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Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2013 Article ID 543760 6 pageshttpdxdoiorg1011552013543760

Clinical StudyRelation between Gastric Cancer and Protein OxidationDNA Damage and Lipid Peroxidation

Yongsheng Ma1 Lin Zhang1 Shengzhong Rong2 Hongyan Qu3 Yannan Zhang4

Dong Chang1 Hongzhi Pan4 and Wenbo Wang1

1 The First Affiliated Hospital of Harbin Medical University No 199 Dongdazhi Street Nangang District HarbinHeilongjiang 150001 China

2 Public Health School Mudanjiang Medical College No 3 Tongxiang Street Aimin District Mudanjiang Heilongjiang 157011 China3TheThird Affiliated Hospital of Harbin Medical University No 150 Haping Road Nangang District HarbinHeilongjiang 150081 China

4 Public Health School Harbin Medical University No 157 Baojian Road Nangang District Harbin Heilongjiang 150081 China

Correspondence should be addressed to Hongzhi Pan panhongzhilaoshi163com and Wenbo Wang 949439265qqcom

Received 28 August 2013 Revised 29 October 2013 Accepted 1 December 2013

Academic Editor Neelam Khaper

Copyright copy 2013 Yongsheng Ma et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

ObjectsThe aim of this study is to evaluate protein oxidation DNA damage and lipid peroxidation in patients with gastric cancerand to investigate the relationship between oxidative stress and gastric cancerMethods We investigated changes in serum proteincarbonyl (PC) advanced oxidation protein products (AOPP) and 3-nitrotyrosine (3-NT) levels as indicators of protein oxidationserum 8-hydroxydeoxyguanosine (8-OHdG) as a biomarker of DNA damage and malondialdehyde (MDA) conjugated diene(CD) 4-hydroxynonenal (4-HNE) and 8-ISO-prostaglandin F

2120572(8-PGF) in serum as lipid peroxidation markers in gastric cancer

(GC) patients and healthy control Results Compared with control a statistically significant higher values of 8-OHdG PC AOPPand 3-NT were observed in the GC patients (119875 lt 005) The products of lipid peroxidation MDA CD 4-HNE and 8-PGFwere significantly lower in the GC patients compared to those of control (119875 lt 005) In addition the products of oxidative stresswere similar between the Helicobacter pylori positive and the negative subgroups of GC patients Conclusions GC patients werecharacterized by increased protein oxidation and DNA damage and decreased lipid peroxidation Assessment of oxidative stressand augmentation of the antioxidant defense system may be important for the treatment and prevention of gastric carcinogenesis

1 Introduction

Gastric cancer (GC) is one of the most frequent diseasesin human population It is the fourth frequent cancer andthe second most common cause of deaths from cancer inthe world accounting for a large proportion of cancer casesin China in recent years [1] The pathogenesis of GC is notunderstood completely Nutritional microbial and geneticfactors acting in a multistep and multifactorial process havebeen proposed [2] where oxidative stress was involved in thepossible mechanisms of GC development

Reactive oxygen species (ROS) are the normal product ofa variety of essential biochemical reactions and the genera-tion of ROS is an unavoidable consequence of aerobic life

Normal level of ROS has physiological functions whereasit is toxic to the cells at high level Overproduction of ROSthrough either endogenous or exogenous insults is harmfulto living organisms and is termed oxidative stress [3]

Oxidative stress can damage cellular macromoleculesleading to DNA and protein modification and lipid perox-idation [4] Oxidative stress produced by free radicals hasbeen associated with the development of several diseasessuch as cardiovascular cancer and chronic inflammation [3ndash7] It is also known that ROS could be excessively formedin chronic diseases of the gastrointestinal tract and toxic tonormal cells which might contribute to the increased risk ofcancer [6 7] but the precise mechanisms are still remainingto be elucidated

2 Oxidative Medicine and Cellular Longevity

The aim of this study is to investigate the relationshipbetween oxidative stress and GC We used spectrophotomet-ric assay and enzyme-linked immunosorbent assay (ELISA)to detect the levels of lipid peroxidation products suchas malondialdehyde (MDA) conjugated dienes (CD) 4-hydroxynonenal (4-HNE) and 8-Iso-prostaglandin F

2120572(8-

PGF) and measured the concentration of protein damageproducts including advanced oxidation protein products(AOPP) protein carbonyl (PC) and 3-nitrotyrosine (3-NT)in patients with gastric cancer and healthy control subjectsIn addition we determined 8-hydroxydeoxyguanosine (8-OHdG) content the DNA damage produced in GC patientsand healthy control too In the present study we provideevidence for the first time indicating the relationship betweenprotein carbonyl and the risk of gastric cancer

