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metabolic activities and exogenously by number (ROS), and reactive oxygen metabolites such as
of harmful compounds, tobacco and tobacco superoxide anions (O2), hydrogen peroxide (H2O2),
products. In human cells, mitochondria are the hydroxyl radicals (OH), malondialdehyde, and
major intracellular source of reactive oxygen species nitric oxide are involved in the multistep process of
(ROS) generation.[1,2] These molecules are highly carcinogenesis. The free radicals increase the level
254 Journal of Cancer Research and Therapeutics - April-June 2012 - Volume 8 - Issue 2
Kumar, et al.: Oxidative stress and cancer
of proteins degradation products such as kinins and activate related parameters (ROS, RNS, GSH), antioxidant capacity (TAC)
the arachidonic acid.[3] The free radical-induced oxidative stress and oxidative DNA adduct (8-OHdG) in plasma of patients
is associated with a variety of chronic degenerative diseases, with SCCHN and their respective controls to understand the
including cancer, diabetes, cardiovascular diseases, and relationship between the levels of oxidants and antioxidants
Alzheimers disease as well as in aging. The free radicals can in pathogenesis of the disease.
induce several kinds of DNA damage including strand breakage,
base modification and DNAprotein cross-linkage. They can MATERIALS AND METHODS
react with cell membrane fatty acids and form lipid peroxides.
Patients
8-Hydroxy-2-deoxyguanine (8-OHdG) is one of the major This casecontrol study was approved by the ethical
oxidative modified DNA base products, which may lead to G:C committees concerned institute and medical university for
to T:A transversions.[4,5] The 8-OHdG was first reported by Kasai clinical research. The protocol confirmed to the provisions of
et al.[6] to be formed on interaction of hydroxyl radical (OH) and declaration of Helsinki in 1995. Prior to collection of samples,
singlet oxygen photodynamic action with DNA. Several studies an informed consent was obtained from the study subjects,
have reported the higher content of 8-OHdG in cancer tissue in for inclusion in the study and subject anonymity was ensured.
comparison to normal tissue. Another risk factor which can also A total of 100 newly diagnosed patients with biopsy proven
contribute to head and neck cancer is the human papilloma virus SCCHN prior to any chemoradiotherapy and 90 healthy control
(HPV). The HPV infects the epithelial cells of skin and mucosa, subjects were included. The mean age of SCCHN patients was
such as the mouth, throat, tongue, tonsils, vagina, penis and 53 (range, 2575 years) and mean age of the controls was 51
anus. Infection with the virus occurs when these areas come into (range, 2260 years). Subjects having regular smoking habits
contact with the virus, allowing it to transfer between epithelial and smoking index (cigarettes/day 365) of more than
cells. It is recognized as the major risk factor in about 60% of 730[15] and regular smokeless tobacco chewers with chewing
head and neck cancer, particularly among young subjects with no index more than 365[16] (CY = frequency of tobacco chewed/
tobacco or alcohol history.[7,8] All HPV-positive cases express viral kept/day 365) were considered in the category of smokers
E6 and E7 oncoproteins which lack specific DNA binding activity, and tobacco chewers, respectively. All the subjects belonged
but can still associate with transcription factor complexes, such to the same socioeconomic group [Table 1]. Two milliliters
as p53 and E2F and alter their transcriptional activity. The high- of the blood sample was collected in 3.4% sodium citrate
risk HPV E6 proteins target p53 for proteosomal degradation, (pH 7.6) vial. The blood samples were immediately kept in ice
whereas E7 expression results in p53 stabilization, but inhibits till further use. One milliliter blood was centrifuged at 2500
its transcriptional activity.[9] rpm for 15 min at 4 C, to separate plasma and remaining one
ml was used for DNA isolation.
On the other hand, all organisms possess a range of enzymatic
and nonenzymatic antioxidant systems, which neutralize a free
radical molecule to a non-free-radical molecule. The enzymes Table 1: Clinical and the socio-demographic details of the
subjects
included in the antioxidant system are glutathione peroxidase,
glutathione reductase, catalase, thioredoxin reductase, N = 90
Healthy individuals
superoxide dismutase, heme oxygenase and biliverdin
Age
reductase. The nonenzymatic part includes antioxidants Median 51
and free radical scavengers, such as -tocopherol (vitamin Range 2560 years
E), vitamin C, phytochemicals, carotenoids and glutathione. Tobacco habituates 84
Non-habituates 6
Glutathione (GSH) is a ubiquitous thiol-containing tripeptide
Habituates
(L--glutamyl-L-cysteinylglycine), which plays a central role Type
in cell. It is a critical factor in protecting organisms against Smokers 32
toxicity and disease since it provides reducing capacity Chewers 14
for several reactions and plays an important role in the Smokers + chewers 38
HNSCC patients N = 100
detoxification of hydrogen peroxide and other free radicals.[10] Age
GSH degrades hydrogen peroxide and singlet oxygen before Median 53
they are converted to a hydroxyl radical. Pastore et al.[11] Range 2575 years
suggested that GSH in the nucleus is involved in mechanisms Tobacco habituates 93
Non-habituates 7
that are necessary for DNA repair and expression. Habituates
Type
In recent past, the role of oxidant and antioxidant system Smokers 34
in development of cancer has gained importance. Higher Chewers 15
Smokers+chewers 44
oxidants and lower antioxidant activities in blood of cancer Site
cases suggest their significance in progression of disease.[12-14] Oral region (Tongue, buccal mucosa, cheek, 54
etc.)
