High Metastaticgastric and Breast Cancer Cells Consume Oleic Acid in an AMPK Dependent Manner
et al. (2014) High Metastaticgastric and Breast Cancer Cells Consume Oleic Acid in an AMPK Dependent Manner. PLoS
ONE 9(5): e97330. doi:10.1371/journal.pone.0097330
High Metastaticgastric and Breast Cancer Cells Consume Oleic Acid in an AMPK Dependent Manner
Shuai Li 0
Ti Zhou 0
Cen Li 0
Zhiyu Dai 0
Di Che 0
Yachao Yao 0
Lei Li 0
Jianxing Ma 0
Xia Yang 0
Guoquan Gao 0
Arun Rishi, Wayne State University, United States of America
0 1 Department of Biochemistry, Zhongshan School of Medicine, SunYat-sen University , Guangzhou , China , 2 Department of Physiology, University of Oklahoma, Health Sciences Center, Oklahoma City, Oklahoma, United States of America, 3 Key Laboratory of Functional Molecules from Marine Microorganisms (Sun Yat-sen University), Department of Education of Guangdong Province , Guangzhou , China , 4 China Key Laboratory of Tropical Disease Control (SunYat-sen University), Ministry of Education , Guangzhou , China
Gastric cancer and breast cancer have a clear tendency toward metastasis and invasion to the microenvironment predominantly composed of adipocytes. Oleic acid is an abundant monounsaturated fatty acid that releases from adipocytes and impinges on different energy metabolism responses. The effect and underlying mechanisms of oleic acid on highly metastatic cancer cells are not completely understood. We reported that AMP-activated protein kinase (AMPK) was obviously activated in highly aggressive carcinoma cell lines treated by oleic acid, including gastric carcinoma HGC-27 and breast carcinoma MDA-MB-231 cell lines. AMPK enhanced the rates of fatty acid oxidation and ATP production and thus significantly promoted cancer growth and migration under serum deprivation. Inactivation of AMPK attenuated these activities of oleic acid. Oleic acid inhibited cancer cell growth and survival in low metastatic carcinoma cells, such as gastric carcinoma SGC7901 and breast carcinoma MCF-7 cell lines. Pharmacological activation of AMPK rescued the cell viability by maintained ATP levels by increasing fatty acid b-oxidation. These results indicate that highly metastatic carcinoma cells could consume oleic acid to maintain malignancy in an AMPK-dependent manner. Our findings demonstrate the important contribution of fatty acid oxidation to cancer cell function.
. These authors contributed equally to this work.
Epidemiological and animal studies have demonstrated an
association between fatty acids (FA) or obesity and the cancer
tumourigenesis and metastasis [1,2]. Advanced gastric cancer and
breast cancer have a clear tendency towards metastasis and
invasion to the microenvironment, which is predominantly
composed of adipocytes [3,4]. Oleic acid is the most common
monounsaturated FA in human adipocytes and other tissues .
Relatively little is known regarding whether highly metastatic
gastric and breast cancer cells could adapt to the highly fatty acid
culture and gain a survival/growth advantage by metabolic
transformation to utilise oleic acid as an energy source.
Studies from recent decades have reported accumulating
evidence of metabolic reorganisation during cancer development
in various tumour types . One of the first biochemical hallmarks
of cancer cells to be identified were the marked changes in
metabolism . Tumour cells gain a survival/growth advantage
by adapting their metabolism to respond to environmental stress, a
process known as metabolic transformation. The best-known
aspect of metabolic transformation is the Warburg effect .
Recently, several lines of evidence implicate fatty acid oxidation
(FAO) as an important contributor to metabolic transformation
, indicating that fatty acid metabolism might contribute to
cancer cell function. With most cancer researchers focusing on
glycolysis, glutaminolysis and fatty acid synthesis, the relevance of
fatty acid oxidation (FAO) to cancer cell function has not been
carefully examined. In particular, little is known regarding the
biochemical pathways by which oleic acid influences tumour
One of the fundamental requirements of all cells is the
balancing of ATP consumption and generation . Tumour
cells typically undergo metabolic transformation modulated by
AMPK . AMPK is a highly conserved sensor of cellular
energy status that exists in the form of heterotrimeric complexes
containing a catalytic a-subunit combined with regulatory b and
c-subunits [21,22]. In response to energy depletion, AMPK
activation promotes metabolic changes to maintain cell
proliferation and survival by directly phosphorylating rate-limiting
enzymes in metabolic pathways, modifying the signal transduction
cascades and gene expression . FAO induction downstream
from AMPK activation might be a survival or growth strategy
employed by some cancer cells subjected to metabolic stress .
