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ORIGINAL PAPER
Reactive oxygen species mediate thymoquinone-induced apoptosisand activate ERK and JNK signaling
Nahed El-Najjar • Manal Chatila • Hiba Moukadem •
Heikki Vuorela • Matthias Ocker • Muktheshwar Gandesiri •
Regine Schneider-Stock • Hala Gali-Muhtasib
� Springer Science+Business Media, LLC 2009
Abstract Thymoquinone (TQ), a component of black
seed essential oil, is known to induce apoptotic cell death
and oxidative stress, however, the direct involvement of
oxidants in TQ-induced cell death has not been established
yet. Here, we show that TQ inhibited the proliferation of a
panel of human colon cancer cells (Caco-2, HCT-116,
LoVo, DLD-1 and HT-29), without exhibiting cytotoxicity
to normal human intestinal FHs74Int cells. Further inves-
tigation in DLD-1 revealed that apoptotic cell death is the
mechanism for TQ-induced growth inhibition as confirmed
by flow cytometry, M30 cytodeath and caspase-3/7 acti-
vation. Apoptosis was induced via the generation of reac-
tive oxygen species (ROS) as evidenced by the abrogation
of TQ apoptotic effect in cells preincubated with the strong
antioxidant N-acetyl cysteine (NAC). TQ increased the
phosphorylation states of the mitogen-activated protein
kinases (MAPK) JNK and ERK, but not of p38. Their
activation was completely abolished in the presence of
NAC. Using PD98059 and SP600125, specific ERK and
JNK inhibitors, the two kinases were found to possess pro-
survival activities in TQ-induced cell death. These data
present evidence linking the pro-oxidant effects of TQ with
its apoptotic effects in colon cancer and prove a protective
role of MAPK.
Keywords Apoptosis � Colon cancer � ROS �Oxidative stress � MAPK � Thymoquinone
Introduction
A few natural compounds are potent anticancer agents
that offer a non-toxic means for cancer intervention.
Understanding the processes leading to the inhibition
of carcinogenesis by these compounds requires a clear
identification of their molecular targets. Nigella sativa
Linn. (Ranunculaceae), commonly known as black seed or
black cumin, is an annual plant traditionally used in the
Indian subcontinent, Arab countries and Europe for culi-
nary and medicinal purposes [1, 2]. Thymoquinone (TQ)
has been shown to be the active component responsible
for the seed’s biological effects.
There is growing interest in the therapeutic potential of
TQ in different research fields, particularly in cancer
therapy. TQ was found to be a potent inhibitory drug in
colon cancer cells [3–5], p53-null myeloblastic leukemia
cells [6], laryngeal carcinoma cells [7, 8], pancreatic cells
[9], and prostate cancer cells [10]. Recently, TQ was
reported to be a neuroprotective agent in SH-SY5Y human
neuroblastoma cells [11], as well as an anti-inflammatory
and immune stimulatory mediator [2, 12–15]. Although the
exact mechanisms of TQ action are not fully elucidated,
recent reports have shown that TQ induces apoptosis by
N. El-Najjar � M. Chatila � H. Moukadem �H. Gali-Muhtasib (&)
Department of Biology, American University of Beirut,
Beirut, Lebanon
e-mail: [email protected]
N. El-Najjar � H. Vuorela
Division of Pharmaceutical Biology, Faculty of Pharmacy,
University of Helsinki, Helsinki, Finland
M. Ocker
Institute for Surgical Research, Philipps University Marburg,
Marburg, Germany
M. Gandesiri � R. Schneider-Stock
Experimental Tumorpathology, Institute for Pathology,
University Erlangen-Nuremberg, Erlangen, Germany
123
Apoptosis
DOI 10.1007/s10495-009-0421-z
Administrator
Sticky Note
但是,TQ中直接导致细胞死亡的氧化剂还没有确定。在这里,我们显示TQ抑制了人Caco-2的增殖,而没有对人小肠FHs74Int细胞显示细胞毒性。在DLD-1细胞上进一步的研究显示,细胞凋亡是一个由TQ导致的生长抑制的机制,这一点被流动细胞计数等证实了。细胞凋亡是经过产生的ROS导致的,证据是强抗氧化剂NAC可以在细胞预培养中消除TQ的细胞凋亡作用。TQ可以增加MAPK的JNK和ERK的磷酸化水平,但p38没有作用它们的活化可以被NAC完全消除。用ERK和JNK的特异性抑制剂PD98059和SP600125,这两种激酶被发现可以在TQ诱导的细胞死亡中有促存活作用。这些数据与TQ在结肠肿瘤中的促氧化效果导致细胞死亡。证实了MAPK的保护性角色。
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p53-dependent [16] and p53-independent [6, 17] pathways.
Therefore, we set forth to determine the molecular path-
ways by which TQ elicits its antineoplastic activities in
colon cancer cell lines.
Several lines of evidence suggest that TQ has potent
anion scavenging abilities in different models [18, 19].
However, the oxidant/antioxidant ability of TQ depends on
the milieu where it is present. As a quinone, TQ can be
reduced by a variety of reductases to yield semiquinone
(one reduction) or thymohydroquinone (two reductions).
While the latter molecule is reported to have antioxidant
effects [20], semiquinone acts as a pro-oxidant by the
generation of reactive oxygen species (ROS).
