Chapter 3

Cytotoxic potential of juglone and

its underlying mechanisms:

in vitro studies

Cell Biology International, 2009; 33: 1039-49

Chapter 3

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ABSTRACT

In the present study, an attempt was made to investigate the cytotoxic and

potential of juglone against a panel of human and murine tumor cell lines grown in

vitro. This study also aimed to elucidate the mechanisms underlying the cytotoxic

potential of juglone against B16F1 melanoma cells.

From the MTT assay, it was found that, among the cell lines tested, juglone

was found to be most effective against MCF7 human breast cancer cells with an IC50

value of about 3.8 and 2.7 µM after 24 and 48 h respectively. The second most

sensitive cell line was B16F1 mouse melanoma with an IC50 value of 7.5 and 6.9 µM

after 24 and 48 h respectively. The other tested cell line (ACHN - human renal

carcinoma and A549 – human lung carcinoma) were much more resistant with IC50

values > 10 µM. Treatment of melanoma cells with juglone resulted in a

concentration-dependent decrease in the cell survival with a corresponding increase

in the lactate dehydrogenase levels. Further, juglone treatment caused a significant

increase in the genotoxicity as evidenced by the concentration-dependent elevation in

the micronucleated binucleate cells (MNBNC) as well as Olive Tail Moment (OTM)

values. Moreover, results of the micronuclei study indicated division delay in the

proliferating cell population of juglone treated group by showing decrease in the

cytokinesis blocked proliferation index. A significant (P<0.05 to P<0.001)

concentration-dependent decrease in the intracellular glutathione levels and an

increase in dichlorofluorescein (DCF) fluorescence after juglone treatment confirm

the ability of juglone to generate intracellular reactive oxygen species, thereby

leading to an alteration in the oxidative balance of the cells. Further, the juglone-

induced apoptotic and necrotic mode of cell death was demonstrated by

oligonucleosomal ladder formation, microscopic analysis, increase in the hypodiploid

fraction (sub Go peak in DNA histogram) as well as increase in percentage of

AnnexinV(+)/PI(+) cells analyzed by flow cytometry. To conclude, cytotoxic effect of

juglone in melanoma cells may be attributed to multiple mechanisms such as

induction of oxidative stress, cell membrane damage (LDH assay) and genotoxic

effect, ultimately leading to cell death by both apoptosis and necrosis.

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3.1. INTRODUCTION

It is long-established that plants possess diverse principles, which are of

immense nutritional and medicinal value. Numerous anticancer screening studies have

been conducted using traditional medicinal plants in an attempt to discover new

therapeutic agents that are devoid of toxic side effects generally known to be

associated with current chemotherapeutic agents. Currently, research is focused more

on developing anti-cancer drugs and their derivatives isolated from natural sources,

especially phytochemicals with improved anticancer potential and minimal systemic

toxicity.

Naturally occurring quinones represent a large group of quinoid compounds

that are known to possess a spectrum of pharmacological activities, including

anticancer activity (Babula et al., 2007; Babula et al., 2009). Although, there are

many clinically important anticancer agents containing quinone nucleus such as

anthracycline, mitoxantrones and saintopin, many other quinones are still being tested

for their anticancer activity (Kim et al., 2006). Among the natural compounds, plant-

derived quinones have been extensively studied for their potential as

cytotoxic/anticancer agents. Plumbagin (5-hydroxy-2-methyl-1, 4-napthoquinone)

derived from the roots of Plumbago zeylanica, is one such compound. Earlier studies

have given sufficient insight into the anticancer and radiosensitizing properties of

plumbagin (Naresh et al., 1996; Prasad et al., 1996; Devi et al., 1998; Tiwari et al.,

2002). Besides, other natural napthoquinones such as lapachone, menadione, lawsone,

shikonin etc. are also known to possess promising cytotoxic/anticancer properties

(Nutter et al., 1991; Kamei et al., 1998; Wu et al., 2004; Reinicke et al., 2005).

Although, DNA represents the main target for quinoid antitumor agents, the

exact contribution of the quinone moiety to the cytotoxic effect is not clear. However,

most of them belong to the groups of DNA intercalating and/ or alkylating age nts

and/or topoisomerase inhibitors (Rowley and Halliwell, 1983; Yamashita et al.,

1991). In general, quinone toxicity has been attributed to its ability to undergo

reversible oxidation-reduction reactions as well as to its electrophilic nature leading to

free radical formation (Bachur et al., 1979; Giulivi and Cadenas, 1994).

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Juglone (5-hydroxy-1, 4-Napthoquinone), a structural analogue of plumbagin

is a pigment that occurs as a natural product in the roots, leaves, nut-hulls, bark and

wood of black walnut (Juglans nigra L.), European walnut (Juglans regia L.) and

butternut (Juglans cinerea L.) (Family: Juglandaceae) (Botanical dermatology

databse, 1999). The herbal preparations of walnut have been extensively used in folk

medicine for the treatment of acne, inflammatory diseases, ringworm, bacterial, viral,

fungal infections and also cancer (Duke and Ayensu, 1985; Blumenthal, 1998).

Herbal preparation of walnut is reported to suppress the growth of spontaneous

mammary adenocarcinoma in Swiss albino mice (Bhargava and Westfall, 1968).

Walnut contains juglone as the principal component, with other constituents such as

alpha-hydrojuglone (1,4,5-trihydroxynaphthalene) and its glycoside beta-

hydrojuglone, along with caffeic acid, ellagic acid, hyperin, and kaempferol. Sugie

and co-workers (1998) reported the ability of juglone to reduce the formation of

azoxymethane induced intestinal tumors in F344 rats and concluded that juglone

could be a promising chemopreventive agent. Although few groups have studied the

in vitro cytotoxic activity of juglone against cancer cell lines (Segura-Aguilar et al.,

1992; Cenas et al., 2006; Li et al., 2010; Ji et al., 2011), the exact mechanism remains

ambiguous. Therefore, the present study was aimed to investigate the cytotoxic

potential of juglone against a panel of human and murine tumor cell lines and to

elucidate its underlying mechanisms using a relatively chemo-resistant cell line

(B16F1 melanoma) growing in vitro.

3.2. MATERIALS AND METHODS

3.2.1. Chemicals and reagents

Juglone was purchased from Sigma (St. Louis, MO, USA) and dissolved in

dimethyl sulfoxide (DMSO), diluted with Eagle’s minimum essential medium (MEM)

to get required concentrations (the final concentration of DMSO was less than 0.02

%). All drug solutions were prepared freshly before use (considering the lack of

stability of juglone in the medium for longer periods). The chemicals, 3-(4’,5’-

dimethylthiazol-2’-yl)-2,5-diphenyl- tetrazolium bromide (MTT), 5,5'-dithiobis (2-

nitrobenzoic acid) (DTNB), 2’,7’-dicholorofluorescein diacetate (DCFH-DA),

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annexin-V-FITC apoptosis detection kit, RNase A, Proteinase-K, Nonidet P40,

sodium dodecyl sulphate (SDS), Cytochalasin-B, glutathione, ethidium bromide,

agarose (normal melting as well as low melting), ethylenediamine tetra acetic acid

(EDTA), MEM, l-glutamine, gentamycin sulfate, fetal calf serum and DMSO were

obtained from Sigma (St. Louis, MO, USA). Cytochalasin-B was dissolved in DMSO

at a concentration of 1 mg/ml, stored at -80°C and diluted with PBS immediately

before use.

