Tumor Biol.

(2015) 36:3429–3439
DOI 10.1007/s13277-014-2978-6

RESEARCH ARTICLE

Reactive oxygen species, glutathione, and thioredoxin influence

suberoyl bishydroxamic acid-induced apoptosis in A549 lung
cancer cells
Bo Ra You & Suhn Hee Kim & Woo Hyun Park

Received: 2 September 2014 / Accepted: 11 December 2014 / Published online: 24 December 2014
# International Society of Oncology and BioMarkers (ISOBM) 2014

Abstract Suberoyl bishydroxamic acid (SBHA) as a histone Abbreviations

deacetylase (HDAC) inhibitor can induce apoptosis through SBHA Suberoyl bishydroxamic acid
the formation of reactive oxygen species (ROS). However, HDAC Histone deacetylase
there is no report about the regulation of ROS and antioxidant ROS Reactive oxygen species
enzymes in SBHA-treated lung cancer cells. Here, we inves- MMP (ΔΨm) Mitochondrial membrane potential
tigated the toxicological effects of SBHA on the regulations of MTT 3-(4,5-Dimethylthiazol-2-yl)-
ROS, glutathione (GSH), and antioxidant enzymes, especially 2,5-diphenyltetrazolium bromide
thioredoxin (Trx) in A549 lung cancer cells. SBHA inhibited PI Propidium iodide
the growth of A549 cells in time- and dose-dependent man- FITC Fluorescein isothiocyanate
ners, and it induced apoptosis which accompanied by the loss NAC N-acetyl cysteine
of mitochondrial membrane potential (MMP; ΔΨm). SBHA H2DCFDA 2′,7′-Dichlorodihydrofluorescein
significantly increased ROS levels including O2•− level at 72 h diacetate
whereas it decreased ROS levels at the early time points DHE Dihydroethidium
(30 min to 3 h). SBHA also induced GSH depletion at 24 GSH Glutathione
and 72 h. N-acetyl cysteine (NAC; a well-known antioxidant) CMFDA 5-Chloromethylfluorescein diacetate
prevented apoptotic cell death and GSH depletion via decreas- Trx Thioredoxin
ing ROS in SBHA-treated A549 cells. In addition, SBHA TrxR Thioredoxin reductase
changed the levels of antioxidant-related proteins, especially Cu/Zn SOD Copper zinc superoxide dismutase
Trx1. The expression and activity of Trx1 in A549 cells were MnSOD Manganese superoxide dismutase
reduced by SBHA. While the downregulation of Trx1 en-
hanced cell death, ROS level, and GSH depletion in SBHA-
treated A549 cells, the overexpression of Trx1 decreased ROS Introduction
level in these cells without the prevention of cell death and
GSH depletion. In conclusion, SBHA-induced A549 cell Histone acetylation is important for transcription [1]. The acet-
death was influenced by changes in ROS and GSH levels. ylation of lysine residues in histones induces euchromatin
The basal status of Trx1 among other antioxidant proteins was structure, which leads transcriptional factor easily to bind to
closely correlated with the survival of A549 cells. promoter regions of genes [2]. The acetylation and
deacetylation of histones are mediated by two enzymes, histone
Keywords Suberoyl bishydroxamic acid . Reactive oxygen acetyltransferase (HAT) and histone deacetylase (HDAC), re-
species . Apoptosis . Thioredoxin . Glutathione spectively [2]. Previous studies have demonstrated that the
activity and expression of HDAC are upregulated in many
B. R. You : S. H. Kim : W. H. Park (*) cancers including pancreatic and prostate cancer [3–5].
Department of Physiology, Medical School, Research Institute for
Therefore, HDAC inhibitors have been considered as novel
Endocrine Sciences, Chonbuk National University, JeonJu 561-180,
Republic of Korea anti-cancer drugs [6]. In fact, vorinostat and romidepsin are
e-mail: parkwh71@chonbuk.ac.kr HDAC inhibitors that have been used for the treatment of
3430
cutaneous T-cell lymphoma [7]. HDAC inhibitor can induce
apoptosis via generating reactive oxygen species (ROS) in
solid tumor and leukemia cells [8–11].