Ample evidence proves that ROS production, by
numerous anticancer agents, is responsible for apoptosis
induction in different types of cancer such as cervical [21],
pancreatic [22], gastric [23], breast [24], as well as colon
cancer and leukemia [25, 26].
ROS causing oxidative stress are known to activate
members of the MAPK family [27, 28]. The latter ones are
important mediators of signal transduction, and play a key
role in the regulation of many cellular processes, such as
cell growth and proliferation, differentiation, and apopto-
sis. The MAP kinase signaling pathway mainly consists of
three subfamilies: extracellular signal-regulated kinase
(ERK), c-jun N-terminal kinase (JNK), and p38 MAP
kinase [29]. In general, ERK delivers a survival signal,
while JNK and p38 correlate with apoptosis induction
under stressful conditions [30–32]. Several chemothera-
peutic drugs have been shown to activate JNK and p38
MAPK and their activation is implicated in apoptosis [26].
Once activated, ERK, JNK, and p38 modulate the phos-
phorylation of transcription factors ultimately leading to
changes in gene expression profiles which encode for
defense against cellular oxidative stress [33].
In this study, we investigated the involvement of ROS
generation and the subsequent activation of the MAPK
pathway in TQ’s antineoplastic effects in colon cancer
cells. Evidence is provided to link the apoptotic effects of
TQ with its pro-oxidant effects and confirms a protective
role of ERK and JNK mitogen-activated protein kinases.
Materials and methods
Cell culture
Colon cancer HCT-116, LoVo, DLD-1, and HT-29 were
grown in RPMI 1640 ? HEPES. FHs74Int cells were
grown in Hybricare medium (ATCC, Manassas, Virginia,
USA) supplemented with 30 ng/l EGF (Biosource, Cama-
rillo, California, USA). Caco-2 cells were cultured in
DMEM:F12 (1:1) with nonessential amino acids. All cells
were maintained at 37�C in a humidified atmosphere of 5%
CO2, 95% air, supplemented with 1% Penicillin–Strepto-
mycin (100 U/ml), and 10% fetal bovine serum (Invitro-
gen, Carlsbad, California, USA). In all experiments (except
ELISA assays), cells were seeded at 105 cells/ml and
exposed to TQ (MP Biomedical, Strasbourg, France) at
40–50% confluency. For experiments involving inhibitors,
cells were pre-treated with 5 mM N-acetyl cysteine (NAC,
Sigma, St. Louis, Missouri, USA) for 2 h, 50 lM PD98059
(Cell Signaling Technology, Beverly, USA) for 2 h, 20 lM
SP600125 (Sigma) for 1.5 h or with 100 lM Dicumarol
(Acros Organics, New Jersey, USA) for 1 h prior to TQ.
TQ was prepared in methanol and the final methanol
concentration on cells was less than 1%.
Cell proliferation and viability assays
Inhibition of cell proliferation by TQ was measured by the
Cell Titer 96 non-radioactive cell proliferation kit (Pro-
mega Corp, Madison, Wisconsin, USA). The proliferation
assay is an MTT-based method that measures the ability of
metabolically active cells to convert tetrazolium salt into a
blue formazan product, and its absorbance is recorded at
570 nm. Proliferation was studied 24 and 48 h post-treat-
ment. Briefly, cells were plated in 96-well plates and
treated with different concentrations of TQ in the presence
or absence of NAC, PD98059, SP600125, or Dicumarol.
The IC50 represents the concentration at which 50% of the
cells are viable.
The CytoTox 96 (viability) assay done in FHs74Int at
24 h quantitatively measures the lactate dehydrogenase
(LDH), a stable cytosolic enzyme that is released upon cell
lysis. Released LDH in culture supernatants is measured
with a coupled enzymatic assay which results in the con-
version of a tetrazolium salt into a red formazan product,
the absorbance of which is recorded at 490 nm.
Cell cycle analysis
The distribution of cells in the different phases of the cell
cycle was evaluated by flow cytometry. Cells were plated
in 60-mm tissue culture dishes. Cells were trypsinized
1 day post-treatment, washed with PBS, and fixed with
70% ethanol at least for 2 h at -20�C. Fixed cells were
washed with PBS, incubated with 200 lg/ml RNase A
(Sigma) for 1:15 h at 37�C, and stained with propidium
iodide (PI) (Molecular Probes, Eugene, Oregon, USA). The
stained cells were analyzed by a FACScan flow cytometer,
and the percentage of cells in preG1, G0/G1, S, and G2/M
phases was determined using the Cell Quest Histogram
analysis program. Cells that were less intensely stained
than G1 cells in flow cytometric histograms were consid-
ered as apoptotic cells and marked as preG1.
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Evaluation of apoptosis
Apoptosis induction was analyzed by M30 cytodeath,
mitochondrial membrane potential analysis and caspase-3
activity as described below.
M30 cytodeath assay
Early induction of apoptosis was evaluated with the M30
cytodeath antibody, which recognizes a specific caspase
cleavage site with cytokeratine 18 that is not detected in
native cells (Roche Diagnostic Corporation, Mannheim,
Germany). Briefly, 1 day post-treatment, cells were
washed with PBS, and fixed with ice cold methanol for at
least 30 min at -20�C. After washing with PBS ? 0.1%
tween, fixed cells were incubated with M30 working
solution for 30 min at room temperature. The stained cells
were analyzed using FACScan flow cytometer or by fluo-
rescent microscope Leica DM6000B) using 20-fold
magnification.
