Juglone induces cell death of Acanthamoeba through increased production of reactive oxygen species

Accepted Manuscript Juglone induces cell death of Acanthamoeba through increased production of reactive oxygen species Bijay Kumar Jha, Hui-Jung Jung, Incheol Seo, Seong-Il Suh, Min-Ho Suh, Won-Ki Baek PII:

S0014-4894(15)30038-2

DOI:

10.1016/j.exppara.2015.09.005

Reference:

YEXPR 7128

To appear in:

Experimental Parasitology

Received Date: 30 January 2015 Revised Date:

15 June 2015

Accepted Date: 3 September 2015

Please cite this article as: Jha, B.K., Jung, H.-J., Seo, I., Suh, S.-I., Suh, M.-H., Baek, W.-K., Juglone induces cell death of Acanthamoeba through increased production of reactive oxygen species, Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.09.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

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Juglone induces cell death of Acanthamoeba through increased production of reactive

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oxygen species

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Bijay Kumar Jha1, Hui-Jung Jung1, Incheol Seo1, Seong-Il Suh1, Min-Ho Suh1, and Won-Ki

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Baek1*

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Korea

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Department of Microbiology, Keimyung University School of Medicine, Daegu, Republic of

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*Corresponding authors. Mailing address: Department of Microbiology, Keimyung University

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School of Medicine, 1095 Dalgubeol-daero, Dalseo-gu, Daegu 704-701, Republic of Korea.

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Phone: +82 53 580 3843. Fax: +82 53 580 3788. E-mail: [email protected]

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Abstract

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Juglone (5-hydroxy-1,4-naphthoquinone) is a major chemical constituent of Juglans

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mandshruica Maxim. Recent studies have demonstrated that juglone exhibits anti-cancer, anti-

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bacterial, anti-viral, and anti-parasitic properties. However, its effect against Acanthamoeba has

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not been defined yet. The aim of this study was to investigate the effect of juglone on

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Acanthamoeba. We demonstrate that juglone significantly inhibits the growth of Acanthamoeba

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castellanii at 3-5 µM concentrations. Juglone increased the production of reactive oxygen species

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(ROS) and caused cell death of A. castellanii. Inhibition of ROS by antioxidant N-acetyl-L-

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cysteine (NAC) restored the cell viability. Furthermore, our results show that juglone increased

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the uptake of mitochondrial specific dye. Collectively, these results indicate that ROS played a

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significant role in the juglone-induced cell death of Acanthamoeba.

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Keywords: Acanthamoeba, Juglone, Reactive oxygen species

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1. Introduction Acanthamoeba is a free living amoeba widely distributed in the environment that can cause

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granulomatous amoebic encephalitis (GAE) and Acanthamoeba keratitis (AK) (Marciano-Cabral

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and Cabral, 2003). GAE is a fatal infection of the brain and spinal cord that occurs among

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immunocompromised persons who are infected with Acanthamoeba whereas AK is the serious

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and rare infection of the cornea that occurs among contact lens wearers. The life cycle of

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Acanthamoeba includes trophozoite and cyst stages, and trophozoites are the infective form to

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cause diseases. The genus Acanthamoeba is classified into 17 genotypes (T1-T17), and most of

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the infections are caused by genotype T4 (Booton et al., 2005, Siddiqui and Khan, 2012).

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Infections caused by Acanthamoeba have increased over the past few years. The infection caused

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by Acanthamoeba is increasing over the last few years. This increasing prevalence of infection is

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due to the increased user of contact lens wearers and better diagnostic approaches (Lorenzo-

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Morales et al., 2013). The treatment of Acanthamoeba keratitis includes the combination therapy

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of chlorhexidine (0.02%) and polyhexamethylene biguanide (PHMB 0.02%) (Clarke et al., 2012).

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Chlorhexidine is effective against cysts and trophozoites whereas PHMB has shown to be more

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effective against trophozoites (Kumar and Lloyd, 2002). Voriconazole and miltefosine

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combination therapy has been recently used to treat GAE (Webster et al., 2012). An

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immunocompromised patient with GAE was successfully treated with the combination of

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surgical excision of the lesion and the administration of sulphadiazine and fluconazole (Seijo

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Martinez et al., 2000).

