Oleanolic Acid Suppresses Aerobic Glycolysis in Cancer

Cells by Switching Pyruvate Kinase Type M Isoforms

Jia Liu1,2., Ning Wu1., Leina Ma4, Ming Liu1, Ge Liu1,2, Yuyan Zhang1, Xiukun Lin1,3*
1 Institutes of Oceanology, Chinese Academy of Sciences, Qingdao, China, 2 Graduate School, University of Chinese Academy of Sciences, Beijing, China, 3 Department of
Pharmacology, Capital Medical University, Beijing, China, 4 Department of Molecular Biology, School of Medicine and Pharmacy, Ocean University of China, Qingdao,
China

Abstract
Warburg effect, one of the hallmarks for cancer cells, is characterized by metabolic switch from mitochondrial oxidative
phosphorylation to aerobic glycolysis. In recent years, increased expression level of pyruvate kinase M2 (PKM2) has been
found to be the culprit of enhanced aerobic glycolysis in cancer cells. However, there is no agent inhibiting aerobic
glycolysis by targeting PKM2. In this study, we found that Oleanolic acid (OA) induced a switch from PKM2 to PKM1, and
consistently, abrogated Warburg effect in cancer cells. Suppression of aerobic glycolysis by OA is mediated by PKM2/PKM1
switch. Furthermore, mTOR signaling was found to be inactivated in OA-treated cancer cells, and mTOR inhibition is
required for the effect of OA on PKM2/PKM1 switch. Decreased expression of c-Myc-dependent hnRNPA1 and hnRNPA1 was
responsible for OA-induced switch between PKM isoforms. Collectively, we identified that OA is an antitumor compound
that suppresses aerobic glycolysis in cancer cells and there is potential that PKM2 may be developed as an important target
in aerobic glycolysis pathway for developing novel anticancer agents.
Citation: Liu J, Wu N, Ma L, Liu M, Liu G, et al. (2014) Oleanolic Acid Suppresses Aerobic Glycolysis in Cancer Cells by Switching Pyruvate Kinase Type M
Isoforms. PLoS ONE 9(3): e91606. doi:10.1371/journal.pone.0091606
Editor: Ming Tan, University of South Alabama, United States of America
Received November 6, 2013; Accepted February 12, 2014; Published March 13, 2014
Copyright: 2014 Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by national innovative drug development projects of (2014ZX-09102043-001). The study was also supported in part by
National Foundation of Natural Sci. of China (81302906, 81273550 and 41306157; http://www.nsfc.gov.cn/Portal0/default152.htm). The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: linxiukun@yahoo.com
. These authors contributed equally to this work.

compromise tumor growth [6]. Therefore, PKM2 is believed to

a promising target in the field of cancer therapy. However, the
compounds that can increase the ratio of PKM1 to PKM2 have
not been found.
Oleanolic acids (OA) is distributed widely in many plants, and it
has been well documented that OA displays anti-tumor activity to
a range of human cancer cells. Previous study in our laboratory
has shown that treatment of human pancreatic pan-28 cancer cells
leading to apoptosis via mitochondrial mediated apoptotic
pathway [7]. Another study also reveals that OA can inhibit
metastasis on glioma cells [8]. However, the target of OA has not
been well identified. In the present study, we found that OA can
suppress aerobic glycolysis by suppressing PKM2 expression and
affected PKM mRNA splicing through mTOR/c-Myc/hnRNP
signaling.

