Growth inhibitory and adjuvant therapeutic potential of aqueous extract of Triticum aestivum on MCF-7 and HeLa cells
Aim: The purpose of the present study is to evaluate the potent growth inhibitory effects of aqueous wheatgrass extract (AWE) alone and in combination with cisplatin on human breast and cervical cancer cells. Materials and Methods: The cytotoxic potential of AWE alone and in combination with cisplat...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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| Цитувати: | Growth inhibitory and adjuvant therapeutic potential of aqueous extract of Triticum aestivum on MCF-7 and HeLa cells / A. Hussain, T.M. Gheewala, A.J. Vas, K. Shah, P. Goala, S. Khan, S. Hinduja, C. Sharma // Experimental Oncology. — 2014. — Т. 36, № 1. — С. 9-16. — Бібліогр.: 57 назв. — англ. |
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irk-123456789-1453122019-01-21T01:24:02Z Growth inhibitory and adjuvant therapeutic potential of aqueous extract of Triticum aestivum on MCF-7 and HeLa cells Hussain, A. Gheewala, T.M. Vas, A.J. Shah, K. Goala, P. Khan, S. Hinduja, S. Sharma, C. Original contributions Aim: The purpose of the present study is to evaluate the potent growth inhibitory effects of aqueous wheatgrass extract (AWE) alone and in combination with cisplatin on human breast and cervical cancer cells. Materials and Methods: The cytotoxic potential of AWE alone and in combination with cisplatin was evaluated on human breast and cervical cancer cells (MCF-7 and HeLa) by cell viability assay. Further, the mode of cell death induced by AWE was determined by nuclear morphological examination and cell cycle analysis. These effects were then correlated with the expression of genes involved in apoptosis and proliferation (cyclin D1 and Bax) by RT-PCR. Results: AWE showed dose- and time dependent selective cytotoxicity towards the cancerhighlighting its safe profile. Lower dose combinations of AWE and cisplatin induced increased growth inhibition compared with the individual drugs on both cell lines (combination index < 1) indicating strong synergistic interactions. AWE was found to induce apoptosis and arrested the cells at G0–G1 phase of the cell cycle which correlated with the modulation of expression of bax and cyclin D1 in a time-dependent manner in MCF-7 and HeLa cells. Conclusion: These results suggest that the anti-cancer potential of AWE may be due to apoptosis induction and its anti-proliferative properties. This study also provides the first evidence demonstrating synergism between AWE and cisplatin, which may enhance the therapeutic index of prevention and/or treatment of human breast and cervical cancer. Key Words: Bax, cisplatin, combination, cyclin D1, synergistic, wheatgrass. 2014 Article Growth inhibitory and adjuvant therapeutic potential of aqueous extract of Triticum aestivum on MCF-7 and HeLa cells / A. Hussain, T.M. Gheewala, A.J. Vas, K. Shah, P. Goala, S. Khan, S. Hinduja, C. Sharma // Experimental Oncology. — 2014. — Т. 36, № 1. — С. 9-16. — Бібліогр.: 57 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/145312 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions |
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Original contributions Original contributions Hussain, A. Gheewala, T.M. Vas, A.J. Shah, K. Goala, P. Khan, S. Hinduja, S. Sharma, C. Growth inhibitory and adjuvant therapeutic potential of aqueous extract of Triticum aestivum on MCF-7 and HeLa cells Experimental Oncology |
| description |
Aim: The purpose of the present study is to evaluate the potent growth inhibitory effects of aqueous wheatgrass extract (AWE) alone and in combination with cisplatin on human breast and cervical cancer cells. Materials and Methods: The cytotoxic potential of AWE alone and in combination with cisplatin was evaluated on human breast and cervical cancer cells (MCF-7 and HeLa) by cell viability assay. Further, the mode of cell death induced by AWE was determined by nuclear morphological examination and cell cycle analysis. These effects were then correlated with the expression of genes involved in apoptosis and proliferation (cyclin D1 and Bax) by RT-PCR. Results: AWE showed dose- and time dependent selective cytotoxicity towards the cancerhighlighting its safe profile. Lower dose combinations of AWE and cisplatin induced increased growth inhibition compared with the individual drugs on both cell lines (combination index < 1) indicating strong synergistic interactions. AWE was found to induce apoptosis and arrested the cells at G0–G1 phase of the cell cycle which correlated with the modulation of expression of bax and cyclin D1 in a time-dependent manner in MCF-7 and HeLa cells. Conclusion: These results suggest that the anti-cancer potential of AWE may be due to apoptosis induction and its anti-proliferative properties. This study also provides the first evidence demonstrating synergism between AWE and cisplatin, which may enhance the therapeutic index of prevention and/or treatment of human breast and cervical cancer. Key Words: Bax, cisplatin, combination, cyclin D1, synergistic, wheatgrass. |
| format |
Article |
| author |
Hussain, A. Gheewala, T.M. Vas, A.J. Shah, K. Goala, P. Khan, S. Hinduja, S. Sharma, C. |
| author_facet |
