Mezencev et al. J. Nat. Med. 2011; 65(3-4):488-499.

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The final publication is available at Springer via http://dx.doi.org/10.1007/s11418-011-0526-x

Camalexin induces apoptosis in Jurkat T-leukemia cells by increased concentration of reactive oxygen species and activation of caspases-8 and -9.

Roman Mezencev*1

, Taylor Updegrove1, Peter Kutschy

2, Mária Repovská

2, and John F.

McDonald1

1 Georgia Institute of Technology, School of Biology, 310 Ferst Dr., Atlanta, GA 30332

2 P. J. Šafárik University, Faculty of Science, Institute of Chemical Sciences, Moyzesova 11, Košice, Slovakia

* corresponding author

Abstract

Camalexin, a major indole phytoalexin of Arabidopsis thaliana, accumulates in various

cruciferous plants in response to environmental stress and reportedly displays antimicrobial

activities against various plant pathogens. However, its cytotoxicity against eukaryotic cells

and potential as a prospective drug for human diseases has been examined only in a limited

context. Our data demonstrate the time- and concentration-dependent cytotoxicity of

camalexin on human T-leukemia Jurkat cells in the micromolar range, and the lower potency

of cytotoxic effects on human lymphoblasts and primary fibroblasts. Cytotoxicity of

camalexin is enhanced by the glutathione-depleting agent buthionine sulfoximine and

completely blocked by pan caspase inhibitor Z-VAD-FMK. Treatment of Jurkat cells with

camalexin resulted in activation of caspase-8, caspase-9, caspases-3/7, and apoptosis that was

detected by the presence of a sub-G1 population of cells, externalization of phosphatidyl

serine and decreased mitochondrial membrane potential. Staining with 2',7'-

dichlorodihydrofluorescein diacetate and dihydroethidium bromide displayed increased

concentration of ROS early in camalexin-treated Jurkat cells, prior to the onset of apoptosis

while staining with MitoSOXTM

dye identified mitochondria as a source of increased ROS.

Our data suggest that this phytochemical, which has a wide range of predicted

pharmacological activities, induces apoptosis in Jurkat leukemia cells through increased ROS

followed by dissipation of mitochondrial membrane potential and execution of caspase-9-

and caspase-8-initiated apoptosis. This is, to the best of our knowledge, the first report on

antileukemic activity and mode of action of this unique indole phytoalexin.

Keywords

camalexin; leukemia; reactive oxygen species; ROS; apoptosis; phytoalexin

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Introduction

Camalexin [3-(thiazol-2-yl)-1H-indole] (Fig. 1) is an indole phytoalexin that accumulates in

various cruciferous plants, e.g. Camelina sativa, Arabidopsis thaliana, and Capsella bursa-

pastoralis, in response to plant pathogen attack or abiotic stress [1-3]. It displays

antimicrobial activity against some bacterial and fungal plant pathogens, including

Pseudomonas syringae, Alternaria brassicicola, Alternaria brassicaeae and Leptosphaeria

maculans reportedly via its cell membrane disrupting effect [4]. In addition, camalexin exerts

cytostatic/cytotoxic effects on the human protozoan pathogen, Trypanosoma cruzi [5], and on

the human breast cancer cell line SKBr3 (4-day treatment, IC50=2.7 μM) where it displays

higher potency than the traditional anticancer agents cisplatin (IC50=7.4 μM) and melphalan

(IC50=13.0 μM) [6]. In spite of the fact that camalexin as well as other phytoalexins may

impact human and animal health as constituents of conventional or enhanced animal feed or

human food [7-8], or as a lead compound for future development of structurally related drugs,

information on its effects on mammalian cells is rather limited, and it has been researched

predominantly with regard to plant chemical defensive mechanisms. For this reason, we have

further investigated the cytotoxic properties of this phytochemical on cultured human T-

leukemia Jurkat cells (cancer cell line) as well as on human lymphoblasts (immortalized cell

line) and human skin fibroblasts (non-malignant primary cell culture), and characterized its

mode of cytotoxic action.

Material and Methods

Chemicals

Camalexin (CAM) was synthesized following Ayer et al. [9] and its stock solution (40 mM in

absolute ethanol) was kept at -80 C before use. DL-buthionine-(S,R)-sulfoximine (BSO),

staurosporine (STA) and etoposide (ETO) were obtained from Sigma-Aldrich (St. Louis,

MO, USA); N-benzyloxycarbonyl-valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone (Z-

VAD-FMK) was from R&D Systems, Inc (Minneapolis, MN, USA), and tris (2-

carboxyethyl) phosphine hydrochloride (TCEP) was obtained from Thermo Fisher Scientific

Inc. (Rockford, IL, USA). Stock solutions of these compounds were prepared as follows:

BSO – 40 mM in sterile H2O; STA - 100 g/mL in DMSO; ETO – 40 mM in DMSO; Z-

VAD-FMK - 20 mM in DMSO; TCEP – 100 mM in H2O. 2',7'-dichlorodihydrofluorescein

diacetate (DCFH-DA), dihydroethidium bromide (DHE) and MitoSOXTM

were obtained from

Cayman Chemical (Ann Arbor, MI, USA), Polysciences, Inc (Warrington, PA), and

Invitrogen Corporation (Carlsbad, California, USA), respectively.

Cell cultures

Acute T-lymphoblastic leukemia Junkat cells [10], clone E6-1, were obtained from ATCC

(Manassas, Virginia, USA) and maintained in RPMI 1640 medium supplemented with fetal

bovine serum (10%), L-glutamine (2 mM), penicillin (100 IU/mL), streptomycin (100

μg/mL) and amphotericin B (0.25 μg/mL) in an atmosphere of humidified air with 5% CO2.

