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Cancer Letters 266 (2008) 6–11

www.elsevier.com/locate/canlet

Mini-review

Oxidative stress, DNA methylation and carcinogenesis

Rodrigo Franco a,b,1, Onard Schoneveld b,1, Alexandros G. Georgakilas c,
Mihalis I. Panayiotidis d,*
a
Laboratory of Cell Biology and Signal Transduction, Biomedical Research Unit, FES-Iztacala, UNAM, Mexico, USA
b
Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health,
Research Triangle Park, NC 27709, USA
c
Department of Biology Thomas Harriot College of Arts and Sciences, East Carolina University, Greenville, NC 27858, USA
d
School of Public Health, University of Nevada-Reno, MS-274, Reno, NV 89557, USA

Received 12 February 2008; received in revised form 12 February 2008; accepted 14 February 2008

Abstract

Transformation of a normal cell to a malignant one requires phenotypic changes often associated with each of the ini-
tiation, promotion and progression phases of the carcinogenic process. Genes in each of these phases acquire alterations in
their transcriptional activity that are associated either with hypermethylation-induced transcriptional repression (in the
case of tumor suppressor genes) or hypomethylation-induced activation (in the case of oncogenes). Growing evidence sup-
ports a role of ROS-induced generation of oxidative stress in these epigenetic processes and as such we can hypothesize of
potential mode(s) of action by which oxidative stress modulates epigenetic regulation of gene expression. This is of outmost
importance given that various components of the epigenetic pathway and primarily aberrant DNA methylation patterns
are used as potential biomarkers for cancer diagnosis and prognosis.
Ó 2008 Elsevier Ireland Ltd. All rights reserved.

Keywords: Oxidative stress; Free radicals; Reactive oxygen species; Oxidative DNA damage; DNA methylation; Epigenetics; Gene
expression; Cancer

1. Introduction oxygen species (ROS) and/or a reduction in antiox-

Oxidative stress has long been known to be
involved in the pathophysiology of many human
diseases including, but not restricted, to cancer.
The term ‘‘oxidative stress”, refers to a cell’s state
characterized by excessive production of reactive
*
Corresponding author. Tel.: +1 775 682 7082; fax: +1 775 784
1340.
E-mail address: mpanagiotidis@unr.edu (M.I. Panayiotidis).
1
Both authors have contributed equally to the preparation of
the manuscript.
idant defenses responsible for their metabolism.
This generates an imbalance between ROS produc-
tion and removal in favor of the former.
Cancer is a multistage process and often involves
‘‘alterations” or ‘‘changes” in the transcriptional
activity of genes associated with many critical cellu-
lar processes for tumor development including pro-
liferation, senescence, inflammation, metastasis, etc.
Furthermore, there are known to be both genotoxic
and non genotoxic mechanisms contributing to
malignant transformation. Generally, genotoxic
mechanisms involve changes in genomic DNA

