Ageing Research Reviews 12 (2013) 376–390

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Ageing Research Reviews

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Review

Oxidative stress and cancer: An overview

Venus Sosa a , Teresa Moliné a , Rosa Somoza a , Rosanna Paciucci b , Hiroshi Kondoh c ,
Matilde E. LLeonart a,∗
a
Oncology and Molecular Pathology Group, Pathology Department, Institut de Recerca Hospital Vall d’Hebron, Passeig Vall d’Hebron 119-129, 08035 Barcelona, Spain
b
Unitat de Recerca Biomèdica, Institut de Recerca Hospital Vall d’Hebron, Passeig Vall d’Hebron 119-129, 08035 Barcelona, Spain
c
Geriatric Medicine Department, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Schogoin, Sakyo-ku, Kyoto 606-8507, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Reactive species, which mainly include reactive oxygen species (ROS), are products generated as a
Received 23 May 2012 consequence of metabolic reactions in the mitochondria of eukaryotic cells. In normal cells, low-level
Received in revised form 16 October 2012 concentrations of these compounds are required for signal transduction before their elimination. How-
Accepted 16 October 2012
ever, cancer cells, which exhibit an accelerated metabolism, demand high ROS concentrations to maintain
Available online 31 October 2012
their high proliferation rate. Different ways of developing ROS resistance include the execution of alter-
native pathways, which can avoid large amounts of ROS accumulation without compromising the energy
Keywords:
demand required by cancer cells. Examples of these processes include the guidance of the glycolytic path-
Cancer
Oxidative stress
way into the pentose phosphate pathway (PPP) and/or the generation of lactate instead of employing
Glycolysis aerobic respiration in the mitochondria. Importantly, ROS levels can be used as a thermostat to monitor
Cancer stem cells the damage that cells can bear. The implications for ROS regulation are highly significant for cancer ther-
Therapy apy because commonly used radio- and chemotherapeutic drugs influence tumor outcome through ROS
modulation. Moreover, the discovery of novel biomarkers that are able to predict the clinical response
to pro-oxidant therapies is a crucial challenge to overcome to allow for the personalization of cancer
therapies.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction pollutants, smoking and alcohol), and exercise. Reactive species

Aerobic respiration generates energy in the mitochondria of
eukaryotic cells, and as a result of this oxidative metabolism, several
compounds are produced. Most of these compounds are benefi-
cial; however, less than 5% of them can be toxic for the cell if
their concentration increases. These normally low-concentration
compounds that are derived from oxidative metabolism are nec-
essary for certain subcellular events, including signal transduction,
enzyme activation, gene expression, disulfide bond formation dur-
ing the folding of new proteins in the endoplasmic reticulum, and
the control of the caspase activity that is activated during the apop-
totic mechanism.
Sources of internal oxidative stress include peroxisomes and
enzymes, particularly the detoxifying enzymes from the P450 com-
plex, xanthine oxidase, and the nicotinamide adenine dinucleotide
(NADPH) oxidase complexes, which include the Nox family. Most
of these enzymes act in the mitochondria, which is the main
source of oxidative stress. External sources of oxidative stress
include UV radiation, chemical compounds (e.g., environmental
∗ Corresponding author. Tel.: +34 93489 4169; fax: +34 93274 6708.
E-mail addresses: matilde.lleonart@vhir.org, melleona@ir.vhebron.net
(M.E. LLeonart).
can be classified into four groups based on the main atom
involved: ROS, reactive nitrogen species (RNS), reactive sulfur
species (RSS) and reactive chloride species (RCS) (Bannister, 2007).
Of all the compounds derived from oxidative metabolism, ROS
are the most abundantly produced. Their half-lives range from
a few nanoseconds to hours, depending on the stability of the
molecule. ROS include superoxide anion (O2 − ), hydrogen perox-
ide (H2 O2 ), hydroxyl radical (OH− ), singlet oxygen (1 O2 ) and ozone
(O3 ) (Simic et al., 1989). ROS and RNS are produced during intracel-
lular metabolic processes, such as the electron transport chain. The
most abundant RNS is nitric oxide (NO− ), which is able to react with
certain ROS, including the peroxynitrite anion and ONOO− , which
is produced by the interaction between the superoxide anion and
nitric oxide; nitric oxide is later converted into peroxynitrous acid
and ultimately into a hydroxyl radical and nitrite anion (NO2 − ).
The damage that these ROS can cause to the cell not only
depends on their intracellular concentration but also on the equi-
librium between the ROS and the endogenous antioxidant species.
When the pro-oxidant/anti-oxidant equilibrium is lost, oxidative
stress is generated, altering and damaging many intracellular
molecules, including DNA, RNA, lipids and proteins (Veskoukis
et al., 2012). These reactive species cause nicks in the DNA and mal-
functions in the DNA repair mechanism. DNA oxidation by these
reactive species generates 8-hydroxy-2 -deoxyguanosine, which is

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 V. Sosa et al. / Ageing Research Reviews 12 (2013) 376–390
a product that is able to generate mutations in DNA in a process that
enhances aging and carcinogenesis (Matsui et al., 2000). Moreover,
the cell membrane is rich in polyunsaturated lipids that are suscep-
tible to oxidation by reactive species. Reactive species liberate lipid
peroxidation reactions and consequently increase the permeability
of the cell membrane, which could lead to cell death (Halliwell and
Chirico, 1993). Proteins are the most affected by a cellular environ-
ment with a high concentration of reactive species. Proteins suffer
from the generation and accumulation of carbonyl groups (i.e., alde-
hydes and ketones) and thiol groups (–SH) that may be converted
into sulfur reactive radicals (Levine, 2002). Due to this oxidation-
induced modification, there is an alteration in the protein structure
and, consequently, changes or loss of protein function.
Natural antioxidants are the cell’s defense mechanisms that
scavenge reactive species, and they can be classified into differ-
ent groups according to their properties: endogenous antioxidants,
natural antioxidants and synthetic antioxidants. Endogenous
antioxidants include glutathione, alpha-lipoic acid, coenzyme Q,
ferritin, uric acid, bilirubin, metallothionein, l-carnitine, melatonin,
enzymatic superoxide dismutase (SOD), catalase (CAT), glutathione
peroxidases (GPXs), thioredoxins (TRX) and peroxiredoxins (PRXs).
PRXs are a ubiquitous family of antioxidant enzymes (PRX I-VI) that
also control cytokine-induced peroxide levels and mediate signal
transduction in mammalian cells. For example, PRX III scavenges
up to 90% of H2 O2 , and PRX V behaves more effectively as a scav-
enger of peroxynitrite. Natural antioxidants coexist in a delicate
balance with oxidative inputs. Other antioxidants can be obtained
from the diet, such as ascorbic acid (Vitamin C), tocopherol (Vita-
min E), ␤-carotene (Vitamin A), lipoic acid, uric acid, glutathione
and polyphenol metabolites. Examples of synthetic antioxidants
include N-acetyl cysteine (NAC), tiron, pyruvate, selenium, buty-
lated hydroxytoluene, butylated hydroxyanisole, and propyl gallate
(Yoshida et al., 2003).
Oxidative stress is important from a biomedical point of view
because it is related to a wide variety of human diseases, such
as neurodegenerative disease (e.g., Alzheimer’s, Parkinson’s, and
amyotrophic lateral sclerosis), inflammatory disease (e.g., rheuma-
toid arthritis), cardiovascular disease (e.g., muscular dystrophy),
allergies, immune system dysfunctions, diabetes, aging and can-
cer. For example, inflammatory cells release chemical mediators of
inflammation, particularly ROS, in swollen tissue, which also affects
normal cells. When this is a chronic process, the extremely high
ROS levels saturate the cell defense mechanisms (i.e., antioxidants),
and intracellular molecules become seriously damaged, affecting
surrounding neighboring cells.
The mechanisms and pathways involved in oxidative stress are
conserved in mammalian cells. ROS can promote many aspects of
tumor development and progression, which can be classified into
the following biological processes: (a) cellular proliferation (e.g.,
extracellular-regulated kinase 1/2 (ERK1/2) activation and ligand-
independent RTK activation), (b) evasion of apoptosis or anoikis
(e.g., Src, NF-␬B and phosphatidylinositol-3 kinase (PI3K)/Akt acti-
vation), (c) tissue invasion and metastasis (e.g., metalloproteinase
(MMP) secretion into the extracellular matrix (ECM), Met overex-
pression, and Rho-Rac interaction), and (d) angiogenesis (e.g., the
release of vascular endothelial growth factor (VEGF) and angiopoi-
etin).
Regarding cellular proliferation, oxidative stress affects sev-
eral biochemical pathways (from epidermal growth factor receptor
(EGFR) to mTOR) that involve key signaling proteins, such as nuclear
factor erythroid 2-related factor 2 (Nrf2), kelch-like protein 19
(Keap1), Ras, Raf, mitogen activated protein kinases (MAPK) such as
ERK1/2, MEK, p38␣, c-Jun N-terminal kinase (JNK), c-myc, p53 and
PKC (Matsuzawa and Ichijo, 2008; Nguyen et al., 2009; Wiemer,
2011). Among them, Nrf2 is considered to be the master regula-
tor of the antioxidant response, but others are also important. For
377
example, p38␣ acts as a key sensor of oxidative stress, and its redox-
sensing function is essential in the control of tumor development
(Luo et al., 2011). In contrast to other MAPKs, p38␣ suppresses
tumorigenesis by blocking proliferation or promoting apoptosis.
