Combination of carboplatin and intermittent normobaric hyperoxia synergistically suppresses benzo[a]pyrene-induced

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1 ORIGINAL ARTICLE Koren J Intern Med 218;33: Comintion of cropltin nd intermittent normoric hyperoxi synergisticlly suppresses enzo[]pyrene-induced lung cncer He Yon Lee, In Kyoung Kim, Hye In Lee, Hw Young Lee, Hye Seon Kng, Chng Dong Yeo, Hyun Hui Kng, Hw Sik Moon, nd Sng Hk Lee Division of Pulmonry, Criticl Cre nd Sleep Medicine, Deprtment of Internl Medicine, The Cncer Reserch Institute, College of Medicine, The Ctholic University of Kore, Seoul, Kore Received : Novemer 2, 216 Revised : April 14, 217 Accepted : My 24, 217 Correspondence to Sng Hk Lee, M.D. Division of Pulmonry, Criticl Cre nd Sleep Medicine, Deprtment of Internl Medicine, College of Medicine, St. Pul s Hospitl, The Ctholic University of Kore, 18 Wngsn-ro, Dongdemun-gu, Seoul 2559, Kore Tel: Fx: E-mil: mdlee@ctholic.c.kr Bckground/Aims: We explored the effects of intermittent normoric hyperoxi lone or comined with chemotherpy on the growth, generl morphology, oxidtive stress, nd poptosis of enzo[]pyrene (B[]P)-induced lung tumors in mice. Methods: Femle A/J mice were given single dose of B[]P nd rndomized into four groups: control, cropltin (5 mg/kg intrperitonelly), hyperoxi (95% frction of inspired oxygen), nd cropltin nd hyperoxi. Normoric hyperoxi (95%) ws pplied for 3 hours ech dy from weeks 21 to 28. Tumor lod ws determined s the verge totl tumor numers nd volumes. Severl mrkers of oxidtive stress nd poptosis were evluted. Results: Intermittent normoric hyperoxi comined with chemotherpy reduced the tumor numer y 59% nd the lod y 72% compred with the control B[]P group. Intermittent normoric hyperoxi, either lone or comined with chemotherpy, decresed the levels of superoxide dismutse nd glutthione nd incresed the levels of ctlse nd 8-hydroxydeoxygunosine. The Bx/Bcl- 2 mrna rtio, cspse 3 level, nd numer of trnsferse-medited dutp nick end-leling positive cells incresed following tretment with hyperoxi with or without chemotherpy. Conclusions: Intermittent normoric hyperoxi ws found to e tumoricidl nd thus my serve s n djuvnt therpy for lung cncer. Oxidtive stress nd its effects on DNA re incresed following exposure to hyperoxi nd even more with chemotherpy, nd this my led to poptosis of lung tumors. Keywords: Apoptosis; Cropltin; Hyperoxi; Lung neoplsms; Oxidtive stress INTRODUCTION Tumors growing in hypoxic environments re more prone to vigorous growth nd metstsis, which re ssocited with poor prognoses [1,2]. Hyperric oxygen enhnces tumor oxygention nd is used in comintion with oth chemotherpy nd rdiotherpy to tret vrious solid tumors [3-6]. Hyperoxi is tumoricidl [7-9]; however, ny effect thereof on lung cncer hs received little ttention, nd the mechnism of the effect is poorly understood [1-13]. Hyperric oxygen (ppliction of pure 1% oxygen under pressure) increses solule oxygen levels in tumor tissue [14]. However, hyperric oxygen pplied s Copyright 218 The Koren Assocition of Internl Medicine This is n Open Access rticle distriuted under the terms of the Cretive Commons Attriution Non-Commercil License ( y-nc/4./) which permits unrestricted noncommercil use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. pissn eissn

