International Journal of

Molecular Sciences

Article
A Novel Tetraenoic Fatty Acid Isolated from
Amaranthus spinosus Inhibits Proliferation and
Induces Apoptosis of Human Liver Cancer Cells
Arijit Mondal 1, *, Tanmoy Guria 2 , Tapan Kumar Maity 2 and Anupam Bishayee 3, *
1 Department of Pharmaceutical Chemistry, Bengal College of Pharmaceutical Sciences and Research,
Durgapur 713 212, West Bengal, India
2 Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700 032, West Bengal, India;
tguria.ju@gmail.com (T.G.); jutkmaity@yahoo.com (T.K.M.)
3 Department of Pharmaceutical Sciences, College of Pharmacy, Larkin Health Sciences Institute,
Miami, FL 33169, USA
* Correspondence: arijit_ncp@rediffmail.com (A.M.); abishayee@ularkin.org or abishayee@gmail.com (A.B.);
Tel.: +91-96-7412-4916 (A.M.); +1-305-760-7511 (A.B.)

Academic Editor: Maurizio Battino

Received: 9 May 2016; Accepted: 12 September 2016; Published: 22 September 2016

Abstract: Amaranthus spinosus Linn. (Family: Amaranthaceae) has been shown to be useful in
preventing and mitigating adverse pathophysiological conditions and complex diseases. However,
only limited information is available on the anticancer potential of this plant. In this study,
we examined the antiproliferative and pro-apoptotic effects of a novel fatty acid isolated from
A. spinosus—(14E,18E,22E,26E)-methyl nonacosa-14,18,22,26 tetraenoate—against HepG2 human
liver cancer cells. We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay to determine cell viability, flow cytometry assay for cell cycle analysis, and Western blot
analysis to measure protein expression of Cdc2), cyclin B1, Bcl-2-associated X protein (Bax), and B-cell
lymphoma 2 (Bcl-2). The MTT assay showed that the fatty acid markedly inhibited the proliferation
of HepG2 cells in a dosage-dependent fashion, with a half maximal inhibitory concentration (IC50 )
value of 25.52 µmol/L. This antiproliferative result was superior to that of another known fatty acid,
linoleic acid (IC50 38.65 µmol/L), but comparable to that of standard anticancer drug doxorubicin
(IC50 24.68 µmol/L). The novel fatty acid also induced apoptosis mediated by downregulation of
cyclin B1, upregulation of Bax, and downregulation of Bcl-2, resulting in the G2 /M transition arrest.
Our results provide the first experimental evidence that a novel fatty acid isolated from A. spinosus
exhibits significant antiproliferative activity mediated through the induction of apoptosis in HepG2
cells. These encouraging results may facilitate the development of A. spinosus fatty acid for the
prevention and intervention of hepatocellular carcinoma.

Keywords: Amaranthus spinosus; fatty acid; hepatocellular carcinoma; antiproliferative effect;

apoptosis; cell cycle regulation

1. Introduction
Cancer has become an enormous global health burden, because there are approximately
12.7 million cancer cases and 7.6 million cancer deaths every year [1,2]. Chemotherapy has been
considered as the most efficient method for cancer therapy, but most cancer patients suffer from
toxicity and side effects associated with chemotherapy.
Hepatocellular carcinoma (HCC), the most predominant pattern of liver cancer, is the sixth most
common cancer and the third leading cause of cancer mortality worldwide [3]. The incidence of HCC
in the United States has dramatically increased by more than 70% in the past 25 years [4]. According to

Int. J. Mol. Sci. 2016, 17, 1604; doi:10.3390/ijms17101604 www.mdpi.com/journal/ijms

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Int. J. Mol. Sci. 2016, 17, 1604 2 of 9

