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: amro@aub.edu.lb

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

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.

Apoptosis

123

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

Apoptosis

123

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)

Apoptosis

123

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)

Apoptosis

123

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)

Apoptosis

123

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

Apoptosis

123

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

123

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

123

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

123

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.

References

1. Ali BH, Blunden G (2003) Pharmacological and toxicological

properties of Nigella sativa. Phytother Res 17:299–305. doi:

10.1002/ptr.1309

2. Marsik P, Kokoska L, Landa P, Nepovim A, Soudek P, Vanek T

(2005) In vitro inhibitory effects of thymol and quinones of

Nigella sativa seeds on cyclooxygenase-1- and -2-catalyzed

prostaglandin E2 biosyntheses. Planta Med 71:739–742. doi:

10.1055/s-2005-871288

3. Norwood AA, Tan M, May M, Tucci M, Benghuzzi H (2006)

Comparison of potential chemotherapeutic agents, 5-fluoruracil,

green tea, and thymoquinone on colon cancer cells. Biomed Sci

Instrum 42:350–356

4. Gali-Muhtasib H, Kuester D, Mawrin C et al (2008) Thymoquinone

triggers inactivation of the stress response pathway sensor CHEK1

and contributes to apoptosis in colorectal cancer cells. Cancer Res

68:5609–5618. doi:10.1158/0008-5472.CAN-08-0884

5. Gali-Muhtasib H, Ocker M, Kuester D et al (2008) Thymoqui-

none reduces mouse colon tumor cell invasion and inhibits tumor

growth in murine colon cancer models. J Cell Mol Med 12:330–

342. doi:10.1111/j.1582-4934.2007.00095.x

6. El-Mahdy MA, Zhu Q, Wang QE, Wani G, Wani AA (2005)

Thymoquinone induces apoptosis through activation of caspase-8

and mitochondrial events in p53-null myeloblastic leukemia

HL-60 cells. Int J Cancer 117:409–417. doi:10.1002/ijc.21205

7. Womack K, Anderson M, Tucci M, Hamadain E, Benghuzzi H

(2006) Evaluation of bioflavonoids as potential chemotherapeutic

agents. Biomed Sci Instrum 42:464–469

8. Richards LR, Jones P, Hughes J, Benghuzzi H, Tucci M (2006)

The physiological effect of conventional treatment with epi-

gallocatechin-3-gallate, thymoquinone, and tannic acid on the

LNCaP cell line. Biomed Sci Instrum 42:357–362

9. Tan M, Norwood A, May M, Tucci M, Benghuzzi H (2006)

Effects of (-)epigallocatechin gallate and thymoquinone on pro-

liferation of a PANC-1 cell line in culture. Biomed Sci Instrum

42:363–371

10. Rooney S, Ryan MF (2005) Modes of action of alpha-hederin and

thymoquinone, active constituents of Nigella sativa, against

HEp-2 cancer cells. Anticancer Res 25:4255–4259

11. Martin TM, Benghuzzi H, Tucci M (2006) The effect of con-

ventional and sustained delivery of thymoquinone and levodopa

on SH-SY5Y human neuroblastoma cells. Biomed Sci Instrum

42:332–337

12. Tekeoglu I, Dogan A, Demiralp L (2006) Effects of thymoqui-

none (volatile oil of black cumin) on rheumatoid arthritis in rat

models. Phytother Res 20:869–871. doi:10.1002/ptr.1964

13. El Gazzar M, El Mezayen R, Marecki JC, Nicolls MR, Canastar

A, Dreskin SC (2006) Anti-inflammatory effect of thymoquinone

in a mouse model of allergic lung inflammation. Int Immuno-

pharmacol 6:1135–1142. doi:10.1016/j.intimp.2006.02.004

14. El Mezayen R, El Gazzar M, Nicolls MR, Marecki JC, Dreskin

SC, Nomiyama H (2006) Effect of thymoquinone on

Apoptosis

123

cyclooxygenase expression and prostaglandin production in a

mouse model of allergic airway inflammation. Immunol Lett

106:72–81. doi:10.1016/j.imlet.2006.04.012

15. El Gazzar MA, El Mezayen R, Nicolls MR, Dreskin SC (2007)

Thymoquinone attenuates proinflammatory responses in lipopo-

lysaccharide-activated mast cells by modulating NF-kappaB

nuclear transactivation. Biochim Biophys Acta 1770:556–564.

doi:10.1016/j.bbagen.2007.01.002

16. Gali-Muhtasib H, Diab-Assaf M, Boltze C et al (2004) Thymo-

quinone extracted from black seed triggers apoptotic cell death in

human colorectal cancer cells via a p53-dependent mechanism.

Int J Oncol 25:857–866

17. Roepke M, Diestel A, Bajbouj K et al (2007) Lack of p53 aug-

ments thymoquinone-induced apoptosis and caspase activation in

human osteosarcoma cells. Cancer Biol Ther 6:160–169

18. Nagi MN, Mansour MA (2000) Protective effect of thymoqui-

none against doxorubicin-induced cardiotoxicity in rats: a pos-

sible mechanism of protection. Pharmacol Res 41:283–289. doi:

10.1006/phrs.1999.0585

19. Badary OA, Taha RA, Gamal el-Din AM, Abdel-Wahab MH

(2003) Thymoquinone is a potent superoxide anion scavenger.