2 The Koren Journl of Internl Medicine Vol. 33, No. 3, My 218 tretment for lung cncer my cuse cute lung injury [15]. In tumor-ering mice exposed to either normoric or hyperric conditions, tumors exhiited growth retrdtion, reductions in vsculr density, decrese in glnd size nd enhnced cell deth. The development of these effects did not seem to require hyperric conditions [9]. We explored whether intermittent normoric hyperoxi ws tumoricidl in mice with lung tumors nd whether hyperoxi enhnced the effect of cropltin chemotherpy. We mesured erly indictors of oxidtive stress nd poptosis to determine the mechnisms responsile for the tumoricidl effects. METHODS Animls nd chemicls Femle A/J mice (6 to 7 weeks old) were purchsed from the Institute of Medicl Science (University of Tokyo, Tokyo, Jpn). Mice were fed the AIN-93M diet nd housed in pthogen-free niml qurters. Fresh food ws provided every 2 to 3 dys nd the wter ottles were chnged twice weekly. The diet nd drinking wter were provided d liitum. The mice were mintined t room temperture (2 C ± 2 C) t reltive humidity of 5% ± 1% with 1 to 15 ir chnges/hour under n lternting 12-hour/12-hour light/drk cycle. The study ws pproved y the Institutionl Animl Cre nd Use Committees in Deprtment of Lortory Animl, St. Pul's Hospitl, The Ctholic University of Kore (SPH ). Benzo[]pyrene (B[]P, purity > 99%) nd cropltin were purchsed from Sigm (St. Louis, MO, USA). Cropltin ws dissolved in doule-distilled wter. Experimentl design The experimentl design is illustrted in Fig. 1. After 1 week of cclimtion, the mice were divided into two groups: (1) control nd (2) orl gvge with 1 mg/kg B[]P. The B[]P group received B[]P in.1 ml mounts of corn oil once week for 3 consecutive weeks. In ech group, the mice were rndomized into four sugroups (n = 6 per group): (1) control, (2) cropltin (5 mg/kg intrperitonelly), (3) hyperoxi (95% frction of inspired oxygen [FiO 2 ]), nd (4) cropltin nd hyperoxi. Mice Control Control Cropltin Hyperoxi Cropltin + Hyperoxi Bp Group 1 Group 2 Group 3 Group 4 B()P 1 mg/kg orl gvge Control Cropltin Hyperoxi Cropltin + Hyperoxi Group 5 Group 6 Group 7 Group 8 Cropltin (5 mg/kg IP) Scrifice (wk) 95% Hyperoxi (3 hr/dy) 95% Hyperoxi (3 hr/dy) Cropltin (5 mg/kg IP) Scrifice (wk) 95% Hyperoxi (3 hr/dy) 95% Hyperoxi (3 hr/dy) Figure 1. Study design for evluting the effect of cropltin or hyperoxi tretment ginst enzo[]pyrene (B[] P)-induced lung tumorigenesis in the A/J mouse. Beginning t ge 6 to 7 weeks, groups of femle A/J mice (groups 5, 6, 7, nd 8) were treted y orl gvge weekly for 3 weeks with 1 mg/kg B[]P in.1 ml corn oil. The mice in groups 2, 4, 6, nd 8 were treted y intrperitonelly (IP) injection t 21 nd 22 weeks with 5 mg/kg cropltin. Mice in groups 3, 4, 7, nd 8 were given 95% normoric hyperoxi for 3 hours dily, from 21 to 28 weeks. Mice were scrificed t 28 weeks. were given 5 mg/kg cropltin vi single intrperitonelly injections t 21 nd 22 weeks. Hyperoxi (95%) ws pplied for 3 hours/dy from weeks 21 to 28 [16]. Body weights were recorded weekly throughout the experiment. All mice were scrificed t 28 weeks. A computer-controlled Biospherix Oxycycler system (Reming Bioinstruments, Redfield, NY, USA) ws used to mintin the oxygen level t constnt 95%. The tmosphere ws circulted continully, nd the oxygen concentrtion monitored. The cron dioxide level ws mintined t <.5% y djusting the extent of chmer lek. Mice exposed to room ir nd oxygen were evluted t the sme time. Lung tumor evlution The gross morphology nd numer of lung tumor nodules were recorded fter scrifice; we counted the tumor numer nd clculted the tumor volume. Size ws mesured using Thorpe cliper (Biomedicl Reserch Instruments, Rockville, MD, USA). Nodules 1.5 mm in dimeter were mesured using the nked eye. Nodules 542

