat showed
259
cytotoxicity to HCE cells (Fig. 5). Although organic peroxides are widely used in
260
various oxidation processes, tBHP has been documented to be a better alternative for
261
H2O2 and has been frequently utilized in oxidative stress studies (15). The effect of
262
tBHP combined with vorinostat on Acanthamoeba will provide important information to
263
develop safe and optimized contact lens care system and treatment for Acanthamoeba
１３
264
keratitis.
265
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267 268
ACKNOWLEDGEMENT This work was supported by grants from the National Research Foundation of Korea (NRF) (2016R1D1A1B03933863, and 2018R1A6A1A03025124).
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Jasim H, Knox-Cartwright N, Cook S, Tole D. 2012. Increase in Acanthamoeba
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Nichols JJ, Chalmers RL, Dumbleton K, Jones L, Lievens CW, Merchea MM,
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Szczotka-Flynn L. 2019. The Case for Using Hydrogen Peroxide Contact Lens
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Paul M, Setlow B, Setlow P. 2007. Killing of spores of Bacillus subtilis by tert-butyl hydroperoxide plus a TAML activator. J Appl Microbiol 102:954-962.
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Richon VM, Garcia-Vargas J, Hardwick JS. 2009. Development of vorinostat: current applications and future perspectives for cancer therapy. Cancer Lett 280:201-210.
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Spector A, Ma W, Sun F, Li D, Kleiman NJ. 2002. The effect of H2O2 and tertiary butyl
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Eye Res 75:573-582.
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Stevenson RW, Seal DV. 1998. Has the introduction of multi-purpose solutions
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contributed to a reduced incidence of Acanthamoeba keratitis in contact lens
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wearers? A review. Cont Lens Anterior Eye 21:89-92.
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Zhao W, Feng H, Sun W, Liu K, Lu JJ, Chen X. 2017. Tert-butyl hydroperoxide (t-
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BHP) induced apoptosis and necroptosis in endothelial cells: Roles of NOX4 and
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FIGURE LEGENDS
328 329
Figure 1. Amoebicidal effect and cytotoxicity of H2O2, tBHP and vorinostat. To
330
investigate the amoebicidal effect of H2O2, tBHP and vorinostat on Acanthamoeba,
331
Acanthamoeba trophozoites were treated with 12.5 ~ 200 µM H2O2 (A), 12.5 ~ 200 µM
332
tBHP (C) or 0.1 ~ 10 µM vorinostat (E) for 24 h. To determine the cytotoxicity of H2O2,
333
tBHP and vorinostat to HCE cells, HCE cells were treated with 50 and 500 µM H2O2
334
(B), 50 and 500 µM tBHP (D) or 10 µM vorinostat (F) for 24 h. *, **: Asterisks denote
335
statistically significant differences (*P<0.05 and **P<0.01) between untreated cells
336
(control) and each agent-treated cells
337 338
Figure 2. Amoebicidal effects of H2O2 and tBHP combined with vorinostat. To enhance
339
the amoebicidal effect of H2O2 and tBHP, 10 µM vorinostat was combined with them.
340
Acanthamoeba were treated with 50 ~ 200 µM H2O2 (A), 50 ~ 200 µM H2O2 with 10
341
µM vorinostat (B), 50 ~ 200 µM tBHP (C), or 50~200 µM tBHP with 10 µM vorinostat
342
(D) for 24 h. After incubation, morphological changes were observed under the
343
microscope (400x) (A ~ D). Panels (E) and (F) summarizes the amoebicidal effects
344
observed in (B) and (D). The amoebicidal effects of the combinations (H2O2 with
345
vorinostat, and tBHP with vorinostat) were compared to H2O2 or tBHP treatment alone
346
(G and H). **: Asterisks denote statistically significant differences (**P<0.01) between
347
control and cells treated with the three combinations.
