Báo cáo khoa học: 2-Amino-nonyl-6-methoxyl-tetralin muriate activity against Candida albicans augments endogenous reactive oxygen species production – a microarray analysis study doc

2-Amino-nonyl-6-methoxyl-tetralin muriate activity against
Candida albicans augments endogenous reactive oxygen
species production – a microarray analysis study
Rong Mei Liang
1,2
, Xiao Lan Yong
2
, Yun Ping Jiang
2
, Yong Hong Tan
3
, Bao Di Dai
1
,
Shi Hua Wang
3
, Ting Ting Hu
2
, Xi Chen
2
, Nan Li
2
, Zhao Hui Dong
2
, Xiao Chun Huang
2
, Jun Chen
2
,
Yong Bing Cao
1

and Yuan Ying Jiang
1
1 Drug Development Center, School of Pharmacy, Second Military Medical University, Shanghai, China
2 Department of Clinical Pharmacy, General Hospital of Chengdu Military Command Region, Chengdu, China
3 Department of Pharmacy, General Hospital of Chengdu Military Command Region, Chengdu, China
Introduction
Candida infections have become a serious medical
problem, because of the high incidence and mortality
in AIDS patients, transplant recipients and other
immunosuppressed individuals [1–4]. Despite conti-
nuous expansion of the arsenal of antifungal drugs,
the antifungal drugs available cannot meet the increas-
ing requirements for managing infections in medically
complex patients.
2-Amino-nonyl-6-methoxyl-tetralin muriate (10b), a
2-aminotetralin derivative, was synthesized as an anti-
fungal agent with a novel chemical structural (Fig. 1)
[5]. Tetralin derivatives are known to be potentially
Keywords
10b; action mechanism; Candida albicans;
microarray analysis; ROS
Correspondence
Y. B. Cao and Y. Y. Jiang, Drug
Development Center, School of Pharmacy,
Second Military Medical University, 325
Guohe Road, Shanghai 200433, China
Fax: +86 021 6549 0641
Tel: +86 021 8187 1357;
+86 021 8187 1275
E-mail: ;

(Received 21 October 2010, revised 14
December 2010, accepted 18 January 2011)
doi:10.1111/j.1742-4658.2011.08021.x
Candida infections have become an increasingly significant problem, mainly
because of the widespread nature of Candida and drug resistance. There is
an urgent need to develop new classes of drugs for the treatment of oppor-
tunistic Candida infections, especially in medically complex patients. Previ-
ous studies have confirmed that 2-amino-nonyl-6-methoxyl-tetralin muriate
(10b) possesses powerful antifungal activity in vitro against Candia albicans.
To clarify the underlying action mechanism, an oligonucleotide microarray
study was performed in C. albicans SC5314 without and with 10b treat-
ment. The analytical results showed that energy metabolism-related genes,
including glycolysis-related genes (PFK1, CDC19 and HXK2), fermenta-
tion-related genes (PDC11, ALD5 and ADH1) and respiratory electron
transport chain-related genes (CBP3, COR1 and QCR8), were downregu-
lated significantly. Functional analysis revealed that 10b treatment
increased the generation of endogenous reactive oxygen species, and
decreased mitochondrial membrane potential, ubiquinone–cytochrome c
reductase (complex III) activity and intracellular ATP levels in C. albicans
SC5314. Also, addition of the antioxidant ascorbic acid reduced the anti-
fungal activity of 10b significantly. These results suggest that mitochondrial
aerobic respiration shift and endogenous reactive oxygen species augmenta-
tion might contribute to the antifungal activity of 10b against C. albicans.
This information may prove to be useful for the development of new
strategies to treat Candida infections.
Abbreviations
AA, ascorbic acid; CFU, colony-forming unit; DCFH-DA, 2,7-dichlorofluorescein diacetate; FI, fluorescence intensity; MCZ, miconazole;
ROS, reactive oxygen species; DW
m

, mitochondrial membrane potential; 10b, 2-amino-nonyl-6-methoxyl-tetralin muriate.
FEBS Journal 278 (2011) 1075–1085 ª 2011 The Authors Journal compilation ª 2011 FEBS 1075
applicable to psychiatry, and dialkylated tetralin
derivatives are the most effective [6]. Aminotetralins,
including 8-hydroxy-2-(di-n-propylamino)-tetralin and
7-hydroxy-2-(di-n-propylamino)-tetralin, behave as
preferential agonists at serotonin (5-hydroxytrypta-
mine)1A and dopamine D3 and D2 receptors [7]. The
former affects intracranial self-stimulation, and the lat-
ter possess anxiolytic properties. However, there are
few reports from other laboratories describing the anti-
fungal activities of 2-aminotetralin derivatives. Our
previous studies [5,8] indicated that 10b possessed high
antifungal activity. In an oophorectomized female SD
rat model of Candida albicans infections, 10b consis-
tently exhibited better antifungal activity than itraco-
nazole [5]. Also, 10b significantly reduced the ergostrol
content by inhibiting the activity of sterol C-14 reduc-
tase, encoded by ERG24 in the ergosterol biosynthetic
pathway [8]. However, C. albicans ERG24 is not
required for growth. The erg24 mutant of C. albicans
is capable of growth under normal aerobic conditions
on standard defined and enriched media. There is a
suggestion that the significant level of ergosta-8,14,22-
trienol accumulated by C. albicans erg24 mutants may
be the element that allows growth under normal condi-
tions [9], implying that the major action mechanism of
10b against C. albicans is not correlated with inhibition
of the activity of sterol C-14 reductase in the ergosterol
biosynthetic pathway.