Drug Chem Toxicol 26:87–98

20. Mansour MA, Nagi MN, El-Khatib AS, Al-Bekairi AM (2002)

Effects of thymoquinone on antioxidant enzyme activities, lipid

peroxidation and DT-diaphorase in different tissues of mice: a

possible mechanism of action. Cell Biochem Funct 20:143–151.

doi:10.1002/cbf.968

21. Lin YT, Yang JS, Lin SY et al (2008) Diallyl disulfide (DADS)

induces apoptosis in human cervical cancer Ca Ski cells via

reactive oxygen species and Ca2?-dependent mitochondria-

dependent pathway. Anticancer Res 28:2791–2799

22. Zhang R, Humphreys I, Sahu RP, Shi Y, Srivastava SK (2008) In

vitro and in vivo induction of apoptosis by capsaicin in pancreatic

cancer cells is mediated through ROS generation and mitochon-

drial death pathway. Apoptosis 13:1465–1478. doi:10.1007/

s10495-008-0278-6

23. Qian X, Li J, Ding J, Wang Z, Duan L, Hu G (2008) Gliben-

clamide exerts an antitumor activity through reactive oxygen

species-c-jun NH2-terminal kinase pathway in human gastric

cancer cell line MGC-803. Biochem Pharmacol 76:1705–1715.

doi:10.1016/j.bcp.2008.09.009

24. Xiao D, Powolny AA, Singh SV (2008) Benzyl isothiocyanate

targets mitochondrial respiratory chain to trigger reactive oxygen

species-dependent apoptosis in human breast cancer cells. J Biol

Chem 283:30151–30163. doi:10.1074/jbc.M802529200

25. Pan MH, Gao JH, Lai CS et al (2008) Antitumor activity of

3,5,40-trimethoxystilbene in COLO 205 cells and xenografts in

SCID mice. Mol Carcinog 47:184–196. doi:10.1002/mc.20352

26. Feng R, Ni HM, Wang SY et al (2007) Cyanidin-3-rutinoside, a

natural polyphenol antioxidant, selectively kills leukemic cells by

induction of oxidative stress. J Biol Chem 282:13468–13476. doi:

10.1074/jbc.M610616200

27. Navarro R, Busnadiego I, Ruiz-Larrea MB, Ruiz-Sanz JI (2006)

Superoxide anions are involved in doxorubicin-induced ERK

activation in hepatocyte cultures. Ann NY Acad Sci 1090:419–

428. doi:10.1196/annals.1378.045

28. Matsuzawa A, Ichijo H (2005) Stress-responsive protein kinases

in redox-regulated apoptosis signaling. Antioxid Redox Signal

7:472–481. doi:10.1089/ars.2005.7.472

29. Kyosseva SV (2004) Mitogen-activated protein kinase signaling.

Int Rev Neurobiol 59:201–220. doi:10.1016/S0074-7742(04)

59008-6

30. Ku BM, Lee YK, Jeong JY et al (2007) Ethanol-induced oxida-

tive stress is mediated by p38 MAPK pathway in mouse hippo-

campal cells. Neurosci Lett 419:64–67. doi:10.1016/j.neulet.

2007.03.049

31. Wang X, Martindale JL, Liu Y, Holbrook NJ (1998) The cellular

response to oxidative stress: influences of mitogen-activated

protein kinase signalling pathways on cell survival. Biochem J

333(Pt 2):291–300

32. Benhar M, Dalyot I, Engelberg D, Levitzki A (2001) Enhanced

ROS production in oncogenically transformed cells potentiates

c-Jun N-terminal kinase and p38 mitogen-activated protein kinase

activation and sensitization to genotoxic stress. Mol Cell Biol

21:6913–6926. doi:10.1128/MCB.21.20.6913-6926.2001

33. Owuor ED, Kong AN (2002) Antioxidants and oxidants regulated

signal transduction pathways. Biochem Pharmacol 64:765–770

34. Khalife KH, Lupidi G (2008) Reduction of hypervalent states of

myoglobin and hemoglobin to their ferrous forms by thymoqui-

none: the role of GSH, NADH and NADPH. Biochim Biophys

Acta 1780:627–637. doi:10.1016/j.bbagen.2007.12.006

35. Karczewski JM, Peters JG, Noordhoek J (1999) Quinone toxicity

in DT-diaphorase-efficient and -deficient colon carcinoma cell

lines. Biochem Pharmacol 57:27–37

36. Cullen JJ, Hinkhouse MM, Grady M et al (2003) Dicumarol

inhibition of NADPH: quinone oxidoreductase induces growth

inhibition of pancreatic cancer via a superoxide-mediated

mechanism. Cancer Res 63:5513–5520

37. Martindale JL, Holbrook NJ (2002) Cellular response to oxidative

stress: signaling for suicide and survival. J Cell Physiol 192:1–15.