Human immortalized lymphoblasts GM 15851 [11] were procured from Coriell (Camden,

NJ, USA) and maintained in RPMI 1640 medium with fetal bovine serum (15%), L-

glutamine (2 mM), penicillin (100 IU/mL), streptomycin (100 μg/mL) and amphotericin B

(0.25 μg/mL) in an atmosphere of humidified air with 5% CO2. Human primary skin

fibroblasts were obtained from Coriell Cell Repositories (Camden, NJ, USA, Catalog ID

AG13153) and maintained in Eagle’s MEM supplemented with fetal bovine serum (15%), 26

mM HEPES, 2 mM L-glutamine, penicillin (100 IU/mL), streptomycin (100 μg/mL) and

amphotericin B (0.25 μg/mL) in an atmosphere of humidified air with 5% CO2. For plating

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onto 96-well plates, a single cell suspension of fibroblasts (passage 13) was prepared by

harvesting and dissociation using Accutase Cell Detachment Solution (Thermo Electron

Corporation, Louisville, CO, USA) for 10 minutes at 37C and filtration through the cell

strainer with 40 m nylon mesh.

Cytotoxicity assay

The cytostatic and cytotoxic activity of camalexin on cultured Jurkat, human lymphoblasts

and human primary fibroblast cells was evaluated using the in vitro toxicology assay kit

TOX-8 (Sigma-Aldrich, St. Louis, MO, USA) [12]. 100 µL of cell suspension were plated in

96-well black-walled plates at 100,000 cells/mL (Jurkat, human lymphoblasts) or 50,000

cells/mL (human fibroblasts) in Eagle’s MEM medium supplemented with 5% FBS, L-

glutamine (2 mM), penicillin (100 IU/mL), streptomycin (100 μg/mL) and amphotericin B

(0.25 μg/mL), and grown at 37C in humidified air with 5% CO2 for 24h. Thereafter, 100 µL

of the tested compounds dissolved in growth medium at a concentration 2-fold of the desired

final concentration were added in quadruplicates and cells were incubated for a specific time.

After that 20 μL of the TOX-8 reagent were added to each well and incubated for the next 2

hours (Jurkat cells and human fibroblasts) or 3 hours (human lymphoblasts). The increase of

fluorescence was measured at a wavelength of 590 nm using an excitation wavelength of 560

nm. The emission of control wells (no drug treatment) after the subtraction of a blank was

taken as 100% and the results for treatments were expressed as a percentage of the control.

IC50 values (concentrations of tested agents that inhibited growth of Jurkat or fibroblast cell

cultures after 72-hour incubation to 50% of the untreated control) were determined by non-

linear regression of log-transformed data using a normalized response-variable slope model

(GraphPad Prism 5.01; GraphPad Software, Inc.) and expressed as mean ± SD. Time

dependence of cytostatic/cytotoxic effects of camalexin was determined for a single

concentration of camalexin (50 M) on Jurkat cells at various incubation times. For

elucidation of the mode of action, cytotoxicity of camalexin on Jurkat cells was also tested in

combinations with BSO (-glutamylcysteine synthetase inhibitor) and Z-VAD-FMK (a cell-

permeant pan caspase inhibitor). In all cytotoxicity assays, test was performed in 4 wells for

each treatment. For each 4 treatment wells there were 4 untreated control wells in the same

column of the 96-well plate. Experiments were performed 2 times and consistency between

plates was observed.

Caspase activity

Activity of caspase-8 and caspase-9 was measured by the homogeneous Caspase-Glo 8 or

Caspase-Glo 9 Assay (Promega, Madison, WI), respectively. These initiator caspases were

determined based on cleavage of their specific luminogenic substrates, Z-LETD-

aminoluciferin (caspase-8) and Z-LEHD-aminoluciferin (caspase-9) in the presence of the

proteasome inhibitor MG-132 [13]. Activities of effector caspase-3 and caspase-7 were

measured using the homogeneous luminescent Caspase-Glo 3/7 Assay (Promega, Madison,

WI) based on caspase 3/7-catalyzed cleavage of Z-DEVD-aminoluciferin [14]. In all these

assays, 50 μL (150,000 cells/mL) of Jurkat cells in Eagle’s MEM medium supplemented with

5% FBS, L-glutamine (2 mM), penicillin (100 IU/mL), streptomycin (100 μg/mL) and

amphotericin B (0.25 μg/mL) were plated onto 96-well white-walled plates at 150,000

cells/mL and grown at 37C in humidified air with 5% CO2 for 24h. Thereafter, 50 µL of

camalexin in full medium were added so that the final concentration was 100 μM and the

plate then was incubated for the next 72 hours. Assays were performed following

manufacturer’s recommendations with following controls: wells with medium only (no-

treatment); 10 μM staurosporine (positive control for caspase-9 and caspase-3/7 activation),

and 25 μM etoposide (positive control for caspase-8) [15]. All positive controls were applied

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during the last 6 hours of incubation. Activities of caspases were determined by imaging

using the Typhoon 8600 Variable Mode Imager in chemiluminescence scan mode. For

detection of each caspase activity, camalexin treatments were performed in 4 wells; untreated

controls were performed in 2-4 wells and positive controls in 2 wells. Luminescence

corresponding to individual wells was quantified using image analysis software Science Lab

2005 Multi Gauge Ver. 3.0 (Fujifilm, USA). The experiment was performed 2 times and

consistency between results was confirmed.

Apoptosis via externalized PS

Apoptosis through detection of externalized phosphatidyl serine (PS) was determined in

Jurkat cells treated with camalexin (100 M) or solvent control by flow cytometry (BD

LSRII, BD Biosciences, San Jose, CA, USA) using Annexin V-FITC Apoptosis Detection

Kit (Calbiochem, San Diego, CA, USA). Upon induction of apoptosis, PS normally localized

in the internal part of the plasma membrane, becomes surface-exposed and available to bind

to the annexin V-FITC conjugate [16]. Early apoptotic cells are detected as Annexin V-

positive and propidium iodide (PI)-negative (FITC+/PI

-); late apoptotic and necrotic cells are

detected as FITC+/PI

+ and non-apoptotic cells as FITC

-/PI

- events. Jurkat cells were treated

with camalexin or solvent control in full RPMI 1640 medium for 8, 24 and 48 hours and the

assay was performed following the manufacturer’s rapid binding protocol. Experiments were

performed 3 times and a representative result is displayed in the form of PI vs Annexin V-

FITC fluorescence scattergrams.