This review is focused on the relationship between oxidative
stress and cancer from both the molecular and clinical point of
views.
2. Metastasis-related processes that influence oxidative
stress
2.1. Epithelial-mesenchymal transition (EMT)
An important event that leads to metastasis is EMT, which is a
biological process by which epithelial cells undergo biological and
chemical alterations that permit the development of a more aggres-
sive mesenchymal phenotype (Mani et al., 2008). These changes
are associated with an increase in ECM proteins, which provide
the cells with migratory properties and allow them to move to
other regions of the body via the bloodstream. There are vari-
ous ROS-associated signaling pathways that are implicated in the
EMT process. Among these pathways, the proteins Smad (activated
by tumor growth factor ␤ (TGF-␤)), Snail, E-cadherin, ␤-catenin,
integrin, matrix metalloproteinases (MMPs), hepatocyte growth
factor receptor (HGFR/c-Met), AP1 (activated by PKC activator),
v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets-1) and
transforming growth factor ␤-activated kinase 1 (TAK1) are mostly
activated in a ROS-dependent manner (Haorah et al., 2007; Ni et al.,
2007; Omori et al., 2012). One of the first studies that established
a direct connection between ROS and EMT is related to TGF-␤
signaling. TGF-␤ activation provokes an increase in intracellular
ROS in a process that is associated with the phosphorylation of
Smad2, p38␣ and ERK1/2 (Rhyu et al., 2005). Moreover, intra-
cellular ROS may regulate EMT through a mechanism involving
NF-␬B in strict collaboration with hypoxia-inducible factor 1 (HIF-
1␣) and cyclooxygenase-2 (COX-2). The role of ROS as a crucial
EMT mediator is further corroborated by MMP-3 (matrix metal-
loproteinase 3), which is also known as Stromelysin-1. MMP-3 is
an enzyme that is involved in the breakdown of the extracellular
matrix, which plays a key role in tumor metastases by degrading
collagen, fibronectin, and laminin. MMP-3 has been reported to be
upregulated in certain tumors, such as breast cancer, and is an EMT
inducer in transgenic mice. In mice, MMP-3 secretion is associ-
ated with Snail upregulation, E-cadherin loss and ␤-catenin nuclear
translocation events, which, in turn, are dependent on the small
GTP-binding protein Rac 1 (Rac1b) (Radisky et al., 2005; Rhyu et al.,
2005). In addition to MMP-3, other metalloproteinases, including
MMP-2 and MMP-9, play important roles in Rac1b stimulation
to influence ROS. Most of these proteins are oncogenic in mouse
models, and their overexpression contributes to human tumorige-
nesis.
Other cellular pathways involved in metastasis are also targeted
by ROS, such as Wnt/TCF (T cell factor), integrin-mediated MAPK
signaling, protein kinase C (PKC), protein tyrosine phosphatases,
and p21-activated kinase 1 (PAK-1) signaling pathways (Almeida
et al., 2007; Lee and Esselman, 2002). For example, ROS oxidize cys-
teine residues in the PKC protein and inactivate protein tyrosine
phosphatases, which are important molecules in tumor cell inva-
sion. ROS also regulate PAK-1, which is involved in Rac-associated
cytoskeleton remodeling, which is directly linked to metastasis and
angiogenesis (Diebold et al., 2010). Proteins such as Snail, TGF-
␤, Twist, Wnt/␤-catenin and Zinc finger E-box-binding homeobox
1 (Zeb), which induce EMT, also modulate the tumor microen-
vironment. Rac1b enhances the expression of Zinc finger protein
SNAI1 (Slug), a transcriptional inhibitor of E-cadherin that can ease
378 V. Sosa et al. / Ageing Research Reviews 12 (2013) 376–390
tumorigenesis by activating the disheveled-3-mediated Wnt/␤-
catenin signaling pathway (Esufali et al., 2007). Aberrant activation
of the Wnt/␤-catenin pathway promotes tumorigenesis in several
cancer types and mainly has effects in breast and colorectal cancers
(Jeong et al., 2012).
In particular, cancer stem cells (CSCs) are rare cells that have
many characteristics in common with stem cells (SCs), including
an indefinite potential for self-renewal and differentiation; thus,
they have been proposed as the cells that drive tumorigenesis (Reya
et al., 2001). Evidence for EMT has been described for CSCs (Chen
et al., 2011). This link between SC/CSC markers and the EMT phe-
nomenon has implications in cancer therapy and cancer remission
as a whole. The relationship between CSCs and ROS will be dis-
cussed elsewhere.
2.2. Angiogenesis
Angiogenesis, which is the process of new blood vessel forma-
tion from pre-existing vessels, is an important biological process
for tumor growth and metastasis (Ushio-Fukai and Nakamura,
2008; Folkman, 1995). Tumors produce many pro-angiogenic fac-
tors, such as vascular endothelial growth factor (VEGF) and its
receptor (VEGFR), MMPs, angiopoietin-1, fibroblast growth factor
(FGF), interleukin-8 (IL-8), platelet-derived growth factor (PDGF)
and TGF-␤. Specifically, VEGF, which is highly upregulated in most
human cancers, has recently emerged as the crucial rate-limiting
protein in the regulation of angiogenesis. Hypoxic conditions, ROS
production, growth factors, and cytokines all increase VEGF levels
in many human tumors (Fei et al., 2009). Anti-angiogenic therapies
have especially focused on using antibodies and tyrosine kinase
inhibitors to block VEGF and VEGFR2 (Carmeliet and Jain, 2000;
Folkman, 1995).
Other ROS-generating enzymes, such as NADPH oxidases (e.g.,
Nox: Nox1-5), activate redox signaling pathways that ultimately
lead to angiogenesis (Arnold et al., 2001; Tojo et al., 2005). For
example, Nox1 is overexpressed in colon and prostate cancers, and
it induces the production of H2 O2 , which augments VEGF, VEGFR
and MMP levels (Lim et al., 2005). Nox4 and Nox5 are increased
in melanoma and prostate cancers, respectively, and act in a simi-
lar manner as Nox1 (Govindarajan et al., 2007). ROS derived from
these oxidases result in VEGFR2 autophosphorylation, which pro-
motes the induction of transcription factors and genes involved
in angiogenesis. These reports underscore the important role for
ROS in the pathophysiological states involved in neovasculariza-
tion, tumor development and progression.
2.3. Hypoxia
Hypoxia is a pathological condition in which cells are deprived of
an adequate oxygen supply, despite proper blood perfusion in a tis-
sue. Cancer cells have high proliferation rates, which often generate
a more hypoxic environment if new blood vessels are not rapidly
generated. To avoid hypoxic conditions, cancer cells must upregu-
late pro-angiogenic genes because the stimulation of angiogenesis
creates a new blood supply. Under normoxia, HIF-1␣ and HIF-2␣
proteins are ubiquitinated through an oxygen-dependent interac-
tion with the von Hippel-Lindau protein (pVLH) and degraded by
the 26S proteasome. However, under hypoxic conditions, HIF-1␣
and HIF-2␣ proteins are not ubiquitinated, leading to their accu-
mulation, translocation into the nucleus, dimerization with HIF-1␤
and transactivation of target genes. Moreover, in cancer cells, Nox-
derived ROS enzymes are involved in the induction of the HIF-1␣
subunit by activating the PI3K/Akt/p706K and the MEK/ERK path-
ways (Shi et al., 2005; Skinner et al., 2004). The MAPK signaling
downstream of Ras has been shown to lead to HIF-1␣ phosphory-
lation via a process that stimulates its translational activity. Cancer
cells adapt to hypoxic conditions, which occur in the core of most
solid tumors, by upregulating HIF-1␣ and other pro-angiogenic
genes (e.g., VEGF and erythropoietin) and transcription factors that
are involved in cell survival, invasion, and glycolysis (Liao and
Johnson, 2007).
Lastly, glioblastoma CSCs express HIF-2␣, while HIF-1␣ is
expressed under more severe hypoxic conditions (Soeda et al.,
2009). These two proteins have non-redundant biological roles
because they regulate unique target genes. Importantly, hypoxia
induces the expression of genes related to SC functions, such as
Sox2 and Oct-4, which define a stemness signature (McCord et al.,
2009). The relevance of this issue at the therapeutic level comes
from the fact that CSCs possess a higher resistance to hypoxia com-
pared to other cancer cells, which is fact that may explain their
enhanced radio- and chemoresistance.
3. The relationship between oncogenes and tumor
suppressor genes and oxidative stress
Genetic factors play key roles in the transforming events that
lead to cancer development. The high metabolism of cancer cells is
generally associated with an increase in ROS; however, such levels
are less deleterious in cancer cells than they would be in normal
cells. For example, although the ROS level increases by a modest
degree, tumorigenic cells can induce a new redox balance, resulting
in cellular adaptation and proliferation. This is a striking feature of
cancer cells that allows for the acquisition of resistance to oxidative
stress inducers relative to normal cells. The resistance to oxidative
stress is one of the major adaptive advantages that permit cancer
cells to increase their metabolic rate and proliferation and to escape
free radical damage. However, this adaptive response to high doses
of ROS is not enough to explain the high metabolic rate of cancer
cells.