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Juglone (5-hydroxy-1,4-naphtoquinone), a major constituent of Juglans mandshruica Maxim, has

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shown a strong anticancer activity against cancer cells in vitro (Paulsen and Ljungman, 2005,

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Zhang et al., 2012). It has been shown that juglone has multiple effects on cells such as DNA

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damage, inhibition of transcription and induction of cell death (Paulsen and Ljungman, 2005).

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Juglone selectively inhibits the enzymatic activity of peptidyl-prolyl cis/trans isomerase NIMA

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interacting 1 (Pin1), a homologous of yeast ESS1, and also blocks the transcription by RNA

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polymerase II (Chao et al., 2001). It has been reported that ESS1, an essential yeast PPIase, has

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important role in the growth of different kinds of fungus (Devasahayam et al., 2002, Hanes et al.,

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1989, Joseph et al., 2004).

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Recent studies suggest that juglone cause cell death through mitochondrial participation along

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with the production of increased reactive oxygen species (ROS) and reduced glutathione (GSH)

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depletion (Inbaraj and Chignell, 2004, Ji et al., 2011, Seshadri et al., 2011). Reactive oxygen

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species are the metabolic by-products of aerobic metabolism which regulate cell death and

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proliferation. Moreover, mitochondrial NADH dehydrogenase subunit 1 (ND1) is the first

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enzyme (Complex I) of the mitochondrial electron transport chain that involves in ROS

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production. Alteration in electron transport chain resulting from mitochondrial dysfunction

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associates with the production of excessive amount of ROS and subsequent cell death. Here, we

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show that juglone treatment caused concentration-dependent toxicity in Acanthamoeba through

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generation of ROS.

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2. Materials and methods 2.1. Acanthamoeba culture

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A. castellanii was purchased from the American Type Culture Collection (ATCC 30011;

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Manassas, VA, USA). Trophozoites of A. castellanii were inoculated in PYG media (20 g/liter

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proteose peptone, 1 g/liter yeast extract, 0.1 M glucose, 4 mM MgSO4, 0.4 mM CaCl2, 3.4 mM

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sodium citrate, 0.05 mM Fe (NH4)2(SO4)2, and 2.5 mM each of Na2HPO4 and KH2PO4; PH of the

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media was adjusted to 6.5), and incubated at 25°C. Fresh PYG media was changed before the

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day of the experiment to collect more than 95% trophozoites.

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2.2. Measurement of cell viability

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Cell death was analyzed by measuring the permeability of the plasma membrane to trypan blue.

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A. castellanii trophozoites at 4 × 105 cells were seeded in 6-well tissue culture plates containing 2

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ml PYG medium. After overnight incubation, the cells were treated with different concentrations

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of juglone (1, 2, 3, 4, and 5 µM) in PYG media, and incubated for 6 h at 25°C. Cells were

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collected and centrifuged at 1000 × g for 5 min. Supernatant was removed and cell pellet was

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resuspended in phosphate buffered saline (PBS). Then, cells were stained with trypan blue with a

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final concentration of 0.2% for 10 min at room temperature. Stained cells were examined under

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microscope using Zeiss Axiovert 25 inverted microscope.

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2.3.Measurement of lactate dehydrogenase (LDH)

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Loss of cellular membrane integrity was determined by measuring the enzymatic activity of

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lactate dehydrogenase (LDH) released from the cells to the medium using LDH cytotoxicity

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detection kit (Takara, MK401, Otsu, Japan). Trophozoites at 4 × 105 cells were seeded in 6-well 5

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tissue culture plates containing 2 ml PYG medium. Juglone at different concentrations were

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treated for 6 h. Cell suspensions were centrifuged at 1000 × g for 5 min. Culture supernatant was

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collected, and LDH released into the medium was determined according to the manufacturer’s

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instructions. Absorbance was measured using Victor3 Wallac ELISA microplate reader

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(PerkinElmer Life and Analytical Sciences, Boston, MA, USA) at 490 nm. Cells of the positive

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control were treated with 0.1% Triton X-100.

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2.4.Measurement of ROS

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Intracellular generation of ROS was measured using DCFDA (which is oxidized by ROS to

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DCF). Approximately 4 × 105 A. castellanii trophozoites were cultured in 6-well plate for 24 h.