Mitochondrial membrane potential

The mitochondrial respiratory chain generates transmembrane potential (ΔΨm) and its

disruption observed during apoptosis can be detected with the lipophilic cationic dye

5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). JC-1

selectively enters into mitochondria with high ΔΨm and reversibly forms J-aggregates with

intense red fluorescence, while in cells with low ΔΨm remains in monomeric form that

displays green fluorescence only [17]. Cells treated with camalexin (100 µM) or solvent

control for 8, 24 and 48 hours were processed using the Mitochondrial Apoptosis Detection

Kit (GenScript, Piscataway, NJ, USA) following manufacturer’s protocol for flow cytometric

assay format. Experiments were performed 3 times and representative results are displayed in

the form of PerCP-Cy5 vs Annexin V-FITC fluorescence scattergrams.

Cell cycle analysis

Distribution of camalexin and control-treated Jurkat cells in various phases of cell cycle was

determined based on cellular DNA content by flow cytometry [18]. Jurkat cells were cultured

in RPMI 1640 medium supplemented with 5% FBS, L-glutamine (2 mM), penicillin (100

IU/mL), streptomycin (100 μg/mL) and amphotericin B (0.25 μg/mL), at 37C in humidified

air with 5% CO2 were treated with 100 μM camalexin or solvent control for 48h. Thereafter,

cells were pelleted at 200 g for 5 minutes, washed twice with ice cold PBS, resuspended in

ice cold PBS at 2106

cells/mL, and slowly added to an equal volume of cold absolute

ethanol. After storing for 24 hours at -40C, cells were centrifuged at 200 g/10 min/4C,

washed with cold PBS and resuspended in 500 L of solution containing 0.2 mg/mL DNAse-

free RNAse A and 20 g/mL PI in a 0.1% solution of Triton-X100 in PBS. After 15 min

incubation at 37C cell cycle analysis was performed using a BD LSRII benchtop flow

cytometer (BD Biosciences). Cell cycle distribution was determined by deconvolution of

DNA content histograms, after discrimination of doublets and other cellular aggregates, by

FlowJo 7.6 software (Tree Star, Inc., Ashland, OR, USA) using the Dean-Jet-Fox (DJF)

model that fits G1 and G2 phases with Gaussian curves and S phase with a second degree

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polynomial [19]. The cell cycle analysis experiment was performed 3 times and

representative data are displayed in Figure 9A. Data summarizing cell cycle distribution in all

3 experiments are presented in Figure 9B.

Determination of cellular ROS

Intracellular levels of hydrogen peroxide and some reactive nitrogen species (nitric oxide and

peroxynitrite) in camalexin and solvent control treated cells were determined by FACS using

the fluorogenic probe DCFH-DA, which diffuses into cells where it is deacetylated by

cellular esterases and rapidly oxidized by ROS to highly fluorescent 2’,7’-

Dichlorofluorescein (DCF) [20]. Detection of intracellular superoxide was performed with

dihydroethidium bromide (DHE). DHE is specifically oxidized by superoxide to a DNA-

binding fluorophore hydroxyethidium [21-22].

In all ROS detection experiments, Jurkat cells were plated onto 6-well plates at cell densities

of 500,000 cells/mL and treated with 100 M camalexin or solvent control for 6 hours. After

the treatment, cells were pelleted, washed with PBS, resuspended in DMEM with 30 M of

DCFH-DA or DHE and incubated for 30 min in 5% CO2 at 37C. After the incubation, cells

were pelleted, washed with PBS, resuspended in PBS and analyzed by FACS using FITC or

PE channels in the BD LSRII benchtop flow cytometer. Experiments were performed 3 times

and representative results of FACS are presented in the form of fluorescence distribution

histograms.

Determination of mitochondrial superoxide

Mitochondrial superoxide is selectively detected using fluorogenic live cell-permeant

mitochondria-targeted dye MitoSOXTM

[23]. Jurkat cells were plated onto 6-well plates at

cell densities of 500,000 cells/mL and treated with 100 M camalexin or solvent control for 6

hours. Thereafter, cells were washed two times with HBBS/Ca/ Mg containing 1% BSA and

stained with 5 M solution of MitoSOX in HBBS/Ca/Mg containing 1% BSA for 10 minutes

at 37C. Stained cells were washed 3 times with warm buffer, re-suspended in the buffer,

analyzed by FACS and visualized by fluorescent microscopy.

Determination of cellular glutathione

Jurkat cells were plated at density 350,000 cells/mL in 12-well plates in DMEM/F12 medium

(glutathione-free) supplemented with 10% FBS and antibiotic/antimycotic and treated with

camalexin (100 μM) or solvent control for 12 hours. Reduced glutathione (GSH) and total

cellular glutathione (GSH+GSSG) in camalexin and solvent control-treated cells were

quantified using GSH-Glo Glutathione Assay (Promega). In this assay the luciferin derivative

Luc-NT is converted in the presence of GSH and glutathione S-transferase (GST) to luciferin

that in a coupled reaction catalyzed by firefly luciferase generates a luminescent signal. The

assay was performed following manufacturer’s instructions for suspension cell cultures. Total

cellular glutathione was determined after reduction of GSSG to GSH with TCEP (final

concentration 1 mM). Luminescence signal after subtraction of blanks (net RLU) was

normalized to the number of viable cells determined by resazurin-based cell viability assay

TOX-8 (Sigma-Aldrich). All experiments were performed in triplicates. BSO was used as a

positive control for glutathione depletion at a final concentration of 25 μM. Camalexin, BSO

and solvent control treatments were performed in 3 replicated wells and the experiment was

performed 2 times.

In silico predictions

The virtual screening tool Miscreen (Molinspiration Cheminformatics, Slovensky Grob,

Slovakia) [24] was employed to perform the ligand-based virtual screening with camalexin.