Introduction
Aerobic glycolysis, also known as Warburg effect, has been
established as a hallmark of cancer cells [1]. Almost all types of
cancer cells change their metabolism by increasing glycolysis and
suppressing mitochondrial oxidative phosphorylation, even under
normoxic conditions (therefore defined as aerobic glycolysis) [2].
Many glycolytic intermediates are indispensible for the synthesis of
molecules that is essential for cellular structures and functions,
such as nucleotides, amino acids and lipids. Thus, highly
proliferating cancer cells meet their requirements for cellular
building materials by switching their metabolism from oxidative
phophorylation to glycolysis, although ATP production is less
efficient in glycolytic process.
PKM gene encodes two protein kinases, PKM1 and PKM2, and
these kinases are also responsible for the conversion of phosphoenolpyruvate (PEP) to pyruvate which can be used for lactic acid
production or enterring mitochondrial oxidative phosphorylation.
PKM1 and PKM2 are generated by exclusive mRNA splicing
(exon 9 for PKM1 and exon 10 for PKM2) [3]. http://en.
wikipedia.org/wiki/PKM2 - cite_note-Corcoran-5PKM1 is catalytically more active than PKM2. PKM2 is expressed in all cells
with a high rate of nucleic acid synthesis [4]. Cancer cells utilize
PKM2 to accumulate the intermediates for the synthesis of nucleic
acid and protein and maintain aerobic glycolysis [5]. Increasing
PKM2 activity or switching mRNA splicing from PKM2 to PKM1
is able to suppress the Warburg effect, and consequently,
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Methods and Materials

Cell Culture and Chemical Compounds
Human prostate carcinoma cell line, PC-3, and human breast
cancer cell line, MCF-7, were purchased from American Type
Culture Collection C (ATCC). PC-3 cells were cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS; GIBCOBRL) at 37uC under a humidified 5% CO2 condition. MCF-7 cells
were cultured in Dulbeccos modified Eagles medium (DMEM).

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Oleanolic Acid Targets PKM2 to Treat Cancer

Figure 1. OA suppresses the aerobic glycolysis in cancer cells. Glucose consumption (A), lactic acid production (B) and oxygen consumption
(C) were assessed in PC-3 and MCF-7 cells treated with 50 or 100 mg/ml OA for 6 or 12 hr respectively. The details of methodology have been
described in the section of Materials and Methods. The ratio of each group to control was presented here. The bars showed the average values of
three independent experiments (Mean 6 SD). *, P,0.05; **, P,0.01.
doi:10.1371/journal.pone.0091606.g001

incubation for 15 min, the mixture was added to the cell cultures
and incubated for certain times.

Oleanolic acid (OA) was purchased from Sigma Aldrich

(O5504, St. Louis, MO). OA was prepared in DMSO at the
concentration of 10 mg/ml as stock solution.

Immunoblotting Assays
Cell Transfection

Cells were harvested by centrifugation at 1000 g for 10 min and

lyzed with M-PER Mammalian Protein Extraction Reagent
(Thermo Scientific, IL). Protein concentrations were determined
using the BCA protein assay kit (Thermo Scientific, FL). The total
protein was separated with electrophoresis with 1012% polyacrylamide gel and transferred onto 0.45 mm nitrocellulose
membranes. Membranes were incubated in blocking solution
(PBS, 0.1% Tween-20, and 5% nonfat dry milk powder) for 2 hr
at room temperature, and then, incubated with primary antibodies
(1:1000). After incubation overnight, the membranes were washed
with PBS containing 0.1% Tween-20 for three times, and then,

Plasmids, including pWZL Neo Myr Flag PKM2 and pMXshcMYC were obtained from Addgene (Cambridge, MA). pcDNA
Flag GFP (Flag-GFP) was preserved in our laboratory and was
used as control. Cell transfection was performed using Lipofectamine 2000 (Invitrogen, CA) according to the manufacturers
instructions. Briefly, PC-3 or MCF-7 cells were plated onto a 96well plate. When the cells are cultured to approximately 85%
confluence, the media were replaced with OPTI-MEM. Then,
3 mg plasmids and 30 ml lipofectamine reagent was mixed in a
tube containing 1500 ml OPTI-MEM by vigorous vortexing. After
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Oleanolic Acid Targets PKM2 to Treat Cancer

incubated for another 1 hr to visualize PKM2. 49,69-diamidino-2phenylindole (DAPI) was used to stain the nuclei. The fluorescence
was detected under a confocal microscope (CLS-2SS, Thorlabs).