The aim of this study was to evaluate oxidantantioxidant Neck region (Larynx, Pharynx, etc.) 46
Journal of Cancer Research and Therapeutics - April-June 2012 - Volume 8 - Issue 2 255
Kumar, et al.: Oxidative stress and cancer
Total antioxidant capacity (TAC), glutathione (GSH), reactive after 10 min incubation at 37C, the absorbance was measured
oxygen species (ROS) and reactive nitrogen species (RNS) were at 550 nm on a plate reader (BMG FLUOstar Omega). For the
analyzed in plasma of all the subjects. quantification of total nitrite in plasma, sodium nitrite solution
was used as standard.
Total antioxidant capacity
Total antioxidant capacity was measured using an Antioxidant Oxidative DNA damage
Assay Kit (Cayman Chemical Company, USA). Theoretically, the The oxidative DNA damage (8-OHdG) was quantified using a
assay relies on the ability of antioxidants in the sample to DNA Damage Quantification Kit (BioVision, USA). After treating
inhibit oxidation of ABTS (2,2-azino-di-(3-ethybenzthiazoline DNA containing abasic sites (AP) with aldehyde reactive probe
sulphonate)) by metmyoglobin, which can be monitored by reagent (ARP), AP sites were tagged with biotin residues which
reading the absorbance at 750 nm. The capacity of antioxidants can be quantified using the avidinbiotin assay followed by
in the sample to prevent ABTS oxidation is compared with colorimetric detection.
that of Trolox and quantified as millimolar Trolox equivalents.
Genomic DNA from whole blood was isolated using the lab
Ten microliters of plasma (20 times diluted) was taken in protocol of Laura-Lee Boodram, Department of Life Sciences,
a microplate and the procedure followed according to the The University of West Indies, with minor modifications.
manual. The absorbance of ABTS oxidation was measured at To 400 l of whole blood, an equal volume of buffer A (0.32
750 nm on a plate reader (BMG FLUOstar Omega). M sucrose, 10 mM TrisHCl, 5 mM MgCl2, 1% Triton X-100,
adjusted to pH 7.6) and two volumes of cold sterile distilled
Glutathione water were added and after gently vortexing for 30 s, it was
Glutathione in plasma was evaluated by using o-phthaldialdehyde kept on ice for 3 min. Following centrifugation at 3500 rpm
(OPT).[17] OPT reacts with both glutathione amino and sulphydryl for 15 min at 4C, the pellets were dissolved in 800 l buffer A
groups, yielding a cyclic highly fluorescent product. Briefly, 25 and 1200 l cold sterile distilled water. The sample was again
l each of plasma was added in a microplate and the volume centrifuged at 3500 rpm for 15 min at 4C and the pellets
was made up to 200 l with HEPES buffer (0.1 M, pH 7.4). Ten (white or cream in color) were re-suspended in 1 ml buffer B
microliters of o-phthaldialdehyde (OPT, 100 mM)) was then (20 mM TrisHCl, 4 mM Na2EDTA, 100 mM NaCl, adjusted to
added and after 10 min incubation at 37C, the fluorescence pH 7.4) and 100 l of 10% SDS. Ten microliters of Proteinase
was measured at 360 nm excitation and 460 nm emission on a K (20 mg/ml, freshly prepared) was then added and further
plate reader (BMG FLUOstar Omega). Glutathione reduced was incubated overnight at 37C.
used as standard, for quantification of GSH.