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The cells were treated with PYG media containing juglone for 6 h. Cells were centrifuged and

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washed twice with 1 ml PBS in an Eppendorf tube. The cells were treated with DCFDA (10 µM

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final concentration) for 30 min at room temperature. The cells were centrifuged and washed once

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with PBS. The cell pellets were resuspended with PBS and the cell suspensions were transferred

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into FALCON FACS tubes. The fluorescence emission from DCF was analyzed using a BD

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FACSCantoTMII flow cytometer using excitation at 488 nm and emission at 520 nm. The data

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were analyzed with BD FACSDiva software 6.0 (BD Biosciences, San Diego, CA, USA).

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2.5. Flow cytometry analysis for the uptake of mitotracker dye

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Approximately 4 × 105 A. castellanii trophozoites were cultured in 6-well plate for 24 h. The

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cells were treated with PYG media containing juglone for 6 h. Cells were centrifuged and

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washed twice with 1 ml PBS in an Eppendorf tube. Cells were resuspended in PBS, and stained

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with 1 µM MitoTracker Orange CMTMRos (Molecular probe, Invitrogen) for 15 min at 25°C.

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Stained cells were washed twice with PBS and sedimented, and the pellet was resuspended in 1 6

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ml of PBS. The cell suspension was transferred to a FALCON FACS tube and changes in

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fluorescence intensity were analyzed with a BD FACSCantoTMII flow cytometer using excitation

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at 578 nm and emission at 599 nm. The data were analyzed with BD FACSDiva software 6.0

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(BD Biosciences, San Diego, CA, USA).

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2.6.Statistical analysis

The data were expressed as mean ± S.D. Statistical analysis was performed by using Student’s t-

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test (two-tailed). P < 0.05 was considered statistically significant.

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3. Results 3.1. Juglone causes cytotoxicity on Acanthamoeba trophozoites

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Juglone has been reported to have anti-microbial and anti-cancer effects of various cells (Fischer

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et al., 2012, Ji et al., 2011, Noumi et al., 2010, Xu et al., 2012). However, the effect of juglone in

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Acanthamoeba has not been known yet. To investigate whether juglone shows a cytotoxic effect

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against Acanthamoeba, cell viability was measured after 1-5 µM treatment with juglone for 6 h.

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As shown in Fig. 1A and 1B, juglone induced significant cell death of Acanthamoeba. The

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vehicle control and low concentrations of juglone (1 and 2 µM) did not induce cell death.

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However, incubation with juglone at higher concentrations (3, 4, and 5 µM) displayed a dose-

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dependent cell death (46%, 93%, and 96%, respectively). We checked the effect of Juglone in

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time dependent manner. As 5 µM Juglone caused extensive cell death, 3 µM of Juglone was

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treated to Acanthamoeba for various time periods (3, 6, and 12h), and cell viability was

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determined. As shown in Fig. 2A, increased cell death was observed as the time of Juglone

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treatment was increased. Again, we attempted to show that Juglone caused irreversible cell death

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by incubating Juglone-treated cells in PYG media for 24 h, assuming that further incubation in

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Juglone-free PYG media could revive the cells if there is no actual cell death by Juglone. As

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shown in Fig. 2B, incubation of Juglone-treated cells could not grow in PYG media, suggesting

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that Juglone caused actual cell death. To observe the relative cytotoxicity of Juglone, we treated

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mammalian cells with Juglone. As Acanthamoeba is the causative agent of granulomatous

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amoebic encephalitis, we selected human primary glioblastoma cell line U87MG to evaluate

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mammalian cell cytotoxicity. As shown in Fig. 2C, the cytotoxicity of U87MG cells was found

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much lower than the cytotoxicity of Acanthamoeba (selectivity index = 8.6).

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3.2. Lactate dehydrogenase (LDH) activity was increased after juglone treatment

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It has been reported that trypan blue can enter inside viable cells due to the increased membrane

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permeability (Tran et al., 2011). To show that the juglone-mediated cell death is actual cell death

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and not due to the increased membrane permeability, we performed LDH assay in the culture

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supernatant. Intracellular release of LDH to culture medium is an indicator of irreversible cell

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death. Acanthamoeba were grown in PYG media along with various concentrations of juglone

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for 6 h and the activity of released LDH in culture medium was determined. Elevated LDH

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activity was observed at concentrations of 2, 3, 4, and 5 µM significantly in the culture medium

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(Fig. 3). Taken together with Fig.1, 2A and 2B, these data indicate that juglone has cytotoxic

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effects on Acanthamoeba.