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This tool generates substructure features (fragments) from a training set of active and inactive

compounds and subsequently builds a bioactivity model using Bayesian statistics. These

models calculate an activity score for each screened molecule as a sum of the activity

contributions of its fragments. A higher score means a higher probability that the compound

is active. For this report, activity scores of camalexin as a GPCR ligand, ion channel

modulator, kinase inhibitor or nuclear receptor ligand were determined using Molinspiration

Cheminformatics models available online (http://www.molinspiration.com. Accessed

October 2010) and compared with those for average organic molecules.

Statistics

When not specified otherwise, significance of differences between means of two-class data

was tested by two-tailed Welch’s t-test. The trend between cell viability and time of cell

exposure to camalexin was determined by linear regression analysis and expressed as slope

SD and p-value of F-test for significance of difference of slope from 0. Significance of

differences among sample means in the time course cytotoxicity experiment was tested by

one-way ANOVA followed by the Tukey-Kramer procedure to test significance of

differences between selected pairs of sample means. In all tests, differences were considered

significant if p-value < 0.05.

Results

Camalexin exerts cytotoxic effects on Jurkat cells at an intermediate micromolar range and

IC50 value for 72-hour exposure equals to 52.9±2.52 μM (mean±SD) (Fig 2A). The cytotoxic

effect of camalexin increases with exposure time (Fig 3) and the trend is statistically

significant with slope 0.4560.0987 and p-value 0.0191 (the difference between 99 h and 121

h is not statistically significant). The potency of the cytotoxic effect of camalexin in human

cultured lymphoblasts is lower (IC50 = 84.27.5 M; p=0.0042) and in human primary

fibroblasts much lower (243.7±12.7 μM; p<0.0001) than that in Jurkat cells (Fig 2B and 2C).

The cytotoxicity of camalexin on Jurkat cells was increased by co-treatment with BSO at

concentrations of 50 and 100 M (Fig 4A), while Z-VAD-FMK at 30 M completely

blocked cytotoxicity of camalexin at 50 M (Fig 4B). Consistent with this finding, assays

based on cleavage of specific luminogenic substrates demonstrated activation of caspase-8,

caspase-9, and caspases -3 and -7 (Fig 5A and 5B) upon treatment of Jurkat cells with

camalexin. Activation of caspases is not sufficient evidence for apoptosis, since they also

function in other cellular processes, including T-cell proliferation and cell cycle regulation

[25]. For this reason, the role of apoptosis in camalexin-induced cytotoxicity of Jurkat cells

was further demonstrated by complementary methods. FACS analysis of Annexin V-

FITC/PI-stained cells shows that Jurkat cells treated with 100 M camalexin display early

apoptosis (meanSD: 23.93.18% of treated vs 4.801.21% of untreated cells; N=3;

p=0.0104) and late apoptosis/necrosis (meanSD: 34.13.46% of treated vs 3.001.07% of

untreated cells; N=3; p=0.0045) after 48-h (Fig 6), but not after 8-h treatment (data not

shown). In contrast, primary human fibroblasts display relatively limited apoptosis after 72-

hour treatment with 50 and 200 μM camalexin, which is consistent with their reduced

sensitivity to the cytotoxic effects of camalexin (Fig 7).

Dissipation of mitochondrial membrane potential, an early event in the apoptotic process that

precedes caspase activation and PS externalization, is detected in JC-1-stained Jurkat cells

after 24- and 48-h treatment with camalexin but not after 8-h (Fig 8) with respective

proportions of apoptotic cells 32.65.31%, 53.48.54% and 7.551.59% (meanSD; N=3).

The proportion of apoptotic cells detected by JC-1 staining in untreated controls was

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7

8.371.72 (meanSD; N=9). The proportion of cells with decreased mitochondrial potential

after 48-h treatment, shown in Figure 8 (52.9%), is consistent with the proportion of Annexin

V-FITC positive cells shown in Figure 6 (Q2 + Q3 = 55.3%), which corresponds to the

combined proportion of early and late apoptotic/necrotic cells. These data show that the

mitochondrial membrane potential is disrupted in both early apoptotic and late

apoptotic/necrotic cells resulting from camalexin treatment of Jurkat cells. Cell cycle

distribution analysis provides an additional line of evidence for apoptotic cell death in

camalexin-treated Jurkat cells. Apoptotic cells are characterized by fractional DNA content

and correspond to the sub-G1 population on the DNA content frequency distribution

histograms (Fig 9A). Our data suggest that the proportion of apoptotic (sub-G1) Jurkat cells

increased upon 48-hour treatment with 100 μM camalexin from 4.10.69% to 33.74.08%

(meanSD; N=3; p=0.0002) (Fig 9B). These values are similar to the proportion of early

apoptotic cells after 48-h treatment, identified by Annexin V-FITC and PI binding

(4.801.21% and 23.93.18%) (Fig 6). In addition, cell cycle distribution analysis reveals

that increase in proportion of apoptotic cells in camalexin-treated Jurkat cells compared to

untreated control is associated with statistically significant decrease in the proportion of S-

and G2/M-phase cells (Fig 9).

Increased concentration of ROS was found in camalexin-treated Jurkat cells after 6-h

treatment using both DCFH-DA and DHE probes (Fig 10). Staining with MitoSOX suggested

intramitochondrial generation of superoxide in Jurkat cells treated with camalexin (Fig 11).

Significant dissipation of mitochondrial membrane potential or externalization of PS were not

detected after 8-h treatment with camalexin; therefore, the increase of ROS occurred prior to

the onset of apoptosis.

Cellular reduced glutathione (GSH) and total cellular glutathione (GSH + GSSG) levels were

not decreased even after 12-hour treatment of Jurkat cells with 100 μM camalexin (Fig 12),

and GSH/(GSH+GSSG) ratio was the same for camalexin-treated and solvent (control)-

treated cells (0.894 and 0.901, respectively).

