Levels of biological markers of nitric oxide in serum of patients with squamous cell carcinoma of the oral cavity

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1 Interntionl Journl of Orl Science (213) 5, ß 213 WCSS. All rights reserved /13 ORIGINAL ARTICLE Levels of iologicl mrkers of nitric oxide in serum of ptients with squmous cell crcinom of the orl cvity Wiolett Rtjczk-Wron 1, Ew Jlonsk 1, Bozen Antonowicz 2, Dorot Dzieminczyk 3 nd Stnislw Zyt Growsk 3 The im of the study ws determintion of the levels of nitric oxide (NO) nd its iologicl mrkers such s mlonyldildehyde (MDA) nd nitrotyrosine in the serum of ptients with squmous cell crcinom (SCC) of the orl cvity nd identifiction of the reltionships etween NO nd those mrkers. These studies were performed on ptients with SCC of the orl cvity efore nd fter tretment. Griess rection ws used for the estimtion of the totl concentrtion of NO in serum. The nitrotyrosine level in serum ws ssessed with n enzyme-linked immunosorent ssy (ELISA) kit, nd MDA level using spectrophotometric ssy. Higher concentrtions of NO in lood serum were determined in ptients with stge IV of the disese efore tretment in comprison to the control group nd ptients with stges II nd III of the disese. Moreover, higher concentrtions of MDA nd nitrotyrosine were determined in the serum of ptients in ll stges of the disese in comprison to helthy people. After tretment, lower concentrtions of NO in the serum of ptients with stge IV of the disese were oserved in comprison to the mounts otined prior to tretment. In ddition, lower levels of nitrotyrosine in the serum of ptients with ll stges of the disese were recorded, wheres higher concentrtions of MDA were determined in these ptients in comprison to results otined efore tretment. The compounds formed with the contriution of NO, such s MDA nd nitrotyrosine, my led to cncer progression in ptients with SCC of the orl cvity, nd contriute to formtion of resistnce to therpy in these ptients s well. Moreover, the lck of reltionship etween concentrtions of NO nd MDA, nd etween NO nd nitrotyrosine in serum suggests tht the process of lipid peroxidtion nd nitrtion in ptients with SCC does not just depend on NO. Interntionl Journl of Orl Science (213) 5, ; doi:1.138/ijos ; pulished online 23 August 213 Keywords: mlonyldildehyde; nitric oxide; nitrotyrosine; squmous cell crcinom of the orl cvity INTRODUCTION Over 9% of hed nd neck cncers re squmous cell crcinoms (SCCs), which re the most common mlignnt cncer of the orl cvity. Ech yer, round 48 new cses of orl cncer re dignosed worldwide. In ddition, simultneous or progressive development of nother tumor occurs in round 2% of ptients. 1 2 Development of SCC is conditioned y oth externl (cigrette smoke, mechnicl injuries, virl nd fungl infections) nd internl (genetic nd immunologicl defects) fctors. Rective oxygen nd nitric species, including nitric oxide (NO), re lso included in these fctors. 1,3 The results from mny clinicl nd experimentl studies hve demonstrted tht the role of NO in the crcinogenic process depends on compound concentrtion. Low levels of NO cn promote tumor development, wheres its high concentrtion cts cytotoxiclly on tumor cells. 4 This hs een confirmed y the studies of Ohshi et l. 5 nd Sumitni et l., 6 who hve demonstrted poptotic chnges in tumor cells in the presence of n NO donor. They hve suggested tht NO cn result in regression of cncer through inhiition of DNA repliction nd prevention of mitochondril respirtion in tumor cells. Especilly interesting from the clinicl point of view of orl cncers re the dt presented y Shng et l., 7 who demonstrted tht high concentrtion of NO is n importnt inhiitory fctor for the growth of orl SCC through the induction of poptosis. On the other hnd, the procrcinogenic role of NO ws lso identified. Some erlier pulictions hve demonstrted tht NO t oth low nd high concentrtions my promote crcinogenesis through the ctivtion of the trnscription hypoxi-inducile fctor 1 (HIF-1). 