After overnight incubation with Proteinase K, 250 l of 6
M NaCl was added and after vigorous shaking for 15 s, the
Reactive oxygen species
samples were centrifuged at 2500 rpm for 15 min. Pellets
The oxygen free radicals were measured with the help of
weres discarded and the supernatant was taken in a separate
2,7,-dichlorofluorescein diacetate (DCF-DA), a fluorescent
tube and double volume of cold ethanol (100%) was added to
probe, which on interaction with ROS yields highly
it, inverting the tube seven to eight times to precipitate DNA.
fluorescent DCF.[18] In cells DCF-DA diffuses through the cell
The precipitated DNA was resuspended in 200 l of TrisHCl,
membrane and is subsequently deacetylated by intracellular pH 8.5 and was kept at 37C to dissolve. The NanoDrop
esterases to nonfluorescent DCF-H, while in the cell free Spectrophotometer (ND 1000 V3.3.1) was used to measure the
system, DCF-DA on treatment with 0.1M NaOH for 30 min amount and purity of DNA. Five microliters of a highly purified
at room temperature gets converted into nonfluorescent DNA sample (0.1 g/l), isolated from blood, were taken in a
DCF-H, which reacts with free radicals and produce microcentrifuge tube, mixed with 5 l of ARP solution and
fluorescent DFC.[19,20] Briefly, 25 l of plasma was added in incubated for 1 h at 37C, to tag AP sites of DNA. Assay was
a microplate, and the volume was made up to 200 l with carried out according to the manual provided. 40 ARP-DNA
PBS. Twenty-five microliters of freshly prepared DCF-H Standard (40 ARP sites per 105 bp) was used for quantification
(500 M final concentration) was then added to each well and of AP sites in samples to determine the level of DNA damage.
after 1 h incubation at 37 C, the florescence was measured
at 485 nm excitation and 528 nm emission on a plate reader Statistical analysis
(BMG FLUOstar Omega). 2,7-Dichlorofluorescein was used The data were statistically analyzed using SPSS statistical
as standard, for quantification of total ROS. software (Version 12). Students t-test was performed to
compare levels between controls and patients. Pearsons
Reactive nitrogen species correlation was carried out to study the association between
The nitrite present in plasma reacts with sulfanilamide and the various oxidants and antioxidants. Differences between
N-(naphthyl)ethylenediamine to produce a red color.[21] Briefly, groups and variables were analyzed for significance using an
10 l of plasma was added in a microplate and the volume one-way ANOVA test using GraphPad PRISM 5 software (CA,
made up to 100 l with PBS. Fifty microliters of Griess Reagent USA). The difference was considered statistically significant
I and of Griess Reagent II were then added to each well and when P value were 0.05 or less.
256 Journal of Cancer Research and Therapeutics - April-June 2012 - Volume 8 - Issue 2
Kumar, et al.: Oxidative stress and cancer
**
another set of comparisons, between control smokers versus
15. 00 1.64
*
9.20 0.57
***
2.54 0.38
3.20 0.35
1.40 0.23
1.63 0.28
Table 3: Levels of the plasma oxidative stress determinants in controls and SCCHN cases
Non-habituates Smokers Chewers Smokers + chewers
Control Case Control Case Control Case Control Case
TAC mM (mean SE) 1.83 0.10 1.50 0.17 1.67 0.03* 1.24 0.02* 1.55 0.03 1.26 0.02 1.48 0.04* 1.33 0.05*
GSH M (mean SE) 5.12 0.48 3.16 0.95 4.40 0.52* 2.48 0.61* 4.06 0.77* 2.37 0.78* 3.87 0.42* 2.33 0.47*
ROS M (mean SE) 2.49 0.65 8.47 1.02 3.24 0.72* 9.22 1.44* 3.35 1.39* 9.97 1.61* 3.87 0.88* 10.71 1.42*
RNS M (mean SE) 31.74 0.30 40.09 1.28 35.98 7.54* 65.17 3.80* 38.43 2.37* 66.77 14.34* 43.33 3.01* 76.67 8.18**
8-OHdG AP sites/105 5.83 0.39 9.11 0.21 7.33 0.71 13.28 2.36 9.55 0.77 14.12 1.59 10.84 0.88 16.08 2.82
(mean SE)
**P < 0.001, *P < 0.05, vs. control by one-way ANOVA
Journal of Cancer Research and Therapeutics - April-June 2012 - Volume 8 - Issue 2 257
Kumar, et al.: Oxidative stress and cancer
habituated patients, no statistical significance was achieved On the other hand, the antioxidant system includes a
in any of the case groups [Table 3]. The overall redox profile variety of antioxidants including vitamins, Carotenoids,
of blood clearly demonstrates oxidative stress determinants flavonoids and glutathione. They scavenge free radicals
including 8-OHdG levels, to be highly altered in SCCHN. and protect the cells from harmful oxidants. Depletion
in levels of antioxidants in the body may lead to harmful
DISCUSSION consequences including cancer. The association between
cancer and inadequate levels of antioxidant capacity has been
In this study, we assess the levels of oxidants as well as reported.[14,23,36-38] Glutathione, another antioxidant, is also
antioxidants in human blood plasma, which forms the frontline found to be lower in cancer patients in comparison to
defense to encounter various oxidants present in tobacco healthy controls.[23,39] Farias et al.[36] claimed that there is
smoke, tobacco chewing, alcohol and food. It is evident that no significant difference in levels of GSH in breast cancer
the higher level of oxidants inside the body or cell may lead patients. The glutathione participates as nonenzymatic
to serious consequences, several neurodegerative, parasitic antioxidant. The other nonenzymatic antioxidants include
diseases and cancer.[12-14,22,23] The imbalance between the levels ascorbic acid (vitamin C), -tocopherol (vitamin E), carotenoids
of oxidants and antioxidants may implicate in the pathogenesis and flavonoids. Another antioxidant studied in this study, is
of SCCHN. The basic concept is that the free radicals damage glutathione, present in all mammalian tissues and is a critical
cellular materials which could result in activation or altering factor in protecting organisms against toxicity and disease.
