Oral Oncology 50 (2014) 1018

Contents lists available at ScienceDirect

Oral Oncology
journal homepage: www.elsevier.com/locate/oraloncology

Review

Oxidative and antioxidative mechanisms in oral cancer and precancer: A

review
Sheetal Korde Choudhari a,, Minal Chaudhary b, Amol R. Gadbail c, Aparna Sharma d, Satyajit Tekade e
a

Dept of Oral Pathology & Microbiology, Yerala Dental College and Hospital, Kharghar, Mumbai 410 210, India
Dept of Oral Pathology & Microbiology, Sharad Pawar Dental College, Sawangi, Wardha, Maharashtra 442 001, India
c
Dept of Oral Pathology & Microbiology, Sharad Pawar Dental College, Sawangi, Wardha, Maharashtra 442 001, India
d
Dept of Oral Pathology, VSPM Dental College and Hospital, Nagpur, Maharashtra 440 019, India
e
Dept of Oral Pathology & Microbiology, Modern Dental College & Research Centre, Gandhi Nagar, Indore, Madhya Pradesh 453112, India
b

a r t i c l e

i n f o

Article history:
Received 12 August 2013
Received in revised form 16 September 2013
Accepted 19 September 2013
Available online 11 October 2013
Keywords:
Reactive oxygen species
Reactive nitrogen species
Oral cancer
Oral precancer
Oxidants
Antioxidants
Oxidative stress
Oxidative damage
Potentially malignant disorders
Free radicals
Cancer biomarkers
Enzymatic antioxidants
Non-enzymatic antioxidants
Oral carcinogenesis
Head and neck cancer

s u m m a r y
Development of cancer in humans is a multistep process. Complex series of cellular and molecular
changes participating in cancer development are mediated by a diversity of endogenous and exogenous
stimuli and important amongst this is generation of reactive oxygen species (ROS). Reactive radicals and
non-radicals are collectively known as ROS. These can produce oxidative damage to the tissues and hence
are known as oxidants in biological system. Many researchers have documented the role of ROS in both
initiation and promotion of multistep carcinogenesis. To mitigate the harmful effects of free radicals, all
aerobic cells are endowed with extensive antioxidant defence mechanisms. Lowered antioxidant capacity
or the oxidant-antioxidant imbalance can lead to oxidative damage to cellular macromolecules leading to
cancer. Oral cavity cancer is an important cancer globally and tobacco is the primary etiological factor in
its development. Tobacco consumption exposes the oral epithelium to toxic oxygen and nitrogen free
radicals that can affect host antioxidant defence mechanisms. Elevated levels of ROS and Reactive Nitrogen Species (RNS) and lowered antioxidants are found in oral precancer and cancer. Protection can be
provided by various antioxidants against deleterious action of these free radicals. Treatment with antioxidants has the potential to prevent, inhibit and reverse the multiple steps involved in oral carcinogenesis.
This review is an attempt to understand the interesting correlation between ROS and RNS mediated cell
damage and enzymatic and non-enzymatic defence mechanisms involved in oral cancer development
and its progression and the use of antioxidants in oral cancer prevention and treatment.
2013 Elsevier Ltd. All rights reserved.

Introduction
The paradox of aerobic life is that higher eukaryotic aerobic
organisms cannot exist without oxygen, yet oxygen is inherently
dangerous to their existence [1]. It was towards the end of eighteenth century that oxygen emerged as the paragon among the
elements which sustained life, promoted physical health and
stimulated mental vigour. But too much of even the best is bad
as was shown by Paul Bert in 1878 that oxygen in high concentrations could damage brain, lungs and other organs [2]. Todays
concept of oxygen toxicity is not restricted only to hyperbaric

Corresponding author. Address: B-15, Mandovi, Chheda Nagar, Chembur(W),

Mumbai 4400089, Maharashtra, India. Tel.: +91 9819331220; fax: +91
02224446586.
E-mail addresses: kordesheetal@yahoo.co.in (S.K. Choudhari), minal53@yahoo.
com (M. Chaudhary), gadbail@yahoo.co.in (A.R. Gadbail), appu4in@yahoo.co.in
(A. Sharma), satyajitraje@gmail.com (S. Tekade).
1368-8375/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.oraloncology.2013.09.011

oxygen but primarily focuses on the stress caused by oxygen

metabolites (oxygen free radicals or reactive oxygen species)
generated as an integral part of our daily life. At present number
of researchers have documented role of free radicals and reactive
oxygen species (ROS) in number of pathophysiological states
including cancer. These intermediates of oxygen reduction attack
DNA and other cellular components such as lipids, proteins,
leaving behind reactive species that in turn can couple to DNA
bases [3]. The accumulation of DNA damage through disrepair
or incomplete repair may lead to mutagenesis and consequently
cancerous transform cation.
Harmful effects of ROS are balanced by non-enzymatic and
enzymatic antioxidants [4]. Depleted antioxidant defence mechanisms leads to oxidative damage to normal cells and tissues. Disruption of the delicate balance between oxidants/antioxidants in
body plays a causative role in carcinogenesis. Enhanced ROS or
reactive notrosative species (RNS) or both along with concomitant
decrease in antioxidants is seen in various cancers including head