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3.3.Juglone increased intracellular generation of ROS

To get a closure insight into the mechanism of the cell death induced by juglone, we assayed

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ROS production. We measured intracellular ROS through the quantification of fluorescence

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intensity of dichlorofluorescein (DCF). Since high concentration of juglone causes rapid cell

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death that could affect the measurement of ROS, we selected 2 µM concentration of juglone to

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quantify intracellular ROS. As shown in Fig. 4A and 4B, flow cytometric data revealed that

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juglone at 2 µM caused increased ROS generation after 6 h treatment in comparison with control.

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3.4.NAC protected the cells against juglone-induced cell death

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To determine whether the increase of ROS production is a cause of juglone-induced cell death,

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we first examined whether juglone-induced ROS is depleted by the treatment of NAC. As shown

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in Fig. 4C, NAC treated cells decreased the amount of ROS which was similar to the level with

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control. Next, we pretreated cells with 5 mM of NAC for 1 h followed by juglone at 1-5 µM for

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6 h. As shown in Fig. 5, treatment of juglone increased cell death dose-dependently, and the co-

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treatment of NAC rescued the cells against juglone-induced cell death. These results suggest that

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ROS is a major cause of juglone-induced cell death.

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3.5.Juglone induced mitochondrial alteration

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We sought to determine whether juglone affects the mitochondrial membrane potential. We used

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the mitochondrial membrane potential-sensitive dye MitoTracker Orange CMTMRos to stain the

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mitochondria of juglone untreated and treated cells. The uptake of MitoTracker Orange was used

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to measure mitochondrial membrane potential (Kessel, 2014). This probe irreversibly stains

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mitochondria and the uptake is more after loss of mitochondrial membrane potential. As shown

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in Fig. 6, the uptake of MitoTracker Orange was high in juglone-treated cells. Collectively, these

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results suggest that increased ROS concentration could be related with the dysfunction of

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mitochondria in juglone-treated cells.

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4. Discussion Juglone has recently been gaining importance because of its pharmacological activity. Recent

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studies revealed that juglone has antimicrobial properties against different kinds of bacteria

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including Streptococcus mutans, Streptococcus sanguis, Porphyromonas gingivalis, and

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Prevotella intermedia (Cai et al., 2000). It is also been reported that juglone contains

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antileishmanial activity and inhibits the growth of promastigotes (Serakta et al., 2013). The

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present study is the first report to demonstrate that juglone causes significant cell death of

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Acanthamoeba trophozoites. Acanthamoeba is widely present in the environment such as soil,

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water source (lakes, river, and ponds), and air. Several disinfectants including sodium

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hypochlorite, ethanol, hydrogen peroxide and peracetic acid

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trophozoites of Acanthamoeba (Coulon et al., 2010). The observed effects of juglone in our

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study suggest the use of juglone as an alternative disinfectant in the health care settings as it does

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not induce encystation (data not shown).

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Previous studies have shown that juglone induces ROS generation in human peripheral blood

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lymphocytes and leukemia cells (Seshadri et al., 2011, Xu et al., 2012). The result of our study is

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also similar with the previous studies showing that juglone induced increased production of ROS

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in Acanthamoeba. Moreover, ROS is involved as a major cause of cell death in juglone-treated

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cell, as NAC pretreatment protected the cells from oxidative cell death. Excessive production of

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ROS such as superoxide radical, hydrogen peroxide, and hydroxyl radical causes oxidative stress

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and ultimately cell death (Fleury et al., 2002). Therefore, our study suggests that juglone-

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mediated cell death of Acanthamoeba is due to the increased production of ROS. It has been

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demonstrated that juglone undergoes one-electron reduction and form semiquinone through the

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enzymes such as microsomal NADPH-cytochrome P450 reductase, microsomal NADH-

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have been used to kill the

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cytochrome b5 reductase, and NADH-ubiquinone oxidoreductase (Monks et al., 1992). These

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semiquinones of juglone react with molecular oxygen to produce superoxide (O2 ̄) and thereby

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hydrogen peroxide (H2O2), which may be the source of ROS in the juglone-treated

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Acanthamoeba.