2 Material and Methods

21 Subjects This is an age-and sex-matched case-controlstudyThirty patients with newly diagnosed GC are recruitedfor the experiment group among whom 17 patients werepositive for Helicobacter pylori (Hp+) and 13 negative (HPminus)Diagnosis of gastric cancer was confirmed by histopathologi-cal examination and histopathology Tumors were classifiedaccording to their predominant histological pattern intointestinal (119899 = 13) diffuse (119899 = 6) and adenocarcinoma(119899 = 11) The pathologic stage (TNM) was determinedaccording to the newly revised classification of the AmericanJoint Committee on Cancer (AJCC) and the InternationalUnion Against Cancer (UICC) Seven patients had AJCCStage II tumors and ten patients had AJCC Stage III tumorsThe remaining thirteen patients had UICC Stage III orIV gastric adenocarcinoma Urease test and histopathologicexamination were carried out to determine the infection ofHelicobacter pylori (HP) If the urease test and histologywere positive the diagnosis of HP infection was confirmedIf both tests were negative the patient was considered HpminusExclusion criteria included subjects who had been takingantioxidant supplements and regularly used acetylsalicylicacid and other nonsteroidal anti-inflammatory drugs Otherexclusion criteria included diabetes mellitus hypertensionarteriosclerosis severe liver disease cataract renal failureand dialysis All patients were recruited from the ThirdAffiliated Hospital of the Harbin Medical University Bothgroups belonged to the same ethnic group

Thirty healthy age-matched subjects who came to thesame hospital for an annual checkup were also included ascontrols The control subjects were in good health as deter-mined by a medical history questionnaire physical examina-tion and normal results of clinical laboratory tests None ofthe control subjects were taking any medication All subjectsgave written informed consent to participate in this study

22 Blood Sampling Venous peripheral blood samples(10mL) were collected from each subject after 12 h of over-night fastingThe samples were placed on ice and centrifugedat 3500 rpm and 4∘C for 15min and the supernatants werestored at minus80∘C and determination was accomplished within

3months All samples from each patient were run in the sameassay

23 Laboratory Analysis AOPP were quantified as describedby Witko-Sarsat et al [8] PC concentrations in plasmawere measured with spectrophotometric assay described byReznick and Packer [9] CD and MDA were measured byspectrophotometric assay [4] Serum 8-OHdG was mea-sured with ELISA method following the makerrsquos instructions(Highly Sensitive 8-OHdG Check Japan Institute for theControl of Aging Fukuroi Shizuoka) 3-NT (Hycult Biotech-nology BV) 4-HNE and 8-PGF (Cell Biolabs Inc) weremeasured with ELISA following the makerrsquos instructionsBriefly 50ndash100120583L of serum was added into each well of themicrotiter plate and incubated for 2 h at 37∘C Secondaryantibody was added to the plate and incubated at 37∘C for1 h the unbound enzyme-labeled secondary antibody wasremoved and then the antibodies binding with the platewere identified using a substrate that contained 331015840551015840-tetramethylbenzidine Absorbance was measured using aspectrophotometric plate reader (BIO-RAD) atwavelength of450 nm Quantification of the 8-OHdG 3-NT 8-PGF and 4-HNE were done by comparing the optical densities of eachsample to that of an internal standard known at variousconcentrations

24 Other Biochemical Parameters Blood glucose triglyc-erides and cholesterol were determined using routine clinicalchemical assays

25 Statistical Analysis Data are presented as mean plusmn SDAll experimental data in this study were statistically analyzedwith SAS 913The statistical significance was evaluated usingunpaired Studentrsquos t-test Results were considered significantwhen 119875 lt 005

3 Results

31 Baseline Characteristics Theclinical characteristics of theGC patients and control subjects are shown in Table 1 Ageand gender of the patients were not significantly differentfrom those of the controls Initial clinical laboratory testvalues such as glucose cholesterol triglycerides and bloodpressure were also not significantly different between the twogroupsWhat ismore all the test values were in normal range

32 Results of Protein Oxidative Damage and DNA DamageProducts in Serum As shown in Table 2 the concentrationof AOPP in GC patients was increased significantly than thatof control (2575 plusmn 575 versus 2229 plusmn 527 119875 lt 005) Serum3-NT also significantly increased in GC patients comparedwith normal subjects (11775 plusmn 3712 versus 9285 plusmn 1447119875 lt 001) so was serum PC (216 plusmn 068 versus 173 plusmn 075119875 lt 005) and serum 8-OHdG (1634 plusmn 830 ngmL versus1229 plusmn 572 ngmL 119875 lt 005)

33 Results of Products of Lipid Peroxidation in Serum Asshown in Table 3 there was a significant decrease in serum

Oxidative Medicine and Cellular Longevity 3

Table 1 Demographic and clinical characteristics of patients andcontrols

Factors Control Patients119873 30 30Age (years) 625 plusmn 84 614 plusmn 77

Sex (FM) 1713 1911Cholesterol (mmolL) 492 plusmn 063 506 plusmn 068

Triglycerides (mmolL) 106 plusmn 033 113 plusmn 041

Glucose (mmolL) 462 plusmn 087 456 plusmn 065

Systolic blood pressure (mmHg) 1245 plusmn 90 1247 plusmn 93

Diastolic blood pressure (mmHg) 765 plusmn 66 778 plusmn 73

Smoking habitCurrent smokers 8 6Nonsmokers 16 14Ex-smokers 6 10

Alcohol intake (YN) 1614 1713Physical exercise (YN) 1812 1020History of family (YN) 624Data are means plusmn SD