S.K. Choudhari et al. / Oral Oncology 50 (2014) 1018

and neck cancer [510]. To examine the involvement of oxidative

stress and depleted antioxidant defence mechanisms in oral carcinogenesis, a literature search using online databases (Pubmed,
MEDLINE, Scopus) was done using the search terms such as oxidative stress and cancer, role of oxidative stress in cancer, oxidative
stress and oral cancer, antioxidants, antioxidants in oral cancer,
and antioxidants in cancer prevention, and all relevant articles
were reviewed and included. Role of oxidants in oral precancer
and cancer as well as preventive and therapeutic importance of
antioxidants, an outlook on modulation of oxidants and antioxidants in cancer therapeutics along with their utility as cancer biomarkers is discussed in this review.
Introduction to oxidative stress, ROS and RNS
In 1954, Gerschman and colleagues for the rst time proposed
that damaging effects of oxygen could be attributed to formation
of oxygen free radicals [11]. Free radical is a molecule or molecular
fragment containing an unpaired electron in the valence shell (i.e.
radical) and capable of existing independently (i.e. free) [12]. ROS
include both free radicals as well as non-radical derivatives of oxygen [13]. The relation between free radicals and disease can be explained by the concept of oxidative stress elaborated by Sies [14].
He dened oxidative stress as an imbalance between oxidants and
antioxidants in favour of oxidants, potentially leading to damage
[15]. Products of biological damage are referred as biomarkers of
oxidative stress [16].
Nitrosative stress
Nitric Oxide(NO) is an important biologic signalling molecule.
Several different forms of NO synthase(NOS) enzymes generate
nitrogen based radicals. Nitrosative stress (NS) is dened as the ratio of nitrosants to antioxidants as >1 similar to oxidative stress,
but with involvement of RNS. This process involves a variety of
oxygennitrogen species causing excessive oxidation and/or nitrosylation compared to antioxidation or reduction [17]. Nitrosative
stress has been implicated in cellular damage or alterations in normal cell signalling pathways.
Chemistry and sources of ROS/RNS
Free radical contains an odd number of electron, which makes it
unstable, short lived and highly reactive. Generally, it reacts with
the nearest stable molecule, stealing its electron to gain stability.
The attacked molecule looses its electron, it becomes a free radical
itself, beginning a chain reaction cascade resulting in disruption of a
living cell [12]. Most ROS are generated as by-products during mitochondrial electron transport. In addition they are formed as necessary intermediates of metal catalyzed oxidation reactions. RNS are
formed from interactions of NO with O2 or O2 resulting in formation
of dinitrogen trioxide (N2O3) and peroxynitrite (ONOO ).
Free radicals are formed by [12]:
A. Covalent bond cleavage of normal molecule or atom: Homolytic cleavage of molecule leads to formation of free radicals.
Such type of cleavage requires high energy input. Whereas,
in hetrolytic cleavage, one of the atom retains both the
bonding electrons and another takes none resulting in formation of ionic species.
B. Electron transfer: is a common important source of free radical generation in biological system. (i) Oxidation reaction:
By loss of single electron from a normal molecule. (ii) Reduction reaction: By addition of single electron to a normal
molecule.

11

Sources of oxidative stress

ROS can be produced from endogenous and exogenous substances. Potential endogenous sources include mitochondria, cytochrome P450 metabolism, peroxisomes, and inammatory cell
activation [18]. Exogenous sources include environmental agents
such as non-genotoxic carcinogens, various xenobiotics, ultrasound and microwave radiation [19,20].
Various ROS and RNS are given in Table 1. They have dual nature, on one hand they are necessary for normal cellular functions
but when in excess they can cause cellular damage and can lead
to cancer.