ROS regulate many cellular events such as gene expres-
sion, differentiation, and cell proliferation [12]. However,
excessive production of ROS can cause cell damage by oxi-
dizing DNA, lipid, and protein [13]. Therefore, various anti-
oxidants including glutathione (GSH), superoxide dismutase
(SOD), catalase, and thioredoxin (Trx) exist to reduce ROS
levels in cells. GSH is a main non-protein antioxidant in the
cell, which supplies electrons for enzymes such as GSH
peroxidase [14]. GSH is crucial for cell proliferation and
apoptosis, which can protect cells from oxidative stress [15,
16]. SOD metabolizes superoxide anion (O2•−) to hydrogen
peroxide (H2O2) [17]. There are three kinds of isoforms;
extracellular (ecSOD), intracellular (Cu/ZnSOD), and mito-
chondrial (MnSOD) [17]. Catalase is an antioxidant enzyme
containing a tetrameric heme, converting H2O2 into water and
oxygen [18]. The TRX system consists Trx, Trx reductase
(TrxR), and NADPH, which is an important enzymatic net-
work to maintain the homeostasis of cellular redox [19]. Trx is
a small protein with two redox-active cysteine residues in its
active center [20]. When Trx is oxidized, it is reduced back to
dithiol by NADPH-dependent enzyme, TrxR [20]. While
Trx1 and TrxR1 are usually localized in the cytoplasm, Trx2
and TrxR2 are located in the mitochondria [20]. Especially,
Trx1 and TrxR1 are overexpressed in many cancer cells
including lung cancer [21–23]. Therefore, the modulation of
TRX system can be a promising target for cancer therapy.
Lung cancer is a major reason of cancer-related death in
developed countries. The carcinogenesis of lung cancer is
related to excessive inflammation mediated by ROS originat-
ed from airborne and blood borne. ROS influence genetic and
epigenetic changes thereby modulating cellular proliferation
and differentiation [24]. We recently demonstrated that
suberoyl bishydroxamic acid (SBHA) as a HDAC inhibitor
attenuates the growth of A549 lung cancer cells via a caspase-
dependent apoptosis [25]. In addition, SBHA induced apopto-
sis in HeLa cells via ROS-independent manner [26].
However, there is no report about the regulation of ROS and
antioxidant enzymes in SBHA-treated lung cancer cells.
Therefore, in the present study, we investigated the toxicolog-
ical effects of SBHA on the regulations of ROS, GSH, and
antioxidant enzymes, especially Trx in A549 lung cancer
cells.
Materials and methods
Cell culture
The human lung adenocarcinoma A549 cells from the American
Type Culture Collection (ATCC, Manassas, VA, USA) were
Tumor Biol. (2015) 36:3429–3439
maintained in a humidified incubator containing 5 % CO2 at
37 °C. A549 cells were cultured in RPMI-1640 supplemented
with 10 % fetal bovine serum (FBS) and 1 % penicillin-
streptomycin (Gibco BRL, Grand Island, NY, USA). Cells were
routinely grown in 100-mm plastic tissue culture dishes (Nunc,
Roskilde, Denmark) and harvested with a solution of trypsin-
EDTA (Gibco BRL) while in a logarithmic phase of growth.
Reagents
SBHA was purchased from Calbiochem (San Diego, CA, USA)
and was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich
Chemical Company, St. Louis, MO, USA) at 100 mM as a stock
solution. NAC was also obtained from Sigma-Aldrich Chemical
Co. and dissolved in buffer [20 mM HEPES (pH 7.0)] at
100 mM as a stock solution. Based on the previous study [27],
cells were pretreated with NAC for 1 h prior to treatment with
SBHA. DMSO (0.05 %) was used as a control vehicle.
Growth inhibition assay
The effect of SBHA on cell growth was determined by measur-
ing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT; Sigma-Aldrich Chemical Co.) dye absorbance of
living cells as previously described [28]. In brief, 5×103 cells
were seeded in 96-well microtiter plates (Nunc) for MTT assays.
After exposure to the designated doses of SBHA with or without
adTrx1 for the indicated hours, 20 μL of MTT solution [2 mg/ml
in phosphate buffered saline (PBS)] were added to each well of
96-well plates (Nunc). The plates were incubated for four addi-
tional hours at 37 °C. The medium in the plates was withdrawn
by pipetting, and 200-μl DMSO was added to each well to
solubilize the formazan crystals. The optical density was mea-
sured at 570 nm using a microplate reader (Synergy™ 2,
BioTek® Instruments Inc., Winooski, VT, USA).
Detection of intracellular ROS levels
Intracellular ROS levels were detected by means of an
oxidation-sensitive fluorescent probe dye, 2′,7′-
dichlorodihydrofluorescein diacetate (H2DCFDA, Invitrogen
Molecular Probes, OR; Ex/Em = 495/529 nm) and
dihydroethidium (DHE, Invitrogen Molecular Probes; Ex/
Em=518/605 nm) as previously described [29, 30]. DHE is
highly selective for O2•− among ROS. In brief, 1×106 cells in
a 60-mm culture dishes (Nunc) were incubated with the des-
ignated doses of SBHA with or without 2-mM NAC, TRX
system-related siRNAs, or adTrx1 for the indicated times.