MDA in cancer patients (507 plusmn 189 nmolmL) comparedwith normal subjects (632plusmn188 nmolmL119875 lt 005) Serumconjugated dienes in cancer patients have also a significantdecrease than those in controls (0534 plusmn 0239 versus 0739 plusmn0239 119875 lt 001) So was 4-HNE (1393 plusmn 750 versus 1813 plusmn795119875 lt 005) and 8-PGF (3011plusmn1265 versus 3832plusmn1082119875 lt 001)

34 Results of Oxidative Stress Products in Helicobacter PyloriInfection Positive and Negative Subgroups We divided the 30patients into 2 groups according toHP infection As shown inTable 4 products of oxidative stress were similar between theHP infection positive and negative subgroups of the gastriccancer patients (119875 gt 005)

4 Discussion

Gastric carcinoma is one of the most common neoplasms inthe worldThere are a lot of pathological factors such as ROSinvolved in the process of cancer initiation and progressionas evidenced by the fact that oxidative stress plays animportant role in the molecular mechanism of gastric cancer[3 4 6 7] Exposure of proteins to reactive oxygen or nitrogenspecies results in modification of amino acid residues alter-ing the protein structure and function [10 11] Biomarkers ofprotein oxidation are often applied when a battery of markersof oxidative stress status is being studied In this study wedetected the contents of PC AOPP and 3-NT which havebeen applied as biomarkers of protein oxidation Althoughproteins are major targets for oxidative and nitrative damagein vivo it is widely recognized that oxidation of proteins playsan essential role in the pathogenesis of cancers [10]

Oxidation of proteins can lead to the formation ofoxidized amino acids and altered amino acid side chainscontaining reactive carbonyls [12] The major product of

peroxynitrite attack on proteins is an addition of a nitrogroup to the orthoposition of tyrosine residues to producenitrotyrosine 3-NT has been used as a biomarker of nitrativedamage in vivo [13 14] The presence of 3-NT in somecarcinomas was previously reported [15] In our study serum3-NT level was significantly higher in the GC patients Thisresult is constant with other researchersrsquo findings Goto et al[16] and Bancel et al [17] found that 3-NT levels are elevatedin gastric cancer patients

Protein carbonyl is themost popular biomarker for severeoxidative protein damage [18] It can be generated throughoxidative cleavage of proteins by either the 120572-amidationpathway or by oxidation of glutamyl side chains leadingto the formation of a peptide in which the N-terminalamino acid is blocked by an 120572-ketoacyl derivative [10] Theprevious studies showed that cancer patients have higherlevels of PC [4 19] But Hoque et al found that there was nosignificant change in prostate cancer patients of serum PC ina large nested case-control study [20] In this study we foundincreased PC levels in gastric cancer Our results show thatPC is a potential biomarker available for assessing oxidativestress in gastric cancer

AOPP products of the action of free radicals on proteinswere described by Witko-Sarsat et al [8] for the first timeAOPP is defined as dityrosine containing cross-linked pro-tein products and are considered to be reliable biomarkersto estimate the degree of protein oxidation [21] They areelevated in patients with renal insufficiency and they reachthe highest levels in patients after renal replacement therapy[22] Increased levels were also found in cancer patientswhere they correlated with markers of oxidative stress [2324] We found that the level of AOPP was significantly higherin GC patients In accordance with our findings Noyan etal [25] showed that AOPP concentration was significantlyhigher in the serum specimens from gastric cancer than thosefrom the normal control

In the present study we have determined the proteinoxidation products of 3-NT AOPP and PC All the proteinoxidation products were significantly higher in GC patientsThismeans that there was severe protein oxidation damage ingastric cancer patients

ROS can cause strand breaks and base modifications inDNA For example oxidation of guanine residues to 8-OHdGis an oxidized nucleoside of DNA that is the most frequentlydetected and studied DNA lesion It has been regarded asa novel biomarker for oxidative DNA damage in vivo andappears to play a crucial role inmutagenesis Elevated levels of8-OHdG were demonstrated in some carcinomas includinggastric cancer [15 26ndash28] Farinati et al [27] and Hahmet al [28] showed a significant increase of 8-OHdG in HP-positive and -negative gastric cancer patients Interestinglythe levels of 8-OHdG in gastric mucosa have been shown todecrease substantially after the eradication of HP infectionIn our research we also found that there was increased serum8-OHdG in gastric cancer Nevertheless the greater oxidativeDNA damage in the GC as a possible result of impairedantioxidant activity implies an important role for oxygen freeradicals in stomach carcinogenesisTherefore the contents of


