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Alpha-linolenic acid regulates the growth of breast and cervical cancer cell lines through regulation of NO release and induction of lipid peroxidation

Authors:
  • IRSHA (Bharati Vidyapeeth Deemed University)

Abstract and Figures

In the present work, we have analyzed the effect of the essential fatty acid, alpha linolenic acid (ALA) on nitric oxide release as well as induction of lipid peroxidation in breast (MCF-7 and MDA-MB-231) and cervical (SiHa and HeLa) cancer cell lines. ALA-treated cells showed a dose-dependent decrease in cell viability in both breast and cervical cancer cell lines without affecting the viability of non-cancerous transformed HEK 293 cells. Both types of cancer cells treated with ALA demonstrated a significant reduction in nitric oxide (NO) release with a simultaneous increase in lipid peroxidation (LPO). This was followed by a decrease in the mitochondrial membrane potential as well as activation of caspase 3 leading to apoptosis. Thus, ALA regulated the growth of cancer cell lines through induction of lipid peroxidation and modulation of nitric oxide release resulting in apoptosis
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In the present work, we have analyzed the effect of the
essential fatty acid, alpha linolenic acid (ALA) on ni-
tric oxide release as well as induction of lipid peroxi-
dation in breast (MCF-7 and MDA-MB-231) and cer-
vical (SiHa and HeLa) cancer cell lines. ALA-treated
cells showed a dose-dependent decrease in cell viabil-
ity in both breast and cervical cancer cell lines without
affecting the viability of non-cancerous transformed
HEK 293 cells. Both types of cancer cells treated with
ALA demonstrated a significant reduction in nitric
oxide (NO) release with a simultaneous increase in
lipid peroxidation (LPO). This was followed by a de-
crease in the mitochondrial membrane potential as well
as activation of caspase 3 leading to apoptosis. Thus,
ALA regulated the growth of cancer cell lines
through induction of lipid peroxidation and modu-
lation of nitric oxide release resulting in apoptosis.
Research Article
Rashmi Deshpande, Prakash Mansara, Snehal Suryavanshi and Ruchika Kaul-Ghanekar
Interactive Research School for Health Affairs (IRSHA), Bharati Vidyapeeth University, Katraj-Dhankawadi, Pune-Satara
Road, Pune-411043, Maharashtra, India
Received on April 19, 2012; Accepted on December 29, 2012; Published on February 18, 2013
Correspondence should be addressed to Ruchika Kaul-Ghanekar; Phone: +91 20 24366929/24366931, Fax: +91 20
24366929/24366931, Email: ruchika.kaulghanekar@gmail.com
Alpha-linolenic acid regulates the growth of breast and cervical cancer
cell lines through regulation of NO release and induction of lipid
peroxidation
Introduction
Alpha-linolenic acid (ALA 18:3), an omega-3 (n-3)
fatty acid, is an essential fatty acid (EFA) that cannot
be synthesized by the human body and thus must be
obtained from dietary sources. ALA is found mostly in
certain plant foods that include walnuts, rapeseed
(canola), several legumes, flaxseed and green leafy
vegetables (Barceló-Coblijn & Murphy 2009,
Bougnoux et al. 2010). ALA is the parent n3 fatty acid
and gets converted into longer chain fatty acids such as
Eicosapentaenoic (EPA 20:5, ω3) and Docosahex-
aenoic acid (DHA 22:6, ω3) that are well known for
their various functions including cardio-protection,
anti-inflammatory, anticancer as well as brain develop-
ment (Allayee et al. 2009, Burdge et al. 2005, Stark et
al. 2008, Zhao G et al. 2004). There is a vast amount
of research on EPA and DHA derived from fish oil;
however, very few studies have been conducted on
ALA present in plants.
A number of studies have reported that essen-
tial fatty acids selectively kill tumor cells through the
generation of free radicals as well as lipid peroxidation
(Das et al. 1999, 2000). Free radicals in the form of
ROS (reactive oxygen species) and RNS (reactive ni-
trogen species) are known to cause oxidation of
biomembranes as well as modulation of inter- and in-
tracellular signaling networks resulting in changes of
cell proliferation, differentiation and apoptosis (Das
2002, Sun et al. 2012). Nitric oxide, a measure of
RNS, is an endogenously produced free radical that
has been known to either promote or inhibit lipid per-
oxidation (Hogg & Kalyanaraman 1999). The enzyme
responsible for the conversion of L-arginine to NO is
the nitric oxide synthase (NOS) that exists in three ma-
jor isoforms; inducible (NOS II/iNOS), endothelial
(NOS III/eNOS) and neuronal NOS (NOS I/nNOS). In
cancer cells, it has been shown that increased NO gen-
erated by iNOS contributes to tumor angiogenesis by
the up-regulation of vascular endothelial growth factor
(VEGF), which may increase tumor metastasis
(Nakamura et al. 2006). Increased release of NO in the
cervix has also been shown to be associated with HPV
infection in cases of cervical cancer (Rahkola et al.