in the United States has dramatically increased by more than 70% in the past 25 years [4]. According
to the American Cancer Society, an estimated 35,660 new instances of liver cancer (including
intrahepatic
the American bile Cancerduct cancers)
Society, are anticipated
an estimated 35,660 new to take placeofin
instances thecancer
liver United States during
(including 2015,
intrahepatic
approximately
bile duct cancers) three-quarters
are anticipated of which
to take areplace
expectedin thetoUnited
be HCC. StatesLiver cancer
during incidence
2015, rates are
approximately
around three times
three-quarters higher
of which areinexpected
adult males to bethan
HCC. in Liver
women, and incidence
cancer have doubled ratesinareeach sex over
around threethe past
times
two decades
higher in adult[5]. The than
males majority of HCC
in women, andcases
haveare attributable
doubled in each tosexunderlying
over the past infections caused [5].
two decades by
hepatitis
The B and
majority of CHCC
virusescases [6].are
In attributable
India, 70% toto80% of all HCCs
underlying are related
infections causedto theby hepatitis
hepatitis BB virus
and
C(HBV),
virusesabout 15%
[6]. In are linked
India, 70% toto80% hepatitis C virusare
of all HCCs (HCV),
related andto 5%
the to both HBV
hepatitis and (HBV),
B virus HCV. Incidence
about 15% of
HCC
are in India
linked occurs inCindividuals
to hepatitis virus (HCV), between
and 5% 40 toto both
55 years,
HBVand andalso HCV. above 60 years.
Incidence of Eighty
HCC inpercent
India
of all HCCs
occurs in India between
in individuals occur with 40 toliver cirrhosis
55 years, andin alsotheabove
background,
60 years.and Eighty60%percent
of all these
of all cases
HCCsare in
hepatitis
India occur B-positive
with livercarriers
cirrhosis [7].Moreover,
in the background,several and other 60%riskof factors,
all thesesuch casesasare alcohol consumption,
hepatitis B-positive
obesity, [7].
carriers iron overload,
Moreover, environmental
several pollutants,
other risk factors, such and dietary
as alcohol carcinogensobesity,
consumption, (i.e., aflatoxins
iron overload, and
nitrosamines) have
environmental been proven
pollutants, and dietaryto play active roles
carcinogens (i.e.,in HCC etiology
aflatoxins [8]. Still, nohave
and nitrosamines) proven
beeneffective
proven
systemic
to chemotherapy
play active roles in HCC is available
etiology [8].for HCC patients.
Still, no proven effective systemic chemotherapy is available
for HCCConsidering
patients. the lack of effective treatment and the grave prognosis of HCC, chemoprevention
(use Considering
of natural and synthetic
the lack agentstreatment
of effective to reverse, andsuppress,
the grave or preventofcarcinogenesis)
prognosis HCC, chemoprevention has been
considered
(use of naturalto and
be the best strategy
synthetic agents to inreverse,
reversing the current
suppress, morbidity
or prevent and mortality
carcinogenesis) associated
has been with
considered
this disease [9]. Emerging studies provide compelling evidence that
to be the best strategy in reversing the current morbidity and mortality associated with this disease [9]. various phytoconstituents,
derived from
Emerging studiesdietary
provide and medicinalevidence
compelling plants, thatexhibit significant
various promise in derived
phytoconstituents, the prevention
from dietary and
management
and medicinalof HCC exhibit
plants, [10]. significant promise in the prevention and management of HCC [10].
Amaranthus spinosus Linn. (Family:
Amaranthus (Family:Amaranthaceae),
Amaranthaceae),commonly commonly known
known as as
“spiny
“spinypigweed”
pigweed” or
“spiny
or “spinyamaranth”,
amaranth”, is used
is usedas vegetable
as vegetable and cultivated
and cultivated throughout
throughoutIndia, India,
Sri Lanka, and many
Sri Lanka, andtropical
many
countries
tropical [11]. Traditionally,
countries [11]. Traditionally,this plant has been
this plant has been used usedin in
diabetic
diabeticcomplications,
complications,asas aa diuretic,
analgesic,antipyretic,
analgesic, antipyretic, antileprotic
antileprotic agent,agent,
and inand in the treatment
the treatment of bronchitis of andbronchitis
piles [12].andA. piles
spinosus[12].
is
A. spinosus
reported is reported
to possess to possess[13],
antidepressant antidepressant
anti-inflammatory [13], anti-inflammatory
[14], antimalarial [15], [14], antimalarial [16],
antiandrogenic [15],
antiandrogenic [16],
antihyperlipidemic, andantihyperlipidemic,
spermatogenic activities and [17].spermatogenic
Administration activities [17]. extract
of A. spinosus Administration
restored the of
A. spinosus
altered extract restored
hematological parameters the inaltered
pigs [18] hematological
and normalized parameters
biochemical in pigs
changes[18]inand normalized
the epididymis
biochemical
of rats [19]. changes
Amaranthine,in the epididymis
isoamaranthine, of rats hydroxycinnamates,
[19]. Amaranthine, isoamaranthine,
rutin, quercetin, hydroxycinnamates,
and kaempferol
rutin, quercetin,
glycosides are some andofkaempferol
the phytochemicalglycosides are some identified
constituents of the phytochemical
in A. spinosus. constituents
Accordingidentified
to previous in
A. spinosus.
studies, leavesAccording
of A. spinosus to possess
previous studies, antitumor
significant leaves ofand A. cytotoxic
spinosus activities
possess significant antitumor
[20,21]. However, the
and cytotoxic
active constituents activities [20,21].
responsible forHowever,
such activities the were
activenot constituents
specified. In responsible
this respect,for wesuch
haveactivities
recently
were not
isolated andspecified.
purified one In this
novelrespect, we have recently isolated and purified
fatty acid—namely,(14E,18E,22E,26E)-methyl one novel fatty
nonacosa-14,18,22,26
acid—namely,(14E,18E,22E,26E)-methyl
tetraenoate (Figure 1)—from the whole plant nonacosa-14,18,22,26
of A. spinosus, and reported tetraenoate (Figure activity
antidiabetic 1)—from the
of this
whole plant of A. spinosus,and reported antidiabetic activity of this
fatty acid [22]. Nevertheless, the potential anticancer effect of the aforementioned fatty acid has yetfatty acid [22]. Nevertheless, the
potential
to anticancer
be examined, effect
to our of the aforementioned
knowledge. Thus, in the fatty present acid has yet
study, weto be examined,
have investigated to the
our effect
knowledge.
of the
Thus, in theisolated
previously presentfatty
study,acidweagainst
have investigated
the growththe effect ofhuman
of HepG2 the previously
liver cancerisolated
cellsfatty
and acid against
defined the
the growth mechanisms
underlying of HepG2 human liver cancer cells and defined the underlying mechanisms of action.
of action.

