乳癌（Breast Cancer）?
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Thymoquinone Inhibits
Tumor Growth and Induces Apoptosis in a Breast Cancer Xenograft Mouse Model:
The Role of p38 MAPK and ROS
Chern Chiuh Woo,1 Annie Hsu,1 Alan Prem Kumar,1,2,3,4 Gautam Sethi,1,2,* and Kwong Huat Benny Tan1,*
Jin Q. Cheng, Editor
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Abstract
Due to narrow therapeutic window of
cancer therapeutic agents and the development of resistance against these
agents, there is a need to discover novel agents to treat breast cancer. The
antitumor activities of thymoquinone (TQ), a compound isolated from Nigella
sativa oil, were investigated in breast carcinoma in vitro and in
vivo. Cell responses after TQ treatment were assessed by using different
assays including MTT assay, annexin V-propidium iodide staining, Mitosox
staining and Western blot. The antitumor effect was studied by breast tumor
xenograft mouse model, and the tumor tissues were examined by histology and
immunohistochemistry. The level of anti-oxidant enzymes/molecules in mouse
liver tissues was measured by commercial kits. Here, we show that TQ induced
p38 phosphorylation and ROS production in breast cancer cells. These inductions
were found to be responsible for TQ’s anti-proliferative and pro-apoptotic
effects. Moreover, TQ-induced ROS production regulated p38 phosphorylation but
not vice versa. TQ treatment was found to suppress the tumor growth and
this effect was further enhanced by combination with doxorubicin. TQ also
inhibited the protein expression of anti-apoptotic genes, such as XIAP,
survivin, Bcl-xL and Bcl-2, in breast cancer cells and breast tumor xenograft.
Reduced Ki67 and increased TUNEL staining were observed in TQ-treated tumors.
TQ was also found to increase the level of catalase, superoxide dismutase and
glutathione in mouse liver tissues. Overall, our results demonstrated that the
anti-proliferative and pro-apoptotic effects of TQ in breast cancer are
mediated through p38 phosphorylation via ROS generation.
Introduction
In the last decade, numerous papers
have reported that thymoquinone (TQ), a compound isolated from Nigella
sativa oil, was able to suppress a range of carcinomas including breast,
liver, prostate and colorectal carcinoma [1]. Many potential targets which TQ
regulates for its anticancer activities have been identified including p53 [2,3], p73 [4], STAT3 [5], NF-κB [6], PPAR-γ [7] and reactive oxygen species (ROS)
[4,8]. In addition, the combination of
TQ with conventional medicine can result in greater anticancer effect, for
example in NCI-H460 non-small cell lung cancer cells [9] and U266 multiple myeloma cells [5]. Moreover, TQ can even
protect against the toxicity caused by conventional medicine, for example, to
ameliorate the nephrotoxicity induced by cisplatin in rodents [10] and the cardiotoxicity
of doxorubicin in mice [11]. However, the detailed molecular
mechanisms of the antineoplastic effects of TQ are yet to be elucidated, and
the potential therapeutic effects of TQ in breast carcinoma are also not clear.
The p38 pathway plays a number of
roles including regulation of apoptosis, cell cycle progression, cell growth
and differentiation. A number of diseases have been found to be associated with
p38 signaling, namely rheumatoid arthritis [12], cardiovascular disease [13] and Parkinson’s disease
[14]. Many studies suggest that the p38
pathway may play an important role in cancer as a tumor suppressor. p38 MAPK
was shown to up-regulate p16 expression, which in turn inhibits cyclin D1/cdk4
activity [15]. p38 MAPK can stabilize HBP1
protein by phosphorylating it [16], whereby HBP1 can then negatively regulate
cell cycle genes, including cyclin D1 and N-myc [17,18]. It had been shown that several
chemotherapeutic agents, such as nocodazole, taxol, vincristine and
vinblastine, can induce p38 MAPK activation and mitotic cell cycle arrest [19]. The p38 inhibitor was
found to reverse nocodazole-induced apoptosis [19]. Moreover, phospho-p38 is almost
undetectable in most solid tumors including breast, lung, liver, gastric, renal
and ovarian cancers, while this protein is relatively higher expressed in
normal organs [20]. Together, these findings explain
the potential role of p38 MAPK in anticancer therapy. The agent that can modulate
p38 pathway could thus be a solution to tumor malignancy.
