تفاصيل البحث
Thymoquinone induces apoptosis and increase ROS in ovarian cancer cell line
ورقة منشورة
6/18/2016 12:00:00 AM
كرسي المعلم محمد بن لادن لأبحاث الإعجاز العلمي في الطب النبوي
Abstract: Nigella sativa is also known for its properties as a traditional herbal healing for many ailments. In this study, the anticancer properties
of thyomquinone (TQ), the active ingredient of N. sativa, were studied using ovarian cancer cell line (Caov-3 cells). The anti-proliferative activity
of TQ was determined using MTT and the apoptosis was investigated using Flowcytometry and Annexin-V Assays. Multiparameteric cytotoxi-
city bioassays were used to quantify the changes in cell permeability and mitochondrial membrane potential. Reactive oxygen species (ROS)
and apoptosis-involved cell markers were examined to verify cell death mechanism. The MTT-assay showed that TQ induces anti-proliferative
activity on Caov-3 with an IC50 of 6.0±0.03 μg/mL, without any cytotoxic activity towards WRL-68 normal hepatocytes. A significant induction
of early phase of apoptosis was shown by annexin-V analysis. Treatment of Caov-3 cells with TQ induces decreases in plasma membrane per-
meability and mitochondrial membrane potential. Visible decrease in the nuclear area was also observed. A significant decrease is observed in
Bcl-2 while Bax is down-regulated. TQ-triggered ROS-mediated has found to be associated with Hsp70 dysregulation, an indicator of oxidative
injury. We found that TQ induced anti-cancer effect involves intrinsic pathway of apoptosis and cellular oxidative stress. Our results considered
collectively indicated that thyomquinone may be a potential agent for ovarian cancer drug development.
Key words: Thymoquinone, cell death, ovarian cancer, oxidative stress.
Introduction
In the ancient time of medicine, black cumin [Ni-
gella sativa L. (Ranunculaceae)] is considered as an
important remedy for a variety of ailments. The seeds
have been used by traditional healers in the Middle East
and Southeast Asian countries for a variety of diseases
(1, 2). The attractiveness of this plant was highly impro-
ved by the ideologys’ believes in the herb as a cure for
multiple diseases. N. sativa (black seed) oil contains
an abundance of thymoquinone, nigellone (dithymo-
quinone), conjugated linoleic acid, damascenine, me-
lanthin, nigilline, and tannins (3, 4). Thymoquinone
(Figure 1A), extracted from the seed oil of N. sativa,
has been demonstrated to possess in vivo and in vitro
anti-neoplastic effects (5, 6).This natural compound has
been shown to protect through antioxidant mechanism,
exert anti-inflammatory effects, and trigger apoptosis of
the tumor cells (7).
Ovarian cancer is the second most widespread gy-
necologic female cancer and the deadliest in terms of
absolute number (8). Symptoms of this malignant di-
sease include pelvic pain, bloating, lack of appetite and
frequent urination (9, 10). It has many complications
such as spread of the cancer to other organs, progres-
sive function loss of various organs, accumulation of
abdominal fluids and intestinal obstructions (11). Ova-
rian cancer usually has a comparatively poor prognosis.
The five-year survival rate for all stages of ovarian can-
cer is 47% (12). Treatment regularly involves chemo-
therapy and surgery, and sometimes radiotherapy (13).
An alternative treatment with larger safety margins are
needed to avoid the side effects of chemotherapy. Medi-
cinal plants with rich ethno-knowledge are considered
on the biggest source of natural anticancer agents (14).
In this study we have chosen Nigella sativa as source
of natural anticancer agent(s). The reputation of black
seed was highly promoted by religious believes in the
herb as a cure for multiple diseases. Reports from the
Prophet Muhammad (PBUH) of Islam emphasize the
significance of black cumin (15, 16). There are few stu-
dies focused specifically and in general regarding the
anti-cancer mechanism of TQ in ovarian cancer in vitro
(17-20). Therefore, the current study was designed to
investigate the anticancer mechanisms of thymoqui-
none against ovarian cancer in vitro focused on ROS
and apoptosis mechanism.
