Fertility and Infertility Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran
Department of Endodontics, Dental Research Center, Isfahan University of Medical Sciences, Isfahan, Iran
Fertility and Infertility Research Center
Kermanshah University of Medical
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Evaluation of Hydro-Alcoholic Extract of Trifolium Pratens L. for
Its Anti-Cancer Potential on U87MG Cell Line.
Glioblastoma multiforme is the most malignant form of brain tumors. Trifolium pratense L. has been suggested
for cancer treatment in traditional medicine. Here we have investigated the effects of T. pratense extract on glioblastoma
multiforme cell line (U87MG).
Materials and Methods
In this experimental study the effect of T. pratense extract on cell viability was investigated
using trypan blue staining, MTT assay, and lactate dehydrogenase activity measurement. Apoptosis and autophagy
cell death were detected by fluorescent staining. Nitric oxide (No) production was measured using Griess reaction.
Expression levels of some apoptotic and autophagic-related genes were detected using real-time polymerase chain
reaction (PCR). The combination effects of T. pratense extract and temozolomide (TMZ) were evaluated by calculating
the combination index and dose reduction index values.
After treatment with T. pratense extract, the cell viability was significantly reduced in a time- and dose-
dependent manner (P<0.05). Apoptosis and autophagy of U87MG cells were significantly increased (P<0.05). Also, T.
pratense extract significantly decreased NO production (P<0.05) by U87MG cells. Combination of TMZ and T. pratense
extract had a synergistic cytotoxic effect.
T. pratense showed anti-cancer properties via induction of apoptosis and autophagy cell death.
Glioblastoma multiforme (GBM) is the most aggressive
and most common type of the malignant astrocytic brain
tumors. Surgical resection (which is usually incomplete
because of the proximity of the tumor to vital brain
structures), radiotherapy, and chemotherapy are the currently
used conventional treatments (1). DNA alkylating agents are
among the oldest class of anti-cancer drugs still commonly
used, which play an important role in the different types of
tumor treatments (2).
Temozolomide (TMZ), an imidazole derivative, is an oral
chemotherapy agent commonly used to control the growth
of GBM tumors. Due to its lipophilic properties, it readily
passes the blood brain barrier and spontaneously hydrolyzes
under physiological conditions to its active form. It can
methylate DNA at the O6 and N7 positions of guanine. These
methylated bases disturb DNA replication and cell cycle,
therefore activating apoptosis death pathway. However,
GBM are among the most resistant tumors to chemotherapy
treatment, because of cell DNA repair system.
The most important mechanism of TMZ resistance is the
DNA repair enzyme O6-methylguanine methyltransferase
(MGMT), which removes the cytotoxic O6-methylguanine
and counteracts the effect of TMZ. GBM patients survive,
on average, between 12 and 15 months, despite conventional
therapy (3). So it seems necessary to identify new strategies
to treat this kind of cancer. Currently, many attempts have
been made to overcome drug resistance, using combination
therapy with multiple anti-cancer agents. Different anticancer
agents affect different targets and cell subpopulations
and therefore can enhance the therapeutic effects, reduce dose
and side effects and prevent or delay the induction of drug
resistance. Recent studies have shown that combination of
TMZ with some herbal agents enhances its effectiveness on
glioblastoma cells (4).
For over 40 years, natural products, in either unmodified
or synthetically modified forms, have played an important
role in cancer therapy. In fact, over 60% of currently used
chemotherapy drugs have been isolated from natural products,
mostly of plant origin (5). In the 1950s the potential of using
natural products as anti-cancer drugs was confirmed by U.S
National Cancer Institute (NCI), and from that time there is
a growing interest in discovery of naturally occurring anticancer
drugs. Some of such drugs that are used against cancer
include vinca alkaloids (vincristine, vinblastine, vindesine,
vinorelbine), taxanes (paclitaxel, docetaxel), podophyllotoxin
and its derivatives (etoposide, teniposide), camptothecin
and its derivatives (topothecan, irinothecan), anthracyclines
(doxorubicin, daunorubicin, epirubicin, idarubicin) and
According to other studies the mechanisms of plants
for anti-cancer properties are numerous and most of
them cause apoptotic cell death induction via intrinsic
or extrinsic mechanisms, and CASPASE- and/or P53-dependent
or independent pathways. Also, anti-cancer
potentials of some plants are through induction of
autophagy, necrosis-like programmed cell death, mitotic
catastrophe, and senescence (7).