were incubated with horseradish peroxidase-conjugated secondary

antibody (IgG goat anti-rabbit or anti-mouse; 1:2000; Bio-Rad,
CA) for 1 hr at room temperature. The protein bands were
visualized with SuperSignal West Dura Extended Duration
Substrate (Thermo Scientific, IL). The intensity of the blots were
quantified using ImageJ software.
The primary antibodies used in our study were as follows:
PKM1 (#7067, Cell Signaling Technology, Danvers, MA), PKM2
(#4053, Cell Signaling Technology, Danvers, MA), b-tubulin
(#2146, Cell Signaling Technology, Danvers, MA), PhosphomTOR (Ser2448) (#2971, Cell Signaling Technology, Danvers,
MA); mTOR (#2983, Cell Signaling Technology, Danvers, MA);
c-Myc, (sc-40, Santa Cruz Biotechnology, Dallas, Texas); hnRNP
A1 (#8443, Cell Signaling Technology, Danvers, MA); hnRNP
A2 (#9304, Cell Signaling Technology, Danvers, MA).

Metabolic Assays
Assays of O2 content, pyruvate kinase (PK) activity, glucose
uptake, lactate production, and cellular respiration were used to
study the changes in metabolic switch in cancer cells treated with
OA. The Pyruvate Kinase (PK) Assay Kit (ab83432, Abcam) and
OX-500 O2 Microsensor (Unisense, Denmark) were used to
determine PK activity and O2 content, respectively, in PC-3 and
MCF-7 cell treated with OA, following the manufacturers
instructions.
To measure the glucose uptake, PC-3 and MCF-7 were seeded
at a density of 56103 cells/well in 96-well plates. After incubation
overnight, the cells were transfected with certain plasmids or
treated with MHY1485. Then, the cells were treated with 50 or
100 mg/ml OA, respectively. After incubation for 6 or 12 hr, the
culture media were harvested by centrifugation at 3000 g for
15 min. The quantity of glucose consumption of the tested cancer
cells was detected using Glucose Uptake Colorimetric Assay Kit I
(#K676, BioVision, Milpitas, CA) according to the manufacturers

Immunofluorescence Analysis
PC-3 and MCF-7 cells (56104/well) were plated onto 24-well
plates, and then, OA (100 mg/ml) was added. After 6 hr
incubation, the cells were fixed with 4% Polyoxymethylene for
0.5 hr, followed by incubation with PKM2 antibody (1:1000) for
2 hr. The corresponding secondary antibody was added and

Figure 2. OA treatment affects the expression profile of PKM isoforms in cancer cells in a dose- and time-dependent manner. PKM1
and PKM2 expression level was evaluated by immunoblotting assays in PC-3 and MCF-7 cells treated with 10, 25, 50,100 mg/ml OA respectively for
12 hr (A) or 100 mg/ml OA for 0.5, 1, 2, 3, 6, 12 hr respectively (B). b-tubulin was used as endogenous references. (C) The PKM2 levels (Green) were in
PC-3 and MCF-7 cells treated with 100 mg/ml OA for 12 hr respectively, as determined by immunfluorescent staining. The nuclei (Blue) were stained
with DAPI. The merged pictures were shown in the bottom of the panel. The fluorescence intensity was quantified with ImageJ software. The ratio of
each group to control was presented here. The bars represented the average values of 5 randomly selected experiments.
doi:10.1371/journal.pone.0091606.g002

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Oleanolic Acid Targets PKM2 to Treat Cancer

Figure 3. PKM2 overexpression abolishes the effect of OA on aerobic glycolysis in cancer cells. (A) OA-treated PC-3 cells were
transfected with pWZL Neo Myr Flag PKM2 (Flag-PKM2) or control vector (Flag-GFP). Immunoblot assays were performed to determine the expression
of PKM2 protein. b-tubulin was used as endogenous references. Glucose consumption (B), lactic acid production (C) and oxygen consumption (D)
were assessed in PC-3 cells treated with 100 mg/ml OA for 12 hr. The details of methodology was described in the section of Materials and Methods.
The ratio of each group to control was presented here. The bars showed the average values of three independent experiments (Mean 6 SD). *, P,
0.05; **, P,0.01.
doi:10.1371/journal.pone.0091606.g003

plated onto a bottom layer containing 0.6% agar and covered with
a top layer containing 0.6% agar. The cells were cultured in a 6well plate and the media were changed weekly. After incubation
for 10 days, crystal violet was added and the number of colonies
was counted and analyzed with ImageJ software.