Figure 1.
Figure 1. Structure
Structure of
of the
the fatty
fatty acid
acid isolated
isolated from
from the
the chloroform
chloroformfraction
fractionof
ofAmaranthus
Amaranthusspinosus.
spinosus.

2. Results
2. Results

2.1. The
2.1. The Purified
Purified Fatty
Fatty Acid
Acid Exerts
Exerts an
an Antiproliferative
AntiproliferativeEffect
Effect
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT)
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT) assay indicated that the fatty fatty acid
acid
isolated from
isolated from A.
A. spinosus
spinosus markedly
markedly inhibited
inhibited the
the proliferation
proliferation of
of HepG2
HepG2 cells
cells in
in aa dosage-dependent
dosage-dependent
fashion (Figure
fashion (Figure 2),
2), and
and the
the half
half maximal
maximal inhibitory
inhibitory concentration
concentration (IC
(IC50
50) value waswas found
found toto be
be
25.52 µmol/L
25.52 µmol/L (Figure 2). On the otherhand, linoleic
On the otherhand, linoleic acid (another fatty acid) and doxorubicin
acid) and doxorubicin
(a standard
(a standard anticancer
anticancer drug)
drug) showed
showed growth
growth inhibitory
inhibitory effects
effects in
inHepG2
HepG2cellscells(Figure
(Figure2),
2),with
withICIC5050
Int. J. Mol. Sci. 2016, 17, 1604 3 of 9
Int. J. Mol. Sci. 2016, 17, 1604 3 of 9
Int. J. Mol. Sci. 2016, 17, 1604 3 of 9
values of 38.65
values andand
of 38.65 24.68 µmol/L,respectively.
µmol/L,
24.68 Thisoutcome
respectively. This outcomereflects
reflects a promising
a promising anticancer
anticancer activity
activity
of values
of the
the of 38.65
purified
purified and
fatty 24.68
fatty acid
acid µmol/L,
against respectively.
againstHCC
HCC Thistooutcome
comparable
comparable reflects
doxorubicin,
doxorubicin, butasuperior
but promising anticancer
to to
superior linoleic activity
acid.
linoleic acid.
of the purified fatty acid against HCC comparable to doxorubicin, but superior to linoleic acid.