Trifolium pratense L., a member of Leguminosae or
Fabaceae family, is a short-lived biennial plant, which has
been used as a fodder crop for its nitrogen fixation potential,
increases soil fertility and is considered as a health food
for humans. It is probably native to Europe, Western Asia,
and northwest Africa, but it has been naturalized in other
continents (8). Many isoflavones extracted from T. pratense
are available nowadays as dietary supplements (9). This plant
has also been suggested in traditional medicine as a treatment
for some human diseases such as whooping cough, asthma,
eczema and certain eye diseases (10). A study documented
the chemical profile of Trifolium pratense L. extract using
the high-performance liquid chromatography-ultraviolet
(HPLC-UV) chromatogram. The results showed that
Trifolium pratense L. extract was composed of isoflavones,
flavonoids, pterocarpans, coumarins and tyramine (11). Its
main isoflavones are biohanin A, formononetin, daizdein,
genistein, pratensein, prunetin, pseudobaptigenin, calycosin,
methylorobol, afrormosin, texasin, irilin B and irilone (12).
Despite current remarkable progress in cancer therapeutics,
it remains the leading cause of death in the world. So the
discovery and development of new therapeutic strategies
seems to be necessary. Although Trifolium pratense L.
has been suggested for cancer treatment in traditional
medicine, but there are currently no literature reports about
anti-cancer potentials of this plant. Therefore, the present
study was performed to determine the effects of T. pratense
hydroalcoholic extract on a glioblastoma cell line.
Materials and Methods
Cell line and reagents
For this in vitro experimental study, human GBM cell
line (U87MG) was obtained from the National Cell Bank
of Iran (NCBI). TMZ, trypsin, 3-(4, 5-dimethylthiazol2-
yl)-2, 5-diphenyltetrazolium bromide (MTT), acridin
orange (AO), ethydium bromide (EB) and propidium
iodide (PI) were purchased from Sigma-Aldrich
Chemical Co (St. Louis, MO, USA). Dulbecco’s modified
eagle medium/Ham’s F12 nutrient mixture (DMEM/
F12) and fetal bovine serum (FBS) were purchased from
Gibco (Gaithersburg, MD, USA). All experiments were
performed in triplicates and were repeated independently
at least three times. The study was approved by Ethical
Committee of Kermanshah University of Medical
Sciences, Kermanshah, Iran (Code: kums.res.1395.46).
Preparation of crude extracts
T. pratense seeds were cultured in spring of 2017 in a farm
and identified in terms of species by a botanist (Kermanshah
University of medical sciences, Kermanshah, Iran). Aerial
parts of the plants were dried and powdered, and 15g of the
powder were dissolved in 150 mLof 70% ethanol for 48 hours
in the dark. Then it was filtered through filter paper (Watman,
grade 42) and dried to allow for evaporation of the alcohol
at room temperature. Finally, the powder was dissolved in a
serum-free cell culture medium, and passed through a 0.22
µm filter (13).
Cell culture and treatment
U87MG cell line was grown in cell culture flasks containingDMEM/F12 supplemented with 10% FBS and no antibiotics.
Cells were maintained at 37.C in a humidified chamber
containing 5% CO2 (14). TMZ were dissolved in DMSO at astock concentration of 100 mM and stored at -20.C until use.
The cell line was treated with T. pratense extract (6.25, 12.5,
25, 50, 100, 200 and 400 µg/mL).
Trypan blue dye exclusion
U87MG cells were seeded in 24-well plates at 7×104
cells per well and incubated overnight. Then, the cell
culture medium was replaced with fresh serum-free
medium containing various concentrations of T. pratenseextract. The cells were incubated for 24, 48 and 72 hours.
Subsequently, the cells were harvested by trypsinization
and were resuspended in phosphate-buffered saline
(PBS). The cell suspension was then mixed with an equal
volume of 0.4% trypan blue solution prepared in PBS.
The number of live cells (unstained) over the total number
of cells was calculated as the percentage of viability (15).