348
１９
349
Figure 3. Detection of apoptosis. To measure apoptotic cell death of Acanthamoeba,
350
Acanthamoeba trophozoites were treated with 50 µM H2O2 (B), 50 µM H2O2 with 10
351
µM vorinostat (C), 50 µM tBHP (D), or 50 µM tBHP with 10 µM vorinostat (E) for 24
352
h. Untreated cells were used as normal control (A). Cells were subsequently stained
353
with annexin V and propidium iodide (PI), and these cells were assessed using flow
354
cytometry. The upper right quadrant (Q2-UR) in each flow cytometry plot indicates
355
apoptosis.
356 357
Figure 4. DNA and cell cycle analysis. To analyze the cell cycle of Acanthamoeba,
358
asynchronous cultured Acanthamoeba was stained with PI, and were subjected to flow
359
cytometry (A). DNA replication cycle of Acanthamoeba was divided into three
360
segments: M1, M2, and M3. After treatment of 50 µM H2O2 (B), 50 µM H2O2 with 10
361
µM vorinostat (C), 50 µM tBHP (D), or 50 µM tBHP with 10 µM vorinostat (E) for 24
362
h, the percentage of cells with fractional DNA content was estimated.
363 364
Figure 5. Cytotoxicity of the two combinations to HCE cells. To evaluate the
365
cytotoxicity of two combinations to HCE cells, HCE cells were treated with 50 µM
366
H2O2, 50 µM H2O2 with 10 µM vorinostat, 50 µM tBHP, and 50 µM tBHP with 10 µM
367
vorinostat for 24 h (A and B). The cytotoxicity of the combinations were compared to
368
those of control (untreated) and cells receiving single treatment. *, **: Asterisks denote
369
statistically significant differences (*P<0.05 and **P<0.01) between control and each
370
treated cells.
２０
▪ The combination of 50 µM tBHP and 10 µM vorinostat showed high amoebicidal
effect on Acanthamoeba, and low cytotoxicity on HCE cells. ▪ Vorinostat combined with H2O2 or tBHP increased amoebicidal effect on Acanthamoeba by cell cycle arrest. ▪ The effect of tBHP combined with vorinostat on Acanthamoeba will provide important information to develop safe and optimized contact lens care system and treatment for Acanthamoeba keratitis.
S0014-4894(19)30282-6
DOI:
https://doi.org/10.1016/j.exppara.2020.107833
Reference:
YEXPR 107833
To appear in:
Experimental Parasitology
Received Date: 24 June 2019 Revised Date:
6 December 2019
Accepted Date: 10 January 2020
Please cite this article as: Lee, H.-A., Moon, E.-K., Quan, F.-S., Combination of tert-butyl hydroperoxide with vorinostat induces cell death of Acanthamoeba through cell cycle arrest, Experimental Parasitology (2020), doi: https://doi.org/10.1016/j.exppara.2020.107833. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.
Author statement
Hae-Ahm Lee: Methodology, Formal analysis, Investigation Eun-Kyung Moon: Conceptualization, Validation, Writing – Original draft preparation Fu-Shi Quan: Writing – Reviewing and Editing
1 2
Title: Combination of tert-butyl hydroperoxide with vorinostat induces cell death of Acanthamoeba through cell cycle arrest
3 4 5 6
RT: Combination of tBHP with vorinostat
7 8 9
Authors: Hae-Ahm Lee1, Eun-Kyung Moon2, and Fu-Shi Quan1, 2, *
10 11
Address: 1Medical Research Center for Bioreaction to Reactive Oxygen Species and
12
Biomedical Science Institute, School of Medicine, Graduate school, Kyung
13
Hee University, Seoul, Republic of Korea, 2 Department of Medical Zoology,
14
Kyung Hee University School of Medicine, Seoul, Republic of Korea
15 16
17
* Corresponding author
18
Fu-Shi Quan: Department of Medical Zoology, Kyung Hee University School of
19
Medicine, Seoul, Republic of Korea
20 21
Tel: +82-2-961-2302
22
Fax: +82-2-961-0278
23
E-mail: [email protected]
24
１
25
ABSTRACT
26
Safety precautions prior to contact lens usage is essential for preventing
27
Acanthamoeba keratitis. Contact lens disinfecting solutions containing 3% hydrogen
28
peroxide (H2O2) are known to exert amoebicidal effect against Acanthamoeba. Yet,
29
these solutions need to be neutralized to prevent ocular irritation, which consequently
30
may result in incomplete disinfection. In this study, amoebicidal effect of tert-butyl
31
hydroperoxide (tBHP) was investigated and its efficacy was compared to those of
32
hydrogen peroxide (H2O2). H2O2 and tBHP showed dose dependent amoebicidal effect,
33
however high concentration of these compounds demonstrated cytotoxicity in human
34
corneal epithelial (HCE) cells. To reduce their cytotoxicity, the concentrations of both
35
compounds were diluted to 50 µM and subsequently combined with 10 µM vorinostat to
36
enhance amoebicidal effect. Addition of vorinostat induced high amoebicidal effect
37
against Acanthamoeba trophozoites, even at low concentrations of H2O2 or tBHP.
