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Silver nanoparticles induce apoptotic cell death in

Candida albicans through the increase of hydroxyl radicals

In-sok Hwang1, Juneyoung Lee1, Ji Hong Hwang1, Keuk-Jun Kim2 and Dong Gun Lee1

1 School of Life Sciences and Biotechnology, Kyungpook National University, Buk-gu, Daegu, Korea

2 Department of Clinical Pathology, Tae Kyeung College, Gyeongsan-si, Korea

Keywords

antifungal activity; apoptosis;

Candida albicans ; hydroxyl radicals; silver

nanoparticles

Correspondence

D. G. Lee, School of Life Sciences and

Biotechnology, College of Natural Sciences,Kyungpook National University, Daehak-ro

80, Buk-gu, Daegu 702-701, Korea

Fax: +82 53 955 5522

Tel: +82 53 950 5373

E-mail: [email protected]

(Received 25 November 2011, revised 19

January 2012, accepted 8 February 2012)

doi:10.1111/j.1742-4658.2012.08527.x

Silver nanoparticles have been shown to be detrimental to fungal cells

although the mechanism(s) of action have not been clearly established. In

this study, we used Candida albicans cells to show that silver nanoparticles

exert their antifungal effect through apoptosis. Many studies have shown

that the accumulation of reactive oxygen species induces and regulates the

induction of apoptosis. Furthermore, hydroxyl radicals are considered an

important component of cell death. Therefore, we assumed that hydroxylradicals were related to apoptosis and the effect of thiourea as a hydroxyl

radical scavenger was investigated. We measured the production of reactive

oxygen species and investigated whether silver nanoparticles induced the

accumulation of hydroxyl radicals. A reduction in the mitochondrial mem-

brane potential shown by flow cytometry analysis and the release of cyto-

chrome c from mitochondria were also verified. In addition, the apoptotic

effects of silver nanoparticles were detected by fluorescence microscopy

using other confirmed diagnostic markers of yeast apoptosis including

phosphatidylserine externalization, DNA and nuclear fragmentation, and

the activation of metacaspases. Cells exposed to silver nanoparticles

showed increased reactive oxygen species and hydroxyl radical production.

All other phenomena of mitochondrial dysfunction and apoptotic fea-

tures also appeared. The results indicate that silver nanoparticles possess

antifungal effects with apoptotic features and we suggest that the hydroxyl

radicals generated by silver nanoparticles have a significant role in mito-

chondrial dysfunctional apoptosis.

Introduction

It has been known since ancient times that silver and

its compounds are effective antimicrobial agents

[1,2]. In the 19th century, microbial infections were

treated with 0.5% AgNO3, which was also used for

the prevention of infections in burns. When the era

of antibiotics began with the discovery of penicillin,

the use of silver slowly declined [3]. Currently,

due to the appearance of micro-organisms insensitive

to conventional drugs, the use of silver for treating

infections has once again gained importance. How-

ever, the use of silver ions has one major drawback;

they are easily inactivated by complexation and pre-

cipitation and the use of silver ions has therefore

been limited [4].

Here, silver nanoparticles (nano-Ag), which are not

electrocharged, can be a valuable alternative to ionic

Abbreviations

DHR-123, dihydrorhodamine; DiOC6(3), 3,3¢-dihexyloxacarbocyanine iodide; FITC, fluorescein isothiocyanate; H2O2, hydrogen peroxide;

HPF, 2-[6-(4¢-hydroxy) phenoxy-3H -xanthen-3-on-9-yl]-benzoic acid; nano-Ag, silver nanoparticles; MIC, minimum inhibitory concentration;

PI, propidium iodide; ROS, reactive oxygen species; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

FEBS Journal 279 (2012) 1327–1338 ª 2012 The Authors Journal compilation ª 2012 FEBS 1327

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silver [5]. Nano-Ag are clusters of silver atoms that

range in diameter from 1 to 100 nm and are attracting

interest as antibacterial and antimicrobial agents. In

particular, because of recent advances in research on

metal nanoparticles, nano-Ag have received special

attention as a possible antimicrobial agent. Nano-Ag

are known to be a nontoxic and safe antibacterialagents for the human body. In addition, nano-Ag have

also been reported to possess antifungal activity [6],

anti-inflammatory properties [7], antiviral activity [8]

and anti-angiogenic activity [9]. Although the antimi-

crobial effects of nano-Ag are well known, their

mechanisms of action have been addressed only spo-

radically in the literature. Recent studies have shown

that nano-Ag interact with three main components

of micro-organisms to produce the antimicrobial

effect: the membrane or cell wall [6,10], DNA [11]