It has been reported recently that omental adipocytes promote
homing, migration and invasion of ovarian cancer cells .
Adipocyte-ovarian cancer cell coculture led to the direct transfer of
lipids from adipocytes to ovarian cancer cells and promoted
tumour growth, suggesting that there is a link in cancer cells
between the adaptation to consume exogenous energy and the
ability to migrate. It is unknown whether oleic acid provides an
energy source for other highly metastatic carcinoma cells; if so,
there is a question regarding the basis of molecular mechanism.
It is essential to elucidate the molecular mechanisms by which
oleic acid regulates the malignant behaviour of high metastatic
cancer cells. To clarify the unknown problems, we based our work
on the premise that assessing the influence and mechanism of oleic
acid on cancer cells would provide a better understanding of fatty
acid metabolism and the molecular mechanisms present in gastric
and breast cancer cells.
Materials and Methods
Fatty acid-free BSA was obtained from Wako Pure Chemical
Industries, Ltd., and oleic acid, AICAR and Compound C were
purchased from Sigma.
Cancer Cell Lines
The HGC-27, AGS, SGC7901, BGC823 human cancer cell
lines were obtained from the Cell Bank of the Chinese Academy of
Science. The MDA-MB-231 and MCF-7 cells were obtained from
the American Type Culture Collection (ATCC). The MCF-7 and
MDA-MB-231 cells were maintained in DMEM supplemented
with 10% heat-inactivated foetal calf serum (FCS), and the
HGC27, AGS, SGC7901 and BGC823 cells were maintained in RPMI
1640 supplemented with 10% FCS. The cells were grown in
monolayer cultures at 37uC in a humidified atmosphere of 95% air
and 5% CO2. When BSA-bound fatty acids were added to the
serum-free culture medium, the final concentration of BSA was
adjusted to 0.5%.
Cell Viability Assay
The cells in 48-well plates at a density of 10,000 cells per well
were treated with different stressors. The cell viability was
measured using the 3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyl
tetrazolium bromide (MTT) assay (Roche Co.), according to the
BrdU Incorporation Assay
The cell proliferation was determined by measuring the BrdU
incorporation using a BrdU incorporation assay (Roche Molecular
Biochemicals), according to the manufacturers instructions.
Briefly, 5,000 cells/well seeded in a 96-well plate were
pulselabelled for 2 h with 10-uM BrdU. The cells were incubated for
30 min with a diluted, peroxidase-conjugated anti-BrdU antibody.
The absorbance values were measured at 450 nm using an ELISA
reader (Bio-Rad iMark).
The cell migration assays were performed using Transwell
chambers (8-mm pore size polycarbonate membrane, Corning). A
total of 50 K cells were plated into the insert in 200 ml serum-free
medium containing BSA or 400 mM BSA-bound oleic acid and
allowed to migrate from the upper compartment to the lower
compartment toward a 15% FBS gradient for the indicated time
period. After the migration, the non-migratory cells on the upper
membrane surface were removed by scrubbing, and the
membrane was fixed in buffered 4% paraformaldehyde and
stained with 0.1% crystal violet at room temperature. The
migrated cells were then enumerated. The migration values were
expressed as the average number of migrated cells per microscopic
field over six fields per assay from three independent membrane
The cells were harvested and lysed for the total protein
extraction. The protein concentration was determined using a
BioRad DC protein assay kit (Bio-Rad Laboratories) according to the
manufacturers protocol. The aliquots of equal amounts of protein
from the cell lysate were subjected to western blot analysis. The
antibodies used in this study include those specific for
phosphoThr172-AMPKa (Cell Signaling Technology, Danvers, MA,
#4188s), AMPKa (CST, #2532s), phospho-Ser96-ACC (CST,
#3661S), ACC (CST, #3676s), ATGL (CST, #2138S), caspase9
(CST,#9502s), caspase3 (CST, #9662s), CPT1a (Proteintech
group, 15184-1-AP), MCAD (Proteintech group, 55210-1-AP),
GAPDH(Santa Cruz, sc-365062) and b-actin (Sigma,MO,
USA,A5441). The densitometry was performed using ImageJ
software (developed by Wayne Rasband, National Institutes of
Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/
index.html) and normalised by the GAPDH or b-actin levels.