3.2.2. Cell line and culture

B16F1 (murine melanoma cells), MCF7 and MDAMB-231 (Human breast

cancer cells), ACHN (human renal carcinoma cells), A549 (Human lung carcinoma

cells) were obtained from the National Centre for Cell Sciences (Pune, India) and

maintained in our laboratory. All cell types (except MDAMB-231) were routinely

grown in Eagle’s minimum essential medium (MEM) supplemented with 10% fetal

bovine serum, 1% l-glutamine and 80 μg/ml gentamycin sulfate. Cells were cultured

in 75 cm2 flasks with loosened caps and incubated in a 5% CO2 in humidified air at 37

°C (NuAire, Plymouth, MN, USA). MDAMB-231 was grown in Leibovitz L15

medium in an atmosphere devoid of CO2.

3.2.3. Cytotoxicity assays

3.2.3.1. MTT assay

The cytotoxic potential of juglone against various tumor cells was quantified

using MTT assay. This test, first described by Tim Mosman (Mosmann, 1983), is a

colorimetric assay that measures the reduction of yellow 3-(4,5-dimethythiazol-2-yl)-

2,5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase.

The MTT enters the cells and passes into the mitochondria where it is reduced to an

insoluble, dark purple colored formazan product (Figure 3.1). These crystals are

largely impermeable to cell membranes, thus resulting in its accumulation within

healthy cells. These cells are then solubilized with an organic solvent (like DMSO)

and the released, solubilized formazan reagent is measured spectrophotometrically

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Since reduction of MTT can only occur in metabolically active cells, the level of

activity is a measure of the viability of the cells.

Based on this principle, MTT assays have been extensively used for various

medical, microbiological and toxicological approaches including in vitro assessment

of chemotherapeutic agents against tumor cells, assessment of radio sensitivity,

bacterial enterotoxins, pesticides and antibiotics and the detection of lymphokines

(Carmichael et al., 1988; Price and McMillan, 1990).

Figure 3.1. Mitochondrial dehydrogenase mediated reduction of yellow colored MTT

to dark purple colored formazan

In the present study, an attempt was made to evaluate the effect of juglone on

the viability of a panel of human and murine tumor cell lines including MCF7,

MDAMB-231, ACHN, A549, B16F1. Initial experiments were carried out to

determine the most suitable number of cells to be seeded into each well. Based on

these studies, 5 x 103 cells per well were plated in 96 well plate and incubated. Also,

cell blank (no cells) and drug blank (no drug) were included for each 96-well plate.

Twenty-four hours later, cells were divided into following groups.

Group 1: Cells in this group were treated with vehicle control for 24 and 48 h; Group

2: Cells in this group were treated with different doses (5, 10, 15 and 20 µM) of

juglone for 24 and 48 h; Group 3: These cells were treated with positive control

(doxorubicin hydrochloride).

At the end of the incubation time, cells were washed with PBS and incubated

further for 4 h with 100 µl (1 mg/ml) of MTT (dissolved in a medium (MEM) devoid

of serum and phenol red). Washing with PBS was followed by the addition of DMSO

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(100 µl), gentle shaking for 10 min so as to ensure complete dissolution of the

formazan crystals. Absorbance values were then recorded at 540 nm using the

microplate spectrophotometer system (Biotek ELx 800, USA). Each treatment was

completed in quadruplicate (with each experiment being repeated at least two times)

and IC50 (concentration of drug that inhibits cell growth by 50%) values were

determined from the concentration Vs percent viability curve. The percent viability

was calculated as follows:

Percent viability Test absorbance Blank absorbance

ontrol absorbance Blank absorbance 100

3.2.3.2. Lactate dehydrogenase (LDH) leakage assay

Lactate dehydrogenase is an enzyme present in the cytoplasm of most cell

types. Upon cell membrane damage, this cytoplasmic enzyme is released from

damaged cells into the extracellular medium, which can be measured

spectrophotometrically. The amount of enzyme activity correlates to the proportion of

the damaged cells.

In biological systems, LDH is known to catalyze the inter-conversion of

pyruvate and lactate with concomitant inter-conversion of NADH and NAD+. The

reduced coenzyme NADH absorbs at 339 nm, whereas the oxidized form NAD,

lactate and pyruvate do not (Figure 3.2). Based on this principle, the progress of this

reaction can be followed by measuring the decrease in the light absorption at 339 nm.

Alternatively, the enzyme can also be measured by monitoring the reverse reaction

under slightly alkaline conditions which however suffers from being much slower

than the reaction starting with pyruvate.

Figure 3.2. Role of lactate dehydrogenase enzyme in two-way conversion of lactate

to pyruvate

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In the present study, LDH released in the medium was measured according to

the procedure of Wrobleski and Ladue (Wroblewski and Ladue, 1955). A fixed

number (5 × 105) of exponentially growing cells were seeded into several individual

25 cm2 culture T-flasks and allowed to grow. Twenty four hours later, several of such

individual cell cultures were used for the experiments and they were divided into

following groups

Group 1: Cells in this group was treated with vehicle (0.02 % v/v DMSO); Group 2:

Cells in this group were treated with different concentrations of juglone (5, 10, 15 and

20 µM) for 1 h

At the end of the treatment, the whole medium from cell culture flask of

control and treated groups was removed and collected separately. The tubes

containing media were centrifuged at 1500 rpm for 10 min and 50 µl of the medium

was transferred to individual tubes containing Tris EDTA NADH buffer followed by

10 min incubation at 37 °C and the addition of pyruvate solution. The absorbance was

read at 339 nm using a UV-Vis spectrophotometer (UV-260, Shimadzu Corp, Tokyo,

Japan) and the data was expressed as units/liter (U/l).

3.2.3.3. Clonogenic assay

Clonogenic assay or colony formation assay is an in vitro cell survival assay

which is based on the ability of a single cell to grow into a colony (defined to consist

of at least 50 cells) and is often regarded as the “gold standard” cellular-sensitivity

assay. The assay essentially tests every cell in the population for its ability to undergo

“unlimited” division. Only a fraction of seeded cells retains the capacity to produce

colonies. Considering the reliability and the utility of this assay, it is among the most

widely used experimental approach in pharmaceutical, clinical and basic research for

testing the effects of drugs/genes/radiation on the growth and proliferative

characteristics of cells in vitro (Langdon, 2004; Franken et al., 2006).

Clonogenic death induced by juglone was measured using colony-forming

assay according to the method of Puck and Marcus (Puck and Marcus, 1955). A fixed

number (5 × 105) of exponentially growing cells were seeded into several individual

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25 cm2 culture T-flasks and allowed to grow. Twenty four hours after culture

initiation, several individual cell cultures were randomly divided into following

groups

Group 1: Cells in this group was treated with vehicle (0.02 % v/v DMSO); Group 2:

Cells in this group were treated with different concentrations of juglone (5, 10, 15 and

20 µM) for 1 h

At the end of various treatments, cells from above groups were trypsinized,

single cell suspension of cells were counted using a hemocytometer and appropriate

numbers of viable cells was seeded into culture petri dishes in triplicate and were left

undisturbed for 10 - 12 days for colony formation. The colonies formed were then

washed with phosphate buffer saline (PBS), fixed, stained with crystal violet (%) and

the viable colonies (containing 50 or more cells) were manually counted. The plating

efficiency (PE) and surviving fraction (SF) were then calculated as follows.

Plating efficiency (PE) Number of colonies counted

Number of cells seeded 100

Surviving Fraction (SF) Number of colonies counted

Number of cells seeded (PE 100)

3.2.4. Genotoxicity assays

Since the measurement of DNA damage was extremely difficult beyond 10

µM concentration of juglone, genotoxicity assays were carried out using lower

concentrations (in the range of 0 -10 µM). A fixed number (5 × 105) of exponentially

growing cells were seeded into several individual 25 cm2 culture T-flasks and allowed

to grow. Twenty four hours after culture initiation, several of such individual cell

cultures were used for the experiments and they were divided into following groups

Group 1: Cells in this group was treated with vehicle (0.02 % v/v DMSO); Group 2:

Cells in this group were treated with different concentrations of juglone (0.5, 1, 2.5, 5

and 10 µM) for 1 h

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At the end of the treatment, cells from above groups were dislodged by mild

trypsin EDTA treatment and micronucleus as well as comet assays were carried out

from the same stock of cells.