Cells were then washed in PBS and incubated with 20-μM
H2DCFDA or DHE at 37 °C for 30 min.
Dichlorodihydrofluorescein (DCF) and DHE fluorescences
were detected using a FACStar flow cytometer (Becton
Dickinson, Franklin Lakes, NJ, USA). ROS and O2•- levels
Tumor Biol. (2015) 36:3429–3439
were expressed as mean fluorescence intensity (MFI), which
was calculated by the CellQuest software (Becton Dickinson).
Detection of intracellular GSH levels
Cellular GSH levels were analyzed using 5-
chloromethylfluorescein diacetate (CMFDA, Invitrogen
Molecular Probes; Ex/Em=522/595 nm) as previously de-
scribed [30]. In brief, 1×106 cells were incubated in a 60-
mm culture dishes (Nunc) with the designated doses of SBHA
with or without 2-mM NAC, TRX system-related siRNAs, or
adTrx1 for the indicated times. Cells were then washed with
PBS and incubated with 5-μM CMFDA at 37 °C for 30 min.
The fluorescence intensity of chloromethylfluorescein (CMF)
was measured by using a FACStar flow cytometer. Negative
CMF staining (GSH-depleted) of cells is expressed as the
percents of (−) CMF cells.
Annexin V-FITC/PI staining for cell death detection
Apoptosis was determined by staining cells with annexin V-
fluorescein isothiocyanate (FITC, Invitrogen Corporation,
Camarillo, CA, USA; Ex/Em=488/519 nm) and propidium
iodide (PI; Sigma-Aldrich; Ex/Em=488/617 nm) as previous-
ly described [29, 31]. In brief, 1×106 cells in a 60-mm culture
dish (Nunc) were incubated with the designated doses of
SBHA with or without 2-mM NAC, TRX system-related
siRNAs, or adTrx1 for the indicated times. Cells were washed
twice with cold PBS and then added in 500 μl of binding
buffer (10 mM HEPES/NaOH pH 7.4, 140 mM NaCl,
2.5 mM CaCl2) at a concentration of 1×106 cells/ml. Five
microliters of annexin V-FITC and PI (1 μg/ml) were then
added to these cells, which were analyzed with a FACStar
flow cytometer (Becton Dickinson). Viable cells were nega-
tive for both PI and annexin V; apoptotic cells were positive
cells displayed both high annexin V and negative for PI
whereas late apoptotic dead cells display both high annexin
V and PI labeling. Nonviable cells, which underwent necrosis,
were positive for PI and negative for annexin V.
Western blot analysis
The expression of proteins was evaluated using Western blot
analysis, as previously described [32]. In brief, 1×106 cells in
a 60-mm culture dish (Nunc) were incubated with the desig-
nated doses of SBHA for 72 h. The cells were then washed in
PBS and suspended in 5 vol. of lysis buffer (20 mM HEPES
pH 7.9, 20 % glycerol, 200 mM KCl, 0.5 mM EDTA, 0.5 %
NP40, 0.5 mM DTT, 1 % protease inhibitor cocktail).
Supernatant protein concentrations were determined using
the Bradford method. Supernatant samples containing 30 μg
total protein were resolved by 12.5 % SDS-PAGE gels de-
pending on the sizes of target proteins, transferred to
3431
Immobilon-P PVDF membranes (Millipore, Billerica, MA,
USA) by electroblotting, and then probed with anti-Trx1,
anti-TrxR1, anti-Trx2, anti-TrxR2, anti-Cu/ZnSOD, anti-
MnSOD, anti-catalase, and anti-β-actin antibodies (Santa
Cruz Biotechnology, Santa Cruz, CA, USA). Membranes
were incubated with horseradish peroxidase-conjugated sec-
ondary antibodies. Blots were developed using an ECL kit
(Amersham, Arlington Heights, IL, USA). Quantitative data
were obtained using an imaging densitometer (ImageJ version
1.33 software, NIH).
Measurement of Trx1 activity
The Trx1 activity was assessed using the ProteoStat™
Thioredoxin-1 assay Kit according to manufacturer’s instruc-
tions (EnZo Life Science, Plymouth Meeting, PA, USA). In
brief, 1×106 cells in 60-mm culture dish (Nunc) were incu-
bated with the indicated doses of SBHA for 72 h. The cells
were then washed in PBS and suspended in 5 vol. of lysis
buffer (R&D systems, Inc., Minneapolis, MN, USA). Protein
concentrations were determined using the Bradford method.