The AMPKa1 siRNA and a nonspecific siRNA (control) were
purchased from RiboBio. According to the manufacturers
instructions, the transfections were performed at approximately
60% confluency using Lipofectamine 2000 (Invitrogen). For each
transfection reaction, 20 nM AMPKa1 siRNA or control siRNA
was used for the preparation of the siRNA-transfection complexes
at room temperature for 20 min. The transfections were
performed in 0.5-(12-well plate) or 1.5-mL (6-well plate)
serumfree medium for 8 hr. After incubation, the transfection complexes
were removed and replaced with their corresponding media. The
transfection efficiency (80% 90%) was determined by western
blotting analysis. The cells were used for western blotting analysis
and cell viability 2472 hr after transfection.
Oil Red O Staining and Triglyceride Assay
The lipid droplets were stained by Oil red O. A stock solution
was prepared in 2-propanol (0.3%), and a working solution was
freshly prepared by diluting the stock solution with water (3:2).
After fixation, the cells were washed twice in PBS and stained with
Oil red O and hematoxylin for 15 min and 1 min, respectively.
The cells were washed with PBS, and images were obtained under
a light contrast microscope. The intracellular triglycerides were
assayed using a triglyceride assay kit (GPO-POD; Applygen
Technologies, Inc., Beijing, China) according to the
manufacturers recommended protocol.
Determination of Oxygen Consumption
Five million cells were resuspended in 1 ml of fresh warm
medium pre-equilibrated with 21% oxygen and placed in a sealed
respiration chamber equipped with a thermostat control, micro
stirring device and Clark-type oxygen electrode disc (Oxytherm,
Hansatech Instrument, Cambridge, UK). The oxygen content in
the cell suspension medium was constantly monitored for 10 min,
and the oxygen consumption rate was recorded.
The intracellular level of ATP was measured with an ATPlite
assay kit (Beyotime Biotech). The cells were incubated in a
serumfree medium with or without 400 mM of OA for 48 h, washed with
PBS three times, lysed in ATP extraction buffer and centrifuged in
4uC. ATP was measured by luminometric methods using
commercially available luciferin/luciferase reagents on a
luminometer (TD-20/20; Turner Designs) according to the
manufacturers instructions. The data were normalised to total protein.
The data are presented as the means 6 SD. SPSS 13.0 software
was used for the statistical evaluation using one-way ANOVA for
comparison of more than two groups, and LSD-t test, Dunnetts
T3 or Tamhanes T2 were used for the multiple comparisons.
Students t test was used for the comparison of the two groups. P
values of less than 0.05 were considered significant.
The Opposite Effects of Oleic Acid on the Cell Viability of
Various Cancer Cell Lines
To better clarify the role of oleic acid in tumour growth, we
examined the effects of oleic acid on the cell viability of human
gastric carcinoma cell lines and breast cancer cell lines,
respectively. As shown in Fig. 1A, 400 mM of BSA-bound oleic
acid stimulated obvious cell viability in HGC-27 cells after 24, 48,
and 72 hours of exposure. A decrease in cell viability was observed
in the AGS, SGC7901 and BGC823 cells treated with OA. In the
MDA-MB-231 cells, OA had an effect on cell viability similar to
that of the HGC-27 cells, more profoundly with the 72-hours
treatment. We observed an inhibitory effect of OA on cell viability
in the MCF-7 cells (Fig. 1B).
For a better assessment of the reason for the cell viability change
induced by OA, we first determined the cell proliferation using
BrdU ELISA. The assessment shows that cell proliferation was
significantly higher in the OA groups compared with the control
group in the HGC-27 and MDA-MB-231 cells; OA exhibited a
slight inhibition of cell proliferation in the SGC7901 and MCF-7
cells (Fig. 1C). The Hoechst staining and flow cytometry analysis
indicated that OA afforded protection against apoptosis in the
HGC-27 and MDA-MB-231 cells whereas it induced apoptosis in
the SGC7901 cells and MCF-7 cells. Mechanically, the level of
cleaved caspase3 and cleaved caspase9 were changed in the
OAtreated cells compared to that in the control group, suggesting that
mitochondrial apoptotic pathway might be involved in this
progress (Figure S1).