ROS are oxygen-containing reactive
molecules or ions, which are formed via incomplete one electron reduction of
oxygen [21]. Although studies on the effect of
ROS in oncology are not fully understood, there are reports suggesting that ROS
can promote tumorigenesis through Ras-Raf-MEK-ERK pathway, or suppress
tumorigenesis via p38 pathway [21]. It has been reported that ROS,
via Ras, can activate ERK1/2, where ERK1/2 plays important roles in
tumorigenesis such as cell growth and apoptosis prevention [22,23]. In
contrast, ROS was shown to activate p38 MAPK for apoptotic cell death in human
cervical cancer cells [24]. The p53/ROS/p38α cascade, whereby
p38α can be activated via p53-mediated ROS production, plays an essential role
in cisplatin-induced apoptosis in HCT116 colorectal cancer cells [25]. Interestingly, there
was a study which suggested that ROS is tumor-promoting, and that p38
MAPK-induced apoptosis is initiated in response to ROS accumulation. This
response is believed to play an important role in inhibiting tumor initiation
during oxidative stress [26].
Despite the identification of
various targets for TQ, the effect of TQ on MAPKs, particularly p38, still
remains unexplained. The present work seeks to explain the role of p38 MAPK on
the anticancer effects of TQ in breast cancer cells and in the breast tumor
xenograft mouse model. We also investigate the role of ROS and its interaction
with p38 MAPK. We believe the results will add significant knowledge to the
potential use of TQ in breast cancer therapy, in particular its effects on
growth inhibition and apoptosis.
Materials
and Methods
Chemicals and Antibodies
Trypsin EDTA, trypan blue, thiazolyl
blue tetrazolium bromide (MTT), thymoquinone and N-acetylcysteine were
purchased from Sigma-Aldrich (St. Louis, MO, USA), while doxorubicin was
purchased from Euroasian Chemical Private Ltd. (Mumbai, India). SB203580 was
purchased from Promega (WI, USA). RPMI1640 and fetal bovine serum were purchased
from Hyclone (Loughborough, UK). Antibiotic-antimycotic mixture was purchased
from Gemini Bio-products (West Sacramento, CA, USA). Dimethyl sulfoxide was
purchased from MP Biomedicals (Solon, OH, USA). BD matrigel was purchased from
BD Biosciences (Franklin Lakes, NJ, USA). Antibodies to Bcl-2, Bcl-xL, Ki67,
XIAP, JNK, p-JNK, ERK, p-ERK and PARP were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA), while survivin, p38, p-p38 and β-actin
were purchased from Cell Signaling (Beverly, MA, USA). Chicken anti-rabbit IgG
HRP-conjugated, chicken anti-mouse IgG HRP-conjugated, chicken anti-rabbit IgG
TR-conjugated antibodies, p38 siRNA and control siRNA-A were purchased from
Santa Cruz Biotechnology.
Cell lines
MCF-7 and MDA-MB-231 breast cancer
cell lines were purchased from ATCC (Manassas, VA, USA). These cell lines were
cultured in RPMI1640 medium supplemented with 10% fetal bovine serum and 1%
antibiotic-antimycotic. All cell culture were maintained at 37 °C and 5% CO2
in a humidified atmosphere.
3:
(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay
The anti-proliferative effect of TQ
was assessed by MTT assay. TQ was dissolved in PBS containing 0.5% DMSO for all
in vitro studies. Briefly, breast cancer cells were seeded (104
cells/well) in a 96-well microtiter plate followed by overnight incubation.
After appropriate treatment, 10 μl MTT solution (5 mg/ml) was added to each
well for 4 h. The mixture was removed carefully via pipette, and the remaining
formazan crystals formed were dissolved by 100 μl DMSO. After 30 mins, the
absorbance of each well was read at 570 nm with an absorbance reader (Tecan
Infinite M200, Mannedorf, Switzerland).
Annexin V-propidium
iodide analysis
The level of apoptosis of cancer
cells was assessed with Annexin V-propidium iodide kit from Miltenyi Biotec
(Bergisch Gladbach, Germany). Briefly, breast cancer cells were seeded (2.6 X
105 cells/well) in a 6-well microtiter plate followed by overnight
incubation. After appropriate treatment, the cells were trypsinized, washed,
and incubated with Annexin V-FITC solution for 15 mins under dark condition.