8 This process is time- nd cell type-dependent. HIF-1 is sujected to destiliztion nd protesoml degrdtion in normoxic conditions, while in hypoxi conditions, its overexpression is oserved. An overexpression of HIF-1 trnscription fctor, s one of the mechnisms of cncer cells dpttion to the conditions of oxygen deficiency, is connected inter li to chnges in intercellulr metolism. 9 It hs een oserved tht high HIF-1 expression in SCC cells of the orl cvity is correlted with their incresed resistnce to rdio- nd chemotherpy tht my result in n increse in primry cncer tumor nd dditionlly fvor secondry foci formtion. 1 One consequence of n incresed concentrtion of NO my e its direct effect on the uilding components of cells in n orgnism. 11 It hs een estlished tht NO plys n essentil role in the process of lipid peroxidtion. This is free rdicl chin process in which there 1 Deprtment of Immunology, Medicl University of Bilystok, Bilystok, Polnd; 2 Deprtment of Orl Surgery, Medicl University of Bilystok, Bilystok, Polnd nd 3 Mxillofcil nd Plstic Surgery Clinic, Medicl University of Bilystok, Bilystok, Polnd Correspondence: Dr W Rtjczk-Wron, Deprtment of Immunology, Medicl University of Bilystok, Wszyngton 15A, Bilystok , Polnd E-mil: rwiolett@um.edu.pl Received 22 Decemer 212; ccepted 26 June 213

2 142 Levels of iologicl mrkers of NO is oxidtion of polyunsturted ftty cids or unsturted ftty cid moieties, included in the composition of phospholipids the min uilding component of cell memrnes. Unlike proteins nd nucleic cids, the process of lipid peroxidtion is chrcterized y chin rection tht results in the genertion of lrge numer of peroxides of unsturted ftty cids or other lipids. 11 One of the finl products of lipid peroxidtion is mlonyldildehyde (MDA) tht contins two rective ldehyde groups which cn rect with two different molecules (R 1 NH 2 nd R 2 NH 2 ) nd cn sew them into products with chrcteristic structure (R 1 N5CH CH5NH R 2 ) clled Schiff ses N,N9-mino imino-propene. 11 It hs een proven tht ldehydes formed s result of lipid peroxidtion re less rective thn free rdicls nd therey cn diffuse to significnt distnces in cells; therefore, they ply the role of the secondry meditors of dmge cused y rective oxygen nd nitrogen species. Aldehydes rect mostly with thiol nd mine groups of proteins, lipids, mino sugrs nd nitrogenous ses of nucleic cids. They modify physicl properties of cell memrnes y incresing their permeility in respect of H 1 ions nd other polr sustnces This cuses chnges in electric potentils on oth sides of the memrne, resulting in loss of integrtion of the intrcellulr memrnes nd the plsmtic memrne nd inhiition of ctivity of memrne enzymes nd crrier proteins. 14 NO is lso included mong the min fctors responsile for nitrifiction of the phenol groups of tyrosine in tissues nd lood proteins. Nitrotyrosine, which is formed in this process, cn cuse loss of the iologicl function of lood proteins nd result in pthologicl chnges. Nitrotyrosine concentrtion cn e helpful mrker for the evlution of NO ction under in vivo conditions. Moreover, nitrotyrosine, ecuse of its longer hlf-life, my e etter indictor of the incresed production of NO thn metolites of NO The im of the study ws the determintion of the totl concentrtion of NO, MDA nd nitrotyrosine in the serum of ptients with SCC of the orl cvity nd identifiction of the reltionship etween these prmeters, which could extend knowledge out the role of NO nd mrkers of NO ctivity during the pthogenesis of cncer in the studied group of ptients. MATERIAL AND METHODS We exmined 24 ptients with SCC of the orl cvity treted in the Mxillofcil nd Plstic Surgery Clinic, Medicl University of Bilystok. Assys were performed efore the tretment nd 3 weeks fter surgicl removl of the tumor mss. Study results were nlyzed tking into ccount clinicl stge of the disese ccording to tumour node metstsis stging system (TNM clssifiction system) (Tle 1). 