U87MG cells were cultured in a 96-well plates at a
density of 1.5×104 cells per well and were allowed to attachovernight.
Then media containing different concentrationsof the extract were added to separate
wells. After 24, 48 and 72 hours of treatment at 37°C and 5% CO2, the media were
removed and 30 µL of MTT solution (5 mg/mL) was addedto each well, then incubated for
4 additional hours. Then 100
µL of dimethyl sulfoxide (DMSO) was added to dissolvethe formazan crystals produced
by living cells at room
temperature for 10 minutes with gentle shaking. The opticaldensity (OD) of resulting
solutions was measured using anELISA reader at 570 nm with a reference wavelength of
630 nm. The percentage of cell viability was calculated
according to the following formula (16):
The half maximal inhibitory concentration (IC50) values
of T. pratense extract were obtained by nonlinear regression
using GraphPad Prism 5 (GraphPad Software Inc, San Diego,
Lactate dehydrogenase assay
U87MG cells were seeded in 24-well plates and incubated
overnight. Culture media (500 µl) containing different
concentrations of T. pratense extract were added to separate
wells, and the plates were incubated for 24, 48 and 72 hours.
Then, 100 µl of medium from each sample was transferred to
another plates and lactate dehydrogenase (LDH) activity was
measured using Cytotoxicity Detection Kit (Roche Chemical
Co., Germany) according to the manufacturer’s procedures.
Finally, the OD at 490 nm with a reference wavelength of 690
nm for each sample was measured (17).
Nitric oxide measurement
Griess reaction was used for evaluation of the effect of T.
pratense extract on nitric oxide (NO) production by U87MG
cells. After treatment with the different concentrations of the
extract for 48 hours, the culture medium from each sample
was collected. In order to remove the proteins, 100 µl of each
sample was mixed with 6 mg of zinc sulfate and centrifuged
at 10000 g for 10 minutes at 4ºC. Then 100 µl of each
supernatant was mixed with 100 µl vanadium (III) chloride.
Immediately Griess reagents [50 µl 2% sulfanilamide and 50
µl 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride]
were added and the samples were incubated for 30 minutes at
37ºC. The OD was measured by a microplate reader at 540 nm
with a reference wavelength of 630 nm. The concentrations of
NO were calculated from a sodium nitrite standard curve (18).
Median effect analysis for TMZ and T. pratense
The method proposed by Chou was used to determine andquantify the nature of TMZ and T. pratense extract interaction
(synergistic, additive, or antagonistic) in a combination
treatment. The combination of TMZ and T. pratense extract
was prepared in constant concentration ratio (5.57:1) basedon their corresponding IC50 values in serial dilutions above
and below the IC50 value of each agent, and then the MTTassay was performed. The combination index (CI) and dosereduction index (DRI) were calculated using CompuSynsoftware (ComboSyn, Inc., Paramus, NJ, USA). The CIvalues were interpreted as additive (CI=1), synergistic(CI<1) and antagonistic (CI>1). The DRI values representthe degree, to which the concentration of a compound can bereduced when used in combination with another compound,
to maintain an equivalent effect. Finally, Fa is the fraction ofcell death ranging from 0 (no cell killing) to 1 (100% cell
Apoptosis was evaluated by labeling the 3´- hydroxyl
termini in DNA fragments using an In Situ Cell Death
Detection Kit, AP (Roche Diagnostics, Germany) according
to the manufacturer’s instructions. After 48 hours of treatment
with T. pratense extract in a 96 well plate, the cells were fixed
with a freshly prepared paraformaldehyde solution (4% in
PBS, pH=7.4) for 20 minutes at room temperature. Then the
cells were rinsed with PBS and permeabilized with a 0.1%
Triton X-100 solution in 0.1% sodium citrate for 5 minutes on
ice (4°C). The cells were rinsed twice with PBS, and 50 µL of
the TUNEL reaction mixture (label and enzyme solution) was
added to each well, followed by incubation in a humidified
chamber for 1 hour at 37°C. For differential staining of the
cells a PI staining solution was used. The plate was incubated
for 5 minutes at room temperature. The cells were then
rinsed three times with PBS and analyzed under a fluorescentmicroscope (Nikon Corporation, Japan). All the mentionedstages are performed in the dark. The apoptotic index of the
cells was calculated as follows (14):
Apoptotic index (%)=(number of apoptotic cells/total
number of cells)×100
Acridin orange/ethydium bromide double staining
For observation of the intact, apoptotic and necrotic cellsunder the
fluorescent microscope, AO/EB double stainingwas performed. AO passes
through the plasma membrane ofcells and emits a green fluorescent light.