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Recent studies have revealed several mitochondrial functions of Acanthamoeba. The

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mitochondria of Acanthamoeba consist of plant type respiratory chain and contain external and

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internal NADH dehydrogenases (Antos-Krzeminska and Jarmuszkiewicz, 2014, Gawryluk et al.,

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2012). Also, uncoupling protein in Acanthamoeba maintains the mitochondrial proton gradient

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(Jarmuszkiewicz et al., 1999). ROS production in the cell depends on the mitochondrial proton

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gradient and mitochondrial membrane potential (Rousset et al., 2004). Accordingly, it is

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reasonable to suggest that the increase of ROS after juglone treatment might have linked with the

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mitochondrial proton gradient and altered electron transport chain. Therefore, further studies are

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needed to explore the mitochondrial involvement and the mechanism of ROS-dependent cell

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death of Acanthamoeba by juglone.

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Juglone is well-known for an inhibitor of peptidyl-prolyl cis/trans isomerase (PPIases) domain of

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Pin1 that accelerates the cis/trans isomerization of peptide bonds preceding prolyl residues

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(Hennig et al., 1998). Pin1 modulates the activity of protein that is phosphorylated to proline

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directed serine or threonine residue (pSer/Thr-Pro). Recent studies have shown that Pin1 and its

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yeast homologue ESS1 are essential for the survival of Candida albicans (Devasahayam et al.,

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2002), Aspergillus nidulns (Joseph et al., 2004) and Saccharomyces cerevisiae (Gemmill et al.,

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2005, Hanes et al., 1989); and has been isolated from different organisms including Neurospora

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crassa (Kops et al., 1998), Drosophila melanogaster (Maleszka et al., 1996), and Trypanosoma

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cruzi (Erben et al., 2007). Therefore, our study does not exclude the possibility of the juglon’s

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inhibitory function of Pin1 in Acanthamoeba. However, the function of Pin1 in Acanthamoeba

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has yet to be determined.

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In conclusion, we suggest that juglone could be a potential disinfectant candidate to kill

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Acanthamoeba as well as candidate for the treatment of Acanthamoeba infections. However,

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further studies are needed to understand the cidal mechanisms of juglone in Acanthamoeba and

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the use of juglone as treatment purpose.

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Acknowledgement

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This research was supported by the National Research Foundation of Korea (NRF) Grant funded

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by the Korean Government (MSIP) (No. 2014R1A5A2010008).

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Conflict of Interest

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The authors have declared that no conflict of interests exists.

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Ji, Y. B., Qu, Z. Y., and Zou, X., 2011. Juglone-induced apoptosis in human gastric cancer SGC7901 cells via the mitochondrial pathway. Experimental and Toxicologic Pathology 63, 69-78. Joseph, J. D., Daigle, S. N., and Means, A. R., 2004. PINA is essential for growth and positively influences NIMA function in Aspergillus nidulans. Journal of Biological Chemistry 279, 32373-32384. Kessel, D., 2014. Reversible effects of photodamage directed toward mitochondria. Photochemistry and Photobiology 90, 1211-1213. Kops, O., Eckerskorn, C., Hottenrott, S., Fischer, G., Mi, H., and Tropschug, M., 1998. Ssp1, a site-specific parvulin homolog from Neurospora crassa active in protein folding. Journal of Biological Chemistry 273, 31971-31976. Kumar, R. and Lloyd, D., 2002. Recent advances in the treatment of Acanthamoeba keratitis. Clinical Infectious Diseases 35, 434-441. Lorenzo-Morales, J., Martin-Navarro, C. M., Lopez-Arencibia, A., Arnalich-Montiel, F., Pinero, J. E., and Valladares, B., 2013. Acanthamoeba keratitis: an emerging disease gathering importance worldwide? Trends in Parasitology 29, 181-187. Maleszka, R., Hanes, S. D., Hackett, R. L., de Couet, H. G., and Miklos, G. L., 1996. The Drosophila melanogaster dodo (dod) gene, conserved in humans, is functionally interchangeable with the ESS1 cell division gene of Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences U S A 93, 447-451. Marciano-Cabral, F. and Cabral, G., 2003. Acanthamoeba spp. as agents of disease in humans. Clinical Microbiology Reviews 16, 273-307. Monks, T. J., Hanzlik, R. P., Cohen, G. M., Ross, D., and Graham, D. G., 1992. Quinone chemistry and toxicity. Toxicology and Applied Pharmacology 112, 2-16. Noumi, E., Snoussi, M., Hajlaoui, H., Valentin, E., and Bakhrouf, A., 2010. Antifungal properties of Salvadora persica and Juglans regia L. extracts against oral Candida strains. European Journal of Clinical Microbiology & Infectious Diseases 29, 81-88. Paulsen, M. T. and Ljungman, M., 2005. The natural toxin juglone causes degradation of p53 and induces rapid H2AX phosphorylation and cell death in human fibroblasts. Toxicology and Applied Pharmacology 209, 1-9. Rousset, S., Alves-Guerra, M. C., Mozo, J., Miroux, B., Cassard-Doulcier, A. M., Bouillaud, F., and Ricquier, D., 2004. The biology of mitochondrial uncoupling proteins. Diabetes 53 Suppl 1, S130-135. Seijo Martinez, M., Gonzalez-Mediero, G., Santiago, P., Rodriguez De Lope, A., Diz, J., Conde, C., and Visvesvara, G. S., 2000. Granulomatous amebic encephalitis in a patient with AIDS: isolation of Acanthamoeba sp. Group II from brain tissue and successful treatment with sulfadiazine and fluconazole. Journal of Clinical Microbiology 38, 3892-3895. Serakta, M., Djerrou, Z., Mansour-Djaalab, H., Kahlouche-Riachi, F., Hamimed, S., Trifa, W., Belkhiri, A., Edikra, N., and Hamdi Pacha, Y., 2013. Antileishmanial activity of some plants growing in Algeria: Juglans regia, Lawsonia inermis and Salvia officinalis. African Journal of Traditional, Complementart, and Alternative Medicines 10, 427-430. Seshadri, P., Rajaram, A., and Rajaram, R., 2011. Plumbagin and juglone induce caspase-3dependent apoptosis involving the mitochondria through ROS generation in human peripheral blood lymphocytes. Free Radical Biology & Medicine 51, 2090-2107. Siddiqui, R. and Khan, N. A., 2012. Biology and pathogenesis of Acanthamoeba. Parasites & Vectors 5, 6.