Figure
Figure 2. vitro
2. In In vitro cytotoxiceffects
cytotoxic effectsof
ofpurified
purified fatty
fatty acid
acidfrom
fromA.A.spinosus, linoleic
spinosus, acid,
linoleic andand
acid, doxorubicin
doxorubicin
in HepG2 cells. HepG2 cells were seeded in a 96-well plate (10,000 cells/well) 24 h prior to the addition
in Figure
HepG2 cells.
2. In vitroHepG2 cells
cytotoxic were
effects seeded fatty
of purified in a acid
96-well
fromplate (10,000linoleic
A. spinosus, cells/well) 24 doxorubicin
acid, and h prior to the
of each test material. Following 48h incubation with the extract, cell proliferation was determined by
addition
in HepG2 of cells.
eachHepG2
test material.
cells wereFollowing
seeded in a48h incubation
96-well withcells/well)
plate (10,000 the extract,
24 h cell
priorproliferation was
to the addition
the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT) assay. Each data point represents
of each test by
determined material. Following 48h incubation with the extract, cell proliferation
the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT) wasassay.
determined
Each by
data
mean ± standard deviation based on quadruplicate determinations in three to five independent
the represents
point 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
mean ± standard deviation based on quadruplicate(MTT) assay.determinations
Each data pointinrepresents
three to five
experiments.
mean ± standard
independent deviation based on quadruplicate determinations in three to five independent
experiments.
2.2.experiments.
The Purified Fatty Acid Induces Apoptosis
2.2. The Purified Fatty Acid Induces Apoptosis
2.2. TheToPurified
confirm thatAcid
Fatty the fatty acidApoptosis
Induces induces cellular apoptosis, the proportion of cells in sub-G1 phase
To confirm
was evaluated. that
As the fatty acid
depicted induces
in Figure 3, thecellular apoptosis,
dual parameter the proportion
fluorescent dot plot of cellsthe
shows in sub-G phase
viable 1cell
To confirm thatQ3 thequadrant
fatty acid inducesV−/propidium
cellular apoptosis, the proportion ofincells in apoptosis
sub-G1 phase
waspopulation
evaluated.inAsthe depicted (annexin
in Figure 3, the dual parameter iodide (PI)−),
fluorescent thedot
cells
plot early
shows the viable in cell
was
theevaluated.
Q4 quadrant As(annexin
depictedV+/PI−),
in Figureand 3, the
thecells
dualatparameter fluorescent dot plot
the late apoptotic/necrotic stage shows
in the the quadrant
viable cell
population in the Q3 quadrant (annexin V−/propidium iodide (PI)−), the cells in Q2
early apoptosis
population
(annexin in the Q3 quadrant
V+/PI+). (annexin
Figure 3A V+/PI (annexin
shows − the V−/propidium iodide (PI)−), the cells in early apoptosis in
in the Q4 quadrant ), mean
and the apoptotic
cells atpopulation (annexin V+/PI−) of HepG2
the late apoptotic/necrotic stage in cells,
the Q2
the Q4 quadrant
which (annexin (annexin
was 2.5%, V+/PI+). V+/PI−),
47%, and 68% and
for the the cells at the late apoptotic/necrotic stage in the Q2 quadrant
quadrant Figure 3Acontrol,
shows 25 theand 60 µmol/L
mean apoptotic of the fatty acid-treated
population (annexin HepG2
V+/PI cells
−) of
(annexin V+/PI+). Figure
in 24 h, respectively. The3A shows
results the mean
strongly apoptotic
indicated population
that the fatty acid (annexin
possessedV+/PI−) of HepG2
substantial cells,
apoptosis-
HepG2
which
cells, which wasand
was 2.5%,
2.5%, 47%, and 68% for25the control, 25 and 60 µmol/L of the fatty acid-treated
inducing effect 47%, 68% cells.
on the cancer for theAscontrol,
portrayedand 60 µmol/L
in Figure 3A, ofin the fatty acid-treated
untreated cells, 2.5% of HepG2 cells
the cells
HepG2
inwere cells in 24 h,
24 h, respectively. respectively.
The results The results
strongly strongly
indicated indicated that the fatty acid possessed substantial
annexin V+/PI−, whereas 1.1% of the cellsthat the fatty
were annexin acidV+/PI+
possessed substantial
(double apoptosis-
positive). After
apoptosis-inducing
inducing effect effect on the cancer cells. As portrayed in Figure 3A, in untreated cells, 2.5%
cells of
treatment withon25 the
andcancer cells.
60 µmol/L ofAs theportrayed in Figure
fatty acid for 24 h, the 3A, in untreated
double positive cells, 2.5% of
cell populations thewere
thewere
cellsannexin
found were
to be annexin
4%V+/PI−,
(FigureV+/PI
whereas−, 28.4%
3B) and whereas of 1.1%
1.1% (Figure of respectively.
the 3C),
cellsthe cells
were were annexin
annexin V+/PI+ V+/PI+ (double positive).
(double positive). After
After treatment with 25 and 60 µmol/L of the fatty acid for 24 h, the double
treatment with 25 and 60 µmol/L of the fatty acid for 24 h, the double positive cell populations were positive cell populations
were found
found to beto4%be (Figure
4% (Figure 3B) 28.4%
3B) and and 28.4% (Figure (Figure 3C), respectively.
3C), respectively.

Figure 3. Flow cytometric analyses of A. spinosus fatty acid-induced apoptosis in HepG2 cells using
annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI). Quadrants: Q3 (normal cells), Q4
(apoptotic cells), Q2 (late apoptotic/necrotic cells). The mean apoptotic population for (A) control
Figure
3. 3.Flow
Flowcytometric
cytometricanalyses
analyses of
of A.
A. spinosus
spinosus fatty acid-induced apoptosis in HepG2 cells using
Figure
(normal cells); (B) 25 µmol/L; and (C) 60 µmol/L of fatty acid-induced
fatty acid-treated apoptosis
HepG2 cells inin
24HepG2 cells using
h, respectively.
annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI). Quadrants: Q3 (normal cells), Q4
annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI). Quadrants: Q3 (normal cells),
(apoptotic cells), Q2 (late apoptotic/necrotic cells). The mean apoptotic population for (A) control
Q4 (apoptotic cells), Q2 (late apoptotic/necrotic cells). The mean apoptotic population for (A) control
(normal cells); (B) 25 µmol/L; and (C) 60 µmol/L of fatty acid-treated HepG2 cells in 24 h, respectively.
(normal cells); (B) 25 µmol/L; and (C) 60 µmol/L of fatty acid-treated HepG2 cells in 24 h, respectively.
Int. J. Mol. Sci. 2016, 17, 1604 4 of 9