38
Cellular damage induced by combined treatment of H2O2 or tBHP with vorinostat in
39
Acanthamoeba were determined by assessing cell cycle arrest and apoptosis via FACS
40
analysis. While 50 µM H2O2 combined with 10 µM vorinostat showed 36.26%
41
cytotoxicity on HCE cells during 24 h exposure, 50 µM tBHP with 10 µM vorinostat did
42
not show cytotoxicity on HCE cells. These findings suggest that the application of tBHP
43
and vorinostat for Acanthamoeba keratitis treatment and contact lens disinfection
44
system is highly plausible.
45 46 47
Key Words: Acanthamoeba, tBHP, vorinostat
48
２
49
INTRODUCTION
50
Acanthamoeba keratitis (AK) caused by the pathogenic Acanthamoeba is a rare
51
disease, but its incidence is increasing (Auran et al., 1987). The risk factor of AK
52
infection is associated with contact lens usage and poor lens hygiene (Jasim et al., 2012;
53
Maycock and Jayaswal, 2016). Diagnosis of AK is difficult and often delayed. Limited
54
effective treatment options, as well as difficulties arising from highly drug-resistant cyst
55
stage of Acanthamoeba also remains a challenge that needs to be addressed. Most of the
56
currently used agents for AK treatment are biguanides (0.02% polyhexamethylene
57
biguanide or 0.02% chlorhexidine) and diamidine (0.1% propamidine or 0.1%
58
hexamidine) (Lorenzo-Morales et al., 2015; Carrijo-Carvalho et al., 2017), whose potent
59
side effects are the results of drug toxicity and sensitive reaction of the corneal cell.
60
Approximately 90% of AK infections occur in contact lens wearers (Jasim et
61
al., 2012; Maycock and Jayaswal, 2016). Therefore, correct contact lens use and
62
disinfection can reduce AK incidence. Current trend in contact lens care system is the
63
use of multi-purpose solution (MPS) for cleansing and disinfection (Stevenson and Seal,
64
1998). Most of the MPSs contain polyhexamethylene biguanide, and they are effective
65
against microorganisms. However, they showed cytotoxicity in human corneal epithelial
66
(HCE) cells and its efficacy was insufficient to thwart Acanthamoeba infections,
67
particularly those arising from cysts (Beattie et al., 2003; Moon et al., 2016). Hydrogen
68
peroxide (H2O2) is known to be an effective antimicrobial agent, and 3% H2O2 solution
69
is used widely for contact lens disinfection (Gasset et al., 1975; Nichols et al., 2019).
70
H2O2-derived free radicals can serve as either essential components of cells or
71
detriments, which can destroy integral cell components (Lowe and Brennan, 1987).
72
Because HCE cells are also susceptible to the oxidative effects of H2O2, lens care
３
73
solution containing H2O2 require complete neutralization (Nichols et al., 2019). Varying
74
consequences may arise depending on the neutralization process, as extremely rapid
75
H2O2 neutralization reaction can result in inadequate disinfection, whereas ocular
76
irritation may occur from incomplete neutralization (Kilvington and Winterton, 2017).
