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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

  • 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:
Alpha-linolenic acid regulates the growth of breast and cervical cancer
cell lines through regulation of NO release and induction of lipid
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.
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
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
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,
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.
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.
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.
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.
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
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
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... Our findings show downregulation of fatty acid biosynthesis following olaparib treatment, with a reduction in phospholipid levels, including lysophosphatidylcholines and glycerolphosphocholines, in all cell lines. Poly-unsaturated fatty acids (PUFAs) have previously been implicated in MCF7 and MDA-MB-231 cell apoptosis through the induction of lipid peroxidation and altered cellular redox state [55]. Moreover, elevated PUFA levels have been associated with the proteolytic cleavage of PARP and its inhibition, leading to cell death [56]. ...
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Histone deacetylase 8 (HDAC8) has emerged as a promising drug target for cancer therapeutics development. HDAC8 has been reported to regulate cancer cell proliferation, invasion and promote metastasis through modulation of cell cycle associated proteins. Of late, phytocompounds have been demonstrated to exhibit anticancer and anti-HDAC8 activity. Here, we have shown the HDAC8 inhibitory potential of an active phytocompound from HC9 (herbal composition-9), a polyherbal anticancer formulation based on the traditional Ayurvedic drug, Stanya Shodhan Kashaya. HC9 was recently reported to exhibit anticancer activity against breast cancer cells through induction of cell cycle arrest, decrease in migration and invasion as well as regulation of inflammation and chromatin modulators. In silico studies such as molecular docking, molecular dynamics (MD) simulation and binding free energy analyses showed greater binding energy values and interaction stability of MA with HDAC8 compared to other phytocompounds of HC9. Interestingly, in vitro validation confirmed the anti-HDAC8 activity of MA. Further, in vitro studies showed that MA significantly decreased the viability of breast and prostate cancer cell lines, thereby confirming its anticancer potential.
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Ethnopharmacological relevance Anethum sowa Roxb. ex Fleming (Syn. Peucedanum sowa Roxb. ex Fleming, Family: Apiaceae) is a pharmacologically important as aromatic and medicinal plant. Various parts of this plant are used in traditional medicine systems for carminative, uterine and colic pain, digestion disorder, flatulence in babies, appetite-stimulating agent and used to treat mild flue and cough. The essential oil is used for aromatherapy. It is also used as a spice for food flavouring and culinary preparations in many Asian and European countries. Aim of the review This review aims to provide a comprehensive and critical assessment from the reported traditional and pharmaceutical uses and pharmacological activities of the extracts, essential oil and phytoconstituents with emphasis on its therapeutic potential as well as toxicological evaluation of A. sowa. Materials and methods Online search engines such as SciFinder®, GoogleScholar®, ResearchGate®, Web of Science®, Scopus®, PubMed and additional data from books, proceedings and local prints were searched using relevant keywords and terminologies related to A. sowa for critical analyses. Results The literature studies demonstrated that A. sowa possesses several ethnopharmacological activities, including pharmaceutical prescriptions, traditional applications, and spice in food preparations. The phytochemical investigation conducted on crude extracts has been characterized and identified various classes of compounds, including coumarins, anthraquinone, terpenoids, alkaloid, benzodioxoles, phenolics, polyphenols, phenolic and polyphenols, fatty acids, phthalides and carotenoids. The extracts and compounds from the different parts of A. sowa showed diverse in vitro and in vivo biological activities including antioxidant, antiviral, antibacterial, analgesic and anti-inflammatory, Alzheimer associating neuromodulatory, cytotoxic, anticancer, antidiabetes, insecticidal and larvicidal. Conclusion A. sowa is a valuable medicinal plant which is especially used in food flavouring and culinary preparations. This review summarized the pertinent information on A. sowa and its traditional and culinary uses, as well as potential pharmacological properties of essential oils, extracts and isolated compounds. The traditional uses of A. sowa are supported by in vitro/vivo pharmacological studies; however, further investigation on A. sowa should be focused on isolation and identification of more active compounds and establish the links between the traditional uses and reported pharmacological activities with active compounds, as well as structure-activity relationship and in vivo mechanistic studies before integrated into the medicine. The toxicological report confirmed its safety. Nonetheless, pharmacokinetic evaluation tests to validate its bioavailability should be encouraged.