Int. J. Mol. Sci. 2016, 17, 1604 4 of 9

2.3. Purified Fatty Acid Exhibits Cell Cycle Arrest

2.3.
Int.Purified
J. Mol. Sci.Fatty Acid
2016, 17, Exhibits Cell Cycle Arrest
1604 4 of 9
The cell cellcycle
cycleconsists ofoffour distinct phases,
phases, which are cell
cell growth
growth(G (G1 1phase),
phase),DNA DNAsynthesis
synthesis
2.3.The
Purified Fatty consists
Acid Exhibits four
Cell distinct
Cycle Arrest which are
(S (S
phase),
phase), cell division (G2 2 phase), and mitosis and DNA replication (M phase). The alteration inin
cell division (G phase), and mitosis and DNA replication (M phase). The alteration cellcell
cycle
cycleinduced
The cellby
induced bythe
thefatty
cycle fattyacid
consists of was
acid wasassessed
four distinct by
assessedphases, flow
by flow cytometry
which are cell
cytometry using
growth
using PIstaining
PI staining
(G ofofHepG2
1 phase), HepG2
DNA cells
synthesis
cells after
after
2424 (Sh.
h. phase),
It cell division (G 2 phase), and mitosis and DNA replication (M phase).
It was found that the mean percentage of cells in the G2 phase increased from 10% ± 1.31% toto
was found that the mean percentage of cells in the G 2 phase increased The
fromalteration
10% ± in cell
1.31%
14% cycle
± induced
1.06%,
14% ± 1.06%, 66%by±the
66% fatty acid
± 1.03%,
1.03%, and was
and 70%assessed
70% 0.99%by
±±0.99% flow
after
after cytometry
treatment
treatment using
with
with PI
10,staining
0,0,10, 25,25,and of60HepG2
and60 µmol/L
µmol/L cells after
fatty
fatty acid,
acid,
24 h.
respectively It was found that the mean percentage of cells in the G 2 phase increased from 10% ± 1.31% to
respectively(Figure(Figure4). 4). Our
Our results
results indicated
indicated thatthat the
the fatty
fattyacid
acidtreatment
treatmentcould couldarrest
arrestcells
cellsininthethe
G2G/M14%phases,
± 1.06%, 66% ±might 1.03%, and 70% ± 0.99% after treatment of with 0, 10, 25, and 60and µmol/L fattyprocesses
acid,
2/M phases,which which might be ascribable
ascribable to to the
the destruction
destruction DNA
of DNA replication
replication mitosis
and mitosis processes
respectively (Figure 4). Our results indicated that the fatty acid treatment could arrest cells in the
ofofHepG2
HepG2cells. cells.This
Thisgrowth
growthwas wasaccompanied
accompanied by a reduction
reduction in in the
thenumber
numberofofGG0/G 0 /G 1 phase
1 phase cells.
cells.
G2/M phases, which might be ascribable to the destruction of DNA replication and mitosis processes
of HepG2 cells. This growth was accompanied by a reduction in the number of G0/G1 phase cells.

Figure4. 4.Cell
Figure Cellcycle
cycle distribution
distribution of HepG2 cells
cells with
with or
or without
withouttreatment withA.A.spinosus
treatmentwith spinosusfatty
fatty
acid at
acid various
at various
Figure concentrations
4. Cell concentrations after
after
cycle distribution 24
of 24 h. The
h. The
HepG2 percentage
percentage
cells of cells in each
of cells treatment
with or without phase
in each phase was
with was estimated
fattybyby
estimated
A. spinosus
flow
flowcytometry.
acidcytometry.
at various concentrations after 24 h. The percentage of cells in each phase was estimated by
flow cytometry.
2.4.
2.4. PurifiedFatty
Purified FattyAcid
AcidAlters
Altersthe
theExpression
Expression of
of Apoptosis-Associated
Apoptosis-Associated Proteins
Proteins
2.4. Purified Fatty Acid Alters the Expression of Apoptosis-Associated Proteins
Theexpression
The expressionlevelslevelsofofseveral
several proteins
proteins related
related to to the
the cell
cellcycle
cycleininHepG2
HepG2cellscellstreated
treatedwith
with
The expression levels of several proteins related to the cell cycle in HepG2 cells treated with
A.A. spinosusfatty
spinosus fattyacid
acidwere
wereevaluated
evaluated usingusing Western
Westernblot blotanalyses.
analyses.ItItisisknown
known that cell
that division
cell divisioncycle
cycle
A. spinosus
protein fatty acid
2 homolog wereand
(Cdc2) evaluated using
cyclinB1B1 have Western blot analyses. with
It is known that cell division cycle
protein 2 homolog (Cdc2) and cyclin have a aclose
closerelationship
relationship with GG2 /M2/M arrest.
arrest. B-cell
B-cell lymphoma
lymphoma 2
protein
2 (Bcl-2) 2 homolog (Cdc2) and cyclin B1 have a close relationship with G /M arrest. B-cell lymphoma
is an is an anti-apoptotic molecule, and Bcl-2-associated
X proteinX(Bax) protein (Bax) apoptosis.
promotes HepG2
apoptosis.
2
(Bcl-2) anti-apoptotic molecule, and Bcl-2-associated promotes cells
2 (Bcl-2)
HepG2 cellsiswere
an anti-apoptotic
treated with the molecule,
fatty acidand Bcl-2-associated
at different X protein
concentrations (0, 10,(Bax) promotes
25, and 60 µmol/L)apoptosis.
for 24 h.
were treated
HepG2was
with
cellsused
the
were as
fatty with
treated
acid the
at different
fattyAs
concentrations
aciddepicted
at different
(0, 10, 25,(0,
concentrations
and 60 µmol/L) for 24for
h. 24
β-Actin
β-Actin internal control. in Figure 5, the level10,of25, and 60
Cdc2 wasµmol/L)
not altered, h.
the
wasβ-Actin
used aswas internal
used control.
as As
internal depicted
control. As in Figure
depicted 5,
in the level
Figure 5, of Cdc2
the levelwas
of not
Cdc2 altered,
was not the expressions
altered, the
expressions of cyclin B1 and Bcl-2 were decreased, and the expression of Bax increased after treatment
of cyclin B1 and Bcl-2
expressions were decreased, and the expression of Bax increased after treatment with the
with the fatty of cyclin
acid. B1 and
A. spinosus Bcl-2 wereregistered
fatty acid decreased, and results
these the expression of Bax increased
in a dosage-dependent after treatment
fashion.
fattywith
acid.theA. spinosus
fatty acid. A.fatty acidfatty
spinosus registered these results
acid registered in a dosage-dependent
these results in a dosage-dependent fashion.
fashion.