Materials and Methods
Chemicals and Reagents
Thymoquinone (>99% purity) was purchased from
Sigma (St. Louis, MO, USA). Caov-3 ovarian cancer
cell line (ATCC®HTB-75TM;Organism:Homo sapiens/
Tissue:ovary/Disease:adenocarcinoma) and WRL-68
(normal hepatocytes) used in this study were obtained
from ATTC, USA (ATCC®HTB-75TM;Organism: Homo
sapiens /Tissue:ovary/ Disease: adenocarcinoma). RPMI
Received January 4, 2016; Accepted May 22, 2016; Published May 30,
2016
* Corresponding author: Siddig Ibrahim Abdelwahab, Substance Abuse
Research Centre, Jazan University, P.O. Box 114 Jazan, 45142, Saudi Arabia.
Email: siddigroa@yahoo.com
Copyright: © 2016 by the C.M.B. Association. All rights reserved.
Taha et al. Cell. Mol. Biol.2016, 62 (6): 97-101
ISSN: 1165-158X
doi: 10.14715/cmb/2016.62.6.18
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M. M. E. Taha et al. 2016 | Volume 62 | Issue 6 Anticancer effect of Thymoquinone.
1460, fetal bovine serum (FBS) and streptomycin-peni-
cillin were procured from Bioscience Ltd. (Wokingham,
UK). Phosphate buffered saline (PBS), ethanol (95%),
3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium
bromide (MTT) was from Santa Cruz Biotechnology
(Santa Cruz, CA, USA).
Cell Culture and Viability Assay
Caov-3 and WRL-68 cell lines were cultured in a
humidified chamber with 5% CO2
at 37°C. MTT assay
was used to study the proliferation and survival of can-
cer cells with or without TQ-treatment. In this assay,
cells were seeded on a 96-well microplate at 2x105
cells/
mL in 100 μL culturing medium. They were plated in
triplicate. Concentrations of TQ (0-50μg/mL) were
prepared by serial dilution and were transferred to the
cells in the 96-well plate and incubated for 24 h. After
incubation, the viability of the cells was assessed using
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT, 5 mg/mL); 20 μL were added to the
cells in a dark place, and the cells were then covered
with aluminium foil and incubated for 4 h. After incu-
bation, all of the media were removed and 100 μL of
DMSO were added to the cells in order to solubilise the
formazan crystals. Subsequently, the absorbance was
read at a wavelength of 570 nm using a micro-plate rea-
der. The potency of cell growth inhibition for the test
agent was expressed as an IC50 value.
Multiple Cytotoxicity Assay
Cellomics Multiparameter Cytotoxicity-3 Kit was
carried out as clarified earlier in details (21). This kit
allows instant and direct quantification in the same cell
of different autonomous parameters that scrutinize cell
health, including nuclear size, morphological changes,
mitochondrial membrane potential changes, alterations
in cell permeability and cell loss. Multiparameter Cyto-
toxicity-3 Plates were examined using the CellRepor-
terTM cytofluorimeter system (Gentix/Molecular de-
vices, United Kingdom).
Image Acquisition and Cytometric Analysis
CellReporterTM cytofluorimeter system is a compu-
ter-based and automated fluorescence imaging micros-
cope that automatically recognizesflouro-stained cells
and reports the strength and allocation of fluorescence
in individual cells. The Array-Scan HCS system scans
numerous microfields in individual microwells to ob-
tain and evaluate images of single cells according to
distinct algorithms. In each microwell, 1,000 cells were
analyzed. Automatic repeated focusing was carried out
in the nuclear channel to guarantee focusing in spite of
staining intensities in the other channels. Images were
obtained for each fluorescence channel, using Array-
Scan HCS appropriate filters. Images and data regar-
ding strength and consistency of the fluorescence wit-
hin each cell, as well as the average fluorescence of the
cell population within the well were kept in a Microsoft
SQL database for easy recovery. Data were captured,
extracted, and analyzed with Data Acquisition and Data
Viewer software (Gentix/Molecular devices).