doi:10.1002/jcp.10119

38. Asche C (2005) Antitumour quinones. Mini Rev Med Chem

5:449–467

39. Brewer J, Benghuzzi H, Tucci M (2006) Effects of thymoquinone,

lycopene, and selenomethione in the presence of estrogen on the

viability of SiHa cells in vitro. Biomed Sci Instrum 42:37–41

40. Brenner B, Koppenhoefer U, Weinstock C, Linderkamp O,

Lang F, Gulbins E (1997) Fas- or ceramide-induced apoptosis is

mediated by a Rac1-regulated activation of Jun N-terminal

kinase/p38 kinases and GADD153. J Biol Chem 272:22173–

22181

41. Guyton KZ, Gorospe M, Kensler TW, Holbrook NJ (1996)

Mitogen-activated protein kinase (MAPK) activation by butyl-

ated hydroxytoluene hydroperoxide: implications for cellular

survival and tumor promotion. Cancer Res 56:3480–3485

42. Ikeyama S, Kokkonen G, Shack S, Wang XT, Holbrook NJ

(2002) Loss in oxidative stress tolerance with aging linked to

reduced extracellular signal-regulated kinase and Akt kinase

activities. FASEB J 16:114–116. doi:10.1096/fj.01-0409fje

43. Petrache I, Choi ME, Otterbein LE et al (1999) Mitogen-activated

protein kinase pathway mediates hyperoxia-induced apoptosis in

cultured macrophage cells. Am J Physiol 277:L589–L595

44. Wang X, Martindale JL, Holbrook NJ (2000) Requirement for

ERK activation in cisplatin-induced apoptosis. J Biol Chem

275:39435–39443. doi:10.1074/jbc.M004583200

45. Brand A, Gil S, Seger R, Yavin E (2001) Lipid constituents in

oligodendroglial cells alter susceptibility to H2O2-induced apop-

totic cell death via ERK activation. J Neurochem 76:910–918

46. Ishikawa Y, Kitamura M (2000) Anti-apoptotic effect of quer-

cetin: intervention in the JNK- and ERK-mediated apoptotic

pathways. Kidney Int 58:1078–1087. doi:10.1046/j.1523-1755.

2000.00265.x

47. Yi T, Cho SG, Yi Z et al (2008) Thymoquinone inhibits tumor

angiogenesis and tumor growth through suppressing AKT

and extracellular signal-regulated kinase signaling pathways.

Mol Cancer Ther 7:1789–1796. doi:10.1158/1535-7163.MCT-

08-0124

48. Selimovic D, Hassan M, Haikel Y, Hengge UR (2008) Taxol-

induced mitochondrial stress in melanoma cells is mediated by

activation of c-Jun N-terminal kinase (JNK) and p38 pathways

via uncoupling protein 2. Cell Signal 20:311–322. doi:

10.1016/j.cellsig.2007.10.015

Apoptosis

123

49. Kang YH, Lee SJ (2008) The role of p38 MAPK and JNK in arsenic

trioxide-induced mitochondrial cell death in human cervical cancer

cells. J Cell Physiol 217:23–33. doi:10.1002/jcp.21470

50. Sanchez AM, Malagarie-Cazenave S, Olea N, Vara D, Chiloe-

ches A, Diaz-Laviada I (2007) Apoptosis induced by capsaicin in

prostate PC-3 cells involves ceramide accumulation, neutral

sphingomyelinase, and JNK activation. Apoptosis 12:2013–2024.

doi:10.1007/s10495-007-0119-z

51. Engelbrecht AM, Niesler C, Page C, Lochner A (2004) p38 and

JNK have distinct regulatory functions on the development of

apoptosis during simulated ischaemia and reperfusion in neonatal

cardiomyocytes. Basic Res Cardiol 99:338–350. doi:10.1007/

s00395-004-0478-3

52. Dougherty CJ, Kubasiak LA, Prentice H, Andreka P, Bishopric

NH, Webster KA (2002) Activation of c-Jun N-terminal kinase

promotes survival of cardiac myocytes after oxidative stress.

Biochem J 362:561–571

53. Kane RC, Farrell AT, Saber H et al (2006) Sorafenib for the

treatment of advanced renal cell carcinoma. Clin Cancer Res

12:7271–7278. doi:10.1158/1078-0432.CCR-06-1249

54. Richly H, Schultheis B, Adamietz IA et al (2008) Combination of

sorafenib and doxorubicin in patients with advanced hepatocel-

lular carcinoma: results from a phase I extension trial. Eur J

Cancer. doi:10.1016/j.ejca.2008.10.039

55. Mross K, Steinbild S, Baas F et al (2007) Results from an in vitro

and a clinical/pharmacological phase I study with the combina-

tion irinotecan and sorafenib. Eur J Cancer 43:55–63. doi:

10.1016/j.ejca.2006.08.032

Apoptosis

123