Detection of reactive oxygen metabolites in malignant and adjacent normal tissues of patients with lung cancer.

0.24 MB·English

by  Okur, Hacer Kuzu

Okuretal.WorldJournalofSurgicalOncology2013,11:9
http://www.wjso.com/content/11/1/9 WORLD JOURNAL OF
SURGICAL ONCOLOGY
RESEARCH Open Access
Detection of reactive oxygen metabolites in
malignant and adjacent normal tissues of
patients with lung cancer
Hacer Kuzu Okur1*, Meral Yuksel2, Tunc Lacin3, Volkan Baysungur3 and Erdal Okur4
Abstract
Background: Different types of reactive oxygen metabolites (ROMs)are knownto be involved incarcinogenesis.
Several studies have emphasized the formation of ROMs in ischemic tissues and in cases of inflammation. The
increased amountsof ROMs intumor tissues can either be because of their causative effects or because they are
produced bythe tumor itself. Our study aimed to investigateand compare the levels of ROMs intumor tissue and
adjacent lung parenchyma obtained from patients withlung cancer.
Methods: Fifteenpatients(all male, mean age 63.6 ± 9years) with non-small cell lung cancer were enrolled inthe
study. Allpatients were smokers.Of the patients with lung cancer, twelve had epidermoid carcinoma and threehad
adenocarcinoma.During anatomical resection ofthelung, tumor tissue and macroscopically adjacent healthy lung
parenchyma (control) that was 5cm away from thetumor were obtained. The tissues were freshly frozen and
stored at−20°C. The generation of ROMs was monitored using luminol- and lucigenin-enhanced
chemiluminescence (CL) techniques.
Results: Bothluminol(specificfor.OH,H O ,andHOCl-)andlucigenin(selectiveforO.-)CLmeasurementswere
2 2 2
significantlyhigherintumortissuesthanincontroltissues(P<0.001).LuminolandlucigeninCLmeasurementswere
1.93±0.71and2.5±0.84timesbrighter,respectively,intumortissuesthanintheadjacentparenchyma(P=0.07).
Conclusion:Inpatientswithlungcancer,allROMlevelswereincreasedintumortissueswhencomparedwiththe
adjacentlungtissue.Becausetheincreaseinlucigeninconcentration,whichisduetotissueischemia,ishigherthan
theincreaseinluminol,whichisdirectlyrelatedtothepresenceandseverityofinflammation,ischemiamaybemore
importantthaninflammationfortumordevelopmentinpatientswithlungcancer.
Keywords:Reactiveoxygenmetabolites,Lungcancer,Oxidativestress
Background and activation of leucocytes, which produce high levels of
Lung cancer, which is the most common cause of cancer reactive oxygen metabolites (ROMs) and nitric oxide [3,4].
deaths worldwide among both men and women, accounts Lung tissue is protected against these oxidants by a var-
for 28% of cancer deaths and approximately 6% of all iety of antioxidant mechanisms such as superoxide dis-
deaths[1].Tobaccosmokeisthemaincauseoflungcancer mutases. An imbalance between oxidant and antioxidant
andisresponsiblefor87%ofalllungcancersintheUnited levels induces oxidative stress in lung tissues [5]; thus,
States[1,2]. Therisk increases withthe amountof tobacco excessive and inappropriate production of endogenous
used and the length of time for which it has been used. and/or exogenous reactive oxygen species and nitric
Cigarette smoke, a major source of exogenous oxidants, oxide is implicated in the pathogenesis of lung cancer
leads to chronic airway inflammation with accumulation [1,6,7]. Free radicals are well-known mutagenic agents
and cause genotypic changes that may lead to the devel-
*Correspondence:[email protected] opmentofcancer[8].