Different Effects of Oleic Acid on Cell Migration
To further determine the action of OA on cell migration, we
used Transwell chamber assays to gauge the migratory ability of
the cells treated with OA. The cells were plated into the insert in a
serum-free medium containing BSA or 400 mM of BSA-bound
oleic acid and allowed to migrate from the upper compartment to
the lower compartment toward a 15% FBS gradient. OA
prompted an obvious increase in the migration of the HGC-27
and MDA-MB-231 cells, whereas this process was reduced in the
SGC7901 cells (Figure 2).
Effects of Oleic Acid on the Activation of AMPK Signalling
and Downstream Enzymes
Exogenous OA transported into the cells is converted into
acylCoA, which is esterified with glycerol to yield inert triacylglycerols
(TGs) or b-oxidation [25,26].
Based on these findings, we hypothesised that the disparity in
the cellular biological function treated by OA between the high
metastatic cancer cells (HGC-27, and MDA-MB-231) and low
metastatic cancer cells (SGC7901, MCF-7) is ascribed to the
difference in the ability of intracellular TG lipolysis. We detected
the expression of adipose triglyceride lipase (ATGL), which
catalyses the initial step of lipolysis, converting TGs to
diacylglycerols (DGs). ATGL was completely stimulated in all the cells
treated by OA compared to the control cells (Fig. 3A),
demonstrating that lipolysis was not the distinction separating the high
metastatic cancer cells from the low metastatic cancer cells.
AMPK, a fuel sensor, plays an important role in regulating fatty
acid b-oxidation and is involved in the different effects OA has on
cell function. We performed a comprehensive analysis of the
expression of AMPK and a downstream gene by western blotting
in various cancer cells. Phosphorylation of AMPKa was selectively
up regulated by OA in the HGC-27 and MDA-MB-231 cells, and
there was no obvious change in the SGC7901 and MCF-7 cells
(Fig. 3A & B).
Carnitine palmitoyl transferase 1(CPT1a), which catalyses the
transport of long-chain fatty acids (LCFAs) into mitochondria for
b-oxidation, is highly inhibited by the malonyl CoA that is
catalysed in mitochondria by acetyl-CoA carboxylase (ACC).
AMPK could phosphorylate and inactivate ACC, thus the
ACCdependent malonyl CoA levels fall, releasing the inhibition of
CPT1a, which facilitates the mitochondrial entry of the LCFAs for
b-oxidation [21,27]. As shown in Fig. 3A, OA significantly
increased the phosphorylation of ACC in the HGC-27 cells and
mildly affected the ACC in the MDA-MB-231 cells. The
expression of CPT1a was up regulated consistently by OA in
those two cell lines (Fig. 3A & B).
Consistently, the oxygen consumption rate of the
MDA-MB231 cells in the medium containing OA was significantly higher
compared to that of the cells grown in the control medium. The
oxygen consumption rate of the MCF-7 cells grown in the medium
containing OA did not differ significantly from that of the control
cells (Figure S2). These results suggested that AMPK activation
participated in the utilisation of OA through b-oxidation and
maintained a metabolic advantage.
The Different Actions of OA on Cellular Behaviour are
To further confirm that AMPK activation was involved in the
utilisation of OA for growth, survival and migration in the cancer
cell lines, 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside
(AICAR) and Compound C were used. As shown in Figure 4A,
the addition of Compound C to the HGC-27 and MDA-MB-231
cells suppressed the facilitation of growth by OA. The treatment
with AICAR restrained the growth inhibition by OA in the
SGC7901 and MCF-7 cells.
We designed siRNA specific to the AMPK catalytic a1-subunit
to reduce the protein levels because AMPK is activated by
phosphorylation of Thr172 on the catalytic a-subunit. The
western blot assay confirmed an obvious reduction in the protein
level in the MAD-MB-231 cells (Figure S3). As depicted in
Figure 4B, consistent with the exposure to Compound C, silencing
of the AMPK catalytic a-subunit by treatment with OA caused a
reduction of cell viability after 72 hours.
There was a question regarding whether the alteration of cell
proliferation and migration relied on AMPK activation in the cells
treated by OA. As shown in Figures 4 C&D, Compound C elicited
a 1.0 to 1.5-fold decrease in cell proliferation and migration in the
OA-treated HGC-27 and MAD-MB-231 cells, whereas the
reduction of cell proliferation and migration in the OA-treated
SGC7901 and MCF-7 cells was compensated for by AICAR.