After washing, the cells were analyzed with flow cytometry (CyAnTM
ADP from Beckman Coulter, Brea, CA, USA) immediately after propidium iodide
solution was added.
Western blot analysis
The protein expressions of genes of
interest in breast cancer cells and breast tumor tissues were measured by
Western blot. Briefly, the cells were seeded (2.6 X 105 cells/well)
in a 6-well microtiter plate followed by overnight incubation. After
appropriate treatment, the cells were trypsinized followed by whole cell lysate
extraction. For in vivo study, the tumor tissues were homogenized for
tissue lysate extraction. Both cell lysate and tissue lysate were centrifuged
and the supernatants were collected. After protein estimation with Bio-Rad
protein assay (Hercules, CA, USA), a calculated volume of lysate was mixed with
laemmli sample buffer, whereby the mixture was resolved by 10% or 12% SDS/PAGE
gel and then electroblotted onto a nitrocellulose membrane. The membrane was
probed with primary antibody (1:1000) for overnight incubation at 4°C, and then
washed and incubated with HRP-conjugated secondary antibody (1:10000) for 1 h
at room temperature. The membrane was examined for its chemiluminescence by ECL
(GE Healthcare, Little Chalfont, Buckinghamshire, UK). Densitometric analysis
of the scanned blots was measured using ImageJ software and the results were
expressed as fold change relative to the control after normalization to β-actin.
ROS measurement
The ROS level of cancer cells was
measured by flow cytometry after Mitosox staining (Invitrogen, Carlsbad, CA,
USA). Briefly, breast cancer cells were seeded (2.6 X 105 cells/well)
in a 6-well microtiter plate followed by overnight incubation. After
appropriate treatment, the cells were trypsinized and washed with PBS buffer
before mixing with Mitosox-added serum-free medium. The cells were then
incubated under dark condition for 15 mins at 37°C before analysis with a flow
cytometer (BD LSRII, Franklin Lakes, NJ, USA).
PathScanR Phospho-p38
MAPK (Thr180/Tyr182) Sandwich ELISA Kit
The p-p38 MAPK level of cancer cells
was examined with PathScanR Phospho-p38 MAPK (Thr180/Tyr182) Sandwich
ELISA Kit (Cell Signaling, Beverly, MA, USA). The experimental procedures were
carried out according to the manufacturer’s protocol. Briefly, breast cancer
cells were seeded (2.6 X 105 cells/well) in a 6-well microtiter
plate followed by overnight incubation. After appropriate treatment, the cells
were lysed followed by centrifugation. The resulting supernatant was added into
the wells supplied by the manufacturer. After 4 h incubation at 37°C, the wells
were washed with buffer for 4 times. Detection antibody was then added for 1 h
at 37°C. The washing step was repeated, followed by incubation for 30 mins with
HRP-Linked secondary antibody at 37°C. The washing step was again repeated,
followed by incubation for 10 mins with TMB substrate at 37°C. STOP solution
was then added into each well for 5 mins. The absorbance was read at 450 nm
with an absorbance reader (Tecan Infinite M200, Mannedorf, Switzerland).
Gene silencing using
siRNA
The protein expression of p38/p-p38
was suppressed by siRNA silencing. Briefly, breast cancer cells were seeded
(1.7 X 105 cells/well) in a 6-well microtiter plate followed by
overnight incubation. The cells were then tranfected with 30 nM of p38 siRNA or
control siRNA-A using Oligofectamine tranfection reagent (Invitrogen, Carlsbad,
CA, USA) for 6 h according to the manufacturer’s protocol. Serum-added medium
was then added for at least 24 h before exposure to appropriate treatment.
In vivo experiment
Female nude mice (BALB/c
OlaHsd-foxn1) were purchased from Biological Resource Centre (BRC, Biopolis,
Singapore). The animal protocol was approved by The NUS Institutional Animal
Care and Use Committee (IACUC No. 065/11). Upon arrival, the nude mice were
kept in individual disposable cages with ventilation, and given food and water ad
lib. After acclimatisation over 7 days, each mouse was injected subcutaneously
with 107 MDA-MB-231 human breast cancer cells (resuspended in
matrigel-added serum free medium) at the right flank region. When the tumor
size was about 100 mm3 (Volume = ? X width2 X length),
the mice were divided into different treatment groups (n=5) as following.