1DepartmentofPulmonology,SureyyapasaChestDiseaseandThoracic
Although it is difficult to quantitate ROMs because of
SurgeryTrainingandResearchHospital,Basibuyuk-Maltepe,Istanbul34726,
Turkey theirshort-livedandreactivenature,thechemiluminescence
Fulllistofauthorinformationisavailableattheendofthearticle
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Okuretal.WorldJournalofSurgicalOncology2013,11:9 Page2of5
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(CL)methodusedinthepresentstudyisasimpleandre- awiderangeofapplications,especiallythosethatinvolve
producible technique. The two CL probes, luminol and monitoringofreactiveoxygenspecies[10].
lucigenin, differ in their selectivity. Lucigenin is particu- In our experiment, tissues were thawed and washed
larly sensitive to the superoxide radical (O.-), whereas with saline. Luminescence of the tissue samples was
2
luminol detects hydrogenperoxide(H O ),hydroxyl radi- recorded at room temperature using a Junior LB 9509
2 2
cals (.OH), hypochlorite (ClO-), peroxynitrite (ONOO-), luminometer(EG&GBerthold,BadWildbad,Germany)in
andlipidperoxylradicals[9]. thepresenceofenhancers.Tissuespecimenswerecutinto
Detection of high levels of ROMs, which are known to two pieces and placed into tubes containing PBS-HEPES
be increased by ischemia or inflammation, in tumor tis- buffer (0.5 mol/L phosphate buffered saline containing 20
sues may be attributable either to their role in the eti- mmol/L HEPES, pH 7.2). ROMs were quantitated after
ology of cancer or to the presence of the tumor itself. addition of the enhancer (lucigenin or luminol) to a final
The present study was designed to explore the intensity concentration of 0.2 mmol/L. After the measurements
of oxidative stress in patients with lung cancer by asses- weremade,thetissueswereremovedfromthetubes,dried
sing the generation of ROMs in tumor and normal lung on filter papers, and weighed. All chemiluminometric
parenchyma and comparing the levels of metabolites in countswereobtainedat1-minintervalsfor5min,andthe
both tissues. resultswereexpressedasareasunderthe curve(AUCs)of
relativelightunits(rlu)for5minpermgoftissue[9].The
Methods calculationwasbasedontheintegrationofthecurveusing
Patients thetrapezoidalrule(alinearapproximation).
Fifteen consecutive patients with non-small cell lung
carcinoma who were operated on in our clinic were Statisticalanalysis
includedinthestudy.Allpatientswere smokers andhad Thevariousdegreesofoxidativestressindifferenttypesof
no preoperative neoadjuvant treatment and no previous lung tumors, including epidermoid carcinomaand adeno-
history of carcinoma of any type. Patients with tumors carcinoma,andadjacentlungtissueswerestatisticallyana-
less than 2 cm in size or endobronchial tumors were not lyzed. All data are expressed as means ± SEM. Groups of
included because obtaining a 1-cm sample from such a datawerecomparedusingapairedttest.Theresultswere
small tumor might disturb the pathological examination. consideredsignificantwhenthePvaluewaslessthan0.05.
After routine preoperative evaluation and staging, the Calculations were performed using GraphPad Prism 3.0
patientsunderwentanatomicallungresection(eitherlobec- (GraphPadSoftware,SanDiego,CA,USA).
tomyorpneumonectomy)togetherwithmediastinallymph
node dissection. Informed consent of the patients and Results and discussion
approval of the ethics committee of Marmara University We examined a total of 15 patients, all of whom were
MedicalSchoolwereobtained. male with a mean age of 63.6 ± 9 years (range 48 to 74
years).Patients’characteristicsaresummarizedinTable1.