EB only passes fromthe plasma membrane of cells when cytoplasmic membraneintegrity
is lost, and emits a red fluorescent light. EB emissiondominates over AO. Therefore,
live cells show uniform
green nuclei, early apoptotic cells have yellow nuclei withfragmented chromatin,
late apoptotic cells have fragmentedchromatin and orange nuclei,; and necrotic
cells have solidorange nuclei (19).
U87MG cells were cultured in 24-wellplates and treated with T. pratense
extract. After 48 hours,
cells were stained with mixture of AO/EB dye containing100 µg/ml of AO and
100 µg/ml of EB in PBS and observed
under a fluorescent microscope (4).
Detection of acidic vesicular organelles
Autophagy induction was investigated by detection of acidicvesicular organelles (AVOs), which consist predominantlyof autophagosomes and autolysosomes. U87MG cells weregrown in the absence or presence of T. pratense extract for
48 hours in 24 well plates. Then the cells were stained with1 µg/ml AO (in PBS) for 20 minutes and were observedunder a fluorescent microscope. The percentage of the cellsgoing through autophagy was calculated using the following
% of autophagic cells=(the number of cells with AVOs/
the total number of stained cells)×100
Real-time polymerase chain rection
The effects of various concentrations of T. pratense
extract on P53, CASPASE 3, BAX, BCL-2, LC3, ATG-7 and
BECLIN-1 mRNA expression were analyzed by real-timepolymerase
chain reaction (PCR). Total RNA from GBMcells, treated with T. pratense extract for 48 hours, was
prepared by total RNA isolation kit (DENAzist, Iran) and
the quantity and quality of the extracted RNA were tested by
Nano drop and gel electrophoresis. The complementary DNA
(cDNA) synthesis was carried out using cDNA synthesis kit
(Vivantis Technologies, Selangor DE., Malaysia). Real-time
PCR was performed using SYBR Premix Ex Taq technology
(Takara Bio Inc., Japan) on the Applied Biosystems
StepOne Real Time PCR System (Life Technologies, USA).
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) wasserved as
an internal control and the fold change in relativeexpression of each target
mRNA was calculated on the basisof comparative Ct (2-ΔΔct) method. Thermal cycler conditionswere 15 minutes at 50°C for cDNA synthesis, 10 minutes at95°C followed by 40 cycles of 15 seconds at 95°C to denaturethe DNA, and 45 seconds at 60°C to anneal and extend the
template. The primer sequences were as follows:
All data are presented as mean ± SD of three independentexperiments. Statistical evaluation was done using one-wayanalysis of variance (ANOVA) with SPSS version 16.0(SPSS Inc., Chicago, IL, USA) software, and differences
were considered to be statistically significant when P<0.05.
The results of trypan blue staining and MTT assay after24, 48, and 72 hours showed a significant difference amongthe groups treated with T. pratense extract (6.25, 12.5, 25,50, 100, 200 and 400 µg/ml) compared to the control group(P<0.05). Increasing the dose significantly decreased cellviability (Fig .1A, B,, P<0.05). So T. pratense extract reduced
U87MG cell viability in dose- and time-dependent manner.
The IC50 values for 24-, 48- and 72-hour treatments were
398.37, 109.19 and 21.06 µg/ml, respectively.
Measurement of LDH activity in cell culture medium
revealed that T. pratense extract significantly increased
LDH release in dose- and time-dependent manners (Fig .1C,,
P<0.05). Therefore, cell death mediated by T. pratense extract
is accompanied by plasma membrane damage.
Nitric oxide levels
The effects of different concentrations of T. pratense
extract on U87MG cells after 48 hours of treatment
showed a dose-dependent decrease in NO production. The
difference compared to the control group was significant
with the 12.5 µg/ml (P=0.02), 25 µg/ml (P=0.00), 50 µg/
ml (P=0.00), 100 µg/ml (P=0.00), 200 µg/ml (P=0.00)
and 400 µg/ml (P=0.00) doses (Fig .1D,).