Virtual screening results demonstrate that camalexin is not likely to act as a ligand of G

protein-coupled receptors, a kinase inhibitor or a nuclear receptor ligand, while its activity as

an ion channel modulator is inconclusive (Table 1).

Discussion

Camalexin, a major indole phytoalexin of A. thaliana, previously has been investigated with

regard to its biosynthesis, regulation, antimicrobial properties and degradation by plant

pathogens [26-29]. However, its cytotoxicity against eukaryotic cells and potential as a

prospective drug for human diseases has been examined only in a limited context [5-6]. Since

camalexin might possess some therapeutic activity, including antibacterial, antifungal,

antiviral, anticancer, antidepressive, antiparkinsonic, antiallergic, antihypertensive and

analgesic, as predicted by binary QSAR models from GeneGo MetaDrug system

(http://www.genego.com/metadrug.php. Accessed October 2010) [30] we decided to further

explore its cytotoxicity on human cancer and non-cancerous cells. We found that camalexin

exhibited cytotoxic effects on leukemia Jurkat cells with higher potency than on non-

leukemic human lymphoblasts and human primary fibroblasts. Complete suppression of

camalexin-induced cytotoxicity in Jurkat cells by Z-VAD-FMK suggests that caspase-

mediated apoptosis is an exclusive mode of cell death under these conditions without

involvement of non-apoptotic cell death such as necrosis and autophagy. Furthermore, our

cytotoxicity data do not demonstrate switch to necrosis [31] or autophagy [32] upon

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inhibition of caspases by Z-VAD-FMK, a phenomenon observed by other investigators in

similar experiments. Enhanced cytotoxicity of camalexin in combination with BSO is

consistent with the role of ROS in cytotoxicity of this phytochemical and suggests that GSH

may have a protective effect against it. BSO enhances the cytotoxicity of various anticancer

agents, including cisplatin, melphalan, doxorubicin [33], treosulfan [34], etoposide,

vincristine [35], as well as arsenic trioxide (ATO) [36]. Chemosensitization by BSO

generally involves depletion of cellular glutathione, followed by suppression of drug

detoxification, and decreased multidrug resistance-associated protein (MRP)-mediated drug

efflux. BSO-mediated chemosensitization of different cancer cell types to ATO is associated

with enhanced generation of ROS [36-37], that likely reflects an impaired redox state by BSO

with the increase in reactive oxygen species generated by ATO. Our data suggest that, similar

to ATO [38-39], camalexin induces generation of ROS followed by caspase-dependent

apoptosis. Furthermore, cytotoxicity of both camalexin and ATO is not induced by

glutathione depletion [40] and both these agents appear to induce mitochondrial generation of

ROS [41]. Considering these facts, camalexin appears to display substantial similarity in its

mode of cytotoxic action to ATO, an approved drug for the treatment of relapsed or

refractory acute promyelocytic leukemia [42].

The mode of action (MOA) of camalexin is not yet understood in detail and there are no

published reports on molecular mechanisms of camalexin on mammalian cells.

Transcriptomics analysis on Alternaria brassisicola ascomycete identified several functional

categories of genes induced by camalexin, including lipid, fatty acids and sterol metabolism,

cellular transport, cell wall structure and function, protein fate, and oxidative burst, stress and

defense [43]. However, the relevance of these data to the understanding of camalexin MOA

on mammalian cells is rather limited. Since breast cancer cells SKBr3 sensitive to camalexin

over express topoisomerase IIα, it has been speculated that topoisomerase II is the target of

camalexin [6]. However, camalexin up to 200 M does not display any inhibitory activity on

topoisomerase II-catalyzed relaxation of supercoiled plasmids in vitro (data not presented)

using Topoisomerase II alpha assay kit (USB Corporation, Cleveland, OH). Thus, the

antiproliferative effect of camalexin most likely does not include inhibition of topoisomerase

II. Camalexin also does not appear to interact directly with DNA, since such interaction

should result in the changed stability of the dsDNA duplex that may be recognized by

dsDNA melting experiments [44], and we did not identify any alterations by analyses of

melting curves of calf thymus DNA treated with camalexin in vitro (data not shown).

In conclusion, camalexin triggers apoptotic cell death of Jurkat cells by generation of ROS. It

is well known that ROS may promote cell proliferation or cell death depending on

circumstances that include intensity and cellular location of generated ROS, as well as status

of the antioxidant defense system [45]. In addition to ATO, many other anticancer agents

increase production of ROS in cancer cells, including cisplatin, paclitaxel, 2-

methoxyestradiol, doxorubicin, bleomycin and 5-fluorouracil, and oxidative stress is

responsible, at least in part, for their anticancer activity or toxic side effects [45-46]. These

agents generate ROS by various mechanisms, including the microsomal monooxygenase

system, xanthine oxidase, Fenton and Haber-Weiss reactions, the mitochondrial electron

transport chain, and inhibition of superoxide dismutase [47]. Our results imply camalexin-

induced mitochondrial superoxide generation, that likely results from targeting one or more

possible ROS generating or ROS detoxifying systems, such as Complex I (NADH-

ubiquinone oxidoreductase), Complex III, MnSOD, glutathione system and others. Our

results further suggest that camalexin does not inhibit -glutamylcysteine synthetase, the rate-

controlling enzyme for GSH synthesis, and that camalexin may also inhibit ROS scavenging

by glutathione system, since the total cellular glutathione and GSH/(GSH+GSSG) ratio

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9

remained unchanged in camalexin-treated cells with increased ROS. The specific source of

mitochondrial ROS and components of the signaling pathway connecting camalexin-induced

ROS with apoptosis of Jurkat cells remain to be elucidated. Understanding may result in

development of new cancer therapeutic strategies and possibly also new drugs using

camalexin as a lead compound. Camalexin contains a biaryl scaffold considered to be a

privileged structure for drug discovery due to its wide range of different pharmacological

activities, including antifungal, anti-inflammatory, anticancer and follicle-stimulating

hormone (FSH) agonistic effects [48].

Disclosure Statement

Authors declare that no actual or potential conflict of interest exists in relation to this article.