Collectively, these data suggested that AMPK activation is critical
for the cancer cell survival and growth induced by OA.
Figure 1. Effects of oleic acid on cell survival in various human cancer cell lines. Human gastric cancer cell lines (A) and breast cancer cell
lines (B) were starved for 12 hours and then incubated with 0.5% BSA as the control or 400 mM BSA-bound oleic acid for the indicated time period
(hours). The cell viability was determined by an MTT assay and expressed as a percentage of the control cells. The values are the mean 6 SD, n = 3;
**p,0.01 vs. BSA at the indicated time points. Students t test. (C) The cells were incubated with 0.5% BSA or 400-mM BSA-bound oleic acid for the
indicated time points, and the cell proliferation was examined using BrdU ELISA. The values are the mean 6 SD, n = 3; *p,0.05 and **p,0.01 for OA
compared with BSA.Students t test.
The Activation of AMPK is a Key Factor to Utilising OA
and Providing ATP for Cancer Cell Growth and Survival
We assessed whether AMPK has a function in regulating the
lipid accumulation induced by OA. Consistent with other
observations , the lipid droplet material stained by Oil red
O increased in the OA-treated MDA-MB-231 (Fig. 5A), HGC-27
and SGC7901 cells compared to the control cells (Figure S4),
suggesting that cancer cells are able to esterify oleate for storage as
TG. The lipid droplets in the OA-treated MDA-MB-231 cells
were diffused with small granules, indicating that the TG stored in
lipid droplets was simultaneously hydrolysed and provided with
energy. Consistent with the Oil red O staining, the cellular
triglyceride content assay indicated that OA induced TG
accumulation significantly for the initial 6 hours, which was
consumed during the next 30 hours in incubation with fresh
medium in the absence of OA (OA+Con). Compound C
attenuated the TG consumption compared to the control group
(OA+Con), as shown in Figure 5B. This finding demonstrated that
the inhibition of AMPK signalling impeded the utilisation of OA
and fatty acid metabolism in an obvious manner.
To further elucidate the mitochondrial respiratory chain
activity, we measured the oxygen consumption rate in the cells
with different treatments. This result indicated that OA stimulated
the oxygen consumption whereas Compound C reduced the
oxygen consumption in the MDA-MB-231 cells, suggesting that
AMPK might participate in the intracellular fatty acids flux into
mitochondria for b-oxidation (Fig. 5 C&D).
We determined whether AMPK activation is directly
attributable to the ATP production influenced by the OA stress in our cell
models. The intracellular levels of ATP in the various treated
groups of different cell lines were measured (Fig. 5E). As shown in
figure 5E, the inhibition of the AMPK signalling by Compound C
attenuated the increase of ATP production in the HGC-27 and
MDA-MB-231 cells treated with OA, whereas the AICAR
compensated for the ATP level by activating the AMPK signalling
in the SGC7901 and MCF-7 cells.
These data support our hypothesis that the activation state of
AMPK is a switch in the cells to respond to OA by mediating fatty
AMPK Regulated the Key Enzymes Involved in
boxidation in OA- treated-high Metastatic Cancer Cells
To understand the accurate molecular mechanism by which
AMPK activation determined the effects of OA on cellular
function, we examined the status of AMPK activation and the
expression of related enzymes regarding the FFA metabolism. It
was parallel to the data shown in Figure 3 and demonstrated that
OA activated the AMPK signalling by up-regulating the
Figure 2. Effects of oleic acid on cell migration of gastric carcinoma cells and breast cancer cells. Cells treated with 0.5% BSA or 400 mM
BSA-bound oleic acid were allowed to migrate from upper compartment to lower compartment toward a 15% FBS gradient for 12 hours in HGC-27
and SGC7901 cells and 6 hours in MDA-MB-231 cells. (A) After then the migrated cells were fixed, stained, and photographed (magnification 100 x)
and (B) the migrated cells were then enumerated and normalised to the control, #p,0.05 for OA compared with BSA.Students t test.
phosphorylation of AMPKa and ACC. This up regulation is
accompanied by an increase in the expression of CPT1a, whereas
this induction was obviously attenuated by Compound C in the
HGC-27 and MDA-MB-231 cells. Apparently, AMPK was not
stimulated in the MCF-7 or SGC7901 cells in the presence of OA.