instructions. The absorption density (OD) at 412 nm was read

using a BioRad680 microplate reader (Bio-Rad, Hercules, CA).
The production of lactic acid was determined using Lactate
Colorimetric Assay Kit II (#K627, BioVision, Milpitas, CA) as the
manufacturers protocols. Briefly, PC-3 and MCF-7 cells were
treated with pWZL Neo Myr Flag PKM2, pMXs-hcMYC and
pcDNA Flag GFP for 72 hr or MHY1485 for 1 hr, respectively.
OA was added at the indicated concentrations and incubated for
the indicated times. Then the culture media were collected by
centrifugation at 3000 g for 15 min, mixed with 45 ml of Lactate
Assay Buffer. The absorbance at 450 nm was read using a
BioRad680 microplate reader (Bio-Rad, Hercules, CA).
The oxygen consumption was examined using Oxygen Consumption Rate Assay Kit (#600800, Cayman Chemical Company, Ann Arbor, MI) following the manufacturers instruction.
VICTOR X3 Multilabel Plate Reader (Perkin Elmer) was used to
detect the OD value at 650 nm. The oxygen consumption rate
was shown as lifetime versus time (ms/hr).

Statistical Analysis
The experiments except immunoblot assays were performed for
at least three times. All values were expressed as means 6 SD, and
compared at a given time point by unpaired, two-tailed t test. Data
were considered to be statistically significant when p,0.05 (*) and
p,0.01 (**).

Results
OA Inhibits Aerobic Glycolysis in Cancer Cells
Aerobic glycolysis is characterized by increased glucose uptake
and lactate production, and compromised oxygen consumption in
cancer cells with high availability to oxygen. Therefore, we
measured the effect of OA on these activities of cancer cell.
Reduced glucose consumption was found in several types of cancer
cells when OA was added to the cultures (Fig. 1A). The
production of lactic acid in these cancer cells was also found to be
decreased in cells treated with OA (Fig. 1B). Furthermore, OA

Colony Formation Assay

PC-3 or MCF-7 cells were plated onto a 6-well plate, and
transfected with plasmids, including pWZL Neo Myr Flag PKM2
and pcDNA Flag GFP. After incubation for certain times, the cells
were trypsinized, suspended in DMEM containing 10% FBS,
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Oleanolic Acid Targets PKM2 to Treat Cancer

Figure 4. mTOR suppression is required for OA-induced metabolic switch. (A) The levels of phosphorylated mTOR was examined in PC-3
and MCF-7 cells treated with 50 or 100 mg/ml OA for 6 or 12 hr respectively as determined by immunoblot assays. b-tubulin was used as endogenous
references. (B) Immunoblot analysis of PKM1 and PKM2 expression was performed in PC-3 cells incubated with OA (100 mg/ml) or/and a mTOR
activator MHY1485 (2 mM). Glucose consumption (C), lactic acid production (D) and oxygen consumption (E) were detected in PC-3 cells treated with
OA (100 mg/ml) and/or MHY1485 (2 mM) for 12 hr. The ratio of each group to control was presented here. The bars showed the average values of
three independent experiments (Mean 6 SD). *, P,0.05; **, P,0.01.
doi:10.1371/journal.pone.0091606.g004

enhanced the consumption of oxygen in cancer cells (Fig. 1C).

The above data showed that OA was able to induce the metabolic
alteration of cancer cells by suppressing aerobic glycolysis.
In addition, we compared O2 content in cancer cells with or
without OA treatment. The data showed that OA has no influence
on the concentration of oxygen in the cell culture, thereby
excluding the possibility that OA lead to metabolic alteration by
affecting normic conditions (Figure S1).

of cells. PKM2 has been found to be highly expressed in all

proliferating cells, such as tumor cells [4]. To identify the
mechanisms that OA affected aerobic glycolysis, we subsequently
evaluated the changes in the expression level of PKM1 and PKM2
in cancer cells treated with OA. The immunoblot analysis revealed
that OA was able to decrease PKM2 abundance, and increase
PKM1 expression, in both PC-3 and MCF-7cancer cell lines, in a
dose- and time-dependent manner (Fig. 2A and 2B). The
reduction in PKM2 expression was also found in Immunofluorescent staining experiments. The treatment of cancer cells with OA
down-regulated the PKM2 expression significantly in both PC-3
and MCF-7 cells (Fig. 2C). Furthermore, PK activity was shown
to be elevated in OA-treated cancer cells, along with the switch
from PKM2 to PKM1 (Figure S2A and S2B).