Zinc, ω-3 polyunsaturated fatty acids (PUFAs) and vitamin D are essential nutrients for health, maturation and general wellbeing. Extensive literature searches have revealed the widespread similarity in molecular biological properties of zinc, ω-3 PUFAs and vitamin D, and their similar anti-cancer properties, even though they have different modes of action. These three nutrients are separately essential for good health, especially in the aged. Zinc, ω-3 PUFAs and vitamin D are inexpensive and safe as they are fundamentally natural and have the properties of correcting and inhibiting undesirable actions without disturbing the normal functions of cells or their extracellular environment. This review of the anticancer properties of zinc, ω-3 PUFAs and vitamin D is made in the context of the hallmarks of cancer. The anticancer properties of zinc, ω-3 PUFAs and vitamin D can therefore be used beneficially through combined treatment or supplementation. It is proposed that sufficiency of zinc, ω-3 PUFAs and vitamin D is a necessary requirement during chemotherapy treatment and that clinical trials can have questionable integrity if this sufficiency is not checked and maintained during efficacy trials.
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Endothelial dysfunction may underline the pathobiology of human essential hypertension. Endothelial cells produce vasodilator and platelet anti-aggregator substances: prostacyclin (PG12) and nitric oxide (NO). It is possible that in hypertension there is an alteration in the levels of PG12 and NO and so also in the concentrations of oxy radicals and antioxidants, which can regulate the synthesis, release and action of PG12 and NO. Also, dietary factors such as sodium, potassium, calcium, magnesium, zinc, selenium, vitamin A,C, and E and essential fatty acids and their products such as eicosanoids can influence blood pressure, cardio- and cerebrovascular diseases and the concentrations of blood lipids and atherosclerosis. These observations suggest that there may possibly be a close interaction between these dietary factors, the metabolism of essential fatty acids, NO, and PG12 and the role of endothelium in human essential hypertension. These seemingly disparate factors may interact with each other to maintain the integrity of endothelium such that it can produce adequate amounts of NO and PG12 and other vasodilators to keep blood pressure within the normal range. Any deficiency in any one of these factors, either dietary or endogenous, or alterations in their interaction(s) among them, may lead to endothelial dysfunction and the development of hypertension. Based on these ideas, it is suggested that decreases in the activities of delta-6-desaturase and delta-5-desaturase, the rate limiting steps in the metabolism of essential fatty acids, may predispose to the development of insulin resistance. This suggestion may explain the high incidence of insulin resistance type II diabetes mellitus, lipid abnormalities and other features of syndrome X in South East Asians in whom EFA metabolism is defective. This hypothesis, if confirmed, could possibly pave the way for the development of newer therapeutic strategies in the treatment of hypertension, type II diabetes mellitus, hyperlipidaemias and their attendant complications.
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Eicosapentaenoic acid and docosahexaenoic acid (EPA/DHA), n-3 polyunsaturated fatty acids (PUFAs), have a variety of biological activities including anti-inflammatory and anticancer effects. We hypothesized that their peroxidized products contributed in part to anti-inflammatory effects. In the liver, the production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been implicated as one of the factors in hepatic inflammation and injury. We examined whether the peroxidation of EPA/DHA influences the induction of iNOS and NO production in proinflammatory cytokine-stimulated cultured hepatocytes, which is in vitro liver inflammation model. Peroxidized EPA/DHA inhibited the induction of iNOS and NO production in parallel with the increased levels of their peroxidation, whereas unoxidized EPA/DHA had no effects at all. Peroxidized EPA/DHA reduced the activation of transcription factor, NF-κB, and the expression of the iNOS antisense transcript, which are involved in iNOS promoter transactivation (mRNA synthesis) and its mRNA stabilization, respectively. These findings demonstrated that peroxidized products of EPA/DHA suppressed the induction of iNOS gene expression through both of the transcriptional and posttranscriptional steps, leading to the prevention of hepatic inflammation.