Hussain, A. Gheewala, T.M. Vas, A.J. Shah, K. Goala, P. Khan, S. Hinduja, S. Sharma, C. |
| author_sort |
Hussain, A. |
| title |
Growth inhibitory and adjuvant therapeutic potential of aqueous extract of Triticum aestivum on MCF-7 and HeLa cells |
| title_short |
Growth inhibitory and adjuvant therapeutic potential of aqueous extract of Triticum aestivum on MCF-7 and HeLa cells |
| title_full |
Growth inhibitory and adjuvant therapeutic potential of aqueous extract of Triticum aestivum on MCF-7 and HeLa cells |
| title_fullStr |
Growth inhibitory and adjuvant therapeutic potential of aqueous extract of Triticum aestivum on MCF-7 and HeLa cells |
| title_full_unstemmed |
Growth inhibitory and adjuvant therapeutic potential of aqueous extract of Triticum aestivum on MCF-7 and HeLa cells |
| title_sort |
growth inhibitory and adjuvant therapeutic potential of aqueous extract of triticum aestivum on mcf-7 and hela cells |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2014 |
| topic_facet |
Original contributions |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/145312 |
| citation_txt |
Growth inhibitory and adjuvant therapeutic potential of aqueous extract of Triticum aestivum on MCF-7 and HeLa cells / A. Hussain, T.M. Gheewala, A.J. Vas, K. Shah, P. Goala, S. Khan, S. Hinduja, C. Sharma // Experimental Oncology. — 2014. — Т. 36, № 1. — С. 9-16. — Бібліогр.: 57 назв. — англ. |
| series |
Experimental Oncology |
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| fulltext |
Experimental Oncology 36, 9–16, 2014 (March) 9
GROWTH INHIBITORY AND ADJUVANT THERAPEUTIC
POTENTIAL OF AQUEOUS EXTRACT OF TRITICUM AESTIVUM
ON MCF-7 AND HeLa CELLS
A. Hussain, T.M. Gheewala, A.J. Vas, K. Shah, P. Goala, S. Khan, S. Hinduja, C. Sharma*
Department of Biotechnology, Manipal University, P.O. Box 345050, Dubai, United Arab Emirates
Aim: The purpose of the present study is to evaluate the potent growth inhibitory effects of aqueous wheatgrass extract (AWE) alone
and in combination with cisplatin on human breast and cervical cancer cells. Materials and Methods: The cytotoxic potential of AWE
alone and in combination with cisplatin was evaluated on human breast and cervical cancer cells (MCF-7 and HeLa) by cell viabil-
ity assay. Further, the mode of cell death induced by AWE was determined by nuclear morphological examination and cell cycle
analysis. These effects were then correlated with the expression of genes involved in apoptosis and proliferation (cyclin D1 and Bax)
by RT-PCR. Results: AWE showed dose- and time dependent selective cytotoxicity towards the cancerhighlighting its safe profile.
Lower dose combinations of AWE and cisplatin induced increased growth inhibition compared with the individual drugs on both cell
lines (combination index < 1) indicating strong synergistic interactions. AWE was found to induce apoptosis and arrested the cells
at G0–G1 phase of the cell cycle which correlated with the modulation of expression of bax and cyclin D1 in a time-dependent man-
ner in MCF-7 and HeLa cells. Conclusion: These results suggest that the anti-cancer potential of AWE may be due to apoptosis
induction and its anti-proliferative properties. This study also provides the first evidence demonstrating synergism between AWE
and cisplatin, which may enhance the therapeutic index of prevention and/or treatment of human breast and cervical cancer.
Key Words: Bax, cisplatin, combination, cyclin D1, synergistic, wheatgrass.
Cancer is a multistep and multifactorial disorder,
involving aberrations in the genetic and epigenetic
makeup of the cells, influenced directly and indirectly
from the environmental factors which account for
almost 90–95% of all cancer cases [1, 2]. Numerous
phytonutrients, such as sulforaphane, genistein, res-
veratrol, curcumin, β-elemene etc. derived from edible
plants are the foremost prospective agents reported
to interfere with different stages of carcinogenesis and
many other health conditions, including cardiovascular
diseases, diabetes etc. Mounting scientific reports
on these dietary agents corroborate that a diet rich
in fruits and vegetables could reduce 7–31% of all
cancers worldwide [3–5]. These phytonutrients act
through multiple signalling pathways which make these
agents important for cancer prevention and therapy.
Concurrent therapies, utilizing these nutrients along
with other standard cancer treatment modalities like
chemotherapy, are one of the current thrusted areas
to enhance the therapeutic index by synergistic or ad-
ditive interactions between these drugs [6, 7].
Wheat (Triticum aestivum), one such phytonutri-
ent, is an important part of human diet, and epide-
miological studies have suggested its protective role
against many chronic diseases including thalassemia,
cancer etc [8–10]. A number of studies have used
wheat in various forms such as whole grain, wheat-
grass etc. (5–10 day old grass of the common wheat
plant) [10–12]. Wheatgrass contains selenium, laetrile,
vitamins C and E, beta carotene, ferulic acid and va-
nilic acid, phenolic compounds including flavonoids,
the concentrations of which increase with the ger-
mination period [11, 12]. It has been shown to inhibit
the metabolic activity of carcinogens and has anti-
mutagenic and antioxidant activities and can reduce
chemotherapy associated side-effects [13–18].