2009, Wei et al. 2009).
Nitric oxide has been reported to be a potent
inhibitor of the lipid peroxidation chain reaction and
has been shown to inhibit peroxidase enzymes that are
potential initiators of the former process. Conversely,
in the presence of superoxide, nitric oxide forms per-
oxynitrite that can initiate lipid peroxidation and oxi-
dize lipid soluble antioxidants (Gago-Dominguez et al.
Abstract
Journal of Molecular Biochemistry (2013) 2, 6-17 © The Author(s) 2013. Published by Lorem Ipsum Press.
2005, Hofseth et al. 2008, Hogg & Kalyanaraman
1999, Xu et al. 2002). The cell membranes contain
high concentrations of polyunsaturated fatty acids and
are thus susceptible to peroxidation, which is a critical
mechanism leading to growth inhibition and cell death
(Gago-Dominguez et al. 2007).
In the present study, we analyzed the effect of
ALA, an omega 3 fatty acid, on breast and cervical
cancer cell lines in terms of cell viability, nitric oxide
generation as well as status of lipid peroxidation. We
observed that ALA decreased the viability of both
breast and cervical cancer cells in a significant manner,
albeit, at higher doses and higher exposure times. In-
terestingly ALA regulated the growth of both types of
cancer cells through an increase in lipid peroxidation
and a reduction in nitric oxide generation. This re-
sulted in loss of the mitochondrial membrane potential
of the cells leading to apoptosis through activation of
the caspase 3 pathway.
Materials and Methods
Reagents
Tissue culture plasticware was purchased from BD Bio
-sciences, CA, USA. Alpha linolenic acid, fatty acid-
free bovine serum albumin (BSA) and 3-(4, 5-
dimethylthiazol-2-yl)-2, 5-diphenylthiazoliumbromide
(MTT) were purchased from Sigma-Aldrich, St. Louis,
MO, USA. Dulbecco's Modified Eagles Medium
(DMEM), Penicillin and streptomycin were obtained
from Gibco BRL, USA. Fetal bovine serum was pur-
chased from Moregate Biotech, Australia. L-
Glutamine, BHT and TBA were obtained from Hime-
dia Corporation, Mumbai, India. Sulfanilamide was
purchased from Qualigens, N-[1-napthyl] ethylenedia-
mine (NEDD) was purchased from SRL and TCA was
purchased from Merck.
Cell culture
The human breast adenocarcinoma (MCF-7 and MDA
MB231) and cervical cancer (SiHa and HeLa) cell
lines as well as the human embryonic kidney cell line
(HEK293) used in this study were obtained from the
National Centre for Cell Science (NCCS), Pune, India.
The cells were grown in DMEM containing 2 mM L-
glutamine supplemented with 10% fetal bovine serum
and 100 U/ml of penicillin-streptomycin. They were
incubated in a humidified 5% CO2 incubator at 37°C.
Conjugation of Alpha linolenic acid with BSA
ALA was reconstituted in 200 µL of ethanol. For con-
jugation, ALA (10mM) was added to fatty acid-free
bovine serum albumin (BSA) (3mM) to obtain a ~3:1
ratio of ALA: BSA (Mahadik et al. 1996). The conju-
gated omega fatty acids were incubated at 37˚C for 30
min in a CO2 incubator and stored at -20˚C. Before use
they were diluted to the required concentration with
10% DMEM.