and microbial proteins [10]. In addition, there is

substantial evidence that nano-Ag produce reactive

oxygen species (ROS) [12]. The accumulation of

intracellular ROS is well known as an important reg-

ulator of apoptosis accumulating in the early apopto-

sis phase [13]. Subsequently, the level of intracellular

ROS accumulation increases, which initiates mito-

chondrial fragmentation [14]. Some other studies

have shown that hydroxyl radicals are linked to cell

death [15]. Because apoptosis is one of the mecha-

nisms of cell death, we investigated whether there

are any connections between apoptosis and hydroxyl

radicals.

Candida albicans is probably one of the most suc-

cessful opportunistic pathogens in humans. Underconditions of a weakened immune system, colonizing

C. albicans can become opportunistic, causing recur-

rent mucosal infections and life-threatening contagious

infections with high mortality rates. Furthermore,

the number of known multidrug resistant bacteria

and fungi is increasing rapidly. Thus, the development

of more effective antifungal therapies is of great

importance. Understanding the mechanisms and deci-

sions of cell death in fungi may provide new develop-

ments in the search for diverse novel antifungal

nanoparticles.

According to previously reported studies, nano-Ag

possess antifungal effects and cell-cycle analysis has

shown significantly arrested cell cycles during the

G2 ⁄ M phase [6]. There are many studies showing

G2 ⁄ M-phase-mediated apoptosis [16]. For these rea-

sons, we investigated whether nano-Ag could exert

apoptotic cell death in C. albicans and found a rela-

tionship between mitochondrial dysfunction and

hydroxyl radicals, which was induced by nano-Ag,

during apoptotic cell death.

Results

Intracellular ROS accumulation

In a previous study, nano-Ag showed anticandidal

activity against C. albicans (Fig. 1). This substance

exhibited a minimum inhibitory concentration (MIC)value of 2 lgÆmL)1, which was as efficient as that of

3 mm hydrogen peroxide (H2O2) on C. albicans (data

not shown). We used H2O2 as a positive control to

determine programmed cell death [16].

ROS are continuously formed because of cellular

oxygen metabolism. Recent studies have suggested that

the accumulation of ROS induces and regulates the

induction of apoptosis in metazoans and yeasts [17].

Therefore, to determine the production and accumula-

tion of intracellular ROS induced by nano-Ag, we

chose to use the ROS-sensitive dye dihydrorhodamine

(DHR-123), which has been used previously as a gen-

eral indicator of cellular ROS levels. Multiple ROS

directly oxidize DHR-123 to the highly stable, fluores-

cent derivative rhodamine-123 in such a way that an

increase in the fluorescent signal reflects ROS produc-

tion [18]. Cells treated with nano-Ag exhibited high

ROS levels compared with untreated cells. In the posi-

tive control, there was a significant increase in the

amount of fluorescence when the cells were treated

with H2O2 (Fig. 2).

First, we investigated the activity of nano-Ag

for chemically generated ROS. The iron-catalyzed

Fig. 1. Transmission electron micrograph of the nano-Ag used in

this work. The bar marker represents 20 nm.

Silver nanoparticles induce apoptotic cell death I.-s. Hwang et al.

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Haber–Weiss process is known to be a promoter of

oxygen radicals under aerobic conditions. Ferritin, the

iron storage protein, is the principal reservoir for iron

within the cell [19]. For this reason, we used ferrous

perchlorate as a positive control in solely aqueous

solution. To detect hydroxyl radicals (•OH) formed

in the Fenton reaction, we used the fluorescent dye

2-[6-(4¢-hydroxy)phenoxy-3H -xanthen-3-on-9-yl]-benzoic

acid (HPF). The fluorescence intensity did not increase

upon the addition of H2O2 alone, but did increase

substantially upon the addition of nano-Ag or ferrous

perchlorate in the presence of H2O2. The results clearly

show that nano-Ag could transmute H2O2 into •

OH(Fig. 3A). We thought that nano-Ag induces apoptotic

cell death through the formation of highly ROS such

as •OH.