Analysis of mitochondrial membrane potential
Apoptosis-associated mitochondrial potential loss (Dwm)
was determined by staining DLD-1 cells with 25 nM 3,30
dihesiloxalocarbocyanine Iodide (DiOC6(3) (Molecular
Probes, Eugene, Oregon, USA). Briefly cells were pre-
treated with 50 lM PD98059 (1.5 h) and 20 lM SP600125
(2 h) followed or not with 40 lM TQ. Cells were then
collected, incubated with 25 nM DiOC6(3) for 30 min at
37�C and read by FACScan flow cytometry.
Caspase-3/7 activity assay
Cell lysates from DLD-1 cells treated with 40 lM TQ with
and without NAC, SP600125 and PD98059 were prepared,
and caspase 3 activity was measured 24 h later according
to the manufacturer’s protocol (Caspase-Glo� 3/7 Assay,
Promega Corp, Madison, WI, USA). Briefly, 50 lg of total
protein were incubated with equal volume of the caspase-
3/7 mixture, and incubated at room temperature for 3 h,
after which the luminescence was measured by a micro-
plate reader.
Intracellular ROS generation by DCFH
The level of ROS was examined using 20,70-dichlorodihy-
drofluorescein diacetate (DCFH-DA) (Acros Organics,
New Jersey, USA). This molecule passively diffuses into
the cells and is cleaved and oxidized in the intracellular
environment to the green fluorescence emitting compound,
20,70-dichlorofluorescein (DCF). Cells were treated at 50%
confluency with 40 lM TQ for 30 min in the presence and
absence of NAC. Attached cells were harvested, washed,
and incubated with 10 lM H2DCFDA for 30 min at 37�C.
Cells were then washed, resuspended in 19 PBS and ROS
generation was then determined by flow cytometric anal-
ysis. To rule out hydrogen peroxide generation in phenol
red containing media, the assay was repeated by using 19
PBS instead of media. In this latter protocol cells were
treated when 50% confluent with 100 lM H2DCFDA for
30 min at 37�C prepared in 19 PBS followed by 40 lM
TQ for 30 min in the presence and absence of NAC. Cells
were then washed, lysed in 90% DMSO/10% PBS for
10 min in the dark and DCF fluorescence was determined
using a fluorescent plate reader with 485 nm excitation and
520 nm emission wavelengths. ROS production by TQ in
DLD-1 and Caco-2 cells was similar using both protocols.
The data presented in Fig. 4a, b is representative of 3
independent experiments done in 19 PBS.
Western blot analysis
DLD-1 cells were plated in 100-mm tissue culture dishes
and treated with 40lM TQ for different durations in the
presence or absence of 5 mM NAC (2 h), 20 lM
SP600125 (1.5 h), 50 lM PD98059 (2 h). Cellular protein
extracts were prepared in 29 SDS-lysis buffer (0.25 M
Tris–HCl; pH 6.8, 20% glycerol, 4% SDS) and in 1:100
Protease inhibitor (Roche Applied Science, Penzberg,
Germany). Protein extracts were quantified using the DC
Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules,
California, USA) according to the manufacturer’s protocol.
Protein samples were mixed with 10% b-mercaptoethanol
and 29 sample buffer containing bromophenol blue for gel
electrophoresis. An equal amount of protein lysate was
placed on 12% SDS–PAGE for 2 h at 90 V. After elec-
trophoresis, proteins were transferred onto polyvinylidene
difluoride (PVDF) membrane (Amersham, Arlington, IL)
in transfer buffer under 30 volts overnight at 4�C. After
transfer, the membrane was immunoblotted with appro-
priate primary and secondary antibodies (all obtained from
Santa Cruz, California, USA; except GAPDH), and was
reacted with enhanced chemiluminescence reagent and
exposed to X-ray films for different time periods. Equal
loading was then verified through re-probing the membrane
with the primary antibody for GAPDH (Biogenesis, Poole,
UK) or actin. Band quantification was performed using the
Labworks software (Ultraviolet Products, Upland, Canada).
The cellular activation of signaling ELISA (CASE) kit
DLD-1 cells were plated at 1.5 9 105 cells/ml in 96-well
plate (in triplicate). Following overnight starvation in
serum-free medium, cells were treated with 40 lM TQ for
30 min after which cell culture medium was removed and
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cells were fixed with 100 ll of 4% fixing buffer. The rel-
ative extent of target protein phosphorylation (p-ERK,
p-JNK, p-p38) was determined using CASE kits according
to the manufacturer’s procedures (SuperArray Bioscience
Corporation, Frederick, Maryland, USA).
Statistical analysis
Results are expressed as means ± standard deviation (SD).
Statistical analysis was performed using SPSS Student
Version 11.0 Software Package. Comparisons between
different treatments were evaluated using a one-tailed
Student’s t-test. The level of significance was set at 0.05.
* P \ 0.05, ** P \ 0.01.
Results
TQ selectively inhibits the proliferation of human colon
cancer cells
To study TQ antiproliferative effects, we used the normal
intestinal cell line FHs74Int and a panel of human colon
cancer cell lines having different p53 status. As shown in
Fig. 1, TQ inhibited the growth of HT-29, HCT-116, DLD-1,
Lovo, and Caco-2 in a dose- and time-dependent man-
ner. The IC50 after 24 h of TQ incubation were 30 lM in
HCT-116, 42 lM in DLD-1, 38 lM in Lovo, and 15 lM in
Caco-2. The IC50 after 48 h of TQ incubation were 110 lM
in HT-29, 14 lM in HCT-116, 23 lM in DLD-1, 28 lM in
Lovo, and 12.5 lM in Caco-2. HT-29 cell line was the least
sensitive to TQ-induced growth inhibition, while Caco-2
cells were most sensitive to the drug. Interestingly, in
response to 24 h incubation, no significant growth
inhibition or toxicity was observed in FHs74Int human
normal intestinal cells for TQ doses up to 60 lM (Fig. 1f).
