Arch. Pharm. Res.
DOI 10.1007/s12272-015-0550-6
REVIEW
ROS and energy metabolism in cancer cells: alliance for fast
growth
Sang Won Kang Sunmi Lee Eun Kyung Lee
Received: 2 November 2014 / Accepted: 5 January 2015
The Pharmaceutical Society of Korea 2015
Abstract In normal cells, the cellular reactive oxygen
species (ROS) level is proportional to the activity of
mitochondrial electron transport and tightly controlled by
endogenous antioxidant system. However, energy metab-
olism and ROS homeostasis in cancer cells are much dif-
ferent from those in normal cells. For example, a majority
of cellular glucose is metabolized through aerobic glycol-
ysis (Warburg effect) and the pentose phosphate path- concentration in normal cells (Wang et al. 2008). In con-
way. Cancer cells harbor functional mitochondria, but
many mutations in nuclear DNA-encoded mitochondrial
genes and mitochondrial genome result in the mitochon-
drial metabolic reprogramming. The other characteristic of
cancer cells is to maintain much higher ROS level than
normal cells. Ironically, cancer cells overexpress the ROS-
producing NADPH oxidase and the ROS-eliminating
antioxidant enzymes, both of which enzyme systems share
NADPH as a reducing power source. In this article, we
review the complex connection between ROS and energy
metabolisms in cancer cells.
Keywords Reactive oxygen species  Energy
metabolism  Antioxidant enzyme  NADPH oxidase
Introduction
Reactive oxygen species (ROS) include the superoxide
anion (O2-), hydrogen peroxide (H2O2), and hydroxyl
radical (OH). Among these species, the superoxide anion
S. W. Kang (&)  S. Lee  E. K. Lee
Department of Life Sciences, Research Center for Cell
Homeostasis, Ewha Womans University, Seoul 120-750,
Republic of Korea
e-mail: kangsw@ewha.ac.kr
can be generated by either mitochondrial electron leakage
or NADPH oxidases (NOX) (Fig. 1). The free electrons
that are leaked from mitochondrial electron transport chain
reduces the dissolved oxygen to the superoxide anion,
similar to the Fenton reaction. The extent of mitochondrial
electron leakage is proportional to the usability of the
electron transport chain depending on NADH/FADH2
trast, NOX directly produces the superoxide anion by
reduction of the molecular oxygen using the NADPH-
derived electrons (Bedard and Krause 2007). The super-
oxide anion can be converted to H2O2 by another free
electron or by a superoxide dismutase (SOD)-catalyzed
reaction. Conversion of H2O2 to a hydroxyl radical is
detrimental to the cells because the radical is the most
reactive among ROS and hence attacks cellular macro-
molecules, including proteins, DNAs, and membrane lip-
ids. The irreversible chemical modification of protein by
the hydroxyl radicals involves cross-linking and aggrega-
tion (Guptasarma et al. 1992). Unlike the hydroxyl radicals,
the superoxide anion and hydrogen peroxide are mild
chemical species reversibly oxidizing proteins and DNAs.
The ROS-mediated oxidation of macromolecules can be
measured in the cells by various methods, such as the
detection of protein carbonyls and lipid peroxidation by
immunoassays (Shacter 2000). In order to prevent such
unwanted damages, aerobic cells are equipped with a set of
peroxidase enzymes, including peroxiredoxin (Prx), gluta-
thione peroxidase (Gpx), and catalase, that reduce hydro-
gen peroxide to water (Fig. 2). Hence, a broad distribution
of the peroxidase enzymes is an evolutionary advantageous
strategy for aerobic cells to prevent the formation of
hydroxyl radicals throughout cellular compartments. Like
NOX enzymes, the reducing power for the activity of the
peroxidase enzymes also comes from NADPH. Thus, the
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 S. W. Kang et al.

2e-

2e - Leakage

e- e- e-

Fig. 1 Cellular ROS hemeostasis. Free unpaired electrons are leaked
while the two electrons liberated from NADH are transported along
the mitochondrial electron transport chain and react with the
molecular oxygen to produce the superoxide anion. Alternatively,
the superoxide anions can be produced by the catalytic activity of
NADPH oxidases in many different locations, including plasma
membrane, mitochondria, and nucleus. The superoxide anion is then
converted to the hydrogen peroxide spontaneously or by superoxide
dismutase. Before its conversion to hydroxyl radicals, H2O2 is
reduced to water by cellular antioxidant peroxidases (peroxiredoxin
and glutathione peroxidase) in use of NADPH-driven electrons