We examined •OH formation with HPF, which is

oxidized by •OH with high specificity, because hydro-

xyl radicals have been suggested to be a crucial com-

ponent of apoptosis in many studies [20]. Consistent

with the increase in intracellular ROS, the level of

intracellular •OH was markedly increased in nano-Ag-

treated cells (Fig. 3B). These results indicate that ROS

induced by nano-Ag accumulated in the interior of

C. albicans cells, and most were converted into the

strong oxidant •OH, considered to be a significant fac-

tor in aging and apoptosis in yeast cells. To demon-

strate that thiourea acts as an •OH scavenger, we also

treated cells exposed to nano-Ag with thiourea. Thio-

urea significantly reduced •OH formation in nano-Ag

treated cells (Fig. 3B,c). We used thiourea in subse-

quent experiments to show the effect of decreased

hydroxyl radicals on mitochondria-mediated apoptotic

cell death.

Measurement of mitochondrial membrane

potential (DWm)

In many systems, apoptosis is associated with loss of

the mitochondrial inner membrane potential (DWm),

which may be regarded as a limiting factor in the

apoptotic pathway. Reduction of DWm is among thechanges encountered during the early reversible stages

of apoptosis and is preceded by cytochrome c release

in several cell types [21,22].

To investigate whether nano-Ag decreased DWm, we

used the mitochondria-specific voltage-dependent dye

3,3¢-dihexyloxacarbocyanine iodide, DiOC6(3), which

aggregates inside healthy mitochondria and fluoresces

green. When the mitochondrial membrane depolarizes,

the dye no longer accumulates and is distributed

throughout the cell, resulting in a decrease in green flu-

orescence. The results show that nano-Ag-treated cells

had a decreased DWm, which was in agreement with

the pattern induced by H2O2 treatments as the positive

control (Fig. 4A). However, cells that were treated

with nano-Ag and thiourea did not undergo substan-

tial changes (Fig. 4A,a).

We performed the mitochondrial DWm assay with

JC-1 to verify our results. JC-1 has advantages over

other cationic dyes in that it can selectively enter the

mitochondria and reversibly change color from red to

green as the membrane potential decreases. In healthy

cells with high mitochondrial DWm, JC-1 spontaneously

forms complexes known as J-aggregates with intense

red fluorescence. However, in apoptotic or unhealthy

cells with low DWm, JC-1 remains in the monomericform, which shows only green fluorescence [23]. The

ratio of green to red fluorescence is dependent only on

the membrane potential and not on other factors such

as mitochondrial size, shape and density, which may

influence single-component fluorescence signals. Flow

cytometric analysis of JC-1 fluorescence is best per-

formed using 2D green versus red fluorescence plots.

As shown in Fig. 4B, both nano-Ag and H2O2 treat-

ments induced a significant decrease in DWm, whereas

the combined treatment with nano-Ag and thiourea

appeared to have only a slight effect. Therefore, the

results suggest that nano-Ag induced the breakdown of

DWm, which is a critical step in cells undergoing apop-

tosis, and the loss of mitochondrial permeability. This

result suggests that restriction of •OH formation helps

maintain the balance of the mitochondrial membrane.

Cytochrome c release

Translocation of cytochrome c from the mitochondria

to the cytosol is a pivotal event in apoptotic cell death.

Fig. 2. Flow cytometric analysis of ROS accumulation in nano-Ag

(blue) and H2O2 (red solid line) treated C. albicans cells stained with

DHR-123.

I.-s. Hwang et al. Silver nanoparticles induce apoptotic cell death


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Cytochrome c, a soluble protein electrostatically bound

to the outer face of the inner mitochondrial mem-

brane, is an essential component of the respiratory

chain acting as an electron carrier between the cyto-

chrome bc1 and cytochrome c oxidase complex [24].

We assumed that cytochrome c would be detected

in the cytosol because of the results of the previ-

ous mitochondrial membrane potential assay. In this

regard, we investigated whether nano-Ag-treated

cells could induce cytochrome c release from the mito-

chondria. A large amount of cytochrome c was

detected in the cytosolic buffer medium following the

nano-Ag-treated cells, although cytochrome c rarely

appeared in supernatants that were additionally treated

with thiourea (Fig. 4C). These results show that

nano-Ag induced the release of cytochrome c from the

mitochondria and suggest that the mitochondria of

nano-Ag-treated cells, which suppressed the formation

of •OH by thiourea, are not directly affected by the •OH.

Annexin V–propidium iodide double staining

The early stages of apoptotic phenomenon can be

detected with fluorescein isothiocyanate (FITC)–

Annexin V staining, which binds to phosphatidylserine

with high affinity in the presence of Ca2 + [25], com-

bined with the membrane-impermeable dye propidium

iodide (PI). Phosphatidylserine is only distributed in

the inner leaflet of the lipid bilayer of the plasma

membrane, which is maintained by the ATP-binding

Fig. 3. (A) Detection of hydroxyl radicals in

the Fenton reaction using HPF (final 5 lM;

0.1% dimethylformamide as a cosolvent).