Genetic alterations in genes that affect cancer cells are able to
directly or indirectly modulate ROS. Apart from the protection pro-
vided by specific antioxidant enzymes, such as SOD, CAT, GPXs,
TRXs and PRXs, the master regulator of the antioxidant response
is the transcription factor Nrf2. Nrf2 modulates the expression
of hundreds of genes, including not only the familiar antioxidant
enzymes but also a large number of genes that control several
processes, including immune and inflammatory responses, tissue
remodeling and fibrosis, carcinogenesis, and metastasis (Hybertson
et al., 2011). ROS levels are tightly controlled and predominantly
regulated by Nrf2 and its repressor protein Keap1. Moreover, when
treated with oxidation-inducing drugs, mice that lack Nrf2 develop
more severe intestinal inflammation than controls, with increased
aberrant crypts, suggesting a function for Nrf2 in the prevention
of inflammation and carcinogenesis (Khor et al., 2006). Initially, it
was thought that Nrf2 was capable of regulating oxidative stress
levels by modulating the production of Antioxidant Response Ele-
ment (ARE)-regulated antioxidant enzymes. However, alternative
mechanisms for Nrf2 activation were further described, and they
are dependent upon kinase pathways, such as MAPK, PI3K, and
other pathways (Yu et al., 1999; Kang et al., 2002). Importantly,
somatic mutations that disrupt the Nrf2-Keap1 interaction were
recently identified in cancer patients (Singh et al., 2006; Kim et al.,
2010b). In breast cancer, the tumor suppressor gene breast can-
cer gene 1 (BRCA1) is mutated in 40–50% of hereditary breast
cancers and absent or expressed at low levels in 30–40% of spo-
radic breast cancers (Rosen et al., 2003). BRCA1 is a caretaker gene
that is responsible for repairing DNA, and it is able to upregulate
several genes involved in the antioxidant response by controlling
the activity of the transcription factors Nrf2 and Nf␬B (Bae et al.,
2004; Benezra et al., 2003). In addition, Nrf2 induces cytoprotective
enzymes, such as GST, GPx and oxidoreductases, which help cells
 V. Sosa et al. / Ageing Research Reviews 12 (2013) 376–390
to combat oxidative stress (Banning et al., 2005; Kohle and Bock,
2007). Apart from the inhibitory action of BRCA1 on ROS genera-
tion, BRCA1 also reduces the levels of protein nitration due to RNS
accumulation in cells, and it enhances DNA repair processes that
ultimately help to cope with oxidative stress. In breast cancer cells,
the redox factor 1/AP endonuclease 1 (Ref1/APE1) also participates
in reducing the generation of ROS (Seo and Kinsella, 2009).
One of the most important pathways related to oxidative stress
and cancer is the Ras pathway (Yagoda et al., 2007). In humans,
the Ras gene family consists of three small G proteins, Ha-, N-
and Ki-ras, which participate in cell signaling (Malumbres and
Barbacid, 2003; Yagoda et al., 2007). Important for its relationship
with ROS is the fact that the main mechanism of Ras activation
in tumors involves point mutations (i.e., at specific amino acids
that result in changes at positions 12, 13 and 61). Approximately
30% of human tumors contain activating mutations in the Ras fam-
ily of oncogenes, rendering a protein constitutively active (Bos,
1989). Several studies have demonstrated that mutant Ras leads
to an increase in ROS levels, which causes DNA damage and con-
tributes to transformation (Maciag et al., 2004; Rai et al., 2011).
Mutant RasVal12 positively regulates the NADPH oxidase system
Nox4-p22phox , which produces H2 O2 , in a process accompanied
by ␥-H2A.X (foci of heterochromatin). Accordingly, knockdown
of NADPH oxidase decreases the RasVal12 -induced DNA damage
response. Therefore, the NADPH oxidase Nox4 is a critical medi-
ator of oncogenic RasVal12 -induced DNA damage (Weyemi et al.,
2012). Cells that overexpress oncogenic Ras display increased mito-
chondrial mass and ROS accumulation. The ROS generated by the
respiratory chain in the mitochondria and by the Nox enzymes in
the cytoplasm are particularly important. In fact, Nox proteins are
now considered to be oncogenic proteins, and mitochondrial dys-
function is associated with tumorigenesis (Graham et al., 2010).
Mitochondrial dysfunction is also accompanied by DNA damage, a
drop in ATP levels, and protein kinase AMPK activation. The consti-
tutive activation of mutant K-rasVal12 in non-transformed epithelial
cells leads to a highly significant increase in the levels of peroxides
and an increase in the amount of DNA strand breakage compared
with control cells. Peroxides may also be directly generated by COX-
2 because COX-2 activity correlates with K-ras activity (Wang et al.,
2009). COX-2, one of the rate-limiting enzymes in the metabolism
of arachidonic acid, is reported to be involved in the pathogenesis
of many human tumors. Both peroxide generation and DNA single-
strand breaks are significantly reduced by pre-treatment with the
COX-2 specific inhibitor SC58125. Therefore, the dominant onco-
genicity of mutant K-rasVal12 can be influenced by several genes,
such as COX-2 and/or HIF-1␣, which is a transcription factor that
is activated in response to low oxygen concentrations, and its ulti-
mate influence is on DNA damage.
Moreover, the overexpression of oncogenic proteins (i.e., Raf,
Mos, MEK, Myc, Cyclin E and Telomerase reverse transcriptase
(hTERT)) and the silencing of tumor suppressor genes (i.e., p53,
p21CIP1 and phosphatase and tensin homolog (Pten)) can induce
senescence by increasing ROS. Pten loss and Ras/MAPK activation
cooperate to promote EMT and metastasis that are initiated from
prostate progenitor cells (Mulholland et al., 2012). Pten-deficient
glioblastoma cells, with high levels of Akt and ROS, undergo senes-
cence, and such high ROS levels are indispensable for the observed
phenotype (Lee et al., 2011). While the conventional oncogenic role
of hTERT has been linked to its ability to provide immortalization
in human cells by acting through the telomeres, evidence is accu-
mulating that supports the contribution of non-canonical hTERT
activity in cancer (Indran et al., 2010). To that end, hTERT has been
implicated in redox-mediated events, and its expression has been
shown to impact the cellular redox status via the recruitment of
mitochondria, which are a critical intracellular source of ROS. Fur-
ther evidence supporting the role of mitochondria in hTERT biology
379
comes from findings that hTERT is localized in the mitochondria and
the ability of hTERT inhibitors to induce mitochondrial-dependent
apoptosis in target cells (Indran et al., 2010).
From a genetic point of view, many genes are implicated in the
modulation of energy metabolism and cancer. For example, p53,
one of the most frequently mutated genes in human cancers, mod-
ulates the balance between glycolysis and the utilization of the
respiratory chain in the mitochondria. A major mediator of this
effect is cytochrome oxidase deficient homolog 2 (SCO2), whose
expression is critical for regulating the COX complex, the main
site of oxygen utilization in eukaryotic cells (Matoba et al., 2006).
Reduced SCO2 synthesis may cause low respiration and high gly-
colytic rates.
Sirtuins are a class of proteins that possess histone deacety-
lase activity and are implicated in aging, transcriptional regulation,
apoptosis, stress resistance, energy efficiency, and alertness during
low-calorie situations. Sirtuins are NAD-dependent deacetylases
whose enzymatic activity is regulated by the ratio of NAD to NADH.
High NAD levels activate sirtuins, while high NADH levels inhibit
them. Of the three mitochondrial sirtuins, Sirt3 is the most studied,
and it has been linked to longevity in humans, acting as a tumor
suppressor protein (Kim et al., 2010a). Kim and colleagues demon-
strated that with the expression of a single oncogene, such as Myc
or Ras in Sirt3 (−/−) murine embryonic fibroblasts (MEFs), the cells
exhibited increased glycolysis, decreased oxidative phosphoryla-
tion, and increased ROS. Moreover, the absence of Sirt3 increases
tumorigenesis in cancer cells in a ROS-dependent manner (Bell
et al., 2011). This process is accompanied by HIF-1␣ target gene
activation under hypoxic conditions.
To identify new genes implicated in tumorigenesis, our group
has performed functional genome-wide screens using the retro-
viral delivery of cDNA libraries. By these means, we found that
non-glycosylated membrane associated protein 17 (MAP17) was
associated with tumor malignancy due to its capacity to promote
ROS production. In fact, we demonstrated that antioxidant treat-
ment is useful in decreasing the tumorigenic properties that these
cells acquired (Guijarro et al., 2007). Another gene identified was
phosphoglycerate mutase 1 (PGM), which codifies for an enzyme
involved in glycolysis and was independently found to be related to
ROS (Kondoh et al., 2005; Guijarro et al., 2007). The action of PGM
was also found to decrease ROS levels by significantly stimulating
the immortalization of murine cells, thereby increasing glycoly-
sis (Kondoh et al., 2005). Furthermore, inherited defects in DNA
repair genes correlate with increased oxidative stress and prema-
ture aging (Barzilai et al., 2002). This is the case for ATM deficiency
in patients with ataxia telangiectasia, which is a disease related to
premature aging and cancer. Unraveling the status of oncogenes
and tumor suppressor genes related to ROS modulation in cancer
patients promises to be beneficial in the development of successful
therapies.