3.2.4.1. Micronucleus assay

To evaluate genotoxic effects of chemical substances in vitro, various methods

are available that differ in their sensitivity, their practicability, and, finally, the genetic

end points considered. The micronucleus assay is one of the methods used to detect

chromosomal aberrations in proliferating cell culture systems (Fenech et al., 2003)

with several advantages including sensitivity as well as uncomplicated realization and

evaluation of results.

Micronuclei represent chromosome fragments or whole chromosomes which

are not incorporated into the main nuclei at mitosis and they consequently appear only

in dividing eukaryotic cells. In order to score micronuclei exclusively in cells that

have completed at least one nuclear division only, the cytokinesis block method is

applied in this test version, which prevents the cytoplasmic division after nuclear

division by use of cytochalasin B.

In the present study, micronucleus assay was performed as reported by Fenech

and Morley (Fenech and Morley, 1985) with minor modifications (Rao et al., 2006).

At the end of various treatments, the medium containing the drug was replaced with

fresh medium containing cytochalasin-B (final concentration 3 µg/ml). After

incubating for 36 h, cells were dislodged with trypsin EDTA treatment, subjected to

mild hypotonic treatment (0.75 % ammonium oxalate for 3 min) and fixed using

Carnoy's fixative (3: 1, Methanol: Acetic acid). The cells were centrifuged again, re-

suspended in a small volume of fixative and spread onto pre-cleaned slides and

stained with 0.01 % acridine orange (prepared in Sorensen's buffer pH 6.8) and

observed under a fluorescent microscope (Photomicroscope III, Carl Ziess, Germany)

using 40X neofluar objective. Slides were coded to avoid observer bias and 1000

binucleate cells with well-preserved cytoplasm were scored from each group (for the

presence of micronuclei) according to the established criteria (Fenech et al., 2003).

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The data regarding the cell proliferation was also collected, by counting the

frequencies of mono-, bi-, tri- and tetranucleate cells and the cytokinesis blocked

proliferation index (CBPI) was calculated according to previously described method

(Surralles et al., 1995) as follows

BP M 2M 3(M M )

Total

Where MI = Total mononucleates; MII = Total binucleates; MIII = Total trinucleates;

MIV = Total tetranucleates (MIII and MIV are equally considered to be in their third

cell cycle).

3.2.4.2. Single cell gel electrophoresis or comet assay

The comet assay or single-cell gel test is a microgel electrophoresis technique

that measures DNA damage at the level of single cells. A small number of cells

suspended in a thin agarose gel on a microscope slide is lysed, electrophoresed, and

stained with a fluorescent DNA binding dye. Cells with increased DNA damage

display increased migration of chromosomal DNA from the nucleus toward the anode,

which resembles the shape of a comet. In the alkaline version of the assay which is

most often used, DNA strand breaks (both single strand and double strand) and alkali-

labile sites become apparent, and the amount of DNA migration indicates the amount

of DNA damage in the cell (indicated by the increase in tail length, decrease in the

head intensity and Olive tail moment). The comet assay combines the simplicity of

biochemical techniques for detecting DNA single-strand breaks and/or alkali- labile

sites with the single-cell approach typical of cytogenetic assays. Over the past decade,

the comet assay, or single-cell gel electrophoresis (SCGE) has become one of the

standard methods for assessing DNA damage, with applications in genotoxicity

testing, human biomonitoring and molecular epidemiology, ecogenotoxicology, as

well as fundamental research in DNA damage and repair. The assay attracts adherents

by its simplicity, sensitivity, versatility, speed, and economy.

After various treatments, cells from above groups were processed for the

comet assay under alkaline conditions, as described by Singh and co-workers (1988)

with minor modifications of Collins and co-workers (1997). About 10 µl of cell

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suspension containing about 10,000 cells was mixed with 190 µl of 0.75 % low

melting point agarose (LMA) at 37 °C and layered onto a frosted slide pre-coated with

1.5 % normal agarose layer (prepared in Ca2+ and Mg2+ free PBS) and immediately

covered with a cover glass and kept at 4 °C to allow solidification of the agarose. A

third layer of 0.75% LMA without the cells was then made in such a way that the

sample cells are sandwiched between two layers of agarose. Subsequently, the cells

were lysed in a lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton

X-100 and 10 % DMSO, pH 10) for 1 h. After washing in re-distilled water, the slides

were placed on a horizontal gel electrophoresis chamber. The chamber was filled with

electrophoretic buffer (1 mM EDTA, 300 mM NaOH, pH 13) and slides were placed

in this buffer at 4°C for 20 min to allow the DNA to unwind. Electrophoresis was then

carried out for 20 min at a voltage of 0.7 V/cm and a current of 300 mA. All

preparative steps were conducted in the dark to prevent additional DNA damage.

Next, the slides were rinsed with neutralization buffer (0.4 M Tris; pH 7.4) and

stained with ethidium bromide. The stained cells were then observed and

photographed at 20X magnification using a fluorescence microscope (Olympus

BX51, Olympus Microscopes, Tokyo, Japan) equipped with a 515 535 nm excitation

filter, a 590 nm barrier filter and a CCD camera (Cool SNAP-Procf Digital Color

Camera Kit Version 4.1, Media Cybergenetics, Silver Spring, Maryland, USA).

Images of a minimum of 100 cells per treatment were analyzed using the Komet

software (Version 5.5, Kinetic Imaging Ltd., Bromborough, UK). The mean Olive tail

moment (OTM) was used as a parameter to assess the level of DNA damage.

Tail Moment (OTM) ead mean Tail % DN

100

3.2.5. Measurement of oxidative stress induction

3.2.5.1. Glutathione (GSH) assay

Glutathione (GSH, γ-glutamyl-cysteinylglycine) is a ubiquitous tripeptide that

is formed by the ATP dependent condensation of glutamic acid and cysteine,

catalyzed by g-glutamylcysteinyl synthetase. Glycine is then added by glutathione

synthetase to form GSH. In addition to donating an electron during the reduction of

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hydroperoxides to the respective alcohols (or water in the case of hydrogen peroxide),

GSH functions as a co-substrate in the metabolism of several xenobiotics catalyzed by

glutathione S-transferases. It is also a co-factor for several metabolic enzymes and is

involved in intracellular transport, functions as an antioxidant and radioprotectant and

facilitates protein folding and degradation.

The spectrophotometric assay method for glutathione (GSH) involves

oxidation of GS by the sulfhydryl reagent 5,5′-dithio-bis(2-nitrobenzoic acid)

(DTNB) to form the yellow derivative 5′-thio-2-nitrobenzoic acid (TNB), measurable

at 412 nm. The glutathione disulfide (GSSG) formed can be recycled to GSH by

glutathione reductase in the presence of NADPH (Figure 3.3). Considering the

simplicity, convenience, sensitivity and accuracy of this method, it is extensively used

to measure GSH levels in whole blood, plasma, serum, lung lavage fluid,

cerebrospinal fluid, urine, tissues and cell extracts and can be extended for drug

discovery/pharmacology and toxicology protocols to study the effects of drugs and

toxic compounds on glutathione metabolism (Rahman et al., 2007).