When AICAR was added to the medium, the level of the
phosphorylated AMPKa and ACCwas increased, as well as the
CPT1a and MCAD levels, which catalysed crucial steps in the
mitochondrial fatty acid oxidation (Fig. 6A). To confirm further
the implication of the AMPK signalling in the OA action, we
transfected the AMPKa1-siRNA in the HGC-27 and
MAD-MB231 cells, and then treated the cells with OA or BSA, as the
control. As shown in Figure 6B, silencing the AMPKa caused a
reduction in the expression of phosphorylated AMPKa and ACC
with CPT1a and MCAD.
Although the expression of ATGL was completely stimulated in
all the cells treated by OA compared to the control cells (Fig. 3A),
it was not influenced by the AMPK status (Fig. 6A, 6B), suggesting
that ATGL, at least at the protein level, was not regulated by
AMPK in the conditions of the OA treatment.
These data suggested that AMPK is critical for OA stimulation
of cancer cell survival and growth through the promotion of fatty
The data presented in this study demonstrated that oleic acid
selectively promotes cell proliferation and migration in high
metastatic cancer cells whereas it has inhibitory effects in low
metastatic cancer cells. Although for decades it has been well
accepted that AMPK activation suppresses cell growth and
proliferation [29,30], new evidence that AMPK activation
promotes certain human cancer cell growth and survival emerged
recently in such as prostate and ovarian cancer and in glioma cells
[23,30,31]. Our data reinforced this new concept with additional
information elucidated the unique characteristics of energy
metabolism in high metastatic cancer cells encountering metabolic
stress from oleic acid exposure, which was mediated by AMPK
activation that resulted in enhanced fatty acid oxidation.
The physiological roles of oleic acid in health and disease in
humans are minimally investigated and understood.
HighFigure 3. The AMPK pathway was selectively activated in various human cancer cells treated with oleic acid. (A) The cells were
incubated with 0.5% BSA or 400 mM of BSA-bound oleic acid for 48 hours. The levels of phosphorylated AMPK (Thr172), the total AMPK, pACC (Ser79),
the total ACC, CPT1a and ATGL were determined by western blot analysis using 50 mg of the total proteins from each sample. (B) The Quantification
of Protein expression by densitometry from three independent experiments normalised to GAPDH. The values are expressed as the percent of control
cells, given as the mean 6 SD, n = 3; **p,0.01 for OA compared with BSA.Students t test.
metastatic cancer cells and low metastatic cancer cells had
opposite responses to oleic acid treatment with respect to cell
survival and migration. As shown, the HGC-27 and
MDA-MB231 cells grew faster in the presence of oleic acid (Fig. 1&2). There
is increasing evidence that cancer cells show specific alterations in
different aspects of lipid metabolism. The changes in lipid
metabolism affect numerous cellular processes, including cell
growth, proliferation and motility . Metabolically, the high
metastastic cancer cells setting from low metastastic cancer cells
are endowed with rapid growth and high migration ability, which
are because of the distinct metabolism trait. We concluded that
high metastatic cancer cells with high-level consumption for
energy utilise OA more efficiently than their counterpart cells.
The molecular differences induced by OA in these two cell
groups are unclear. AMPK is a key regulator of energy
homeostasis within cells. On activation, AMPK switches off the
ATP-consuming biosynthetic pathways (e.g., fatty acid synthesis)
and activates the ATP-generating metabolic pathways (e.g., fatty
acid oxidation) to preserve the ATP levels for cell survival .
This study was the first to unmask AMPK activation as a possible
mechanism for the intrinsic correlation between OA and different
cell fates. Mechanically, we found increased AMPK activation,
based on Thr172 phosphorylation, in the high metastatic cancer
cells exposed to OA, which exhibited the promotion of cell
proliferation and migration. As shown, the total and
phosphorylated AMPKa and ACC amounts as well as CPT1a were
remarkably high in the HGC-27 and MDA-MB-231 cells,
although the ATGL level was up-regulated in these two cell
groups (Fig. 3). The role of fatty acid oxidation in the altered
energy metabolism of cancer cells is less clear and might have been
influenced by the culture conditions to which the cells were
exposed. Our data suggested that b-oxidation rather than lipolysis
is the main change in the microenvironment with OA; the data
support a possible functional link between AMPK and the altered
energy metabolism in cancer cells. The selective AMPK activation
in high metastatic cancer cells results in energy metabolism
plasticity or adaptation. This assumption was further sustained by
the data obtained by inhibition or stimulation of AMPK activity.