The effect of T. pratense extract treatment on
Cancer cells were treated with a combination of TMZ
and T. pratense extract for 48 hours (Fig .1E, F,). Cell
viability reduction by TMZ and T. pratense extract
combination was greater than either TMZ or T. pratense
extract alone (Fig .1G,). In addition, CI and DRI values
were calculated and listed in Table 1. The results showed
that the CI values obtained in all tests were <1, indicating
a synergistic effect. The DRI values for TMZ were >1
indicating a dose reduction for a given therapeutic effect.
The apoptosis index of U87MG cells treated with
various concentrations of T. pratense extract for 48
hours showed that T. pratense increased apoptosis
significantly in a dose-dependent manner (P<0.05).
Apoptotic cell death was quantified and presented as
percentage (Fig .2,).
Acridin orange/ethydium bromide staining
Morphological changes in apoptotic cells including
cell shrinkage, chromatin condensation and nuclear
fragmentation were detected using fluorescent dyes.
Live cells with normal morphology were abundant in
the control group, whereas early apoptotic cells were in
cultures treated with 6.25 and 12.5 µg/ml (Fig .3,). Both
early and late apoptotic cells were observed in cultures
treated with 25, 50, 100 and 200 µg/ml, and in the 400
µg/ml group most of the cells were in the late stage.
Therefore, apoptosis increased in U87MG cells treated
with T. pratense extract in a dose-dependent manner.
Combination index (CI) and dose reduction index (DRI) values for temozolomide (TMZ) and Trifolium pretense extract combination
Fa; Fraction affected.
- The effects of T. pratense extract on U87MG cells. Cell proliferation was determined using A. The MTT assay, B. Trypan blue staining, C. Lactate
dehydrogenase (LDH) release from U87MG cells was measured colorimetric, D. Nitric oxide (NO) production was evaluated by Griess reaction, E. The effect
of T. pratense extract, F. TMZ, and G. their combination on viability of U87MG cells after 48 hours of treatment were evaluated by MTT assay. The data are
expressed as the percentage of control cells as the means ± SD. *; P<0.05 and **; P<0.01 compared with control.
- Apoptosis induction potential of T. pratense
extract in U87MG cells was evaluated using TUNEL staining. A. Control cells, B. In the presence of 6.25
µg/ml, C. 12.5 µg/ml, D. 25 µg/ml, E. 50 µg/ml, F. 100 µg/ml, G. 200 µg/ml, H. 400 µg/ml of T. pratense
extract, I. Positive control, J. Negative control, K.
Columns mean percentage of apoptotic cells from three independent experiments. Negative control, positive control and control cells were treated with
label solution without enzyme solution, DNase and serum free medium, respectively. The data are expressed as the percentage of the control cells as the
means ± SD. *; P<0.05 and **; P< 0.01 compared with control.
- U87MG cells were stained with AO/EB and observed under fluorescent microscope. A. Control group, B. In the presence of 6.25, C. 12.5, D. 25, E.
50, F. 100, G. 200, and H. 400 µg/ml of T. pratense extract.
Acidic vesicular organelles detection
pratense increased autophagy significantly in a dose-
The percentage of autophagy in U87MG cells treated dependent
manner (P<0.05). Autophagy cell death were
with T. pratense extract for 48 hours showed that T. quantified and
presented as percentage (Fig .4,).
- The effect of T. pratense
extract on autophagy was investigated by
AO staining in GBM cells. A. Control cells, B. In the presence of 6.25, C.
12.5, D. 25, E. 50, F. 100, G. 200, H. 400 µg/ml of T. pratense
I. Columns mean percentage of autophagic cells from three independent
experiments. Red dots indicate autophagic vesicles. The data are
expressed as the percentage of the control cells as the means ± SD. *;
P<0.05 and **; P<0.01 compared with control.