Figure
Figure5.5.Western
Western blot
blot analysis
analysisofof expression
ofexpression
expressionof of proteins
of proteins involved in
proteins involved
involved in apoptosis
apoptosis andGG 2/M arrest of
Figure 5. Western blot analysis in apoptosisand /M
and 2G arrest of
2 /M arrest of
HepG2 cells exposed to A. spinosus fatty acid. HepG2 cells were treated with several concentrations
HepG2 cells exposed to A. spinosus fatty acid. HepG2 cells were treated with several concentrations
HepG2 cells exposed to A. spinosus fatty acid. HepG2 cells were treated with several concentrations
(0–10
(0–10µmol/L)
µmol/L)ofoffatty
fattyacid
acidfor
for24
24h,
h,which
which altered
altered the expression of
the expression ofvarious
variousproteins.
proteins.
(0–10 µmol/L) of fatty acid for 24 h, which altered the expression of various proteins.
Int. J. Mol. Sci. 2016, 17, 1604 5 of 9

3. Discussion
Bioactive plant extracts are becoming useful resources to develop less toxic and more effective
drugs to manage cancer progression. Cytotoxicity and antitumor effects of different extracts from
A. spinosus have recently been reported [20,21], but the active constituents responsible for such
activities have not been identified. Accordingly, the present work was initiated to probe into a possible
anticarcinogenic effect and the associated cellular and molecular mechanisms of action of a novel fatty
acid isolated from the chloroform fraction of A. spinosus.
The isolated and purified fatty acid from A. spinosus was found to be involved in inhibiting the
proliferation of HepG2 cells, based on results from the MTT cytotoxicity assay. The fatty acid-induced
apoptosis in HepG2 cells was confirmed by annexin V/fluorescein isothiocyanate (FITC)/PI staining
utilizing a flow cytometric technique. Our results showed that the fatty acid induced significant cell
cycle alteration and arrested cancer cells at the G0 /G1 interface, which indicates the antiproliferative
potency of the A. spinosus fatty acid.
It is well known that cell cycle dysregulation is a characteristic of tumor cells. To investigate
a useful anticancer target, it is essential to study the proteins that actually mediate the critical events
of the cell cycle. During G2 /M transition, the cyclin-dependent kinases (CDKs) are activated, which,
in turn, mediate the cell cycle after binding to a specific regulatory subunit, called cyclin. Together,
these cyclin/CDK complexes are known to be a cell cycle regulator. The B-type cyclins, together with
Cdc2, control the mitotic entry process. Without the synthesis of cyclin B prior to the G2 /M transition,
Cdc2 is not activated, and subsequently, cells cannot enter mitosis. Thus, the cell cycle will arrest
at the G2 phase. Accordingly, these complexes (cyclin B/Cdc2) are called the M phase-promoting
factor. Eventually, degradation of cyclin B by ubiquitin is the essential step for cells to exit mitosis.
Our results indicated that the fatty acid could arrest HepG2 cells in the G2 /M phase, suggesting
that the fatty acid might contribute some effect to the dissociation of the cyclin B1/Cdc2 complex.
The Western blot analysis exhibited that the expression of cyclin B1 was decreased, but that of Cdc2
remained unaffected.
Overall results suggested that the fatty acid was capable of arresting the cells at the G2 phase and
preventing them from entering the mitosis process. Generally, apoptosis is regulated biologically via
two major pathways: the extrinsic pathway and the intrinsic pathway [19]. The intrinsic mitochondrial
pathway is controlled by Bcl-2 family proteins. In humans, more than 20 members of this family
have been identified so far, including proteins that suppress apoptosis (Bcl-2, Bcl-xL, Mcl-1 and A1),
and proteins that promote apoptosis (e.g., Bax, Bak, Bad and Bid) [23]. The pro-apoptotic and
anti-apoptotic proteins of the Bcl-2 family may turn on and off apoptosis because of the formation of
heterodimers among these proteins [24–26]. The heterodimerization results in mutual neutralization
of the bound pro- and anti-apoptotic proteins. For this reason, anti-apoptotic Bcl-2 family proteins are
important players in cancer cell survival and are recognized as relevant targets in cancer treatment.
In HepG2 cells treated with the tested fatty acid, the level of Bax was increased, and concomitantly
that of Bcl-2 was decreased, suggesting that the fatty acid induced apoptosis through upregulation of
Bax and downregulation of Bcl-2.