77
For successful contact lens care, an investigation researching simple yet effective lens
78
disinfection system would be necessary.
79
Oxidative stress caused by reactive oxygen species (ROS) induce cellular
80
damages to DNA, proteins, and lipids (Kehrer, 1993). Tert-butyl hydroperoxide (tBHP)
81
is a representative oxidant that induces mitochondrial dysfunction and cell death
82
(Prakash and Kumar, 2014). Two stressors, H2O2 and tBHP enhanced cell damage and
83
ROS generation. Previous studies have documented apoptosis and necroptosis induction
84
by tBHP in endothelial cells (Zhao et al., 2017). The combination of tBHP plus
85
cetyltrimethyl ammonium bromide and a tetra-amido macrocyclic ligand is effective in
86
killing Bacillus subtilis spores (Paul et al., 2007). While the research of amoebicdal
87
effects of H2O2 against Acanthamoeba is progressing well, the effect of tBHP in
88
Acanthamoeba remains unknown.
89
Histone deacetylase (HDAC) inhibitors are chemical compounds that inhibit
90
histone deacetylases, and they have shown to induce cell cycle arrest, differentiation, or
91
apoptosis in tumor cells (Dokmanovic and Marks, 2005; Mei et al., 2004). Vorinostat is
92
a one of the numerous types of HDAC inhibitor and their effects on cell growth,
93
differentiation, and apoptosis have been reported (Bubna, 2015). Its application as a
94
monotherapeutic agent or in combination with other drugs have been documented.
95
In this study, we demonstrated the amoebicidal effect and its mechanism in
96
tBHP compared to that of H2O2. Furthermore, we investigated the effect of tBHP
４
97
combined with vorinostat to Acanthamoeba trophozoites and HCE cells.
98
５
99 100
MATERIALS AND METHODS Cell cultures
101
Acanthamoeba castellanii Castellani was obtained from the American Type
102
Culture Collection (ATCC 30868). Acanthamoeba trophozoites were cultured axenically
103
in PYG medium (20 g proteose peptone, 1 g yeast extract, 0.1M glucose, 4 mM MgSO4,
104
0.4 mM CaCl2, 3.4 mM sodium citrate, 0.05 mM Fe(NH4)2(SO4)2, 2.5 mM Na2HPO4,
105
and 2.5 mM K2HPO4 in 1 liter of distilled water with the final pH adjusted to 6.5) at
106
25°C incubator. Human corneal epithelial (HCE) cells (ATCC PCS-700-010) were
107
cultured in endothelial cell growth medium kits (KGM BulletKit; Lonza, Portsmouth,
108
NH, USA) in a humidified incubator containing 5% CO2 at 37°C.
109
110
Amoebicidal effects on Acanthamoeba
111
Acanthamoeba trophozoites were seeded into 96-well culture plates at a density
112
of 1 × 104 cells/well. To determine the effect of H2O2 (12.5, 25, 50, 100, and 200 µM)
113
tBHP (12.5, 25, 50, 100, and 200 µM), vosinostat (0.5, 1, and 10 µM), combination of
114
H2O2 and 10 µM vorinostat, and combination of tBHP and 10 µM vorinostat,
115
monolayers of amoebae were incubated with aforementioned reagents for 24 h at 25°C.
116
After incubation, 10 µl of cell counting kit-8 (CCK-8) was added to each well and the
117
plate was incubated for 6 h at 25°C. The sample absorbance was measured at the 450
118
nm wavelength against blank which contained medium only. The cell viability was
119
calculated according to the following formula: (%) = (OD of sample - OD of
120
blank)/(OD of control - OD of blank) × 100%. All assays were performed in triplicate.
６
121
122
Cytopathic effects (CPE) on human corneal cells
123
HCE cells were seeded into 96-well culture plates at a density of 1 × 104
124
cells/well. The next day, HCE cell monolayers were incubated with H2O2 (50 and 500
125
µM), tBHP (50 and 500 µM), vorinostat (10 µM), combination of 50 µM H2O2 and 10
126
µM vorinostat, or combination of 50 µM tBHP and 10 µM vorinostat at 37°C in 5%
127
CO2 for 24 h. After incubation, CPE on HCE cells were assessed using the
128
aforementioned CCK-8. All assays were performed in triplicate.