Table 2 Serum levels of protein oxidation and DNA damage products

Group AOPP (120583molmL) 3-NT (ngmL) Protein carbonyl (nmolmL) 8-OHdG (ngmL)Control 2229 plusmn 527 9285 plusmn 1447 173 plusmn 075 1229 plusmn 572

Patients 2575 plusmn 575lowast

11775 plusmn 3712lowastlowast

216 plusmn 068lowast


Data are means plusmn SD lowast119875 lt 005 compared to controls lowastlowast119875 lt 001 versus healthy subject

Table 3 Serum levels of lipid peroxidation products

Group MDA (nmolmL) 8-PGF (pgmL) 4-HNE (nmolmL) CD (OD 233 nm)Control 632 plusmn 188 3832 plusmn 1082 1813 plusmn 795 0739 plusmn 0239





Data are means plusmn SD lowast119875 lt 005 versus healthy subject lowastlowast119875 lt 001 versus healthy subject

Table 4 The results of oxidative stress products in H pylori infec-tion patients

HP infectionNegative (Hpminus)

HP infectionPositive (Hp+)

119899 13 17MDA (nmolmL) 490 plusmn 149 520 plusmn 218

CD (OD 233 nm) 0551 plusmn 0192 0521 plusmn 0265

4-HNE (nmolmL) 1465 plusmn 984 1337 plusmn 535

8-PGF (pgmL) 2874 plusmn 811 3115 plusmn 1543

AOPP (120583molmL) 2599 plusmn 601 2556 plusmn 572

PC (nmolmL) 213 plusmn 081 218 plusmn 058

3-NT (ngmL) 11449 plusmn 4456 12025 plusmn 3151

8-OHdG (ngmL) 1536 plusmn 772 1709 plusmn 888

Data are means plusmn SD

8-OHdG in serum could act as a sensitive biomarker for GCpatients too

Lipid peroxidation is initiated by free radical attackon membrane lipids generating large amounts of reactiveproducts which have been implicated in tumor initiation andpromotion In this study we selected a series of products oflipid peroxidation MDA CD 8-PGF and 4-HNE which arewidely used sensitive and applicable for large scale studies

MDA is a product of the breakdown of mainly unsatu-rated fatty acids into their essential chains through oxidationwhich is the most studied free radical reaction But thedata reported in the literature on MDA levels in differenthuman cancer types remains controversial Some researchersreported that lipid peroxidation increased and MDA leveladvanced in cancer patients Arivazhagan et al [29] reportedincreased erythrocyte MDA levels in patients with gastriccancer Khanzode et al [30] and Bakan et al [31] reportedincreased plasmaMDA levels in gastric cancer patients com-pared with those in control subjects However some otherreports are not consistent with these findings Punnonen etal [32] found increased serum MDA levels but decreasedtissue MDA levels in human breast cancer Chang et al[4] and Biasi et al [33] reported decreased serum MDAlevels in human colon cancer Murugan et al [34] reportedsignificantly decreased serum MDA levels in N-methyl-N1015840-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcino-genesis in rat model Inconsistent with some but consistent

with other findings our results show a decrease of serumMDA levels in patients compared with those in controlsubjects [30 31]

CD is the initial formation of the lipid peroxideMuruganet al [34] reported significantly decreased serum CD levelsin MNNG-induced gastric cancer in rats Chang et al [4]revealed significantly decreased serumCD levels in colorectalcancer patients 4-HNE is the major 120572 120573-unsaturated alde-hydes produced by membrane lipid peroxidation and it canbe a potential indicator of lipid peroxidation Murugan etal [34] also reported significantly decreased serum 4-HNElevels inMNNG-induced gastric carcinogenesis in rat modelIn the present study we found that there were decreased CDand 4-HNE in serum in GC patients compared with controlsubjects

The prostaglandin 8-PGF is an isoprostane that is pro-duced by the nonenzymatic peroxidation of arachidonic acidin membrane phospholipids [35] 8-PGF is a major iso-prostane that is chemically stable andmeasurable in biofluidsand is a reliable biomarker for assessing the rate of lipidperoxidation of subjects Other than the widely appreciatedbiomarker Camphausen et al [36] found it unattainableto detect an increased 8-PGF in the urine of patients withprostate cancer compared with normal controls We foundthat the level of 8-PGFwas significantly lower compared withcontrol As far as we know our study represents the firstreport to test the relationship between serum 8-PGF and therisk of gastric cancer