The present study was designed to evaluate the po-
tent growth inhibitory effects of aqueous wheatgrass
extract (AWE) alone and in combination with cisplatin
on human breast cancer, MCF-7 and human cervical
cancer, HeLa cell lines. Further, its effect on the modu-
lation of genes involved in proliferation and apoptosis
were analyzed to understand the chemopreventive
mechanism of wheatgrass.
MATERIALS AND METHODS
Cancer cell lines.The effect of AWE alone and
in combination with cisplatin was studied on human
breast carcinoma cell line (MCF-7) and human cer-
vical carcinoma cell line (HeLa) obtained courtesy
of Dr. Tahir Rizvi, UAE University, Al Ain, UAE. The cells
were maintained in DMEM supplemented with
10% FBS, penicillin (100 units/mL), and incubated
at 37 °C in a humidified atmosphere with 5% CO2.
Lymphocytes were isolated from healthy non-smoking
donors using HiSep Media (HiMedia, India) as per
the manufacturer’s instructions and were maintained
in RPMI media (Sigma, USA) [6].
Preparation of drug solutions. AWE was pre-
pared as previously described [19]. Briefly, wheat
seeds were purchased from the local market, washed
with tap water, followed by distilled water. The seeds
were soaked in distilled water for 9 h and transferred
to containers with soil. The wheatgrass was collected
on day 11. Only wheatgrass of uniform size, shape and
without injuries were selected. The wheatgrass was
washed, wiped and cut into small pieces. A 30% (w/v)
aqueous extract was prepared (stock) using a clean
Received: December 23, 2013.
*Correspondence: E-mail: chhaviaiims@gmail.com
Abbreviations used: AWE — aqueous wheatgrass extract;
CI — combination index.
Exp Oncol 2014
36, 1, 9–16
10 Experimental Oncology 36, 9–16, 2014 (March)
mortar and pestle to make a homogenized paste.
The extract was centrifuged at 15,000 rpm for 20 min
at 4 oC and filtered using 0.2 μm filter. The purified
extract was stored at -20 oC in aliquots until further
use. Further dilutions were made from the 30% stock
in complete medium to required concentrations be-
tween 1–25% for the treatment of MCF-7 cells, HeLa
cells and lymphocytes.
A stock solution of 3.3 mM of cisplatin (Cadila
Pharmaceuticals Ltd, India) was used to make drug
dilutions of varying concentrations(1–200 μM) in com-
plete medium.
Cell viability assay. The anti-proliferative acti-
vity of AWE (1–25%) and cisplatin (1–200 μM) alone
or in combination (1 and 5% AWE; 1 and 5 μM cis-
platin) on MCF-7 cells, HeLa cells and lymphocytes
was evaluated using the MTT (3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide) assay,
as previously described [20]. In short, the cells were
plated at a density of ~1 • 104 cells/well in 96-well
plates in triplicates. Next day the culture medium was
removed and replaced by varying concentrations
of AWE (1–25%) or cisplatin (1–200 μM) in complete
medium or by the combination doses and incubated
for 24 and 48 h. The MTT assay was performed and
the optical density (OD) was measured at 570 nm.
The percent viability was calculated as (OD of the drug-
treated sample/OD of the non-treated sample) x 100,
considering that the colorimetric signal is directly
proportional to the number of viable cells. This assay
allows measurement of growth rate and conversely,
when metabolic events lead to apoptosis or necrosis,
the reduction in cell viability. The EC50 (50% effective
concentration) values were calculated from the dose–
response curves.
Calculation of combination effects of cisplatin
and AWE. Calculations of combination effects were
based on the method of Chouand Talalay (1984) and
were expressed as a combination index (CI) [21].
CI analysis provides qualitative information on the na-
ture of drug interaction, and CI, a numerical value, was
calculated according to the following equation:
CI = CA,x/ICx,A + CB,x/ICx,B,
where, CA,x and CB,x are, respectively, the concentra-
tions of drugs A and B used in combination to achieve
x% drug effect. ICx,A and ICx,B are the concentrations
for single agents to achieve the same effect. A CI value
< 1, =1, or > 1 represents, respectively, synergy, ad-
ditivity, and antagonism of cisplatin and wheatgrass,
respectively.
Detection of apoptosis in MCF-7 and HeLa
cells after treatment with AWE
Microscopic examination. Morphological
changes in MCF-7 and HeLa cells were observed
on treatment with AWE and cisplatin at different con-
centrations (5, 15 and 25%) for 24 and 48 h using
a normal inverted microscope (Labomed, USA).
The untreated cells were used as negative control.