Group 1: Vehicle control saline
water (i.p.), 6 days per week.
Group 2: TQ 4 mg/kg (i.p.), 6 days
per week.
Group 3: TQ 8 mg/kg (i.p.), 6 days
per week.
Group 4: Dox (doxorubicin) 2.5 mg/kg
(i.p.), once per week.
Group 5. TQ 4 mg/kg (i.p.), 6 days
per week + Dox 2.5 mg/kg (i.p.), once per week.
TQ and doxorubicin were dissolved in
saline water containing 5% DMSO. The tumor volume and body weight were measured
twice per week. After two weeks of treatment, the mice were euthanized with CO2
asphyxiation. Tumor tissues were collected for histological,
immunohistochemical and Western blot analysis, while liver tissues were
collected for enzymatic assays.
Hematoxylin and Eosin
(H&E) staining
The tumor tissues were placed in 10%
neutral buffered formalin solution (Sigma-Aldrich, St. Louis, MO, USA) before
being processed and paraffinized. The samples were sectioned and stained with
H&E solution (Merck, Germany). The tissue section was examined and
photographed with a fluorescence microscope (Olympus BX51, Shinjuku, Japan).
TUNEL staining
The level of apoptosis of tumor
tissues was assessed by TUNEL staining (Promega, WI, USA). The experimental
procedures were carried out according to the manufacturer’s protocol. Briefly,
the tissue section was deparaffinized before rehydration with decreasing
concentrations of ethanol. After washing with 0.85% NaCl and PBS, the tissue
section was fixed with 4% formaldehyde for 15 mins. Following washing with PBS,
the tissue section was covered with Proteinase K solution for 8-10 mins. After
another PBS wash, the tissue section was again fixed with 4% formaldehyde for 5
mins. Following PBS wash, the tissue section was covered with equilibrium buffer
for 5-10 mins before addition of TdT reaction mixture. After incubation under
dark condition for 1 h, the tissue section was incubated with SSC solution for
15 mins, followed by a final PBS wash. After DAPI counterstain, the tissue
section was examined and photographed with a fluorescence microscope (Olympus
BX51, Shinjuku, Japan). Average number of fluorescence dots of three images
from each treatment group was calculated.
Ki67 immunohistochemistry
The tissue section was
deparaffinized before undergoing antigen retrieval step with citrate buffer.
The tissue section was next blocked with 2% fetal bovine serum for 20-30 mins,
and then incubated with rabbit anti-human Ki67 antibody (1:200) for 1 h at room
temperature. After rinsing with PBS, the tissue section was incubated with
chicken anti-rabbit IgG TR-conjugated antibody (1:500) for 1 h under dark
condition. Following DAPI counterstain, the tissue section was examined and
photographed with a fluorescence microscope (Olympus BX51, Shinjuku, Japan). Average
number of fluorescence dots of three images from each treatment group was
calculated.
Catalase assay
The catalase level in mouse liver
tissues was measured using the catalase assay kit from Cayman Chemical (Ann
Arbor, Michigan, USA). The experimental procedures were carried out according
to the manufacturer’s protocol. Briefly, the liver tissues were homogenized in
cold buffer (50 mM potassium phosphate, 1 mM EDTA, pH 7). The supernatant was
collected after centrifugation. The sample was mixed with diluted assay buffer
and methanol in a 96-well microtiter plate. The reaction was initiated by
adding diluted hydrogen peroxide for 20 mins with constant shaking. Diluted
potassium hydroxide was then added followed by catalase purpald. The plate was
incubated immediately for 10 mins with constant shaking. Catalase potassium
periodate was then added followed by 5 mins incubation with constant shaking.
The absorbance was then read at 540 nm with an absorbance reader (Tecan
Infinite M200, Mannedorf, Switzerland).
Superoxide dismutase
(SOD) assay
The SOD level in mouse liver tissues
was measured using the SOD assay kit from Cayman Chemical (Ann Arbor, Michigan,
USA). The experimental procedures were carried out according to the
manufacturer’s protocol. Briefly, the liver tissues were homogenized in HEPES
buffer (20 mM HEPES buffer, 1 mM EGTA, 210 mM mannitol, 70 mM sucrose, pH 7.2).