Considering that there are some contradictory resultsof lipid peroxidation reported in cancer in this study weselect a series of different products of lipid peroxidationincluding MDA CD 4-HNE and 8-PGF to investigate therelationship between GC and lipid peroxidation We foundfor the first time the lipid peroxidation products proneto the same declining trend with all the products of lipidperoxidation being decreased significantly in serumof gastriccancer patients compared to healthy control We proposethat the decreased levels of MDA CD 4-HNE and 8-PGF inGC patients may relate to tumor aggressiveness which is inline with the hypothesis proposed by Slater et al [37] whoassumed that rapidly dividing cells tend to set an oxidant-antioxidant status favorable to their growth Rapidly prolif-erating tumor cells show resistance to lipid peroxidation andoverexpress antioxidant enzymes Peroxidation is deleterious


for cell proliferation through the production of reactive oxy-gen species with the exact mechanism remaining unraveledFurther studies are needed to conclusively determinate thecorrelation between the lipid peroxidation and cancer

Helicobacter pylori related gastritis is characterized byincreased free radical production and peroxidative damage[38 39] Production of ROS by gastric cells and phagocytesinduced by HP has been shown in vitro and increased levelsof ROS in the gastric mucosa have been verified in HP-infected patients [40 41] A positive association has beendemonstrated between HP infection and gastric adenocarci-noma with increased oxidative stress [42]

We therefore investigated whether HP infection was alsorelated to a progressive accumulation of oxidative mutagenicevents that could play a major role in gastric carcinogenesisThe 8-OHdG levels were significantly higher in GC than inthe control group but significant difference was not detectedbetweenHP-positive and -negative samples in spite of slightlyhigher 8-OHdG levels in the HP-positive GC patients Theresult is in line with the findings of Farinati et al [43] andNoyan et al [25] where the level of AOPP was increasedsignificantly in the gastric cancer patients as comparedwith nongastric cancer patients However there were nosignificant differences between the anti-HP IgG positive andnegative patients with gastric cancer And we also found thatthe level of AOPP was not significantly different between theHP-positive and -negative gastric cancer patients Goto etal [16] demonstrated that 3-NT in the gastric mucosa wassignificantly higher in the HP-positive group than in the HP-negative group But our results showed that there was nosignificant difference in oxidative stress parameters betweenthe HP-positive and -negative gastric cancer patients Thesecontradictory results suggest that the relationship betweenHP infection and gastric adenocarcinoma with increasedoxidative stress still remains unclear Although approximatelyhalf of the world population are infected withHP a cancerousoutcome of infection is very rare Hence whether HP infec-tion is a major causative factor for GC remains questionableaccording to the discrepancies among the current literature

5 Conclusions

In summary the results obtained here confirm that oxidativestress is involved in gastric cancer though there was nosignificant difference in oxidative stress parameters betweenthe HP-positive and -negative gastric cancer patients Wefound that the gastric cancer patients had increased levels ofprotein oxidation and DNA damage versus control subjectsbut decreased lipid peroxidation products The mechanismremains to be fully revealed It would beworthwhile to furtherstudy the correlation between these biomarkers and levelsof lipid peroxidation and GC Assessment of oxidative stressand augmentation of the antioxidant defense systems may beimportant for the treatment and prevention of gastric cancer

Authorsrsquo Contribution

Yongsheng Ma and Lin Zhang contributed equally to thiswork

Acknowledgment

This study was supported by the Heilongjiang Province Post-doctoral Foundation Grant LBH-Z10075

References

[1] D M Parkin F Bray J Ferlay and P Pisani ldquoGlobal cancerstatistics 2002rdquo Cancer Journal for Clinicians vol 55 no 2 pp74ndash108 2005

[2] P Correa ldquoA new paradigm for human gastric carcinogenesisrdquoJournal of Clinical Gastroenterology vol 30 pp 381ndash385 2000

[3] M Valko C J Rhodes J Moncol M Izakovic and M MazurldquoFree radicals metals and antioxidants in oxidative stress-induced cancerrdquo Chemico-Biological Interactions vol 160 no 1pp 1ndash40 2006

[4] D Chang F Wang Y-S Zhao and H-Z Pan ldquoEvaluation ofoxidative stress in colorectal cancer patientsrdquo Biomedical andEnvironmental Sciences vol 21 no 4 pp 286ndash289 2008

[5] T DOberley ldquoOxidative damage and cancerrdquoAmerican Journalof Pathology vol 160 no 2 pp 403ndash408 2002

[6] Y Lin S Kikuchi Y Obata and K Yagyu ldquoSerum copperzincsuperoxide dismutase (CuZn SOD) and gastric cancer risk acase-control studyrdquo Japanese Journal of Cancer Research vol 93no 10 pp 1071ndash1075 2002

[7] M Yasuda F Takesue S Inutsuka M Honda T Nozoe and DKorenaga ldquoPrognostic significance of serum superoxide dismu-tase activity in patients with gastric cancerrdquoGastric Cancer vol5 no 3 pp 148ndash153 2002

[8] V Witko-Sarsat M Friedlander C Capeillere-Blandin et alldquoAdvanced oxidation protein products as a novel marker ofoxidative stress in uremiardquo Kidney International vol 49 no 5pp 1304ndash1313 1996

[9] A Z Reznick and L Packer ldquoOxidative damage to proteinsspectrophotometric method for carbonyl assayrdquo Methods inEnzymology vol 233 pp 357ndash363 1994