OA Induces the Switch from PKM2 to PKM1 and Increase

PK Activity in Cancer Cells
Pyruvate kinase muscle isozyme 2 (PKM2), whose enzymatic
activity is less active than PKM1, is responsible for the conversion
of phosphopyruvate into pyruvate in the glycolytic process.
PKM2, especially in its dimeric form, accumulates glycolytic
intermediates and channels them into the biosynthesis of
nucleotide and amino acids, thereby contributing to the growth

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Oleanolic Acid Targets PKM2 to Treat Cancer

Figure 5. The switch from PKM2 to PKM1 by OA results from the decrease in Myc, hnRNPA1 and hnRNPA2 expression. (A) The
expression of c-Myc, hnRNPA1 and hnRNPA2 was determined by immunoblot analysis in PC-3 and MCF-7 cells treated with 50 or 100 mg/ml OA for 6
or 12 hr respectively. (B) The expressionof c-Myc, hnRNPA1 and hnRNPA2 expression was determined by immunoblot analysis in PC-3 cells treated
with OA (100 mg/ml) or/and MHY1485 (2 mM) respectively. (C) The level of c-Myc, hnRNPA1, hnRNPA2, PKM1 and PKM2 was determined by
immunoblot assays in PC-3 cells treated with plasmids pMXs-hcMYC (Flag-cMyc) in the presence or absence of OA. b-tubulin was used as
endogenous references.
doi:10.1371/journal.pone.0091606.g005

mTOR by MHY1485 protected cancer cells from OA-mediated

decline in PKM2 expression and increase in PKM1 levels
(Fig. 4B). Our further study showed that the glucose consumption, the lactate production and the oxygen consumption rate are
also restored in cells treated with MHY1485 (Fig. 4C, 4D and
4E). The result reveals that mTOR is responsible for the OA
induced the switch of PKM isoforms and metabolism.

The Increase in PK Activity Caused by PKM2/PKM1 Switch

Mediates OA Suppression of Aerobic Glycolysis
Based on the finding that the switch from PKM2 to PKM1
occurs in cancer cells treated with OA, we subsequently
investigated the role of increaed PK activity in OA suppression
of aerobic glycolysis. PC-3 cells were transfected with PKM2- or
GFP-expressing vector prior to OA stimulation (Fig. 3A). OAinduced increase in PK activity was rescued by PKM2 overexpression in PC-3 cells (Figure S3). The results also showed that
the glucose consumption, the lactate production as well as the
oxygen consumption were all restored in cells overexpressing
PKM2 (Fig. 3B, 3C and 3D). The results suggest that increased
PK activity due to PKM2/PKM1 switch is required for the
inhibitory effect of OA on aerobic glycolysis in cancer cells.

OA-induced mTOR Suppression Induces the Switch of

PKM Isoforms by Inhibiting c-Myc-dependent hnRNPA1
and hnRNPA2 Expression
mTOR has been demonstrated to elevate PKM2 level by
stimulating c-myc-dependent hnRNPA1 and hnRNPA2 expression [9], both of which are responsible for the exclusive splicing of
PKM mRNA [3]. We subsequently determined the influence of
OA on the expression of hnRNPA1 and hnRNPA2. The results
showed that OA can reduce the expression level of c-Myc, as well
as its target gene hnRNPA1 and hnRNPA2 expression (Fig. 5A).
mTOR activation mediated by MHY1485 was able to restore the
expression of c-Myc, hnRNPA1, and hnRNPA2 in OA-treated
cancer cells (Fig. 5B). Furthermore, transfection of OA-treated
cancer cells with plasmids expressing c-Myc abrogated the effect of
OA on the change in PKM1 and PKM2 expressions (Fig. 5C).
The results suggest that OA can induce PKM2/PKM1 switch via