The supernatant was collected after centrifugation. The sample was added to
diluted radical detector in a 96-well microtiter plate. The reaction was
initiated by adding diluted xanthine oxidase. The plate was incubated
immediately for 20 mins with constant shaking. The absorbance was then read at
450 nm with an absorbance reader (Tecan infinite M200, Mannedorf, Switzerland).
Glutathione assay
The glutathione level in mouse liver
tissues was measured using the glutathione assay kit from Cayman Chemical (Ann
Arbor, Michigan, USA). The experimental procedures were carried out according
to the manufacturer’s protocol. Briefly, the liver tissues were homogenized in
cold buffer (50 mM phosphate, 1 mM EDTA, pH 6-7). The supernatant was collected
after centrifugation. The sample was first deproteinated by triethanolamine.
The sample was added to assay cocktail in a 96-well microtiter plate. The plate
was incubated immediately for 25 mins under dark condition with constant
shaking. The absorbance was then read at 405 nm with an absorbance reader
(Tecan Infinite M200, Mannedorf, Switzerland).
Statistical analysis
Statistical analysis was performed
by one way analysis of variance (ANOVA). A p-value of less than 0.05 was
considered to be statistically significant.
Results
MAPKs protein phosphorylation
after TQ treatment
We first determined whether TQ can
induce any effect in MAPKs protein phosphorylation in breast cancer cells.
Western blot analysis demonstrated that TQ significantly up-regulated the
phosphorylation of various MAPKs in MCF-7 cells (Figure
1A).
The increase of JNK and p38 protein phosphorylation was found to be maximal at
12 h. On the other hand, the increase of ERK protein phosphorylation peaked at
4 h and gradually decreased till 12 h.
TQ induces MAPKs protein
phosphorylation, particularly p38, in breast cancer cells.
The specific p38
inhibitor (SB203580) abrogates TQ-induced p38 phosphorylation
Having determined the potential
effect of TQ on p38 MAPK, we further investigated the specificity of this
effect on both MCF-7 and MDA-MB-231 breast cancer cell lines. Both cell lines
were pre-treated with 10 μM SB203580 followed by TQ treatment. In both cell
lines, TQ was found to induce the phosphorylation of p38, and this induction
was reversed by SB203580 treatment (Figure
1B).
In addition to western blot, a p38 MAPK ELISA kit (as described in Materials
and Methods) was also used to measure the p-p38 level in TQ-treated cells. We
found that TQ significantly increased the p-p38 level in both cell lines after
exposure to 40 μM TQ for 12 h (Figure
1C).
This increase was also significantly reversed by SB203580 treatment.
The involvement of p38
MAPK in TQ-induced anti-proliferative and pro-apoptotic effects
MCF-7 and MDA-MB-231 cells were
treated with increasing doses of TQ for 24 h with or without SB203580
treatment. The results from MTT assay indicated that SB203580 treatment
significantly reversed the anti-proliferative effect of TQ in both cell lines,
at least partially (Figure
2A).
We also examined whether the level of p38 phosphorylation interfered with
TQ-induced apoptosis. We found that SB203580 treatment significantly reversed
TQ-induced increased percentage of Annexin V positive cells (Figure
2B).
As shown in Figure
2C,
the cleaved-PARP protein in both cell lines was increased after TQ treatment,
and this increase was reversed when the cells were pre-treated with SB203580.
We also investigated the protein expression of various
anti-apoptotic/pro-survival genes such as survivin, XIAP, Bcl-xL and Bcl-2. In
both cell lines, we found that TQ suppressed the protein expression of these
genes, however, these suppressions were not all reversed by SB203580 treatment
(Figure
2D).
We found that the decrease of XIAP in MCF-7 cells by TQ could be reversed by
SB203580 treatment. On the other hand, the decrease of survivin and Bcl-2 by TQ
was reversed by SB203580 treatment in MDA-MB-231 cells.
The role of p38 MAPK on
TQ-induced anti-proliferative and pro-apoptotic effects in breast cancer cells.
N-acetylcysteine (NAC)
prevents TQ-induced ROS production
There are papers reporting that TQ
mediates ROS production as a mechanism to induce apoptosis and growth
inhibition in various cancer cells [8,27,28] except breast cancer.