Fig. 2 Reaction mechanism of

peroxidase enzymes. Gpx1
Glutathione peroxidase (Gpx) Gpx2 2H2O
catalyzes the reduction reaction Gpx3
of H2O2 into water using Gpx4
electrons from NADPH by GSSG NADP+
Gpx5
coupling with via glutathione
reductase (GR) and glutathione
(GSH); whereas, the
Gpx GR
peroxiredoxin (Prx) does the
same reaction using NADPH by GSH NADPH
coupling with thioredoxin
reductase (TrxR) and
thioredoxin (Trx) SOD Catalase Pentose
O2- H 2O 2 2H2O+O2 phosphate
pathway

Trxred NADP+ Malic enzyme

Prx TrxRs

Prx1 Trxox NADPH

Prx2
Prx3
Prx4
Prx5 2H2O

maintenance of ROS homeostasis exclusively relies on the
energy metabolism, such as pentose phosphate pathway
(PPP) for the NADPH supply. It is well-established that the
cancer cell metabolism is distinct from normal cells. In
cancer cells, glucose is metabolized through aerobic
glycolysis (Warburg effect) and the PPP. Cancer cell
mitochondria is functional, but metabolize only *5 % of
glucose (Vander Heiden et al. 2009). The tricarboxylic acid
(TCA) cycle is altered because of the mutation of the key
enzymes, such as isocitrate dehydrogenase (IDH). Such

123
ROS and energy metabolism in cancer
abnormal respiratory metabolic pathways influence energy
balance and thereby the ROS balance in cancer cells. In
this review, we introduce the basic principle on the ROS-
mediated protein regulation and discuss the connection
between ROS and energy metabolism focusing on the
redox-dependent protein regulation.
Oxidation-dependent regulation of signaling protein
function by ROS
H2O2 can oxidize the cysteinyl thiol to the cysteinyl sulfenyl
group on proteins. The cysteinyl sulfenyl group is further
oxidized into the sulfenic/sulfonic acid or form disulfide
with other thiol group. The disulfide is formed intramolec-
ularly or in a mixed disulfide. All these cysteine oxidation
processes are reversible by the activity of redox proteins,
such as sulfiredoxin and thioredoxin (Fig. 3). Such H2O2-
mediated reversible oxidation of proteins was intensively
studied in the receptor-mediated signal transduction. The
first pioneer work was the H2O2-mediated tyrosine phos-
phorylation in the PDGF-treated smooth muscle cells
(Sundaresan et al. 1995). This work opens a redox signaling
field and makes the researchers focused on protein tyrosine
phosphatase (PTP), which has a reactive cysteine residue in
active site. In PTPs, H2O2 oxidizes the thiolate group of the
active-site cysteine residue to the sulfenyl group, which in
turn forms sulfenylamide linkage by reacting with the
peptidyl a-amino group (van Montfort et al. 2003; Salmeen
et al. 2003). After a decade, it has been shown that Prx II is
an endogenous peroxidase enzyme that regulates the level of
intracellular H2O2 and PDGFRb phosphorylation induced
by PDGF (Choi et al. 2005). A new finding was that the
intracellular H2O2 under the control of Prx II affect the
phosphorylation of only two selective tyrosine residues on
PDGFRb. This study further indicates that a membrane-
Cys-SOH
+ H 2O2
ATP NADPH
Cys-SO2/3H Srx Cys-SH Trx Cys-S-S-R
Fig. 3 Reversible oxidation of cysteine sulfhydryl on proteins. The
cysteine sulfhydryl group (SH) can be ionized into thiolate anion via
deprotonation in a hydrophobic pocket and then react with H2O2. The
resulting cysteine sulfenyl group (SOH) is further oxidized to
sulfinic/sulfonic group (SO2/3H) or a mixed disulfide with other
cysteine SH, typically GSH or a neighboring resolving cysteine. The
cysteine sulfinic acid is reduced by sulfiredoxin (Srx) in a ATP-
dependent manner; whereas, the disulfide is reduced by thioredoxin
(Trx) in a NADPH-dependent manner
associated PTP integrates the site-selective phosphoryla-
tion. The PTPs are well known to be redox regulated by
H2O2; however, the substrate specificity of PTPs remains in
debate (Tonks 2006, 2005). Therefore, identification of such
a site-specific PTP toward PDGFRb may change a paradigm
on the mechanism of PTP action. In other cases, the cysteine
sulfenyl group forms mixed disulfide linkage with either a
neighboring cysteine sulfhydryl group, also known as
resolving cysteine (CR), or glutathione. The intramolecular
formation of cysteine sulfenyl or disulfide linkage in a
protein is sufficient to control its activity via a structural
change. For example, in the case of E. coli transcription
factor OxyR, the reactive cysteine oxidation results in a
structural change by the formation of intramolecular disul-
fide between two cysteine residues separated by 17 A (Choi
et al. 2001). The peroxidatic cysteine residue in the 2-Cys
Prx enzyme with fully-folded conformation reacts with
H2O2 and then forms intramolecular disulfide with CR res-
idue buried 14 A away (Wood et al. 2003). Furthermore, the
cumulative peroxidatic reaction in the Prx enzymes causes
the hyperoxidation of the active-site cysteine residue into
cysteine sulfinic and sulfonic acids (Yang et al. 2002). Such
hyperoxidation turns out to be reversible by sulfiredoxin in
an ATP-dependent manner (Jeong et al. 2006; Woo et al.
2003; Biteau et al. 2003).
Recently, accumulating evidence supports the signifi-
cant role of the cysteine oxidation in the signaling proteins.
A Src family kinase Lyn was activated by the H2O2-
dependent oxidation of a cysteine residue Cys466 (Yoo
et al. 2011). A DNA damage response kinase ATM forms
several disulfide bonds upon the H2O2 oxidation. Among
them, the Cys2991-containing intermolecular disulfide is
essential for the ATM activation (Guo et al. 2010). A key
endothelial growth factor receptor VEGFR2 is oxidized at
Cys1206 when endogenous Prx II is absent and subsequently
inactivated by the formation of an intramolecular disulfide
linkage (Kang et al. 2011). In most cases, the importance of
the cysteine oxidation is proven by site-directed mutagen-
esis: however, there is no structural evidence showing the
molecular mechanism underlying how the cysteine oxida-
tion changes function or activity of mammalian signaling
proteins.
Connection between ROS and energy metabolism
The best example of ROS-dependent regulation of met-
abolic enzymes is glyceraldehyde-3-phosphate dehydro-
genase (GAPDH) and PKM2 (Fig. 4). GAPDH contains
many free cysteine thiols including several active-site
thiols. The cysteine thiols in GAPDH can be oxidized by
different types of ROS, such as superoxide, H2O2, nitric
oxide, and peroxynitrite (Rodacka et al. 2014; Hwang
123
 S. W. Kang et al.