Real-time polymerase chain reaction
Expression of some apoptosis- and autophagy-related
genes was evaluated using real time PCR. P53 was
upregulated in cells that were treated with T. pratense
extract (Fig .5A,). The results of real time PCR also
suggested a downregulation of BCL-2 and upregulation
of BAX mRNA expression after 48 hours exposure to T.
pratense extract (Fig .5B, C,). Exposure of U87MG cells to
T. pratense extract led to increased mRNA expression of
CASPASE 3 gene (Fig .5D,). Also, the extract increased the
LC 3, BECLIN-1 and ATG-7 mRNA levels (Fig .5E-G,).
Thus, T. pratense extract induced apoptotic and autophagy
in U87MG cells at the transcriptional level.
- Expression levels of some apoptotic and autophagic factors in
U87MG cells after treatment with different concentration of T. pratense
extract for 48 hours was evaluated by real time PCR. A. P53 (tumorsuppressor), B. BAX (pro-apoptotic), C. BCL-2 (anti-apoptotic), D. CASPASE
3 (required enzyme for execution of apoptosis), E. BECLIN-1 (key positive
autophagic regulator), F. ATG-7 (essential autophagy gene), and G. LC3
(essential for autophagosome formation). The data are expressed in terms
of percent of control cells as the means ± SD. *; P<0.05 and **; P<0.01
compared with control.
The aim of the current study was to evaluate the
effects of T. pratense extract on GBM cells. First, the
potentials of seven different concentrations of this extract
to promote cell death were tested. Our results showed
that T. pratense hydroalcoholic extract decreased cell
viability in a time- and dose-dependent manner. We also
investigated whether T. pratense extract could have a
therapeutically beneficial effect when administered in
combination with TMZ (conventional chemotherapy
agent for GBM). Interestingly, the results of this study
showed that T. pratense extract increased the cytotoxicity
of TMZ and a combination of the extract with TMZ
demonstrated synergistic effects on U87MG cell line
proliferation with CI values between 0.27 and 0.46. The
mean CI of all tests in the present study was 0.37. In other
words, TMZ and T. pratense extract acted synergistically
to reduce the viability of GBM cells. This combination
also resulted in a noticeable dose reduction for TMZ and
reduced its IC50 to about 4.27 fold smaller. TMZ, like
many other chemotherapeutic drugs, produces different
types of general side effects such as moderate to severe
lymphopenia or abnormally low levels of white blood
cells. Therefore, a dose reduction of TMZ for therapy is
clinically very important.
Further, this research showed that T. pratense extract
induced both apoptosis and autophagy in U87MG cells.
Apoptosis is a programmed cell death and characterized
by morphological and biochemical. This kind of cell death
acts as a homeostatic mechanism. Cells with defective or
inefficient apoptosis pathway are enabling to survive even
under oxidative stress or hypoxia. Induction of apoptosis
can be an appropriate strategy, by which anti-cancer
agents destroy tumor cells (20). Autophagy is a catabolic
process that is essential for development, differentiation,
survival and homeostasis, and allows cells to degrade and
recycle of cellular components via lysosomal enzyme. A
number of studies have indicated that anti-cancer agents
induce autophagy in human cancer cells (21).
In the present study, our findings showed that the
number of AVO-containing cells was increased in a dose-
dependent manner, indicating the induction of autophagy.
Also, the number of TUNEL-positive cells was increased
in a dose-dependent manner, indicating the induction of
apoptosis. At a molecular level, the mRNA expression
of some autophagy- and apoptosis-related genes were
significantly changed by T. pratense extract treatment in
GBM cells. T. pratense extract treatment increased the
expression of P53, BAX, CASPASE 3, LC3, BECLIN-1
and ATG-7. The expression level of BCL-2 was reduced
by T. pratense extract treatment. These results were in
agreement with the findings of AVO and TUNEL staining.
P53 is a tumor suppressor protein, involved in both
apoptosis and autophagy cell death. P53 increases the
expression of BAX and reduces the expression of BCL-2
genes. The ratio of pro-apoptotic BAX to anti-apoptotic
BCL-2 protein controls the intrinsic pathway of apoptosis
(22). Increased BAX /BCL-2
ratio up-regulates CASPASE
3 expression and induces apoptosis cell death (23). P53
induces autophagy through TOR inhibition and also
through transcriptional activation of DRAM (24). P53 can
also induce autophagy by regulation of LC3. LC3 is an
essential protein in autophagy pathway (25).
the other essential protein in autophagy pathway that has
an impotent roll in autophagosome formation (26). Also
ATG-7 is another autophagy-promoting gene involving in
regulation of autophagy (27).