The present study was a continuation of our previ-
ous studies, in an attempt to clarify the mechanism of
action of 10b against C. albicans through analyzing the
gene expression profiles of C. albicans SC5314 without
and with 10b treatment, using oligonucleotide micro-
array assays and real-time RT-PCR assays. It was
found that a series of differentially expressed genes
were involved in energy metabolism, oxidoreduction
and other biological functions. In addition, measure-
ments of endogenous reactive oxygen species (ROS)
generation, mitochondrial membrane potential (DW
m
),
intracellular ATP concentration, respiratory electron
transport chain complex III (ubiquinone–cytochrome c
reductase) activity and the effect of antioxidant ascor-
bic acid on the antifungal activity of 10b suggested
that the antifungal activity of 10b against C. albicans
might be related to mitochondrial aerobic respiration
shift and endogenous ROS augmentation.
Results
Response of gene expression to 10b exposure
A total of 957 differentially expressed genes were found
upon exposure to 10b; the expression of 457 genes was
decreased, and the expression of 500 genes was
increased. Forty-five downregulated genes and nine
upregulated genes were found to be related to energy
metabolism. Of the 45 downregulated genes, 34 were
involved in glycolysis (e.g. PFK1, CDC19 and HXK2),
fermentation (e.g., PDC11, ALD5 and ADH1), the

respiratory electron transport chain (e.g. CBP3, COR1
and QCR8) and ROS scavenging (e.g. GPX2). Of the
nine upregulated genes, five were related to fermenta-
tion (e.g. ADH3 and ADH5) and ROS scavenging (e.g.
GPX1, SOD5 and SOD6). An additional 29 upregulated
genes and 15 downregulated genes were concerned with
lipid metabolism. Of the 29 upregulated genes, nine
were directly linked to ergosterol biosynthesis. In addi-
tion, 93 (20.35%) of the 457 downregulated genes were
related to translation, whereas only two genes in this
category were upregulated, suggesting that the transla-
tion level might be lower in SC5314 cells exposed to 10b
than in controls (Tables S1 and S2).
Validation of microarray data by real-time
RT-PCR
Knowing that augmentation of endogenous ROS pro-
duction was directly related to the antifungal activity
of some antifungal drugs [10–12], we were particularly
interested in energy metabolism-related genes. There-
fore, real-time RT-PCR analysis was designed to detect
genes related to energy metabolism. Real-time RT-
PCR reactions were performed in triplicate with
independent RNA isolations. As shown in Fig. 2, the
expression levels of glycosis-related genes, PFK1,
PFK2, HXK2 and CDC19, decreased significantly by
30.30-fold, 43.48-fold, 20.83-fold and 20.41-fold,
respectively; the expression levels of fermentation-
related genes, ALD5, ADH1 and PDC11, decreased
significantly by 15.63-fold, 2.20-fold and 83.33-fold
respectively; and the expression levels of genes coding

for mitochondrial respiratory chain complex III, CBP3
(333.33-fold), COR1 (9.62-fold), QCR2 (8.70-fold),
QCR8 (5.43-fold), CYT1 (11.24-fold) and RIP1 (12.5-
fold), were also markedly decreased. Also, the
expression level of GPX2 decreased significantly, by
12.05-fold, whereas the expression levels of SOD5 and
GPX1 increased by 38.686-fold and 5.433-fold, respec-
tively. In addition, the expression levels of ADH3 and
ADH5, two fermentation-related genes, also increased
CH
3
O
NH(CH
2
)
8
CH
3
.HCl
Fig. 1. Chemical structure of 10b.
10b and endogenous reactive oxygen species R. M. Liang et al.
1076 FEBS Journal 278 (2011) 1075–1085 ª 2011 The Authors Journal compilation ª 2011 FEBS
significantly, by 10.382-fold and 3.212-fold, respec-
tively.
ROS production in C. albicans treated with 10b
Before measurement of the level of ROS production,
drug concentrations inhibiting growth to 80% of con-
trol levels were estimated by interpolation, and this
concentration for 10b was 0.5 lgÆmL
)1