To understand the observed cell growth inhibition, we
further evaluated the drug effects on cell cycle distribution.
TQ induces apoptosis in DLD-1 cells but not in HT-29
cells
Cell cycle analyses were carried out in DLD-1, a cell line
showing sensitivity (IC50 = 42 lM) to TQ. DLD-1 cells
were treated with 40 lM TQ for 24 or 48 h, and harvested
for flow cytometric analysis of DNA content by PI staining.
TQ caused a significant increase in the percentage of cells
in the preG1 phase of the cell cycle in time- dependent
manner (Fig. 2a): at 40 lM TQ 24 h from 2.5 to 18.8%,
and 48 h from 4.0 to 31.2%.
To understand and confirm the nature of cell death,
apoptosis induction was determined using the M30 cyto-
death antibody which recognizes a specific caspase cleav-
age site with cytokeratine 18, a hallmark of early apoptosis
induction, that is not detected in native cells.
As shown in Fig. 2b, 40 lM TQ caused a significant
shift of the peak (sign of apoptosis) in time-dependent
manner: 21.4 and 36.1% at 24 and 48 h, respectively. The
time-dependent increase in apoptosis by TQ was further
confirmed by the M30 immunofluorescent images showing
clear cytoplasmic signals for M30 antibody after TQ
treatment (Fig. 2c). At 24 h, the percentage of preG1 cells
obtained in response to 40 lM TQ (18.8%) (Fig. 2a) cor-
related well (R2 = 0.86) with the extent of apoptosis
observed using the M30 cytodeath assay (21.4%) (Fig. 2b).
A third line of evidence of TQ-induced apoptosis in DLD-1
cells was obtained by measuring the caspase-3/7 activity:
a 2.5- and 4-fold increase in caspase-3/7 activity was
Fig. 1 Effect of TQ on the proliferation of the human colon cancer
cell lines, HT-29 (a), HCT-116 (b), DLD-1 (c), Lovo (d), and Caco-2
(e) and on human normal intestinal cells (FHs74Int) (f). Cells were
plated in 96-well plates at 105 cells/ml and treated with \ 0.1%
methanol (control) or TQ. Cell proliferation was determined by the
Cell Titer96 non-radioactive cell proliferation assay as described in
‘‘Materials and methods’’. Results are expressed as percentages of
methanol-treated cells. Each value is the mean ± SD of two separate
experiments each done in triplicates. A one-tailed t-test was used for
each TQ concentration (* P \ 0.05, ** P \ 0.01)
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observed at 24 h and 48 h after 40 lM TQ, respectively
(Fig. 2d).
On the other hand, in the relatively resistant HT-29 cell
line, treatment with 40 lM TQ caused no increase in the
percentage of cells in the preG1 phase of the cell cycle
(Fig. 3a) and this was further confirmed by the lack of shift
in the M30 cytodeath peaks and the nearly complete
absence of cytoplasmic fluorescence in the M30 immuno-
fluorescence analysis (Fig. 3b, c).
Oxidative stress mediates TQ’s antineoplastic and pro-
apoptotic effects
ROS is an important mode of action of many chemother-
apeutic agents. Knowing that quinones undergo redox
cycling in the presence of oxygen to produce ROS, we
sought to explore whether cell death induced by TQ is due
to its pro-oxidant effects.
To confirm the role of ROS and to determine their direct
involvement in TQ-induced cell death, DLD-1 and Caco-2
cells were pre-treated with the strong antioxidant NAC in
the presence and absence of TQ, and growth inhibition was
measured by cell proliferation assay. Pre-treating the cells
with 5 mM NAC for 2 h totally reversed the inhibitory
effects of the drug on cell proliferation and viability was
restored to 100% (Fig. 4a, b). The exact same effect was
observed with HCT-116 (data not shown). These data are
in accordance with our finding that TQ treatment elicited a
strong ROS production as measured by the extent of DCF
fluorescence in DLD-1 and Caco-2 cells which was
Fig. 2 TQ induces apoptosis in DLD-1 cells. a DLD-1 cells were
treated with 40 lM TQ and harvested for FACScan flow cytometry
after 24 and 48 h. The distribution of cell cycle phases with different
DNA contents was determined using FACScan flow cytometry. The
percentages of cells in the preG1, G0/G1, S, and G2/M phases were
determined using Cell Quest and are indicated at the top right of each
figure. Each value is the mean ± SD of 2 independent experiments
done in duplicate. b, c Cells were processed as above and early
apoptosis induction characterized by caspase cleavage was
determined using the M30 cytodeath detection kit and measured by
flow cytometry (b) and fluorescent microscopy (c). The percentage of
apoptotic cells was scored using Cell Quest. Each value is the
mean ± SD of 2 independent experiments done in duplicate. d TQ
induces caspase-3 cleavage. DLD-1 cells were treated as above. 50 lg
of proteins were incubated with equal volume of caspase-3/7 reagent
and luminescence was measured by a microplate reader. Each value is
the mean ± SD of 2 independent experiments done in duplicate. A
one-tailed t-test was used for each TQ concentration (** P \ 0.01)
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inhibited in the presence of NAC by 40 and 60%, respec-
tively (Fig. 4a, b). To decipher the link between ROS
production and apoptosis induction, DLD-1 cells were pre-
treated with NAC prior to TQ, and apoptosis induction was
measured 24 h later by the M30 cytodeath assay. As evi-
dent from Fig. 4c, NAC pre-treatment completely abol-
ished TQ’s apoptotic effect in DLD-1 cells. The percentage
of apoptosis decreased from 21.4% in TQ treated alone to
0.7% in TQ ? NAC (Fig. 4c) confirming that TQ’s effect
is mediated via ROS production. The latter effect was
further confirmed by measuring the caspase-3 activity in
DLD-1 cells treated with TQ in the presence or absence of
NAC. TQ induced 2-fold increase in caspase-3 activity, an
effect which was inhibited by 55% upon NAC pre-treat-
ment (Fig. 4d). HT-29 cells treated with TQ, however, did
not elicit any oxidant shift as compared to control (Fig. 5a).