Acknowledgement

This work was supported by Deborah Nash Harris Endowment Fund; Ovarian Cycle

Foundation, the Ovarian Cancer Institute, and the Slovak Research and Development Agency

under the contract No. APVV-0514-06. Authors thank Dr. DeEtte Walker for reviewing the

manuscript and Dr. Kenneth Scarberry for his help with the artwork.

References

1. Browne LM, Conn KL, Ayer WA, Tewari JP (1991) The camalexins: new

phytoalexins produced in the leaves of Camelina sativa (Cruciferae). Tetrahedron

47:3909–3914.

2. Jimenez LD, Ayer WA, Tewari JP (1997) Phytoalexins produced in the leaves of

Capsella bursa-pastoris (shepherd’s purse). Phytoprotection 78: 99–103.

3. Tsuji J, Jackson EP, Gage DA, Hammerschmidt R, Somerville SC (1992) Phytoalexin

accumulation in Arabidopsis thaliana during the hypersensitive reaction to

Pseudomonas syringae pv syringae. Plant Physiol 98:1304–1309.

4. Glawischnig E (2007) Camalexin. Phytochemistry 68:401-406.

5. Mezencev R, Galizzi M, Kutschy P, Docampo R (2009) Trypanosoma cruzi:

antiproliferative effect of indole phytoalexins on intracellular amastigotes in vitro.

Exp Parasitol 122:66-69.

6. Moody CJ, Roffey JRA, Stephens MA, Stratford IJ (1997) Synthesis and cytotoxic

activity of indolyl thiazoles. Anti-Cancer Drugs 8:489-499.

7. Beier RC, Nigg HN (2001) Toxicology of naturally occurring chemicals in food. In:

Hui YH, Smith RA, Spoerke DG, Jr (eds) Foodborne Disease Handbook, Volume 3,

Plant Toxicants. Marcel Dekker Inc., New York, 2001, pp. 37-186.

8. Boue SM, Cleveland TE, Carter-Wientjes C, Shih BY, Bhatnagar D, McLachlan JM,

Burow ME (2009) Phytoalexin-enriched functional foods. J Agric Food Chem

57:2614-2622.

9. Ayer WA, Craw PA, Ma Y, Schiang M (1992) Synthesis of camalexin and related

phytoalexins. Tetrahedron 48:2919-2924.

Mezencev et al. J. Nat. Med. 2011; 65(3-4):488-499.

10

10. Schneider U, Schwenk HU, Bornkamm G (1977) Characterization of EBV-genome

negative "null" and "T" cell lines derived from children with acute lymphoblastic

leukemia and leukemic transformed non-Hodgkin lymphoma. Int J Cancer 19:621-

626.

11. Willis JH, Isaya G, Gakh O, Capaldi RA, Marusich MF (2008) Lateral-flow

immunoassay for the frataxin protein in Friedreich's ataxia patients and carriers. Mol

Genet Metab 94:491-497.

12. O’Brien J, Wilson I, Orton T, Pognan F (2000) Investigation of the Alamar Blue

(resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J

Biochem 267:5421-5426.

13. Hickey TE, Majam G, Guerry P (2005) Intracellular survival of Campylobacter jejuni

in human monocytic cells and induction of apoptotic death by cytholethal distending

toxin. Infect Immun 73:5194-5197.

14. Ren YG, Wagner KW, Knee DA, Aza-Blanc P, Nasoff M, Deveraux QL (2004)

Differential regulation of the TRAIL death receptors DR4 and DR5 by the signal

recognition particle. Mol Biol Cell 15:5064–5074.

15. Boesen-de Cock JG, de Vries E, Williams GT, Borst J (1998) The anti-cancer drug

etoposide can induce caspase-8 processing and apoptosis in the absence of CD95

receptor-ligand interaction. Apoptosis 3:17-25.

16. Reutelingsperger CP, van Heerde WL (1997) Annexin V, the regulator of

phosphatidylserine-catalyzed inflammation and coagulation during apoptosis. Cell

Mol Life Sci 53:527-532.

17. Cossarizza A, Baccarani-Contri M, Kalashnikova G, Franceschi C (1993) A new

method for the cytofluorimetric analysis of mitochondrial membrane potential using

the J-aggregate forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-

tetraethylbenzimidazol carbocyanine iodide (JC-1). Biochem Biophys Res Commun

197: 40-45.

18. Nunez R (2001) DNA measurement and cell cycle analysis by flow cytometry. Curr

Issues Mol Biol 3:67-70.

19. Fox MH (1980) A model for the computer analysis of synchronous DNA distributions

obtained by flow cytometry. Cytometry 1:71-77.

20. Possel H, Noack H, Augustin W, Keilhoff G, Wolf G (1997). 2,7-

Dihydrodichlorofluorescein diacetate as a fluorescent marker for peroxynitrite

formation. FEBS Letters 416:175-178.

21. Tarpey MM, Wink DA, Grisham MB (2004) Methods for detection of reactive

metabolites of oxygen and nitrogen: in vitro and in vivo considerations. Am J Physiol

Regul Integr Comp Physiol 286:R431-44.

22. Peshavariya HM, Dusting GJ, Selemidis S (2007) Analysis of dihydroethidium

fluorescence for the detection of intracellular and extracellular superoxide produced

by NADPH oxidase. Free Radic Res 41:699-712.

23. Mukhopadhyay P, Rajesh M, Yoshihiro K, Haskó G, Pacher P (2007) Simple

quantitative detection of mitochondrial superoxide production in live cells. Biochem

Biophys Res Commun 358:203-208.

Mezencev et al. J. Nat. Med. 2011; 65(3-4):488-499.

11

24. Mazumdera J, Chakraborty R, Sena S, Vadrab S, Dec B, Ravi TK (2009) Synthesis

and biological evaluation of some novel quinoxalinyl triazole derivatives. Der

Pharma Chemica 2:188-198.