3 Lee HY, et l. Hyperoxi suppress lung tumor growth < 1.5 mm in dimeter were microscopiclly ctegorized s either.5 mm in dimeter or otherwise. Tumor volume ws clculted using the formul: (d1 d2 d3).5236, where dn represent the three orthogonl dimeters. A portion of ech tumor ws fixed in 4% prformldehyde prior to histologicl nlysis. Broncholveolr lvge fluid smpling Mice were nesthetized vi intrperitonelly injection of mixture of zoletil (3 mg/kg; Zoletil 5, Virc, Crros, Frnce) nd rompun (1 mg/kg; Rompun, Byer Helth- Cre, Leverkusen, Germny), nd the trche were exposed nd cnnulted with silicone tuing ttched to 23-guge needle on tuerculin syringe. Broncholveolr lvge (BAL) fluid ws withdrwn fter instilltion of 8 µl sterile phosphte-uffered sline (PBS) through the trche into the lung. The totl cell counts in BAL fluid were determined using hemocytometer. The BAL fluid ws cytospun (5 minutes t 75 g) onto microscope slides nd stined with Diff-Quick (Sysmx, Koe, Jpn). The percentges of mcrophges, lymphocytes, eosinophils, nd neutrophils were clculted y counting 5 leukocytes on rndomly selected regions of the slides under light microscope. The superntnts were stored t 7 C. Histopthology The left lung ws fixed in 1% formlin, emedded in prffin wx, nd cut into 4 μm thick sections using microtome. Deprffinized tissue sections were sujected to hemtoxylin nd eosin stining nd exmined under microscope. Lung tumors were clssified s ppillry denoms, solid/lveolr denoms, or undifferentited crcinoms. Antioxidnt enzyme ssys Lung tissues were excised, weighed, nd homogenized in the ssy uffers from vrious kits. Within 1 week of collection, ll homogentes were sujected to enzyme-linked immunosorent ssy (ELISA) to determine the levels of superoxide dismutse (SOD), totl glutthione peroxidse (GSH), nd ctlse (ebioscience, Sn Diego, CA, USA) following the mnufcturer s instructions. Mesurement of 8-OHdG nd cspse 3 levels The serum levels of the oxidtive stress iomrker 8-hydroxy-2 -deoxygunosine (8-OHdG) were mesured y competition ELISA employing monoclonl ntiody (Oxford Biochemicl Reserch, Oxford, MI, USA) ccording to the mnufcturer s instructions. Opticl densities t 45 nm were mesured using microplte reder. Vlues were clculted in ng/ml using the stndrds included in the kit. Cspse 3 levels were ssyed using commercil kit (BioVision, Milpits, CA, USA). Snp-frozen lung tissue ws homogenized in uffer nd nlyzed ccording to the mnufcturer s instructions. Opticl densities t 45 nm were mesured using microplte reder. Cspse 3 mrna expression ws mesured y quntittive reverse trnscription polymerse chin rection (PCR) nd the level of cleved cspse 3 protein ws mesured using Western lot nlysis. Western lot nlysis Lung tissues were homogenized in rdio immunoprecipittion ssy cell lysis uffer contining mixture of protese inhiitors (GenDEPOT, Brker, TX, USA) nd centrifuged t 13, rpm for 15 minutes t 4 C. Proutein concentrtions were determined using BCA proetein ssy kit (Thermo Scientific/Pierce Biotechnology, Rockford, MA, USA). Equl mounts of protein (4 μg) were loded onto 15% SDS-PAGE gel, electrophoresed, nd trnsferred to PVDF memrne (Millipore, Billeric, MA, USA). The memrne ws locked with 5% nonft milk contining.1% Tween-2 t room temperture for 2 hours nd incuted with the primry ntiody (ginst Bcl-2, Bx, cspse 3, or β-ctin [Cell Signling Technology Inc., Dnvers, MA, USA]) t 1:1, dilution t 4 C overnight. The memrne ws then wshed with Tris-uffered sline contining.1% Tween-2 nd incuted with 1:2, dilution of got nti-rit secondry ntiody for 2 hours t room temperture. The lot ws developed using the ECL-Western Blotting nlysis system (GE Helthcre Life sciences, Buckinghmshire, UK) nd exposed to X-ry film. Quntittive reverse trnscription polymerse chin rection Totl RNA ws extrcted from lung tissue using TRIzol regent (Invitrogen, Crlsd, CA, USA) ccording to the 543

4 The Koren Journl of Internl Medicine Vol. 33, No. 3, My 218 mnufcturer s recommendtions, nd RNA qulity nd quntity were evluted using the Nnodrop spectrophotometer (Nnodrop Technologies, Wilmington, DE, USA). First-strnd cdna synthesis ws performed using rndom hexmers nd the PrimeScript RT regent kit (TAKARA Bio, Otsu, Jpn) ccording to the mnufcturer s protocol. Quntittive PCR ws performed using 2 SYBR FAST qpcr Mster Mix (KAPA Biosystems, Wilmington, MA, USA). The PCR conditions were 95 C (3 minutes), followed y 4 cycles t 95 C (3 seconds) nd 6 C (3 seconds); we drew the stndrd denturtion curves. The following primers were used: Bx forwrd 5 -GGCT- GGACACTGGACTTCCT-3, reverse 5 -GGTGAGGACTC- CAGCCACAA-3 ; Bcl-2 forwrd 5 -TTCGCAGAGATGTC- CAGTCA 3, reverse 5 -TTCAGAGACAGCCAGGAGAA-3 ; cspse 3 forwrd 5 -TGTCATCTCGCTCTGGTACG-3, reverse 5 -AAATGACCCCTTCATCACCA-3 ; nd β-ctin (control) forwrd 5 - GACGGCCAGGTCATCACTAT-3, reverse 5 -CGGATGTCAACGTCACACTT-3. All ssys were performed in triplicte using the CFX96 Touch Rel-Time PCR Mchine (Bio-Rd, Foster City, CA, USA), nd fold chnges in expression were derived using the comprtive cycle threshold method. Terminl trnsferse-medited dutp nick end-leling (TUNEL) ssy Apoptosis ws evluted using the TUNEL ssy ccording to the mnufcturer s instructions (Promeg, Mdison, WI, USA). Briefly, 4 µm thick sections of prffin emedded tissue on glss slides were deprffinized nd hydrted. After wshing in PBS, equilirtion uffer nd terminl deoxynucleotidyl trnsferse ws dded to ll slides followed y incution t 37 C for 1 hour. The slides were immersed in sline sodium citrte nd counterstined, followed y severl wshes with distilled wter. The numers of TUNEL-positive cells (identified y rown nuclei) were counted microscopiclly in the entire field of ech section t 2 mgnifiction. Sttisticl nlysis Dt were sujected to one-wy nlysis of vrince (ANOVA) followed y Dunnett s multiple rnge test using GrphPd Prism version 5. (GrphPd Softwre, Sn Diego, CA, USA). All dt re expressed s men ± stndrd devition, nd p vlue <.5 ws considered to reflect sttisticl significnce. RESULTS Body weight Over the experimentl period, ll nimls were weighed periodiclly. All nimls were weighed periodiclly. All tolerted oxygen exposure well; no mortlity due to hyperoxi ws pprent. Mice treted with oth cropltin nd hyperoxi exhiited the smllest weight gins y 24 weeks in oth the norml control nd B[]P groups (oth p <.5). B[]P reduced the ody weight somewht reltive to tht of norml controls, ut the difference ws not significnt. The ody weight reduction of B[]- P mice treted with oth hyperoxi nd cropltin persisted the entire 28 weeks (p <.5) (Fig. 2). Lung tumor numers We mesured the