Since NAC is a thiol compound which may react with
TQ [34], we investigated whether the loss of TQ growth
inhibition in cells pre-treated with NAC is due to a direct
complexation of the two molecules. Mixing TQ and NAC
in vitro at 37�C and subjecting the mixture to HPLC
analysis resulted in only 30% loss of TQ due to binding
with NAC, however, the majority of TQ (70%) was still
available (data not shown). These findings suggest that the
loss of TQ activity in the presence of NAC is not due to a
complex forming between the two molecules but rather to
the inhibition of TQ-induced ROS generation.
We then sought to understand the mechanism of drug
resistance in HT-29 cells. These cells are known to express
high levels of DT-diaphorase [35], an enzyme that cata-
lyzes the two-electron reduction of quinones (oxidized
form) to hydroquinones (reduced form)[36]. If the high
levels of DT-diaphorase are responsible for drug resistance,
then inhibiting this enzyme should sensitize HT-29 cells to
TQ. Enzyme levels were reduced by treatment with the
specific DT-diaphorase inhibitor dicumarol. Pre-incubation
of HT-29 cells with dicumarol sensitized them to TQ and
reduced the IC50 from 95 to 63 lM (Fig. 5b). Therefore,
the high levels of DT-diaphorase enzyme in HT-29
appeared to be partly responsible for their resistance.
TQ activates members of the MAPK family in DLD-1
Oxidant stress is known to activate members of the
MAPK family, ERK, JNK, and p38 by phosphorylation
[37]. To elucidate the link between ROS production by
Fig. 3 HT-29 cells are resistant to apoptotic cell death by TQ. a HT-
29 cells were treated with 40 lM TQ and harvested for FACScan flow
cytometry after 24 and 48 h. The distribution of cell cycle phases with
different DNA contents was determined using FACScan flow
cytometry. The percentages of cells in the preG1, G0/G1, S, and
G2/M phases were determined using Cell Quest and are indicated at
the top right of each figure. Each value is the mean ± SD of 2
independent experiments done in duplicate. b, c Cells were processed
as above and early apoptosis induction characterized by specific
caspase cleavage site within cytokeratin 18 was determined using the
M30 cytodeath detection kit and measured by flow cytometry (b) or
by fluorescent microscope using counter staining with DAPI (c)
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TQ and the subsequent activation of members of the
MAPK, we examined the effect of TQ treatment on the
phosphorylation state of ERK, JNK, and p38 expression
by western blotting and ELISA. Interestingly, a 12-fold
increase in the level of p-ERK proteins was observed as
early as 15 min (Fig. 6a), but no significant changes were
found in total ERK1 and ERK2 protein (Fig. 6a). Like-
wise, there was a 14-fold upregulation of p-JNK expres-
sion without any significant modulation of the total JNK1
and JNK 2 protein (Fig. 6a). The phosphorylation of JNK
and ERK was lost after 24 h (Fig. 7c). No changes in
p-p38 and total p38 protein expression were observed in
response to TQ (Fig. 6a). The upregulation in the phos-
phorylated state of the JNK and ERK was further con-
firmed by ELISA. As early as 30 min, TQ caused
3–4 fold increase in the relative extent of p-ERK and
p-JNK phosphorylation, whereas again p-p38 did not
show any activity induction (Fig. 6b).
Fig. 4 TQ causes ROS production and NAC abrogates growth
inhibition by TQ in Caco-2 (a) and DLD-1 (b) cells. For proliferation
assay, cells were pre-treated with 5 mM NAC for 2 h after which TQ
(20, 40, and 60 lM) was added and cell proliferation was determined
24 h post-treatment. For ROS production, cells were incubated with
40 lM TQ prepared in 1 9 PBS or in DMEM for 30 min in the
presence or absence of NAC. ROS production was assessed by DCFH
assay and similar effects were obtained upon using phenol free PBS
or DMEM. The data presented are representative of 3 independent
experiments done in 1 9 PBS. c DLD-1 cells were pre-treated with
NAC for 2 h after which TQ was added. Apoptotic cell death was
assessed by M30 cytodeath assay as described in ‘‘Materials and
methods’’. d Caspase-3 activation in TQ treated DLD-1 cells in the
presence and absence of NAC. Proteins were extracted as described in
‘‘Materials and methods’’. 50 lg of proteins were incubated with
equal volume of caspase-3/7 reagent and luminescence was measured
by a microplate reader. Each value is the mean ± SD of 2
independent experiments done in duplicate. Asterisks (**) indicate
values that are significantly different (Student’s t test, P \ 0,01) as
compared to cells treated with TQ alone
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To provide further evidence that MAPK phosphoryla-
tion is due to ROS produced by TQ, DLD-1 cells were pre-
treated with NAC, and the expression levels of p-ERK and
p-JNK were determined. Interestingly, p-ERK and p-JNK
upregulation was completely abrogated in the presence of
NAC (Fig. 6a). In fact, NAC pre-treated cells showed ERK
and JNK phosphorylation levels similar to the control.