17 Chrcteristic of ptients ws presented in the Tle 1. for one week fter surgery received Morphini Sulfs (1 mg every 6 h for 3 dys; Polf Trchomin S.A., Wrsw, Polnd), prcetmol (1 mg every 12 h for 7 dys, Perflgn; Bristol-Myers Squi, Uxridge, UK) for postopertive pin nd ntiiotics cephlosporin (1 g every 12 h for 7 dys, Trfzolin; Polf Trchomin S.A., Wrsw, Polnd). All femles nd mles prticipted in the study were cigrette smokers for over 1 yers. sujects (n515) were non-smoking helthy volunteers ged from 3 to 6 yers (men6 s.d.: yers). None of the ptients nd control suject hd concomitnt diseses such s dietes mellitus, liver disese or rheumtoid rthritis. The study ws pproved y the Ethics Committee of the Medicl University of Bilystok (R-I-3/87/26). Tle 1 Clinicl chrcteristic of ptients with squmous cell crcinom of the orl cvity Chrcteristics Numer of ptients in TNM clssifiction Stge II Stge III Stge IV Mle/femle 7/1 6/2 8/ Men ge Site of cncer Orl cvity fundus 1/ 2/ 4/ Tongue 1/ 1/1 / Tongue1orl cvity 1/ 1/1 / undus Cheek mucos 1/1 / 1/ Inferior gingivl 1/ 1/ 2/ Lower lip 2/ 1/ 1/ Blood smpling with SCC of the orl cvity nd helthy control sujects were recruited into the study fter otining their informed consent. were not smoking cigrettes within 24 h prior to lood collection. Five milliliters of fsting lood ws collected y venous rm puncture under septic conditions. Serum ws otined y centrifugtion t 2 g for 5 min of lood smples tken without nticogulnt. Serum ws kept t 2 6C until the nlysis dte. Determintion of totl NO (NO 3 2 /NO 2 2 ) concentrtion in serum Nitrite (NO 2 2 ) nd nitrte (NO 3 2 ) re stle finl products of NO metolism nd my e used s indirect mrkers of NO presence. Totl NO concentrtion is commonly determined s sum of nitrite nd nitrte concentrtions. NO concentrtion ws determined using n indirect method sed on mesurement of nitrite concentrtion in serum ccording to Griess s rection. 18 In the smples nlyzed, nitrte were reduced to nitrite in the presence of cdmium (Sigm- Aldrich, Steinheim, Germny), nd then converted to nitric cid tht gve color rection with Griess s regent (Sigm-Aldrich, Stinheim, Germny). Nitrite concentrtions were determined y spectrophotometric nlysis t 54 nm (UVN-34 ASYS Hitech GmH microplte reder; Biogenet, Eugendorf, Austri) with reference to stndrd curve. NO products were expressed s mmoles. Determintion of MDA concentrtion in serum The evlution of MDA in serum ws done y the method of Buege nd Aust. 18 The rection is sed on fusion of lipids oxidtion products with thiorituric cid leding to cretion of colored product. In the first step, 2 ml of 5% trichlorocetic cid ws dded to 2 ml of serum, nd then mixed until precipittion of proteins nd centrifugted t 5 g for 5 min. Following ddition of 16 ml of thiorituric cid to 16 ml of superntnt collected fter centrifugtion, smples were incuted in wter th t 9 6C. After 3 min, the colored product y the rection of thiorituric cid with MDA ws mesured colorimetriclly t 533 nm using DU SERIES 6 spectrophotometer (Beckmn Coulter, Bre, CA, USA). The otined results were expressed s nmoles. Determintion of nitrotyrosine concentrtion in serum The nitrotyrosine level in serum ws ssessed y sndwich enzymelinked immunosorent ssy (ELISA) using commercilly ville kit (Nitrotyrosine ELISA kit; Hycult Biotechnology.v., PB Uden, The Netherlnds). Before performing the ssy, serum ws diluted 1 times with dilution uffer (protein-stilized phosphte-uffered sline, Interntionl Journl of Orl Science

3 Levels of iologicl mrkers of NO 143 contining 2-chlorocetmide s preservtive). Nitrotyrosine stndrds or serum (1 ml) were pipetted into n ntiody-coted 96- well plte nd incuted t room temperture for 1 h. The wells were then wshed four times with wsh uffer (contining Tween-2), 1 ml of nti-humn nitrotyrosine ntiody ws dded nd the smples were gin incuted for 1 h t room temperture. The plte ws wshed four times, 1 ml of streptvidin-peroxidse conjugted ws then pplied for 1 h t room temperture. After finl wshing, 1 ml of tetrmethylenzidine sustrte ws dded nd llowed to develop for 2 3 min in the drk t room temperture. After stopping the rection with