4. Materials and Methods

4.1. Chemicals and Reagents

Linoleic acid was procured from Hi-Media Laboratories Pvt. Ltd. (Mumbai, Maharastra, India),
and doxorubicin was obtained from Biochem Pharmaceutical Industries (Mumbai, Maharastra,
India). Dulbecco’s modified Eagle medium (DMEM), L-glutamine, fetal bovine serum (FBS),
penicillin–streptomycin, and trypsin-ethylenediaminetetraacetic acid (EDTA) were obtained from
Gibco BRL (Grand Island, NY, USA). MTT azocasein, sodium dodecyl sulfate (SDS), Tris, glycine,
β-mercaptoethanol (β-ME), CoomassieBrilliant Blue R-250, acrylamide/bisacrylamide, Brij-35 solution,
propidium iodide (PI), Triton X-100, and ribonucleaseA were obtained from Sigma-Aldrich Chemical Co.
Int. J. Mol. Sci. 2016, 17, 1604 6 of 9

(St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) and Tris-EDTA (TE) buffer were purchased from
Merck Co. (Darmstadt, Germany). Antibodies to Cdc2, cyclin B1, Bax, Bcl-2, and β-actin, as well
as horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). All other chemicals and reagents (analytical grade) were
purchased from Merck (Bangalore, Karnataka, India) and Thermo Fisher Scientific International, Inc.
(Mumbai, Maharastra, India).

4.2. Plant Material, Extraction, and Isolation

The whole plant was collected in the month of June 2011, and was identified by Dr. V.P. Prasad,
Scientist “D”, Botanical Survey of India, Howrah, West Bengal, India. A voucher specimen
(No. CNH/18/2011/Tech.II/419) was deposited in the Department of Pharmaceutical Technology,
Jadavpur University, Kolkata, West Bengal, India. The freshly collected plant material was washed,
shade-dried, and then milled to coarse powder by a mechanical grinder for further studies.
The powdered plant material (2 kg) was extracted with methanol at room temperature for 48 h.
The extract was separated out and dried under reduced pressure at 45 ◦ C to afford a crude methanol
extract (59.46 g). A part of the methanol extract (50 g) was suspended in water and fractioned
successively with chloroform, ethyl acetate, and n-hexane. Then, the extracts were evaporated under
reduced pressure in a rotatory evaporator and lyophilized to give remainders of the chloroform
(7.76 g), ethyl acetate (7.06 g), and n-hexane (20.12 g) fractions. All the fractions were evaluated for
anticancer activity. The chloroform fraction was found to be more potent than the ethyl acetate and
n-hexane fractions. Hence, the chloroform fraction was further exploited, which contributed to the
isolation of a novel fatty acid. The isolated compound was characterized as (14E,18E,22E,26E)-methyl
nonacosa-14,18,22,26 tetraenoate, based on spectrographic analysis (infrared, proton nuclear magnetic
resonance, and mass spectrometry). The detailed isolation procedure and structural elucidation of this
fatty acid were reported in our previous publication [22].

4.3. Cell Culture

The HepG2 human liver carcinoma cell line was purchased from the National Centre for Cell
Science (Pune, Maharastra, India). Cells were grown and maintained in DMEM supplemented with 10%
fetal bovine serum, 100 µg/mL of penicillin, and 100 µg/mL of streptomycin. Cells were maintained
at 37 ◦ C in a humidified atmosphere of 5% CO2 in air. When the cells were 60%–70% confluent,
the medium was aspirated, the cells were washed with phosphate-buffered saline (PBS), and fresh
DMEM with or without antibiotic was added. Control plates were replenished with fresh medium and
also incubated at similar conditions, as stated above.