Experimentaldesign In all patients, the lucigenin and luminol CLvalues were
Immediately after completion of lung resection and re- higher in cancerous lung tissues than in adjacent lung tis-
moval of the surgical specimen, two tissue samples were sues. The luminol CL level in tumor tissues was signi-
obtained from each patient: the first sample was obtained ficantlyhigherthanthatincontroltissues(4.32±0.38rlu/
from the lung tumor and the second was obtained from mg tissue vs. 2.26 ± 0.22 rlu/mg tissue, P <0.001)
adjacent lung parenchyma (for the control group) at least (Figure 1a). Similarly, the lucigenin CL level showed a
5 cm away from the gross margin of the lung tumor. The marked increase in the tumor tissues as compared to con-
tissues were then washed with saline (0.9% NaCl) to re- troltissues(4.49±0.44rlu/mgtissuevs.1.91±0.12rlu/mg
moveblood,freshlyfrozen,andstoredat−20°Cuntilthey tissue,P<0.001)(Figure1b).
wereexamined. In this study, we investigated the role of oxidative
stress in lung cancer. We demonstrated that ROM
Chemiluminescencemeasurement levels, as measured by the CL method, were significantly
To assess the role of ROMs in this study, CL derived higher in the cancerous tissues of lung cancer patients
from luminol and lucigenin was measured as an indica- than incontroltissues obtainedfrom thesame patients.
tor of radical formation. CL measurements are based on Theantitumordefensemechanismsofthebodyresultin
light emission with specific enhancers such as luminol, continuous oxidative stress and inflammatory responses,
lucigenin, luciferase, and horseradish peroxidase. The which contribute to the various types of oxidative damage
benefits of this method include rapidity, ultrasensitive observed in lung cancer [11]. It may be hypothesized that
detection limits, and broad applicability. In clinical re- chronic injury to the epithelium caused by the production
search, the sensitivity of this method has led to its use in ofROMsmayleadtolungcancer.Freeradicalsthatplaya
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Table1Characteristicsofpatients
Age(years) Locations TNM Histologicaltype
1 59 RUL TNM Adenoca
2 0 0
2 73 RUL TNM Epidca
1 1 0
3 71 LLL TNM Epidca
1 1 0
4 65 RUL TNM Epidca
1 0 0
5 58 RUL TNM Epidca
1 1 0
6 65 RUL TNM Adenoca
2 1 0
7 60 RLL TNM Epidca
1 1 0
8 74 RML TNM Epidca
2 0 0
9 49 LLL TNM Adenoca
2 1 0
10 72 LLL TNM Epidca
1 1 0
11 78 LUL TNM Epidca
2 0 0
12 53 LLL TNM Epidca
2 0 0
13 67 LUL TNM Epidca
2 1 0
14 62 RUL TNM Epidca
2 0 0
15 48 LLL TNM Epidca
1 1 0
Adenoca,adenocarcinoma;Epidca,epidermoidcarcinoma;LLL,leftlower
lobe;LUL,leftupperlobe;RLL,rightlowerlobe;RML,rightmediallobe;
RUL,rightupperlobe;TNM,tumor,node,metastasis(classificationsystem).
role in carcinogenesis may originate from cigarettesmoke,
air pollution, or activated phagocytes, the latter of which
areaconsequenceofchronicinflammatorydiseases[12].
In a report that examined lipid peroxidation in whole
blood samples obtained from patients with lung cancer,
the authors reported that the level of malondialdehyde
(MDA, a lipid peroxidation product) in patients with
early-stage (stage I and II) lung cancer was not signifi-
cantly altered compared to the control group [7]. How-
Figure1Lucigenin(a)andluminol(b)chemiluminescence(CL)
ever, in patients with advanced-stage lung cancer, the levelsinthecontrolandtumorgroups.Rlu,relativelightunits.