129
130
Detection of apoptosis and cell cycle arrest
131
Apoptotic cell death was detected using FITC Annexin V Apoptosis Detection
132
Kit (BD Bioscience, San Jose, California, United States). All experiments were
133
conducted following the manufacturer's protocol. Briefly, A. castellanii trophozoites
134
were seeded into 6-well culture plates at a density of 5×105 cells per well. After
135
overnight culture, monolayers of amoebae were incubated with 0.0002% PHMB, 50 µM
136
H2O2, combination of 50 µM H2O2 and 10 µM vorinostat, 50 µM tBHP, or combination
137
of 50 µM tBHP and 10 µM vorinostat for 24 h at 25°C. Amoebae were washed with
138
PBS to remove excess drug, and re-suspended in 1x binding buffer at a concentration of
139
1×106 cells/ml. Amoebae (1×105 cells/100 µl) were transferred to a new tube and
140
incubated with 5 µl of FITC annexin V and 5 µl of propidium iodide (PI) for 15 min at
141
RT in the dark. Finally, 400 µl of 1x binding buffer was added, and cells were analyzed
142
by a fluorescent microscope (Leica DMi8, Wetzlar, Germany) and flow cytometry using
143
BD Accuri C6. To investigate the cell cycle arrest, amoebae were treated as above for
７
144
apoptosis assessment. Samples were fixed with 70% ethanol for 30 min, and washed
145
twice with PBS. Afterwards, cells were treated with ribonuclease (final conc. 10 µg/ml)
146
for 10 min at 37°C, and stained with PI for 30 min at RT in the dark. Stained cells were
147
analyzed by flow cytometry using BD Accuri C6.
148 149
Statistical analysis
150
Data are presented as mean ± SD from three independent experiments.
151
Statistical analysis was performed using Student's t-test. A p value of < 0.05 was
152
considered statistically significant.
153
８
154
RESULTS
155
H2O2 and tBHP showed amoebicidal effect on Acanthamoeba trophozoites
156
Cell viability assay using CCK-8 showed that H2O2 decreased Acanthamoeba
157
viability in a dose dependent manner (Fig. 1A). However, 50 µM of H2O2 that showed
158
amoebicidal effect also demonstrated 34.98% of cytotoxicity against HCE cells (Fig.
159
1B). Amoebicidal effects of tBHP were noticeable at 50, 100, and 200 µM (Fig 1C).
160
Although miniscule levels of amoebicidal effect were induced by 50 µM tBHP, its
161
cytotoxicity to HCE cells were significantly low (Fig 1D). Neither Acanthamoeba nor
162
HCE cell viabilities were affected by vorinostat concentrations of 0.1 ~ 10 µM (Fig. 1E
163
and F).
164
165
Vorinostat increased the amoebicidal effects of H2O2 and tBHP
166
To increase the amoebidical effects of H2O2 and tBHP, 10 µM vorinostat was
167
combined with them. Upon addition of vorinostat, morphological changes were
168
observed in Acanthamoeba treated with 50 ~ 200 µM H2O2 or tBHP (Fig. 2B and D).
169
Cell viability assay confirmed increased amoebicidal effects at low concentrations of
170
H2O2 and tBHP when vorinostat was combined (Fig. 2E and F). Cyst formation was not
171
observed from these samples. Compared to single treatment of H2O2 and tBHP, the
172
combination of 50 µM H2O2 with 10 µM vorinostat showed increased amoebicidal
173
effect from 25.12% to 34.89%, and the combination of 50 µM tBHP with 10 µM
174
vorinostat showed drastically increased amoebicidal effects from 2.63% to 34.58% (Fig.
175
2G and H). These results suggest that vorinostat enhanced the amoebicidal effects of
９
176
low concentration of H2O2 and tBHP.