Thus, our findings suggest that there is a ROS-dependent
induction of p-ERK and p-JNK by TQ.
ERK and JNK activation protect DLD-1 from
TQ-mediated oxidative stress and apoptosis
In an effort to characterize the role played by ERK and
JNK in TQ-induced cell death, DLD-1 were pre-treated for
2 h with the specific ERK 1/2 inhibitor PD98059 or 1.5 h
with the specific JNK inhibitor SP600125, and cell prolif-
eration was assessed 24 h post TQ treatment. As shown in
Fig. 7a, treatment of DLD-1 with either inhibitor did not
affect the proliferation of the cells. Interestingly, inhibition
of both ERK and JNK pathways sensitized DLD-1 cells to
TQ’s antiproliferative effect (Fig. 7a). ERK and JNK
inhibition led, respectively, to 20 and 40% decrease in
DLD-1 proliferation at 40 lM TQ (Fig. 7a). To decipher
the link between MAPK activation, and apoptosis induc-
tion, DLD-1 cells were pre-treated with, PD98059, and
SP600125 prior to TQ, and apoptosis induction was mea-
sured 24 h later by the M30 cytodeath assay. As evident
from Fig. 7b, MAPK inhibition potentiated TQ’s apoptotic
effect. Figure 7b shows that PD98059 and SP600125,
which have negligible apoptogenic activity, act synergis-
tically with 40 lM TQ to increase the percentage of
apoptotic DLD-1 cells; while a 2-fold increase in the per-
centage of apoptotic cells was observed in DLD-1 treated
with TQ ? PD98059, a 3-fold increase was observed in
TQ ? SP600125 (Fig. 7b). As seen in Fig. 7c, the levels of
p-JNK and p-ERK are completely inhibited with 50 lM
Fig. 5 HT-29 cells are resistant to ROS generation by TQ. a Cells were
incubated with TQ for 30 min then harvested, incubated with 10 lM
DCF-DA dye for 30 min and fluorescence was read using flow
cytometry. b The DT-diaphorase inhibitor dicumarol sensitizes HT-29
cells to TQ’s antineoplastic effects. HT-29 were treated for 1 h with
100 lM dicumarol prior to TQ (20, 40, 60 and 80 lM), and cell
proliferation was determined 24 h post-treatment. Cell proliferation
was determined by the Cell Titer96 non-radioactive cell proliferation
assay as described in ‘‘Materials and methods’’. Results are expressed as
percentages of methanol-treated cells. Each value is the mean ± SD of
2 separate experiments each done in triplicates. A one-tailed t-test was
used for each TQ concentration (* P \ 0.05, ** P \ 0.01)
Fig. 6 Impact of ROS and NAC on MAPK activation. a Represen-
tative Western blots show the time dependent increase in phosphor-
ylation of ERK1/2 and JNK. Cells were incubated in 40 lM TQ for
15 min, 1, 4 or 12 h. Additional treatments included pre-incubation
with NAC for 2 h prior to TQ for 1 or 12 h. Quantification was assessed
using Labworks software (Ultraviolet Products, Upland, Canada). b TQ
activates p-ERK, p-JNK but not p-p38. DLD-1 were plated, serum-
starved and treated with 40 lM TQ for 30 min. Phospho- (JNK, p38,
ERK) and total (JNK, p38, ERK) levels were measured using the CASE
Cellular activation of Signalling ELISA kits. Following incubation
with primary and secondary antibodies, the amount of bound antibody
in each well was determined using a developing solution and an ELISA
Plate Reader. The absorbance readings were then normalized to relative
cell number as determined by a cell staining solution
Apoptosis
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PD98059 and 20 lM SP600125 while the levels of total
ERK and JNK are not affected.
Apoptosis induction was further confirmed by measur-
ing the caspase-3 activity in DLD-1 cells treated with TQ
with and without the inhibitors. TQ induced a 2-fold
increase in caspase-3 activity (Fig. 7d). PD98059 and
SP600125 combined with TQ induced more activation of
the caspase-3 activity as compared to TQ alone (Fig. 7d).
The percentage increased from 2.2-fold in TQ treated alone
to 3.6-fold in TQ ? SP600125 (Fig. 7d). This has been
further shown by the increase in the loss of the mito-
chondrial potential in the combined treatment as compared
to TQ alone (Fig. 7e).
Discussion
The mechanism of quinone cytotoxicity is attributed
mainly to their ease of reduction and therefore their ability
to act as oxidizing or dehydrogenating agents. In biological
systems, quinones can undergo one or two electron
reduction by cellular reductases, leading to the corre-
sponding semiquinones or hydroquinones, respectively.