In this study, we demonstrated the effect of TQ on ROS production in breast
cancer cells, and its effect on cell proliferation and apoptosis. MCF-7 cells
were treated with 40 μM TQ for various time periods ranging up to 6 h. As shown
in Figure
3A, TQ
significantly induced ROS production as early as 30 mins, and this induction
was time-dependent up to 3 h after TQ treatment. TQ-induced ROS production was
reversed by pre-2 h treatment with NAC, a strong antioxidant (Figure
3B).
The role of ROS in the
anti-proliferative and pro-apoptotic effects induced by TQ in breast cancer cells.
The role of ROS in
TQ-induced anti-proliferative and pro-apoptotic effects
We also investigated whether ROS
level can interfere with TQ-induced growth inhibition. We found that the
anti-proliferative effect of TQ in MCF-7 cells was reversed by NAC in a
dose-dependent manner (Figure
3C).
Next, we pre-treated MCF-7 cells with 5 mM NAC for 2 h before exposure to TQ
for 12 h. Both TQ-induced increased percentage of Annexin V positive cells (Figure
3D)
and cleavage of PARP protein (Figure
3E)
were reversed by NAC treatment, indicating apoptosis reversal through ROS
reduction. We also examined whether the level of ROS inter-relates with the
protein expression of various anti-apoptotic/pro-survival genes. We found that
the decrease of survivin, XIAP, Bcl-xL and Bcl-2 protein expression by TQ were
all reversed by NAC treatment (Figure
3F).
p38 MAPK gene silencing
reversed TQ-induced apoptosis
As shown in Figure
4A,
both p-p38 and p38 protein expressions were reduced with p38 siRNA
transfection. Moreover, we found that TQ-induced PARP-cleavage (Figure
4B)
and increased percentage of Annexin V positive cells (Figure
4C)
were both partially reversed by p38 siRNA transfection, which confirmed the
role of p-p38 in TQ-induced apoptosis.
Effect of p38 siRNA gene
silencing on the apoptotic effect of TQ, and investigation on the relationship
between p38 and ROS.
ROS regulates p38
phosphorylation
Since TQ was shown to affect ROS and
p38 pathways, we investigated the relationship between ROS and p38 MAPK. MCF-7
cells were pre-treated with 10 μM SB203580 for 1 h before exposure to 40 μM TQ
for 1 h or 3 h. We found that SB203580 treatment did not make any significant
changes on TQ-induced ROS level (Figure
4D).
This indicates that p38 phosphorylation level did not affect the level of ROS.
Next, we pre-treated MCF-7 cells with 5 mM NAC for 2 h before exposure to 40 μM
TQ for 12 h. Through Western blot analysis, we found that NAC treatment
reversed TQ-induced p38 phosphorylation (Figure
4E).
Furthermore, the results from the p38 MAPK ELISA kit also showed that NAC
treatment could significantly reverse TQ-induced p-p38 level (Figure
4F).
These results indicate that TQ-induced ROS regulates the phosphorylation of p38
in MCF-7 cells.
TQ inhibits breast tumor
growth in nude mice
MDA-MB-231 breast cancer cells were
injected subcutaneously into the right flank region of female nude mice to
develop breast tumor xenograft. As shown in Figure
5A,
the tumor volume of vehicle group was increased aggressively after 2 weeks
(from about 100 mm3 to about 330 mm3). Treatment with 4
mg/kg TQ, 8 mg/kg TQ and 2.5 mg/kg Dox significantly slowed the tumor growth,
though they did not completely eliminate the tumors or return to the start
level. The combined treatment (4 mg/kg TQ + 2.5 mg/kg Dox) slowed tumor growth
more significantly than either agent alone. Although the combined treatment did
not completely eliminate the tumor, it could maintain the tumor growth at the
start level throughout the 2 weeks period of treatment. We also found that TQ
treatment slightly reduced mouse body weight although no visible adverse effect
was observed (Figure
5B).
Less than 9% body weight reduction was observed for all TQ-treated groups
including 4 mg/kg TQ, 8 mg/kg TQ and the combined treatment group.
TQ suppresses breast
tumor growth in nude mice, and increases levels of anti-oxidant enzymes/molecules
in liver tissues.