Cell Viability Assay
Cell viability was measured using the MTT assay in
breast (MCF-7 and MDA-MB-231) and cervical can-
cer cell lines in the presence of different concentrations
of ALA and compared with the non-cancerous trans-
formed cell line, HEK 293. The cells were seeded at a
density of 1 × 105 cells/ml in 96-well plates (TPP,
Europe/Switzerland) and grown for 24 h. ALA was
added at different concentrations: 0-320μM for 24, 48
and 72 h. The MTT solution (5 mg/ml) was added to
each well and the cells were cultured for another 4 h at
37°C in a 5% CO2 incubator. The formazan crystals
formed were dissolved in 90 μl of SDS-DMF (20%
SDS in 50% DMF) (Singh et al. 2009). After 15 min,
the amount of colored formazan derivative was deter-
mined by measuring optical density (OD) at 570 nm
using the ELISA micro plate reader (Bio-Rad, Hercu-
les, CA).
Nitric Oxide assay
The concentration of NO was indirectly determined in
culture supernatants as nitrite, a major stable product
of NO. The cells (breast and cervical cancer cell lines)
were seeded at a density of 1x105 cells/ml in 96-well
plates (TPP, Europe/Switzerland) for 24 h and then
incubated with different concentrations of ALA (0-80
μM) for different time intervals (24, 48 and 72 h). The
NO levels were estimated by the Griess reaction
(Udenigwe et al. 2009). Briefly, 100 µL of culture su-
pernatant was mixed with an equal volume of Griess
reagent [1% sulfanilamide and 0.1% N-(1-naphthyl)-
ethylenediamine in 5% phosphoric acid) and incubated
at room temperature for 10 min. The absorbance at 540
nm was measured with the ELISA micro plate reader
(Bio-Rad, Hercules, CA). Nitric oxide concentration
was determined using sodium nitrite (NaNO2) as a
standard.
RT-PCR
The total cellular RNA from control as well as cells
treated with different concentrations of ALA (0-80
μM) was extracted by a one-step acid guanidine
isothiocyanate-phenol method using the TRI reagent
(Invitrogen). RNA was precipitated with isopropanol
and the concentration was estimated using Nanodrop
(Eppendorff BioPhotometer plus). 10 mg of total RNA
were used for each RT-PCR reaction. 50 units of
Moloney murine leukemia virus reverse transcriptase
(MMuLV) (Bangalore Genei, Bangalore, India) were
7 Journal of Molecular Biochemistry, 2013
added in a typical 50 μl reaction (10 μg RNA, 5X first-
strand buffer, 1 mM DTT, 2.5 mM dNTPs, 50 ng/μl
random primers and 15 U/μl RNAse i) and incubated
for 1 h at 40°C followed by incubation at 95°C for 5
min. The purified cDNA template was amplified using
different sets of primers. The primers used were β-
actin-F: 5'-taccactggcatcgtgatg-gact-3'; β-actin-R: 5’-
tttctgcatcctgtcggaaat-3'; iNOS-F: 5'- caga-
taagtgacataagtga-3'; iNOS-R: 5'- ctatctttgttgttgtccttg-3'.
PCR was performed in 25 μl volume in which 1X PCR
buffer, 2.5 mM dNTPs, 1.5 mM MgCl2, 1 U of Taq
polymerase and 100 ng of the specific primers were
added. A brief initial denaturation at 95°C for 5 min
was followed by 35 cycles with the following steps:
95°C for 1 min, annealing at 42°C for 1 min and exten-
sion at 72°C for 1 min. RT-PCR products were then
separated on a 1.2% agarose gel and visualized by
staining with ethidium bromide. The intensities of the
bands corresponding to the RT-PCR products were
quantified using phosphor imager (Alpha Imager, Al-
pha Innotech) and normalized with respect to the β-
actin product.
Lipid peroxidation assay (thiobarbituric acid reac-
tive substance)
The generation of thiobarbituric acid reactive sub-
stances (TBARS) was measured following a published
protocol (Ding et al. 2006) with minor modifications.
Briefly, the cells were seeded at a density of
4x105cells/ml in 6-well plates (TPP, Europe/
Switzerland) and were grown for 24 h, followed by
treatment with different concentrations of ALA (0-80
μM). After 24 h of treatment, cells were harvested and
resuspended in 120 µl of 1X PBS. They were homoge-
nized on ice for 10 min using a micro-pestle and then
centrifuged at 10,000 rpm for 10 min. Following this,
100 mM butylated hydroxytoluene (1.5 µL), 15% Tri-
chloroacetic acid (50 µL), 0.25 mM butylated hy-
droxytoluene (50 µL), 0.375% thiobarbituric acid (50
µL) and 8.5% SDS (20 µL) were added. The samples
were then vortexed for 5 min. This mixture was incu-
bated at 80 oC for 120 min and the reaction was
Journal of Molecular Biochemistry, 2013 8
Figure 1. ALA alters the viability of breast and cervical cancer cell lines. All cell lines were treated with different doses of ALA
(0-320µM) for 24, 48 and 72 h. ALA alters the cell viability of MCF-7 (A) and MDAMB231 (B) breast cancer cells as well as
of SiHa (C) and HeLa (D) cervical cancer cells at 24, 48 and 72 h. All the data are presented as mean ± SEM of three independ-
ent experiments. p < 0.05 indicates statistically significant differences compared to the control untreated group.