Mechanism of action of ROS in cancer

Cancer development is characterised by cumulative action of
multiple events occurring in single cell and can be described by
three stages: initiation, promotion and progression. ROS is involved in all these stages. The effect of oxidative stress at a certain
stage of carcinogenesis is directly proportionate to the type and the
reactivity of radicals involved. Initiation results when a normal cell
sustains a DNA mutation that, when proceeded by a round of DNA
synthesis, results in xation of the mutation, producing an initiated
cell. Initiation of cancer by ROS is supported by presence of oxidative DNA modications in cancer tissues [22]. The promotion stage
is characterized by clonal expansion of initiated cells, by induction
of cell proliferation and/or inhibition of apoptosis [19]. Oxidative
stress is strongly involved in this stage. ROS can stimulate expansion of mutated cell clones by temporarily modulating the genes
which are related to proliferation or cell death [23] and by regulating activity of certain transcription factors such as NFjB, Nrf2, HIF,
and p53 [24] which control cell growth and oncogenesis [23,25]. It
can lead to NFjB activation, with subsequent induction of genes
encoding for proteins that inhibit apoptosis [26]. It can also act
at signal-transduction level to exert pro-survival functions. Oxidative stress can activate ERK/MEK and PI3K/AKT pathways. This
could result in inactivation of proapoptotic proteins and upregulation of antiapoptotic genes [27]. A low level of oxidative stress can
stimulate cell division in promotion stage and thus promotes tumour growth [28]. This implies that ROS production during this
stage is the main mechanism of ROS-related tumour promotion.
ROS also contributes to the last stage of carcinogenesis, Progression. In this stage, generation of large amounts of ROS may contribute to mutate, inhibit antiproteisases, upregulate matrix
metalloproteinases (MMPs) [29,30] and injure local tissues [31].
Increased levels of oxidatively modied DNA bases may contribute
to genetic instability and metastatic potential of tumor cells in
fully developed cancer [32]. ROS is reported to be crucial for triggering angiogenic response, which is important in cancer metastasis [33]. This suggests that ROS is involved in all these stages of
carcinogenesis. ROS, which are formed through various events
and pathways, react with and damage cellular components and
contribute to neoplastic transformation [34,35]. Here is an overview of this (Fig. 1).

ROS mediated damage to biomolecules (Table 2) and its role in

carcinogenesis
Oxidative nuclear and mitochondrial DNA damage
DNA is highly sensitive to ROS attacks. Permanent modication
of genetic material resulting from this represents rst step involved in mutagenesis and carcinogenesis. Elevated levels of oxidative DNA lesions have been noted in many tumors, strongly

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S.K. Choudhari et al. / Oral Oncology 50 (2014) 1018

(5) Exposure of cells to H2O2, and perhaps other oxidants, suppress DNA repair in addition to inducing damage [42].
Reduced repair will result in elevated DNA lesions and an
increased risk of disease.

Table 1
Various reactive oxygen and nitrogen species [13,21].
Reactive oxygen species

Reactive nitrogen species

Superoxide anion (oxygen with an unpaired

electron) O2 
Hydrogen peroxide (H2O2)
Hydroxyl radical (OH)

Nitric oxide (NO)

Organic hydroperoxides (ROOH)

Alkoxy (RO) and peroxy (ROO) radicals
Hypochlorite anion (OCl )
Peroxynitrite (OONO )
Ozone (O3)

Peroxynitrite (OONO )
Nitrosoperoxycarbonate
(ONOOCO2 )
Nitrogen dioxide (NO2 )
Dinitrogen trioxide (N2O3)
Dinitrogen tetraoxide (N2O4)

Table 2
Types of antioxidants.
Enzymatic
antioxidants

Non-antioxidant enzymes

Superoxide
dismutase
Catalase
Glutathione
peroxidise

Antioxidant vitamins E(tocopherols), C(ascorbic acid)

and A(carotenoids)
Thiol antioxidants
Antioxidant minerals selenium, copper, manganese,
zinc
Melatonin
Flavenoids
Phytochemicals
Others coenzyme Q, dietary polyphenols

ROS/RNS mediated DNA damage may participate in carcinogenesis via activation of protooncogenes and inactivation of tumor
suppressor genes. In terms of oxidative DNA damage, major interest has focused on modications of DNA bases [3]. One of the most
frequent base modications is 8-hydroxy-deoxyguanosine (8-oxodG). This base modication formation increases by 3550% in
individuals using tobacco smoke a well known carcinogenic
source of ROS [20]. Accumulation of 8-nitroguanine, which is a
potentially mutagenic DNA lesion, and 8-oxodG is found in tissues
of patients with oral lichen planus (OLP) [43,44] oral squmaous cell
carcinoma (OSCC) [43]and leukoplakia [45], whereas no immunoreactivity was observed in normal oral mucosa [43]. Kawanishi
et al. from their study concluded that formation of 8-nitroguanine
and 8-oxodG may contribute to development of oral cancer from
OLP and leukoplakia [46]. They also demonstrated that iNOSdependent DNA damage may lead to p53 accumulation in OLP, leukoplakia and OSCC [46]. All these ndings suggest that oxidative
and nitrosative DNA damage may be responsible for initiation
and promotion of oral carcinogenesis and can be used as potential
biomarkers to evaluate the risk of oral cancer in potentially malignant disorders.
Mitochondrial DNA damage

implicating its role in cancer aetiology [3638]. ROS/RNS can have

following DNA damaging effects [39]:
(1) Cause structural alterations in DNA, e.g. base pair mutations,
rearrangements, deletions, insertions and sequence amplication. ROS can produce gross chromosomal alterations and
thus could be involved in inactivation or loss of second wildtype allele of a mutated proto-oncogene or tumour-suppressor gene that can occur during tumour promotion and progression, allowing expression of mutated phenotype.
(2) Affects cytoplasmic and nuclear signal transduction
pathways.
(3) Modulates activity of proteins and genes that respond to
stress and which act to regulate genes that are related to cell
proliferation, differentiation and apoptosis.
(4) RNS such as NO2 , ONOO , N2O3 and HNO2 are mutagenic
agents, with the potential to produce nitration, nitrosation
and deamination reactions on DNA bases [40,41].