0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.canlet.2008.02.026
 R. Franco et al. / Cancer Letters 266 (2008) 6–11
sequences that ultimately lead to mutations whereas
non-genotoxic include mechanisms (other than
directly affecting DNA) capable of modulating gene
expression. ROS have been implicated at all stages
of the carcinogenic process by involving both types
of mechanisms [1].
On the other hand, the term ‘‘epigenetics” refers
to altered levels of a gene’s transcriptional activity
without directly affecting its primary DNA nucleo-
tide sequence. It involves alterations in DNA meth-
ylation patterns that together with specific histone
modifications (methylations, acetylations, deacety-
lations, etc.) contribute to a transcriptional inactive
chromatin state [2]. In this respect, epigenetic regu-
lation of gene expression can be viewed as a non
genotoxic mechanism for promoting tumor
formation.
Throughout this review article, we will present
the evidence for the involvement of oxidative stress
in carcinogenesis, consider the evidence for its role
in inducing DNA methylation changes and assess
the importance of these changes in the multistage
process of human carcinogenesis.
2. Sources of ROS
There are both endogenous and exogenous
sources of ROS generation. Endogenous sources
include those of: (1) mitochondrial oxidative phos-
phorylation, (2) P450 metabolism, (3) peroxisomes
and (4) activation of inflammatory cells. It has been
postulated that during oxidative phosphorylation
1–2% of molecular oxygen is converted to ROS
primarily through a series of sequential one-, two-
and three-electron reductions giving rise to superox-
ide, hydrogen peroxide and hydroxyl radical
formation consecutively [3]. In addition, activation
of P450 metabolism has been proposed as a very sig-
nificant source of ROS formation by mechanisms
that involve: (1) redox cycling, (2) peroxidase-cata-
lyzed drug oxidations and (3) ‘‘futile cycling” of
cytochrome P450. In particular, induction of P450
2E1 and 2B has been proposed to contribute signif-
icantly to ROS formation primarily through metab-
olism of ethanol and phenobarbital, respectively.
Furthermore, in a number of studies using chemi-
cals such as peroxisome proliferators, an increased
production of hydrogen peroxide was observed that
contributed to increased levels of oxidative stress
and their association with cancer induction. Finally,
inflammatory cells like neutrophils and macro-
phages are perhaps the most significant sources of
7
endogenous ROS formation. More specifically, acti-
vated macrophages through ‘‘respiratory burst”
give rise to various ROS and primarily superoxide
and hydrogen peroxide. For instance, activation of
specialized macrophages in liver known as Kupffer
cells has been implicated in tumor promotion
through ROS release [1].
On the other hand, exogenous sources of ROS
generation include those of various xenobiotics
(with a variable degree of potency), chlorinated
compounds, various transition metals (participating
in Fenton-type chemical reactions) and radiation,
all of which have been documented to cause ROS-
induced damage to cellular macromolecules
(DNA, RNA, lipids and proteins) both in vitro
and in vivo [1].
3. Involvement of oxidative stress in carcinogenesis
In general, the multistage process of carcinogen-
esis involves the distinct phases of initiation, promo-
tion and progression. The cellular and molecular
events underlying each of these phases include
DNA damage, increased proliferation, deficient cell
death and further genetic instability, respectively
[1,4].
Oxidative DNA damage can trigger tumor initia-
tion. More specifically, studies using ionizing radia-
tion have shown multiple hydroxyl radical-induced
genotoxic by- products in the bases as well as the
deoxyribose backbone of DNA. Among these by-
products, the most characterized one is 8-hydroxy-
2-deoxyguanosine (8-OHdG) [5,6]. This DNA lesion
has been shown to be mutagenic by causing G ? T
transversions in both bacterial and mammalian cells
[7]. However, there are a number of studies indicat-
ing that oxidative DNA damage could not account
entirely by itself for tumor development as they
have shown that elevated levels of 8-OHdG do not
reflect increased cancer rates [8,9].
So, what other mechanisms could account for the
involvement of ROS in carcinogenesis? As it was
mentioned earlier on, ROS can affect a number of
cellular processes critical in tumor development
such as cell proliferation, senescence, inflammation,
metastasis, etc. In terms of cell proliferation, ROS
have been shown to modulate cell cycle regulation
through modulation of various cell cycle proteins
including p53 [10] and ATM (ataxia telangiectasia
mutated) [11]. ROS have also been shown to modu-
late the cell death (senescence) process [12] by acting
either as an anti-senescence stimulus [13] or through
8 R. Franco et al. / Cancer Letters 266 (2008) 6–11
the specific induction of AIF (apoptosis-inducing
factor) which suppresses apoptosis and conse-
quently maintains the transformed phenotype of a
cancer cell [14]. On the other hand, inflammation
has long been known to act as a trigger in cancer
development. For example, chronic hepatitis B viral