Figure 3.3. Principle reaction involved in the estimation of glutathione using DTNB

method

A fixed number (5 × 105) of exponentially growing cells were seeded into

several individual 25 cm2 culture T-flasks and allowed to grow. Twenty four hours

after culture initiation, several of such individual cell cultures were used for the

experiments and they were divided into following groups

Group 1: Cells in this group was treated with vehicle (0.02 % v/v DMSO); Group 2:

Cells in this group were treated with different concentrations of juglone (5, 10, 15 and

20 µM) for 1 h.

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At the end of the treatment, cells from above groups were dislodged by mild

trypsin EDTA treatment, washed with PBS and lysed at 4 °C for 2 h using 5 % w/v

metaphosphoric acid (chilled) to extract the cellular GSH. The suspension was then

centrifuged at 13,000 rpm for 5 min and GSH levels were determined using the

standard method (Moron et al., 1979). The supernatant was mixed with 0.2 M sodium

phosphate buffer (pH 8.0) and 0.04 mM DTNB [5,5'-dithiobis (2-nitrobenzoic acid)]

and kept at room temperature for 10 min. The absorbance of the samples was

recorded against reagent blank at 412 nm in an ultraviolet-visible (UV-Vis) double

beam spectrophotometer. The GSH levels were determined from a standard curve

prepared with known concentrations of GSH under similar conditions.

3.2.5.2. Measurement of intracellular ROS using DCFH-DA assay

The fluorogenic compound 2’, 7’-dichloroflourescin diacetate (DCFH-DA)

has been utilized extensively as a marker for oxidative stress, and is suggested to

reflect the overall oxidative status of the cell (Wang and Joseph, 1999). In this assay,

DCFH-DA which is freely permeable diffuses passively through the cellular

membrane where the intracellular esterases cleave off the acetate moieties leaving the

non- fluorescent 2’, 7’ dichlorofluorescin (D F ). In the presence of intracellular

hydrogen peroxide and peroxidases, DCFH get oxidized to highly fluorescent 2’, 7,

dichlorofluorescein (DCF) which can be measured at 525 nm (Figure 3.4). The

intensity of green fluorescence thus produced is proportional to the amount of

intracellular H2O2 (Myhre et al., 2003).

Figure 3.4. Reaction leading to the detection of reactive oxygen species by DCFH-

DA assay

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The effect of juglone on the intracellular ROS levels was studied using DCFH-

DA fluorescence assay as previously described (Bai and Cederbaum, 2003). The

exponentially growing cells were treated with different concentrations of juglone for

one hour followed by incubation with 5 µM DCFH-DA in MEM for 30 min in the

dark. The cells were then washed, resuspended in PBS, and the fluorescence intensity

was measured using fluorescence microplate reader (InfiniteM200, TECAN) at an

excitation and emission wavelength of 488 nm and 525 nm respectively. Results were

expressed as arbitrary units of DCF fluorescence per 105 cells.

3.2.6. Apoptosis/necrosis assays

To evaluate the mode of cell death (apoptosis and/or necrosis) induced by

juglone against B16F1 melanoma cells, a fixed number of cells (2x106) were

inoculated into several individual 100 cm2 petri plates and were allowed to grow for

24 h. Cultures were then divided into following groups:

Group 1: cells of this group were treated with vehicle control; Group 2: cells of this

group were treated with 5 and 10 µM juglone for 12 and 24 h

At the end of various treatments, both the media (containing floating cells) as

well as the attached cells were collected and were processed for fluorescence

microscopy, DNA fragmentation assay and flow cytometric analysis (univariate DNA

analysis as well as Annexin-V/PI dual staining).

3.2.6.1. Microscopic examination of nuclear morphology

Acridine orange/Ethidium bromide (AO/EtBr) staining was used to study the

juglone- induced morphological alterations in melanoma cells. At the end of the

treatment times, cells were washed with cold PBS and stained with AO/EtBr (1:1 v/v)

at a final concentration of 100 µg/ml and observed under a fluorescent microscope

(Olympus BX51, Singapore). According to this staining method, live cells with intact

membrane have green staining of the nucleus; cells undergoing early apoptosis with

intact membrane but fragmented DNA, showing green staining in the nucleus as well

as the cytoplasm, with a visible marginalization of the nuclear contents; cells in late

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apoptotic phase show areas stained orange (due to membrane damage) in the

cytoplasm as well nuclear sites where the chromatin looks condensed, distinguishing

them from necrotic cells; whereas in necrotic cells (having membrane damage) a

uniform orange staining of the nucleus (intact without chromatin condensation) and

cytoplasm is seen (Renvoize et al., 1998).

3.2.6.2. Assessment of DNA fragmentation by agarose gel electrophoresis

The biochemical hallmark of apoptosis is the fragmentation of the genomic

DNA, an irreversible event that commits the cell to die. In many systems, this DNA

fragmentation has been shown to result from activation of an endogenous Ca2+ and

Mg2+ dependent nuclear endonuclease. This enzyme selectively cleaves DNA at sites

located between nucleosomal units (linker DNA) generating mono- and

oligonucleosomal DNA fragments. These DNA fragments reveal, upon agarose gel

electrophoresis, a distinctive ladder pattern consisting of multiples of an

approximately 180 bp subunit (Wyllie, 1980).

In the present study, the effect of juglone on the DNA fragmentation was

evaluated according to previously described method (Herrmann et al., 1994). At the

end of various treatments, the cell suspension was centrifuged and the cell pellets

were then treated for 1 min with lysis buffer (1% NP-40 in 20 mM EDTA, 50 mM

Tris-HCl, pH 7.5; 10 µl per 106 cells, minimum 50 µl). After centrifugation for 5 min

at 1600 x g the supernatant was collected and the extraction was repeated with the

same amount of lysis buffer. The supernatants (and the resuspended nuclei as control

for the complete recovery of the apoptotic DNA fragments) were brought to 1% SDS

and treated for 2 h with RNase A (final concentration 5 µg/µl) at 56 °C followed by

digestion with proteinase K (final concentration 2.5 µg/µl) for at least 2 h at 37 °C. At

the end of extraction, DNA was precipitated overnight at -20 °C by adding half

volume of 10 M ammonium acetate and 2.5 volumes of 100 % ethanol. The DNA was

recovered by centrifugation at 13,000 rpm for 15 min at 4 °C. The supernatant was

discarded and the DNA pellet was washed with chilled 70% ethanol and centrifuged

as previously described. The dried DNA pellet was then dissolved in sterile water and

resolved on a 1.5 % agarose gel. DNA was finally visualized and photographed under

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UV illumination after staining with ethidium bromide. Untreated cells processed in

the same way served as control.

3.2.6.3. Analysis of DNA content by flow cytometry

The nuclear DNA content of a cell can be quantitatively measured at high

speed by flow cytometry. Initially, a fluorescent dye that binds stoichiometrically to

the DNA is added to a suspension of permeabilized single cells or nuclei. The

principle is that the stained material has incorporated an amount of dye proportional

to the amount of DNA. The stained material is then measured in the flow cytometer

and the emitted fluorescent signal yields an electronic pulse with a height (amplitude)

proportional to the total fluorescence emission from the cell. Thereafter, such

fluorescence data are considered a measurement of the cellular DNA content. Samples

should be analyzed at rates below 1000 cells per second in order to yield a good signal

of discrimination between singlets or doublets (Nunez, 2001).

Juglone- induced apoptosis was assessed flow cytometrically using propidium

iodide staining (Chang et al., 1999). At the end of treatment, cells (along with the

media) were harvested and fixed using 70 % ice-cold ethanol overnight at 4 °C. The

cells were then washed twice with cold phosphate-buffered saline (PBS), subjected to

RNase A treatment (100 µg/ml) at 37 °C for 1 h. The cellular DNA was then stained

with PI (50 µg/mL in PBS) for 15 min at 4 °C in dark and analyzed by FACSCalibur

flow cytometer using CellQuest software (Becton Dickinson, San Jose, CA, USA). At

least 10,000 events were acquired, cell debris was excluded by gating and the

percentage of sub-Go cell population (hypo-diploid fraction) was determined using

Win MDI version 2.9 software.