stopped by cooling on ice for 10 min. The samples
were centrifuged at 10,000 rpm for 10 min and the su-
pernatant from each tube was transferred to a 96-well
plate. The optical density was measured at 540 nm us-
ing the ELISA plate reader (Bio-Rad, Hercules CA).
TBARs were calculated using 1, 1, 3, 3-
tetraethoxypropane (TMP) as a standard. The result-
ing TBAR values were normalized by the protein con-
centration of each sample that was estimated by the
Bradford reagent (Bio-Rad Laboratories Inc, CA,
USA).
Mitochondrial membrane potential
Both breast and cervical cancer cell lines were seeded
at a density of 1×105 cells/ml in a black 96-well plate
and incubated at 37oC in a CO2 incubator. The next
day, the cells were treated with different concentra-
tions of ALA (0-80 μM) and were incubated in a CO2
incubator at 37°C for 24 h. The following day, the me-
dium was removed and the cells were washed with 1X
PBS and incubated with 2.5 μg/ml JC-1 staining solu-
tion (Sigma-Aldrich, St. Louis, MO) for 1 h in the dark
(Wang et al. 2009). Fluorescence readings were meas-
ured using the Fluostar Omega microplate reader
(BMG Labtech) at 520 nm for JC-1 monomers and at
590 nm for JC-1 aggregates.
Immunoblotting
Cell extracts were prepared from controls as well as
cells treated with different concentrations of ALA (0-
80 μM). Briefly, the cell pellets were resuspended in
40 μl lysis buffer containing 50 mM Tris (pH 7.4), 5
mM EDTA, 0.5% NP40, 50 mM NaF, 1 mM DTT, 0.1
mM PMSF, 0.5μg/ml leupeptin (Pro-pure Amersco,
Solon, USA), 1 μg/ml pepstatin (Amresco, Solon,
USA), 150 mM NaCl, 0.5μg/ml aprotinin (Amersco,
Solon, USA), a protease inhibitor cocktail (Roche,
Lewes, UK) and incubated on ice for 1 h with intermit-
tent mixing. The extract was centrifuged for 20 min at
4°C at 12000 rpm. Protein concentration was estimated
9 Journal of Molecular Biochemistry, 2013
Figure 2. ALA reduces the nitric oxide release as well as iNOS expression in breast cancer cell lines. Cells were treated with
different concentrations of ALA (0-80 µM) for 24, 48 and 72 h and the nitric oxide release was measured in MCF7 (A) and
MDA-MB-231 (B) cells. ALA treatment reduces the iNOS expression at mRNA level in MCF-7 (C) and MDAMB231 (D)
cells. β-actin was used as the loading control. Densitometric analysis of iNOS expression is shown (E). Values are represented
as mean ± SEM of five independent experiments, each conducted in triplicates. *p < 0.05 indicates statistically significant differ-
ences compared to the untreated control cells.
using the Bradford reagent (Biorad Laboratories Inc,
CA, USA). Equal amounts of protein were loaded on a
10% SDS-polyacrylamide gel and transferred electro-
phoretically to an Amersham Hybond-P PVDF mem-
brane (GE Healthcare, UK) in sodium phosphate
buffer (pH 6.8). The membrane was blocked in 5%
BSA in TBST and incubated at room temperature for 1
h with rabbit polyclonal antibody for caspase 3 and
mouse monoclonal antibody for tubulin (Santacruz,
CA, USA) at 1:500 and 1:2000 dilutions, respectively.
The membrane was washed in TBST and incubated
with donkey anti-rabbit IgG HRP conjugate at 1:5000
(for caspase) and donkey antimouse IgG HRP conju-
gate at 1:3000 (for tubulin) dilutions. Proteins were
visualized using a chemiluminescence kit (Amersham
ECL Advance western blotting detection kit, GE
Healthcare, UK) and densitometric analysis of X-ray
films was performed on the Alpha Imager using the
Alpha Ease FC software, Alpha Innotech.