tumor numer nd volume t week 28 (Fig. 3). The mrked increses in tumor numer nd volume in the B[]P control group were significntly reduced y cropltin nd hyperoxi tretment. The tumor numer decresed y 59% nd the tumor volume y 72% (p <.5 nd p <.1, respectively). Mouse weight (g) CON CAR H CAR + H + H c Week Figure 2. Body weight chnge of nimls during exposure to cropltin or hyperoxi in enzo[]pyrene (B[]P)-induced lung cncer mouse model. The ody weight ws mesured weekly. Vlues from the cges per group were verged for ech time point. Significnt reduction of ody weight y hyperoxi or cropltin ws oserved t 28 weeks. CON, control group; CAR, cropltin group; H, hyperoxi group. p <.5 compred with the CON group. p <.5. c p <.5 compred with the group. c 544

5 Lee HY, et l. Hyperoxi suppress lung tumor growth BAL nlysis We counted totl inflmmtory cell numers in BAL fluid. Fig. 4A shows tht cell numers incresed fter cropltin or hyperoxi tretment in norml controls, nd lymphocyte numers incresed significntly fter tretment with cropltin nd hyperoxi (p <.1). Fig. 4B shows tht the totl cell counts incresed in ll B[]P groups compred with norml controls. Administrtion of cropltin nd hyperoxi to B[]P-treted mice significntly reduced the cell counts compred with the B[]P control group (p <.5). No significnt differences in the numers of inflmmtory cells (lymphocytes nd neutrophils) ws evident mong the B[]P-treted groups. Histopthologicl chnges Fig. 5 shows the histologicl profiles of lung sections from the control nd experimentl groups. Control lungs hd norml rchitecture nd smll uniform nuclei. The B[]P control group exhiited loss of rchitecture nd lveolr dmge, evidenced y hyperchromtic nuclei in cells of the lveolr wlls. B[]P groups treted with cropltin or hyperoxi exhiited reduced levels of lveolr dmge nd hyperchromtic nuclei in lveolr cells. The cropltin nd hyperoxi treted group exhiited reduced tumor urden with ner-norml lung rchitecture. 2 Lung tumors/mouse Tumor volume (mm 3 ) 1 5 A + H + H Figure 3. Effect of cropltin or hyperoxi tretment on the lung tumor volume in enzo[]pyrene (B[]P)-induced lung cncer mouse model. Lung tumor development ws evluted y counting (A) tumor numer nd (B) tumor volume. The results re the men ± SE. CON, control group; CAR, cropltin group; H, hyperoxi group. p <.5 nd p <.1, compred with the group. B BAL cell counts (x 1 4 /ml) A 1 5 c c c CON CAR H CAR + H,e Totl cell Mcrophge Lymphocyte Neutrophil BAL cell counts (x 1 4 /ml) H Totl cell Mcrophge Lymphocyte Neutrophil Figure 4. Numer of inflmmtory cells in roncholveolr lvge (BAL) fluid in enzo[]pyrene (B[]P)-induced lung cncer mouse model. BAL cells were isolted nd totl nd differentil cells were counted: (A) norml control mouse group; (B) B[] P-induced lung cncer mouse group. The results re the men ± SE. CON, control group; CAR, cropltin group; H, hyperoxi group. p <.5, p <.1, nd c p <.1 compred with the group. d p <.5 nd e p <.1 compred with the B[] P + H group. B,d 545