177 178
H2O2 induced apoptosis in Acanthamoeba
179
To investigate the effects of two combinations on Acanthamoeba, apoptosis
180
analysis was performed (Fig. 3). Acanthamoeba treated with 50 µM H2O2 induced
181
20.7% apoptosis (Fig. 3B), and the combination of 50 µM H2O2 with 10 µM vorinostat
182
induced 18.3% apoptosis (Fig. 3C), while untreated control cells showed 13.2%
183
apoptosis (Fig. 3A). The 50 µM tBHP showed 8.1% apoptotic cell death, and the
184
combination of 50 µM tBHP with 10 µM vorinostat showed 9.0% apoptosis in
185
Acanthamoeba (Fig. 3D and E). The addition of vorinostat did not affect apoptosis by
186
H2O2 and tBHP. These results suggest that 50 µM H2O2 induced apoptosis in
187
Acanthamoeba, whereas 50 µM tBHP did not induce apoptotic cell death in
188
Acanthamoeba.
189 190
tBHP combined with vorinostat arrested the cell cycle of Acanthamoeba
191
To determine whether these combinations arrest the cell cycle of
192
Acanthamoeba, flow cytometry was performed (Fig. 4). We divided the DNA
193
replication cycle of Acanthamoeba into three segments: M1, M2, and M3 (Fig. 4).
194
Because Acanthamoeba trophozoites in this study were examined in asynchronous
195
culture, majority of the amoebae were in a single phase of the cycle. We supposed that
196
M2 segment corresponded to G2 phase, which were set to be approximately 90%.
197
Population changes and cell distribution among the three segments were investigated
198
after treatment with the four aforementioned combinations. Acanthamoeba exposed to
１０
199
50 µM H2O2 showed reduced M2, and increased M1 and M3 population compared to
200
control (Fig. 4B). The addition of 10 µM vorinostat to H2O2 increased M3 population
201
(Fig. 4C). Acanthamoeba treated with 50 µM tBHP alone revealed reduced M2 and M3,
202
but increased M1 populations compared to control cells (Fig. 4D). Combining 50 µM
203
tBHP with 10 µM vorinostat significantly increased M1 population, whereas M3
204
population was reduced (Fig. 4E). These findings suggested that H2O2 combined with
205
vorinostat induced cell cycle arrest at G2/M phase (Fig. 4C, M3 segment), and tBHP
206
combined with vorinostat induced cell cycle arrest at G1 phase (Fig. 4E, M1 segment).
207 208
Combining tBHP with vorinostat did not show cytotoxicity to HCE cells
209
To determine the cytotoxicity of two combinations in HCE cells, HCE cells
210
were treated with 50 µM H2O2, 50 µM H2O2 combined with 10 µM vorinostat, 50 µM
211
tBHP, and 50 µM tBHP combined with 10 µM vorinostat (Fig. 5). The combination of
212
50 µM H2O2 and 10 µM vorinostat showed 36.26% cytotoxicity to HCE cells, which
213
was similar level to that of 50 µM H2O2 (Fig. 5A). The combination of 50 µM tBHP and
214
10 µM vorinostat showed slight cytotoxicity (6.98%) to HCE cells (Fig. 5B). The
215
addition of 10 µM vorinostat did not increase the cytotoxicity by 50 µM tBHP, and this
216
combination was considered to be non-toxic to HCE cells.
217
１１
218
DISCUSSION
219
In this study, we report that oxidant tBHP has amoebicidal effect on
220
Acanthamoeba trophozoite. The combination of low concentration of tBHP (50 µM) and
221
vorinostat (10 µM) showed high amoebicidal effect on Acanthamoeba, and low
222
cytotoxicity on HCE cells (Fig. 2 and 5).
223
Although
vorinostat
alone have
not
shown
amoebicidal
effect
on
224
Acanthamoeba, it showed strong amoebicidal effect when combined with tBHP or H2O2.