Statistical analysis
All experiments were performed in triplicates and re-
peated at least three times. The data are presented as
mean ± SD. Statistical analysis was conducted with the
Graph Pad 4 prism program using one-way ANOVA.
The p values used for comparisons were < 0.05.
Results
ALA alters the cell viability of breast and cervical
cancer cell lines
Omega 3 fatty acids including, ALA, have been known
to inhibit the growth of cancer cells (Das et al. 1998,
Horia & Watkins 2005, Kim et al. 2009, Sagar & Das,
1995). In the present report, we have treated breast
(MCF7 and MDA-MB-231) and cervical (SiHa and
HeLa) cancer cell lines with different concentrations (0
-320 μM) of ALA for 24, 48 and 72 h. It was observed
that in breast cancer cell lines, there was an apprecia-
Journal of Molecular Biochemistry, 2013 10
Figure 3. ALA reduces nitric oxide as well as iNOS levels in cervical cancer cell lines. The cells were treated with different
concentrations of ALA (0-80 µM) for 24, 48 and 72 h and the nitric oxide release was measured in SiHa (A) and HeLa (B) cells.
ALA treatment reduces the iNOS expression at mRNA level in SiHa (C) and HeLa (D). β-actin was used as the loading control.
Densitometric analysis of iNOS expression has been shown (E). Values are represented as mean ± SEM of five independent
experiments, each conducted in triplicates. *p < 0.05 indicates statistically significant differences compared to the untreated con-
trol cells.
ble decrease in cell survival (p<0.0001) above 80 µM
of ALA at 24 h (Figure 1A and B). However, in
MCF7 cells, the viability was found to significantly
decrease at 48 h post-ALA treatment at the relatively
lower dose of 20 μM, whereas at 72 h, the cell viability
was similar to that observed at 24 h (Figure 1A). On
the other hand, MDA-MB-231 cells showed a decrease
in viability above 80 μM at 48 h that was observed at
24 h as well. However, the percentage viability was
reduced at 48 h compared to that at 24 h. There was no
appreciable difference in MDA-MB-231 cells post-
ALA treatment at 72 h compared to the 24 h treatment
period (Figure 1B). These data suggest that both breast
cancer cell lines seem to be more sensitive to ALA
treatment at 48h.
In the cervical cancer cell line SiHa, ALA was
found to decrease the viability after a 160 µM dose at
24 h (p<0.0001). However, after incubating the cells
with ALA for 48 h and 72 h, the viability started de-
creasing at 20 μM and 40 μM, respectively (Figure
1C). In HeLa cells, ALA decreased the cell viability
after 40 and 160 μM at 24 and 48h, respectively. How-
ever, at 72h, there was a significant decrease in viabil-
ity at a lower dose (20 μM) (Figure 1D). These results
suggest that cervical cancer cells respond to ALA
treatment at higher exposure times (beyond 24h) at
lower concentrations. Interestingly, in HEK 293 cells
(non-cancerous), ALA did not show any toxicity up
until 320 μM, implying that it was specific only for the
cancerous cell lines (Figure 1S).
ALA decreases the nitric oxide levels in breast and
cervical cancer cell lines
Nitric oxide plays a dual role in cancer; it can either
promote or suppress it (Crowell et al. 2003, Lechner et
al. 2005). To test the effect of ALA on nitric oxide
levels, both the breast and cervical cancer cell lines
were treated with different concentrations of ALA (0-
80 μM) at different time intervals (24-72h). It was ob-
served that at 40 and 80 μM, ALA significantly re-
duced the levels of NO by ~3.0 and ~2.8-fold
(p<0.05), respectively, in MCF7 cells in 24h (Figure
2A). Similarly, in MDA-MB-231 cells, there was a
~3.4 and 3.6-fold (p<0.05) decrease in NO at 40 and
80 μM concentration of ALA, respectively, in 24h
(Figure 2B). On incubating the cells until 48 and 72h,
the trend in the decrease in NO was similar to that ob-
served at 24h, however, it was more significantly de-
creased in 24h of treatment. The intracellular nitric
oxide levels were also measured using the fluorescent
dye DAF-FM; both cell lines showed an appreciable
decrease (Figure 2Sa). The decrease in NO was further
confirmed by analyzing the effect of ALA on iNOS
expression at mRNA level that showed a significant
dose dependent decrease compared to the untreated
control cells in both MCF-7 (Figure 2C) and MDA-
MB-231 (Figure 2D) cell lines. Densitometric analysis
of iNOS expression was performed using the phos-
phorimager (Figure 2E) and normalized with respect to
β-actin as an internal control.