OA Suppression of mTOR Mediates the Alteration in the

Expression Profiles of PKM Isoforms and Metabolic
Switch in Cancer Cells
We were interested in how OA induced the decrease in the
switch from PKM2 to PKM1 in cancer cells. mTOR signaling has
been demonstrated to participate in the switch of PKM isoforms in
cancer cells [9]. Therefore, we detected the changes in active
status of mTOR in OA-treated cancer cells. Immunoblotting
analysis revealed that mTOR activation was diminished when OA
was added to the culture of cancer cells (Fig. 4A). Reactivating
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Oleanolic Acid Targets PKM2 to Treat Cancer

Figure 6. OA suppression of aerobic glycolysis contributes to its anti-tumor activity. (A) PC-3 cells was counted 12, 24, 36 and 48 hr after
the treatment of OA (100 mg/ml) or/and MHY1485 (2 mM) using hemacytometry. The average values of three independent experiments were shown
as Mean 6 SD. **, P,0.01. The representative figure was shown for each group. (B) Cells were treated with OA (100 mg/ml) or/and MHY1485 (2 mM)
for 10 days, the number of colonies of PC-3 cells were counted and analyzed using ImageJ software. The representative figure was shown for each
group. (C) OA suppresses the activation of mTOR in cancer cells. This inhibitory effect on mTOR signaling, in turn, abrogated c-Myc/hnRNPA1/
hnRNPA2-dependent PKM2 expression. Consequently, the aerobic glycolysis was inhibited in cancer cells treated with OA.
doi:10.1371/journal.pone.0091606.g006

growth rate and the number of formed colonies in PC-3 cells cotreated with OA and PKM2-expressing vectors (OA+PKM2
group) (Fig. 6A and 6B).
In summary, OA suppresses the activation of mTOR signaling
in cancer cells. In turn, the inhibitory effect of OA on mTOR
pathway leads to the switch from PKM2 to PKM1 expression,
which is mediated by c-Myc/hnRNPA1/hnRNPA2 pathway.
Eventually, aerobic glycolysis was suppressed in OA-treated
cancer cells (Fig. 6C).

c-Myc-dependent hnRNPA1 and hnRNPA2 mTOR signal

pathway.

Reduced Aerobic Glycolysis Partially Accounts for the

Anti-tumor Activity of OA
To address the contribution of impaired aerobic glycolysis to
tumor suppressor activity of OA on cancer cells, we detected the
changes in malignant phenotypes of OA-stimulated cancer cells
which still underwent aerobic glycolysis due to PKM2 overexpression. OA was found to reduce the growth of PC-3 cells
(Fig. 6A). Also, the number of colonies formed in PC-3 cells was
also decreased under the treatment of OA (OA+GFP group)
(Fig. 6B). However, PKM2 restoration significantly abolished the
effect of OA on cancer cells, evidenced by the increase in the

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Discussion
OA, a natural compound widely distributed in plants, has been
documented to possess a variety of bioactivities including antitumor property [10]. The mechanisms of OAs anti-tumor activity
7

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Oleanolic Acid Targets PKM2 to Treat Cancer