Nuclear morphological studies. Apoptosis
induction after treatment with AWE at the EC50 con-
centration (15 and 25% for MCF-7 and HeLa cells,
respectively) for different time intervals (0, 6 and
24 h) was evaluated by the nuclear morphological
changes associated with it using propidium iodide
staining [20]. Briefly, ~106 cells/ml cells were seeded
on glass coverslips and incubated overnight in com-
plete medium at 37 °C. Further, cells were treated with
AWE at its EC50 for a series of time periods (0, 6 and
24 h). At the end of the desired time interval, cells
were fixed in a mixture of acetone: methanol (1:1) at
-20 oC for 10 min, washed with 1X PBS (pH 7.4) twice
and stained with propidium Iodide (10 mg/ml in PBS)
for 30 s in dark at RT. The coverslips were thoroughly
washed with PBS and placed upturned onto a glass
slide with mounting media (DPX). Slides were viewed
at 515 nm under the Progress Fluorescent Micro-
scope (Olympus, USA). The images were captured at
×40 magnification.
Quantification of apoptotic cells by flow cyto-
metry. AWE-induced apoptosis in MCF-7 and HeLa
cells was determined by flow cytometric analysis
as described earlier [20]. After treatment of syn-
chronous cultures of MCF-7 and HeLa cells with AWE
at their respective EC50 for 0 and 24 h, both adherent
and floating cells were harvested, washed with phos-
phate buffered saline (PBS, pH 7.2) and fixed with
ice-cold absolute ethanol at -20 oC overnight. Cells
were then washed with PBS prior to resuspending
in a buffer containing PI (50 mg/ml), 0.1% sodium
citrate, 0.1% Triton X-100 and 100 mg/ml of RNase A.
The cells were analyzed using flow cytometry (Beck-
man Coulter Flow Cytometer FC500, CXP Version 2.2).
The data was analyzed using the Beckman Coulter
KALUZA 1.1 analysis software.
Expression analysis of various genes targeted
by AWE. Reverse transcription-PCR was used to de-
tect the expression of Bax and cyclin D1 in response
to treatment with AWE at EC50 for varying time points
(0, 6 and 24 h). Total RNA extraction from untreated
and AWE-treated MCF-7 and HeLa cells was carried
out as per the manufacturer’s instructions (GenElute
Mammalian Genomic Total RNA Kit, Sigma, USA)
at various time intervals. Further, total RNA was
subjected to first strand synthesis as per manufac-
turer’s protocol (ProtoScript M-MuLVTaq RT-PCR Kit,
New England Biolabs, USA) followed by PCR using
gene-specific primers [20, 22, 23]. в-Actin was taken
as an internal control. The PCR cycle was as fol-
lows: initial denaturation at 95 oC for 5 min, followed
by 35 amplification cycles (denaturation at 94 oC for
30 s, annealing at 55 oC for в-actin, 56 oC for Bax and
54 oC for cyclin D1 for 30 s, and extension at 72 oC for
45 s), with final extension at 72 oC for 7 min. Amplified
products were visualized on a 2% agarose gel conta-
ining ethidium bromide.
Statistical analysis. All data are expressed
as means ± SD of at least 3 experiments. Fisher’s exact
test was adopted for statistical evaluation of the results.
Significant differences were established at p < 0.05.
Experimental Oncology 36, 9–16, 2014 (March) 11
RESULTS
AWE selectively suppresses the viability
of MCF-7 and HeLa cells. The growth suppres-
sive effects of different concentrations of AWE
on MCF-7 cells, HeLa cells and lymphocytes were
evaluated by the MTT assay. AWE treatment at vari-
ous concentrations ranging from 1–25% for 24 and
48 h induced cell death in MCF-7 and HeLa cells
in a dose and time-dependent manner (Fig. 1 a, b).
The EC50 for MCF-7 cells was found to be 15% and
10% whereas for HeLa cells, it was found to be 25 and
15%, respectively, for 24 and 48 h. The experiment was
reproduced at least three times.
0
10
20
30
40
50
60
70
80
90
100
110
1 5 10 15 20 25
Wheatgrass concentration, %
Ce
ll
via
bi
lit
y,
%
24 h
48 h
0
10
20
30
40
50
60
70
80
90
100
110
1 5 10 15 20 25
Wheatgrass concentration, %
Ce
ll
via
bi
lit
y,
%
24 h
48 h
0
10
20
30
40
50
60
70
80
90
100
110
1 5 10 15 20 25
Wheatgrass concentration, %
Ce
ll
via
bi
lit
y,
%
a
b
c
Fig. 1. Selective cytotoxicity of AWE on MCF-7 and HeLa cells
(a, b). MCF-7 and HeLa cells treated with AWE at varying con-
centrations (1–25%) results in dose and time dependent growth
suppression at 24 h (EC50 = 15%, 25%, respectively) and 48 h
(EC50 = 10%, 15%, respectively) whereas treatment of lympho-
cytes with AWE at similar concentrations for 24 h was found
to be minimally cytotoxic (c). Values are means ± SD of three
independent experiments. Each value with AWE treatment differs
from the control value (p < 0.05)
In order to confirm the safety profile of AWE, its ef-
fect was determined on lymphocytes as normal cells.