In the SiHa cervical cancer cell line, it was
observed that ALA significantly reduced the levels of
11 Journal of Molecular Biochemistry, 2013
Figure 4. ALA increases lipid peroxidation in breast and cervical cancer cells. All the cell lines were treated for 24 h. ALA in-
duced lipid peroxidation in breast (A) and cervical cancer (B) cells, as shown by TBARs assay. The TBAR values were normal-
ized by the protein concentration of each sample estimated by the Bradford reagent. Values are represented as mean ± SEM of
five independent experiments. *p<0.001 indicates statistically significant differences compared to the untreated control.
NO by ~2.6 and 2.7-fold (p<0.0001) at 40 and 80 μM,
respectively (Figure 3A). On the other hand, in HeLa
cells, the NO levels were reduced by ~1.7 and 1.8-fold
(p=0.0010) at 40 and 80 μM, respectively, in 24 h
(Figure 3B). In this case, after treating the cells with
ALA for 48 and 72h, the trend in the decrease in NO
was similar to that observed at 24h, however, it was
more significantly reduced after 24h of treatment. The
intracellular nitric oxide levels were measured by us-
ing the fluorescent dye DAF-FM; SiHa showed a sig-
nificant decrease in NO levels compared to HeLa
(Figure 2Sb). Moreover, the decrease in NO was sup-
ported by the corresponding decrease in iNOS expres-
sion at the mRNA level in a dose-dependent manner,
compared to the untreated control cells in both HeLa
(Figure 3D) and SiHa (Figure 3C) cells. Densitometric
analysis of iNOS expression was performed using the
phosphorimager (Figure 3E).
ALA increases lipid peroxidation in breast and cer-
vical cancer cell lines
Since ALA decreased the nitric oxide release in our
study and NO is known to either inhibit or promote
lipid peroxidation (Cauwels et al 2005, Miles et al.
1996), we analyzed the effect of ALA on lipid peroxi-
dation in both breast and cervical cancer cell lines. It
was observed that ALA increased lipid peroxidation at
all doses in both types of cancer cell lines; the increase
was more significant at 40 μM. At this dose, ALA in-
creased the lipid peroxidation by ~1.6 (p=0.0038) and
~2-fold (p=0.0002) in MCF-7 and MDA-MB-231, re-
spectively (Figure 4A). On the other hand, in SiHa
Journal of Molecular Biochemistry, 2013 12
Figure 5. ALA induces apoptosis in breast cancer cells. Breast cancer cells, MCF-7 and MDAMB231, (1 x 105cells/well) were
treated with ALA for 24 h. Decrease in mitochondrial membrane potential was analyzed by MARS data analysis software
2.10R3 (BMG Labtech) (A). All the data are presented as means ± SEM of three independent experiments. p < 0.05 indicate
statistically significant differences compared to the control untreated group. Caspase 3 (17/21 kDa) expression was determined
in ALA treated MCF-7 (B) and MDAMB231 (C) cell lines. The histogram depicts densitometric analysis of western blots of
caspase 3 (D). Values are represented as mean ± SEM of three independent experiments p<0.001 indicate statistically signifi-
cant differences compared to the untreated control cells.
cells, ALA showed a dose-dependent increase in lipid
peroxidation (LPO) wherein at 80 μM concentration, a
~2.6-fold increase in LPO was observed. However, in
HeLa cells, there was a dose-dependent increase in
LPO until 40 μM ALA treatment wherein there was a
~2.2-fold (p=0.0017) increase in lipid peroxidation
(Figure 4B).
ALA induces apoptosis in breast and cervical can-
cer cell lines
The loss of mitochondrial membrane potential
is the hallmark of apoptosis (Wang et al. 2009). Since
ALA decreased NO and increased LPO, we wanted to
analyze whether ALA induced apoptosis in both can-
cer cell types. Thus, we evaluated the effect of ALA on
the mitochondrial membrane potential in both breast
and cervical cancer cells. It was observed that it sig-
nificantly reduced (p<0.0001) the mitochondrial mem-
brane potential in breast cancer cell lines (MCF-7 and
MDA-MB-231) in a dose-dependent manner (Figure
5A). This was supported by a corresponding increase
in the expression of caspase 3 (17/21 kDa) in ALA-
treated MCF7 (Figure 5B) and MDA-MB-231 (Figure
5C) cells compared to the untreated control cells. Den-
sitometric analysis of caspase 3 expression was per-
formed by the phosphorimager (Figure 5D) and nor-
malized with respect to tubulin as an internal control.