25. Schwerk C, Schulze-Osthoff K (2003) Non-apoptotic functions of caspases in cellular

proliferation and differentiation. Biochem Pharmacol 66:1453-1458.

26. Glawischnig E, Hansen BG, Olsen CE, Halkier BA (2004) Camalexin is synthesized

from indole-3-acetaldoxime, a key branching point between primary and secondary

metabolism in Arabidopsis. PNAS 101:8245-8250.

27. Pedras MSC, Jha M, Okeola OG (2005) Camalexin induces detoxification of the

phytoalexin brassinin in the plant pathogen Leptosphaeria maculans. Phytochemistry

66:2609-2611

28. Thomma BP, Nelissen I, Eggermont K, Broekaert WF (1999) Deficiency in

phytoalexin production causes enhanced susceptibility of Arabidopsis thaliana to the

fungus Alternaria brassicicola. Plant J 19:163–171.

29. Zhao J, Last RL (1996) Coordinate regulation of the tryptophan biosynthetic pathway

and indolic phytoalexin accumulation in Arabidopsis. Plant Cell 8:2235-2244.

30. Ekins S, Bugrim A, Brovold L, Kirillov E, Nikolsky Y, Rakhmatulin E, Sorokina S,

Ryabov A, Serebryiskaya T, Melnikov A, Metz J, Nikolskaya T (2006) Algorithms

for network analysis in systems-ADME/Tox using the MetaCore and MetaDrug

platforms. Xenobiotica 36:877-901.

31. Lemaire C, Andreau K, Souvannavong V, Adam A (1998) Inhibition of caspase

activity induces a switch from apoptosis to necrosis. FEBS Letters 425:266-270.

32. Shao Y, Gao Z, Marks PA, Jiang X (2004) Apoptotic and autophagic cell death

induced by histone deacetylase inhibitors. Proc Natl Acad Sci USA 101:18030-18035.

33. Hamilton TC, Winker MA, Louie KG, Batist G, Behrens BC, Tsuruo T, Grotzinger,

KR, McKoy WM, Young RC, Ozols RF (1985). Augmentation of Adriamycin.

melphalan. and cisplatin cytotoxicity in drug-resistant and sensitive human ovarian

carcinoma cell lines by buthionine sulfoximine mediated glutathione depletion.

Biochem Pharmacol 34: 2583-2586.

34. Reber U, Wüllner U, Trepel M, Baumgart J, Seyfried J, Klockgether T, Dichgans J,

Weller M (1998) Potentiation of treosulfan toxicity by the glutathione-depleting agent

buthionine sulfoximine in human malignant glioma cells: the role of bcl-2. Biochem

Pharmacol 55:349-359.

35. Schneider E, Yamazaki H, Sinha BK, Cowan KH (1995) Buthionine sulphoximine-

mediated sensitisation of etoposide-resistant human breast cancer MCF7 cells

overexpressing the multidrug resistance-associated protein involves increased drug

accumulation. Br J Cancer 71:738–743.

36. Kito M, Akao Y, Ohishi N, Yagi K, Nozawa Y (2002) Arsenic trioxide-induced

apoptosis and its enhancement by buthionine sulfoximine in hepatocellular carcinoma

cell lines. Biochem Biophys Res Commun 291:861-867.

37. Pu YS, Hour TC, Chen J, Huang CY, Guan JY, Lu SH (2002) Arsenic trioxide as a

novel anticancer agent against human transitional carcinoma--characterizing its

apoptotic pathway. Anticancer-Drugs 13:293-300.

Mezencev et al. J. Nat. Med. 2011; 65(3-4):488-499.

12

38. Wang TS, Kuo CF, Jan KY, Huang H (1996) Arsenite induces apoptosis in Chinese

hamster ovary cells by generation of reactive oxygen species. J Cell Physiol 169: 256-

268.

39. Chen YC, Lin-Shiau SY, Lin JK (1998) Involvement of reactive oxygen species and

caspase 3 activation in arsenite-induced apoptosis. J Cell Physiol 177:324-333.

40. Wang Y, Xu Y, Wang H, Xue P, Li X, Li B, Zheng Q, Sun G (2009). Arsenic induces

mitochondria-dependent apoptosis by reactive oxygen species generation rather than

glutathione depletion in Chang human hepatocytes. Arch Toxicol 10: 899-908.

41. Han YH, Moon HJ, You BR, Kim SZ, Kim SH, Park WH (2009) The effect of MAPK

inhibitors on arsenic trioxide-treated Calu-6 lung cells in relation to cell death, ROS

and GSH levels. Anticancer Res 29:3837-3844.

42. Cohen MH, Hirschfeld S, Flamm Honig S, Ibrahim A, Johnson JR, O'Leary JJ, White

RM, Williams GA, Pazdur R (2001) Drug approval summaries: arsenic trioxide,

tamoxifen citrate, anastrazole, paclitaxel, bexarotene. Oncologist 6: 4-11.

43. Sellam A, Dongo A, Guillemette T, Hudhomme P, Simoneau P (2007) Transcriptional

responses to exposure to the brassicaceous defence metabolites camalexin and allyl-

isothiocyanate in the necrotrophic fungus Alternaria brassicicola. Mol Plant Pathol 8:

195-208.

44. Žaludová R, Žákovská A, Kašpárková J, Balcarová Z, Kleinwächter V, Vrána O

(1997) DNA interactions of bifunctional dinuclear platinum(II) antitumor agents. Eur

J Biochem 246:508-517.

45. Manda G, Nechifor MT, Neagu TM (2009) Reactive Oxygen Species, Cancer and

Anti-Cancer Therapies. Curr Chem Biol 3:22-46.

46. Hwang IT, Chung YM, Kim JJ, Chung JS, Kim BS, Kim HJ, Kim JS, Yoo YD (2007)

Drug resistance to 5-FU linked to reactive oxygen species modulator 1. Biochem

Biophys Res Commun 359:304-310.

47. Benhar M, Engelberg D, Levitzki A (2002) ROS, stress-activated kinases and stress

signaling in cancer. EMBO Reports 3:420-425.