involve apoptosis induction, cell cycle arrest and metastasis

inhibition [7,8]. However, the effect of OA on the metabolic
pathway in cancer cells is still unclear. In this study, we found that
OA was able to suppress the Warburg effect in cancer cells,
evidenced by increased oxygen consumption, reduced lactate
production and lower glucose uptake (Fig. 1A1C). To our
knowledge, this is the first time to reveal that OA affects the
metabolic switch of cancer cells. Our finding may contribute to the
understanding of OA action on cancer cells and help to the
development of OA and its derivatives with more potent efficacy
for clinical application.
Aerobic glycolysis is believed to be a novel, important and
effective target in cancer chemotherapy. Increasing evidences
indicate that aerobic glycolysis plays a critical role in the onset and
progression of malignant diseases. Several compounds have been
reported to suppress the growth of tumor cells by affecting aerobic
glycolysis[1115]. For instances, the extract from Spatholobus
suberectus was shown to inhibit the activity of lactate dehydrogenase A (LDHA), thereby suppressing the aerobic glycolysis in
breast cancer cells [13]. An anti-inflammatory compound,
curcumin, has recently been demonstrated to reverse Warburg
effect caused by Tumor Necrosis Factor (TNF) stimulation in
breast cancer cells [14]. Our study confirmed that OA also
possesses the inhibitive activity on aerobic glycolysis. The study
increases the understanding of anticancer mechanism of OA.
Many studies have revealed that PKM2 plays a key role in
aerobic glycolysis in cancer cells [3]. In the present study, we
found that the PKM2 expression can be inhibited in cells treated
with OA in a dose- and time-dependent manner (Fig. 2A2C).
More importantly, increased PK activity, due to PKM2/PKM1
switch, mediated the effect of OA on aerobic glycolysis in cancer
cells, since reducing PK activity by overexpressing PKM2 level in
cancer cells abolished the OA-induced metabolic switch (Fig. 3A
3D). OA is therefore identified as the first compound reducing
PKM2 abundance, and exerts its effect on metabolism in cancer
cells through a new target. Therefore, we provided initial
evidences that targeting PKM2 may be a novel strategy to
develop cancer agents, and OA may be a lead compound for
developing anti-cancer drugs targeting PKM2.
Aberrant activation of mTOR signaling is closely implicated in
a variety of cancers [16]. mTOR activation promotes the
resistance of cancer cells to apoptosis induced by both cytokines
and chemotherapeutic agents, accelerates the proliferation of a
wide range of cancer cells, and facilitates the metastasis [16]. Most
of the current mTOR inhibitors suppress the growth of cancer
cells primarily by inducing apoptosis and cell cycle arrest, or
impairing metastasis [1719]. In this study, we found that OA
reduces the expression level of phosphorylated mTOR in cancer
cells (Fig. 4A) associated with its inhibitory activity on aerobic
glycolysis. Recently, mTOR signaling has been proven to

modulate the metabolic switch in cancer cells and this effect is

mediated by the change in PKM2 expression [14,20,21].
Furthermore, mTOR/PKM2 pathway is responsible for, at least,
liver tumorigenesis [22]. These new findings suggest that targeting
metabolism may be a novel strategy of mTOR inhibitor to
suppress the growth of cancers.
Collectively, we proved evidence that OA was able to switch
metabolic patterns from aerobic glycolysis to oxidative phosphorylation through affecting mTOR/c-Myc/PKM2 pathway in
cancer cells. Considering its low toxicity to normal tissues, OA
and its analoges may be a promising candidate agent for
developing anticancer agents targeting metabolism. Our studies
contribute to a better understanding on the mechanism of OAs
antitumor property, and also the study provides primary evidence
that PKM2 can be selected as a critical therapeutic target in
aerobic glycolysis pathway in developing novel anticancer agents.

Supporting Information
Figure S1 OA failed to affect the content of oxygen in
cell culture. PC-3 and MCF-7 cells were treated with or without
OA for 12 hr. The O2 content was detected with at the indicated
time points. The average values of three independent experiments
were shown as Mean 6 SD.
(PPT)
Figure S2 OA incubation induced the elevation in PK
activity in cancer cells. (A) PC-3 and MCF-7 cells were treated
with OA at the indicated doses and PK activity was determined
12 hr after OA treatment. The average values of three
independent experiments were shown as Mean 6 SD. *, P,
0.05, **, P,0.01. (B) The activity of PK was also assessed in
cancer cells exposed with 100 mg/ml OA at the indicated time
points. The average values of three independent experiments were
shown as Mean 6 SD. *, P,0.05, **, P,0.01.
(PPT)
Figure S3 PKM2 overexpression rescue the elevation in

PK activity induced by PKM2/PKM1 switch. PK activity

was evaluated in OA-treated PC-3 cells transfected with PKM2or GFP-expressing vector, 12 hr after the treatment of 100 mg/ml
OA. The average values of three independent experiments were
shown as Mean 6 SD. *, P,0.05.
(PPT)

Author Contributions
Conceived and designed the experiments: JL NW XL. Performed the
experiments: JL NW LM ML GL YZ. Analyzed the data: JL NW.
Contributed reagents/materials/analysis tools: XL. Wrote the paper: JL
NW XL.

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