On the other hand, in SiHa cells, there was a
dose-dependent decrease in the mitochondrial mem-
brane potential that correlated with a dose-dependent
increase in caspase 3 expression. In HeLa cells, the
decrease in mitochondrial membrane potential was
observed more significantly at 20 μM of ALA. How-
ever, there was a significant dose-dependent increase
in caspase 3 expression in HeLa suggesting that ALA
induced apoptosis in cervical cancer cells (Figure 6A).
Densitometric analysis of caspase 3 expression was
performed by the phosphorimager (Figure 6D).
All these results suggested that ALA induced
apoptosis in both breast and cervical cancer cell lines
through activation of caspase 3 and a decrease in the
mitochondrial membrane potential.
13 Journal of Molecular Biochemistry, 2013
Figure 6. ALA induces apoptosis in cervical cancer cells. ALA decreases the mitochondrial potential in the cervical cancer cell
lines SiHa and HeLa. The data was analyzed by the MARS data analysis software 2.10R3 (BMG Labtech). (A) All data are pre-
sented as means ± SEM of three independent experiments. p < 0.05 indicate statistically significant differences compared to the
control untreated group. Caspase 3 (17/21 kDa) expression was determined in ALA-treated SiHa (B) and HeLa (C) cell lines.
The histogram depicts densitometric analysis of western blots of caspase 3 (D). Values are represented as mean ± SEM of three
independent experiments. p<0.001 indicate statistically significant differences compared to the untreated control cells.
Discussion
Omega-3 (n-3) and omega-6 (n-6) polyunsatu-
rated fatty acids (PUFAs) are the essential fatty acids
that are important for human health. Various studies
suggest that the dietary fatty acids play an important
role in carcinogenesis wherein the n-3 fatty acids have
anti-carcinogenic potential and n-6 fatty acids are pro-
cancerous (Barceló-Coblijn & Murphy 2009, Sinclair
et al. 2002, Stark et al. 2008). Alpha-linolenic acid
(C18:3n-3, ALA), the most abundant n-3 PUFA, is an
essential fatty acid in the human diet and is present in
green leaves, oil, seeds (flaxseed, canola, perilla) and
nuts. It has been shown to reduce the growth of various
cancers including breast and cervical cancer (Horia &
Watkins 2005, Kim et al. 2009, Sagar & Das 1995).
Even though epidemiological studies showing a direct
correlationsip between ALA intake and cancer re-
sponse are limited (De Stefani et al. 1998, Franceschi
et al. 1996), there are studies showing association be-
tween low levels of ALA in adipose tissue of patients
with high risk of breast cancer (Klein et al. 2000).
There are reports suggesting that high ALA diets can
inhibit the growth of spontaneous or carcinogen in-
duced mammary tumors (Fritsche et al. 1990, Hirose
et al.1990, Kamano et al. 1989, Munoz et al. 1995).
The average intake of ALA in European countries,
USA and Canada has been shown to range between 0.8
and 2.2 g/d (Burdge & Calder 2005). In the UK the
intake of ALA has risen from a mean of 1.4 g/d in
19878 to 2.1 g/d according to the British Adult Diet
Survey (Henderson et al. 2004). Since, the conversion
of ALA into longer ω-3 PUFAs is generally considered
low, it has been shown that a moderate consumption of
walnuts (4 walnuts/day for 3 weeks) markedly in-
creases the blood levels of ALA and its metabolic de-
rivative, EPA.
In the current study, we have focused on ana-
lyzing the effect of ALA on modulation of growth of
breast and cervical cancer cells in terms of regulation
of nitric oxide and lipid peroxidation. ALA decreased
the nitric oxide levels in both cancer cell types. It is
well-known that high levels of NO have both
genotoxic and angiogenic properties (Nakamura et al.