infection and elevated liver levels of 8-OHdG can
lead to development of hepatocellular carcinoma
[15]. The mode of action by which chronic inflam-
mation can trigger tumor initiation seems to involve
NF-jB (nuclear factor jB) activation which, among
its pleiotrophic effects, can cause inhibition of apop-
tosis [16]. Finally, metastasis is an integral part of
tumor progression during which ROS have been
documented to play a major role [17–19]. In fact,
various studies have shown that metastatic tumor
cells produce higher levels of ROS than primary
malignant cells which together with increasing levels
of ROS metabolizing enzymes and antioxidant com-
pounds greatly reduce metastasis [20–22].
4. The role of epigenetics in carcinogenesis
In general, increased DNA methylation in the
promoter region of genes causes gene silencing and
in this respect can contribute to the multistage pro-
cess of carcinogenesis. In addition, in mammalian
cells, both DNA methylation and chromatin struc-
ture are interconnected in specific ways so that genes
will either be transcribed or repressed. Briefly, the
process is initiated by DNA methyltransferases
(DNMTs) bringing together the DNA methylation
machinery to the chromatin through recruitment
of histone deacetylases (HDACs) and other chro-
matin-binding proteins to promoter sites. In this
regards, the state of the chromatin’s acetylation sta-
tus is important in regulating transcriptional activ-
ity in a way that histone acetylation maintains
chromatin in a transcriptionally active state whereas
histone deacetylation maintains transcriptional
silencing. More specifically, during gene silencing
DNMTs direct the binding of HDACs and other
proteins [methyl-CpG binding proteins (MeCPs);
and methyl-CpG binding domain (MBP)] to hyper-
methylated regions of the chromatin where they
form complexes with other molecules and thus
blocking access of the transcriptional machinery to
the promoter. Specific histone modifications have
been implicated in gene silencing including the fol-
lowing: (1) histone H3 methylation at lysines 9
and 27 and/or deacetylation at lysine 9, (2) histone
H4 methylation at lysine 20 and/or deacetylation
at lysine 16. Among these, the best-characterized
histone modification(s) to date is methylation of his-
tone H3 at lysine 9 and how it ‘‘translates” into
DNA methylation in order to repress transcrip-
tional activation [2,23–25].
But how is DNA methylation involved in regula-
tion of transcription? In mammalian cells, the
enzymes critical for DNA methylation reactions
are DNA methyltransferases 1 (DNMT1), 3a
(DNMT3a), and 3b (DNMT3b). These enzymes
catalyze the transfer of the methyl donor group
from S-adenosylmethionine (SAM) to the 50 -carbon
of cytosines within CpG dinucleotide islands
(regions of 0.5–4.0 kb in length) in genomic DNA.
More specifically, these enzymes are involved in
DNA methylation by means of either maintenance
(DNMT 1) and/or de novo methylation (DNMT
3a and DNMT 3b). Thus, they could potentially
contribute not only to increased promoter methyla-
tion status (through de novo methylation) but also
ensure inheritance of gene silencing (through main-
tenance methylation) both of which could account
for the acquisition of a malignant transformation
phenotype [26,27]. In general, 5-methylcytosine (by
being a heritable modification) has the potential to
alter gene expression by means of: (1) causing an
increase in mutation rate [28] given that the presence
of 5-methylcytosine in the DNA promotes deamina-
tion of cytosine to uracil with 5-methylcytosine
deaminating 2–4 times faster than cytosines [29] or
by (2) silencing genes necessary for controlling gene
proliferation [30,31]. Finally, many genes in tumor
cells have been shown to contain alterations in their
DNA methylation patterns. Two such alterations
have been proposed to be the common epigenetic
mechanisms underlying development of human can-
cer: (1) global hypomethylation occurring early in
the progression phase of neoplasia and (2) regional
hypermethylation of normally unmethylated CpG
islands. Both events can result in the transcriptional
silencing of tumor suppressor gene expression and
the transcriptional activation of oncogenes respec-
tively. In addition, increased expression of DNMT1
has been found in early and late stages of neoplasia
suggesting its involvement in the generation of
abnormal methylation patterns in cancer cells [32].
Finally, genome-wide hypomethylation has been
shown to increase mutation rates and thus leading
to genome instability [33]. It is therefore evident that
alterations in DNA methylation patterns underlie
aberrant gene expression that is associated with
malignant transformation.
 R. Franco et al. / Cancer Letters 266 (2008) 6–11
5. Involvement of oxidative stress in DNA
methylation
Oxidative stress can contribute to tumor develop-
ment not only through genetic but also through epi-
genetic mechanisms. As it was mentioned earlier on,
generation of the hydroxyl radical can cause a wide
range of DNA lesions including base modifications,
deletions, strand breakage, chromosomal rearrange-
ments, etc. Such DNA lesions have been shown to
interfere with the ability of DNA to function as a