Determination of Reactive Oxygen Species (ROS)
The assembly of intracellular reactive oxygen spe-
cies was detected using 2',7'-dichlorofluorescin diace-
tate (DCFH-DA). Briefly, 10 mM floursstain (DCFH-
DA) stock solution (in methanol) was diluted 500-fold
in Hank's Balanced Salt Solution (HBSS) without se-
rum or other additives to give a 20 μM working solu-
tion. After 24 h of exposure to TQ-treatment, the cells
in the black microplate were flushed twice with HBSS
and then kept in 100 μL working solution of DCFH-DA
at 37°C for 30 min. Fluorescence was then determined
at 485-nm excitation and 520-nm emission using a fluo-
rescence microplate reader (Tecan Infinite M 200 PRO,
Männedorf, Switzerland).
Annexin V Assay
Cells (2x105
cells/mL) were treated with the IC-
50concentration for 24, 48 and 72 h. This assay was done
using the Annexin V-FITC Apoptosis Kit (BD Pharmin-
gen, BD, USA). Briefly, TQ-exposed and unexposed
cells (control) were washed and centrifuged to eliminate
the culturing media. Afterward, the cells were rinsed
with binding buffer (1X). The rinsed cells were re-sus-
pended in binding buffer and annexin V, and propidium
iodide (10 μL) were added and the cells were then incu-
bated at room temperature in the dark for 15 min. Flow
cytometric analysis was carried out using a BD FACS-
CantoTM II instrument. The binding buffer supplied by
the manufacturer was used to bring the reaction volume
to at least 500 μL for the flow cytometry analysis. DM-
SO-treated (0.1%, v/v) cells were used as control.
Protein Detection by Western Blotting
Cells at a concentration of 2×105
cells/mL were cultu-
red in RPMI 1640 (PAA, Coelbe, Germany) medium
containing 10% FBS, seeded into a 75-mm culture flask
(TPP Brand) and then treated with IC50 concentration for
3, 6, 12 and 24 h. After incubation, the cells were spun
down at 1,000 rpm for 10 min. The supernatant was dis-
carded and the pellet was washed twice with phosphate
buffered saline (PBS) to remove any remaining media.
Estimation of the packed cell pellet volume was done
and 20 volumes of mammalian cell lysis reagent (Proteo
JET, Fermentas Life Sciences, Burlington, ON, Canada)
were added to 1 volume of packed cells. The cells were
then incubated for 10 min at room temperature on a
shaker (900–1,200 rpm) and centrifugation was done at
16,000–20,000 × g for 15 min to clarify the lysate. The
resultant lysate was then transferred to a new tube and
stored at −70 °C until analysis by sodium dodecyl sul-
fate-polyacrylamide gel electrophoresis (SDS-PAGE).
Statistical Analysis
Results were reported as means ± SEM of at least
three analyses for each sample. Normality and
Homogeneity of variance assumptions was checked.
Statistical analysis was performed according to the
SPSS-16.0 package.
Results and Discussion
TQ induced cell viability inhibition in Caov-3 cells
The cytotoxic effects of TQ on the viability of ova-
rian cancer cell (Caov-3) were determined using the
MTT test. Cellular viability following 24h of contact to
TQ showed considerable inhibition in TQ-exposed cells
99
M. M. E. Taha et al. 2016 | Volume 62 | Issue 6 Anticancer effect of Thymoquinone.
90.00 % viable cells, 6.23% early apoptosis and 4.33%
late apoptosis. As the treatment time increased to 48h
and 72 h, the percentage of both early and late apopto-
tic cells continued to increase substantially (Fig2). The
date showed that the TQ has the ability to induce apop-
tosis time dependently.
Concurrent assessment of cell behavior with nuclear
stain showed that the nuclear intensity, which is directly
corresponding to apoptotic chromatin changes has in-
creased from 69.45 to 138.07 at 48 hr treatment (fig 3 A,
B). Meanwhile, concurrent significant (p<0.05) increase
in the cell permeability was also observed (Fig. 3C).
TQ-induced MMP disruption in Caov-3
The changes in MMP in Caov-3 cells have been
compared to control cells (untreated). The IC50value
of TQ was6.0± 0.03μg/mL (Figure 1B). TQ has been
shown previously to exert in vitro anti-neoplastic effects
(22). TQ did not show any cytotoxic activity on WRL-
68 normal hepatocytes.