The fluorescence intensity was determined

at 515 nm with excitation at 490 nm. Nano-

Ag (lower solid line) and ferrous perchlorate

(upper dotted line) were added at 40 s. (B)Flow cytometric analysis of the formation of

hydroxyl radicals in C. albicans using the

dye HPF. (a) Control, (b) cells exposed to

nano-Ag, (c) cells exposed nano-Ag with

thiourea, (d) cells exposed to H2O2.



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cassette transporters in C. albicans. To determine whether

nano-Ag could induce apoptotic features, the FITC–

Annexin V and PI double-staining method was used.

As shown in Fig. 5, the cell population in the lower

right (LR) quadrant, which corresponds to the percent-

age of early apoptotic cells (Annexin V-positive and

PI-negative), increased to 35.87% and 45.37% after

treating the cells with nano-Ag and H2O2, respectively,

for 1 h. Curiously, the percentage of nano-Ag treated

with thiourea did not increase significantly as when

treated solely with nano-Ag. To show the distinct dif-

ference, we drew a bar graph showing the percentage

of apoptotic cells at the bottom. These results demon-

strate that it is possible for nano-Ag to induce apopto-

tic cell death in C. albicans cells. Hence, it was

confirmed that the generation and accumulation of

intracellular ROS, specifically hydroxyl radicals,

induced by nano-Ag was related to an apoptotic mech-

anism in C. albicans cells.

Measurement of DNA damage

To further confirm the apoptotic features induced in

nano-Ag-treated C. albicans cells, a terminal deoxynu-

cleotidyl transferase-mediated dUTP nick end labeling

(TUNEL) assay was conducted to detect apoptotic

DNA fragmentation by labeling 3¢-OH termini

with modified nucleotides catalyzed by terminal

deoxynucleotidyltransferase. The labeling of breaks in

the DNA by TUNEL, a reliable method for the

identification of apoptotic cells, is utilized to visualize

the apoptotic phenotype of cells [26].

A strong blue fluorescence indicated a greater degree

of typical apoptotic DNA condensation and fragmen-

tation in the nuclei of C. albicans cells exposed to

nano-Ag than in the intact nuclei of normal control

cells. 4¢-6-Diamidino-2-phenylindole staining of the

nano-Ag-treated cells showed the distributed nuclear

fragments (Fig. 6A). Similar results were obtained by

A a

a

a b c d

b c d

b

B

C

Fig. 4. (A) Loss of the mitochondrial inner membrane potential in C. albicans induced by treatment with nano-Ag (a), and H2O2 (b) for 1 h. In

each panel, the untreated control is the black background peak and the red solid lines represent individual treatment with nano-Ag or H2O2

only. Nano-Ag treatment with thiourea is shown by the blue solid lines (a). Cells were stained with DiOC6 and the fluorescence was mea-

sured by flow cytometry. A decrease in fluorescent signal (shift to the left) corresponds with a loss in the mitochondrial membrane potential.

(B) Quantitative mitochondrial membrane potential of C. albicans stained by JC-1 and measured by FACS. The area under the horizontal line

displays cells with decreased membrane potential. (a) Control, (b) cells exposed to nano-Ag, (c) cells exposed to nano-Ag with thiourea, (d)

cells exposed to H2O2. (C) Detection of cytochrome c released from C. albicans mitochondria following the incubation with nano-Ag. Cytosol

was ultracentrifuged and the supernatants were subjected to SDS ⁄ PAGE and western blotting for released cytochrome c . The untreated

control (lane a) or cells cultured in nano-Ag (lane b), nano-Ag treated with thiourea (lane c), and H 2O2 (lane d).



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TUNEL assay staining of the breaks in the DNAnuclear strands during the late stages of apoptosis.

TUNEL-positive cells, which showed a strong green

fluorescence or intense green fluorescent spots, were

observed in the population treated with nano-Ag

(Fig. 6B). In untreated cultures, the nucleus appeared

as a single round spot in the cells (Fig. 6A,a) or did

not show up well against the backgrounds (Fig. 6B,a).

Candida albicans is known to activate programmed

cell death with features reminiscent of apoptosis in

response to a variety of environmental stimuli such as

H2O2 [26–28]. For this reason, we used cells treated

with H2O2 as a positive control. Supporting our obser-

vations, exposure of C. albicans cells to nano-Ag

resulted in apoptotic DNA damage. Furthermore, we

ascertained the oxidative stress-protecting effects of

thiourea.