Supernatant samples containing 20 μg of total protein were
used for determination of Trx1 activity. These are added to
each well in 96-well microtiter plates (Nunc) with the insulin
and dithiothreitol (DTT) at 25 °C for 30 min. The fluorescence
intensity of each well was determined using a fluorescence
reader (Synergy™ 2, BioTek® Instruments Inc.).
Measurement of TrxR activity
The TrxR activity was assessed using the Thioredoxin
Reductase Assay Kit according to manufacturer’s instructions
(Sigma-Aldrich). In brief, 1×106 cells in 60-mm culture dish
(Nunc) were incubated with the indicated doses of SBHA with
for 72 h. The cells were then washed in PBS and suspended in
5 vol. of lysis buffer (R&D systems, Inc.). Protein concentra-
tions were determined using the Bradford method.
Supernatant samples containing 20 μg of total protein were
used for determination of TrxR activity. These are added to
each well in 96-well microtiter plates (Nunc) with 5,5′-
dithiobis (2-nitrobenzoic) acid (DTNB) at 25 °C for 1 h. The
optical density of each well was measured at 412 nm using a
microplate reader (Synergy™ 2, BioTek® Instruments Inc.).
Measurement of cellular SOD and catalase activities
SOD enzyme activity was measured using the SOD Assay
Kit-WST (Fluka Co., Milwaukee, USA), and catalase enzyme
activity was measured using the catalase assay kit from
Sigma-Aldrich as previously described [33]. In brief, 1×106
cells were incubated with the indicated doses of SBHA with
for 72 h. The cells were then washed in PBS and suspended in
5 vol. of lysis buffer (20 mM HEPES, pH 7.9, 20 % glycerol,
3432
200 mM KCl, 0.5 mM EDTA, 0.5 % NP40, 0.5 mM DTT, 1 %
protease inhibitor cocktail). Supernatant protein concentration
was determined by the Bradford method. Supernatant samples
containing 50 μg of total protein were used for determination
of SOD and catalase enzyme activities. These are added to
each well in 96-well microtiter plates (Nunc) with the appro-
priate working solutions (according to the manufacturer’s
instructions) at 25 °C for 30 min. The color changes were
measured at 450 or 520 nm using a microplate reader
(Synergy™ 2, BioTek® Instruments Inc.). The value for the
experimental group was converted to the percentage of control
group.
Transfection of cells with TRX system-related siRNAs
Gene silencing of TRX system was performed as previously
described [31, 34]. A nonspecific control (CTR) siRNA du-
plex [5′-CCUACGCCACCAAUUUCGU (dTdT)-3′], Trx1
siRNA duplex [5′-GCAUGCCAACAUUCCAGUU (dTdT)-
3′], TrxR1 siRNA duplex [5′-GUCGUCUAUGAGAAUG
CUU (dTdT)-3′], Trx2 siRNA duplex [5′-CAAUAUACAC
CACGAGGAU (dTdT)-3′], and TrxR2 siRNA duplex [5′-
GGUCCUCAGAUCUGAUGGA (dTdT)-3′] were purchased
from the Bioneer Corp. (Daejeon, South Korea). In brief, 2.5×
105 cells in six-well plates (Nunc) were incubated in RPMI-
1640 supplemented with 10 % FBS. The next day, cells
(approximately 30–40 % confluence) in each well were
transfected with the control (CTR) or TRX system-related
siRNAs duplex [80 pmol in Opti-MEM (GIBCO BRL)] using
LipofectAMINE 2000, according to manufacturer’s instruc-
tions (Invitrogen, Branford, CT, USA). One day later, cells
were treated with or without 100 μM SBHA for additional
24 h. The transfected cells were used for Western blot analy-
sis, annexin V-FITC staining, MMP (ΔΨm), and ROS and
GSH levels measurements.
Measurement of MMP (ΔΨm)
MMP (ΔΨm) levels were measured by a rhodamine 123
fluorescent dye (Sigma-Aldrich Chemical Company; Ex/
Em=485/535 nm) as described previously [29, 35]. In brief,
1×106 cells in a 60-mm culture dish (Nunc) were incubated
with 100 μM SBHA with or without TRX system-related
siRNAs for the indicated times. Cells were washed twice with
PBS and incubated with rhodamine 123 (0.1 μg/ml) at 37 °C
for 30 min. Rhodamine 123 staining intensity was determined
by flow cytometry (Becton Dickinson). An absence of rhoda-
mine 123 from cells indicates the loss of MMP (ΔΨm) in cells.