ROS mediated deletions and mutations in mitochondrial DNA

(mtDNA), accumulate with age at a higher rate than in nuclear
DNA [47]. mtDNA is more vulnerable to free radical damage because of lack of histone proteins as well as its location is in close
proximity to the respiratory chain [42] and thus is frequently exposed to ROS-induced oxidative damage. Moreover, mtDNA repair
is less complete than chromosomal DNA repair making it an
important contributor to carcinogenesis.
In linking oxidative stress with promotion, it must not be forgotten that biomolecules other than DNA may be oxidatively modied which may have signicant effects in carcinogenesis.
Oxidative damage to proteins
Studies have proved that proteins are major initial cell targets of
ROS [48,49]. Oxidative damage of proteins involves loss of histidine
residues, oxidative scission, introduction of carbonyl groups,
and formation of protein-cantered alkyl, R, alkoxyl, RO, and

NO- RNS

DNA damage &

inhibition of DNA
repair mechanisms

Tumor initiation

Damage to
cellular
components

Inhibition of
apoptosis

Increased
angiogenesis

Upregulation of
MMPs & Downregulation of TIMPs

Tumor progression and

metastasis
Figure 1. Mechanism of action of RNS in cancer.

Immune
suppression

S.K. Choudhari et al. / Oral Oncology 50 (2014) 1018

alkylperoxyl, ROO, radicals [50]. Protein oxidation is connected

with formation of inter- and intra-protein cross linkages [19]. It
may result in fragmentation, cross-linking, and aggregation of proteins. Amino acid residue side chains are very susceptible to attack
by ROS and RNS [51]. Radical-protein interaction can damage functions of some important proteins such as DNA repair enzymes,
which can lead to increased frequency of mutations. Proteins oxidation leads to earlier formation of protein carbonyls in biological
systems [52], and increased levels of protein carbonyls has been
proposed as a sign of disease-associated dysfunction [53].
Advanced oxidative protein products (AOPP), are generated by different oxidation patterns that lead to production of either NO or,
H2O2 which sets a cascade of reactions with potential to damage
cellular micro-molecules. Nayar et al. in their study found high
serum levels of AOPP in patients with speckled leukoplakia and
OSCC as compared to healthy individuals [54]. It is required to nd
whether AOPP and other protein oxidation products can be used as
reliable oral cancer biomarkers.
Oxidative damage to lipids
Cell membranes are very sensitive to ROS damage [55]. Methylene group between two double bonds of polyunsaturated fatty acid
(PUFA) in cell membranes make them more sensitive to oxidation
[3]. ROS induced lipid peroxidation of cell membranes has been
implicated in malignant transformation [56]. It results in formation
of reactive aldehydes, including malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE), which demonstrate high reactivity with
proteins and DNA [19,56,57]. Aldehyde end-products of lipid peroxidation can bind to DNA and are potentially mutagenic [58]. Lipid
peroxides can decompose to a range of mutagenic carbonyl products [59]. MDA has been found to be mutagenic in bacterial and
mammalian cells and carcinogenic in rats [19]. Levels of these lipid
peroxides can serve as markers of cellular damage due to free radicals. Increased levels of lipid peroxidation products such as lipid
hydroperoxides, 4-HNE and MDA have been reported in oral cancer
and precancer patients [6,7,60,61]. This could be due to increased
formation of free radicals which suggest that there may be relationship between free radical activity and malignancy [62].
Oxidative modication of sugars [Advanced glycation endproducts (AGEs)] and ROS mediated damage to extracellular components can also occur. Whether these have any relation with oral
cancer needs to be evaluated. Along with ROS, RNS is also known to
play role in carcinogenesis process.
Mechanism of action of RNS in cancer
NO, an abundant reactive radical, acts as an important oxidative biological signalling molecule in various physiological processes. It becomes genotoxic and mutagenic when generated at
higher concentrations for prolonged periods of time. Reaction of
NO with oxygen or other free radicals generates RNS, which causes
multiple biological effects [63]. NO may mediate DNA damage
through formation of carcinogenic nitrosamines, generation of
RNS and inhibition of DNA damage repair mechanisms. It can thus
be considered a tumour initiating agent [64]. It may also have an
impact on other stages of cancer development by inhibiting apoptosis, promoting angiogenesis, by modulating host defence mechanisms, and by interacting with MMPs (Fig. 1) [65]. NO has also
been implicated in oral carcinogenesis. The high incidence of oral
cancer and precancer has been linked with tobacco chewing and
smoking habits [66,67]. NO radicals released from tobacco-related
compounds were shown to cause nitrosative stress and DNA strand
breaks in immortalized hamster cheek pouch cells [68]. Damage to
genes sustained by elevated ROS/RNS could be one of the