Measurement of metacaspase activation

Caspases are typically activated in the early stages of

apoptosis and they play a central role in the apoptotic

signaling network. Although caspases are not presentin fungi, orthologs of caspases in animals, termed

metacaspases, have been identified in fungi and plants,

and their activity can be assessed using the same detec-

tion marker [29,30]. In order to confirm metacaspase

activation, cells were incubated with the CaspACE

FITC–VAD–FMK in situ marker that binds to

the active site of metacaspases, and detected using a

fluorescence microscope. Cells with intracellular active

metacaspases stained fluorescent green, whereas nona-

poptotic cells appeared unstained. Fluorescence analysis

of the cells treated with nano-Ag showed a significant

green fluorescence in the FITC–VAD–FMK-loaded

cells that was consistent with the positive control trea-

ted with H2O2 (Fig. 6C). In addition, the number of

activated metacaspases decreased, which also reduced•OH formation in thiourea-treated cells (Fig. 6C,c), as

expected. These results suggest that nano-Ag treatment

did initially lead to significant generation of strong

oxidant hydroxyl radicals, which are well-known to be

important regulators of yeast apoptosis, and then the

hydroxyl radicals activated the metacaspases.

Fig. 5. Effect of nano-Ag on the exposition of phosphatidylserine at the cytoplasmic membrane. C. albicans cells. Protoplasts were har-

vested, stained with FITC–Annexin V and PI, and observed with a FACS. The bottom bar graph shows the percentage of apoptotic cells. (A)

Control, (B) cells exposed to nano-Ag, (C) cells exposed nano-Ag with thiourea, (D) cells exposed to H2O2.



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Discussion

Apoptosis is a highly regulated cellular suicide pro-

gram crucial for development and homeostasis in

metazoan organisms, resulting in the removal of

unwanted, mutated, damaged or simply dispensable

cells without an inflammatory reaction occurring

[31,32]. Apoptosis has been accepted as a process that

is not exclusive to multicellular organisms, but rather

is a universal mechanism of cell elimination operating

according to a basic program, including in simpler and

more ancient forms of single-celled eukaryotes. The

full apoptotic program comprises two phases, one of

which has necrotic features [33]. Therefore, we ana-

lyzed the more definitive signs of the apoptosis process

in this study.

ROS, such as O2

, H2O2 and •OH, are considered to

be crucial regulators of aging, and their accumulation

has been proven to play a key role in apoptosis [17].

We used DHR-123 to determine ROS accumulation

during exposure to nano-Ag in C. albicans. Nano-Ag-

treated cells displayed increased intracellular ROS lev-

els compared with untreated cells (Fig. 2). In addition,

ROS damaged iron–sulfur clusters, making ferrous

iron available for oxidation by the Fenton reactionand these events appear to be mediated through the

Tricarboxylic acid cycle and the transient depletion of

NADH [34]. The Fenton reaction leads to •OH forma-

tion, and •OH damages DNA, proteins and lipids,

resulting in cell death. •OH is the neutral form of the

hydroxide ion. •OH is highly reactive and consequently

causes damage to oxidative cells. The Haber–Weiss

reaction generates •OH from H2O2 and superoxide

(O2

) [19]. This reaction can occur in cells and is

therefore a possible source of oxidative stress. The

reaction is very slow, but is catalyzed by iron. For this

reason, we thought it possible that nano-Ag induces•OH formation as an iron catalyst. As expected, the

fluorescence intensity increased substantially upon

the addition of nano-Ag in the presence of H2O2(Fig. 3A). After that, we examined the intracellular

levels of hydroxyl radicals treated with nano-Ag and

tried to learn how the thiourea impacts •OH accumula-

tion in C. albicans cells treated with nano-Ag. We used

thiourea as a scavenger of •OH. Thiourea is a potent•OH scavenger that has an established means of

A

a a

b

c

d

a

b

c

d

b

c

d

B C

Fig. 6. DNA and nuclear fragmentation were shown by 4¢-6-diamidino-2-phenylindole (A) and TUNEL (B) staining. Effect of nano-Ag on the

activity of metacaspase in C. albicans (C). Nano-Ag-treated cells were collected, stained and observed under a fluorescent microscope. (a)

Control, (b) cells exposed to nano-Ag, (c) cells exposed nano-Ag with thiourea, (d) cells exposed to H 2O2.