. The level of
endogenous ROS production induced by 10b was mea-
sured with concentrations of 1, 3 and 9 lgÆmL
)1
.
Because miconazole (MCZ) is known to be an excel-
lent endogenous ROS inducer [10], it was selected as
the positive control to investigate the effect of 10b on
endogenous ROS production in C. albicans SC5314.
As shown in Fig. 3, after treatment with 9 lgÆmL
)1
10b, the level of ROS production increased by 22.7-
fold in a dose-dependent manner. MCZ treatment also
augmented ROS production, but to a lesser extent.
Effects on DW
m
, complex III activity and ATP
content in mitochondria of C. albicans after 10b
treatment
Treatment with 10b caused DW
m
degradation in a
dose-dependent manner (Fig. 4A), which was opposite
to the result obtained for endogenous ROS produc-
tion. Although the generation of ROS is exponentially
dependent on DW
m
[13], dysfunction of proton pumps
can disrupt the positive correlation between endoge-
nous ROS production and DW

m
[14]. Therefore, we
determined the activity of two important proton
pumps, complex III and complex I (NADH–ubiqui-
none reductase), which are the main sources of ROS in
mitochondria. As was expected, complex III activity
decreased in a dose-dependent manner after 10b treat-
ment (Fig. 4B). The inhibitory efficiencies of 10b at
3 lgÆmL
)1
and 9 lgÆmL
)1
were 25.43% and 57.26%,
respectively, after 9 h of exposure. However, no signifi-
cant difference in complex I activity was observed
between the control group and the 10b group (data
not shown). It was therefore assumed that 10b treat-
ment inhibited complex III activity, resulting in proton
pump inactivation and a decrease in DW
m
. Because
intracellular ATP generation is known to be positively
correlated with DW
m
in C. albicans under normal cul-
ture conditions, the intracellular ATP concentration in
cells without and with 10b was also measured. The
results revealed a dose-dependent decrease in intracel-
lular ATP generation, which was consistent with the
result obtained for DW

m
(Fig. 4C).
–10
–9
–8
–7
–6
–5
–4
–3
–2
–1
0
1
2
3
4
5
6
7
ADH1
ALD5
PDC11
CDC19
HXK2
PFK1
PFK2
CBP3
COR1
RIP1

CYT1
QCR8
QCR2
COX4
GPX2
SOD5
GPX1
ADH3
ADH5
Log2 ratio
Fig. 2. Changes in gene expression levels
of 19 energy metabolism-related genes in
10b-treated C. albicans SC5314. The con-
centration of 10b was 3 lgÆmL
)1
. All genes
were examined by real-time RT-PCR with
gene-specific primers. Relative fold change
was calculated with the C
T
value (see
details in Experimental procedures). Results
are the mean ± standard deviations for
three independent experiments.
0
Control MCZ 10b
5000
10 000
15 000
20 000

25 000
Fluorescence intensity
1 µg·mL
–1
3 µg·mL
–1
9 µg·mL
–1
**
*
*
**
*
*
Fig. 3. Endogenous ROS generation in C. albicans SC5314 cells
without and with 10b and MCZ. The concentrations of 10b and
MCZ were 1, 3 and 9 lgÆmL
)1
. ROS levels represent the
mean ± standard deviations for three independent experiments.
Statistically significant differences (as determined by Student’s
t-test, as compared with control): *P < 0.01; **P < 0.05.
R. M. Liang et al. 10b and endogenous reactive oxygen species
FEBS Journal 278 (2011) 1075–1085 ª 2011 The Authors Journal compilation ª 2011 FEBS 1077
Effects of ROS on the antifungal activity of 10b
against C. albicans
To determine whether ROS production was directly
involved in the antifungal effect of 10b, the effect of
an antioxidant on the net level of ROS production and
antifungal activity was observed in 10b-treated cells.

The net ROS production in cells induced by 10b treat-
ment was inhibited by addition of the antioxidant
ascorbic acid (AA) in a dose-dependent manner, with
complete inhibition occurring at 10 mm (Fig. 5). We
then examined whether AA treatment interfered with
the antifungal effect of 10b. In a colony formation
assay, 9 lgÆmL
)1
10b caused a cytostatic effect
(approximately 72% inhibition) at 48 h. AA treatment
prevented the colony-inhibitory effect induced by 10b
in a dose-dependent manner (Fig. 6).
Discussion
To further investigate the mechanism of action of 10b
at a molecular level, an oligonucleotide microarray
study was performed in C. albicans SC5314 without
and with 10b treatment. The results showed that differ-
entially expressed genes were involved in multiple bio-
chemical functions. Many experiments have confirmed
that ROS play a central role in yeast signaling and
apoptotic death [15–19]. In addition, they can damage
a wide range of molecules, including nucleic acids,
proteins and lipids, that are involved in a variety of
key events leading to cell death [20–22]. Damage to
0
20
40
60
80
100