13

mechanism by which cancer arises in long-term tobacco abuse

[6]. NO products and NOS enzymes have been found to be raised
in blood and tissues of oral cancer patients [7,60,69]. Raised levels
of NO products are also noted in serum of oral precancer patients
[7,70] and in healthy individuals with tobacco habit [6,70]. Thus
NO may serve as a biomarker for estimation of oral cancer risk in
patients with potentially malignant disorders or in individuals
with tobacco habit.
Oxidative stress in oral cancer and precancer
Tobacco (smoking and smokeless) use and excessive consumption of alcohol are amongst the major risk factors for oral cancer
[7173]. ROS has been implicated in oral cancer development in
tobacco chewers and smokers [74,75]. Oxidative stress is increased
and antioxidant defences are compromised in patients with oral
cavity cancer [7,60]. In vitro studies have demonstrated that free
radicals are produced when human oral epidermal carcinoma cells
are incubated with smokeless tobacco extract [76]. There is continuous endogenous damage to cellular DNA by free radicals generated by the use of tobacco, and accumulation of such damage
plays signicant role in oral carcinogenesis [77]. Tobacco
chewing and smoking causes oxidant/ antioxidant imbalance
which elevates oxidative stress. This is accompanied by increased
lipid peroxidation, oxidative DNA damage, damage to macro and
micro-molecules of cells and disturbances of antioxidant defence
which can induce malignant process. The heat (generated during
smoking) as well as pH (change during chewing) of body uids
due to tobacco consumption affects formation and stabilization
of free radicals [74]. Furthermore, free radicals produced during
auto-oxidation of areca nut-polyphenols in saliva of tobacco users
are crucial in initiation and promotion of oral cancer [78]. This
establishes the role of ROS in oral cancer in tobacco users.
Areca (betel) nut chewing is the most important environmental
factor for oral cancer in South and Southeastern Asians [79]. Areca
nut is identied as a human group I carcinogen [80]. Areca ingredients induce formation of ROS and DNA adducts [8183]. Alkaline
conditions observed in betel nut chewing are favourable for formation of free radicals [74]. Hsuan-Hsuan Lu in their study conrmed
a signicant increase in ROS for areca nut extract-treated OSCC
cells [84] suggesting role of areca nut in oral carcinogenesis
through generation of ROS as one of the mechanisms.
Alcohol increases oral cancer risk in humans regardless of the
form it takes. Petti and Scully in their extensive study found a
strong association between alcohol-drinking proles and oral cancer mortality [85]. Free radicals are produced in excessive amounts
in alcoholics. Ethanol is oxidised by Cytochrome P450 2E1
(CYP2E1) to acetaldehyde which is further oxidised to acetate.
Chronic ethanol ingestion can induce single nucleotide polymorphism of CYP2E1. Resulting increased CYP2E1 activity leads to increased generation of ROS which leads to lipid peroxidation and
its products such as 4-hydroxynonenal (4HNE), which binds to
DNA to form mutagenic adducts [86]. Vitamin C deciency and
marked tissue depletion of vitamin E found in alcoholics [87,88]
is due to their increased utilization for scavenging free radicals
produced in chronic alcoholics [89]. All this suggest that in alcoholics there is oxidantantioxidant imbalance resulting from increased oxidative stress and weakened antioxidant system which
may result in initiation of oral cancer.
Fig. 2 shows role of ROS in oral carcinogenesis.
Oxidants as biomarkers in oral cancer and precancer
It is important to develop oxidative stress biomarkers as they
can inuence the way we diagnose, classify, and monitor tumour

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S.K. Choudhari et al. / Oral Oncology 50 (2014) 1018

Endogenous sources or
exogenous sources like
Tobacco, alcohol, areca nut, etc

Increased ROS

Oxidant antioxidant imbalance

Disturbances of
antioxidant defence

Oxidative Damage to
DNA

Chemical changes in
bases & changes in
DNA confirmation

Mutations &
genetic instability

Damage to lipids

Damage to proteins

Decreased efficiency of
DNA repair enzymes

Impaired/ imbalanced
DNA repair

Increased lipid
peroxidation

Loss of cellular
integrity and cell
damage

Formation of
mutagenic protein &
DNA adducts

Cancer

Figure 2. Role of ROS in Oral carcinogenesis.