stop solution (contining citric cid, 2. mmol?l 21 ) (1 ml), sornce ws red t 45 nm with UVN-34 ASYS Hitech GmH microplte reder (Biogenet, Eugendorf, Austri). The smple concentrtion ws clculted from the stndrd curve. The otined results were expressed s nmoles. Sttisticl evlution The dt otined were sujected to sttisticl nlysis using Microsoft Excel clcultion sheet nd STATISTICA version 9.1 (SttSoft Inc., Tuls, OK, USA). All dt re presented s men6s.d. Dt distriution normlity ws determined using Kolmogorov Smirnov test. Since the dt were not normlly distriuted, for comprison of vritions etween ssyed groups, Mnn Whitney U non-prmetric tests were pplied to unrelted results. A sttisticl significnce level of P,.5 ws ssumed. A correltion nlysis ws undertken using Person s rnk correltion test. RESULTS Our findings on the ssessed prmeters in SCC ptients nd helthy control sujects re shown in Figures 1 3 nd Tle 2. Figure 1 shows the levels of serum totl concentrtion of NO (NO 3 2 /NO 2 2 ) in control sujects nd SCC ptients. In the serum of ptients with stge IV of the disese efore nd fter tretment, significntly higher vlues for totl NO concentrtions were oserved in comprison to the control group. Totl concentrtions of NO in the serum of ptients with stge IV of the disese efore tretment were higher in comprison to the ptients with stges II nd III of the disese. After tretment, lower concentrtions of totl NO were oserved in ptients with stge IV of the disese in comprison to vlues otined efore tretment. No differences were oserved in totl NO levels in the serum of ptients with stges II nd III of the disese fter tretment in comprison to vlues otined efore tretment nd to the control group. Figure 2 presents the concentrtion of MDA in the control sujects nd SCC ptients. Significntly higher vlues for MDA concentrtions in serum of ll ptients efore nd fter tretment in comprison to the control group were noted. In ddition, higher concentrtions of MDA were noted in serum of ptients fter tretment in comprison to vlues otined efore tretment regrdless of tumor stge. Incresing concentrtions of MDA in serum of ptients efore nd fter tretment following the stge of disese progression were oserved. A significntly higher concentrtion of MDA ws oserved in serum of ptients with stge IV of the disese efore nd fter tretment in comprison to ptients with stges II nd III of the disese. Figure 3 shows the concentrtion of serum of nitrotyrosine in the control sujects nd SCC ptients. Significntly higher vlues for nitrotyrosine concentrtions in the serum of ll ptients efore tretment were demonstrted in comprison to the control group. In ddition, concentrtions of nitrotyrosine in serum of ptients efore the tretment were oserved to increse with the progression of the stge Concentrtions of totl NO/(mmol. L 1 ) Before tretment After tretment in stge II in stge III cd in stge IV Figure 1 Concentrtions of totl NO in the serum of ptients in different stge of disese. NO, nitric oxide., Sttisticl differences with control (P,.5);, sttisticl differences etween ptients efore nd fter tretment (P,.5); c, sttisticl differences etween ptients in stge II nd ptients in stge IV (P,.5); d, sttisticl differences etween ptients in stge III nd ptients in stge IV (P,.5). Concentrtions of MDA/(nmol. L 1 ) Before tretment After tretment in stge II in stge III cd cd in stge IV Figure 2 Concentrtions of MDA in the serum of ptients in different stge of disese. MDA, mlonyldildehyde., Sttisticl differences with control (P,.5);, sttisticl differences etween ptients efore nd fter tretment (P,.5); c, sttisticl differences etween ptients in stge II nd ptients in stge IV (P,.5); d, sttisticl differences etween ptients in stge III nd ptients in stge IV (P,.5). Concentrtions of nitrotyrosine/(nmol. L 1 ) Before tretment After tretment in stge II in stge III cd in stge IV Figure 3 Concentrtions of nitrotyrosine in the serum of ptients in different stge of disese., Sttisticl differences with control (P,.5);, sttisticl differences etween ptients efore nd fter tretment (P,.5); c, sttisticl differences etween ptients in stge II nd ptients in stge IV (P,.5); d, sttisticl differences etween ptients in stge III nd ptients in stge IV (P,.5). Interntionl Journl of Orl Science