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Alpha-linolenic acid (ALA) reduces cardiovascular disease (CVD) risk, possibly by favorably changing vascular inflammation and endothelial dysfunction. Inflammatory markers and lipids and lipoproteins were assessed in hypercholesterolemic subjects (n = 23) fed 2 diets low in saturated fat and cholesterol, and high in PUFA varying in ALA (ALA Diet) and linoleic acid (LA Diet) compared with an average American diet (AAD). The ALA Diet provided 17% energy from PUFA (10.5% LA; 6.5% ALA); the LA Diet provided 16.4% energy from PUFA (12.6% LA; 3.6% ALA); and the AAD provided 8.7% energy from PUFA (7.7% LA; 0.8% ALA). The ALA Diet decreased C-reactive protein (CRP, P < 0.01), whereas the LA Diet tended to decrease CRP (P = 0.08). Although the 2 high-PUFA diets similarly decreased intercellular cell adhesion molecule-1 vs. AAD (-19.1% by the ALA Diet, P < 0.01; -11.0% by the LA Diet, P < 0.01), the ALA Diet decreased vascular cell adhesion molecule-1 (VCAM-1, -15.6% vs. -3.1%, P < 0.01) and E-selectin (-14.6% vs. -8.1%, P < 0.01) more than the LA Diet. Changes in CRP and VCAM-1 were inversely associated with changes in serum eicosapentaenoic acid (EPA) (r = -0.496, P = 0.016; r = -0.418, P = 0.047), or EPA plus docosapentaenoic acid (r = -0.409, P = 0.053; r = -0.357, P = 0.091) after subjects consumed the ALA Diet. The 2 high-PUFA diets decreased serum total cholesterol, LDL cholesterol and triglycerides similarly (P < 0.05); the ALA Diet decreased HDL cholesterol and apolipoprotein AI compared with the AAD (P < 0.05). ALA appears to decrease CVD risk by inhibiting vascular inflammation and endothelial activation beyond its lipid-lowering effects.
A protein isolate was produced from cellulase-treated defatted flaxseed meal followed by hydrolysis with seven proteases and evaluation of the hydrolysates for antioxidant and anti-inflammatory properties. The flaxseed protein hydrolysates (FPH) were processed by ultrafiltration and ion-exchange chromatography to isolate low molecular weight (LMW) and cationic peptide fractions, respectively. The peptides showed antioxidant properties in scavenging 2,2-diphenyl-1-picrylhydrazyl radical, superoxide anion radical, electron-spin resonance-detected hydroxyl radical and nitric oxide. In addition, all peptide fractions inhibited semicarbazide-sensitive amine oxidase activity. Antioxidant activities of these peptides were dependent on the specificity of proteases and size of the resulting peptides. The LMW fractions from pepsin, ficin and papain FPH also inhibited lipopolysaccharide-induced nitric oxide productions in RAW 264.7 macrophages without apparent cytotoxicity; thus, these peptides may act as anti-inflammatory agents. Thus, flaxseed protein hydrolysates may serve as potential ingredients for the formulation of therapeutic products.
A thorough comparative analysis of cytotoxic effect of an aqueous cinnamon extract (ACE) from the bark of Cinnamomum zeylanicum L. (Lauraceae) with that of commercially available cinnamaldehyde was performed on various human as well as mouse cell lines and primary cells. The aqueous cinnamon extract (ACE) proved to be more cytotoxic to cancerous cells at concentrations just above 0.16 mg/mL (containing 1.28 μM cinnamaldehyde) around which the commercial cinnamaldehyde (1.6 μM) had no cytotoxic effect. At a critical concentration of 1.28 mg/mL (containing 10.24 μM cinnamaldehyde), ACE treatment resulted in 35-85% growth inhibition of the majority of the cancerous cells, whereas at a similar concentration (10 μM) commercial cinnamaldehyde treatment resulted in 30% growth inhibition of only SK-N-MC cells with no effect on other cell lines. These results suggest that ACE had a significant inhibitory effect on the majority of cancer cells and thus may prove to be a chemotherapeutic agent.
Beta-eleostearic acid (β-ESA, 9E11E13E-18:3), a linolenic acid isomer with a conjugated triene system, is a natural and biologically active compound. Herein, we investigated effects of β-eleostearic acid on T24 human bladder cancer cells. In this study, results showed that β-eleostearic acid had strong cytotoxicity to induce cell apoptosis, which was mediated by reactive oxygen species (ROS) in T24 cells. The cell viability assay results showed that incubation with β-eleostearic acid concentrations of 10-80μmol/L caused a dose- and time-dependent decrease of T24 cell viability, and the IC(50) value was 21.2μmol/L at 24h and 13.1μmol/L at 48h. Annexin V/PI double staining was used to assess apoptosis with flow cytometry. Treatment with β-eleostearic acid caused massive ROS accumulation and GSH decrease, which lead to activation of caspase-3 and down-regulation of Bcl-2 indicating induction of apoptosis. Subsequently, N-acetyl-l-cysteine (NAC) and PEG-catalase effectively blocked the ROS elevated effect of β-eleostearic acid, which suggested that β-eleostearic acid-induced apoptosis involved ROS generated. Additionally, we found that treating T24 cells with β-eleostearic acid induced activation of PPARγ. A PPARγ-activated protein kinase inhibitor was able to partially abrogate the effects of β-eleostearic acid. These results suggested that β-eleostearic acid can induce T24 cells apoptosis via a ROS-mediated pathway which may be involved PPARγ activation.