progression. It can also help to develop new therapeutic targets

and to augment tumour response to therapy. Sensitive, specic,
and reliable methods are required to detect changes in reactive
species so as to develop better understanding about their role in
carcinogenesis.
Detection of ROS/RNS in biologic systems is often problematic.
They have short half-life (seconds) and there are efcient and
redundant systems to scavenge them. At cellular level specic
ROS can be individually assessed in tissue or quantication of the
oxidative damage of biomolecules in saliva, blood or urine is another way of measuring these biomarkers. Stable, specic, or nonspecic derivatives of these substances can be measured, for e.g.
lipid peroxidation products (isoprostanes), amino acid oxidation
products (meta-tyrosine, ortho-tyrosine, hydroxyl-Leu dityrosine),
peptide oxidation products (oxidized glutathione) [90]. These end
products can be measured by changes in their uorescence, colour,
or luminescence. Some of these biomarkers are studied in oral cancer and precancer as mentioned earlier. But large scale and long
term follow up studies are required in order to get insights into
the mechanisms of oral carcinogenesis as well as to develop new
therapeutic targets. Easier techniques for their measurements,
low cost of evaluation can make them versatile and useful prognostic tool for identication of oral cancer patients with high risk
for recurrence and oral precancer patients with high risk for oral
cancer.
Defence against reactive oxygen species: Antioxidants
Antioxidant refers to any molecule capable of stabilizing or
deactivating free radicals before they attack cells [91]. Humans
have evolved highly complex antioxidant systems (enzymic and
nonenzymic) (Table 2), which work synergistically to protect cells
and organ systems of the body against free radical damage. These
can be endogenous or obtained exogenously. They are abundant in

fruits and vegetables as well as in other foods such as nuts, grains,

some meats, poultry and sh. They are intimately involved in
prevention of cellular damage caused by oxidants, the major mechanism for cancer initiation. An imbalance between antioxidant defence mechanisms and oxidants leads to cell and tissue damage
resulting in cancer. Different enzymatic and non-enzymatic antioxidants with their antioxidant actions and their signicance in
carcinogenesis are given in Table 3.
Antioxidants and carcinogenesis
Under pathological conditions, much larger amounts of free
radicals are formed than normal. To mitigate their harmful effects,
antioxidant defense mechanisms act at different levels. Strategies
in biology designed to evade oxidative stress are prevention, interception, and repair of free radicals or their damage by antioxidants.
A rst line of defense against ROS is protection against their formation, i.e. prevention. Metal chelation is a major means of controlling lipid peroxidation and DNA fragmentation [20]. A strategy of
preventive antioxidation also works by channeling an attacking
species into a less harmful product hence lowering the risk of further damage. The next stage is interception, the process of nal
deactivation. In interception would come the strategies which will
prevent chain reaction of free radicals and formation of non-radical
and non-reactive-end products. Another measure is to change the
direction of radical function from more sensitive target sites to cellular compartments in which oxidative damage would be less deleterious. Such intercepting chain-breaking antioxidants are often
phenolic compounds. Non-enzymatic antioxidants like alphatocopherol is probably the most efcient compound in lipid phase.
It maintains a steady state of peroxyl-radical reduction in the cellular membranes [111]. Carotenoids and oxy-caretenoids can
efciently intercept hydroxyl radical [112]. Three major classes
of antioxidant enzymes, superoxide dismutases, catalases and

S.K. Choudhari et al. / Oral Oncology 50 (2014) 1018

15

Table 3
Antioxidants, their actions, and signicance in carcinogenesis.
Name of antioxidant and its nature
Enzymatic antioxidants
Superoxide dismutase(SOD)

Catalase
Glutathione peroxidise

Non-enzymatic antioxidants
Vitamin C

Vitamin E Alpha Tocopherol most

active form of vitamin E in humans

Thiol antioxidants glutathione

Caretenoids pigments that are found

in plants and microorganisms

Flavenoids low molecular ubiquitous

groups of plant metabolites and are
an integral part of the human diet

Antioxidant action

Signicance in carcinogenesis

Destroys O2 [92]

Cu, Zn-SOD catalyzes dismutation of superoxide anion to
oxygen and water [19]
Has high turnover rates [19]
Conversion of H2O2 to water and molecular oxygen [19]
Selenium-dependent glutathione peroxidise catalyzes
conversion of H2O2 or organic peroxide to water or alcohol
[93]

Mn-SOD has anti-tumour activity [19]

Works in aqueous environment, protects membranes against

oxidation [19,94]
Reduces vitamin E degradation
Enhances detoxication via cytochrome P450 [87]
Provides protection against lipid peroxidation [96]
Major membrane bound antioxidant employed by the cell
[97]
Causes inhibition of free radical formations and activation of
endonucleases [19]
Inhibition of DNA, RNA and protein synthesis in cancer cells
[87]
Major thiol-disulphide redox buffer of the cell [101]
Co-factor for several detoxifying enzymes.
Scavenges hydroxyl radical and singlet oxygen directly,
regenerate Vitamins C and E [101]

Reduces incidence of stomach cancer due to its inhibitory

action in generation of N-nitroso compounds [19]

Quenches singlet oxygen without degradation [19]