Fig. 4 ROS-dependent p53

regulation of energy metabolism
in cancer cells. ATM and
AMPK are activated by the
H2O2 oxidation; whereas, two TIGAR
glycolytic enzymes, GAPDH
and PKM2, are inactivated upon
oxidation F-2,6-bisP Pyruvate
PYGL Glucose
Glycogen
n G-1-P PKM2
GAPDH
DH
H2O2 ATM G-6-P F-1,6-bisP H2O2
p53 G6PD Glycolysis
O2
NOX
TAp73/p63 Pentose
O2-
Phosphate NADPH
Pathway H2O2
Prx
Fatty acid Gpx
Starvation AMPK ACC1/2 H 2O
synthesis

H2O2 Fumarate GSH

et al. 2009; Ishii et al. 1999; Souza and Radi 1998). Such
GAPDH oxidation is also observed in cardiac tissues
under ischemia and reperfusion (Eaton et al. 2002). It is
of importance that the GAPDH oxidation reroutes the
carbohydrate flux from glycolysis to the PPP, which
results in the reduced oxidative stress (Ralser et al.
2007). Given the additional signaling function of GAP-
DH beyond glycolysis (Morigasaki et al. 2008), the redox
regulation of GAPDH via the cysteine oxidation may
play a key role in linking the signaling to the glycolysis.
The oxidation-dependent activation of ATM aforemen-
tioned can increase the activity of glucose-6-phosphate
dehydrogenase (G6PD), which is a key regulatory
enzyme in the PPP, by promoting the Hsp27 phosphor-
ylation and its binding to G6PD (Cosentino et al. 2011).
This also counteracts to the oxidative stress by promoting
the NADPH-mediated antioxidant defenses. Another
exciting example is pyruvate kinase M2 (PKM2) that
catalyzes conversion of phosphoenolpyruvate to pyru-
vate. Unlike PKM1, the PKM2 is allosterically regulated
by glycolytic metabolite fructose-1,6-phosphate (Chris-
tofk et al. 2008). H2O2 inhibits the PKM2 by the cysteine
oxidation and such inhibition changes the glucose flux
into the PPP that generates sufficient NADPH to elimi-
nate ROS (Anastasiou et al. 2011). Vaughn et al. showed
that the oxidized cytochrome c is pro-apoptotic in cancer
cells and the high level of the reduced glutathione (GSH)
by PPP is essential for keeping the cytochrome c in
reduced state (Vaughn and Deshmukh 2008). This result
suggests the importance of glucose flux to the PPP in
cancer cell survival. The glycogen degradation by gly-
cogen phosphorylase was also shown to be critical for
reducing ROS level via PPP, thereby preventing tumor-
igenesis (Favaro et al. 2012). A prospective conclusion is
that the ROS-dependent regulation of energy metabolism
drives the carbohydrate flux to PPP, thereby increasing
the NADPH level to counteract intracellular ROS.
Conversely, some metabolites can regulate the cellular
redox status through ROS-metabolizing systems (Fig. 4).
For example, the accumulation of fumarate by the loss of
fumarate hydratase in cancer cells results in the increase of
cellular ROS (Sullivan et al. 2013). Mechanistically, the
accumulated fumarate reacts with GSH to form succinated
GSH that exhausts cellular NADPH via reaction with GSH
reductase. One-carbon metabolism involving serine, gly-
cine, and folate cycle is hyperactivated in tumorigenesis
(Locasale 2013). Serine is also shown to be required for
cancer cell survival: therefore, the serine starvation induces
the active serine biosynthesis in the cancer cells (Maddocks
et al. 2013). Serine can be converted to the GSH via a
trans-sulfuration pathway associated with methionine
cycle, thereby maintaining cellular antioxidant defense. A
recent quantitative analysis of NADPH flux indicates that
serine-driven one-carbon metabolism including the oxida-
tion of methylene tetrahydrofolate to 10-formyl-tetrahy-
drofolate is coupled to NADP? reduction (Fan et al. 2014),
which suggests a role of one-carbon metabolites in
NADPH generation.