Isolated lymphocytes from a healthy non-smoker adult
were plated in triplicates followed by treatment with
AWE at varying doses (1–25%) for 24 h (see Fig. 1 c).
AWE treatment did not result in a significant decrease
in cell viability of lymphocytes thus indicating that
AWE is differentially cytotoxic to cancer cells but not
to the normal cells. This feature of AWE may provide
a platform for its development as a safe drug for treat-
ment for breast and cervical cancer.
Cisplatin induces cell death in both cancer and
normal cells. The effect of increasing concentrations
(1–200 μM) of cisplatin on the viability of MCF-7, HeLa
cells and lymphocytes was determined by MTT assay.
Cisplatin was found to induce dose dependent cyto-
toxicity in these cells as compared to the untreated
controls. The EC50 of cisplatin on MCF-7 and HeLa cells
was found to be 200 μM and 75 μM, respectively, after
24 h treatment (Fig. 2). Importantly, it was found that
cisplatin reduced the viability of lymphocytes (normal
cells) (see Fig. 2). Thus this study provides an insight
into the observed side-effects of chemotherapy seen
in patients which can be extrapolated in vitro.
0
10
20
30
40
50
60
70
80
90
100
110
1 5 25 35 75 100 200
Cisplatin concentration, μM
Ce
ll
via
bi
lit
y,
%
MCF-7
HeLa
Lymphocytes
Fig. 2. Cisplatin is cytotoxic to both cancer and normal
cells. Cisplatin treatment at varying doses (1–200 μM) for
24 h induced dose-dependent cytotoxicity in MCF-7, HeLa and
lymphocytes. The EC50 of cisplatin was found to be 200 and
75 μM at 24 h in MCF-7 and HeLa cells. Values are means ±
SD of three independent experiments. Each value with cisplatin
treatment differs from the control value (p < 0.05)
Combination treatment of wheatgrass and
cisplatin on HeLaand MCF-7 cells. Combinational
therapy utilizing the currently available chemothera-
peutic drugs with the natural dietary agents is a promi-
sing treatment strategy to reduce the non-specific
cytotoxicity caused by chemotherapeutic agents such
as cisplatin. In this study, a combination of wheatgrass
and cisplatin was evaluated by cell viability assay
to minimize the side effects and potentiate the che-
motherapeutic activity of cisplatin.
Cells (MCF-7/ HeLa) were treated with different
concentrations of cisplatin and wheatgrass alone, and
in combination for 24 h and their viability was evalu-
ated. It was observed that 1 μM of cisplatin (C1) used
in combination with 1% (W1) and 5% (W2) wheatgrass
resulted in a significant decrease in cell viability (85 and
80%, respectively) of MCF-7 cells as compared
to either of the compounds alone (98% for C1, 97%
and 90% for W1 and W2). When these combinations
were used on HeLa cells, the combinations resulted
in 60 and 53% (for C1W1 and C1W2) decrease in cell
12 Experimental Oncology 36, 9–16, 2014 (March)
viability while individual drugs decreased the cell vi-
ability by 84% for C1 and 81 and 75% with W1 and W2,
respectively (Fig. 3). Also, treatment of MCF-7 and
HeLa cells with 5 μM of cisplatin (C2) combined with
W1 and W2 resulted in synergistic decrease in cell vi-
ability (81 and 70% for MCF-7; 50 and 45% for HeLa)
as compared to individual doses (C2 = 93% and 77%
for MCF-7 and HeLa; W1 and W2) (see Fig. 3). Further
combinational index was calculated and it was found
to be less than 1 for all the combinations indicative
of synergistic action of these combinations on both
MCF-7 and HeLa cells.
0
10
20
30
40
50
60
70
80
90
100
110
C1 C2 W1 W2 C1W1 C1W2 C2W1 C2W2
Combinations of cisplatin and wheatgrass
Ce
ll
via
bi
lit
y,
%
MCF-7
HeLa
Fig. 3. Concurrent treatment of MCF-7 and HeLa cells with
sub-lethal doses of cisplatin (C1 and C2) and AWE (W1 and W2)
was found to induce synergistic decrease in cell viability in these
cells (combination index (CI < 1). Each value is a ratio of the level
in the treated cells to that in the untreated control cells. Values are
means ± SD of 3 independent experiments. Each value with cis-
platin and AWE treatment differs from the control value (p < 0.05).
AWE induces cell death via apoptosis
in MCF-7 & HeLa cells
Morphological changes induced by wheat-
grass on MCF-7 & HeLa cells. MCF-7 and HeLa cells
treated with increasing concentrations of wheatgrass
(5, 15 and 25%) for 24 and 48 h were observed under
an inverted microscope and their morphological cha-
racteristics were noted. Treated cells, as compared
to untreated cells, showed typical rounding off of cells,
along with cell shrinkage indicating that wheatgrass
induces cell death by apoptosis in these cells. The in-
crease in these characteristics was in a dose and
time-dependent manner (Fig. 4 a, b).