120
AB
C
10b concentration (µg·mL
–1
)
Ratio (red fluorescence/
green fluorescence)
**
*
*
0
20
40
60
80
100
120
10b concentration (µg·mL
–1
)
Complex III activity
(% of control)
*
**
0
100
200
300
400

500
600
Control 9
13
13
9
Control 1 3 9
10b concentration (µg·mL
–1
)
ATP concentration (nM)
**
*
*
Fig. 4. Mitochondrial functional analysis in
C. albicans SC5314 treated or untreated
with 10b: (A) DW
m
. (B) Complex III activity.
(C) Intracellular ATP level. The concentra-
tions of 10b were 1, 3 and 9 lgÆmL
)1
. DW
m
,
complex III activity and ATP levels represent
the mean ± standard deviations for three
independent experiments. Statistically signif-
icant differences (as determined by Stu-
dent’s t-test, as compared with control):

*P < 0.01; **P < 0.05.
–5000
0
5000
10 000
15 000
20 000
25 000
ROS production (FI)
*
*
**
10b (9 µg·mL
–1
)
–++
AA (m
M)00
2.5
++
510
Fig. 5. Effect of AA on ROS production in 10b-treated C. albicans.
The concentrations of AA were 2.5, 5 and 10 m
M, and that of 10b
was 9 lgÆmL
)1
. Data represent the mean ± standard deviations for
three independent samples. Statistically significant differences (as
determined by Student’s t-test, as compared with control):
*P < 0.01; **P < 0.05.

10b and endogenous reactive oxygen species R. M. Liang et al.
1078 FEBS Journal 278 (2011) 1075–1085 ª 2011 The Authors Journal compilation ª 2011 FEBS
mitochondrial macromolecules may lead to increased
ROS production and further damage to mitochondrial
components, thus causing a ‘vicious downward spiral’
in terms of ROS production and damage accumulation
[23]. The antifungal activities of several compounds,
including ketoconazole, polygodial, histain 5, UK-2A
and UK-3A, are achieved by inhibiting the respiratory
activity of mitochondria [24–27].
Therefore, we were particularly interested in the
striking changes in the expression levels of energy
metabolism-related genes: glycolysis-related genes, fer-
mentation-related genes, respiratory electron transport
chain-related genes, and ROS scavenging-related genes.
According to the Pasteur effect, by which aerobic
oxidation can inhibit glycolysis (alcohol-facient
fermentation) under aerobic circumstances, the tricar-
boxylic acid cycling process might be enhanced follow-
ing exposure to 10b, because of the marked decreases
in the expression levels of the genes participating in
glycolysis, including those coding for rate-limiting
enzymes (HXK2, PFK1, PFK2 and CDC19). Also, sig-
nificantly downregulated genes were also involved in
fermentation (e.g. ADH1, ALD5 and PDC11) and
ROS scavenging (e.g. GPX2). Together, these changes
mean that endogenous ROS generation might be
strongly augmented, as shown in Fig. 7. Interestingly,
we also found that the expression levels of two ROS
scavenging-related genes (SOD5 and GPX1) and two

fermentation-related genes (ADH3 and ADH5) were
notably increased. This might be the result of feedback
control in response to high ROS levels.
Mitochondrial oxidative phosphorylation is a major
ATP synthetic pathway in eukaryotes, where electrons
liberated from reducing substrates are delivered to O
2
via a chain of respiratory proton pumps. These pumps
(complexes I, III and IV) establish a proton electro-
chemical gradient (proton concentration gradient and
DW
m
) across the inner mitochondrial membrane to
store energy for the production of ATP. Endogenous
ROS are derived from mitochondrial respiratory chain
electron leakage. The main source of ROS in mitochon-
dria is the ubisemiquinone radical intermediate (QHÆ),
which is formed during the ubiquinone cycle at the Q
o
site of complex III [28–30]. Complex I is also a source
of ROS, although the mechanism of generation is less
clear than that of complex III. In vitro, electrons enter-
ing complex II can flow backwards through complex I
to make ROS [31]. Experimentally, a large increase in
ROS formation is often seen in the condition known as
reverse electron flow [32]. In this study, we found that
10b treatment promoted ROS generation and decreased
DW
m
and ATP production in mitochondria of C. albi-

cans. The results of our further investigation showed
that 10b inhibited complex III activity. It is therefore
presumed that 10b inhibited mitochondrial complex III
activity, causing a reverse flow of electrons from com-
plex II to complex I, resulting in ROS augmentation.
Simultaneously, 10b blocked electron transport in the
mitochondrial respiratory chain and decreased DW
m
and ATP production (Fig. 8).
AA is a known antioxidant, and interactions
between AA and ROS may attenuate the oxidant effect
of ROS and alleviate ROS-induced damage to the
organism [33]. In this study, addition of AA signifi-
cantly reduced the antifungal activity of 10b against
C. albicans, indicating that the antioxidant could alle-
viate the oxidative damage caused to the organism by
endogenous ROS, allowing C. albicans to survive 10b
treatment. These results also imply that endogenous
ROS augmentation might be a major mechanism of
the activity of 10b against C. albicans.
The results of the present study demonstrate that
10b treatment could augment the production of
endogenous ROS via three different mechanisms in
C. albicans: (a) providing more electrons for the mito-
chondrial respiratory chain by enhancing the tricarbox-
ylic acid cycle; (b) attenuating ROS scavenging; and
(c) enhancing the reverse flow of electrons from
complex II to complex I by inhibiting complex III
activity. Increased ROS production contributes to the
antifungal effect by means of strong oxidative damage