4. Environmental factors
In the last few decades, our view of malignancy has broad-
ened considerably in relation to the contribution of environmental
factors and the tumor microenvironment to cancer development.
Hormones, such as estrogens, are important in the development
and progression of human breast cancer because they can be
transformed into the carcinogenic metabolite 4-hydroxyestradiol
(4-OHE2 and 2-OHE2 ) by cytochrome P450 1B1 (Shao et al., 2008).
Both metabolites can be further oxidized into a reactive quinone
that can react with DNA. The compounds 4-OHE2 and 2-OHE2
and their quinone derivatives are able to generate oxidative stress
(Cavalieri et al., 2006). Hormone-dependent tumors (e.g., estro-
gen, androgen and progesterone) affect cell proliferation through
380 V. Sosa et al. / Ageing Research Reviews 12 (2013) 376–390

Fig. 1. The tumor microenvironment influences angiogenesis and tumor progression. ROS produced in cancer cells are released into the tumor microenvironment and
detected by adjacent fibroblasts (CAFs), initiating the onset of stromal oxidative stress, autophagy and mitophagy due to the activation of key transcription factors (i.e., HIF-
1␣) and signaling proteins (i.e., VEGF) that will ultimately contribute to angiogenesis. Moreover, CAFs also release MMPs and cytokines (i.e., IL-6, IL-10, TGF-␤, CCL2 and CCL5),
which stimulate tumor growth and block the natural immune response against cancer. Additionally, senescent cells can release cytokines into the tumor microenvironment,
which stimulate tumor growth. Immune surveillance can prevent and/or promote the growth and progression of cancer. For example, within the immune system, cytotoxic
CD8+ and CD4+ T cells, along with their characteristically produced cytokine IFN-␥, function as the major anti-tumor immune effector cells (−), whereas cancer-associated
macrophages (CAMs) and their derived cytokines IL-6, TNF, IL-1␤ and IL-23 are generally recognized as dominant tumor-promoting forces (+).

their ligand-receptor interactions, and their expression is crucial
for determining a specific therapy (Allred et al., 2012). The actions
exerted by estrogen and its catabolites are essential processes in
breast carcinogenesis. In its physiological concentration, estradiol
is able to decrease the CAT enzyme and increase the GPX activities
in breast epithelial cells. Thioredoxin and thioredoxin reductase can
alter hydrogen peroxide levels and estrogen activity in estrogen-
responsive breast cancer cells (Hayashi et al., 1997). Oxidative
stress has been shown to participate in the structural modifica-
tion of the estrogen and progesterone receptors, altering the clinical
evolution of patients with endocrine-responsive breast cancer (Yau
and Benz, 2008). The cysteine residues of the zinc finger transcrip-
tion factor Sp1 and the estrogen receptor (ESR1) are highly prone
to being oxidized by ROS, losing their structural and functional
properties, including their DNA-binding activity. For example, this
failure has been observed in 30–35% of ER-positive breast cancers,
and it was found to be related to a loss of the progesterone receptor.
Zinc finger transcription factors are necessary for the estrogenic
stimulation of certain genes, such as the progesterone receptor
(PGR) and Bcl-2, and estrogen stimulation is inhibited by oxidative
stress. This fact would represent a problem for therapeutic inter-
vention because hormone-dependent tumors have the advantage
of being treated by an analog molecule that blocks ligand-receptor
signaling, which downregulates the proliferative cascade.
Regarding the tumor microenvironment, it is important to
consider that cancer cells are surrounded by other cell types
that facilitate tumor growth. Tumor microenvironments consist of
stromal cells (e.g., myofibroblasts and/or cancer-associated fibro-
blasts (CAFs)), vascular cells (e.g., erythrocytes) and immune cells
(e.g., lymphocytes, natural killer cells, antigen-presenting cells and
cancer-associated macrophages (CAMs)). CAFs have been shown to
contribute to tumor growth and affect cancer survival by (i) con-
tributing to the vasculature by secreting VEGF and angiopoietin,
(ii) generating anti-apoptotic factors, (iii) promoting cell motil-
ity and metastasis by secreting chemokines (e.g., CCL2, CCL5) and
MMPs, and (iv) blocking the immune response through the secre-
tion of IL-6, IL-10 and TGF-␤ (Kayamori et al., 2010; Tsuyada et al.,
2012) (Fig. 1). The importance of CAFs in mediating paracrine
signaling comes from the fact that conditioned media from stromal
fibroblasts that are deficient for the Caveolin (Cav-1) gene, a nega-
tive cytokine receptor signaling regulator, promote EMT, inducing
a mesenchymal appearance in human breast cancer-associated
fibroblasts (Sotgia et al., 2009). Other cells in the tumor microen-
vironment can act independently or synergistically with CAFs,
including CAMs and/or senescent fibroblasts. It is striking that pro-
teins produced by senescent cells can have opposite effects if they
act as autocrine or paracrine factors (Fig. 1). Moreover, senescent
cells secrete pro-inflammatory cytokines and proteases that induce
cancer cells to promote tumor aggressiveness. In this context, can-
cer cells become “parasitic”: the ROS produced in cancer cells are
released into the tumor microenvironment and are detected by
adjacent fibroblasts, initiating the onset of stromal oxidative stress,
autophagy and mitophagy due to the activation of key transcription
factors, such as HIF-1␣ and NF-␬B, which contribute to angiogen-
esis (Toullec et al., 2010). Furthermore, cancer cells may use the
nutrients from autophagic stromal fibroblasts to fuel the oxida-
tive mitochondrial metabolism to generate large amounts of ATP
and protect themselves from apoptosis. At the same time, cancer
cells may establish a strong antioxidant defense by overexpressing
proteins, such as PRXs, or by undergoing TP-53-induced glycol-
ysis (TIGAR). In this model, cancer cells released into the tumor
microenvironment induce the death of healthy cells and ultimately
 V. Sosa et al. / Ageing Research Reviews 12 (2013) 376–390 381

Fig. 2. Glucose metabolism has different destinies. Under aerobic conditions, glucose will produce pyruvate, which is used by the mitochondria for respiration and ATP
production. Under anaerobic conditions, glucose will travel into the PPP and/or produce lactate, but its degradative products will not enter into the mitochondria. Glucose
metabolism through the PPP produces a reduced form of NADPH, which maintains glutathione in the reduced state. Under physiological conditions, the principal source
of ROS is mitochondria. During this process, superoxide is generated and immediately converted into H2 O2 by manganese superoxide dismutase (MnSOD), an important
antioxidant enzyme in mitochondria that protects against oxidative stress. H2 O2 must be rapidly eliminated by GPX, PRX or CAT. Glycolysis through the PPP pathway is the
standard response to a lack of oxygen in the cellular microenvironment.

benefit from the discarded metabolites of these cells, which are
captured by the cancer cells and used to maintain their own prolif-
eration at a high rate.
5. Glycolysis versus oxidative phosphorylation: Cancer
bioenergetics
Glycolysis is the initial metabolic pathway of cellular respi-
ration, and it consists of a series of reactions that result in the
conversion of glucose into pyruvic acid with the concomitant pro-
duction of energy (Bar-Even et al., 2012). After glycolysis, there
are two branches of the pathway, depending on whether the
cell chooses to undergo aerobic or anaerobic respiration. Under
standard conditions, in which oxygen (O2 ) is abundant, and for
long-term maintenance, healthy cells choose aerobic respiration.
In aerobic respiration, which takes place in the cytoplasm and
mitochondria, oxygen and glucose are used to generate 38 adeno-
sine tri-phosphate (ATP) molecules and carbon dioxide (CO2 ) is
released and O2 is absorbed. In contrast, in anaerobic respiration,
which occurs only in the cytoplasm, 2 molecules of ATP are syn-
thesized in the electron transport chain, using inorganic molecules
other than O2 ; CO2 is released, but O2 is not required (Fig. 2).
Although anaerobic glycolysis is energetically less efficient, the
ATP generation in this process is faster than that in aerobic gly-
colysis associated with the tricarboxylic acid (TCA) cycle (or Krebs
cycle). Anaerobic glycolysis generates lactate, and due to the low
levels of energy produced (i.e., ATP), only low levels of ROS are
produced, resulting in cellular benefits. In contrast, while aerobic
metabolism enables efficient energy production, high levels of ROS
are also generated, which damage key cellular components. In prin-
ciple, one could expect that, depending on the O2 concentration
in the microenvironment, cells may choose one pathway or the
other. However, cancer cells in particular choose the most conve-
nient way to increase proliferation, which is determined by their
genetic background (oncogenes and tumor suppressor genes) and
the tumor microenvironment, independent of O2 levels.