Nuclear morphological changes induced
by AWE on MCF-7 & HeLa cells. The apoptotic
changes induced by AWE were verified by analyzing
various nuclear morphological characteristics with
or without treatment. Untreated MCF-7 and HeLa
cells appeared uniform in chromatin density with
an intact nucleus. On the other hand, AWE treatment
of MCF-7 (15% AWE) and HeLa (25% AWE) cells for
6 and 24 h resulted in evidences of apoptosis like
chromatin fragmentation, accumulation of nuclear de-
bris, apoptotic bodies and nuclear blebbing (Fig. 5 a,
b).With an increase in duration of AWE exposure, there
was a cumulative accrual of the said features consis-
tent with apoptosis (see Fig. 5 a, b).
ControlDose
5%
15%
25%
24 h
Treated Control
48 h
Treated
ControlDose
5%
15%
25%
24 h
Treated Control
48 h
Treated
a
b
Fig. 4. Morphological changes induced by AWE treatment
in MCF-7 (a) and HeLa cells (b) at varying concentrations. AWE
treatment (at 5, 15 and 25% for 24 and 48 h) of MCF-7 (a) and
HeLa (b) cells results in characteristic rounding off (arrows)
of these cells compared to untreated cells. These changes accu-
mulated with an increase in the dose and time of AWE treatment
(magnification ×100)
0 h 6 h 24 h
a
b
Fig. 5. The nuclear morphological changes induced by AWE
treatment at various time intervals (0, 6 and 24 h) on MCF-7 (a)
and HeLa (b) cells. Untreated MCF-7 and HeLa cells (0 h) show
large and prominent nuclei indicating no significant characte-
ristics of apoptosis (white arrows). MCF-7 (a) and HeLa (b) cells
treated with AWE at their respective EC50 for 6 and 24 h showed
time-dependent increase in nuclear morphological changes
associated with apoptosis such as nuclear condensation and
fragmentation (green arrows) and apoptotic bodies (yellow ar-
rows) (magnification ×400)
Experimental Oncology 36, 9–16, 2014 (March) 13
Effect of AWE on the cell cycle distribution.
MCF-7 and HeLa cells were exposed to AWE at their
respective IC50 concentrations (15 and 25%, respec-
tively) for 0 and 24 h and their cell cycle distribution was
examined by flow cytometry. Flow cytometric analysis
of control and AWE-treated cells showed proper dis-
tribution of cells in the different phases of cell cycle
in untreated cells (0 h), while on treatment with AWE
for 24 h resulted in a significant increase in the number
of cells in the G0–G1 phase of the cell cycle (80% for
MCF-7 and 76% for HeLa). Furthermore, AWE treat-
ment caused a significant inhibition in the movement
of cells into the S-phase (9% for MCF-7 and 11.5% for
HeLa) in comparison to the untreated cells, respec-
tively (Fig. 6).
0
10
20
30
40
50
60
70
80
90
0 h 24 h 0 h 24 h
G0-G1 phase
S phase
G2-M phase
Ce
ll
cy
cl
e
di
st
rib
ut
io
n,
%
MCF-7 HeLa
Fig. 6. Cell cycle arrest by AWE in MCF-7 and HeLa cells. DNA
content in different phases of the cell cycle was measured
using propidium iodide by flow cytometry. MCF-7 and HeLa
cells(~1•106) treated at their respective EC50 doses of AWE for
24 h showed a significant decrease in the proportion of cells
in the G2/M and S phases of the cell cycle while an increase
in the number of cells in the G0–G1 phase indicating that AWE
treatment results in cell cycle arrest in this phase in MCF-7 and
HeLa cells. The histogram shows % analysis of cells in the dif-
ferent phases of the cell cycle from a representative experiment
(out of three individual experiments)
AWE treatment significantly modulates the ex-
pression of Bax and cyclin D1. In order to determine
the molecular targets of AWE on MCF-7 and HeLa
cells, we analyzed the expression of Bax and cyclin
D1 before and after treatment with AWE. β-Actin was
used as an internal control for comparison of samples.
Bax, a pro-apoptotic Bcl-2-family protein, resides
in the cytosol and translocates to mitochondria upon
induction of apoptosis. The expression of Bax was
found to be low in both untreated MCF-7 and HeLa
cells. Upon treatment with AWE, we observed that
there was a significant rise in the expression of Bax
in a time dependant manner in MCF-7 and HeLa cells
(Fig. 7 a, b). Another important gene involved in cell cy-
cle regulation, cyclin D1, functions in the progression
of the cell from G1 to S phase, and is over-expressed
in cancerous cells. In this study, it was also found
to be expressed in high levels in both the untreated
cell lines (see Fig. 7 a, b). Interestingly, the expression
of cyclin D1 significantly decreased in these cells after
treatment with AWE for 6 and 24 h compared to un-
treated cells (see Fig. 7 a, b).
a
b
b-actin
Bax
Cyclin D1
b-actin
Bax
Cyclin D1
1 2 3 4
Fig. 7. MCF-7 (a) and HeLa (b) cells treated with AWE at their
respective EC50 doses for 6 and 24 h show a significant decrease
in the expression of cyclin D1 while a significant upregulation
in the expression of Bax in a time-dependent manner compared
to untreated cells. Lanes 1–4 represent untreated cells, cells
treated with AWE for 6 and 24 h, and negative control for RT-PCR
respectively. β-Actin was used as an internal control
DISCUSSION
Chemoprevention is a promising interventional ap-
proach utilizing mainly phytochemicals that possess
many health benefits mediated directly or indirectly
by modulating signal transduction pathways [7, 24].