TQ-induced apoptosis in Caov-3 cells
To confirm the presence of apoptosis, we examined
the cell with the help of the annexin V. This assay revea-
led the early stages of apoptosis induced by TQ. In the
assay, the healthy cells showed 90.66 % viable cells,
4% early apoptosis and 4.65% late apoptosis, whereas
after 24 hours treatment TQ in Caov-3 cells showed
Figure 1. (A) Nigella sativa seeds (black seeds) and its active compound (thymoquinone). (B) MTT assay.
Figure 2. The effect of thymoquinone on early apoptosis of
CAOV3 ovarian cancer cell line for (A) Untreated (B) 24h, (C)
48h, (D) 72 h and (E) histogram. CaoV3 cells were staining with
FITC-conjugated Annexin V and PI, cells were analyzed by flow
cytometry. The early apoptotic events (Annexin+/PI-) are shown in
lower right quadrant (Q4-1) of each panel. Quadrant (Q2-1) repre-
sents Annexin+/PI+ late stage of apoptosis/dead cells.
Figure 3. Treated cells shown thymoquinone induced increase in
nuclear area intensity and increase in plasma membrane permea-
bility and reduction in mitochondrial membrane potential. (A-C).
(D) time-dependent quantitative analysis of cells treated with TQ
apoptosis parameters. Multiple Cytotoxicity Assay was conducted
using Cellomics Multiparameter Cytotoxicity-3 Kit. Plates were
analyzed using the CellReporterTM cytofluorimeter system.
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M. M. E. Taha et al. 2016 | Volume 62 | Issue 6 Anticancer effect of Thymoquinone.
observed after treatment with TQ. Figure 3A showed
significant (p<0.05) reduction of MMP occurred upon
treatment. Changes of mitochondrial membrane poten-
tial in Caov-3cells treated with TQ 6μg/ml for 24 h and
48 hr had showed a significant reduction of fluorescence
intensity, 179 for 24 hr and 155 for 48 hr, while control
cells was 265 (Fig. 3D).
TQ induced cell death includes increased ROS for-
mation
Reactive oxygen species (ROS) are a diversity of
free radicals and molecules resulting from molecular
oxygen which are continually produced and eradicated
in the cytoplasm and have significant responsibilities in
cellular signaling and homeostasis (23). Disproportio-
nate quantities of these ROS can lead to oxidative injury
to proteins, lipids and nucleic acid (DNA), leading to
tumorigenesis or cellular death (24, 25). As depicted in
Figure 4, TQ exerted a catalyst role in producing more
ROS in a dose-dependent trend, which augmented the
ROS level on CAOV3 cell lines.
Western Blots
The Bcl-2 and Bax family proteins participate ma-
jorly in the induction of apoptosis and its regulation.
These cellular signaling proteins are known to be mem-
brane-bound and their tendency to experience both he-
terodimerization and homodimerization has been pro-
ved to control programmed cell death (26). Apart from
the major machinery, which is composed of a cascade
of caspases that execute the apoptosis program, for the
apoptosis, Bax and Bcl-2 are two master regulators to
regulate the on and off of apoptotic cascade, respecti-
vely (27, 28). To examine the possible mechanism re-
latedto the TQ-induced cell death (apoptosis), we exa-
mined protein expression of Bax, Bcl-2 and Hsp70 in
ovarian cells prior to TQ-induction. Quantification of
these proteins (Bax, Bcl-2 and Hsp70) was normalized
to β-actin. Bcl-2 and Hsp70 reduced significantly while
Bax protein level increased significantly in a concentra-
tion-dependent manner (Figure 6). The ratio of Bax and
Bcl-2 also dramatically augmented in a time-dependent
manner, whereas Bax intensity has been increased from
3 to 18 after 72 hour. Meanwhile Bcl-2 has been de-
creased from 16 to 3.5 intensity unit (29).
The current findings offered a novel imminent into
the pharmacological mechanism of therapeutic poten-
tials of Nigella sativa. Moreover, our result indicates
that TQ induces intrinsic pathway of apoptosis that is
potentially mediated by up-regulating Bax and down-
regulating Bcl-2. These results underlined that thymo-
quinone could be a nominee for an anticancer agent for
the treatment of ovarian tumors.
Acknowledgment
This project is supported by Al-Moalim MA Bin La-
din (MABL) chair for Scientific Miracles of Prophetic
Medicine, College of Medicine, Taibah University, Sau-
di Arabia (research grant no. MABL 37/05).
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