4 144 Levels of iologicl mrkers of NO Tle 2 Person s rnk correltion etween MDA concentrtions nd nitrotyrosine levels in serum of ptients in different stge of disese efore tretment TNM clssifiction Compound in stge II in stge III in stge IV Nitrotyrosine MDA MDA MDA r5.61, P,.5 r5.68, P,.5 r5.75, P,.5 MDA, mlonyldildehyde. of the disese. However, in serum of ptients fter tretment, significntly lower concentrtions of nitrotyrosine were estlished in comprison to vlues otined efore the tretment. Tle 2 presents the Person s rnk correltions nlysis etween the MDA nd nitrotyrosine levels in serum SCC ptients efore tretment. The nlysis of reltionships etween concentrtions of the iologicl mrkers of NO in serum of ptients proved positive significnt correltion etween concentrtions of MDA nd nitrotyrosine in serum of ll ptients efore tretment. However, no sttisticlly significnt correltions were oserved etween the studied prmeters in serum of ll the ptients fter the tretment (dt not presented). DISCUSSION NO, which is produced y mny cells including cells of the immune system, cn ffect the systemic ction of mny different orgns nd tissues, s well s tumor cells. 3,19 The high concentrtions of totl NO oserved in our own studies in the serum of ptients with SCC of the orl cvity efore tretment my ply role in elimintion of tumor cells. There re dt tht indicte tht high levels of NO result in poptosis of cells through direct dmge of DNA, inhiition of proteins nd nucleic cid synthesis. 3,2 High concentrtions of NO in the serum of ptients with SCC of the orl cvity in the dvnced stge of the disese were lso otined y Beevi et l., 21 Rsheed et l. 22 nd Gokul et l. 23 One of the cuses of high concentrtions of totl NO in the serum of ptients in the dvnced stge of the disese could e secretion of NO y cells of the immune system. Jlonsk et l. 24 demonstrted higher expression nd concentrtion of inducile nitric oxide synthse (inos), the enzyme responsile for the genertion of NO, in polymorphonucler cells nd peripherl lood mononucler cells in those ptients, wheres lower expression nd concentrtion of inos in polymorphonucler cells nd peripherl lood mononucler cells independent of the stge of the disese ws recorded in ptients fter tretment. Similr oservtions concerning the increse in the expression nd ctivity of inos in ptients with SCC of the orl cvity in comprison with wek or no ctivity of inos in the mucous memrne of helthy people hve een mde y Gvilnes et l. 25 Our own studies showed tht lower concentrtions of NO in the serum of ptients fter surgicl removl of the tumor mss my result from decresed production of NO through polymorphonucler cells nd peripherl lood mononucler cells, s well s suggest role of tumor cells in the secretion of this molecule. 26 On the other hnd, Connelly et l. 27 reported significntly higher expression of inos in orl squmous crcinom tissue linking it with high levels of NO in its pthogenesis. Thomsen et l. 28 hve demonstrted tht inos inhiition my reduce the growth of xenogrfted tumors in vivo. There re studies tht report the effect of NO upon tumor growth of mutnt p53 tumors y promoting endothelil mitogenesis. 29 Incresed production of NO y cells of the immune system nd tumor cells, esides ffecting vrious systems nd orgns, cn lso influence the process of neongiogenesis. 3 It hs een demonstrted tht NO modultes the relese of vsculr endothelil growth fctor y tumor cells, s well s inititing ctivtion of mtrix metlloproteinses, enzymes influencing the degrdtion of sement memrne nd other cellulr components Another mechnism y which NO might promote crcinogenesis is proly y modulting the production of prostglndins. NO increses the production of prostglndin E2 y enhncing the prostglndin synthse which results in n incresed lekge in tumor vsculture. 