3.2.6.4. Flow cytometric measurement of apoptosis/necrosis using Annexin V-FITC

This test relies on the property of cells to lose membrane asymmetry in the

early phases of apoptosis. In apoptotic cells, the membrane phospholipid

phosphatidylserine (PS) is translocated from the inner leaflet of the plasma membrane

to the outer leaflet, thereby exposing PS to the external environment. Annexin V is a

calcium-dependent phospholipid-binding protein that has a high affinity for PS, and is

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useful for identifying apoptotic cells with exposed PS. Propidium Iodide (PI) is a

standard flow cytometric viability probe and is used to distinguish viable from

nonviable cells. As the plasma membrane becomes increasingly permeable during the

later stages of apoptosis, PI can readily move across the cell membrane and bind to

cellular DNA, providing a means for identifying those cells that have lost membrane

integrity through mechanisms including necrosis. When cells are double stained with

Annexin V-FITC and PI, three different cell populations may be observed: (i) live

cells that do not stain with either Annexin V-FITC or PI, (ii) early apoptotic cells that

stain with Annexin V-FITC only, and (iii) late apoptotic/necrotic cells that stain with

both reagents.

The extent of apoptosis and/or necrosis was flow cytometrically measured

using annexin V-FITC apoptosis detection kit (Sigma-Aldrich, St. Louis, USA) using

the manufacturers’ protocol. t the end of treatment, both floating as well as attached

cells were collected, washed with PBS two times, and then resuspended in 1X binding

buffer (part of the kit) at a density of 1 × 106 cells/ml. The cells were then double

stained in dark for 10 min with the fluorescein isothiocyanate (FITC)- labeled annexin

(5 μl) and propidium iodide (P ) (10 μl) and analyzed flow cytometrically using

FACSCaliburTM. At least 10,000 events were acquired and the percentage of cells in

different quadrants was analyzed using quadrant statistics (WinMDI Version 2.9).

3.2.7. Statistical analysis

The experimental data were expressed as Mean ± SEM. The plating efficiency

of cells was determined and the data points of surviving fraction was fitted on to a

linear quadratic model, SF (surviving fraction) = exp[ (αD ßD2)]. The percentage of

micronucleated binucleate cells (MNBNC) in different treatment groups was

compared with the control by One-way ANOVA using GraphPAD InStat, Software,

USA. The dose response curve for micronuclei was fitted on a linear model (Y =

+X).

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3.3. RESULTS

3.3.1. Juglone induced cytotoxicity

3.3.1.1. MTT assay

The cytotoxic effects of juglone were studied against a panel of human as well

as murine cancer cell lines (Table 3.1). Among the cell lines tested, juglone was found

to be most effective against MCF7 human breast cancer cells with an IC50 value of

about 3.8 and 2.7 µM after 24 and 48 h respectively. The second most sensitive cell

line was B16F1 mouse melanoma with an IC50 value of 7.5 and 6.9 µM after 24 and

48 h respectively. The other cells tested (ACHN and A549) were much more resista nt

with IC50 values > 10 µM. However, considering that all the in vivo studies were to be

performed using the B16F1 melanoma cells, the remaining in vitro studies were also

done using B16F1 melanoma cells. In comparison, doxorubicin hydrochloride (which

was used as positive control in the present study) showed potent cytotoxic effects

against all tested cell lines with B16F1 melanoma being the most sensitive with IC50

values of 0.6 and 0.1 µM after 24 and 48 h respectively. In all the other tested cells,

the IC50 values for doxorubicin were found to be below 10 µM.

Table 3.1. Cytotoxic potential of juglone against various human and murine tumor

cells assessed using MTT assay

Cell lines

IC50 values (µM)

Doxorubicin Juglone

24 h 48 h 24 h 48 h

Murine melanoma ( B16F1) 0.6 0.1 7.5 6.9

Human breast carcinoma (MCF7) 7.2 3.6 3.8 2.7

Human renal carcinoma (ACHN) 4.1 2.8 12.9 11.8

Human lung carcinoma (A549) 4.7 3.1 18.0 16.9

Human breast carcinoma (MDAMB-231) - - >20.0 >20.0

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3.3.1.2. LDH levels

To study the cell membrane damaging potential of juglone against B16F1

melanoma cells, the LDH leakage into the supernatant of culture following 1 h

treatment was measured (Figure 3.5). Treatment with juglone showed a significant

increase in LD levels at all concentrations (5, 10, 15 and 20 μM), in comparison to

vehicle-treated control. The increase in the LDH levels was concentration-dependent

up to a dose of 20 μM and fitted well into linear model with an R2-value of 0.9922.

Figure 3.5. Juglone induced changes in LDH levels in melanoma cells. Significant

levels -* P<0.05; ** P<0.01; *** P<0.001, compared to vehicle-treated controls.

3.3.1.3. Clonogenic survival

Figure 3.6 summarizes the effect of juglone on the clonogenic survival of

melanoma cells. The cell survival curve was fitted using a linear quadratic equation.

The dose response curve for juglone exhibited a definite shoulder region at lower

doses (5 and 10 μM). Treatment of melanoma cells with juglone caused a

concentration-dependent reduction in their colony forming ability, which followed a

polynomial regression pattern. In comparison to vehicle-treated controls, the lowest

concentration of juglone (5 μM) reduced the mean surviving fraction to 0.62 ± 0.10 (a

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37 % reduction) and the highest concentration (20 μM) caused a drop in the mean

surviving fraction to 0.0032 ± 0.0006 (a 99 % drop).

Figure 3.6. Cell survival curve for juglone against melanoma cells assessed using

clonogenic assay. A Control (150 cells seeded); B 10 µM juglone treated (1000

cells seeded); C 20 µM juglone treated (8000 cells seeded).

3.3.2. Juglone-induced genotoxicity

3.3.2.1. Micronuclei induction

Exposure of melanoma cells to juglone resulted in a concentration dependent

increase in the frequency of MNBNC as shown in Figure 3.7. The increase in the

frequency of MNBNC was statistically significant in comparison to vehicle-treated

controls at all tested doses. Treatment of melanoma cells with 0.5, 1, 2.5, 5 and 10 μM

concentrations of juglone resulted in a 1.25, 1.57, 1.84, 3.16 and 4.61-fold increase in

the mean frequency of MNBNC respectively. The increase in the freq uency of

MNBNC with one MN, two MN, multiple MN and total MN fitted well to linear

model with R2-values of 0.9960, 0.9626, 0.9806 and 0.9935 respectively.

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Figure 3.7. Genotoxic effect of juglone assessed by micronucleus assay; typical

representative photographs of acridine orange stained B16F1 binucleate cells A -

Binucleate cell (BNC) without micronuclei, B - BNC with one micronuclei, C - BNC

with two micronuclei, D - BNC with multiple micronuclei.

3.3.2.2. Cytokinesis blocked proliferation index (CBPI)

The information regarding CBPI was also determined following treatment of

melanoma cells with indicated concentrations of juglone (Table 3.2)

Table 3.2. Cytokinesis blocked proliferation indices of melanoma cells following

juglone treatment. Each value represents mean ± SE of three experiments.