The level of anti-oxidant
enzymes/molecules in mouse liver tissues
Since TQ was found to produce ROS in
cancer cells, we also investigated its effect in in vivo model by
measuring antioxidant enzymes/molecules in mouse liver tissues. We found that
the catalase level was significantly increased in TQ-treated groups compared to
the vehicle group (Figure
5C).
The catalase level was significantly higher in the combined treatment group
compared to the either agent alone. On the other hand, SOD level was
significantly higher in the 8 mg/kg TQ and 2.5 mg/kg Dox groups compared to the
vehicle group (Figure
5D).
Though not statistically significant, 4 mg/kg TQ and the combined treatment
groups had higher mean SOD levels compared to the vehicle group. We also found
that glutathione level was significantly higher in TQ alone groups (Figure
5E).
Interestingly, the glutathione level in the 2.5 mg/kg Dox group was lower
compared to the vehicle group. Glutathione level was lower in the combined
treatment group compared to the vehicle group but higher than in the 2.5 mg/kg
Dox group.
Histology,
immunohistochemistry and Western blot analysis of tumor tissues
The tumor tissues were subjected to
H&E staining for structure analysis. The vehicle group displayed high grade
tumor with irregular cell arrangement (Figure
6A).
In contrast, in the drug treatment groups, alterations in cell architecture
were observed as characterized by an increase in cell debris and a decrease in
stroma. In addition, TUNEL staining was carried out to study the level of
apoptosis of tumor tissues. By calculating the number of green fluorescent dots
relative to the vehicle group, we observed about 50% higher TUNEL staining in 4
mg/kg TQ, 8 mg/kg TQ and 2.5 mg/kg Dox groups, and about 150% higher staining
in the combined treatment group, compared to the vehicle group (Figure
6B).
Furthermore, immunohistochemistry of the tumor tissues targeting Ki67 protein,
a cellular marker for proliferation showed that the Ki67 level significantly
reduced in the drug treatment groups, compared to the vehicle group, with the
lowest level in the combined treatment group (Figure
6C).
Western blot analysis of homogenized tumor tissues showed that p-p38 protein
expression was increased in the TQ-treated groups, but not in the Dox alone
group, compared to vehicle (Figure
6D).
The protein expression of anti-apoptotic/pro-survival genes, such as survivin,
XIAP, Bcl-xL and Bcl-2, were decreased in the drug treatment groups, compared
to the vehicle group (Figure
6D).
TQ induces apoptosis in
breast tumor xenograft with down-regulation of anti-apoptotic proteins.
Discussion
In this study, we explored the
potential effects of TQ on ROS and p38 pathways both in vitro and in
vivo. This is the first report to suggest that TQ induced ROS production,
which, in turn, resulted in p38 phosphorylation, contributing to TQ’s
anti-proliferative and pro-apoptotic effects in breast cancer. In the xenograft
mouse model, we showed the ability of TQ to suppress breast tumor growth, and
the combined treatment with doxorubicin to cause significantly higher
suppression. Moreover, TQ was found to increase p-p38 protein expression in
tumor tissues, with down-regulation of XIAP, survivin, Bcl-xL and Bcl-2
anti-apoptotic gene products. TQ treatment also increased catalase, SOD and
glutathione levels in mouse liver tissues.
Recent developments have raised a
question on the role of ROS, whether it protects against or promotes oxidative
stress [29]. Accumulating evidence suggests
that ROS found in various chronic diseases might not be the result of disease
damage, but instead, could be the response of host to the disease. As such,
researchers start using pro-oxidant agents to increase oxidative stress in
cancer cells as a strategy to target resistant tumor cells [30]. For example,
TQ-induced ROS production was found to down-regulate Akt in primary effusion
lymphoma cells [28]. In addition, TQ was shown to
induce ROS-mediated ERK and JNK phosphorylation in human colon cancer cells [8]. These results have led
us to suggest that TQ’s anticancer effects may be mediated upstream at ROS.