34 In our own studies, higher concentrtions of MDA in the serum of ll ptients with SCC of the orl cvity oth efore nd fter the tretment were oserved, which suggests intensified lipid peroxidtion in these ptients. A consequence of high concentrtions of MDA in serum of these ptients could e tht morphologicl nd functionl chnges of mny cells my finlly result in the dysfunction of individul orgns. Prticiption of MDA in the inctivtion of protein synthesis nd processes of repliction nd trnscription of DNA ws identified. It ws oserved tht MDA s rection with nitrogenous ses of nucleic cids could hve mutgenic function. 35 One of the cuses of high concentrtions of MDA in the serum of ptients in the dvnced stge of the disese in comprison to vlues otined in ptients during the erly stges of the disese ws the lowered totl nti-oxidtive ility of peripherl lood, wherey the peripherl lood ws unle to prevent uncontrolled oxidtion. 21,36 This is confirmed y results otined y Sith et l. 37 in ptients with SCC of the orl cvity with stge III of the disese efore nd fter tretment with rdiotherpy. They demonstrted high level of MDA in lood plsm of these ptients in comprison to the control group, with simultneous decrese in the ctivity of nti-oxidtive enzymes in lood cells. Similr results were otined y Korde et l., 38 who oserved high level of MDA nd totl NO in serum of SCC ptients with clinicl stge III/IV. High MDA levels despite low serum NO concentrtions in the serum of ptients with stges II nd III of the disese, cn e explined y the fct tht other compounds such s sulfur dioxide, hydroxyl rdicls nd rdicl ctions exhiit lipid peroxidtion potentil. 18 Moreover, high concentrtions of totl NO together with the higher concentrtions of nitrotyrosine oserved in our own study in ptients with stge IV of the disese ccording to TNM clssifiction confirm the role of this molecule in the nitrtion process in this group of ptients. Nitrtion of tyrosine is mostly n irreversile process tht cn result in the inhiition of tyrosine phosphoryltion nd incresed sensitivity of proteins towrds proteolytic enzymes. 39 It hs een demonstrted tht due to its incresed immunogenicity, nitrotyrosine modultes the inflmmtory process. 4 Incresed concentrtion of nitrotyrosine in the serum of ptients with SCC of the orl cvity cn fcilitte development of tumors. There re pulished dt which indicte the criticl role of inflmmtion in tumor promotion. 41 The reltionships etween concentrtions of MDA nd nitrotyrosine oserved in the present study in the serum of ptients with SCC efore tretment my negtively ffect the rection of host-cncer cells, nd led to impirment of nticncer response. In conclusion, the compounds formed with the contriution of NO, such s MDA nd nitrotyrosine, my led to cncer progression in ptients with SCC of the orl cvity, nd contriute to formtion of resistnce to therpy in these ptients s well. Moreover, the lck of reltionship etween concentrtions of NO nd MDA, nd etween Interntionl Journl of Orl Science

5 Levels of iologicl mrkers of NO 145 NO nd nitrotyrosine in serum suggests tht the process of lipid peroxidtion nd nitrtion in ptients with SCC does not just depend on NO. Further studies evluting the iologicl ctivity of NO could led to the development of new therpeutic methods using selective donors of NO or inhiitors of NO synthse in ptients with SCC of the orl cvity. ACKNOWLEDGEMENTS This work ws funded y the Medicl University of Bilystok, Polnd (project no: F). Puliction of this mnuscript is supported y Open Fund of Stte Key Lortory of Orl Diseses, Sichun University. 1 Chen AY, Myers JN. Cncer of the orl cvity. Dis Mon 21; 47(7): Mssno J, Regteiro FS, Jnurio G et l. Orl squmous cell crcinom: review of prognostic nd predictive fctors. Orl Surg Orl Med Orl Pthol Orl Rdiol Endod 26; 12(1): Moncd S, Higgs EA. Moleculr mechnisms nd therpeutic strtegies relted to nitric oxide. FASEB J 1995; 9(13): Xu W, Liu LZ, Loizidou M et l. The role of nitric oxide in cncer. 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