to the organism. This biochemical process might
be involved in the mechanism of action of 10b against
C. albicans.
0
20
40
60
80
100
120
0 2.5 5 10
AA (mM)
Cell survival (% of 10b-
untreated cell)
**
*
*
Fig. 6. Effect of AA on 10b-induced colony inhibition in C. albicans.
The concentrations of AA were 2.5, 5 and 10 m
M, and that of 10b
was 9 lgÆmL
)1
. Cells were incubated at 30 °C under constant shak-
ing (200 r.p.m.) for 48 h, and the colonies were counted. The rate
of cell survival is represented as a percentage of the survival rate
for cells not treated with 10b. Data represent the mean ± standard
deviations for three independent experiments. Statistically signifi-
cant differences (as determined by Student’s t-test, as compared
with 10b treatment alone): *P < 0.01; **P < 0.05.
R. M. Liang et al. 10b and endogenous reactive oxygen species

FEBS Journal 278 (2011) 1075–1085 ª 2011 The Authors Journal compilation ª 2011 FEBS 1079
Experimental procedures
Strains and culture
C. albicans SC5314 was used throughout this study. The
antifungal reagents used in the present study included 10b
(Department of Medicinal Chemistry, School of Pharmacy
of the Second Military Medical University, Shanghai,
China), MCZ (Pfizer-Roerig Pharmaceuticals, New York,
NY, USA) and stock solutions of various concentrations
in dimethylsulfoxide. C. albicans cells were propagated in
yeast–peptone–dextrose) medium [1% (w ⁄ v) yeast extract,
2% (w ⁄ v) peptone, and 2% (w ⁄ v) dextrose].
RNA isolation and microarray hybridization
The number of cells was adjusted to 1.0 · 10
6
colony-form-
ing units (CFUs)ÆmL
)1
in yeast–peptone–dextrose medium,
divided into two parts: one was exposed to 3 lgÆmL
)1
10b,
and the other was used as the control. The cells were then
grown at 30 °C under constant shaking (200 r.p.m.) for an
additional 5 h for the control and 9 h for the 10b group,
until they reached 0.6–0.9 · 10
7
CFUsÆmL
)1
, as described

by Hughes [34]. Cells were then collected by centrifugation
at 3000 g for 5 min at room temperature, and frozen in
liquid nitrogen. We chose this 10b concentration because
it had an obvious inhibitory effect on C. albicans and
allowed for recovery of a sufficient cellular mass for RNA
extraction.
Total RNA was isolated by the hot phenol method, and
purified with a NecleoSpin Extract II kit (Machery-Nagel
Corp., Du
¨
ren, Germany) [35]. A 7925 C. albicans genome
70-mer oligonucleotide microarray was obtained from Capi-
talBio Corporation (Beijing, China). A 1-lg sample of total
RNA was used for preparing fluorescent dye-labeled cDNA
by linear mRNA amplification [36]. A DNAÆDNA hybrid-
ization protocol was used to replace RNAÆDNA hybridiza-
tion, to reduce cross-hybridization [37]. The labeled cDNAs
were dissolved in 80 lL of hybridization solution [3 · SSC,
0.2% (w ⁄ v) SDS, 5 · Denhardt’s solution, 25% (v ⁄ v) form-
amide], and denatured at 95 °C for 3 min before hybridiza-
tion. A sample of the mixed hybridization buffer was
placed onto a microarray slide and covered with a glass
coverslip. Hybridization was performed with a BioMixer II
Glucose
HXK2
Glc6P
ROS
GPX2
GSSG
PFK1

Cyt c
Glycolysis
PFK2
Fru6P
PGI1
GSH H
2
O or ROH + H
2
O
PFK1
Fru(1,6)P
2
GraP
LAC
Inner
CX Φ
CXΣ CX Υ
Q
e
–
e
–
e
–
e
–
PFK2
FBA1
TPI1