normal cells into malignant ones.[24] The ROS and RNS are Several studies reported that the decrease in GSH is associated
involved in initiation and promotion of carcinogenesis through with the development of cancer.[40,41] However, some of the
DNA damage.[25,26] for example, NO-mediated inhibition of base studies reported an increase in the levels of GSH in cancer
excision DNA repair may potentiate oxidative DNA damage in patients.[42,43] GSH also prevents oxyradical damage, and thus,
cells and could be relevant to carcinogenesis.[27] Likewise Van blood GSH level may serve as an indicator of GSH status and
Wijk et al.[28] suggested that the metabolism of ROS in cancer disease risk in human subjects.
cells is drastically altered with evidence favoring at least
two mechanisms; cancer cells produce large amounts of ROS These findings indicate an imbalance in the oxidant
compared to non-neoplastic cells and secondly, suppression of antioxidant status that results in reduced TAC and GSH and
the antioxidant system in cancer cells. An oxygen free radical enhanced production of ROS and NO2 in head and neck cancer
interaction with DNA can break its strands or delete a base. patients. This discrepancy in the redox status appears to
have a marked effect on DNA oxidation (DNA adduct), one of
The free radical-induced genetic alterations such as mutations the causative factors for oral cancer development. Since the
and chromosomal rearrangement can lead to initiation and alteration in the oxidantantioxidant profile is more prominent
progression of carcinogenesis. Mutations can occur through in those patients with both smoking and chewing habits, it
misrepair or due to incorrect replication, while chromosomal is imperative to believe that lifestyle habits do play a central
rearrangements can result from strand breakage misrepair.[29] The role in the onset of SCCHN.
increase in replication errors can initiate additional oncogene
activation and tumor suppressor gene inactivation, ultimately Our results also support the idea that oxidative stress plays a
contributing to malignancy. Free radical-induced cytotoxicity role in the development of head and neck cancer. The 8-OHdG
may also contribute to the initiation of carcinogenesis by could be a potential biomarker in evaluating the risk of SCCHN.
To develop new approaches of SCCHN prevention, we need
depleting the normal cell population, Promoting clonal
further studies and better understanding of the molecular
expansion of more resistant initiated cells and thus increasing
mechanisms underlying oxidative stress and DNA damage.
the probability of mutation.[30]
ACKNOWLEDGMENTS
Oxidative DNA damage can affect carcinogenesis by
modulation of gene expression and altered gene expression can
The authors are grateful to the Director, CSIR Indian Institute of
lead to the stimulation of growth signals and proliferation.[31]
Toxicology Research, Lucknow, for his keen interest and support in
It is also evident that ROS may stimulate signal transduction
carrying out the study. AK is thankful to UGC, New Delhi, for providing
pathways, for example, protein kinase and poly(ADP
a Senior Research Fellowship. Financial support from CSIR Network
ribosylation), c-Raf-1 and ras pathways.[32,33] In addition to
Project SIP-08 is gratefully acknowledged. CSIR-IITR communication
8-OHdG production, an accumulation of intracellular ROS and/
number is 3014.
or RNS can induce point mutation in the DNA, thus disrupting
the expression and function of several tumor-suppressing
genes such as RAS and p53, which might contribute to the REFERENCES
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The 8-oxoGua induced aberrant modifications in adjacent DNA, ed. New York: Oxford University Press; 1999.
a hypothesized mechanism, can significantly contribute to 2. Richter C, Gogvadze V, Laffranchi R, Schlapbach R, Schweizer M,
the genetic instability and metastatic potential of tumor cells. Suter M, et al. Oxidants in mitochondria: From physiology to diseases.
258 Journal of Cancer Research and Therapeutics - April-June 2012 - Volume 8 - Issue 2
Kumar, et al.: Oxidative stress and cancer
Journal of Cancer Research and Therapeutics - April-June 2012 - Volume 8 - Issue 2 259