2006). Increased NO production catalyzed by the
iNOS enzyme in tumor cells plays a critical role in
tumor angiogenesis, cancer progression and metastasis
(Narayanan et al. 2003). ALA has been reported to
decrease iNOS expression in the LPS-stimulated
macrophage cell line RAW 264.7 (Ren et al. 2007,
Udenigwe et al. 2009). Our results showed that ALA
not only decreased the iNOS expression at the mRNA
level but also reduced the intracellular levels of NO in
both the breast and cervical cancer cell lines. Thus, the
observed decrease in NO in both cancer cell lines by
ALA reinstates its antineoplastic potential.
PUFAs have been shown to initiate free radical
production and the generation of lipid peroxide prod-
ucts, selectively in tumor cells (Das 2002, Sun et al.
2012). Several studies with ALA have shown that it
increased lipid peroxidation in breast cancer cells
(Menéndez et al. 2001, Pardini 2006). Moreover, an
inverse correlation has been reported between lipid
peroxidation and cell proliferation (Das 2002). In line
with this, we found that ALA increased the lipid per-
oxidation with a simultaneous decrease in cell prolif-
eration in both breast and cervical cancer cell lines.
Recently, it was demonstrated that peroxidized prod-
ucts of n-3 PUFAs suppress iNOS induction and NO
production in a peroxidation-dependent manner (Araki
et al. 2011). Thus, the observed decrease in NO in both
types of cancer cell lines may be partly due to NO sup-
pression by peroxidized products of ALA. A strong
association between decreased NO levels and in-
creased lipid peroxidation has been reported in several
papers. For example, it was found that patients suffer-
ing from fibromyalagia had higher serum levels of
TBARS (particularly, malondialdehyde) and lower
levels of nitrite compared to the control groups
(Ozgocmen et al. 2006). Another report has shown that
a decrease in the level of NO in rats treated with al-
loxan-induced diabetes was associated with increased
levels of lipid peroxides (Mohan & Das, 2001). Thus,
increase in lipid peroxides may lead to increased free-
radical generation that may inactivate NO, resulting in
its low levels.
Mitochondria play an important role during
apoptosis (Wang et al. 2009). Reactive oxygen species
can directly activate the mitochondrial permeability
transition and result in loss of mitochondrial mem-
brane potential (ΔΨ), which results in the release of
cytochrome c (cyt c) and activation of the caspase
pathway (Cao et al. 2010, Kim et al. 2005, Lee et al.
2008, Sun et al. 2012). Our results showed that ALA
reduced nitric oxide levels and increased lipid peroxi-
dation in both breast and cervical cancer cells; this
may be responsible for the observed apoptosis (Figure
7). Conversely, increased nitric oxide has been re-
ported to inhibit lipid peroxidation by scavenging lipid
peroxyl radicals (Hogg & Kalyanaraman 1999). In-
creased NO has also been shown to prevent activation
of caspase 3 resulting in inhibition of apoptosis
(Maejima et al. 2005, Mahidhara et al. 2003, Kim et al
1997, Zhou et al. 2005). Thus, the increased LPO in
the presence of ALA leads to a decrease in NO result-
ing into disruption of the mitochondrial membrane
potential and activation of caspase 3 causing apoptosis
(Figure 7). Taken together, our data suggest that ALA
Journal of Molecular Biochemistry, 2013 14
regulates the growth of breast and cervical cancer cells
through regulation of lipid peroxidation as well as ni-
tric oxide generation that may lead to apoptosis.
Conclusion
Omega 3 fatty acids are known to exert anticancer ef-
fects through various mechanisms. One of them is
through the generation of free radicals while another is
through lipid peroxidation (Sun et al. 2012). Most of
the research work to date has analysed the significance
of EPA and DHA in cancer with very few data re-
ported on ALA. Our paper has tried to delineate the
anticancer properties of ALA in terms of its potential
to regulate lipid peroxidation as well as nitric oxide
generation that in turn result in the control of carcino-
genesis. However, more studies are required in the fu-
ture to elucidate the role of PUFAs in governing the
inter-relationship between the nitric oxide and lipid
peroxidation status of cells for the regulation of cancer
growth.
Acknowledgements
This work was supported by funding from the Interac-
tive Research School for Health Affairs (IRSHA),
Bharati Vidyapeeth University. We thank our Director,
Dr P. K. Ranjekar for supporting our work.
Competing interests
The authors declare no conflict of interest.
Author Contributions
RKG designed the study and drafted the manuscript.
RD has carried out the major experiments and contrib-
uted in manuscript writing. PM and SS have helped in
lipid peroxidation and RT-PCR experiments. All the
authors have read and approved the final version of the
manuscript.
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