GrnP
Gri(1,3)P
2
Pyruvate
Inner
membrane
CX Τ
CX

Φ
NADH
e
–
GA3P
PEP
Aldehyde
FADH
2
Aldehy
de
ENO1
GPM1
GA2P
GPM2
O
2
Aldehyde
Acetic acid Ethanol
TCA cycle
Mitochondrion

y
ALD5
Acetic acid
Cytoplasm
Fermentation
ROS
Fig. 7. Central carbon metabolism in C. albicans SC5314. The gray rectangles indicate low expression genes coding for metabolic enzymes
of participated in energy metabolic process after 10b treatment. The black ellipse or circle indicate augmented the tricarboxylic acid (TCA)
cycling process and endogenous ROS generation after 10b treatment. Glc6P, glucose 6-phosphate; Fru6P, fructose 6-phosphate; Fru(1,6)P
2
,
fructose 1,6-bisphosphate; GraP, glyceraldehyde 3-phosphate; GrnP, dihydroxyacetone phosphate; Gri(1,3)P
2
, 1,3-bisphosphoglycerate;
GA3P, 3-phosphoglyceric acid; GA2P, 2-phosphoglyceric acid; PEP, phosphoenolpyruvate; LAC, lactic acid; CX I–V, complexes I–V; Q, ubiqui-
none cycle; Cyt c, cytochrome c.
10b and endogenous reactive oxygen species R. M. Liang et al.
1080 FEBS Journal 278 (2011) 1075–1085 ª 2011 The Authors Journal compilation ª 2011 FEBS
(CapitalBio Corp.). After hybridization, the slides were
washed with washing solution 1 (2 · SSC, 0.2% SDS) and
then with washing solution 2 (2 · SSC) at 42 °C for 4 min.
Self-hybridization of the control sample was used to evalu-
ate the system noise.
Microarray data processing
The microarrays were scanned with a LuxScan 10KAscan-
ner (CapitalBio Corp.) at two wavelengths to detect emis-
sions from both Cy3 and Cy5. The images obtained were
analyzed with luxscan 3.0 software (CapitalBio Corp.).
Normalization was performed on the basis of a Lowess
program. The Cy5 ⁄ Cy3 ratio was calculated for each loca-

tion on each microarray. To minimize artefacts arising from
low expression values, only genes with raw intensity values
of > 800 counts for both Cy3 and Cy5 were chosen for
analysis. Significance Analysis of Microarrays software
(sam) was used to identify significantly differentially
expressed genes. Genes with a false discovery rate < 5%, a
q-value <1% and variation of at least two-fold in C albi-
cans SC5314 following 10b exposure were identified as sig-
nificantly differentially expressed genes in two independent
experiments.
Differentially expressed genes were clustered hierarchi-
cally by gene cluster 3.0 (Stanford University). DNA
sequences were annotated on the basis of the results of
blastn and blastx searches with the sequencing database
of Stanford University (Palo Alto, CA, USA) (http://www-
sequence stanford.edu ⁄ group ⁄ Candida), GenBank (http://
www.ncbi.nlm.nih.gov/BLAST/), and the CandidaDB data-
base (teur. fr ⁄ CandidaDB ⁄ ). All of the
array data have been deposited in the NCBI Gene Expres-
sion Omnibus () and are acces-
sible through Gene Expression Omnibus series accession
number GSE19552.
Microarray data analysis
We used cgd gene ontology slim mapper to cluster these
differentially expressed genes into particular categories by
choosing GO Set Name ‘ Process’ (didagenome.
org/cgi-bin/GO/go TermMapper). The specific functions
of individual genes were determined from the Candida
Genome database ( />Quantitative real-time RT-PCR assay
Real-time RT-PCR was used to confirm the microarray

results for changes in gene expression. First-strand cDNAs
were synthesized from 1 l g of total RNA in a 20-lL reac-
tion volume, using the cDNA synthesis kit for RT-PCR
(TaKaRa Biotechnology, Dalian, China). Real-time PCR
reactions were performed with SYBR Green I (TaKaRa),
using the ABI 7500 Real-Time PCR System (Applied Bio-
systems, CA, USA). Gene-specific primers were designed
with discovery studio gene software (Accelrys, San
Diego, U.S.A). The thermal cycling conditions comprised
an initial step at 95 °C for 1 min, followed by 40 cycles at
95 °C for 10 s, 55 °C for 20 s, and 72 °C for 30 s. Changes
in SYBR Green I fluorescence in every cycle were monitored
by the system software, and the threshold cycle (C
T
) was
measured. With 18S rRNA as the internal control, gene
expression levels of C. albicans SC5314 cells treated with
10b relative to those without treatment were calculated with
the formula 2
)CT
. Primer sequences are listed in Table 1.
ΔΨm
H
+
Cyt c
H
+
H
+
ROS

Intermembrane
space/cytoplasm
Inner
CX
Σ C
X
Υ
CX
Φ
F
Q
QH
.
e
–
e
–
e
–
e
-
ROS
+
membrane
C
CX Τ
C
F
0
F