6 The Koren Journl of Internl Medicine Vol. 33, No. 3, My 218 CON CAR H CAR + H + H Figure 5. Effect of cropltin or hyperoxi tretment on the histopthologic chnges of lung in enzo[]pyrene (B[]P)-induced lung cncer mouse model (H&E, 2). CON, control group; CAR, cropltin group; H, hyperoxi group. 6 GSH totl (uµ) SOD inhiition rte (%) H A 2 + H B C 8-OHdG (ng/ml) Ctlse (U/L) 6 + H 2 1 D + H Figure 6. Chnges in ntioxidtive enzyme ctivities nd level of oxidtive DNA dmge in enzo[]pyrene (B[]P)-induced lung cncer mouse model y cropltin or hyperoxi tretment. The levels of (A) superoxide dismutse (SOD), (B) glutthione (GSH), nd (C) ctlse ctivity s the ntioxidnt enzymes were mesured in the totl lung homogente from mice in ech groups. (D) 8-Hydroxy-2-deoxygunosine (8-OHdG), which hs een regrded s potentil mrker of oxidtive DNA dmge ws lso ssyed in lung tissue. Vlues re expressed s men ± SE. CON, control group; CAR, cropltin group; H, hyperoxi group. p <.5 compred with the group. 546

7 Lee HY, et l. Hyperoxi suppress lung tumor growth Oxidtive stress nd DNA dmge To explore oxidtive stress mong the B[]P groups, we mesured the levels of severl ntioxidnt enzymes in lung tissue. The SOD level ws somewht incresed in the B[]P + cropltin group compred with the B[]P control group, ut the difference ws not significnt. The SOD level ws decresed in the B[]P + hyperoxi group nd further so in the B[]P + cropltin + hyperoxi group, ut the difference ws not significnt (Fig. 6A). The GSH level ws incresed in the B[]P + cropltin group ut decresed in oth the B[]P + hyperoxi group nd the B[]P + cropltin + hyperoxi group compred with the B[]P control group; however, the differences were not significnt (Fig. 6B). Tretment with oth cropltin nd hyperoxi significntly incresed the ctlse level (p <.5) compred with tht in the B[]P control group (Fig. 6C). We ssyed the level of 8-OHdG, mrker of oxidtive dmge, vi ELISA to mesure the extent of DNA dmge. The 8-OHdG level ws significntly higher in the B[]P + cropltin + hyperoxi group thn in the B[]P control group (p <.5) (Fig. 6D). Apoptotic gene expression We mesured the mrna nd protein expression levels of Bx, Bcl-2, nd cspse 3 (Fig. 7). The Bx/Bcl-2 mrna rtio incresed significntly fter B[]P + cropltin + hyperoxi tretment compred with the B[]P control group (p <.1) (Fig. 7A). Although no significnt etween-group difference in the cspse 3 mrna expression level ws evident, its expression tended to increse stedily fter tretment with cropltin or hyperoxi. In the Western lot nlysis, BAX nd C-cspse 3 showed incresed uptke in the + H group compred to the other groups. (Fig. 7B). A significnt increse in the level of cspse 3 ctivity ws evident in the Reltive mrna expression A B 2 1 BAX Bcl-2 C-cspse 3 β-actin BAX + H Reltive mrna expression CON 1..5 Bcl-2 BAX/Bcl-2 Cspse3 + H B[]P BAX/Bcl-2 rtio CON CAR H CAR + H CON CAR H CAR + H H Cspse 3 ctivity (opticl density) Reltive mrna expression H + H Figure 7. Effect of cropltin or hyperoxi tretment on the expression of poptotic genes in enzo[]pyrene (B[]P)-induced lung cncer mouse model. (A) mrna expression of BAX, Bcl-2, BAX/Bcl-2, nd cspse 3 ws exmined y rel-time polymerse chin rection. (B) Protein levels of poptotic genes were exmined using Western lot nlysis. (C) Cspse 3 ctivity in lung tissue ws mesured using enzyme-linked immunosorent ssy. The results re the men ± SE. CON, control group; CAR, cropltin group; H, hyperoxi group. p <.5 nd p <.1 compred with the group. c p <.5 compred with the group. C c 547