123
ROS and energy metabolism in cancer
On the other hand, the cellular signaling pathways for
cancer cell survival directly or indirectly link ROS to
metabolic pathways. One important signaling is the tumor
suppressor p53-dependent regulation of energy metabolism
(Fig. 4). The p53 protein directly binds to G6PD and
inhibits glucose flux via PPP (Jiang et al. 2011). However,
the p53 transcriptional activity lowers the level of fructose-
2,6-bisphosphate, an allosteric regulator of phosphofruc-
tokinase in glycolysis, through its target gene TIGAR and,
therefore, results in a decrease of cellular ROS level
(Bensaad et al. 2006). The p53-related protein TAp73
inversely induces G6PD expression and enhances the PPP
glucose flux (Jiang et al. 2013; Du et al. 2013). Another
p53-related protein TAp63a promotes the glucose flux to
PPP to prevent oxidative stress in osteosarcoma cells
(DAlessandro et al. 2014). Overall, the p53 and its related
proteins regulate the cellular ROS homeostasis through
controlling the glucose flux to PPP. The other key regulator
of metabolic stress is the AMP-dependent protein kinase
(AMPK) that plays an important role in NADPH homeo-
stasis. AMPK is shown to be induced by low oxygen and
glucose starvation similar to the tumor condition (Ladero-
ute et al. 2006) and confers a tolerance to cancer cells
against metabolic stress (Kato et al. 2002). The underlying
mechanism is recently found that AMPK maintains
NADPH level by decreasing the NADPH consumption
through fatty acid biosynthesis by inactivating acetyl-CoA
carboxylases, ACC1 and ACC2, under the condition where
the pentose phosphate pathway is impaired (Jeon et al.
2012). More interesting report was that the AMPK can be
activated by H2O2-dependent oxidation in the cysteine
residues (Zmijewski et al. 2010). This is the only evidence
but it implicates a link between ROS and energy balance.
Targeting ROS-dependent energy metabolism
for cancer prevention
Based on the fact that the cancer cells produce more ROS
than the normal cells, there have been many trials for the
ROS-based cancer therapy (Trachootham et al. 2009).
Examples include the agents that intracellularly produce
ROS or target the antioxidant systems, such as SOD and
GSH. However, there is no case of an SOD or GSH treat-
ment that reduces cancer. The global level of intracellular
ROS in cancer cells is definitely dependent on the mito-
chondrial source (Newmeyer and Ferguson-Miller 2003).
Indeed, a genetic modification on mitochondrial respiratory
chain is often observed in various cancers and linked to the
increased cellular ROS level (Wallace 2012). In addition,
the polymorphism of mitochondrial genes, for example,
NQO1, also accompanies with the elevated ROS level and
can be a prognostic risk factor in various cancers including
breast and prostate cancers (Fagerholm et al. 2008; Zhang
et al. 2014; Ergen et al. 2007). However, targeting mito-
chondrial respiratory chain for cancer prevention is at a high
risk because the mitochondrial ROS is proportionally cor-
related to the respiratory activity even in normal cells (Wang
et al. 2008). Moreover, the cancer cell-specific energy
metabolism is distinct in that the glucose flux through the
PPP rather than the tricarboxylic acid cycle and the respi-
ratory chain is dominant in generating the reducing power
NADPH in cancer cells, especially with p53 mutation
(Vander Heiden et al. 2009). The worst scenario is that
NADPH is used for both ROS production and elimination
via NOX and Prx/Gpx, respectively. Hence, the ROS-based
anti-cancer strategy must be focused on a ROS network
composed of NADPH-dependent oxidases and peroxidases
in a cell type-specific context.
The first isoform of NOX family enzymes, now named as
NOX1, is a homolog of gp91phox (now NOX2 isoform) that