Q
QH
2
Q
NADH
e
–
e
-
e
–
CX V
–
F
1
TCA
NAD
+
NADH
10b
O
2
TCA
cycle
10b
ATP
+
–
Fig. 8. Proposed mechanism of action of ROS augmentation induced by 10b. Stimulation of the tricarboxylic acid cycle (TCA) by 10b treat-
ment enhances electron flow into the mitochondrial respiratory chain, and generates a reverse flow of electrons from complex II to

complex I by inhibiting complex III, which inhibits electron transport and causes a collapse of the proton gradient across the mitochondrial
inner membrane. These events enhance ROS generation and decrease DW
m
and ATP production. Dashed lines indicate the subdued meta-
bolic process. CX I–V, complexes I–V; Q, ubiquinone cycle; Cyt c, cytochrome c.
R. M. Liang et al. 10b and endogenous reactive oxygen species
FEBS Journal 278 (2011) 1075–1085 ª 2011 The Authors Journal compilation ª 2011 FEBS 1081
Measurement of ROS production
The endogenous amount of ROS was measured by a fluori-
metric assay with 2,7-dichlorofluorescin diacetate (DCFH-
DA) (Molecular Probes, Eugene, OR, USA). Briefly, cells
were first adjusted to an attenuance at 600 nm of 1.0 in
40 mL of NaCl ⁄ P
i
, treated without and with 2.5, 5 and
10 mm AA, and grown at 30 °C under constant shaking
(200 r.p.m.) for 1 h. After incubation with 1, 3 and
9 lgmL
)1
10b and MCZ at 30 °C under constant shaking
(200 r.p.m.) for 2 h, cells were collected by centrifugation
at 3000 g for 5 min, suspended in NaCl ⁄ P
i
, and adjusted to
an attenuance at 600 nm of 1.0 in 10 mL of NaCl ⁄ P
i
.
DCFH-DA 20 lm in NaCl ⁄ P
i
was added and incubated at

30 °C under constant shaking (200 r.p.m.) for 9 h. The flu-
orescence intensity (FI) of the resuspended cells were
detected on a Thermo Scientific Varioskan Flash (BMG,
Labtech, Offenburg, Germany), with an excitation wave-
length of 488 nm and an emission wavelength of 525 nm.
ROS production was calculated by subtracting the FI
for cells not treated with DCFH-DA from the FI for cells
treated with DCFH-DA.
Functional analysis on mitochondria – DW
m
,
complex III and complex I activity and
intracellular ATP content
For DW
m
measurement, cells treated without and with 10b
at 30 °C under constant shaking (200 r.p.m.) for 9 h were
washed and adjusted to 1 · 10
6
CFUsÆmL
)1
with NaCl ⁄ P
i
.
After being treated with 10 lgÆmL
)1
5,5¢,6,6¢-tetrachloro-
1,1¢,3,3¢-tetra-ethylbenzimidazolcarbocy-anine iodide (JC-1;
Molecular Probes, Eugene, OR, USA) at 30 °C under con-
stant shaking (200 r.p.m.) for 15 min [38], cells were

washed with NaCl ⁄ P
i
and analyzed on a Thermo Scientific
Varioskan Flash with an excitation wavelength of 485 nm
and an emission wavelength shifting from green ( 525 nm)
to red ( 590 nm). DW
m
was determined from red FI ⁄ green
FI ratio.
For complex III and complex I activity evaluation, cells
treated without and with 10b at 30 °C under constant shak-
ing (200 r.p.m.) for 9 h were adjusted to 5 · 10
8
cells per
mL. Mitochondria were isolated with the Yeast Mitochon-
dria Isolation Kit (GenMed Scientifics INC., Arlington,
U.S.A.) and lysed in EDTA buffer by ultrasound. The mito-
chondrial protein concentration was determined by the Brad-
ford method [39]. Complex III and complex I activity was
determined by colorimetry, with the Mitochondria Com-
plex III and Complex I Activity Assay Kit (GenMed Scienti-
fics INC., Arlington, U.S.A.). The absorbance was measured
on the Thermo Scientific Varioskan Flash at a wavelength of
550 nm and two wavelengths of 340 nm and 380 nm. One
unit of complex III activity was defined as the amount of
enzyme activity that oxidized 1 lmol of reduced ubiquinone
per minute at 30 °C and pH 7.5, and one unit of complex I
activity was defined as the amount of enzyme activity that
oxidized 1 lmol of NADH per minute at 30 °C and pH 7.5.
For intracellular ATP content determination, cells treated

without and with 10b at 30 °C under constant shaking
(200 r.p.m.) for 9 h were adjusted to 1 · 10
6
CFUsÆmL
)1
.
A 100-lL cell suspension was mixed completely with the
same volume of BacTiter-Glo reagent (Promega Corpara-
tion, Madison, WI, USA), and incubated for 10 min at
room temperature. Luminescent signals were determined on
aTD20⁄ 20 luminometer (Turner Biosystem, Sunnyvale,
CA, USA), with a 1-s integration time per sample. The
Table 1. List of primers used for real-time RT-PCR. F, forward; R,
reverse.
Target
genes Primer pairs (5¢-to3¢)
Amplicon
size (bp)
18S F: TCTTTCTTGATTTTGTGGGTGG
R: TCGATAGTCCCTCTAAGAAGTG
150
ADH1 F: ATCCCTGGTCTTATCTTC
R: AACTGGGTAATCCTTGTAG
184
ADH3 F: CTTTATTACCAATCCCTG
R: ATTTCTCAACCGCACC
225
ADH5 F: ATGCCGTATTGACTCCT
R: CTCTTGCCTTATCCTTT
162