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Tran, S. L., Puhar, A., Ngo-Camus, M., and Ramarao, N., 2011. Trypan blue dye enters viable cells incubated with the pore-forming toxin HlyII of Bacillus cereus. PLoS One 6, e22876. Webster, D., Umar, I., Kolyvas, G., Bilbao, J., Guiot, M. C., Duplisea, K., Qvarnstrom, Y., and Visvesvara, G. S., 2012. Treatment of granulomatous amoebic encephalitis with voriconazole and miltefosine in an immunocompetent soldier. The American Journal of Tropical Medicine and Hygiene 87, 715-718. Xu, H. L., Yu, X. F., Qu, S. C., Qu, X. R., Jiang, Y. F., and Sui da, Y., 2012. Juglone, from Juglans mandshruica Maxim, inhibits growth and induces apoptosis in human leukemia cell HL-60 through a reactive oxygen species-dependent mechanism. Food and Chemical Toxicology 50, 590-596. Zhang, W., Liu, A., Li, Y., Zhao, X., Lv, S., Zhu, W., and Jin, Y., 2012. Anticancer activity and mechanism of juglone on human cervical carcinoma HeLa cells. Canadian Journal of Physiology and Pharmacology 90, 1553-1558.

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Figure legends

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Fig. 1. Juglone induces cytotoxicity to Acanthamoeba. (A) Acanthamoeba trophozoites were

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treated with different concentrations (1-5 µM) of juglone for 6 h, and cell viability was assessed

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by trypan blue staining. The data are shown as the mean ± SD from three separate experiments

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(*p<0.05, **p<0.005). (B) Morphology of juglone-treated cells in PYG media was observed at

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20 × magnification using Nikon Eclipse TS100 inverted microscope. The results are

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representative of three separate experiments.

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Fig. 2. Juglone shows irreversible cell death with selective toxicity to Acanthamoeba. (A)

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Acanthamoeba trophozoites were treated with juglone at 3 µM concentration for indicated time

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periods. Cell viability was calculated after staining with trypan blue staining. The data is

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expressed as mean ± SD from three separate experiments (**p<0.005). (B) Acanthamoeba were

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treated with Juglone at 5 µM concentration for 6 h, cells were washed twice with PBS, and the

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cells were suspended with 2 ml of PYG media for 24 h. Cell viability was determined with

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trypan blue staining. The data represents the mean ± SD from three independent experiments

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(**p<0.005). (C) Effect of Juglone on viability of U87MG cells. Cells were treated with

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indicated concentrations of Juglone for 24 h. Cell viability was monitored with MTS assay. Data

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represents mean ± SD of three independent experiments.