Juglone (μM) Frequency / 1000 binucleates

CBPI Mononucleates Polynucleates

0 277.30 ± 2.02 123.00 ± 2.30 1.88 ± 0.001

0.5 352.00 ± 2.88a 113.30 ± 1.85 1.83 ± 0.003d

1 410.30 ± 1.20b 107.00 ± 3.21a 1.80 ± 0.002d

2.5 498.30 ± 2.30c 96.30 ± 3.17b 1.74 ± 0.003d

5 2513.30 ± 15.60d 57.60 ± 2.60d 1.31 ± 0.002d

10 8121.30 ± 25.80d 37.00 ± 3.05d 1.11 ± 0.0004d

Significant levels - a = P<0.05; b = P<0.01; c = P<0.001; d = P<0.0001 in comparison

to vehicle-treated control.

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Treatment of melanoma cells with juglone resulted in a significant

concentration-dependent increase in the frequency of mononucleate cells evidenced

by the 9-fold and 29-fold increase in the number of mononucleate cells (in

comparison to vehicle-treated control) at 5 μM and 10 μM concentrations of juglone

respectively (Table 3.2). Conversely, the frequency of binucleate cells as well as

polynucleate cells decreased in a concentration dependent manner in comparison to

vehicle treated controls. The CBPI, which is considered an index of cell proliferation,

showed a significant decrease, in a concentration-dependent manner.

3.3.2.3. Comet assay

Exposure of melanoma cells to juglone resulted in a concentration dependent

increase in the Olive tail moment (OTM) (Figure 3.8 and Figure 3.9). The increase in

Olive tail moment after treatment with juglone was statistically significant in

comparison to vehicle-treated controls at concentration above 2.5 µM. Treatment with

0.5, 1, 2.5, 5 and 10 μM concentrations of juglone, resulted in a 1.69, 2.44, 5.09, 7.24

and 11.2-fold increase in the OTM values respectively. The concentration-dependent

increase in the OTM values fitted well to linear model with R2-value of 0.9469.

Figure 3.8. Genotoxic effect of juglone assessed using alkaline comet assay.

Significant levels - * - P<0.05; ** - P<0.01; *** - P<0.001 compared to controls

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Figure 3.9. Representative comet images A - Control (Untreated); B juglone treated

(lower doses of 5 µM); C higher damage levels in cells treated with 10 µM juglone.

3.3.3. Juglone induced oxidative stress

3.3.3.1. Juglone induced changes in GSH levels

Experiments were designed to understand the role of juglone- induced

oxidative stress by measuring GSH levels, a major intracellular antioxidant enzyme.

Figure 3.10. Effect of juglone treatment on the intracellular glutathione levels after

treatment with various doses of juglone in melanoma cells. Significant levels - *

P<0.01; ** P<0.001, compared to vehicle-treated controls.

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Figure 3.10 shows the changes in GSH levels upon treatment of melanoma

cells with different doses of juglone. Treatment with juglone resulted in a

concentration dependent drop in GSH levels, which were statistically significant

(P<0.01 to P<0.001) in comparison to vehicle-treated control. Treatment of

melanoma cells with 5, 10, 15 and 20 µM doses of juglone resulted in a 37%, 58 %,

71 % and 83 % drop in mean intracellular GSH levels respectively. The

concentration-dependent drop in GSH levels was linear with an R2-value of 0.97.

3.3.3.2. Juglone induced changes in intracellular ROS levels

The results of DCFH-DA assay are depicted in Figure 3.11. As can be seen

from the figure, there was a significant dose dependent elevation in the DCF

fluorescence (indicative of intracellular ROS levels) up to 10 µM concentrations of

juglone. Exposure of melanoma cells to 2.5, 5, 7.5 and 10 µM doses of juglone caused

a 2.54, 4.71, 8.32 and 10.12 fold increase in the mean DCF fluorescence respectively.

Figure 3.11. Effect of juglone treatment on the intracellular ROS levels after

treatment with various doses of juglone in melanoma cells. Significant levels - *

P<0.05; ** P< 0.01; *** P<0.001, compared to vehicle-treated controls.

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3.3.3.3. Correlation studies between cell survival and GSH levels

The correlation between cell survival and GSH levels was analyzed at

different concentrations of juglone (Figure 3.12 A). It was observed that, as the GSH

levels dropped, there was a corresponding drop in the cell survival. The relationship

between cell survival and GSH levels fitted well into the linear model with an R2-

value of 0.9860 showing an excellent linear relationship.

The analysis of the correlation between GSH depletion and LDH leakage

(Figure 3.12 B) at different concentration of juglone was a lso performed where an

inverse relation between the LDH leakage into the cytoplasm and extent of GSH

depletion was observed which followed a dose dependent fashion. This relationship

fitted well into the linear model with R2-value of 0.9681 indicating a good linear

relationship.

Figure 3.12. Correlation studies after treatment with juglone A) between cell survival

and GSH levels and B) between GSH levels and LDH levels

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3.3.4. Juglone induced Apoptosis/Necrosis

3.3.4.1. Altered nuclear morphology induced by juglone: Fluorescence microscopic

study

The nuclei of untreated control cells were found to be intact, round in shape

and were stained green. On the other hand, cells treated with 5 μM juglone (for 12 h),

had some cells stained green and some stained red. Membrane blebbing, chromatin

condensation as well as nuclear fragmentation was evident in juglone treated cells

indicating apoptotic mode of cell death. t a higher dose of juglone (10 μM), an

increase in the number cells with red colored nuclei (necrotic/late apoptotic) was

observed (Figure 3.13).

Figure 3.13. Morphological changes induced by juglone assessed using Acridine

orange/Ethidium bromide staining a) control showing live (L) cells b) 5 µM Juglone

for 12 h showing early apoptotic (EA) as well as low percentage of necrotic (N) cells

c) 10 µM Juglone for 12 h showing higher percentage of Late apoptotic (LA) and

necrotic (N) cells as well as early apoptotic (EA) cells

3.3.4.2. DNA fragmentation induced by juglone

To further ascertain whether apoptosis could be the mechanism by which

juglone induces cytotoxic effect, melanoma cells were treated with juglone for various

time periods and the extracted DNA was resolved on agarose gel. The results of the

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DNA fragmentation/ladder assay are shown in Figure 3.14. DNA of melanoma cells

which were exposed to juglone revealed typical oligonucleosomal ladder pattern.

DNA laddering was seen as early as 12 h after treatment of melanoma cells with

juglone, and the intensity of these ladders increased in a time dependent manner (data

not shown).

Figure 3.14. Agarose gel of electrophoresis of DNA from melanoma cells Lane 1 3

kb DNA marker (GeNeiTM Low Range DNA Ruler); Lane 2 Untreated cells; Lane

3 and 4 cells treated with 5 and 10 μM juglone for 12 h. DN fragmentations with a

ladder pattern are considered characteristic of apoptosis

3.3.4.3. Increase in sub-G0 fraction induced by juglone

There was a dose- as well as time-dependent increase in the sub-Go

(hypodiploid) fraction (Figure 3.15). fter 12 h exposure to 5 μM and 10 μM juglone,

the percentage of cells having DNA content < 2n were 3.77 % and 50.75 %

respectively. The corresponding values for 24 h exposure were 26.27 % and 74.08 %.

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Figure 3.15. Flow cytometric analysis of DNA content as an index of juglone-

induced apoptosis. Top panel: percentage of apoptotic cells (sub-Go hypodiploid

fraction) after 12 h incubation with juglone; Bottom panel: percentage of apoptotic

cells (sub-Go hypodiploid fraction) after 24 h incubation with juglone.