ALD5 F: AAGCCGCATACCACAA
R: CCAACCACCACAGGAT
197
PDC11 F: GTGCCTTTATTGCTGAT
R: AGATTCTGGGTCGTTTG
182
GPX1 F: TGAAAGGGAAAGTTGTC
R: TCCAAGACTGGGAATGT
217
GPX2 F: ACTCCACAATACAAAGGTT
R: AATACGGGGAAAGTCAC
164
SOD5 F: ACATTGGCGGTTTATC
R: ATTACCTTGAGGAGCA
185
CDC19 F: CTGCTGCTTACGAACA
R: AATGGGTAGACACCTCTG
163
HXK2 F: CGGTTACTATTTGGGAGA
R: TTGGATGGATAAGAGGC
132
PFK1 F: AGTTGGCGGTGGTAAT
R: TTCGTAAACGGCATAA
142
PFK2 F: AGAAACCTGCCTCCTCA
R: CCAACCCTAATCTGTCG
177
CBP3 F: TAATGCCAATGAGAATG
R: TCAGGAGGCACAAACT
132

COR1 F: AACAACAACACCGTCAT
R: TTGGCAAAGTATCGTCT
160
RIP1 F: CGGTCAAGGAAGCAGAA
R: TTGGCAAAGTATCGTCT
110
CYT1 F: GCTATGGCTGAAGAAT
R: CTGGGAAGTAAGGGTT
280
QCR8 F: GCACCACATCCACATA
R: AAATGGAATGGCAACA
183
QCR2 F: CTGGGTGTATCTCATTT
R: CGAAAGTTCCACCTAAT
103
COX4 F: CCTCTTTGATTGGTCCTG
R: TTCACTGATTGGCATTT
137
10b and endogenous reactive oxygen species R. M. Liang et al.
1082 FEBS Journal 278 (2011) 1075–1085 ª 2011 The Authors Journal compilation ª 2011 FEBS
control tube without cells was used to obtain a value for
background luminescence. The signal-to-noise ratio was cal-
culated: signal-to-noise ratio = (mean of signal ) mean of
background) ⁄ standard deviation of background. A stan-
dard curve for ATP increments (from 1 lm to 10 pm) was
constructed. Signals represented the mean of three separate
experiments, and the ATP content was calculated from the
standard curve.
Colony formation assay
Drug sensitivity and the effect of AA on 10b fungicidal

activity were determined by a colony formation assay based
on the macrodilution reference method (M27-A) of the
Clinical and Laboratory Standards Institute (formerly the
National Committee for Clinical Laboratory Standards).
Briefly, cells were adjusted to 1 · 10
6
CFUsÆmL
)1
in
RPMI-1640 medium buffered with Mops, treated without
and with 2.5, 5 and 10 mm of AA at 30 °C under constant
shaking (200 r.p.m.) for 1 h, and then incubated with 10b
for 48 h under the same conditions. After incubation,
20 lL of appropriately diluted solution was used for colony
formation on Sabouraud dextrose agar (Becton Dickinson
Microbiology Systems, Oakvile, ON, Candida).
Statistical analysis
The statistical significance of differences was determined
with Student’s t-test. A P-value of < 0.05 was considered
to indicate significance.
Acknowledgements
We thank W. A. Fonzi for kindly offering the isolate
of C. albicans SC5314, and Y J. Zhou for synthesizing
the 10b used in this study.This work was supported by
the National Natural Science Foundation of China
(30825041, 30500628 and 30630071), the National
Basic Research Project (2005CB523105), National
High Technology Research, and Development Pro-
gram 863 of China (2008AA02Z302).
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Supporting information
The following supplementary material is available:
Table S1. Selected genes that are downregulated in
10b-grown C. albicans SC5314 as compared with
growth without treatment, determined in two indepen-
dent experiments.
10b and endogenous reactive oxygen species R. M. Liang et al.
1084 FEBS Journal 278 (2011) 1075–1085 ª 2011 The Authors Journal compilation ª 2011 FEBS
Table S2. Selected genes that are upregulated in 10b-
grown C. albicans SC5314 as compared with growth
without treatment, determined in two independent
experiments.

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R. M. Liang et al. 10b and endogenous reactive oxygen species
FEBS Journal 278 (2011) 1075–1085 ª 2011 The Authors Journal compilation ª 2011 FEBS 1085