Prevents damage in lipophilic compartments at low oxygen
partial pressure [105]
Immunomodulation, stimulation of increase in numbers of Thelper and NK cells as well as cells with IL-2 receptors [87]
Can scavenge peroxyl radicals, and are effective inhibitors of
lipid peroxidation, can chelate redox-active metals, and thus
prevent catalytic breakdown of H2O2 (Fenton chemistry)
[107]

glutathione (GSH) peroxidises are important detoxifying compounds. Repair of damage caused by oxidants can be the next stage
in protection against oxidants. There are multiple enzyme systems
involved in DNA repair and lipolytic as well as proteolytic enzymes
capable of serving the functions of restitution or replenishment for
DNA damage, membrane damage, and damage to proteins correspondingly [20].
Weakened antioxidative defence mechanisms result in oxidative-antioxidant imbalance. Reduced activities of antioxidants with
concomitant increased levels of oxidative stress have been
reported in different cancers including head and neck
[7,60,113,114]. Low levels of antioxidants have been associated
with increased risk of oral cancer. Various studies have reported
lowered antioxidants or antioxidant capacity in blood and tissues
of oral precancer and cancer [7,10,70,115]. This could be due to
(1) increased utilization of antioxidants to scavenge ROS/RNS, (2)
poor antioxidant defence system in cancerous environment, (3)
inadequate production of antioxidant enzymes, and (4) increased
destruction of antioxidants by reactive oxygen metabolites. Lowered capacity to defence ROS/RNS might be one of the possible
mechanisms operating in the progression of oral cancer [7]. More
studies should be carried out to know the reliability of antioxidants
to be used as oral cancer biomarkers. Considering the protective
role of antioxidants against free radicals, they are being tried in
cancer prevention and therapeutics.

Linked to increased capacity of variety of tumours for

detoxifying H2O2 [19]
Major source of protection against low levels of oxidative
stress [93]

Protective effect in lung and colorectal cancer [95]

c- and d-tocopherols, are cancer preventive [98]
Protective effect against cigarette smoking [98]
Inhibitory activities in lung and colon cancer [98] and
colorectal cancer [99]
Mixed tocopherols have been found to prevent mammary
carcinogenesis in animal studies [100]
N-acetyl-L-cysteine, inhibit spontaneous mutations and
induced mutations in bacteria, prevent in vivo formation of
carcinogen-DNA adducts, and suppresses or delays
development of tumors or preneoplastic lesions in rodents
[102]
May prevent tumor initiation, and reduced thiols may
defend against oxidative stress [103]
Can be useful for predicting the risk of oral carcinogenesis in
healthy tobacco consumers and predicting overall survival of
oral cancer patients. [104]
May prevent or inhibit certain types of cancer, can cause
inhibition of mutagenesis, inhibition of cancer cell growth
[87]
Can act as a pro-oxidant [19,106]
Antitumor, anticancer agents [108,109]. Modify activities of
enzymes involved in carcinogenesis, tumor growth and
metastasis [108]. Flavonoids from grape seed
proanthocyanidins have been reported to inhibit UVradiation-induced oxidative stress [110]

Cancer inhibitory actions of antioxidants

Enzymic and nonenzymic antioxidant systems, work synergistically, and in combination with each other to protect cells and organ
systems against free radical damage and therefore cancer. Cancer
inhibitory properties of antioxidants are based on: [116,117]
1.
2.
3.
4.

Immune mechanisms.
Molecular genetics pathway.
Depression of tumour angiogenesis activity.
Stimulation of cell differentiation.

Antioxidants help in exerting potent immune response by stimulation of cytotoxic cytokines that will destroy cancer cells. In the
hamster buccal pouch cancer model it has been shown that beta
carotene and alpha tocopherol stimulate the migration of cytokine-laden macrophages and lymphocytes to the sites of developing Squamous cell carcinoma [118]. Antioxidant nutrients were
found to stimulate the activity of langerhans cells in hamster carcinogenesis model [119,120]. They act through stimulation of cancer suppressor genes, such as wild type p53 and diminished
expression or dysregulation of oncogenes such as mutant p53
and H-ras. Antioxidant micronutrients inhibit angiogenesis in tumors by inhibiting TGFalpha. Retinoids promote cellular differentiation with resultant apoptosis of neoplastic cells [121].