225
However, there were no significant differences among different concentrations of H2O2
226
or tBHP (Fig. 2E and F). A 3% H2O2 solution can effectively disinfect soft contact
227
lenses (Gasset et al., 1975), but H2O2 is widely regarded as a cytotoxic agent whose
228
levels must be minimized (Halliwell et al., 2000). In this study, to reduce the
229
cytotoxicity of H2O2, the concentration was diluted to 50 µM. One mole solution of
230
H2O2 is equivalent to 3.4% H2O2. By addition of 10 µM of vorinostat to 50 µM H2O2,
231
the amoebicidal effect was increased (Fig. 2).
232
The actual mechanism of action for the combined treatments remains unclear.
233
H2O2 induced apoptosis in Acanthamoeba, while tBHP did not induce apoptotic cell
234
death (Fig. 3). We speculate that the concentration of tBHP (50 µM) was too low to
235
induce apoptosis of Acanthamoeba (Fig. 1C and 3D). Spector et al. found that H2O2-
236
induced cell death was related to the extensive DNA damage caused by peroxide, and
237
tBHP under similar conditions caused little DNA damage (Spector et al., 2002). In line
238
with this notion, results acquired from the present study also showed a pattern similar to
239
this. The addition of vorinostat did not affect induction of apoptosis.
240
The DNA replication cycle of Acanthamoeba has been examined. In synchrony
１２
241
cultured Acanthamoeba, trophozoites appeared to consist of 37-47% G1 (presynthetic
242
gap), 13% S (synthesis), and 40-50% G2 (postsynthetic gap) plus M (mitosis) (Byers et
243
al., 1991). In asynchronous cultures of Acanthamoeba, the cycle appeared to consist of
244
at least 90% G2, essentially no G1, and 8-10% M plus S (Byers et al., 1991).
245
Acanthamoeba treated with 50 µM H2O2 showed increased G1/S and M (Fig. 4B -
246
segments M1 and M3) compared to untreated control cells. After addition of 10 µM
247
vorinostat, M was increased (Fig. 4C - segment M3). Acanthamoeba treated with 50 µM
248
tBHP showed increased G1/S (Fig. 4D - segment M1). After addition of 10 µM
249
vorinostat, G1/S was increased further and M was decreased (Fig. 4E - segments M1
250
and M3). Vorinostat induces cell-cycle arrest at the G1 or the G2/M boundary in a cell-
251
type dependent and/or dose-related fashion (Richon et al., 2009). In Acanthamoeba,
252
vorinostat with H2O2 induced cell cycle arrest at the G2M boundary, and vorinostat with
253
tBHP induced cell cycle arrest at the G1 phase. These results suggest that vorinostat
254
combined with H2O2 or tBHP increased amoebicidal effect on Acanthamoeba by cell
255
cycle arrest. Prospective studies investigating the effects of these combinations on
256
Acanthamoeba cyst and various clinical strains would be of great value.
257
The low concentration of tBHP (50 µM) combined with 10 µM vorinostat did
258
not show cytotoxicity, while 50 µM H2O2 combined with 10 µM vorinostat showed
259
cytotoxicity to HCE cells (Fig. 5). Although organic peroxides are widely used in
260
various oxidation processes, tBHP has been documented to be a better alternative for
261
H2O2 and has been frequently utilized in oxidative stress studies (15). The effect of
262
tBHP combined with vorinostat on Acanthamoeba will provide important information to
263
develop safe and optimized contact lens care system and treatment for Acanthamoeba
１３
264
keratitis.
265
１４
266
267 268
ACKNOWLEDGEMENT This work was supported by grants from the National Research Foundation of Korea (NRF) (2016R1D1A1B03933863, and 2018R1A6A1A03025124).
269
１５
270
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contributed to a reduced incidence of Acanthamoeba keratitis in contact lens
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wearers? A review. Cont Lens Anterior Eye 21:89-92.
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Zhao W, Feng H, Sun W, Liu K, Lu JJ, Chen X. 2017. Tert-butyl hydroperoxide (t-
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BHP) induced apoptosis and necroptosis in endothelial cells: Roles of NOX4 and
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mitochondrion. Redox Biol 11:524-534.