Dr. Otto Warburg discovered that cancer cells display a high gly-
colytic rate, even under high concentrations of O2 (20%), a finding
later named the Warburg effect in his honor (Warburg, 1956; Kim
and Dang, 2006). The molecular mechanism behind this effect still
needs to be completely elucidated. Importantly, the Warburg effect
is exploited in the clinic to detect primary or metastatic tumors in
patients. The positron-emission tomography (PET) scanning tech-
nique is used to detect highly glycolytic areas in the body mass
by measuring 2-[18 F] fluoro-2-deoxy-d-glucose, a glucose analog
that is concentrated in highly proliferative tumorigenic masses
(Pritchard et al., 2012).
This high glycolytic rate, or the Warburg effect, is interpreted
as an accelerated glucose metabolism that is accompanied by an
increase in glycolytic enzymes, including PGM and glucose phos-
phate isomerase (GPI), events not observed in normal cells. A
possible explanation of the Warburg effect lies in the fact that in
normal differentiated cells, the need for division is low. Glucose
metabolism is therefore optimized for efficient energy genera-
tion: glycolysis is linked to the TCA cycle in mitochondria. Feeding
substrates to the TCA cycle is critical for the generation of ATP,
lipids and ROS. We and others suggest that the major role of
glycolysis in cancer cells is to provide substrates to the PPP for
nucleotide synthesis, a great priority considering their high division
rate (Weinberg and Chandel, 2009; Kondoh et al., 2007a). In this
way, the high glycolytic rate of tumor cells confers an advantage,
as they are hardly exposed to oxidative stress by avoiding ROS gen-
eration. Additionally, the production of NADPH by the PPP is used
by the cell in reductive biosynthesis reactions in the prevention
of oxidative stress (Kondoh et al., 2005; Vaughn and Deshmukh,
2008). This result is supported by specific findings, such as the
enhancement of key enzymes of the PPP in cancer cells (Vizan
et al., 2009). Thus, in primary wild-type cells, the Warburg effect not
382 V. Sosa et al. / Ageing Research Reviews 12 (2013) 376–390
only confers protection from oxidative damage, but this high gly-
colytic flux is translated into a longer life span with the avoidance
of ROS-induced senescence, whereas glycolysis inhibition triggers
early senescence (Kondoh et al., 2005). The Warburg effect also
triggers local acidosis, requiring tumors to selectively develop acid
resistance (Gatenby and Gillies, 2004). According to the “parasitic”
cancer cell hypothesis, this acidosis may dramatically affect CAFs,
but not cancer cells, because these cells are prepared to manage
acidosis and hypoxia.
Another example of the Warburg effect and oxidative damage
are fibroblasts that are deficient for cytoplasmic polyadenyla-
tion element binding protein (CPEB) protein, which have fewer
mitochondria compared with wild-type cells, but have a high gly-
colytic rate (Burns and Richter, 2008). Moreover, in p53 null cells,
aerobic respiration is reduced and glycolysis is elevated. Some
downstream genes whose expression is regulated by p53, includ-
ing SCO2, TIGAR, or p53 inducible gene 3 (PIG3), may be responsible
for the changes related to energy metabolism (Matoba et al., 2006;
Won et al., 2012; Kotsinas et al., 2012).
Alternative explanations for the Warburg effect in tumors
include an optimization of glucose usage, the presence of a scarce
metabolite in the tumor microenvironment, or an impairment
in mitochondrial function. Overall, this high glycolytic rate is an
advantage for the Darwinian selection of tumors in vivo; therefore,
glycolytic inhibiting drugs may provide a benefit for cancer therapy
(Gatenby and Gillies, 2004).
In contrast, mitochondrial oxidative phosphorylation
(OXPHOS), the major energy source in aerobic organisms, is a
process by which glucose or other molecules (i.e., glutamine,
glycine, alanine, glutamate, proline and alternatively, fatty acids,
ketone bodies, short chain carboxylic acids, propionate, acetate
and butyrate) are oxidized to CO2 and H2 O (Fig. 2). Electrons
from NADH or FADH2 , which are produced in glycolysis, fatty acid
oxidation, and the citric acid cycle, are transferred to O2 by electron
carriers located in the mitochondrial inner membrane, leading
to the pumping of protons out of the mitochondrial matrix. The
consequent unbalanced proton distribution creates a pH gradient
and a transmembrane electrical potential that generates a proton-
motive force. ATP synthase produces ATP when protons flow back
into the mitochondrial matrix (Berg, 2002). Mitochondrial energy
metabolism, which is necessary to produce ROS, is disrupted in
many cancer cells because mitochondrial DNA mutations exist in
tumorigenic cells. Remarkably, mitochondrial DNA mutations pro-
mote tumorigenesis via alterations in ROS generation, ultimately
influencing the apoptotic response (Park et al., 2009).
A great variety of different cancer bioenergetic signatures have
been described in tumorigenic cells. Some theories suggest that gly-
colysis and OXPHOS cooperate in cancer cells to provide these cells
with the needed energy (Smolkova et al., 2010). Studies of cancer
cells revealed that mitochondrial OXPHOS contributed to 79% of
the required energy; this percentage decreased to 30% in hypoxic
conditions (Guppy et al., 2002; Hervouet et al., 2008). A high
glycolytic rate, which is associated with a rapid tumor growth pat-
tern, leads to increased tumor cell malignancy, and OXPHOS is the
alternative choice for limited glucose conditions. Several key onco-
genes/tumor suppressor genes that are involved in proliferation
participate in the stimulation of glycolytic metabolism, including
HIF-1␣, Ras, c-myc, Src and p53. Changes in the tumor microen-
vironment provoke some of these genes to enhance OXPHOS.
The c-myc gene can provoke aerobic glycolysis and/or OXPHOS,
depending on the tumor cell microenvironment, by increasing the
expression of glycolytic genes or activating the mitochondrial oxi-
dation of glutamine (Wise et al., 2008; Gao et al., 2009). OXPHOS
may also be required for the activation of certain tumor suppressor
proteins, such as Bax and Bak, to control apoptosis (Tomiyama et al.,
2006). H-RasV12 /E1A transformation initially causes an increase in
O2 consumption and superoxide production in a process that is
accompanied by massive cell death. During prolonged culture in
vitro, the cell growth rate gradually increased along with tumor
forming potential, as observed in in vitro anchorage-independent
growth assays and in vivo tumor formation assays in immuno-
deficient mice. Notably, the glucose-to-lactic acid flux increased
with the passage number, while the cellular oxygen consumption
decreased. This conversion in metabolic properties is associated
with a change in mitochondrial NAD+/NADH redox, which is indica-
tive of a decreased mitochondrial TCA cycle and OXPHOS activity.
Moreover, the Ras oncogene increases OXPHOS activity in early
transformed progenitor cells, such as mesenchymal SCs (de Groof
et al., 2009; Funes et al., 2007).
We and others have shown that increased glucose consump-
tion and lactate production, also known as the Warburg effect, are
almost universal hallmarks of solid tumors that favor tumor growth
(Bensinger and Christofk, 2012; Kondoh et al., 2005). However, high
levels of glycolysis in the absence of adequate OXPHOS may not
be as beneficial for tumor growth in general. The knockdown of
p32/hyaluronic acid binding protein 1 (HABP1), a mitochondrial
cell surface protein that is overexpressed in certain cancer cells,
shows a strong shift in cellular metabolism from OXPHOS to glycol-
ysis. This finding suggests that the function of proteins that regulate
the balance between OXPHOS and glycolysis according to cellular
needs, including HABP1, is important for cancer cells to guarantee
their immortalization and malignancy (Fogal et al., 2010).
However, intense glycolysis is not present in all tumor types;
rapidly growing tumors depend more on this process than do
slowly growing ones (Jose et al., 2011). Recent reports have found
that the mitochondria in tumor cells are active at a low capacity
and provide these cells with most of the needed ATP (Bustamante
and Pedersen, 1977). Several cancer cells rely on the OXPHOS path-
way to produce ATP rather than glycolysis (e.g., cervical carcinoma
and glioma cells). Overall, the fact that some cancer cells preferen-
tially use glycolysis as an energy source and are able to change from
aerobic glycolysis to OXPHOS in low-glucose conditions suggests
that tumor cells have the capacity to reprogram their metabolism
to rapidly adapt to genetic and/or microenvironmental changes
(Beckner et al., 2005; Plecita-Hlavata et al., 2008; Rossignol et al.,
2004). For such cases, dual therapy, i.e., the inhibition of glycoly-
sis in combination with mitochondrial respiration inhibiting drugs,
would be more efficient and promises to be more effective than
suppressing glycolysis alone.
6. ROS-regulated microRNAs involved in cancer
MicroRNAs are small (21–24 nucleotides), non-coding, evolu-
tionarily conserved RNA molecules that regulate gene expression
by base pairing with the 3 untranslated region (UTR) of tar-
get mRNAs, causing translational repression or mRNA cleavage
(Krek et al., 2005; Miska et al., 2004). The human genome con-
sists of more than 1000 microRNAs that are involved in a myriad
of processes. MicroRNA expression is associated with cellular
proliferation, death, and development, and microRNAs actively
participate in different pathologies, including cancer. Ionizing
radiation, etoposide, and H2 O2 can induce alterations in global
microRNA expression patterns (Simone et al., 2009). Fibroblasts
exposed to such agents undergo changes in their microRNA expres-
sion, depending on the anticancer agent, the radiation dose,
and the time of exposure. A common signature in response to
exogenous genotoxic agents can be identified. It is noteworthy
that pre-treatment with the free radical scavenger NAC pre-
vented radiation-induced alterations in microRNA expression,
suggesting that microRNAs actively respond to oxidative stress
(Mathe et al., 2012).