Lifestyle and nutritional factors have been recognized to influence breast cancer survival, irrespective of genomic alterations that are the hallmarks of the disease. The biological and molecular mechanisms involved in the effects of dietary polyunsaturated fatty acids and breast cancer response to treatments in clinical and preclinical studies have been reviewed. Among nutrients, rumenic acid, a naturally occurring CLA isomer and n-3 docosahexaenoic acid (DHA) a highly unsaturated fatty acid, have emerged due to their potential to increase cancer treatment efficacy without additional side effects. In this review, we analyze the literature evidence that breast cancer treatment and outcome could be improved through an adjuvant dietary supplementation. Such an original approach would involve two successive phases of breast cancer treatment: an initial sensitization of residual tumor cells to chemotherapy and to radiation therapy with dietary DHA; then a prevention of metastatic re-growth with a prolonged rumenic acid supplementation. Safety is not anticipated to be a critical issue, although it has to be assessed in the long term. Dietary supplements, used in combination to anti-cancer agents, should be provided under medical prescription. Such an original use of fatty acids in breast cancer treatment could provide the lipid field with a new avenue to impact public health.
There is little doubt regarding the essential nature of alpha-linolenic acid (ALA), yet the capacity of dietary ALA to maintain adequate tissue levels of long chain n−3 fatty acids remains quite controversial. This simple point remains highly debated despite evidence that removal of dietary ALA promotes n−3 fatty acid inadequacy, including that of docosahexaenoic acid (DHA), and that many experiments demonstrate that dietary inclusion of ALA raises n−3 tissue fatty acid content, including DHA. Herein we propose, based upon our previous work and that of others, that ALA is elongated and desaturated in a tissue-dependent manner. One important concept is to recognize that ALA, like many other fatty acids, rapidly undergoes β-oxidation and that the carbons are conserved and reused for synthesis of other products including cholesterol and fatty acids. This process and the differences between utilization of dietary DHA or liver-derived DHA as compared to ALA have led to the dogma that ALA is not a useful fatty acid for maintaining tissue long chain n−3 fatty acids, including DHA. Herein, we propose that indeed dietary ALA is a crucial dietary source of n−3 fatty acids and its dietary inclusion is critical for maintaining tissue long chain n−3 levels.
Baicalein was investigated for tumor cell-specific cytotoxicity, apoptosis-inducing activity and signal pathway against the MDA-MB-231 human breast cancer cell line. After the MDA-MB-231 cells had been treated with baicalein, trypan blue exclusion, propidium iodide (PI) assay and 4',6-diamidino-2-phenylindole (DAPI) were used to stain the dead cells and detect apoptosis, respectively. The effects of baicalein on the levels of reactive oxygen species (ROS), Ca2+ and mitochondrial membrane potential (deltapsim) on MDA-MB-231 cells were examined by flow cytometric assays. The ROS caused endoplasmic reticulum (ER) stress, confirmed by the increase of GADD153 and GRP78 in the examined cells. GADD153 and GRP78 increases were also confirmed by confocal laser microscopy examination and indicated that both proteins translocated to the nucleus. The effects of baicalein on the expression of apoptotic-regulated genes, such as Bcl-2 family and caspase, were detected by Western blotting. To further investigate the apoptotic pathway and the role of Ca2+ induced by baicalein, a caspase-3 inhibitor and Ca2+ chelator were used to block caspase-3 activity and Ca2+ in MDA-MB-231 cells. Baicalein induced apoptosis in a time-dependent effect through the inhibition of Bcl-2 expression, increased the levels of Bax, reduced the level of deltapsim, and promoted the cytochrome c release and caspase-3 activation. MDA-MB-231 cells were pretreated with BAPTA which reduced the levels of Ca2+, deltapsim and apoptosis. In conclusion, baicalein induced apoptosis via Ca2+ production, mitochondria-dependent and caspase-3 activation in MDA-MB-231 cells.
Consumption of omega 3 fatty acids is known to have health benefits. For many years, the importance of the only member of the omega 3 family considered to be essential, alpha-linolenic acid (ALA), has been overlooked. Current research indicates that ALA, along with its longer chain metabolites, may play an important role in many physiological functions. Potential benefits of ALA include cardioprotective effects, modulation of the inflammatory response, and a positive impact on both central nervous system function and behavior. Recommended levels for ALA intake have been set, yet the possible advantages of its consumption are just being revealed.