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Fig. 3. LDH released into the culture medium as a result of cytotoxicity. Cell death was analyzed

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by LDH assay after treatment with juglone at the indicated concentrations for 6 h. Data are

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means ± SD from three independent experiments. Cells treated with Triton X-100 (0.1%) were

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considered as positive control.

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Fig. 4. Juglone induces the production of reactive oxygen species. Acanthamoeba trophozoites

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were treated with juglone (2 µM) for 6 h. ROS production was evaluated by staining with DCF-

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DA, and the intensity of DCF fluorescence was quantified with the flow cytometer. Cells were

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treated with vehicles (A), 2 µM juglone alone (B), or 2 µM juglone after 1 h pretreatment of 5

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mM NAC (C).

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Fig. 5. NAC attenuates the juglone-induced cell death. Cells were pretreated with NAC (5 mM)

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for 1 h before the addition of juglone at indicated concentrations for 6 h. (A) Cell viability was

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assessed by trypan blue dye exclusion assay. Data are shown as the means ± S.E. of three

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independent experiments. (B) Morphology of juglone treated and NAC (5 mM) pretreated with

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juglone was observed at 20 × magnification using Nikon Eclipse TS100 inverted microscope.

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Fig. 6. Juglone induces mitochondrial alteration. Cells were treated with indicated concentrations

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of juglone for 6 h, and the uptake of MitoTracker Orange CMTMRos was quantified by flow

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cytometer.

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Figure legends Fig. 1. Juglone induces cytotoxicity to Acanthamoeba. (A) Acanthamoeba trophozoites were

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treated with different concentrations (1-5 µM) of juglone for 6 h, and cell viability was assessed by trypan blue staining. The data are shown as the mean ± SD from three separate experiments (*p<0.05, **p<0.005). (B) Morphology of juglone-treated cells in PYG media was observed at

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20 × magnification using Nikon Eclipse TS100 inverted microscope. The results are representative of three separate experiments.

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Fig. 2. Juglone shows irreversible cell death with selective toxicity to Acanthamoeba. (A) Acanthamoeba trophozoites were treated with juglone at 3 µM concentration for indicated time periods. Cell viability was calculated after staining with trypan blue staining. The data is expressed as mean ± SD from three separate experiments (**p<0.005). (B) Acanthamoeba were

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treated with Juglone at 5 µM concentration for 6 h, cells were washed twice with PBS, and the cells were suspended with 2 ml of PYG media for 24 h. Cell viability was determined with trypan blue staining. The data represents the mean ± SD from three independent experiments

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(**p<0.005). (C) Effect of Juglone on viability of U87MG cells. Cells were treated with indicated concentrations of Juglone for 24 h. Cell viability was monitored with MTS assay. Data

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represents mean ± SD of three independent experiments. Fig. 3. LDH released into the culture medium as a result of cytotoxicity. Cell death was analyzed by LDH assay after treatment with juglone at the indicated concentrations for 6 h. Data are means ± SD from three independent experiments. Cells treated with Triton X-100 (0.1%) were considered as positive control.

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Fig. 4. Juglone induces the production of reactive oxygen species. Acanthamoeba trophozoites were treated with juglone (2 µM) for 6 h. ROS production was evaluated by staining with DCF-

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DA, and the intensity of DCF fluorescence was quantified with the flow cytometer. Cells were treated with vehicles (A), 2 µM juglone alone (B), or 2 µM juglone after 1 h pretreatment of 5 mM NAC (C).

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Fig. 5. NAC attenuates the juglone-induced cell death. Cells were pretreated with NAC (5 mM) for 1 h before the addition of juglone at indicated concentrations for 6 h. (A) Cell viability was

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assessed by trypan blue dye exclusion assay. Data are shown as the means ± S.E. of three independent experiments. (B) Morphology of juglone treated and NAC (5 mM) pretreated with juglone was observed at 20 × magnification using Nikon Eclipse TS100 inverted microscope. Fig. 6. Juglone induces mitochondrial alteration. Cells were treated with indicated concentrations

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of juglone for 6 h, and the uptake of MitoTracker Orange CMTMRos was quantified by flow

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1. Juglone causes cell death of Acanthamoeba. 2. Juglone induces ROS production. 3. Use of antioxidant such as N-acetyl-L-cysteine (NAC) restored the cell viability.