8 The Koren Journl of Internl Medicine Vol. 33, No. 3, My 218 A B C + H D Figure 8. Effect of cropltin or hyperoxi tretment on the poptosis of lung in enzo[]pyrene (B[]P)-induced lung cncer mouse model. A representtive trnsferse-medited dutp nick end-leling (TUNEL) stining of lung sections showed the poptotic sttus (poptotic nuclei, shown s rrows). (A) No TUNEL-positive cells were detected in group. TUNEL-positive cells were present in lung cncer tissue sections in (B) or (C) group. (D) In the B[]P + CAR + H group, significntly incresed numer of TUNEL-positive cells were oserved ( 2). CON, control group; CAR, cropltin group; H, hyperoxi group. B[]P + cropltin + hyperoxi group compred with the B[]P control group (p <.5) nd the B[]P + hyperoxi group (p <.5) (Fig. 7C). Identifiction of poptosis in lung sections The TUNEL ssy ws used to detect cells undergoing poptosis in lungs of B[]P-treted mice treted with cropltin or intermittent normoric hyperoxi (Fig. 8). No TUNEL-positive cells were detected in B[]P-treted control mice. TUNEL-positive cells (poptotic nuclei, indicted y the rrow in Fig. 8) were evident in cncer tissue treted with cropltin lone or hyperoxi lone. The comintion of cropltin nd hyperoxi significntly incresed the numer of TUNEL-positive cells. DISCUSSION We found tht intermittent normoric hyperoxi with cropltin exhiited synergistic ntitumor effect on B[]P-induced lung cncers in mice. Hyperoxi exerts tumoricidl effects on vrious cncers, including mmmry tumors in rts [8,9]. Two studies on mice injected with vrious tumor cell lines nd then exposed to 7% oxygen for 3 weeks showed tht the extents of lung tumors derived from mmmry crcinom MT-7 cells nd lung tumor cell lines were reduced [1,11]. Most previous studies used hyperric oxygen to increse tissue oxygen levels. However, to prevent hyperric hyperoxi-induced lung injury [15], we employed only intermit- 548 tent normoric hyperoxi. According to the normoric oxygen prdox, erythropoietin (EPO; which exhiits mny tissue-protecting effects) levels increse fter normoric hyperoxi, ut n unexpected reduction in the EPO level ws pprent fter hyperric oxygen exposure (2.5 tm) [17]. Hyperric oxygention my trigger more oxidtive stress thn expected sed on the oxygen concentrtion lone; such stress my induce cytokine production [18]. It is likely tht oxidtive stress plys mjor tumoricidl role. Rective oxygen species (ROS) ctivity is elevted during hyperoxi [19]. Production of free oxygen rdicls my exceed the endogenous ntioxidnt cpcity, creting oxidtive stress tht in turn triggers poptosis nd cell deth [2]. Free rdicls seem to exert opposite effects on norml nd tumor cells. When free rdicls ttck norml cells, DNA dmge follows, ut ROS re ssocited with highly eneficil growth inhiition of tumor cells. The dmging effects of ROS re mitigted y severl cellulr ntioxidnt defenses. Common ntioxidnts include enzymes such s SOD, ctlse, GSH-ssocited enzymes (such s glutthione reductse), nd heme oxygense [21]. SOD ( metlloprotein) is considered the first line of defense ginst free rdicls, trnsforming the superoxide rdicl into oxygen nd hydrogen peroxide (H2O2). H2O2 is next metolized to innocuous oxygen nd wter y GSH nd ctlse [22]. We found tht the ddition of hyperoxi to cropltin tretment reduced the SOD level compred with tht of controls;