 V. Sosa et al. / Ageing Research Reviews 12 (2013) 376–390 383

Fig. 3. ROS levels dictate biological outcomes. Different ROS levels determine the cell response and are classified as follows: +(proliferation and differentiation), ++ (adaptative
response: senescence/quiescence), +++ (generalized DNA damage, also including damaged proteins and lipids), and ++++ (cell death). The ability of ROS to chemosensitize
cancer cells depends on the basal ROS levels in such cells. The ROS levels could represent a double-edged sword: ROS activation below a specific threshold promotes survival;
however, if certain limits are reached, such effects are incompatible with cell viability, and the cells will die. Examples of chemotherapeutic treatments with increased ROS
are paclitaxel, doxorubicin, and cisplatin. Radiotherapy also increases ROS. (N, Normal cells; S, Senescent cells; T, Tumor cells; SCs, Stem cells; CSCs, Cancer stem cells).

Several ROS-related microRNAs have been described (Favaro
et al., 2010). miR-210, which is overexpressed in breast and hepa-
tocellular carcinomas (HCC), is associated with invasion and poor
clinical outcome (Rothe et al., 2011). miR-210 is strongly induced
under hypoxic conditions and mediates the metastasis process
(Ying et al., 2011). Among the target mRNAs identified for miR-210
are vacuole membrane protein 1 (VMP1) and iron sulfur scaffold
protein (ISCU), which is necessary for the formation of iron-sulfur
clusters and cofactors for enzymes involved in the TCA cycle, elec-
tron transport, and iron metabolism. Remarkably, ISCU is also a
target of HIF-1␣. Thus, miR-210 helps cancer cells adapt to hypoxic
conditions by regulating mitochondrial function and ROS genera-
tion in situations in which enhanced cell survival and glycolysis
favor proliferation.
miR-128a is downregulated in medulloblastoma, the most
malignant brain tumor in children, with a very poor progno-
sis (Venkataraman et al., 2010). microRNA-128a re-expression
is able to inhibit medulloblastoma proliferation by downregu-
lating the Bmi-1 oncogene, which, in turn, leads to an increase
in p16INF4A expression and indirect modulation of E2F1 activity
(Jacobs et al., 1999; Molofsky et al., 2005). Bmi-1 downregulation
by microRNA-128a alters the redox equilibrium by increasing ROS
in tumor cells and promoting cellular senescence. In turn, Bmi-1
re-expression reverses superoxide generation and the previously
observed genetic and phenotypic effects.
Two members of the microRNA-200 family, miR-141 and miR-
200a, target p38␣, which is one of the main sensors of oxidative
stress (Mateescu et al., 2011). p38␣ inactivation is associated
with ROS accumulation and the subsequent stimulation of antioxi-
dant defenses. Primary high-grade human ovarian carcinomas, the
most lethal gynecologic malignancies, overexpress the microRNA
miR-200a. Concomitant miR-200a overexpression and p38␣ down-
regulation sensitizes ovarian cancer cells to chemotherapeutic ROS
inducers. Therefore, the accumulation of microRNA-200 family
members is associated with an encouraging therapeutic strategy, in
which tumors may be treated with ROS-inducing drugs and maxi-
mal surgery (Hu et al., 2009). In contrast, ovarian tumors with lower
amounts of miR-200a and no p38␣ effects should be treated by
alternative methods that remain to be further elucidated.
7. SCs/CSCs
The immortalizing properties of SCs are a subject of debate.
We and others have demonstrated that SCs are more glycolytic
than primary cells and possess reduced mitochondrial oxygen
consumption, which protects these cells from oxidative stress
(Diehn et al., 2009; Funes et al., 2007; Iyer et al., 1998; Kondoh
et al., 2005).
The importance of CSCs in molecular oncology results from
the fact that they are responsible for tumor relapse, as they are
more resistant to chemo- and radiotherapeutic treatments than
cancer cells (i.e., non-CSCs). CSCs are present in most solid and non-
solid tumors, including leukemia, brain, head and neck, breast and
colon cancers, among others (Al-Hajj et al., 2003; Lapidot et al.,
1994; O’Brien et al., 2007; Piccirillo et al., 2006; Prince et al., 2007;
Ricci-Vitiani et al., 2007; Wulf et al., 2001). In contrast to dif-
ferentiated cancer cells, CSCs generally maintain low ROS levels,
exhibiting redox patterns similar to their corresponding wild-type
SCs (Fig. 3). Several properties have been suggested in the mainte-
nance of the low ROS levels found in CSCs, which appear to occur
through a combination of mechanisms that may be unique to a
given tumor. These properties include: (a) the expression of ROS-
scavenging molecules, (b) more efficient DNA repair responses, (c)
promotion of glycolysis, (d) promotion of autophagy and (e) FoxO
protein upregulation (Diehn et al., 2009; Piao et al., 2012). Some
of these mechanisms are derived from the transforming activity
of certain oncogenes that are unexpectedly linked to a capacity to
maintain elevated intracellular levels of reduced glutathione (GSH),
the main redox buffer. Because GSH maintenance largely relies
on glucose metabolism through the PPP cycle, this may partially
explain the preference for CSCs in glycolytic metabolism and in a
hypoxic microenvironment. For example, human gastrointestinal
CSCs show an enhanced capacity for GSH synthesis and the defense
against ROS by a cystine-glutamate exchange transporter (Ishimoto
et al., 2011). Alternatively, the low ROS levels of certain subsets of
SC (i.e., non-CSC) populations may represent a reflection of their
state of quiescence in vivo.
Interestingly, CD13, a HCC stem cell marker, has a protective
role in regulating ROS levels to avoid DNA damage by controlling
angiogenesis, apoptosis and metastasis (Mishima et al., 2002; Kim
et al., 2012). The EMT that occurs as a result of the activation of
TGF-␤ is associated with CD13+ CSC survival and ROS reduction in
the liver. One possible explanation for the survival of liver CD13+
CSCs after chemotherapeutic treatment is the high expression of
aminopeptidase N, a ROS scavenger enzyme (Kim et al., 2012).
Immunohistochemical analysis revealed the presence of the con-
comitant expression of the CD13 and N-cadherin proteins in tumor
cells that survived chemotherapy. Accordingly, CD13 repression
generates apoptosis and inhibits the self-renewal ability of CSCs
(Kim et al., 2012).
384 V. Sosa et al. / Ageing Research Reviews 12 (2013) 376–390
By upregulating the cells’ antioxidant capacity (in most cases,
by increasing GSH or FoxO), CSCs couple the SC redox phenotype
with radioresistance. Under these circumstances, it is expected that
they will respond to a specific CSC therapy in combination with
pro-oxidant treatment. Importantly, pharmacological depletion of
ROS scavengers in CSCs markedly decreases their clonogenicity and
results in radiosensitization (Diehn et al., 2009). Loss of the FoxO3
gene causes myeloproliferative syndrome due to the increased
accumulation of ROS in hematopoietic progenitors, leading to the
activation of the Akt/mTOR signaling pathway (Yalcin et al., 2010).
Overall, the fact that SCs have high glycolysis levels and low
mitochondrial oxygen consumption suggests that these features
are undoubtedly related to their immortalizing properties (Kondoh
et al., 2007b). This would explain, at least in part, CSCs’ higher
levels of resistance to ROS and radio- and chemoresistance. The
metabolic modulation-associated survival mechanisms employed
by SCs are possibly mimicked by healthy cells to be immortalized
and transformed.
8. How therapy could target the oxidative stress
mechanism
It is clear that oxidative stress plays a role in the develop-
ment of age-associated pathologies, including cancer (Sohal and
Weindruch, 1996). Importantly, cancer therapy is highly associ-
ated with ROS production because most cytotoxic drugs block
DNA replication, causing DNA damage. For example, the overex-
pression of tumor suppressor genes in combination with radiation,
which is known to resume an apoptotic program, provokes massive
cell killing, a process that is blocked by ROS scavenger molecules
(Yacoub et al., 2004).
8.1. Antioxidants
Antioxidants constitute the cellular mechanism of defense
against the consequences of high concentrations of reactive species
(mainly ROS) (Oh et al., 2008). There is evidence that these
molecules prevent tumorigenesis and increase life span (Kovacic
and Jacintho, 2001). Apart from their protective role as preven-
tive agents against cancer development, there is evidence that
antioxidant supplementation during chemotherapy has potential
to reduce dose-limiting toxicities in patients with cancer (Block
et al., 2008).