Infection of cells with adTrx1
Overexpression of Trx1 was performed using an adeno-
viral gene transfer. adLacZ and adTrx1 were obtained
Tumor Biol. (2015) 36:3429–3439
from Dr. Sadoshima J, New Jersey Medical School,
Newark. In brief, 5×105 cells in six-well plates (Nunc)
were incubated in RPMI-1640 supplemented with 10 %
FBS. Cells (approximately 50–60 % confluence) in each
well were infected with the same titer of adLacZ or
adTrx1, which was determined by plague assays. One
day later, cells were treated with or without 100 μM
SBHA for additional 24 h. The infected cells were
collected and used for Western blot analysis, cell
growth, annexin V-FITC staining, ROS and GSH level
measurements.
Statistical analysis
The results represent the mean of at least three independent
experiments (mean±SD). The data were analyzed using Instat
software (GraphPad Prism4, San Diego, CA, USA). The
Student’s t test or one-way analysis of variance (ANOVA)
with post hoc analysis using Tukey’s multiple comparison
tests was used for parametric data. Statistical significance
was defined as p<0.05.
Results
Effect of SBHA on the growth and intracellular ROS
and GSH levels of A549 cells
We first examined the effect of SBHA on the growth inhibi-
tion of A549 cells using MTT assays. After exposure to
SBHA for 24, 48, and 72 h, the growth of A549 cells was
dose- and time-dependently decreased with an IC50 of approx-
imately 50 μM at 72 h (Fig. 1a). Next, changes in intracellular
ROS and GSH levels were investigated in A549 cells at the
early and late time points. SBHA decreased ROS (DCF) level
from the earlier time point of 30 min and the decrease contin-
ued for 180 min (Fig. 1b). However, SBHA significantly
increased ROS (DCF) levels at the late times of 24 and 72 h
(Fig. 1c). SBHA seemed to decrease O2•− level at 24 h, but it
increased the level at 72 h (Fig. 1d). In relation to GSH level,
treatment with 10 or 50 μM SBHA decreased GSH level at the
early time of 30 min whereas 100 μM SBHA increased this
level at this time and the gradual increase sustained for
180 min (Fig. 1e). All the tested doses of SBHA significantly
induced GSH depletion at 24 and 72 h (Fig. 1f).
Effects of NAC on cell death and ROS and GSH levels
in SBHA-treated A549 cells
To determine whether SBHA-induced increase in ROS level
was involved in A549 cell death, we examined the effects of
NAC on cell death and ROS and GSH levels in SBHA-treated
Tumor Biol. (2015) 36:3429–3439
Fig. 1 Effects of SBHA on cell growth and ROS and GSH levels in
A549 cells. Exponentially growing cells were treated with the designated
concentrations of SBHA for indicated times. a The graph shows cellular
growth changes in A549 cells as assessed by MTT assays. b, c ROS and
GSH levels in A549 were measured using a FACStar flow cytometer. The
cells. For this experiment, we chose 50 μM SBHA as a
suitable dose to differentiate the level of cell death in the
presence or absence of NAC. The concentration of 2-mM
NAC was also used as optimal dose in this study since this
dose did not affect cell death in A549 cells (Fig. 2a, b). NAC
significantly prevented apoptotic cell death in SBHA-treated
A549 cells (Fig. 2a, b). Expectedly, NAC attenuated ROS
levels including O2•− in SBHA-treated A549 cells (Fig. 2c,
d) and it also lessened GSH depletion in these cells (Fig. 2e).
Effects of SBHA on antioxidant-related protein levels
and activities in A549 cells
Next, changes in antioxidant-related protein levels were
assessed in SBHA-treated A549 cells. The levels of Trx1
and Trx2 were decreased by SBHA whereas those of TrxR2,
Cu/Zn SOD, MnSOD, and catalase were increased by it
(Fig. 3a). In addition, SBHA slightly increased the level of
TrxR1 in A549 cells (Fig. 3a). In relation to the activities of
3433
graphs indicate DCF (ROS) and CMF levels at the indicated early times.
d, e The graphs show DCF (ROS) and DHE (O2•−) levels compared with
those in control cells, respectively. f The graph shows the percents of (−)
CMF (GSH-depleted) cells. *p<0.05 compared with the control group
antioxidant proteins, all the tested doses of SBHA significant-
ly diminished the activity of Trx1 in A549 cells (Fig. 3b). The
activity of TrxR was also marginally decreased by SBHA
(Fig. 3c). By contrast, treatment with 50 μM SBHA increased
the activities of SOD and catalase in A549 cells (Fig. 3d, e).