NO has been reported to be involved in many
physiological and pathological processes in the brain
and plays a dual and critical role in glioma biology
(28). This research indicated that T. pratense extract
significantly reduced NO production by GBM cells. NO
is a signaling molecule with complex regulatory effects
on both physiological and pathological conditions (29).
So, modulation of NO production in cancer cells can
potentially be a good strategy to achieve anti-glioma
effects. Previous studies have shown that cell proliferation,
vascularization, invasion, chemo-and radiotherapy
sensitivity and immune reactivity in glioma tumors can
be affected by NO concentration (30).
Also, NO is a bifunctional regulator of apoptosis. Proapoptotic
and anti-apoptotic functions of NO have been
reported in various in vivo and in vitro experimental models
(31). Studies have shown that NO can be an important
endogenous inhibitor of apoptosis (32). Among the most
important anti-apoptotic activities of NO are induction
of cytoprotective stress proteins, cGMP-dependent
inhibition of apoptotic signal transduction, suppression
of CASPASE activity and inhibition of cytochrome c
release (31). NO also inhibits CASPASE activation and
apoptotic morphology in neurons. However, to this point,
there has not been any studies on the role of NO in glial
cells apoptosis (33). Our data indicated that T. pratense
extract reduced NO production and may remove the antiapoptotic
effect of NO in U87MG cells.
As previously stated, Trifolium pratense L. extract
was composed of isoflavones, flavonoids, pterocarpans,
coumarins and tyramine. Its main isoflavones are biohanin
A, formononetin, daizdein, genistein, pratensein, prunetin,
pseudobaptigenin, calycosin, methylorobol, afrormosin,
texasin, irilin B and irilone (12). Dietary flavonoids
are the most abundant polyphenols in plant sources.
Several plant-derived flavonoids (silymarin, genistein,
quercetin, daidzein, luteolin, kaempferol, apigenin, and
epigallocatechin 3-gallate) have been reported to have
an anti-proliferative effect on various cancers such as
prostate, colorectal, breast, thyroid, lung, and ovarian.
Their anti-cancer effect is mediated by activation of
apoptosis, cell cycle arrest, inhibition of metabolizing
enzymes, reactive oxygen species formation, vascular
endothelial growth factor and basic fibroblast growth
factor. Also, some flavonoids have been reported to reduce
cancer cells drug resistance (34).
Genistein and daidzein, two member of flavonoid family,
have noticeable anti-proliferation effects against breast
cancer, due to their structural similarity with estrogen.
Anti-cancer effects of quercetin, another member of
flavonoid family against colon cancer and glioma tumors,
is mediated by activation of autophagy signaling pathway.
Nowadays, a variety of these flavonoids are used in dietary
supplements, but none of them have been approved
for clinical use (34). From the pterocarpans family,
indigocarpan has shown anti-proliferative activities in
human cancer cell lines via induction of a CASPASE
-dependent apoptosis pathway (35).
The anti-cancer activity of coumarins is mediated by
various pathways including inhibition of kinase, cell
cycle progression, angiogenesis, heat shock protein-90
(HSP90), telomerase, mitotic activity, carbonic anhydrase,
monocarboxylate transporters, aromatase and sulfatase
(36). Despite considerable
progress in cancer therapy over
the past decades, GBM is still associated with very poor
prognosis, and few patients survive more than 3 years due
to inherent chemo-resistance. Therefore, development of
new treatment strategies is essential for the patients with
GBM. Our data suggests for the first time that T. pratense
extract enhances the anti-neoplastic effect of TMZ in
GBM predominantly by augmentation of apoptosis and
autophagy of cancer cells.
Trifolium Pratens is potentially beneficial for further
development of new chemotherapeutic agents. The
present data open a new possible approach in the cure of
GBM. Future studies are necessary to seek if a combined
treatment with T. pratense extract and TMZ provide better
results in in vivo models.
This study was financially supported by the vice-
chancellor for research of Kermanshah University
of Medical Sciences (Project number: 95080). The
authors would like to thank the staff of the Fertility
and Infertility Research Center and Kermanshah
University of Medical Sciences. There is no conflict
of interest in this paper.