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mitigating the effects of •OH damage in both eukary-

otes and prokaryotes [35–37]. The results showed that

C. albicans cells treated with nano-Ag produced hydro-

xyl radicals, and thiourea was accompanied by a

reduction in •OH formation (Fig. 3B).

Several other studies have linked cytochrome c

release, ROS formation and changes in the mitochon-drial membrane potential to yeast apoptosis [38,39].

During apoptosis, the decrease in DWm is caused by

the opening of membrane pores that are located in the

mitochondrial membrane. Consequently, the decrease

in DWm leads to the translocation and activation of

various proapoptotic factors. Reduction of the mito-

chondrial inner membrane potential (DWm) is among

the changes encountered during the early reversible

stages of apoptosis and is related to cytochrome c

release [21,22]. Thus, we determined DWm. The results

showed that mitochondrial permeability in nano-Ag-

treated cells was damaged by the breakdown of DWm(Fig. 4A,B). By contrast, cells with hydroxyl radical

accumulation inhibited by thiourea did not show sub-

stantial changes. The contents of cytochrome c

released into the cytosol and mitochondrial membrane

depolarization were measured to understand the influ-

ence of substances on the intrinsic pathway. Cyto-

chrome c, which is located in the mitochondrial

membrane, is released into the cytosol during the early

phases of apoptosis and a caspase-cascade is then acti-

vated as a representative of the other apoptotic prote-

ase [40]. As a result of defects in the mitochondrial

electron transport system, cytochrome c is reduced

when it is released into the cytosol because of the lossof the cytochrome c oxidase activity. Upon the release

of cytochrome c into the cytoplasm, the protein binds

to apoptotic protease-activating factor [38]. The release

of cytochrome c requires an increase in the permiabili-

ty of the mitochondrial outer membrane. The increase

in the mitochondrial transmembrane potential, which

has been predicted to promote osmotic matrix swell-

ing, is associated with one model for cytochrome c

release from the mitochondria during apoptosis.

Because the mitochondrial inner membrane, with its

numerous cristae, has a considerably larger surface

area than that of the outer membrane, expansion of

the inner membrane upon matrix swelling can break

the outer membrane, which would be expected to trig-

ger the release of cytochrome c to the cytosol [41].

Treatment with nano-Ag enhanced the content of cyto-

solic cytochrome c in C. albicans cells (Fig. 4C), sug-

gesting that nano-Ag may trigger cytochrome

c-mediated intrinsic apoptosis. As expected, the addi-

tion of thiourea to nano-Ag-treated cells, which do not

produce hydroxyl radicals ordinarily, exhibited reduced

cytochrome c release compared with those treated with

only nano-Ag. Thus, we believe that nano-Ag induces

apoptosis through the formation •OH and that •OH is

important to the apoptotic process.

Furthermore, we investigated a series of normally

apoptotic properties including the exposition of phos-

phatidylserine, DNA and nuclear fragmentation, andthe activity of metacaspases finally.

To discriminate between apoptotic and necrotic

cells, FITC–Annexin V and PI double staining were

used [25]. Candida albicans cells exposed to nano-Ag

stained Annexin V-positive and PI-negative, which was

similar to the response to H2O2, an inducer of apopto-

sis in yeast cells (Fig. 5). However, cells exposed

nano-Ag with thiourea showed decreasing apoptotic

features, which seemed to be protected by the thiourea.

In addition, we treated cells with nano-Ag and

monitored the proportion of cells positively stained for

4¢-6-diamidino-2-phenylindole and TUNEL staining to

study the development of the apoptotic phenotype,

including DNA and nuclei change (Fig. 6A,B). Finally,

cells exposed to nano-Ag exhibited metacaspase

activity, but cells treated with nano-Ag and thiourea

did not show any activity (Fig. 6C). These phenomena

indicate that nano-Ag induces apoptosis in C. albicans

and that highly reactive hydroxyl radicals are impor-

tant to apoptosis triggered by nano-Ag.

In conclusion, this study demonstrated for the first

time that nano-Ag promotes apoptosis in C. albicans

through phosphatidylserine exposure, DNA damage

and the activation of metacaspases. Ultimately, nano-

Ag disrupts the mitochondrial integrity and inducescytochrome c release. Although the mechanisms of

nano-Ag in mitochondria-dependent apoptosis in

C. albicans have not been fully elucidated, this report

supports that nano-Ag induces programmed cell death

through ROS accumulation, especially •OH. As shown

in Fig. 3, nano-Ag had the ability to generate •OH

and cells treated with thiourea decreased •OH produc-

tion. Consequently, the reduction in •OH accumulation

contributed to diminished mitochondrial dysfunction-

mediated apoptosis. We conclude that nano-Ag induce

apoptotic cell death in C. albicans through •OH gener-

ation, which deserves further study to provide elabora-

tion on the apoptosis mechanisms of nano-Ag.