4.4. Cell Viability Determination

Firstly, the MTT assay was performed to evaluate the antiproliferative effect of various test
materials. HepG2 cells were seeded in a 96-well plate at a density of 1.0 × 104 cells/well [27]. For the
untreated cells (control), vehicle DMSO was added instead of the sample or test drug. The novel
fatty acid from A. spinosus, linoleic acid, or doxorubicin at various concentrations (0–60 µmol/L) were
added to each well separately and cultured for 48 h, followed by incubation with 5 mg/mL MTT for
4 h at 37◦ C. The culture medium was removed after centrifugation and replaced with 150 mL DMSO
to dissolve the formazan crystals. The amount of formazan product was evaluated at 490 nm and
570 nm using a microplate reader. The cell viability was assessed as follows:

% cell viability = [absorbance of treated cells/absorbance of untreated cells] × 100

Subsequently, the IC50 value was calculated, setting the viability of untreated cells as 100%.
Int. J. Mol. Sci. 2016, 17, 1604 7 of 9

4.5. Cell Cycle Arrest Analysis

HepG2 cells (1 × 106 ) were fixed with ice-cold 70% ethanol at 4 ◦ C overnight, washed twice
with PBS, then incubated in RNase A/PBS (100 mg/mL) at 37 ◦ C for 30 min. DNA was labeled with
PI (50 mg/mL), and the fluorescence was measured by a fluorescence-activated cell sorting (FACS)
instrument (LSR Fortessa, BD Biosciences, Sparks, MD, USA). Data accumulation and analysis of the
cell cycle distribution were performed using FACS Diva software (BD Biosciences) [28].

4.6. Apoptosis Assay by Annexin V-FITC/PI

Surface exposure of phosphatidylserine in apoptotic cells were quantitatively detected using
annexin V-FITC/PI apoptosis detection kit (BD Biosciences) following the manufacturer’s protocol.
Cells were incubated with the purified fatty acid at the concentration of 60 µmol/L for 24 or 36 h.
The adherent and floating cells were mixed and processed according to the manufacturer’s instructions
and measured with FITC/PI flow cytometry to differentiate apoptotic cells (annexin-positive and
PI-negative) from necrotic cells (annexin- and PI-positive) [23,29].

4.7. Western Blot Analysis

Cells (2 × 105 ) were seeded in 3 cm culture dishes. After 24 h, the cells were incubated with or
without fatty acid (at IC50 ) for ≈20 h. Cells were washed with ice-cold PBS and then lysed in lysis
buffer (50 mM Tris-HCl (pH 7.4), 10 mM EDTA, 1% Triton-100, and 26% urea). Equal measures of
protein (20 mg) in the cell extracts were fractionated by 10% sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE)and then transferred onto nitrocellulose membranes. The membranes
were blocked in the 20 mM Tris-buffered saline–0.1% Tween (TBST) buffer containing 5% skim
milk at 4 ◦ C for 60 min and then probed with specific primary antibodies overnight at the same
temperature. After washing with TBST for 1 h, the membranes were incubated with HRP-conjugated
anti-rabbit/mouse IgG for 1 h at room temperature, and then washed with TBST for 1 h. Finally,
immunoblot signals were determined using an enhanced chemiluminescence (ECL) Western blotting
detection reagent (Thermo Fisher Scientific International, Inc.) [30].

4.8. Statistical Data Analysis

All statistical analyses were carried out using the Minitab (version16.1.0, Minitab Inc.,
Cubic Computing (P) Ltd., Bangalore, Karnataka, India). The results are shown as mean ± standard
deviation for three to five independent experiments.

5. Conclusions
The results of our present investigation clearly indicate that a novel fatty acid from A. spinosus is
a potent growth inhibitor of cultured HepG2 cancer cells. The growth inhibition is linked to the G2 /M
phase cell cycle arrest associated with the downregulation of cyclin B1. The fatty acid also displayed
programmed cell death (apoptosis) associated with the upregulation of Bax and the downregulation
of Bcl-2. These results provide the molecular basis for the observed anticancer effect of the fatty acid
isolated from the dietary plant A. spinosus. Further research is underway on upstream factors leading to
the decrease of cyclin B1 and subsequent cells arrest in the G2 /M phase, as well as signaling pathways
through which this fatty acid promotes HepG2 cells to undergo programmed cell death.

Acknowledgments: We wish to thank the Council of Scientific and Industrial Research-Indian Institute of
Chemical Biology (Kolkata, India) for providing the research infrastructure and instrumental facilities to carry out
the research work.
Author Contributions: Arijit Mondal conceived and designed the experiments; Arijit Mondal and Tanmoy Guria
performed the experiments; Arijit Mondal analyzed the data; Tanmoy Guria and Tapan Kumar Maity contributed
reagents/materials/analysis tools; Arijit Mondal and Anupam Bishayee wrote the paper.
Int. J. Mol. Sci. 2016, 17, 1604 8 of 9

Conflicts of Interest: The authors declare no conflict of interest. The funding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.

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