[10] B S Berlett andE R Stadtman ldquoProtein oxidation in aging dis-ease and oxidative stressrdquo The Journal of Biological Chemistryvol 272 no 33 pp 20313ndash20316 1997

[11] E R Stadtman and B S Berlett ldquoReactive oxygen-mediatedprotein oxidation in aging and diseaserdquo Drug MetabolismReviews vol 30 no 2 pp 225ndash243 1998

[12] S Adams P Green R Claxton et al ldquoReactive carbonylformation by oxidative and non-oxidative pathwaysrdquo Frontiersin Bioscience A vol 6 pp 17ndash24 2001

[13] B Halliwell ldquoWhat nitrates tyrosine Is nitrotyrosine specific asa biomarker of peroxynitrite formation in vivordquo FEBS Lettersvol 411 no 2-3 pp 157ndash160 1997

[14] H Ohshima M Friesen I Brouet and H Bartsch ldquoNitrotyro-sine as a new marker for endogenous nitrosation and nitrationof proteinsrdquo Food and Chemical Toxicology vol 28 no 9 pp647ndash652 1990

[15] S Kondo S Toyokuni Y Iwasa et al ldquoPersistent oxidativestress in human colorectal carcinoma but not in adenomardquo FreeRadical Biology andMedicine vol 27 no 3-4 pp 401ndash410 1999

[16] T Goto K Haruma Y Kitadai et al ldquoEnhanced expressionof inducible nitric oxide synthase and nitrotyrosine in gastricmucosa of gastric cancer patientsrdquoClinical Cancer Research vol5 no 6 pp 1411ndash1415 1999

[17] B Bancel J Esteve J-C Souquet S Toyokuni H Ohshimaand B Pignatelli ldquoDifferences in oxidative stress dependence


between gastric adenocarcinoma subtypesrdquo World Journal ofGastroenterology vol 12 no 7 pp 1005ndash1012 2006

[18] I Dalle-Donne D Giustarini R Colombo R Rossi and AMilzani ldquoProtein carbonylation in human diseasesrdquo Trends inMolecular Medicine vol 9 no 4 pp 169ndash176 2003

[19] F M Nathan V A Singh A Dhanoa and U D PalanisamyldquoOxidative stress and antioxidant status in primary bone andsoft tissue sarcomardquo BMC Cancer vol 11 article 382 2011

[20] A Hoque C B Ambrosone C Till et al ldquoSerum oxidizedprotein and prostate cancer risk within the prostate cancerprevention trialrdquo Cancer Prevention Research vol 3 no 4 pp478ndash483 2010

[21] C J J Alderman S Shah J C Foreman B M Chain and DR Katz ldquoThe role of advanced oxidation protein products inregulation of dendritic cell functionrdquo Free Radical Biology andMedicine vol 32 no 5 pp 377ndash385 2002

[22] V Witko-Sarsat M Friedlander T N Khoa et al ldquoAdvancedoxidation protein products as novel mediators of inflammationand monocyte activation in chronic renal failurerdquo Journal ofImmunology vol 161 no 5 pp 2524ndash2532 1998

[23] F Kosova B Cetin M Akinci et al ldquoAdvanced oxidationprotein products ferrous oxidation in xylenol orange andmalondialdehyde levels in thyroid cancerrdquo Annals of SurgicalOncology vol 14 no 9 pp 2616ndash2620 2007

[24] A S Nayyar M Khan K R Vijayalakshmi B Suman HC Gayitri and M Anitha ldquoSerum total protein albumin andadvanced oxidation protein products (AOPP)mdashimplicationsin oral squamous cell carcinomardquo The Malaysian Journal ofPathology vol 34 no 1 pp 47ndash52 2012

[25] T Noyan H Guducuoglu and M Ilhan ldquoA study of oxidativestress parameters in anti-helicobacter pylorus immunoglobulinG positive and negative gastric cancer patientsrdquo Yonsei MedicalJournal vol 50 no 5 pp 677ndash682 2009

[26] C-S ChangW-N Chen H-H Lin C-CWu and C-JWangldquoIncreased oxidative DNA damage inducible nitric oxide syn-thase nuclear factor 120581B expression and enhanced antiapop-tosis-related proteins in Helicobacter pylori-infected non-cardiac gastric adenocarcinomardquoWorld Journal of Gastroenter-ology vol 10 no 15 pp 2232ndash2240 2004

[27] F Farinati R Cardin P Degan et al ldquoOxidative DNA damageaccumulation in gastric carcinogenesisrdquo Gut vol 42 no 3 pp351ndash356 1998

[28] K B Hahm K J Lee S Y Choi et al ldquoPossibility of chemo-prevention by the eradication of Helicobacter pylori oxidativeDNA damage and apoptosis in H pylori infectionrdquo AmericanJournal of Gastroenterology vol 92 no 10 pp 1853ndash1857 1997