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FIGURE LEGENDS
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Figure 1. Amoebicidal effect and cytotoxicity of H2O2, tBHP and vorinostat. To
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investigate the amoebicidal effect of H2O2, tBHP and vorinostat on Acanthamoeba,
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Acanthamoeba trophozoites were treated with 12.5 ~ 200 µM H2O2 (A), 12.5 ~ 200 µM
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tBHP (C) or 0.1 ~ 10 µM vorinostat (E) for 24 h. To determine the cytotoxicity of H2O2,
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tBHP and vorinostat to HCE cells, HCE cells were treated with 50 and 500 µM H2O2
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(B), 50 and 500 µM tBHP (D) or 10 µM vorinostat (F) for 24 h. *, **: Asterisks denote
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statistically significant differences (*P<0.05 and **P<0.01) between untreated cells
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(control) and each agent-treated cells
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Figure 2. Amoebicidal effects of H2O2 and tBHP combined with vorinostat. To enhance
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the amoebicidal effect of H2O2 and tBHP, 10 µM vorinostat was combined with them.
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Acanthamoeba were treated with 50 ~ 200 µM H2O2 (A), 50 ~ 200 µM H2O2 with 10
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µM vorinostat (B), 50 ~ 200 µM tBHP (C), or 50~200 µM tBHP with 10 µM vorinostat
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(D) for 24 h. After incubation, morphological changes were observed under the
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microscope (400x) (A ~ D). Panels (E) and (F) summarizes the amoebicidal effects
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observed in (B) and (D). The amoebicidal effects of the combinations (H2O2 with
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vorinostat, and tBHP with vorinostat) were compared to H2O2 or tBHP treatment alone
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(G and H). **: Asterisks denote statistically significant differences (**P<0.01) between
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control and cells treated with the three combinations.
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Figure 3. Detection of apoptosis. To measure apoptotic cell death of Acanthamoeba,
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Acanthamoeba trophozoites were treated with 50 µM H2O2 (B), 50 µM H2O2 with 10
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µM vorinostat (C), 50 µM tBHP (D), or 50 µM tBHP with 10 µM vorinostat (E) for 24
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h. Untreated cells were used as normal control (A). Cells were subsequently stained
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with annexin V and propidium iodide (PI), and these cells were assessed using flow
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cytometry. The upper right quadrant (Q2-UR) in each flow cytometry plot indicates
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apoptosis.
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Figure 4. DNA and cell cycle analysis. To analyze the cell cycle of Acanthamoeba,
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asynchronous cultured Acanthamoeba was stained with PI, and were subjected to flow
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cytometry (A). DNA replication cycle of Acanthamoeba was divided into three
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segments: M1, M2, and M3. After treatment of 50 µM H2O2 (B), 50 µM H2O2 with 10
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µM vorinostat (C), 50 µM tBHP (D), or 50 µM tBHP with 10 µM vorinostat (E) for 24
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h, the percentage of cells with fractional DNA content was estimated.
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Figure 5. Cytotoxicity of the two combinations to HCE cells. To evaluate the
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cytotoxicity of two combinations to HCE cells, HCE cells were treated with 50 µM
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H2O2, 50 µM H2O2 with 10 µM vorinostat, 50 µM tBHP, and 50 µM tBHP with 10 µM
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vorinostat for 24 h (A and B). The cytotoxicity of the combinations were compared to
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those of control (untreated) and cells receiving single treatment. *, **: Asterisks denote
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statistically significant differences (*P<0.05 and **P<0.01) between control and each
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treated cells.
２０
▪ The combination of 50 µM tBHP and 10 µM vorinostat showed high amoebicidal
effect on Acanthamoeba, and low cytotoxicity on HCE cells. ▪ Vorinostat combined with H2O2 or tBHP increased amoebicidal effect on Acanthamoeba by cell cycle arrest. ▪ The effect of tBHP combined with vorinostat on Acanthamoeba will provide important information to develop safe and optimized contact lens care system and treatment for Acanthamoeba keratitis.