Reaction of semiquinones with molecular oxygen
results in the concomitant production of ROS to which
the toxicity of quinones is attributed. However, two
electron reductions generate hydroquinones and, in gen-
eral, lead to detoxification [38]. In this study, we showed
Fig. 7 Impact of MAPK activation on TQ-induced apoptosis.
a PD98059 and SP600125 enhance TQ’s antineoplastic effects in
DLD-1. Cells were pre-treated with PD98059 for 1.5 h and with
SP600125 for 1 h, after which TQ (20, 40 and 60 lM) was added and
proliferation was assessed 24 h later by the Cell Titer96 non-
radioactive cell proliferation assay as described in ‘‘Materials and
methods’’. A one-tailed student t-test was used for each TQ
concentration (* P \ 0.05, ** P \ 0.01). b DLD-1 cells were pre-
treated PD98059 for 2 h, and SP600125 for 1.5 h after which TQ was
added. Apoptotic cell death was assessed by M30 cytodeath assay as
described in ‘‘Materials and methods’’. c Representative Western
blots show lack of activation of p-ERK and p-JNK activation in
samples treated with PD98059 and SP600125. d Caspase-3 activation
in TQ treated DLD-1 cells in the presence and absence of PD98059
and SP600125. Proteins were extracted as described in ‘‘Materials and
methods’’. 50 lg of proteins were incubated with equal volume of
caspase-3/7 reagent and luminescence was measured by a microplate
reader. Each value is the mean ± SD of 2 independent experiments
done in duplicate. Asterisks (**) indicate values that are significantly
different (Student’s t test, P \ 0.01) as compared to cells treated with
TQ alone. e MAPK inhibition potentiates the loss of mitochondrial
potential. DLD-1 cells were treated as above and the loss of
mitochondrial potential was measured 24 h later using 25 nM
DiOC6(3) as described in ‘‘Materials and methods’’. Each value is
the mean ± SD of two separate experiments each done in triplicates
Apoptosis
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that TQ exhibits an antiproliferative effect in a variety of
colon cancer cells (Fig. 1). Apoptosis induction was the
hallmark of TQ’s effect in DLD-1 cells as evidenced by
flow cytometric analysis, M30 cytodeath assay, and acti-
vation of caspase-3 (Fig. 2). TQ induced apoptosis by
causing oxidative stress via the generation of ROS
(Fig. 4). That ROS production by TQ is responsible for its
antiproliferative effects is supported by at least three lines
of evidence.
First, high levels of intracellular ROS were generated in
response to TQ as shown by the accumulation of the
fluorescent probe DCF (Fig. 4a, b). Second, the strong
antioxidant N-acetyl cysteine abrogated TQ’s antiprolifer-
ative effect and completely abolished apoptosis induction
in DLD-1 cells as shown by M30 cytodeath assay (Fig. 4c)
and resulted in 55% reduction in caspase-3 activity assay
(Fig. 4d). Third, HT-29 cells which contain high levels of
the quinone-reducing enzyme DT-diaphorase were resis-
tant to TQ and the drug did not elicit ROS production in
these cells (Fig. 5a). Interestingly, the addition of the
DT-diaphorase inhibitor, dicumarol, sensitized HT-29 cells
to drug treatment (Fig. 5b). This indicates that hydroqui-
none, the by-product of diaphorase reduction, is a molecule
that is less active than TQ.
Our results on TQ pro-oxidant effects are in accordance
with our recent study showing that TQ is involved in
mitochondrial ROS generation and exerts antineoplastic
effects in human osteosarcoma cells [17]. To our knowl-
edge, this and our other study [4] are the only reports
providing evidence of ROS involvement in apoptosis in
colon cancer cells following TQ treatment. In fact, several
reports have discussed the antioxidant properties that TQ
possesses: the compound has potent superoxide anion
scavenging abilities, and inhibits iron-dependent micro-
somal lipid peroxidation [19]. The generation of superox-
ide anion by the xanthine/xanthine oxidase system was
inhibited by TQ in a dose-dependent manner. TQ showed
extremely high superoxide anion radical-scavenging abili-
ties in pure chemical systems [18]. Likewise, TQ’s anti-
oxidant effect has been recently reported to be involved in
the treatment and prevention of several types of cancer,
including pancreatic, cervical, and prostate cancer cells [8,
9, 39]. This seeming discrepancy with our results may be
due to the fact that these studies are either conducted in
pure chemical systems or in different cancer cell lines.
Studies conducted in pure chemical systems do not take
into account the multitude of reactions that could be
undertaken by TQ, and thus are not reflective of TQ’s
oxidant/antioxidant properties in cells.
Once incorporated into our colon cancer cells, TQ
undergoes redox-cycling, thus leading to oxidative stress.
Having established that TQ is an oxidative stress-causing
agent in colon cancer cells, we then studied its effects on
the MAPK. The ERK, JNK, and p38 subfamilies have all
been shown to be activated in response to oxidant injury
and therefore might contribute to influencing survival [27,
28]. The involvement of the MAPK in response to oxidant
stress was confirmed in DLD-1 cells in our study: JNK and
ERK, but not p38 kinases, were activated significantly in
the presence of TQ. This correlation was further confirmed
by our data, showing that pre-incubation of DLD-1 cells
with NAC reduced ERK and JNK activation to control
levels.