MDA level was significantly higher. Additionally, the level
of whole blood glutathione (an intracellular antioxidant)
was decreased in patients with both early- and advanced- are released from lung tumor specimens. In an animal
stage lung cancer. In another study, the ROM levels in study, rats were exposed to cigarette smoke, and the
serum samples from patients with small cell carcinoma, luminol-enhanced CL technique was used to measure
adenocarcinoma, and epidermoid carcinoma were higher ROMs in lung and laryngeal tissues [14]. The results indi-
thanthelevelsdetectedincontrolsubjects[13].Ziebaetal. cated that cigarette smoking increased luminol-enhanced
reported that lipid peroxidation was higher in cancerous CL measurements, and vitamin E decreased ROM release
tissue samples than in normal lung parenchyma and significantly.H O canbeconvertedto.OHinthepresence
2 2
demonstrated that thiobarbituric acid-reactive substances, ofmetalionsviatheFentonreactionorbyneutrophilinfil-
which are markers of lipid peroxidation, were positively tration; alternatively, it can be converted to HOCl- in the
correlatedwithbothclinicalstageandspontaneousgener- presence of chloride ions via the myeloperoxidase enzyme.
ationofH O intumortissue[4]. In our study, luminol-enhanced CL amplification can also
2 2
In our study, luminol-enhanced CL measurements were beusedtomeasure.OHandHOCl-.HOCl-isusuallypro-
significantly higher in tumor specimens than in adjacent duced by polymorphonuclear leukocytes at an inflamma-
pulmonary parenchyma tissues. Our method, which is tion site. In a group of patients with non-small cell lung
based on the measurement of spontaneously released cancer,myeloperoxidaseactivitywaslowerandglutathione
ROMs,isalsocapableofmeasuringH O andanothersub- levels were higher in patients with malignant disease [15].
2 2
stances such as hydroxyl radicals (.OH), hypochlorite ions Reports of increased inflammatory markers and C-reactive
(ClO-), peroxynitrite ions (ONOO-), and lipid peroxyl ra- protein [16] as well as an acute phase response in cancer
dicals. Our results show that H O and other compounds patientsindicatethatbothsystemicandlocal(thatis,atthe
2 2
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tumor site) inflammatory reactions are closely associated Authors’contributions
withthepresenceofatumor[4].Inaddition,somestudies HKOandMYdesignedtheresearch,TL,VBandEOperformedtheoperations
andcollectedthetissuesamples.MYperfomedthelaboratory
indicate that the tumor tissue itself could be the source of
measurementsandanalyses.HKOandEOanalysedthedata.HKO,MYand
ROMsandlipidperoxidationproducts[17]. EOwrotethepaper.Allauthorsreadandapprovedthefinalmanuscript.
Ischemia is the major cause of superoxide generation in
Authordetails
cancertissues.Ischemialeadstoglucoseandadenosinetri-
1DepartmentofPulmonology,SureyyapasaChestDiseaseandThoracic
phosphate (ATP) depletion, which in turn leads to a de- SurgeryTrainingandResearchHospital,Basibuyuk-Maltepe,Istanbul34726,
crease in sodium-potassium ATPase (Na-K ATPase) Turkey.2VocationalSchoolofHealthRelatedProfessions,Departmentof
MedicalLaboratory,MarmaraUniversity,HaydarpasaCampus,Istanbul34668,
activity and causes depolarization of the cells. In these
Turkey.3DepartmentofThoracicSurgery,SureyyapasaChestDiseaseand
depolarized cells, intracellular calcium increases, resulting ThoracicSurgeryTrainingandResearchHospital,Basibuyuk-Maltepe,Istanbul
inactivationofthexanthine-hypoxanthinesystemandpro- 34726,Turkey.4DepartmentofThoracicSurgery,AcibademUniversity
MedicalSchool,Istanbul,Gulsuyu_Maltepe,Istanbul34726,Turkey.
duction of superoxide radicals. Superoxide radicals, which
arehighlyreactive,damagemembranephospholipids,cellu- Received:23August2012Accepted:24December2012
lar proteins, and DNA [18]. In our study, the levels of Published:17January2013
lucigenin-enhancedCLwerehigherintumorsamplesthan
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doi:10.1186/1477-7819-11-9
Citethisarticleas:Okuretal.:Detectionofreactiveoxygenmetabolites
inmalignantandadjacentnormaltissuesofpatientswithlungcancer.
WorldJournalofSurgicalOncology201311:9.
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