induces the cell transformation (Arnold et al. 2001; Suh
et al. 1999). The NOX family consists of 7 members, such as
NOX1 thru NOX5 and DUOX1/2. Among the family
members, NOX1 and NOX4 are well-documented related to
the cancer biology (Bonner and Arbiser 2012). Recent evi-
dence indicates a biological function of NOX enzymes in
inflammatory signaling as a partner of toll-like receptors
(Jeon et al. 2010; Yang et al. 2009; Park et al. 2006). Indeed,
the involvement of NOX enzymes in hepatocarcinoma and
ulcerative colitis is frequently reported (Parzefall et al. 2014;
Crosas-Molist et al. 2014; Treton et al. 2014). Thus, there
are much efforts on developing a NOX-targeting agent for
anti-cancer strategy. On the other hand, the expressional
alteration of peroxidase enzymes is also observed in various
cancer types (Lubos et al. 2011; Kang et al. 2005). Since
GPx enzymes are selenium-containing proteins, a correla-
tion between low selenium diet and cancer risk may involve
the GPx-1 activity. Moreover, the GPx-1 expression is
reduced in many cancer types (Cullen et al. 2003; Gladyshev
et al. 1998) and is associated with the DNA damage-related
tumorigenesis (Baliga et al. 2007). In contrast, the Prx
enzymes are highly expressed in most cancer cells (Chae
et al. 1999; Kang et al. 2005). More importantly, the Prx1
and Prx2 regulate the tyrosine phosphorylation induced by
growth factors, such as the platelet-derived growth factor
(PDGF) and epidermal growth factor (EGF), in cell types
such as primary fibroblast and vascular smooth muscle cells
(Woo et al. 2010; Choi et al. 2005). Prx2 also protects the
VEGFR2 kinase activity against the H2O2-dependent inac-
tivation (Kang et al. 2011). A mitochondrial isoform Prx3
exhibits an anti-apoptotic function in cancer cells treated
with tumor necrosis factor-a and staurosporine (Chang et al.
2004). Such experimental evidence suggests that the Prx
enzymes are likely to function as a signal regulator in cancer
cells. Recently, the first chemical inhibitor called
123

adenanthin, a diterpenoid isolated from the leaves of Rab-
dosia adenantha, targeting Prx1 and Prx2 is discovered to
induce the differentiation of acute promyelocytic leukemia
(APL) cells and suppress the tumor growth in vivo (Liu et al.
2012). Although it is shown later to generally target the
disulfide-containing proteins (Soethoudt et al. 2014; Much-
owicz et al. 2014), such evidence is significant in terms of
proposing a potential of Prx as a therapeutic target for cancer
prevention.
Concluding remarks
Since the mitochondrial electron transport chain is a major
ROS production site, its contribution to the intracellular
ROS level may depend on the metabolic flux into TCA and
b-oxidation in cancer cells. The expression of NOX
enzymes is another critical ROS producer because these
enzymes are highly expressed at subcellular compartments
in cancer cells. Such cellular ROS level is then counteracted
by endogenous antioxidant enzymes. Ironically, the ROS-
producing and eliminating enzymes utilize the same
NADPH pool, thereby raising a complexity between the
ROS homeostasis and energy metabolism in cancer cells.
Hence, we believe that the analyses of cancer cell-specific
metabolism along with the ROS production and elimination
systems will bring a new paradigm for cancer treatment.
Acknowledgments This study was supported by a Grant from the
National R&D Program for Cancer Control, Ministry of Health and
Welfare, Republic of Korea (1420280), the National Research Founda-
tion Grants (2014R1A2A1A01006934 & 2012M3A9C5048709), and the
Research Center for Cellular Homeostasis (2012R1A5A1048236) fun-
ded by the Korean government (MSIP).
Conflict of interest None declared.
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