Materials and methods

Reagents and culture conditions

The H2O2 and thiourea used in this study were purchased

from the Sigma Chemical Co. (St. Louis, MO, USA).



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Nano-Ag were stored at 4 C. Candida albicans (ATCC

90028) cells were cultured in YPD broth (Difco, Franklin

Lakes, NJ, USA) containing yeast extract, peptone and

dextrose (50 gÆL)1) with aeration at 28 C.

Preparation of nano-Ag

One hundred grams of solid silver were dissolved in

100 mL of 100% nitric acid at 90 C, and 1 L of distilled

water was added. By adding sodium chloride to the silver

solution, the Ag ions were precipitated and then clustered

together to form monodispersed nanoparticles in an aque-

ous medium. The sizes and morphology of the nano-Ag

were examined by TEM (H-7600; Hitachi Ltd, Tokyo,

Japan). The results showed that nano-Ag was spherical in

form and its average size was 3 nm (Fig. 1). Because the

final concentration of colloidal silver was 60 000 p.p.m.,

this solution was diluted, and then used to investigate the

apoptotic antifungal effects.

Intracellular ROS accumulation

Intracellular ROS production and the accumulation of

hydroxyl radicals (•OH) were measured using the fluores-

cent dye DHR-123 and HPF. In a previous study, nano-

Ag showed significant antifungal activity at low concentra-

tions, which was similar to the level of amphotericin B [6].

Since then, we have determined the most efficient

concentration of H2O2 for the induction of apoptosis [16].

Cells (2 · 108ÆmL)1) were treated with 2 lgÆmL)1 nano-Ag

and 3 mm H2O2 for 1 h at 28 C, based on the MIC value

as a criterion (data not shown). After incubation, the cells

were washed with NaCl ⁄ Pi before being stained with5 lgÆmL)1 DHR-123 and analyzed using a FACSCalibur

flow cytometer (Becton Dickinson, San Jose, CA, USA).

The reactivity of nano-Ag for ROS was compared with

ferrous perchlorate [Fe(ClO4)2], which was used as the

Fenton reaction. We tried to detect •OH formed in the

Fenton reaction, using HPF. Five micromoles of HPF was

added to sodium phosphate buffer (0.1 m, pH 7.4) containing

3 mm H2O2 and then 2 lgÆmL)1 nano-Ag or 100 lm

ferrous perchlorate was added. The •OH formation was

detected as an increase in HPF fluorescence by a Spectro-

fluorometer (Shimadzu RF-5301PC; Shimadzu, Japan) at

490 nm excitation and 515 nm emission wavelength.

The intracellular •

OH accumulation was measured byincubating the cells with 2 lgÆmL)1 nano-Ag and 3 mm

H2O2 in NaCl ⁄ Pi containing 5 lm using the dye HPF for

1 h at 28 C. Subsequently, the cells were washed twice in

NaCl ⁄ Pi and analyzed by flow cytometry [42]. For the •OH

quenching experiments, 150 mm of thiourea was added

simultaneously with nano-Ag. Thiourea has been used

at mm levels in vitro as a •OH scavenger [43]. Thiourea was

used for all subsequent tests.

Measurement of mitochondrial membrane

potential (DWm)

Fungal mitochondrial membrane depolarization was ana-

lyzed by DiOC6(3) staining. Cells (2 · 108ÆmL)1) were har-

vested and incubated with 2 lgÆmL)1 nano-Ag and 3 mm

H2O2 for 1 h at 28

C. Subsequently, the cells were washedwith NaCl ⁄ Pi and incubated with 2 ngÆmL

)1 of DiOC6(3)

for 30 min. Cells were analyzed by flow cytometer.

JC-1 (Molecular Probes, Carlsbad, CA, USA) is a mito-

chondrial dye (5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢-tetraethyl-benz-

imidazolylcarbocyanine chloride) that stains mitochondria

in living cells in a membrane potential-dependent fashion.

JC-1 was also used to confirm the decrease in membrane

potential and the number of mitochondria specifically. Cells

(2 · 108ÆmL)1) were treated with 2 lgÆmL)1 nano-Ag and

3 mm H2O2 for 1 h at 28 C. Treated cells were washed in

NaCl ⁄ Pi, suspended in 200 lL staining solution containing

2 lgÆmL)1 of JC-1 for 20 min at 37 C. The cells were

centrifuged at 500 g for 5 min and then the pellet wasresuspended with 1 mL NaCl ⁄ Pi. Cells were then analyzed

by flow cytometer.