48. Hajduk PJ, Bures M, Praestgaard J, Fesik SW (2000) Privileged Molecules for Protein

Binding Identified from NMR-Based Screening. J Med Chem 43:3443-3447.

Mezencev et al. J. Nat. Med. 2011; 65(3-4):488-499.

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Figure 1 Structural formula of camalexin

Figure 2 Viability of Jurkat (A), human immortalized lymphoblasts (B) and primary human fibroblast

(C) cells treated with different concentrations of camalexin for 72 hours (log c) and expressed as % of solvent-

treated control. Curves fitted by non-linear regression of log-transformed data using a normalized response-

variable slope model. Error bars: standard deviations; N=4.

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Figure 3 Time dependency of cytotoxic effect of camalexin on Jurkat cells. Viability of cells treated

with 50 M of camalexin for different times is expressed as % of solvent treated control. Error bars: standard errors of means. Statistical significance of differences shown between consecutive timepoints only; ** - p < 0.01

(Tukey-Kramer Multiple Comparisons Test).

Figure 4 Cytotoxic effect of camalexin (CAM) in combination with BSO (A) and Z-VAD-FMK (B) on

Jurkat cells (72-h treatment). Error bars: SEM (N=4).

(A) 1: BSO 50 M; 2: BSO 100 M; 3: CAM 50 M; 4: CAM 50M + BSO 50 M; 5: CAM 50 M + BSO

100 M; significance of differences between 3-4 and 3-5: p<0.01 (**);

(B) 1: Z-VAD-FMK 10 M; 2: Z-VAD-FMK 30 M; 3: CAM 50 M; 4: Z-VAD-FMK 10 M + CAM 50 M;

5: Z-VAD-FMK 30 M + CAM 50 M; significance of differences between 3-4 and 3-5: p<0.01 (**);

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Figure 5 Determination of caspase 8, caspase 9, and caspase 3/7 in Jurkat cells treated with camalexin

at 100 M for 72 hours, ETO at 25 M for 6 hours, STA at 10 M for 6 hours, or solvent control for 72 hours. (A) Visualization of luminescent signal for individual wells (B) Quantification of intensity of luminescent

signal; PPC[%] – luminescence intensity signal expressed in % of positive control; error bars: SEM (N=4).

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Figure 6 Determination of apoptosis (via externalization of PS) in Jurkat cells treated with solvent

control (A) or 100 M camalexin (B) for 48 hours. PI and FITC-A signify respective fluorescence intensities. Early apoptotic cells - Q3 (FITC+/PI-); late apoptotic and necrotic cells – Q2 (FITC+/PI+), viable cells – Q4

(FITC-/PI-).

Figure 7 Apoptosis in human primary fibroblast cells treated with 20 M (A) or 200 M (B) camalexin

for 72 hours. Cells were fixed with phosphate buffered formalin and stained with 10 g/mL Hoechst 33342 in H2O. Apoptotic nuclei demonstrate chromatin condensation and fragmentation (white arrows).

Mezencev et al. J. Nat. Med. 2011; 65(3-4):488-499.

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Figure 8 Mitochondrial membrane potential dissipation upon treatment of Jurkat cells with solvent

control (A) or 100 M camalexin (B) for 8 hours (I), 24 hours (II) and 48 hours (III) detected by FACS using

JC-1 staining. Gated population displays reduced fluorescence of J-aggregates and reduced mitochondrial membrane potential.

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Figure 9 (A) Deconvolution of DNA content frequency histograms for Jurkat cells treated for 48 hours

with solvent control (left histogram) or 100 μM camalexin (right histogram) by the Dean-Jet-Fox model. (B)

Distribution of Jurkat cells in G0/G1, S and G2/M phases of cell cycle and sub-G1 apoptotic fraction after 48-h

treatment with 100 μM camalexin (black bars) or solvent control (empty bars) determined by flow cytometry

analysis. P[%] – proportion of cells in specific phases of cell cycle. Error bars: SEM (N=3). P-values for the

significance of differences between solvent and camalexin-treated cells for each cell cycle phase: p<0.05 (*);

p<0.01 (**).

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Figure 10 Detection of ROS using DCFH-DA (I) and DHE (II) in Jurkat cells treated for 6 hrs with 100

M camalexin (solid lines) or solvent control (hatched lines).

Figure 11 Detection of mitochondrial superoxide by MitoSOX staining using fluorescent microscopy (A)

and flow cytometry (B). (A) Cells treated with 100 µM camalexin for 6 hours imaged by fluorescence

microscope using Olympus rhodamine filter (peak excitation 547 nm, emission filter 580 nm long pass),

demonstrate red fluorescence (untreated cells did not display appreciable fluorescence under the same imaging

conditions). (B) Fluorescence of MitoSOX dye upon its oxidation by mitochondrial O2- and binding to DNA

was detected in Jurkat cells treated for 6 hrs with 100 M camalexin (solid lines) or solvent control (hatched lines).

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Figure 12 Normalized intracellular glutathione levels (NGL) expressed as blank-corrected luminescence

signal from GSH assay normalized to the number of viable cells. Reduced glutathione (grey bars) and total

glutathione (empty bars) were determined in Jurkat cells treated for 12-h with solvent control, 100 μM

camalexin and 25 μM BSO (positive control). P-values for the significance of differences between

corresponding solvent and camalexin treated cells (Tukey-Kramer multiple comparisons test): p>0.05 (ns); for

differences involving BSO-treated cells: p<0.001 (***).

Table 1 Activity scores for 4 different pharmacological mechanisms determined by Molinspiration

Cheminformatics models (http://www.molinspiration.com) for camalexin, known active compounds and a

background set of organic molecules.

Activity score -

camalexin

Mean activity score –

active compounds

Mean activity score –

organic molecules

GPCR ligand -0.33 0.5 -0.5

Kinase inhibitor -0.59 0.6 -0.6

Ion channel

modulator

0.05 0.5 -0.5

Nuclear receptor

ligand

-1.70 0.75 -0.75