[29] S Arivazhagan K Kavitha and S Nagini ldquoErythrocyte lipidperoxidation and antioxidants in gastric cancer patientsrdquo CellBiochemistry and Function vol 15 no 1 pp 15ndash18 1997

[30] S S Khanzode S D Khanzode andGN Dakhale ldquoSerum andplasma concentration of oxidant and antioxidants in patientsof Helicobacter pylori gastritis and its correlation with gastriccancerrdquo Cancer Letters vol 195 no 1 pp 27ndash31 2003

[31] E Bakan S Taysi M F Polat et al ldquoNitric oxide levels and lipidperoxidation in plasma of patients with gastric cancerrdquo JapaneseJournal of Clinical Oncology vol 32 no 5 pp 162ndash166 2002

[32] K Punnonen M Ahotupa K Asaishi M Hyoty R Kudoand R Punnonen ldquoAntioxidant enzyme activities and oxidativestress in human breast cancerrdquo Journal of Cancer Research andClinical Oncology vol 120 no 6 pp 374ndash377 1994

[33] F Biasi L Tessitore D Zanetti et al ldquoAssociated changes oflipid peroxidation and transforming growth factor 1205731 levels inhuman colon cancer during tumour progressionrdquo Gut vol 50no 3 pp 361ndash367 2002

[34] R S Murugan K V P C Mohan K Uchida Y HaraD Prathiba and S Nagini ldquoModulatory effects of black teapolyphenols on oxidant-antioxidant profile and expression ofproliferation apoptosis and angiogenesis-associated proteinsin the rat forestomach carcinogenesis modelrdquo Journal of Gastro-enterology vol 42 no 5 pp 352ndash361 2007

[35] S Basu ldquoFatty acid oxidation and isoprostanes oxidative strainand oxidative stressrdquo Prostaglandins Leukotrienes and EssentialFatty Acids vol 82 no 4ndash6 pp 219ndash225 2010

[36] K Camphausen C Menard M Sproull E Goley S Basu andC N Coleman ldquoIsoprostane levels in the urine of patients withprostate cancer receiving radiotherapy are not elevatedrdquo Inter-national Journal of Radiation Oncology Biology Physics vol 58no 5 pp 1536ndash1539 2004

[37] T F Slater K H Cheeseman C Benedetto et al ldquoStudies onthe hyperplasia (ldquoregenerationrdquo) of the rat liver following par-tial hepatectomyChanges in lipid peroxidation and general bio-chemical aspectsrdquo Biochemical Journal vol 265 no 1 pp 51ndash591990

[38] GRDaviesN J Simmonds T R J Stevens et al ldquoHelicobacterpylori stimulates antral mucosal reactive oxygen metaboliteproduction in vivordquo Gut vol 35 no 2 pp 179ndash185 1994

[39] F Farinati G D Libera R Cardin et al ldquoGastric antioxidantnitrites and mucosal lipoperoxidation in chronic gastritis andHelicobacter pylori infectionrdquo Journal of Clinical Gastroenterol-ogy vol 22 no 4 pp 275ndash281 1996

[40] N Ramarao S D Gray-Owen and T F Meyer ldquoHelicobacterpylori induces but survives the extracellular release of oxygenradicals fromprofessional phagocytes using its catalase activityrdquoMolecular Microbiology vol 38 no 1 pp 103ndash113 2000

[41] S-C Baik H-S Youn M-H Chung et al ldquoIncreased oxidativeDNA damage in Helicobacter pylori-infected human gastricmucosardquo Cancer Research vol 56 no 6 pp 1279ndash1282 1996

[42] S Park W S Kim U J Choi et al ldquoAmelioration of oxidativestress with ensuing inflammation contributes to chemopreven-tion of H pylori-associated gastric carcinogenesisrdquo Antioxi-dants and Redox Signaling vol 6 no 3 pp 549ndash560 2004

[43] F Farinati R Cardin M Bortolami et al ldquoOxidative DNAdamage in gastric cancer CagA status and OGG1 gene poly-morphismrdquo International Journal of Cancer vol 123 no 1 pp51ndash55 2008

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Evidence-Based Complementary and Alternative Medicine

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The aim of this study is to investigate the relationshipbetween oxidative stress and GC We used spectrophotomet-ric assay and enzyme-linked immunosorbent assay (ELISA)to detect the levels of lipid peroxidation products suchas malondialdehyde (MDA) conjugated dienes (CD) 4-hydroxynonenal (4-HNE) and 8-Iso-prostaglandin F

2120572(8-

PGF) and measured the concentration of protein damageproducts including advanced oxidation protein products(AOPP) protein carbonyl (PC) and 3-nitrotyrosine (3-NT)in patients with gastric cancer and healthy control subjectsIn addition we determined 8-hydroxydeoxyguanosine (8-OHdG) content the DNA damage produced in GC patientsand healthy control too In the present study we provideevidence for the first time indicating the relationship betweenprotein carbonyl and the risk of gastric cancer