substrate for the DNMTs, resulting in global
hypomethylation [34]. More specifically, X-rays
[35], ultraviolet [36] and c-rays [37] have been shown
to reduce the methyl-accepting ability of DNA.
Also, the presence of 8-OHdG in CpG dinucleotide
sequences has been shown to strongly inhibit meth-
ylation of adjacent cytosine residues [38,39], and
interfere with the ability of restriction nucleases to
cleave DNA [40]. More specifically, in a series of ele-
gant experiments, it was shown that when 8-OHdG
is substituted for either guanine on the HpaII meth-
ylase recognition site (CCGG) such substitution can
inhibit DNA methylation of adjacent cytosines as
well as binding to the methyltransferase. The extent
of this inhibition is dependent on the position of 8-
OHdG [38,39]. In addition, 8-OHdG may not be
recognized by proofreading enzymes and thus may
persist as a mutation resulting in G ? T transver-
sions [41]. Another potentially mutagenic lesion in
ROS-induced DNA damage is O6-methylguanine.
A number of studies have shown that its presence
can inhibit binding of DNA methyltransferases
and therefore can lead to hypomethylation by
means of inhibiting methylation of adjacent cyto-
sine molecules [42,43]. Alternatively, O6-methylgua-
nine can be spontaneously mispaired with thymine
and thus also contribute to DNA hypomethylation
[44]. Finally, single-stranded DNA can signal de
novo methylation and, thus, it may be possible that
formation of single strand breaks by oxidative stress
can contribute to the modifications of DNA methyl-
ation patterns observed in oxidant-transformed cell
lines [45]. These studies suggest that oxidative DNA
damage can affect patterns of DNA methylation
leading to aberrant gene expression and possibly
contributing to the development of malignancy.
As it was mentioned earlier on, DNA methyla-
tion mediates gene expression via binding of MBPs
through DNA–protein and protein–protein interac-
tions followed by recruitment of histone modifying
enzymes. Consequently, these interactions result in
9
chromatin condensation and transcriptional inacti-
vation. Incorporation of 8-OHdG and 5-hydroxym-
ethylcytosine (the oxidation by-product of 5-
methylcytosine) in the MBP recognition sequence
resulted in the significant inhibition of the binding
affinity of MBP [46]. These results provide further
evidence about the mechanism underlying ROS-
induced DNA damage in interfering with DNA
methylation patterns and consequently with tran-
scriptional activation.
Only recently, there have been a number of stud-
ies investigating changes in gene expression levels of
major antioxidant enzymes in an attempt to relate
diminished levels with the pathophysiology underly-
ing human carcinogenesis. In this respect, it has
been shown that decreased expression of the enzyme
manganese superoxide dismutase (MnSOD; which
metabolizes superoxide anion) was associated with
decreased proliferation in human pancreatic carci-
noma [47] and breast cancer [48] cells and that its
inhibition was underlined by DNA hypermethyla-
tion-induced silencing. In addition, in the case of
the metallothionein gene expression (induced by a
variety of oxidative stress stimuli), it was also found
to be silenced through promoter-specific DNA
hypermethylation events in both mouse lymphosar-
coma [49] and rat hepatoma [50] cells. Finally, both
the NAD(P)H: quinone oxidoreductase 1 (NQO1)
and glutathione S-transferase P1 (GSTP1) genes
(phase II xenobiotic metabolizing enzymes) have
been shown to be inactivated via promoter hyper-
methylation in hepatocellular carcinoma [51,52],
breast, renal [53] and prostate [54] cancers, respec-
tively. These studies clearly demonstrate the associ-
ation between repressed expression of key
antioxidant enzymes (in metabolizing ROS genera-
tion) and tumor development by means of promoter
hypermethylation-induced gene silencing.
6. Conclusions and perspectives
It is evident that ROS-induced oxidative stress is
involved in the multistage process of carcinogenesis
by both genetic and epigenetic mechanisms. In par-
ticular, there seems to be a growing interest (by
many investigators) in the involvement of oxidative
stress in the epigenetic regulation of gene expression
and specifically in controlling DNA methylation.
Given the recent findings in detecting circulating
tumor DNA in patients with cancer [55], it becomes
of paramount importance to reveal the molecular
mechanisms underlying aberrant DNA methylation
10 R. Franco et al. / Cancer Letters 266 (2008) 6–11
patterns since potentially they could be used as bio-
markers in cancer prognosis and diagnosis.
Acknowledgements
This work was supported, in part, by the Intra-
mural Research Program of the National Institutes
of Health (NIH)/National Institute of Environmen-
tal Health Sciences (NIEHS) (Onard Schoneveld
and Rodrigo Franco).
Dr. Georgakilas has been supported by funds pro-
vided by the Biology Department of East Carolina
University and a College Research Award (East Car-
olina University). Finally, Dr. Panayiotidis has been
supported by funds provided by the School of Public
Health of the University of Nevada at Reno.
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