Ambit reports indicate their important role in cancer
prevention and treatment owing to their relatively
safe cytotoxicity profile. Standard cancer therapies
include surgery, radiotherapy, chemotherapy etc.,
but their successful therapeutic outcome is limited
due to various side effects and development of multi-
drug resistance thus necessitating a need for alternate
or adjuvant therapies. Hence, phytochemical based
therapeutic strategies may help in minimizing these
side effects as well as prevent the transformation
of precancerous lesions or development of secon-
dary or second primary tumors. Studies are underway
to harness the potential of combinational approaches
utilizing one or more synthetic or natural phytochemi-
cals along with an effective drug such as chemothera-
py to enhance the therapeutic effects of conventional
cancer therapy [7, 24, 25].
The present focused on unraveling the chemopre-
ventive and therapeutic action of wheatgrass (AWE)
and decipher its molecular targets on human cancer
cells. In order to determine the differential cytotoxicity
towards cancer cells, the effect of varying concentra-
tions of AWE was evaluated on MCF-7, HeLa cells and
lymphocytes. It was found that AWE inhibited the cell
14 Experimental Oncology 36, 9–16, 2014 (March)
viability of the cancer cells in a dose and time dependant
manner and its EC50 (effective concentration, the dose
which reduces the viability of cells by 50%) was deter-
mined to be 15 and 10% on MCF-7 cells, and 25 and
15% on HeLa cells after 24 and 48 h treatment, respec-
tively (see Fig. 1 a, b). These results are in line with other
in vitro studies that have shown the growth inhibitory
effects of wheatgrass on several cancer cells including
leukemia, skin, prostate cancer cells [17, 19, 26, 27].
To qualify that AWE can be used as a safe chemopre-
ventive agent, its effect on normal cells (lymphocytes)
at various concentrations was determined for the first
time. It was found that AWE treatment of lymphocytes
for 24 h did not result in a significant toxicity towards
these cells (see Fig. 1 c). This property of selectively
cytotoxicity ofAWE provides evidence that it can be used
as a safe modality for cancer treatment. This is consis-
tent with the previous studies which found that many
phytochemicals such as curcumin, sulforaphane etc.
are minimally toxic to the normal cells [28, 29].
As mentioned before, the combinational strategies
for cancer treatment are considered more favourable
due to their higher efficacy, resulting in better sur-
vival rates. Cisplatin is widely used chemotherapeutic
drug but its efficacy is limited by the associated many
side-effects like nausea and vomiting, nephrotoxicity,
alopecia, etc. because of its high toxicity to normal
cells [30]. In this study, first we analyzed the effect
of cisplatin on MCF-7, HeLa cells and lymphocytes for
24 h. It was found that cisplatin inhibited the growth
of cancer cells in a dose dependant manner with
the EC50 at 200 μM and 75 μM for MCF-7 and HeLa,
respectively (see Fig. 2). Moreover, cisplatin also
induced significant decrease in cell viability of nor-
mal lymphocytes though at higher doses compared
to the cancer cells, thus confirming that its non-
specific cytotoxicity towards cancer and normal cells
which may account for its observed side-effects
in clinical settings (see Fig. 2). Previous studies have
shown that cisplatin is aneffective cytotoxic drug used
in the treatment of several tumour types including lung,
bladder, testicular, and ovarian cancer with associ-
ated side-effects. In addition some patients develop
resistance on prolonged exposure to cisplatin [30–32].
Taking into account the observed non-specific cy-
totoxicity of cisplatin, low-dose combination treatment
using cisplatin and AWE was analyzed on MCF-7 and
HeLa cells. Our results showed that AWE potentiated
the growth inhibitory effects of cisplatin at sub-lethal
doses. Fig. 3 shows that after treatment of MCF-7 cells
with 1 μM of cisplatin (C1) in conjunction with 1%
AWE (W1), there was a synergistic (CI < 1) decrease
in the cell viability (85%) in combination treatment
(C1W1) than achieved with individual doses (98 and
97% for cisplatin and AWE alone respectively). Also,
the other lower dose combinations of cisplatin and
AWE i.e., C1W2, C2W1 and C2W2 (where C2 = 5 μM,
W2 = 5%) showed combination index less than 1 indi-
cating a sy nergistic action of these drugs in combina-
tion at the doses used (see Fig. 3). Similar results were
obtained on treatment of HeLa cells with the same
combinations (C1W1, C1W2, C2W1 and C2W2) (see
Fig. 3). This implies that AWE enhances the efficacy
of cisplatin in killing cancer cells, especially at lower
doses, thereby minimizing the cytotoxicity to normal
cells. Similar studies on various cancer cells using cis-
platin in combination with other agents such as EGCG,
β-elemene, AT-101, honey bee venom, etc. have
shown cisplatin to act in a synergistic manner [33–37].