Effects of TRX system-related siRNAs on cell death, MMP
(ΔΨm), and ROS and GSH levels in SBHA-treated A549
cells
Because the levels of TRX system-related proteins were al-
tered by SBHA, it was considered that changes in these levels
influence cell death and ROS level in SBHA-treated A549
cells. To investigate this possibility, A549 cells were
transfected with either control siRNA or TRX system-related
siRNAs for the downregulation of the corresponding proteins.
For this experiment, the dose of 100 μM SBHA and the
incubation time of 24 h were used as a suitable condition
due to the confluence of experiment cells. As shown in
3434
Fig. 2 Effects of NAC on cell death and ROS and GSH levels in SBHA-
treated A549 cells. Exponentially growing cells were treated with 50 μM
SBHA and/or 2 mM NAC for 72 h. a Annexin V-FITC and/or PI positive
cells were measured with a FACStar flow cytometer. b The graph shows
the percents of annexin V positive cells from a. c, d The graphs show
Fig. 4a, the levels of Trx1, TrxR1, Trx2, and TrxR2 were
completely decreased in A549 cells transfected with each
TRX system-related siRNA. Among TRX system-related
siRNAs, Trx1 siRNA significantly intensified apoptotic cell
death in SBHA-treated and -untreated control A549 cells and
TrxR2 also seemed to increase cell death in those cells
(Fig. 4b). In addition, Trx1 siRNA augmented the loss of
MMP (ΔΨm) in SBHA-treated A549 cells and both Trx2
and TrxR2 siRNAs were also likely to enhance the MMP
(ΔΨm) loss in these cells (Fig. 5a, b). Moreover, Trx1 and
TrxR2 siRNAs slightly induced the loss of MMP (ΔΨm) in
A549 control cells (Fig. 5a, b).
In relation to ROS and GSH levels, all the TRX system-
related siRNAs, especially Trx1 siRNA increased ROS levels
in SBHA-treated A549 cells and those siRNAs also seemed to
increase ROS levels in A549 control cells (Fig. 6a). In addi-
tion, Trx1 siRNA augmented the level of O2•− in SBHA-
treated A549 cells (data not shown). Moreover, Trx1 siRNA
Tumor Biol. (2015) 36:3429–3439
DCF (ROS) and DHE (O2•−) levels compared with those in control cells,
respectively. e The graph shows the percents of (−) CMF (GSH-depleted)
cells. *p<0.05 compared with the control group. #p<0.05 compared with
cells treated with SBHA only
among TRX system-related siRNAs intensified GSH deple-
tion in SBHA-treated A549 cells (Fig. 6b).
Effects of adTrx1 on cell growth, cell death, and ROS
and GSH levels in SBHA-treated A549 cells
Next, we investigated the effects of Trx1 overexpression on
cell growth, death, and ROS and GSH levels in SBHA-treated
A549 cells. For this experiment, A549 cells were infected with
either adLacZ or adTrx1 for the overexpression of Trx1. In
addition, 100 μM SBHA and 24 h were used as a suitable dose
and time due to the confluence of experimental cells. As
shown in Fig. 7a, A549 cells infected with adTrx1 showed
an increase in Trx1 protein level compared with cells infected
with control adLacZ. While administration with adTrx1
slightly attenuated cell growth inhibition by SBHA, this ad-
ministration failed to prevent cell death in SBHA-treated
A549 cells (Fig. 7a, b). The overexpression of Trx1
Tumor Biol. (2015) 36:3429–3439
Fig. 3 Effects of SBHA on antioxidant-related protein levels and activ-
ities in A549 cells. Exponentially growing cells were treated with the
designated concentrations of SBHA for 72 h. a Western blot analysis
shows changes in antioxidant-related proteins. Thirty-microgram samples
of protein extracts from A549 cells were resolved by SDS-PAGE gel,
transferred onto PVDF membranes, and immunoblotted with the indicat-
ed antibodies against Trx1, TrxR1, Trx2, TrxR2, Cu/Zn SOD, Mn SOD,
significantly decreased ROS levels in SBHA-treated and -
untreated A549 cells (Fig. 7c). However, this overexpression
did not alter GSH depletion in SBHA-treated A549 cells
(Fig. 7d).