Extensive investigations were made to examine the
importance of MAPK cascade in the regulation of apop-
tosis during stress conditions. Many of these studies have
provided the general view that activation of the ERK
pathway confers survival signals, which counteracts the
pro-apoptotic signaling associated with JNK and p38
activation [40, 41]. Interestingly, our results on ERK and
JNK activation confirmed a survival role of both MAPK
whereby inhibition of the ERK pathway by PD98059 and
JNK pathway by SP600125 potentiated apoptosis induction
by TQ and increased caspase-3 activity in our cell system.
Our findings implicating ERK activation following TQ
treatment are similar to other studies using H2O2 as an
oxidant [41]. In those studies, pharmacologic agents as
well as molecular alterations resulting in reduced ERK
activation were found to sensitize 3T3 cells to H2O2,
while molecular strategies leading to elevated ERK acti-
vation enhanced survival of cells treated with the oxidant.
Subsequent studies from a number of laboratories con-
firmed these findings in other cell types and with other
agents in which pharmacologic inhibition of ERK
increased H2O2-induced apoptosis in HeLa cells and in
young hepatocytes [31, 42]. ERK activation can also
contribute to apoptosis in response to oxidant injury in
different model system. These include hyperoxia-induced
apoptosis of macrophages [43], cisplatin-induced apopto-
sis of HeLa cells [44], hydrogen peroxide-induced apop-
tosis of oligodendrocytes [45], and mesengial cells [46].
In fact, TQ has been shown to have opposing effects on
ERK phosphorylation (activation vs. inhibition) which
seems to be cell-type specific and stimulus specific.
Whereas TQ was found in our study to cause ERK acti-
vation in colon cancer cells, it inhibited ERK phosphor-
ylation in response to the vascular endothelial growth
factor (VEGF) in human umbilical vein endothelial cells
(HUVEC). Interestingly, the suppression of VEGF-
induced ERK activation contributed to the inhibition of
HUVEC migration, invasion and tube formation [47]. It
would be interesting to determine whether TQ would have
similar inhibitory effects on ERK phosphorylation in
cancer cells exposed to growth factors.
JNK activation is reported as an apoptosis mediator in
the treatment of several types of cancer. These include
Apoptosis
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paclitaxel-induced apoptosis in human melanoma cell lines
A375 and BLM [48], arsenic trioxide-induced apoptosis in
HeLa cells [49], capsaicin-induced apoptosis in PC-3 cells
[50], and in glibenclamide-induced apoptosis in human
gastric cell line MGC-803 [23]. Interestingly, our results on
JNK activation as a protective survival signal, although
they do not support the common role played by JNK in
apoptosis, are in accordance with others who reported a
pro-survival role for JNK. For instance, Engelbrecht et al.,
proved a protective role of JNK in response to reperfusion
in rat neonatal ventricular myocytes subjected to simulated
ischemia/reperfusion [51]. Furthermore, Dougherty and his
co-workers have shown that JNK activation is a pro-sur-
vival factor in neonatal cardiac myocytes subjected to
hypoxic injury [52]. What determines whether ERK and
JNK will act in a pro-apoptotic or anti-apoptotic fashion
remains an important unanswered question, but the dura-
tion of their activation as well as the cell type used may be
determinant factors.
Our proposed mechanism of TQ antineoplastic effect
can be summarized as follows. TQ treatment induced ROS
generation, which increased JNK and ERK in an attempt to
bypass the stress injury. However, ERK and JNK fail to
confer a survival role, and the cells undergo apoptosis. This
is confirmed by the fact that NAC pre-treatment abolishes
apoptosis along with the fact that MAPK inhibition sensi-
tizes the cells to TQ’s apoptotic effect generated by ROS.
Cells pre-treated with ERK and JNK inhibitor prior to
TQ addition resulted in 60 and 80% apoptosis induction,
respectively. These results might be exploited to improve
the antitumor properties of TQ, and provide a rationale for
the use of TQ in combination with kinase inhibitors for the
treatment of colon cancer. Sorafenib, a multikinase inhib-
itor, has been approved by the FDA for the treatment of
renal and hepatic cancer [53]. Sorafenib has been suc-
cessfully used in combination treatment with other anti-
cancer compounds such as doxorubicine [54], and
irinotecan [55]. Combinatorial treatment provided a syn-
ergistic effect as compared to compounds tested alone.
Therefore, further evaluation involving combination treat-
ment of TQ and MAPK inhibitors, such as sorafenib, could
be next used in an attempt to improve TQ’s antitumor
effect.
In conclusion, this study has shown that in human colon
cancer cells, TQ is rapidly absorbed by the cells where it
undergoes redox-cycling and generates ROS. Our data
provide evidence that ROS mediate TQ’s apoptosis
induction, whereby NAC pre-treatment completely abol-
ished TQ’s effect. The produced ROS result in the acti-
vation of p-ERK and p-JNK, which prove to play a
protective role. When inhibiting the activation of ERK and
JNK by PD98059 and SP600125, respectively a further
potentiation of the apoptotic response by TQ was observed.
Acknowledgments We thank members of the Central Research
Science Laboratory at the American University of Beirut, Lebanon for
their help in using the flow cytometer and HPLC. We thank Isabel
Zeittrager and Astrid Taut from Department of Medicine 1, Erlangen,
Germany for their technical assistance. This work was supported by
Deutsche Forschungsgemeinschaft (SCHN477/7-3, SCHN477/7-4)
and by the University Research Board of the AUB and the Lebanese
National Council for Scientific Research. Nahed El-Najjar was partly
supported by the DAAD.
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