3.3.4.4. Juglone induced late apoptosis and necrosis

Phosphotidyl serine translocation from inner part of plasma membrane to outer

part is believed to be an early event in apoptosis. Binding of Annexin V to

phosphotidyl serine in presence of calcium ions, results in green fluorescence. During

late apoptosis or necrosis, owing to increased membrane permeability, PI also enters

the cell and binds to cellular DNA and stains the nucleus red. Results from the present

study (Figure 3.16) shows that juglone treatment resulted in significant elevation in

the percentage of Annexin V-FITC (+)/PI(+) cells (upper right quadrant), both in a

time and concentration dependent manner indicating late apoptotic/secondary necrotic

death. About 30.64% cells were present in upper right quadrant after treatment with 5

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μM juglone for 12 h. This figure drastically increased to 67 % after 24 h incubation

with juglone. owever, at a higher dose (10 μM) of juglone, no significant time-

dependent increase was observed in the percentage of Annexin V-FITC(+)/PI(+) cells.

Figure 3.16. Flow cytometric analyses of apoptosis and necrosis using AnnexinV-

FITC/PI dual staining. Cells in lower right quadrant (AnnexinV-FITC(+)/PI(-))

represents apoptotic population and cells in upper right quadrant (AnnexinV-

FITC(+)/PI(+)) represents late apoptotic/necrotic population. Top panel: cells treated

with indicated concentration of juglone for 12 h; Bottom panel: cells treated with

indicated concentration of juglone for 24 h.

3.4. DISCUSSION

Cancer is one of the most prevalent diseases and can seriously threaten human

health. Despite advancement in diagnostic and operative procedures, cancer still

remains an enigma to researchers. The conventional methods currently used in cancer

treatment (like surgery, chemotherapy, radiotherapy) are indeed good but far from

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ideal. Although a variety of cancer chemotherapeutic drugs have been discovered,

treatment of most human tumors remains largely palliative. In cases where the

primary tumor has been surgically removed, chemotherapy is considered the best

treatment modality to prevent the tumor from metastasizing to distant organs.

However, the fundamental problem of cancer chemotherapy is the severe toxic side

effects of anticancer drugs on healthy tissues (Chabner and Roberts, 2005). In many

cases, cancer has to be treated by the combination of different treatment modalities,

which increase the probability of tumor cell-kill. Nonetheless, there is not yet a

conclusive treatment modality for cancer. Thus it is very essential to look for novel

compounds that possess better anticancer efficacy and acceptable toxicity. The

anticancer activity of plumbagin, a plant naphthoquinone, has been adequately studied

against various cells as well as animal tumor models (Krishnaswamy and

Purushothaman, 1980; Naresh et al., 1996; Prasad et al., 1996; Kini et al., 1997;

Singh et al., 1997; Singh and Udupa, 1997; Devi et al., 1998; Tiwari et al., 2002;

Srinivas et al., 2004). In the present study, an attempt has been made to elucidate the

anticancer activity of juglone (a structural analogue of plumbagin) and to understand

its underlying mechanism.

Walnut has been used in traditional medicines for various ailments and some

extracts of walnut are also reported to possess anticancer properties (Blumenthal,

1998). Juglone is reported to be the main active ingredient in walnut (Duke and

Ayensu, 1985). However, not much has been reported about the mechanisms

underlying the cytotoxic potential of juglone. In the present study, treatment of tumor

cells in vitro with juglone resulted in concentration-dependent loss of viability in all

tested cell types, which was statistically significant in comparison to the vehicle

treated controls, with human breast cancer (MCF7) cells being the most sensitive cell

line. Also, an insignificant time-dependent decrease in the cell viability was observed,

which is similar to earlier study carried out using HaCaT keratinocytes (Inbaraj and

Chignell, 2004). Earlier, Segura-Aguilar and co-workers (1992) compared the effect

of juglone and other quinones on human leukemic (HL-60) cells and doxorubicin-

resistant human leukemic (HL-60R) cells and concluded that multidrug resistance that

develops in the doxorubicin-resistant HL-60R cells had no effect on the cytotoxicity

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of juglone indicating its therapeutic possibilities. Similar observations were also made

by Li and co-workers (2010) where significant reduction in the cell proliferation was

observed in esophageal carcinoma (EC1) cells treated with juglone. More recently, Ji

and co-workers (2011) studied the cytotoxic potential of juglone against human

gastric cancer (SGC-7901) cell lines and observed a dose dependent reduction in cell

viability, which is similar to what was found from the present study, indicating the

cytotoxic potential of juglone.

The estimation of lactate dehydrogenase in cell culture supernatant is known

to provide a quantitative basis for the loss of cell viability and is frequently used to

estimate the cytotoxic effects of various drugs (Cook and Mitchell, 1989; Nehar et al.,

1998). Moreover, some recent studies suggest that LDH estimation may be a more

reliable and accurate marker of cytotoxicity, because damaged cells are fragmented

completely during the course of incubation with test substances (Grivell and Berry,

1996; Haslam et al., 2000). Since LDH is released only when the cell membrane is

damaged, it has in the past been used as an indirect indicator of the loss of cell

membrane integrity (Aalto et al., 1996; Riss and Moravec, 2004; Wolterbeek and van

der Meer, 2005). The LDH leakage assay was used in the present investigation to

confirm the results of MTT assay as well as to study if cell membrane damage could

possibly be the mode of cell death induced by juglone in melanoma cells. Treatment

of melanoma cells with juglone resulted in a significant concentration-dependent

increase in the LDH levels which supported the findings of MTT assay. This indicates

that the cytotoxicity of juglone against melanoma cells could be attributed, at least in

part, to the cell membrane damaging effects of juglone.

Clonogenic assay is an in vitro cell survival assay, which is used to test the

cytotoxic potential of a test agent. Any compound that can cause damage to the

clonogenic potential of the tumor cell could serve as a good anticancer agent. Various

drugs of plant origin like vincristine, taxol have been reported to cause a dose-

dependent reduction in tumor cell survival (Mujagic et al., 1983; Helson et al., 1993).

However, there are no previous reports on the effect of juglone on the clonogenic

survival in melanoma cells. Treatment of melanoma cells with juglone caused a dose-

dependent reduction in the cell survival, which was statistically significant in

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comparison to vehicle-treated controls. The survival curve for juglone against

melanoma cells had a linear coefficient (α) value of 0.00753 ± 0.0154 and with a “p

value” of 0.00012. These results indicate that the juglone severely affects the

clonogenic potential of melanoma cells.

Micronuclei are formed from whole or fragmented chromosomes that lag

behind during the cell division (Fenech, 2007). The micronuclei are expressed only

after one cell division. Therefore, micronuclei analysis is a good indicator of

clastogenesis and mutagenesis. Several plant-derived anticancer agents have been

shown to cause dose-dependent increase in the frequency of MN in various cell lines

(Reinecke et al., 1997), lymphocytes (Gonzalez-Cid et al., 1999) as well as in mouse

bone marrow (Choudhury et al., 2004). Further, several previous studies have also

shown a close association between reduction in clonogenic survival and induction of

micronuclei in various model systems (Geard and Chen, 1990; Mariya et al., 1997). In

order to study if the observed reduction in the clonogenic survival of melanoma cells

after treatment with juglone is related to its genotoxic potential, the cytokinesis

blocked micronucleus assay was performed. Exposure of melanoma cells to juglone

caused a concentration-dependent increase frequency of MN up to 10µM. The

increased micronuclei frequency indicates that a substantial part of the genome is lost

which may have a major impact on the cell division as well as cell death. Another

important feature of this assay is its ability to provide data regarding the proliferative

activity of a cell population. In the present study, treatment of melanoma cells with

juglone caused a significant delay in cellular proliferation as evidenced by the dose-

dependent reduction in CBPI values (Table 3.2). Based on results of the present study,

the genotoxic potential of juglone may have contributed to the observed dose

dependent reduction in clonogenic survival of melanoma cells.