Fig. 4 Effects of TRX system-related siRNAs on cell death in SBHA-
treated A549 cells. A549 cells (approximately 30–40 % confluence) were
transfected with either control (CTR) siRNA or each TRX system-related
siRNAs. One day later, cells were treated with 100-μM SBHA for
additional 24 h. a The expressions of Trx1, TrxR1, Trx2, and TrxR2 were
3435
catalase, and β-actin. The expression levels of antioxidant-related pro-
teins were quantified using a densitometer program, ImageJ software.
The relative levels were annotated as the ratio of each antioxidant-related
protein level to β-actin protein level. b, c The graphs show the activities
of Trx1 and TrxR in samples containing 50 μg total protein. d, e The
graphs show the activities of SOD and catalase in samples containing
50 μg total protein. *p<0.05 compared with the control group
Discussion
Previously, we reported that SBHA induces cell growth
inhibition and caspase-dependent apoptosis in A549
examined by Western blotting. b The graph indicates the percents of
annexin V-FITC positive staining cells in A549 cells. *p<0.05 compared
with the control group. #p<0.05 compared with cells treated with SBHA
only
3436
Fig. 5 Effects of TRX system-related siRNAs on MMP (ΔΨm) in
SBHA-treated A549 cells. A549 cells (approximately 30–40 % conflu-
ence) were transfected with either control (CTR) siRNA or each TRX
system-related siRNAs. One day later, cells were treated with 100-μM
SBHA for additional 24 h. a Rhodamine-negative cells were measured
lung cancer cells [25]. In the present study, we focused
on elucidating the effects of SBHA on A549 cell death
Fig. 6 Effects of TRX system-related siRNAs on ROS and GSH levels
in SBHA-treated A549 cells. A549 cells (approximately 30–40 % con-
fluence) were transfected with either CTR siRNA or each TRX system-
related siRNAs. One day later, cells were treated with 100-μM SBHA for
Tumor Biol. (2015) 36:3429–3439
with a FACStar flow cytometer. b The graph indicates the percents of a
rhodamine 123-negative [MMP (ΔΨm) loss] cells from M1 region of A.
*p<0.05 compared with the control group. #p<0.05 compared with cells
treated with SBHA only
in relation to changes in ROS and GSH levels as well
as the regulations of antioxidant enzymes, especially Trx
additional 24 h. a The graph shows DCF (ROS) levels compared with
those in control cells. b The graph shows the percents of (−) CMF (GSH-
depleted) cells. *p<0.05 compared with the control group. #p<0.05
compared with cells treated with SBHA only
Tumor Biol. (2015) 36:3429–3439
(Fig. 7). Many reports have been demonstrated that
HDAC inhibitors can induce apoptosis via generating
ROS in solid tumor and leukemia cells [8–11]. In fact,
ROS scavengers such as NAC prevent cell death in-
duced by HDAC inhibitors [9–11]. Likewise, ROS
levels including O2•− were significantly increased in
SBHA-treated A549 cells at 72 h. In addition, NAC
attenuated cell death in SBHA-treated A549 cells and
it decreased ROS levels including O2•− in these cells.
These results suggested that SBHA-induced A549 cell
death is mediated by oxidative stress. However, we did
not observe increases in ROS levels in SBHA-treated
A549 cells at the early time points (30 min to 3 h).
SBHA also did not increase O2•− level detected by DHE
at these early time points (data not shown). These
results suggest that SBHA does not affect both mito-
chondrial respiratory transport chain and the activity of
various oxidases to generate ROS including O2•− within
these early time points. It is assumed that the decreased
ROS level in SBHA-treated A549 cells might result
from the regulation of ROS defense system such as
antioxidant proteins and GSH. However, the exact
mechanism on how to increase ROS levels in cells
including A549 cells needs to be further defined.
SBHA altered the levels and activities of many antioxidant
proteins in A549 cells. SBHA decreased the levels of Trx1 and
Trx2 whereas it increased the levels of TrxR2, Cu/ZnSOD,
MnSOD, and catalase in A549 cells. SBHA dose-dependently
attenuated the activity of Trx1 but it increased activities of
Fig. 7 Effects of adTrx1 on cell growth, cell death, and ROS and GSH
levels in SBHA-treated A549 cells. A549 cells (approximately 50–60 %
confluence) were infected with either adLacZ or adTrx1. One day later,
cells were treated with 100-μM SBHA for additional 24 h. The inside
figure indicates the expression level of Trx1 in A549 cells. a The graph
shows cellular growth change. b The graph shows the percents of
3437
SOD and catalase. Interestingly, the expression level of TrxR1
was marginally increased by SBHA whereas the activity of
that was slightly decreased by it. The activity of TrxR1
seemed to be regulated by unknown posttranslational factors
rather than its expression level. In addition, it is possible that
the downregulations of Trx1 and Trx2 by SBHA correspond-
ingly increased ROS levels including O2•− in A549 cells,
consequently inducing oxidative stress to kill A549 cells. On
the other hand, the upregulation of SOD and catalase might be
induced by the increased ROS levels and also that can be
transcriptionally regulated by the inhibition of HDAC by
SBHA. These results imply that antioxidant proteins influence
ROS levels in cells and they can also be influenced by ROS
levels and vice versa. The transcriptional regulations of anti-
oxidant genes by HDAC inhibitors need to be further studied
in relation to redox status in cells.