As shown in Figure 4, in the HGC-27 and MDA-MB-231 cells
with AMPK activation induced by OA, the inhibition of AMPK
0.5% BSA or 400 mM of BSA-bound oleic acid for 24, 48, 72, of 96 hours. The cell viability was determined using an MTT assay and expressed as the
percentage of the control cells at the indicated time points. The values are the mean 6 SD, n = 3; *p,0.05 for OA+ AMPKa1-siRNA compared with
OA+control-siRNA.one-way ANOVA followed by Fishers LSD test.(C) The cells were cultured with 0.5% BSA or 400 mM of BSA-bound oleic acid with
5 mM of Compound C or with 100 mM of AICAR for 48 or 72 hours. The cell proliferation was examined using BrdU ELISA at the indicated time points.
The values are the mean 6 SD, n = 3; # p,0.05 for OA+Compound C or OA+AICAR compared with OA. one-way ANOVA followed by LSD test. (D) The
statistical comparison of the percentage of the control cells in the cell-migration ability (the cells treated with 0.5% BSA or 400 mM of BSA-bound oleic
acid with 5 mM of Compound C or with 100 mM of AICAR were allowed to migrate for 12 hours in the HGC-27 and SGC7901 cells and for 6 hours in
the MDA-MB-231 cells.). All of the statistical analysis values represent the mean of three experiments (the means 6 SD, n = 3, *p,0.05 and **p,0.01
for OA compared with BSA; #p,0.05 and ##p,0.01 for OA+Compound C or OA+AICAR compared with OA. one-way ANOVA followed by LSD test.
Figure 6. AMPK is involved in the OA-mediated protein expressions of fatty acid metabolism enzymes. The gastric carcinoma cells and
breast cancer cells were cultured with 0.5% BSA or 400 mM of BSA-bound oleic acid with 5 mM of Compound C or with 100 mM of AICAR for 48 h. The
levels of pAMPKa (Thr172), the total AMPKa, pACC (Ser79), the total ACC, CPT1a, MCAD and ATGL were determined by western blot analysis using
50 mg of the total proteins from each sample. A representative blot is shown. (B) The cells transfected with control-siRNA or AMPKa1-siRNA (24 hours)
were treated with 0.5% BSA or 400 mM of BSA-bound oleic acid for another 48 hours. The expressions of pAMPKa (Thr172), the total AMPKa, pACC
(Ser79), the total ACC, CPT1a, MCAD and ATGL were determined by western blot analysis.
by Compound C or AMPKa1 siRNA attenuated the increase of
cell growth and migration, and the artificial activation of AMPK
by AICAR in the SGC7901 and MCF7 cells compensated these
inhibitory effects induced by OA.
AMPK contributes to the maintenance of high ATP levels,
which might remain remarkably stable for high energy-dependent
molecular activities. Consistently, we found elevated levels of
cellular ATP with AMPK activation and reduced ATP levels with
inhibition of AMPK by Compound C in the OA-treated HGC-27
and MDA-MB-231 cells, whereas AIACR activated AMPK,
subsequently leading to the ATP production in the SGC7901 and
MCF7 cells (Fig. 5E). The significant increase in oxygen
consumption induced by oleic acid could be reduced by AMPK
inhibition (Fig. 5C&D). ATP production apparently results from
the activation of AMPK and downstream enzymes. To support
this mechanism, the study interpreted the intrinsic link between
AMPK activation and the cellular behaviours. As shown in
Figure 6, Compound C or AMPKa1 siRNA reduced the total and
phosphorylated AMPKa and ACC as well as the subsequent
CPT1a and MCAD levels, and AIACR compensated for these
corresponding changes. One possible explanation for the
difference between high and low malignancy cells would be the inability
of the low malignancy cells to increase AMPKa and ACC gene
expression in response to OA.Thus, it is possible that the
longterm effects promoted by the constitutive AMPK activation
support the adaptation of metastatic cells to the energy pathways
that are predominant in specific metabolic stress and that
contribute to the growth advantage of tumour cells.