Treatment with antioxidants (particularly polyphenols) is effec-
tive in combating tumor cells (Borrelli et al., 2011; Dhar et al.,
2011). When the naturally occurring equilibrium between anti- and
pro-oxidant agents is lost, high ROS levels bypass the antioxidant
function and supplementary antioxidants may be necessary for
therapy against ROS-induced cancer. However, it must be kept in
mind that the supplied synthetic antioxidant concentration is cru-
cial for a correct cellular response; a high concentration could cause
an opposing effect and act as a pro-oxidant. Initially, it was demon-
strated that the overexpression of genes that codify for antioxidant
enzymes was effective in cancer therapy. For example, overex-
pression of SOD2 was found to suppress the malignant phenotype
of human melanoma cells (Church et al., 1993). In addition, several
Nrf2 activators constitute potential therapies for oxidative stress,
inflammation and chemoprevention. In a two-stage mouse skin
carcinogenesis model, a Protandim-supplemented diet (patented
supplement consisting of several low-dose natural Nrf2 activators)
was found to significantly reduce the incidence of skin tumors (Liu
et al., 2009). The increased malignant cell behavior induced by
several oncogenes is associated with an increase in ROS produc-
tion, and the treatment of such malignant cells with antioxidants
results in a reduction in the tumorigenic properties (Guijarro et al.,
2007). Moreover, the status of certain oncogenes/tumor suppres-
sor genes is also important in determining its effectiveness (Zhao
et al., 2006). Synthetic, metal-based antioxidants with biologically
active ligands are potential ROS scavenging molecules, and redox
balance is restored in damaged cells. However, further research
is needed to establish an optimum design of these compounds.
The combination of a metal ion and a redox-active organic ligand
appears to be a promising approach for creating an effective syn-
thetic antioxidant. The carefully controlled titration of the amount
of catalytic antioxidants administered to treat some cancers could
facilitate ROS scavenging, restore the redox balance in tumor cells,
and reduce their growth advantage.
8.2. Pro-oxidant therapy
Given the role of antioxidants in cancer prevention and treat-
ment, it somehow seems contradictory that a pro-oxidant therapy,
which is based on the generation of oxygen radicals, could be used
for cancer annihilation. Although oxidative stress caused by ROS
accumulation promotes tumor growth, it can also increase the sen-
sitivity to treatment. This may be the reason for the limited success
of antioxidant therapies in clinical trials (Appierto et al., 2009;
Mateescu et al., 2011). However, it remains a challenge to clas-
sify the types of tumors that may benefit from pro-oxidant therapy
based on their intrinsic properties. The first attempt to employ
in vivo pro-oxidant agents, such as antitumor agents, was reported
by Nathan and Cohn, who used glucose oxidase as an H2 O2 precur-
sor and obtained a significant decrease in tumor growth (Nathan
and Cohn, 1981). Because cells can develop adaptive responses to
ROS by primarily increasing detoxification enzymes, free radical
research has focused on disease treatment with radical scavengers
that help in coping with the elevated ROS levels (Benhar et al., 2002;
Nathan et al., 1981). Currently, there is a major focus on cancer ther-
apies that increase ROS to reach a threshold that is incompatible
with cell viability, even for a highly tumorigenic cell (Figure 3). In
such a context, it will be necessary to first precisely define the ROS
levels in a particular tumor and to explore its ability to switch from
aerobic glycolysis to OXPHOS, considering that it would be possible
to manipulate it to obtain efficient cell killing in terms of therapy.
Examples of drugs that induce ROS include arsenic trioxide, N-(4-
hydroxyphenyl) retinamide (HPR) and dithiophene (NSC656240).
Moreover, certain commonly used chemotherapeutic agents
induce ROS. The anticancer drug 5-fluorouracil (5-FU) interferes
with DNA replication and consequently with the inhibition of cell
division, which are effects that are highly associated with tumor
cells. Recent in vitro and in vivo data from animal studies and
patient treatment have suggested that the cytotoxicity induced
by the chemotherapeutic drugs 5-FU and oxaliplatin is linked to
increased ROS formation (Afzal et al., 2012).
One of the most exciting topics regarding pro-oxidant therapy
is its effects on CSCs. For example, CD13+ cells reduce ROS-induced
DNA damage after genotoxic chemo-radiation stress, a feature that
protects these cells from apoptosis. In mouse xenograft models,
the combination of a CD13 inhibitor and 5-FU drastically reduced
the tumor volume compared with either agent alone (Haraguchi
et al., 2010). Therefore, the combination of a CSC surface receptor
inhibitor with ROS-inducing therapy (i.e., chemo- or radiotherapy)
may improve the treatment of aggressive tumors, such as liver can-
cer, by precisely eradicating the cell type that permanently feeds
the tumor mass (i.e., CSCs). In this context, Venkataraman et al.
(2010) envisioned a scenario in which miR-128a can be used to
induce ROS in medulloblastoma SCs to make them more radiosen-
sitive.
Paclitaxel, a mitotic inhibitory drug, stabilizes microtubules, and
as a result, it interferes with the normal breakdown of microtubules
during cell division. Paclitaxel also promotes ROS generation
 V. Sosa et al. / Ageing Research Reviews 12 (2013) 376–390
(i.e., O2 − and H2 O2 ) by enhancing the activity of the NADPH
oxidase Nox and slowing tumor growth (Alexandre et al., 2007;
Ramanathan et al., 2005). In breast cancer cells, this treatment
caused the translocation of Rac1, a positive regulatory protein of
Nox. It has been established that the ROS generated by paclitaxel
mainly accumulates outside of the cells, while the intracellular ROS
remains unchanged. This accumulation outside of the cells pro-
vokes lethal damage to bystander cancer cells that have not been
exposed to paclitaxel (Alexandre et al., 2007). This unexpected
cytotoxic bystander effect also occurs with other microtubule-
targeted agents, such as vincristine and taxotere, but not with
5-FU or doxorubicin. This fact represents a novel basis on which
to improve the clinical use of chemotherapeutic agents that not
only consider the tumor cells but also affect neighboring cells. In
most cases, combined therapy appears to be effective for cancer
treatment. For example, it has been demonstrated that the anti-
cancer effects of ionizing radiation combined with arsenic trioxide
increase the therapeutic efficacy compared to individual treat-
ments in human prostate cancer cells. Combined treatment exhibits
enhanced ROS generation compared to each treatment alone,
provoking autophagy and apoptosis by inhibiting the Akt/mTOR
signaling pathways (Chiu et al., 2012).
In contrast, a gene of particular significance, melanoma differen-
tiation associated gene-7/interleukin-24 (mda-7/IL-24), selectively
induces growth suppression and apoptosis in a broad spectrum
of human cancers without eliciting apparent deleterious effects in
normal cells. One exception to the action of this gene is in pancreatic
cancer, due to the lack of the capacity to convert mda-7/IL-24 mRNA
into protein. Lebedeva et al. (Lebedeva et al., 2005) showed that
pancreatic cells, regardless of the status of the K-ras gene, simulta-
neously express mda-7/IL-24 protein and undergo apoptosis when
treated with ROS inducers, demonstrating that tumorigenesis was
effectively suppressed in vivo and in vitro. The specificity of this
action is documented as the ability of ROS inhibitors to block this
killing effect. This example represents a promising combinatorial
approach for pancreatic tumors, which are exceptionally aggressive
and have no long-term effective therapy.
Recently, a small molecule named piperlongumine, which
selectively kills cancer cells, has been described. Piperlongumine
potently inhibits the growth of spontaneously formed malignant
breast tumors and their associated metastases in mice by increas-
ing ROS levels and apoptosis. Importantly, piperlongumine has
little effect on rapidly or slowly dividing primary normal cells (Raj
et al., 2011), which represents a clear advantage of this compound
for therapeutic purposes. Fucoidan, an enzymatic, digested, low
molecular weight compound that is extracted from brown seaweed
and has anti-tumor properties, activates an independent caspase
apoptotic pathway and is implicated in angiogenesis inhibition
(Liu et al., 2012). Such a pathway involves JNK, p38␣ and ERK1/2
phosphorylation in various cancer cells by activating ROS-mediated
MAP kinases and the regulation of bcl-2 protein family-mediated
mitochondrial apoptosis (Zhang et al., 2011). Although this com-
pound has not been tested in patients, it appears to be efficient in
leukemia, breast, colon, and lung cancer cells (Jin et al., 2010).
Overall, a further understanding of the oncogenic/tumor sup-
pressor proteins that are respectively overexpressed or silenced
in human tumors and their relationship with ROS would permit
the classification of tumor types that are susceptible to efficient
eradication by pro-oxidant therapy.
8.3. Glycolysis inhibition
A first choice for inhibiting glycolysis would be the use of non-
hydrolyzable glucose analogs that prevent the initial glycolytic
steps from advancing to the final cascade of metabolic degrada-
tion (e.g., past the hexokinase-mediated phosphorylation step).