The TRX antioxidant system consists of Trx, TrxR, and
NADPH, which is an important enzymatic network to main-
tain cellular redox homeostasis [19]. Especially, Trx1 and
TrxR1 are overexpressions in many cancer cells including
lung cancer [23]. Therefore, TRX system can be a promising
target to treat cancer patients. According to our results, the
regulation of Trx1 by SBHA is considered to be an important
factor on cell survival and ROS level. In fact, it is reported that
suppression of Trx1 can enhance cytotoxicity of anti-cancer
drugs in human liver carcinoma and diffuse large B-cell
lymphoma cells [36, 37]. In the same way, treatment with
Trx1 siRNA significantly intensified cell death and MMP
(ΔΨm) loss in SBHA-treated A549 cells. Interestingly, Trx2
Annexin V-FITC positive staining cells. c The graph shows DCF
(ROS) levels compared with adLacZ-treated control cells. d The graph
shows the percents of (−) CMF (GSH-depleted) cells. *p<0.05 compared
with the control group. #p<0.05 compared with cells treated with SBHA
only
3438
and TrxR2 siRNAs slightly augmented MMP (ΔΨm) loss in
SBHA-treated A549 cells whereas these siRNAs did not
affect cell death in these cells. Therefore, the slight enhance-
ment of MMP (ΔΨm) loss by Trx2 and TrxR2 siRNAs might
not be enough to additionally augment A549 cell death by
SBHA. Because Trx1 and TrxR1 are usually localized in the
cytoplasm and Trx2 and TrxR2 are located in the mitochondria
[20], these two systems can differently play roles in the mainte-
nance of cell survival and mitochondrial integrity. On the whole,
all the TRX system-related siRNAs increased ROS levels in
SBHA-treated A549 cells, especially Trx1 siRNA. In addition,
these siRNAs were likely to increase ROS levels in A549 control
cells. Therefore, the downregulation of TRX antioxidant system
seemed to increase ROS levels in A549 cells. Moreover, the
overexpression of Trx1 significantly decreased ROS levels in
SBHA-treated A549 cells. However, this overexpression did
not prevent apoptosis in SBHA-treated A549 cells but slight-
ly recovered the growth inhibition by SBHA. Taken together,
the basal level of Trx1 has a crucial effect on the survival of
A549 cells, which result is correlated with the report that the
targeted disruption of mouse Trx gene causes early embry-
onic lethality [38]. The overexpression of this protein did not
have an additional effect on the protection of A549 cells
from SBHA regardless of its downregulated role in ROS
level.
GSH is a main non-protein antioxidant in cells. The intra-
cellular GSH content has a decisive effect on anti-cancer drug-
induced apoptosis [9, 26, 35, 39–41]. It is reported that HDAC
inhibitors including SBHA induce GSH depletion in HeLa
cancer cells [9, 26]. Similarly, our result demonstrated that
SBHA significantly induced GSH depletion in A549 in dose-
or time-dependent manners. NAC, which prevented cell death
in SBHA-treated A549 cells, decreased the number of GSH-
depleted cells in these cells. Moreover, administration of Trx1
siRNA enhanced GSH depletion in SBHA-treated A549 cells
whereas the overexpression of Trx1 did not affect GSH de-
pletion in these cells. It is of note that 100 μM SBHA
increased CMF (GSH) level at the early times (30 min to
3 h). The increased GSH level seemed to happen as a defense
effect to protect cells from SBHA insult, consequently de-
creasing ROS levels at these early times.
In conclusion, SBHA induced growth inhibition and death in
A549 lung cancer cells, which were influenced by changes in
ROS and GSH levels. The basal status of Trx1 among other
antioxidant proteins was closely correlated with the survival of
A549 cells.
Acknowledgements This work was supported by the National Re-
search Foundation of Korea (NRF) grant funded by the Korea govern-
ment (MSIP) (No. 2008–0062279) and supported by the Basic Science
Research Program through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education, (2013006279).
Conflicts of interest None
Tumor Biol. (2015) 36:3429–3439
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