9 Lee HY, et l. Hyperoxi suppress lung tumor growth however, it ws not sttisticlly significnt. SOD ctivity is often reduced erly in cncer development, rendering tumors more susceptile to the effects of incresed ROS levels cused y hyperoxic exposure. However t lte stges, SOD ctivity my increse to help fight ginst elevted ROS levels. Therefore this shift in SOD expression my hve cused insignificnt chnge in SOD levels, s in our study [22]. GSH plys criticl role in mintining n pproprite lnce etween oxidnt nd ntioxidnt levels. GSH inctivtes toxic or crcinogenic electrophiles [23]. Therefore, decrese in the GSH level is n indictor of oxidtive stress. However in our study, we could not find vlid chnge in GSH levels mong the groups. This my e due to the consequence of decrese in GSH level in lung tumors nd increse in GSH level in norml lung tissue, cusing insignificnt chnges in the finl outcome. Further studies re needed. The level of ctlse ctivity under physiologicl conditions is low. Ctlse hs lower ffinity for H 2 O 2 thn does GSH, ut ctlse plys n importnt role in diseses ssocited with elevted H 2 O 2 levels [24]. Ctlse decomposes H 2 O 2 to wter nd oxygen without producing free rdicls [25]. We found tht ddition of hyperoxi to cropltin tretment sustntilly incresed the ctlse level in lung homogentes compred with the cropltin tretment lone. Excess free rdicls oxidtively dmge lipids, proteins, nd DNA. The most commonly used mrker of oxidtively modified DNA is 8-OHdG [26]; this dduct is n importnt mrker of crcinogenesis, indicting the extents of G to T nd A to C trnsversions [27]. The 8-OHdG level in mice treted with oth hyperoxi nd cropltin ws meningfully higher thn tht in controls. All of our results suggest tht oxidtive stress, nd the effects thereof on DNA, were incresed following exposure to hyperoxi or cropltin. Furthermore, the oxidtive stress level ws significntly greter fter hyperoxi + cropltin tretment compred with either tretment lone. Both decrese in the GSH level nd n increse in the ROS level enhnce poptosis [28]. Incresed Bx expression indictes greter susceptiility to poptosis; incresed Bcl-2 expression protects ginst poptosis. The Bx/Bcl-2 (mrna or protein) rtio is mesure of the susceptiility to poptosis [29,3]. We found tht the mrna rtio ws incresed significntly in mice treted with cropltin nd hyperoxi. Western lotting lso confirmed the incresed expression of BAX nd cspse 3. Cspse 3, n executioner cspse ctivted vi oth the extrinsic nd intrinsic poptotic pthwys, is n importnt component of poptosis [31]. The cspse 3 level ws incresed significntly fter hyperoxi ws dded to cropltin tretment. Progrmmed cell deth ws evident in tumor tissues fter tretment with oth hyperoxi nd cropltin, nd the numer of TUNEL-positive poptotic cells ws incresed significntly. Thus, the comintion of hyperoxi nd cropltin exerted synergistic effect on poptotic induction. KEY MESSAGE 1. Intermittent normoric hyperoxi with cropltin displys synergistic tumoricidl effect in mouse lung cncer model. 2. Addition of hyperoxi to chemotherpy enhnced oxidtive stress, which is considered to induce cell deth minly vi poptosis. 3. Intermittent normoric hyperoxi my e useful djuvnt therpy for lung cncer. Conflict of interest No potentil conflict of interest relevnt to this rticle ws reported. Acknowledgments This reserch ws supported y Bsic Science Reserch Progrm through the Ntionl Reserch Foundtion of Kore (NRF) funded y the Ministry of Eduction (214R1A1A25826) nd y grnts Clinicl Reserch Lortory of The ctholic University of Kore, St. Pul's Hospitl. REFERENCES 1. Hockel M, Vupel P. Tumor hypoxi: definitions nd current clinicl, iologic, nd moleculr spects. J Ntl Cncer Inst 21;93: Rofstd EK, Gustd JV, Egelnd TA, Mthiesen B, Glppthi K. Tumors exposed to cute cyclic hypoxic stress 549

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