16

S.K. Choudhari et al. / Oral Oncology 50 (2014) 1018

Oral cancer many times evolves through precancerous lesions

and conditions. Treatment at these earlier stages can result in high
cure rate. Antioxidants have the potential to prevent, inhibit and
reverse the multiple steps involved in oral carcinogenesis. Curcumin (diferuloylmethane), a yellow colourings agent present in turmeric, has been linked with suppression of mutagenesis [122]. It
has been demonstrated that it downregulates STE (khaini) or
NNK-induced NF-jB and COX-2 in oral premalignant and cancer
cells in vitro [123]. Rai et al. from their study on oral precancerous
lesions concluded that curcumin mediates its anti-pre-cancer
activities by increasing the levels of vitamins C and E, and by
preventing lipid peroxidation and DNA damage at a dose of up to
8 g/d, without any toxic effects [124]. Decrease in MDA level along
with clinical improvement after oral administration of beta-carotene and vitamin E in oral submucous brosis (OSMF) patients is
reported [125]. Maher et al. also reported clinical improvement
in OSMF patients after giving retinol, vitamin E, D, and B complex
and some minerals [126]. Chitra from her study concluded that alpha-tocopherol plays role in protection against the damage caused
by radiation in OSCC patients treated with radiotherapy [127].
Lycopene, a bright red carotene and carotenoid pigment, is a phytochemical antioxidant. It helps to attenuate free- radical- initiated
oxidative reactions, particularly lipid peroxidation and DNA damage. It has antiproliferative and prodifferentaition properties. Studies showed that lycopene might have benecial effects in
management of oral premalignant lesions [125,128130]. The
curative effect of lycopene in OSMF may be owing to an inhibition
of abnormal broblasts, upregulation of lymphocyte resistance to
stress, and suppression of inammatory response [131]. But to
prove lycopene a promising anticarcinogenic agent for oral cancer,
rigorously designed clinical trials are still necessary. Carotenoids
and tocopherols have been shown to be capable of cancer regression, as well as inhibition and prevention of carcinogenesis [132].
Antioxidants have also been used to enhance the efcacy of
cancer treatment by radiotherapy and chemotherapy. Elango
et al. in their study found a marked improvement in antioxidant
status in oral cancer patients who were supplemented with selenium during radiotherapy [133]. Shariff et al. suggested that
antioxidant supplementation during radiotherapy may serve as
an adjuvant therapy in malignancies offering a good protection
to normal cells that may further reduce the risk of developing
secondary cancers [134]. However, the association of antioxidants and cancer treatment is a controversial issue. There is a
concern that antioxidants might reduce the efcacy of drugs
which damages malignant cells by generation of free radical oxidants. However, in oral cancers which are more solid tumors
where radio and chemotherapy serve as adjuvant therapies, predominance of evidence supports that antioxidants do not conict
with the use of these therapies. And they could be of help in
reducing the side effects associated with treatment. However,
studies with specic antioxidants like vitamin C, E, selenium,
taking larger patient cohort with longer follow up period for
checking therapeutic response are required to derive a proper
conclusion.
Although antioxidant defences are important but antioxidant
therapy against free radicals should be used only with caution
since its effects depend on the stage at which it is introduced.
When used during progression stage of cancer, it might actually
stimulate growth of tumours through enhanced survival of tumour
cells. Another important issue which should be taken into consideration is pro-oxidant character of some antioxidants which may
occur depending on concentration and environment (oxygen pressure) in which they act. Major interest is now focused on possible
anticancer activity of relatively non-toxic antioxidant nutrients
like beta carotene, alpha tocopherol and glutathione, as well as various retinoids.

Conclusions
Free radical generation is a continuous process in biological system which is unavoidable. ROS/RNS, when in excess have an ongoing and usually detrimental effects in humans. They attack and
damage cellular macromolecules and can lead to cancer. Tobacco,
the major risk factor associated with oral cancer, interact with cells
through generation of ROS. ROS in turn can activate various transcription factors resulting in expression of proteins that control
inammation, cellular transformation, tumour cell survival, tumour cell proliferation and angiogenesis and metastasis. While
involvement of oxidants at various stages of malignant transformation is evident, many details regarding role of ROS-induced damage
in aetiology of oral cancer is yet to be discovered. ROS/RNS can
serve as oral cancer biomarkers which could help in knowing early
prognosis and also in structural design of new and more efcient
therapeutic regimes. It is required to evaluate whether ROS/RNS
would reliably identify people with potentially malignant disorders who are at high risk of developing oral cancer. Protection
against ROS/RNS primarily occurs through exogenous dietary antioxidants or through enzymatic cellular defence mechanisms. Many
research papers have demonstrated anticancer activity of retinoids,
carotenoids and tocopherol in oral precancer and cancer. Their
anticancer mechanisms have also been explored to develop preventive and therapeutic strategies against oral cancer. Another
application of antioxidants could be to develop specic, sensitive,
high throughput diagnostic and prognostic markers that can be
used routinely. Application of such cancer predictive biomarkers
within cancer preventive strategies is warranted in view of reducing global head and neck cancer burden. Use of oxidants and antioxidants should be explored with the aim of reduction of incidence
of potentially malignant disorders, clinical regression of these disorders, reduction in incidence of oral cancer, prevention of metastasis and prevention of development of secondary malignancies in
patients cured of primary malignancies. Epidemiological studies
have shown that high intake of antioxidant rich foods is inversely
related to cancer risk. Change in lifestyle with proper intake of balanced diet which will supply the much needed antioxidants, would
denitely help people to have low health risks and enable them to
live longer without disabilities.
Conict of interest
None declared.
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