The two AMPKa variants (AMPK-a1 and AMPK-a2) have a
differential localisation pattern in mammalian cells, with the
AMPK-a1 subunit being localised in the cytoplasm whereas the
AMPK-a2 subunit is localised in the nucleus . The
transfection of cells with AMPK-a1 siRNA, and not AMPK-a2siRNA,
abolished the effects of OA on the cell viability and protein
expressions of phosphorylated ACC, CPT1a and MCAD. The
downstream genes of AMPK, including ACC, that are localised in
the cytoplasm suggest that at least some of the effects that were
observed in the study are primarily because of the alpha-1 activity.
The AMPK system is activated by a large variety of cellular
stresses that deplete ATP, such as glucose deprivation, ischemia,
hypoxia, oxidative stress and hyperosmotic stress.It has been
demonstrated in cultured adipocytes that agents that increase
intracellular cAMP increase the activity of AMPK . Wu has
reported that treatment of bovine aortic endothelial cells (BAECs)
with OA-NO2 induced a significant increase in AMPKThr172
phosphorylation and AMPK activity . Recent reports have
shown that oncogenic Ras, MYC and Pten deletion activates
AMPK and that this activation stimulates cell proliferation
[31,37]. We hypothesise that the intracellular stresses or the
oncogene induced by OA treatment in high metastatic cancer cells
contribute to the activation of AMPK. The accurate mechanisms
involved inthe regulation of AMPK in response to OA warrant
Oleic acid prompted cell proliferation and migration in high
metastatic cancer cells via enhanced b-oxidation mediated by
AMPK activation. Provided that these observations might be
extended to the in vivo situation, it could be postulated that a
microenvironment rich in oleic acid might favour tumour
progression. We reported for the first time that activated AMPK
is involved in OA-induced cell proliferation and migration in
terms of energy metabolism, which offers novel potential targets
for the chemoprevention of human cancer.
Figure S1 Effects of oleic acid on cell apotosis in various
human cancer cell lines. Human gastric cancer cell lines
(HGC-27 and SGC7901), breast cancer cell lines (MDA-MB-231
and MCF-7) were starved for 12 hours, and then incubated with
0.5% BSA as control or 400 mM BSA-bound oleic acid for 48
hours. (A) Cells were stained with Hoechst 33258 and
photographed (magnification 200x). (B) Cells were stained with
AnnexinV and PI respectively and then quantified by flow
cytometry analysis. Values are the mean 6 SD, n = 3;#p,0.05
for OA compared with BSA.Students t test. (C) Caspase 3 and
Caspase 9 were examined by Western blotting analysis with actin
loaded as a control.
Figure S2 The oxygen consumption rate in
MDA-MB231 and MCF-7 cells treated with OA. Cells were incubated
with 0.5% BSA as control or 400 mM BSA-bound oleic acid for 48
hours. Five million cells were resuspended in 1 ml of fresh warm
mediumpre-equilibrated with 21% oxygen and the oxygen content
in thecell suspension medium was constantly monitored for
10 min andoxygen consumption rate was recorded. Values are
the mean 6 SD, n = 3;*p,0.05 for OA compared with
BSA.Students t test.
Figure S3 Representative bolt of pAMPK and AMPK in
control-siRNA and AMPKa1-siRNA cells. MDA-MB-231
and HGC-27 cells at approximately 60% confluency were
transfected with control-siRNA and AMPKa1-siRNA using
Lipofectamine 2000. Transfections were performed in serum-free
medium for 8 hours. After incubation, transfection complexes
were removed and replaced with serum-free medium. (A)The
expressions of pAMPKa and AMPKa were determined by
Western blotting analysis.(B)Quantification of Protein expression
by densitometry from three independent experiments,normalised
to actin. Values are expressed as percent of control cells, given as
mean 6 SD, n = 3; *p,0.05 for AMPKa1-siRNAcompared with
control-siRNA.Students t test.
Figure S4 Oil red O staining in cells treated with OA in
the presence of Compound C or AICAR. HGC-27 and
SGC7901 cells were cultured with 0.5% BSA or 400 mM
BSAbound oleic acid either with 5 mM Compound C or with 100 mM
AICAR. Cells were stained with oil red O and photographed
We thank Adham Sameer A. Bardeesi for proofreading the manuscript.
Conceived and designed the experiments: SL TZ XY GQG. Performed
the experiments: SL TZ. Analyzed the data: SL TZ JXM. Contributed
reagents/materials/analysis tools: CL DC ZYD YCY LL. Wrote the
paper: SL TZ XY GQG.
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