Cytochrome c release

To investigate cytochrome c release from the mitochondria,

isolations of mitochondria were prepared [44]. Candida albi-

cans cells were cultured in 500 mL of YPD medium for 24 h

at 30 C, collected by centrifugation at 500 g, and washed

twice with NaCl ⁄ Pi and once with 1 m sorbitol. These cells

were treated with 2 lgÆmL)1 nano-Ag and 3 mm H2O2 for

2 h at 28 C. The treated cells were lysed with lysis buffer

(150 mm sodium chloride, 1% Triton X-100, 1 mm EDTA,1 mm EGTA, 50 mm Tris, pH 8) and then centrifuged at

2000 g for 10 min to remove the cell debris and unbroken

cells. The supernatants were collected and centrifuged at

40 000 g for 1 h. The supernatants were collected to assay

for cytochrome c released from the mitochondira to the

cytoplasm. The protein content of these supernatants was

estimated using a NanoVue Plus Spectrophotometer (GE

Healthcare, Little Chalfont, Buckinghamshire, UK). Each

sample equivalent to 50 lg of protein was resolved on 12%

SDS ⁄ PAGE. Separated proteins were transferred to a nitro-

cellulose membrane and analyzed by western blotting with

rabbit polyclonal anti-(yeast cytochrome c) [45]. Horseradish

peroxidase-linked goat anti-(rabbit IgG) was used as thesecondary antibody, and enhanced-chemiluminescence sub-

strate was used for the detection of cytochrome c.

Annexin V–PI double staining

Protoplasts of C. albicans were stained with FITC-labeled

Annexin V and PI using the FITC–Annexin V apoptosis

detection kit. Cells (2 · 108ÆmL)1) were digested for 1 h at



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28 C in a potassium phosphate buffer (pH 6.0) containing

20 mgÆmL)1 lysing enzyme and 1 m sorbitol. Protoplasts

were incubated with 2 lgÆmL)1 nano-Ag and 3 mm H2O2for 1 h at 28 C, based on the MIC value as a criterion,

and incubated for 20 min in an Annexin-binding buffer

containing 5 lL FITC–Annexin VÆmL)1 and PI. Protop-

lasts were then examined by a FACSCalibur flow cytometer

(Becton Dickinson).

Measurement of DNA damages

DNA strand breaks in C. albicans cells were analyzed by

TUNEL [46]. Cells (2 · 108ÆmL)1) treated for 2 h with

2 lgÆmL)1 nano-Ag and 3 mm H2O2, were washed in

NaCl ⁄ Pi, permeabilized for 2 min on ice and washed again

with a NaCl ⁄ Pi. DNA ends were labeled with an in situ cell

death detection kit for 1 h at 37 C. The stained cells were

observed with a fluorescence microscope.

Nuclear condensation and fragmentation were analyzed

by 4¢-6-diamidino-2-phenylindole staining [47]. Cells were

treated with 2 lgÆmL)1 nano-Ag and 3 mm H2O2 for 2 h

and then collected. For nuclear staining, cells were washed

twice with NaCl ⁄ Pi, permeabilized in a permeabilization

solution (0.1% Triton X-100 and 0.1% sodium citrate) and

incubated with 1 lgÆmL)1 of 4¢-6-diamidino-2-phenylindole

in the dark for 20 min. Cells were then examined by a fluo-

rescence microscope.

Measurement of metacaspase activation

Activated metacaspases in C. albicans were measured using

the CaspACE FITC–VAD–FMK in situ marker (Pro-

mega). Briefly, each substance treated cell was washed inNaCl ⁄ Pi, suspended in 200 lL staining solution containing

10 lm of CaspACE FITC–VAD–FMK in situ marker

and incubated for 30 min at room temperature in the dark.

Cells were then washed once and suspended in NaCl ⁄ Pi.

Sample analysis was performed with a fluorescence micro-

scope, the Axio Imager A1, and Axio Cam MR5 (Carl Zeiss,

Thornwood, NY, USA).

Acknowledgements

This work was supported by the National Research

Foundation of Korea (NRF) grant funded by the Korea

government (MEST) (No. 2011-0000915) and by theNext-Generation BioGreen 21 Program (No. PJ008158),

Rural Development Administration, Republic of Korea.

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