M.K.; Designed experiments, analysed data, supervised
the research and co-authored the manuscript. M.P.;
Performed all of the experiments and wrote the manuscript.
S.K. ; Edited the manuscript and done gene expression
analysis of this work. All authors read and approved the
Glioblastoma: from molecular pathology to targeted treatment.
Annu Rev Pathol.
DNA damage induced by alkylating agents and repair pathways.
J Nucleic Acids.
Mechanisms of Chemoresistance in Malignant Glioma.
Clin Cancer Res.
Temozolomide-mediated apoptotic death is improved by thymoquinone in U87MG cell line.
Discovery of natural product anticancer agents from biodiverse organisms.
Curr Opin Drug Discov Devel.
Natural sources as potential anticancer agents: a review.
International Journal of Phytomedicine.
Cell death mechanisms of plant-derived anticancer drugs: beyond apoptosis.
Evaluation of introduced and naturalised populations of red clover (trifolium pratense l.) at pergamino eeainta, Argentina.
Genet Resour Crop Evol.
Trifolium pratense L.as a potential natural antioxidant.
Seasonal variation of red clover (Trifolium pratense L., Fabaceae) isoflavones and estrogenic activity.
J Agric Food Chem.
The chemical and biologic profile of a red clover (Trifolium pratense L.) phase II clinical extract.
J Altern Complement Med.
Vojinovic- Miloradov M.
Antioxidant profile of Trifolium pratense L.
Protective effect of tragopogon graminifolius dc against ethanol induced gastric Ulcer.
Iran Red Crescent Med J.
Thymoquinone synergistically potentiates temozolomide cytotoxicity through the inhibition of autophagy in U87MG cell line.
Iran J Basic Med Sci.
Antiproliferatory effects of crab shell extract on breast cancer cell line (MCF7).
J Breast Cancer.
In vitro inhibitory effect of crab shell extract on human umbilical vein endothelial cell.
In Vitro Cell Dev Biol Anim.
Synergistic effect of temozolomide and thymoquinone on human glioblastoma multiforme cell line (U87MG).
J Cancer Res and Ther.
Apoptosis induction of human endometriotic epithelial and stromal cells by noscapine.
J Basic Med Sci.
A simple technique for quantifying apoptosis in 96-well plates.
Apoptosis: a review of programmed cell death.
The role of autophagy in cancer development and response to therapy.
Nat Rev Cancer.
The relationship between BcI-2, BAX and P53: consequences for cell cycle progression and cell.
Mol Hum Reprod.
Increased BAX /BCL-2 ratio up-regulates CASPASE -3 and increases apoptosis in the thymus of patients with myasthenia gravis.
Crosstalk between apoptosis, necrosis and autophagy.
Biochim Biophys Acta.
P53-dependent regulation of autophagy protein LC3 supports cancer cell survival under prolonged starvation.
Proc Natl Acad Sci USA.
Roles of autophagyrelated genes BECLIN-1 and LC3 in the development and progression of prostate cancer and benign prostatic hyperplasia.
Biogenesis and cargo selectivity of autophagosomes.
Annu Rev Biochem.
Role of nitric oxide in biological systems: a systematic review.
J Mazandaran Univ Med Sci.
Nitric oxide as a bioregulator of apoptosis.
Biochem Biophys Res Commun.
The dual role of nitric oxide in glioma.
Curr Pharm Des.
Nitric oxide as a bifunctional regulator of apoptosis.
Nitric oxide inhibits apoptosis by preventing increases in caspase-3-like activity via two distinct mechanisms.
J Biol Chem.
Nitric oxide inhibits CASPASE activation and apoptotic morphology but does not rescue neuronal death.
J Cereb Blood Flow Metab.
Cancer chemoprevention through dietary flavonoids: what’s limiting?.
Chin J Cancer.
Gnana Oli R,
Antioxidant and antiproliferative activity of indigocarpan, a pterocarpan from Indigofera aspalathoides.
J Pharm Pharmacol.
Coumarins as anticancer agents: a review on synthetic strategies,mechanism of action and SAR studies.
Eur J Med Chem.