Many studies have identified potential targets of TQ such as p53 [2,3], p73 [4], STAT3 [5], NF-κB [6] and PPAR-γ [7]. One may raise a
question on whether these targets are also regulated by ROS. A recent review by
Maillet and Pervaiz (2012) explained how ROS production regulates p53 activity
[31]. In addition, STAT3 activation was
found to be mediated via ROS in pulmonary epithelial cells [32] and B lymphocytes [33]. However, this kind of
relationship is not always one way, for example, p53 can act as upstream
regulator of ROS production by binding to promoters such as GPX and PIGs [31]. Therefore, the role of
ROS in TQ-induced apoptosis requires further comprehensive investigation with
well-designed models.
Numerous studies have suggested that
p38 MAPK can act as a tumor suppressor by negatively regulating cell cycle
progression, p53 activation and oncogene-induced premature senescence [34]. It was shown that p38α
regulates the proliferation and differentiation of lung stem and progenitor
cells, and inactivation of this pathway can lead to K-Ras(G12V)-induced
tumorigenesis [35]. Furthermore, p38α protein
expression was found to be approximately 3 times lower in human lung tumors
than in human normal lung tissues [35]. In the present study, we show
that p38 MAPK plays an important role in TQ’s anti-proliferative and
pro-apoptotic effects. In contrast, the study by El-Najjar et al. (2010)
described the ability of TQ to increase the phosphorylation of JNK and ERK, but
not p38, in human colon cancer cells via ROS [8]. Interestingly, the
phosphorylation of JNK and ERK was found to serve as a survival mechanism in
TQ-induced cell death, and the inhibition of these MAPKs can potentiate
TQ-induced apoptosis [8]. Nevertheless, both El-Najjar’s
study and this study have explained the role of ROS as an upstream mediator of
phosphorylation of MAPKs, and this fact should further be explored for its role
in cancer therapeutics.
The antitumor effects of TQ have
been shown in other types of carcinoma including lung [9,36], pancreas [37], prostate [38], gastric [39] and colon [40]. We found that TQ and
doxorubicin in combination suppressed tumor growth more significantly than
either agent alone. A similar finding was also reported for the combination of
TQ and 5-fluorouracil [39], and TQ and cisplatin [9]. Moreover, TQ in
combination with gemcitabine or oxaliplatin produced greater antitumor effect
than either agent alone in the pancreatic tumor xenograft mouse model [37]. These results strongly
suggest the possible use of TQ as a complementary agent to potentiate the
antitumor effect of conventional anticancer drugs.
We found that TQ increased the
levels of catalase, SOD and glutathione in liver tissues of mouse xenograft
model. These enzymes/molecules are generally known for their involvement in
cellular anti-oxidative activities. However, we are not certain whether the
increase of these enzymes/molecules was due to TQ induction or the response of
cellular defense mechanisms against TQ-induced ROS production. TQ has been
shown to reverse the decrease of glutathione peroxidase,
glutathione-S-transferase, catalase, and reduced glutathione in kidney and
liver tissues of streptozotocin nicotinamide-induced diabetic rat [41]. TQ-induced glutathione
level in female Lewis rats with experimental allergic encephalomyelitis is
believed to improve the condition of the disease [42]. In contrast, there was a study
reporting that TQ treatment did not change the level of reduced glutathione or
glutathione-S-transferase in liver and kidney tissues of normal mice [43]. As such, the nature of
TQ as anti-oxidant or pro-oxidant in different models has to be further
explored.
In conclusion, our study provides
evidence for the mechanism of action of TQ in suppressing human breast
carcinoma in both in vitro and in vivo models. We demonstrated
that the anti-proliferative and pro-apoptotic effects of TQ are mediated
through its induction effect on p38 and ROS signaling. Our results also
indicate the anti-tumor effects of TQ in breast tumor xenograft mice and its
ability to potentiate the antitumor effect of doxorubicin. TQ serves as a
promising anticancer agent and further studies may provide important leads for
its clinical application.
Acknowledgments
We thank Shanmugam Muthu Kumaraswamy
for his useful suggestions on animal works in this project.
Funding
Statement
This work was supported by grants
from NUS Academic Research Fund (R-184-000-207-112) and National Medical
Research Council of Singapore (R-184-000-211-213) to GS. (http://www.nuhs.edu.sg/research/funding/funding-opportunities/individual-research-grants/academic-research-fund-tier-1-university-research-committee.html) (http://www.nmrc.gov.sg/content/nmrc_internet/home.html) The funders had no role
in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
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