385
We have previously demonstrated that treatment using the glu-
cose analog 2-deoxyglucose in SCs is able to considerably decrease
their proliferative potential and immortalizing properties (Kondoh
et al., 2007b). Deoxyglucose and two other inhibitors of hexoki-
nase (HK), lonidamide and 3-Bromopiruvate (3-BrPa), have been
successfully tested in pre-clinical trials of malignant tumors (Ko
et al., 2001; Oudard et al., 1995). Importantly, 3-BrPa represents a
novel therapy for liver cancer and other cancer types with poor sur-
vival rates (Geschwind et al., 2002). Along the same line of evidence,
targeting lactate efflux is another avenue to inhibit the glycolytic
pathway and provoke cell death. This has been performed by uti-
lizing the small-molecule alpha cyano-4-hydroxy cinnamic acid
(ACCA) (Colen et al., 2006). In this case, the targeted tumors show
increased sensitivity if combined therapy is given, e.g., the addition
of radiation treatment (Colen et al., 2006).
Bezielle (BZL101), an oral drug presently being tested in human
clinical trials for advanced breast cancer (Chen et al., 2012; Perez
et al., 2010), has proven to be efficient in inhibiting not only gly-
colysis but also oxidative phosphorylation. Bezielle is derived from
Scutellaria barbata, a plant with anti-cancer properties. This drug
has selective cytotoxic effects in vitro in cancer cells and in vivo
in renal, hepatocellular and prostate carcinomas (Cha et al., 2004;
Marconett et al., 2010; Yin et al., 2004). Bezielle’s selectivity relies
on generating large amounts of ROS in tumor cells, causing DNA
damage and hyperactivation of PARP, which mainly targets the
mitochondria. This PARP hyperactivation provokes massive apop-
totic cell death (Zong et al., 2004). PARP hyperactivation in cancer
cells greatly reduces the cellular supply of NAD+ and ATP, which are
needed for the PARylation of several proteins implicated in the DNA
repair mechanism, the regulation of transcription, and cell cycle
progression (Rouleau et al., 2010). A possible cause for the inhibi-
tion of glycolysis observed in tumors treated with Bezielle could
be the limited cytosolic NAD+ concentration (Alano et al., 2010).
Importantly, the small amount of DNA damage that Bezielle causes
in normal cells is restored by the DNA repair mechanism.
Overexpression of the glycolytic enzyme pyruvate kinase M2
(PKM2) promotes anabolic processes and confers a selective advan-
tage to cancer cells, enabling them to sustain antioxidant responses
that lengthen cancer cell survival under intense oxidative stress
(Anastasiou et al., 2011). It has been reported that the high ROS
levels that occur in human lung cancer cells repress PKM2 through
cysteine 358 oxidation, and consequently, glucose is directed to the
PPP cycle. Under these circumstances, the cell is able to detoxify
ROS (Anastasiou et al., 2011). However, lung cancer cells in which
endogenous PKM2 is replaced with Cys358 to Ser358 oxidation-
resistant mutants exhibit increased sensitivity to oxidative stress
and impaired tumor formation in a xenograft model (Anastasiou
et al., 2011). A possible therapeutic approach is the use of small
PKM2 activator molecules that are able to interfere with tumor cell
metabolism, as PKM2 inactivation ultimately increases ROS sensi-
tivity. These results, which have been proven in mouse models, are
promising for the design of future therapies in patients with cancer.
Studies in cell lines that are defective for mitochondrial enzymes
have demonstrated that the oxidative phosphorylation activ-
ity defines the rate of glucose utilization by aerobic glycolysis
(Sanchez-Arago et al., 2010). Importantly, abrogated mitochondrial
respiration leads to a diminished ROS signaling potential in an
efficient response to 5-FU treatment. In contrast, an abrogated gly-
colysis pathway in the same cells with intact mitochondria results
in a more efficient response to 5-FU.
Over the course of years, it has been discovered that glycolytic
inhibition per se has not been an effective chemotherapeutic strat-
egy, due in part to the systemic toxicity of antiglycolytic drugs.
However, combined treatment that involves glycolysis inhibition
and mitochondrial function may concomitantly help to over-
come this issue. Some examples include the mitochondria-targeted
386 V. Sosa et al. / Ageing Research Reviews 12 (2013) 376–390
drugs Mito-Q and Mito-CP, which, in combination with 2-deoxy-d-
glucose, are more effective than inhibiting glycolysis alone (Cheng
et al., 2012).
Along the same lines of evidence, those drugs, which are equally
effective for glycolysis and OXPHOS inhibition, are more efficient
than acting through a single process. For example, 3-BrPA inhibits
cellular ATP production due to its effects on glycolysis and OXPHOS.
It acts on several cellular enzymes that are involved in glycolysis,
such as GAPDH and PGK, and it inhibits mitochondrial respiration
enzymes, including succinate dehydrogenase (SDH). The combined
results of 3-BrPA are responsible for a decrease in viability and
the apoptotic death of HCC cells. These effects, which are linked
to proton leaks, may reflect permeability in the transition pores
of the mitochondrial membrane, which occurs prior to apoptotic
processes.
Earlier studies have demonstrated that curcumin, betulinic acid
(BA), synthetic triterpenoid anticancerigenous drugs, and tolfe-
namic acid are able to reduce bcl2, survivin, hepatocyte growth
factor receptor (c-Met), epidermal growth factor receptor (EGFR),
cyclin D1, VEGF, and its receptors VEGFR1 and VEGFR2 in sev-
eral cancer cell types. BA is a naturally-occurring triterpenoid
and is a selective inhibitor of human melanoma, malignant brain
tumors, and human leukemia cells, but it is apparently inefficient in
epithelial tumors. The BA mechanism mainly works by inhibiting
topoisomerase, a process that is accompanied by caspase activa-
tion, mitochondrial membrane alterations and DNA fragmentation;
therefore, BA belongs to a class of mitochondria-targeted drugs (Li
et al., 2010). Recently, BA has been suggested to act by repressing
the specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4,
which are overexpressed in cancer cells but underexpressed in nor-
mal tissues. In colon cancer cells, the mechanism of BA-induced
repression of the Sp protein is due to the induction of ROS, ROS-
mediated miR-27a repression, and the induction of the Sp repressor
gene ZBTB10 (Chintharlapalli et al., 2011).
Another drug, ethyl 2-((2,3-bis(nitrooxy)propyl)disulfanyl)
benzoate (GT-094), is a novel drug chimera that exhibits nonste-
roidal anti-inflammatory properties, nitric oxide moieties and a
disulfide pharmacophore that possesses cancer chemopreventive
activity (Pathi et al., 2011). GT-094 was found to inhibit cell prolifer-
ation and induce apoptosis (i.e., caspase-dependent PARP cleavage)
in RKO and SW480 colon cancer cell lines, in addition to low-
ering the mitochondrial membrane potential and enhancing ROS
production. These responses are reversed with antioxidant (glu-
tathione) treatment (Pathi et al., 2011). GT-094 has been proven to
act by downregulating genes associated with cell growth (i.e., cyclin
D1, c-Met, EGFR, bcl-2, and survivin) and angiogenesis, along with
some regulators of these genes, including Sp1, Sp3, and Sp4, which
have been found to be overexpressed in different cancer cells. GT-
094 is able to control the expression of these transcription factors
by repressing miR-27a and inducing ZBTB10 (Sp repressor). The
repression of the above mentioned transcription factors is reversed
by a glutathione antioxidant, suggesting that the antitumorigenic
activity of GT-094 in colon cancer cells is partially due to the acti-
vation of the ROS-miR-27a axis (Pathi et al., 2011).
9. Conclusions
There is much evidence that normal cells are affected by sur-
rounding cancer cells, helping the cancer cells to grow, invade
and metastasize. Remarkably, cancer cells are able to reprogram
their metabolism by adapting it to microenvironmental changes.
Research in cancer bioenergetics has acquired a better understand-
ing of the way in which energy metabolism is regulated during
oncogenesis. Compared to normal cells, tumor cells exhibit impor-
tant metabolic changes. Tumor cells show an increase in glucose
uptake due to enhanced glycolysis and increased lactate produc-
tion. Thus, in solid tumors, the Warburg effect could be the result
of an adaptive mechanism that, in combination with angiogen-
esis, allows cells to overcome hypoxic stress. In addition, the
Warburg effect confers protection from ROS for cancer cells in
comparison to normal cells. In highly glycolytic tumors, a poten-
tial therapeutic strategy would consist of inhibiting glycolysis to
obtain a final effect on the re-activation of mitochondrial oxida-
tive metabolism, and ultimately, increased ROS levels. However,
not all tumors exhibit augmented glycolysis. Some tumors prefer
to enhance OXPHOS, particularly under low glucose concentrations
in which glycolysis would be less efficient. In these cases, novel
energy restriction/mimetic agents promise to represent a better
therapeutic choice for reducing human tumor growth. Accumu-
lating evidence shows that ROS levels in tumor cells are key for
determining a specific therapy. There is an urgent need for estab-
lishing biomarkers (i.e., oncogenes, tumor suppressor genes, and
microRNAs) that will predict the response of a pro-oxidant ther-
apy to define the best choice for cancer eradication for each cancer
patient. The association of CSCs and the resistance to therapy solic-
its a critical rethinking of the efficiency of pro-oxidant therapy on
CSCs. Because low ROS levels seem to be critical for maintaining SC
function, specific ROS induction in CSCs should render these cells
sensitive to therapy.
Acknowledgement
Work in the authors’ laboratory is supported by the Healthy
Ministry (FIS Project PI09/02193 to M.E.LL.).
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