Thus, combination treatment approaches of cisplatin
with phytochemicals such as wheatgrass may have
immense prospects for development of therapeutic
strategies to overcome cisplatin associated resistance
and side-effects in human cancers.
Importantly, preventive and therapeutic inter-
ventions ought to induce cell death via induction
of apoptosis and not necrosis which is associated with
extensive tissue damage resulting in an inflammatory
response. The mode of cell death induced by AWE was
firstly observed by microscopic examination of AWE
treated MCF-7 and HeLa cells at various concentra-
tions (5, 15 and 25%) for 24 and 48 h. AWE treated
cancer cells showed characteristic rounding off, cell
shrinkage and detachment from the matrix, indicating
that cell death induced by AWE is through apoptotic
pathway as compared to untreated cells in which these
morphological changes were absent (see Fig. 4 a, b).
Further, to verify the apoptosis induced by AWE
in MCF-7 and HeLa cells, changes in the nuclear mor-
phology were examined. Treatment of these cells with
AWE (15 and 25% for MCF-7 and HeLa respectively) for
varying time points (6 and 24 h) showed accumulation
of characteristics changes in the nuclear morpho-
logy viz., nuclear condensation and fragmentation,
extensive blebbing and presence of apoptotic bodies
in these cells which increased in a time-dependent
manner in comparison to untreated cells marked
by uniform and intact nucleus (see Fig. 5 a, b).
Cell cycle analysis by flow cytometry was per-
formed to confirm the mode of cell death. It was ob-
served that AWE treatment of MCF-7 and HeLa cells
at their respective EC50 concentrations for 24 h resulted
in increased proportion of cells in the G0/G1 phase
of the cell cycle indicating that AWE causes growth
arrest of these cells at G0/G1 phase of the cell cycle
in comparison to the untreated controls (see Fig. 6).
Our studies confirm that AWE induces cell death
in these cells mediated by the apoptotic pathway.
Another study also indicated that wheatgrass induces
apoptosis in human acute promyelocytic leukemia
cells [26]. Other chemopreventive agents have also
been found to induce apoptosis in various cancer
cells [20, 38–42].
The mechanism of apoptosis involves expres-
sion of several genes and pathways. To authenticate
the cell cycle inhibitory and apoptosis-inducing
activity of AWE, the expression of cyclin D1, an onco-
genic G1 cyclin and Bax, a pro-apoptotic gene were
analyzed. Cyclin D1, a key regulator of cell cycle
progression, forms a complex with CDK 4 and 6 (cy-
Experimental Oncology 36, 9–16, 2014 (March) 15
clin dependant kinases) which phosphorylates the
retinoblastoma (Rb) protein, thus allowing the pro-
gression of cells from G1 to S phase in the cell cycle
[43]. The overexpression of cyclin D1 has been linked
to the development and progression of cancer [44,
45]. Treatment of MCF-7 and HeLa cells with 15 and
25% AWE for 6 and 24 h resulted in significant down-
regulation in the expression of cyclin D1 in a time
dependant manner in comparison to untreated cells
which showed a high level of expression of cyclin
D1 (Fig. 7 a, b). Consequently the cell cycle arrest
mediated by AWE in MCF-7 and HeLa cells might
be through the modulation of cyclin D1 expression.
These results are consistent with other studies which
showed that many chemopreventive agents act
by downregulating the expression of cyclin D1 [46–48].
Thus, cyclin D1 may be an important target for cancer
chemoprevention and therapy.
Bax, a member of the bcl-2 family, plays a major
role in apoptosis. Gamut reports suggest that evasion
of apoptosis in cancer cells may be correlated with
the low expression of Bax [49–51]. Untreated MCF-7 and
HeLa cells showed no or feeble expression of Bax while
on treatment of these cells with AWE for 6 and 24 h, the
expression of Bax was significantly upregulated in a time-
dependent manner (see Fig. 7 a, b). Studies have impli-
cated the role of Bax in induction of sensitization towards
therapy as well as in cell cycle arrest and programmed
cell death [52, 53]. Various chemopreventive agents
have been shown to induce aforementioned activities
which have been correlated with increased expression
of Bax [53–57]. Taken together, the pro-apoptotic and
anti-proliferative activity of AWE may be related to its ef-
fect of the expression of Bax.
Conclusively, wheatgrass exhibits tumoricidal
effects and acts as a biological response modifier
in cancer treatment by inducing apoptosis and cell
cycle arrest. These results provide new insights that
wheatgrass may serve as an alternative approach for
cancer prevention and therapy by potentiating avai-
lable treatments. Additional in vitro and in vivo studies
are needed to assess the application of wheatgrass for
therapeutic purposes.
ACKNOWLEDGEMENT
The authors would like to thanks to Dr. Tahir Rizvi,
UAE University, Al-Ain, UAE for providing the cell lines
used in this study.
CONFLICT OF INTEREST
There is no conflict of interest.
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