To study the ability of juglone to induce oxidative stress in melanoma cells,

the GSH and the DCFH-DA assays were performed. GSH is an important intracellular

reducing agent, which is responsible for the maintenance of antioxidant molecules and

the thiol groups on intracellular proteins (Hwang et al., 2002). GSH is also the most

important biomolecule protecting against chemically induced cytotoxicity, by

participating in the elimination of reactive intermediates by conjugation and

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hydroperoxide reduction, or of free radicals by direct quenching (Jewell et al., 1986;

Liu et al., 2002). Therefore, analysis of changes in intracellular GSH status, produced

by toxic compounds may provide important information about the mechanism of

toxicity of the test compound. A significant concentration-dependent drop in GSH

levels was observed after treatment with juglone indicating the induction of oxidative

stress. These results are consistent with a recently reported study where juglone and

plumbagin were shown to cause oxidative stress in keratinocytes (Inbaraj and

Chignell, 2004).

Juglone- induced oxidative stress was further confirmed by measuring the

intracellular levels of ROS using DCFH-DA assay. The result of DCFH-DA assay

gives an idea about the ability of juglone to induce formation of intracellular ROS,

which substantiated the findings of GSH studies. A concentration-dependent depletion

of GSH levels with corresponding elevation in intracellular ROS levels after treatment

with juglone in the present study indicates that oxidative stress could be an important

mechanism by which juglone exerts its toxic activity against melanoma cells as well.

In general, toxicity of quinones is known to depend mainly on two mechanisms such

as, the redox cycling resulting in production of semiquinone radicals and reactive

oxygen species leading to depletion of glutathione in the ce lls (Inbaraj and Chignell,

2004), the former leading to the latter. Further, the structure-activity relationship

studies on the toxicity of naphthoquinones have revealed that 1,4-naphthoquinones

with a hydroxyl group at position 5 (as seen in plumbagin and juglone) or at position

5- and 8- (as in naphthazarin) in the aromatic ring showed an appreciable increase in

the toxicity against rat hepatocytes (Ollinger and Brunmark, 1991) compared to 1,4-

naphthoquinone itself due to increased efficiency of redox cyc ling. On the other hand,

methyl substitution of 1, 4 naphthoquinone at position 2 (as in plumbagin and

menadione) is reported to reduce its cytotoxicity owing to lower rate of redox cycling,

or lower rate of electrophilic addition (Ross et al., 1986).

Correlation studies reveals a strong correlation between surviving fraction and

GSH levels, which suggests that juglone- induced reduction in clonogenic survival,

could be due to GSH depletion/oxidative stress induced by juglone on melanoma

cells. Bravard and co-workers (2002) reported a strong correlation between GSH and

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surviving fraction in case of B α] P cell lines following treatment with γ-radiation (6

Gy). A good linear inverse correlation (R2-value of 0.96) between GSH levels and

LDH levels was also seen, suggesting that GSH depletion/oxidative stress induced by

juglone could be one of the reasons behind cell membrane damaging effects of

juglone.

Apoptosis and/or necrosis are among the key mechanisms by which most

compounds exert their cytotoxic effects, especially anticancer agents (Renvoize et al.,

1998). Overload of intracellular ROS has been known to induce apoptosis or necrosis

or the combination of both (Higuchi, 2003; Pelicano et al., 2004). Many of the other

well-known cytotoxic /anticancer agents belonging to anthracyclins, alkylating agents,

epipodophyllotoxins and camptothecins are known to induce apoptosis through

oxidative stress-mediated mechanisms (Conklin, 2004). Using different assays, an

attempt was made to ascertain whether the cell death induced by juglone in melanoma

cells could be due to apoptosis and/or necrosis. Microscopic staining revealed

membrane blebbing, chromatin condensation and/or fragmentation, which are

considered to be hallmarks of apoptosis. The increase in the number of cells stained

red with or without fragmented DNA at higher doses of juglone suggests the

possibility of late apoptotic as well as necrotic cell death. Also, gel electrophoresis of

extracted DNA revealed characteristic oligonucleosomal ladder, which further

confirms the mechanism of apoptosis. The DNA content analysis using flow

cytometry also revealed prominent (dose- and time-dependent) sub-Go hypo-diploid

fraction (< 2n DNA content), which is again considered to be associated with

apoptosis (Renvoize et al., 1998). Flow cytometric analysis using Annexin V-FITC/PI

dual staining showed the ability of juglone to induce time as well as concentration

dependent increase in the percentage of late apoptotic/necrotic cells. Earlier, Cenas

and co-workers (2006) studied the effect of several chemicals including juglone

against a couple of cancer cell lines in vitro where, juglone was shown to be a potent

inducer of apoptosis. More recently, juglone was shown to induce ROS mediated

apoptosis in human gastric cancer cells (SGC-7901) through a mitochondrial

pathway, involving up-regulation of Bax, down-regulation of Bcl-2 levels, decreased

mitochondrial membrane potential and increased cytochrome C release (Ji et al.,

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2011). In another recent study, juglone was reported to cause apoptotic mode of cell

death in human esophageal cancer cells (EC1) (Li et al., 2010). However, results from

the present study demonstrate that the cell death caused by juglone in melanoma cells

proceeds via a bimodal mechanism involving both apoptosis as well as necrosis.

Traditionally, necrotic cell death is recognized as an unregulated form of cell

death. However, few of the recent studies have shown that necrosis could be self-

determined fate of cell, which could also be induced via several mechanisms such as

stress stimuli, ion channel activation or DNA damage. Moreover, in cases where

apoptotic pathways are deficient or absent (as seen in cancer), necrosis is known to

occur as an alternate form of cell death (Sun et al., 2006). Recent literature illustrates

that, the use of compounds which specifically induce necrosis in tumor cells could

also be a promising therapeutic strategy to counter the defective apoptotic pathways

typically found in cancerous tissue. Other structurally related naphthoquinones with

established anticancer activity such as β- lapachone, shikonin etc are also known to

exert their cytotoxic activity via necrosis as their predominant mechanism (Sun et al.,

2006; Han et al., 2007). In view of this, juglone induced apoptotic and necrotic cell

death observed in this study using chemoresitant melanoma cells may have its clinical

relevance.

In addition, juglone is known to be a potent inhibitor of Pin1 (a unique

peptidyl-prolyl isomerases (PPIases) which accelerates the cis-trans conversion of

peptide bonds preceding prolyl residues, that can cause alterations in protein

conformation) via covalent modification of thiol groups of cysteine residues in

parvulin, one of which is essential for PPIase activity (Chao et al., 2001). Pin1 has

also been reported to play a key role in the regulation of cell cycle function in concert

with proline-directed kinases, phosphatases, and ubiquitin ligases. Given that Pin1 is

overexpressed in many human cancers (Fila et al., 2008) and also the fact that juglone

can act directly on tubulin and inhibit its polymerization, juglone might present an

attractive opportunity for controlling tumor growth.

In conclusion, this study for the first time has shown the potential of juglone to

reduce the clonogenic survival as well as the ability to induce micronuclei in

Chapter 3

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melanoma cells grown in vitro. Besides, the study also demonstrates that juglone-

induced cell death proceeds via multifactorial mechanisms such as induction of

oxidative stress, cell membrane damage and genotoxic effect ultimately resulting in

cell death both by apoptosis and necrosis. Juglone may also be categorized as a

potential anticancer compound based on the criteria established by the National

Cancer Institute (Pisha et al., 1995) that any compound with IC50 value of ≤ 4 µg ml

may be categorized as potential anticancer compound. In view of these present

findings, and the existing reports of its use in traditional folk medicine as an

anticancer agent, juglone deserves further studies to justify its potential as an

anticancer agent, using a spectrum of preclinical models.

Chapter 3

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