2 Material and Methods

21 Subjects This is an age-and sex-matched case-controlstudyThirty patients with newly diagnosed GC are recruitedfor the experiment group among whom 17 patients werepositive for Helicobacter pylori (Hp+) and 13 negative (HPminus)Diagnosis of gastric cancer was confirmed by histopathologi-cal examination and histopathology Tumors were classifiedaccording to their predominant histological pattern intointestinal (119899 = 13) diffuse (119899 = 6) and adenocarcinoma(119899 = 11) The pathologic stage (TNM) was determinedaccording to the newly revised classification of the AmericanJoint Committee on Cancer (AJCC) and the InternationalUnion Against Cancer (UICC) Seven patients had AJCCStage II tumors and ten patients had AJCC Stage III tumorsThe remaining thirteen patients had UICC Stage III orIV gastric adenocarcinoma Urease test and histopathologicexamination were carried out to determine the infection ofHelicobacter pylori (HP) If the urease test and histologywere positive the diagnosis of HP infection was confirmedIf both tests were negative the patient was considered HpminusExclusion criteria included subjects who had been takingantioxidant supplements and regularly used acetylsalicylicacid and other nonsteroidal anti-inflammatory drugs Otherexclusion criteria included diabetes mellitus hypertensionarteriosclerosis severe liver disease cataract renal failureand dialysis All patients were recruited from the ThirdAffiliated Hospital of the Harbin Medical University Bothgroups belonged to the same ethnic group

Thirty healthy age-matched subjects who came to thesame hospital for an annual checkup were also included ascontrols The control subjects were in good health as deter-mined by a medical history questionnaire physical examina-tion and normal results of clinical laboratory tests None ofthe control subjects were taking any medication All subjectsgave written informed consent to participate in this study

22 Blood Sampling Venous peripheral blood samples(10mL) were collected from each subject after 12 h of over-night fastingThe samples were placed on ice and centrifugedat 3500 rpm and 4∘C for 15min and the supernatants werestored at minus80∘C and determination was accomplished within

3months All samples from each patient were run in the sameassay

23 Laboratory Analysis AOPP were quantified as describedby Witko-Sarsat et al [8] PC concentrations in plasmawere measured with spectrophotometric assay described byReznick and Packer [9] CD and MDA were measured byspectrophotometric assay [4] Serum 8-OHdG was mea-sured with ELISA method following the makerrsquos instructions(Highly Sensitive 8-OHdG Check Japan Institute for theControl of Aging Fukuroi Shizuoka) 3-NT (Hycult Biotech-nology BV) 4-HNE and 8-PGF (Cell Biolabs Inc) weremeasured with ELISA following the makerrsquos instructionsBriefly 50ndash100120583L of serum was added into each well of themicrotiter plate and incubated for 2 h at 37∘C Secondaryantibody was added to the plate and incubated at 37∘C for1 h the unbound enzyme-labeled secondary antibody wasremoved and then the antibodies binding with the platewere identified using a substrate that contained 331015840551015840-tetramethylbenzidine Absorbance was measured using aspectrophotometric plate reader (BIO-RAD) atwavelength of450 nm Quantification of the 8-OHdG 3-NT 8-PGF and 4-HNE were done by comparing the optical densities of eachsample to that of an internal standard known at variousconcentrations

24 Other Biochemical Parameters Blood glucose triglyc-erides and cholesterol were determined using routine clinicalchemical assays

25 Statistical Analysis Data are presented as mean plusmn SDAll experimental data in this study were statistically analyzedwith SAS 913The statistical significance was evaluated usingunpaired Studentrsquos t-test Results were considered significantwhen 119875 lt 005

3 Results

31 Baseline Characteristics Theclinical characteristics of theGC patients and control subjects are shown in Table 1 Ageand gender of the patients were not significantly differentfrom those of the controls Initial clinical laboratory testvalues such as glucose cholesterol triglycerides and bloodpressure were also not significantly different between the twogroupsWhat ismore all the test values were in normal range

32 Results of Protein Oxidative Damage and DNA DamageProducts in Serum As shown in Table 2 the concentrationof AOPP in GC patients was increased significantly than thatof control (2575 plusmn 575 versus 2229 plusmn 527 119875 lt 005) Serum3-NT also significantly increased in GC patients comparedwith normal subjects (11775 plusmn 3712 versus 9285 plusmn 1447119875 lt 001) so was serum PC (216 plusmn 068 versus 173 plusmn 075119875 lt 005) and serum 8-OHdG (1634 plusmn 830 ngmL versus1229 plusmn 572 ngmL 119875 lt 005)

33 Results of Products of Lipid Peroxidation in Serum Asshown in Table 3 there was a significant decrease in serum













4 Discussion














































5 Conclusions




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Clinical Study Relation between Gastric Cancer and Protein ...downloads.hindawi.com/journals/omcl/2013/543760.pdf · Clinical Study Relation between Gastric Cancer and Protein Oxidation,