Peroxiredoxin 1 has an anti-apoptotic role via apoptosis signal-regulating kinase 1 and p38 activation in mouse models with oral precancerous lesions

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1 ONCOLOGY LETTERS 12: , 2016 Peroxireoxin 1 hs n nti-poptoti role vi poptosis signl-regulting kinse 1 n p38 tivtion in mouse moels with orl prenerous lesions JIANFEI ZHANG, XINYING JING, WENWEN NIU, MIN ZHANG, LIHUA GE, CONGCONG MIAO n XIAOFEI TANG Beijing Institute of Dentl Reserh, Beijing Stomtologil Hospitl n Shool of Stomtology, Beijing Key Lortory, Cpitl Meil University, Beijing , P.R. Chin Reeive Mrh 17, 2015; Aepte April 8, 2016 DOI: /ol Astrt. Peroxireoxin 1 (Prx1) is importnt in the protetion of ells from oxitive mge n the regultion of ell prolifertion n poptosis. Prx1 is overexpresse in orl prenerous lesions of orl leukoplki (OLK) n orl ner; however, the ssoition etween Prx1 expression n OLK pthogenesis remins unknown. The present stuy investigte the role of Prx1 n its moleulr mehnisms in oxitive stress inue poptosis uring the pthogenesis of OLK. Wil type n Prx1 knokout mie were trete with 50 µg/ml 4 nitroquinoline 1 oxie (4NQO) or 4NQO + H 2 O 2 for 16 weeks to estlish mouse moels with tongue prenerous lesions. Apoptoti ells were etete using terminl eoxynuleotiyl trnsferse UTP nik en leling ssy. The expression of Prx1, poptosis signl regulting kinse 1 (ASK1), phosphor ASK1, p38 n phosphor p38 ws nlyze using immunohistohemil stining, n their mrna expression levels were evlute y reverse trnsription quntittive polymerse hin retion. The present results emonstrte tht 4NQO or 4NQO + H 2 O 2 inue the evelopment of tongue prenerous lesions in Prx1 knokout n wil type mie. Prx1 ws overexpresse in tongue prenerous lesions ompre with norml tongue muos. There ws signifint erese in the egree of moerte or severe epithelil ysplsi, n mil epithelil ysplsi ws lerly elevte, in Prx1 knokout mie trete with 4NQO + H 2 O 2 ompre with wil type mie trete with 4NQO + H 2 O 2. Prx1 suppresse poptosis n upregulte phosphor ASK1 n phosphor p38 expression in tongue prenerous lesions. The present results suggest tht Prx1 suppresses oxitive Corresponene to: Professor Xiofei Tng, Beijing Institute of Dentl Reserh, Beijing Stomtologil Hospitl n Shool of Stomtology, Beijing Key Lortory, Cpitl Meil University, 4 Tintn Xili, Dongheng, Beijing , P.R. Chin E mil: tngxf0401@sohu.om Key wors: orl leukoplki, Prx1 knokout mie, oxitive stress, poptosis, ASK1, p38 stress inue poptosis vi the ASK1/p38 signlling pthwy in mouse tongue prenerous lesions. In onlusion, Prx1 n H 2 O 2 hve oorintion role in promoting the progression of tongue prenerous muos lesions. The present finings provie novel insight into Prx1 funtion n the mehnisms of Prx1 in OLK pthogenesis. Introution Orl leukoplki (OLK) is the most ommon orl prenerous lesion, with glol prevelne of 1% (1) n mlignnt trnsformtion rte of % (2). Following OLK trnsformtion to orl ner, the 5- n 10-yer survivl rtes re 59 n 48%, respetively (3). Currently, the pthogenesis of OLK is unler. Numerous stuies hve emonstrte tht OLK is losely ssoite with smoking, rinking n etel hewing (4 8). Too, etel nut n lohol ll inrese the expression of the oxint H 2 O 2 in sliv n orl muosl ells (9,10), n H 2 O 2 expression t high level my result in oxitive mge of DNA n tivtion of poptoti genes, thus inuing poptosis of ells (11 13). Retive oxygen speies (ROS) is olletive term tht esries O 2 erive non ril speies, inluing H 2 O 2, n O 2 erive free rils, suh s superoxie nion, hyroxyl n peroxyl free rils. At physiologil low levels, ROS funtions s reox messengers in intrellulr signling n regultion. However, exessive ROS inue oxitive moifition of mromoleules, inhiit protein funtions n promote poptosis of ells (14). Peroxireoxins (Prxs) re thio speifi ntioxint enzymes, n my e inue y severl types of oxitive stress onitions. They re ssoite with neutrlizing ellulr hyroperoxies, whih protet ells from oxitive mge. Prxs re often ientifie in mmmls, yest n teri, whih re lssifie s 1 ys Prx n 2 ys Prx on the sis of one or two onserve ysteine resiues. Peroxireoxin 1 (Prx1), s n importnt memer of Prxs, hs two onserve ysteine resiues (15). Current eviene suggests tht Prx1, s simple peroxise, initites the mehnisti swith from peroxise to hperone funtion, mening tht it is losely ssoite with vriety of iologil proesses inluing ell prolifertion, ifferentition n poptosis (16).

2 414 ZHANG et l: ANTI-APOPTOTIC ROLE OF PEROXIREDOXIN 1 IN ORAL PRECANCEROUS LESION MOUSE MODELS Yngw et l (17,18) hve ientifie tht n overexpression of Prx1 is signifintly ssoite with the reurrene of orl squmous ell rinom (OSCC). Previous stuies y the present uthors hve onfirme tht Prx1 expression n 8 hyroxy 2' eoxygunosine (8 OHG) expression levels re elevte in humn OLK tissues, n n inrese in 8 OHG is onsistent with the expression of Prx1 (19). This result inites tht there is signifint ssoition etween Prx1 n oxitive mge in the progression of OLK. Whether Prx1 is importnt in OLK remins unknown, n the mehnism ssoite with Prx1 n poptosis or oxitive stress remins unler. Apoptosis signl regulting kinse 1 (ASK1) is serine threonine protein kinse tht funtions s mitogen tivte protein kinse (MAPK), whih tivtes Jun N terminl kinse (JNK) n p38 MAPK signling ses. ASK1 my e tivte y vrious stresses n is ritil in the regultion of signling in response to oxitive stress, whih is mjor ontriutor to ell eth (20 22). Kim et l (23) hve emonstrte tht Prx1 plys negtive role in regulting ASK1 inue poptosis. However, to the est of our knowlege, there is no eviene tht revels similr results in vivo. In the present stuy, 4 nitroquinoline 1 oxie (4NQO) ws use to estlish prenerous lesion moel in wil type n Prx1 knokout mie, to investigte the poptoti role of Prx1 in orl prenerous lesions se on the hypothesis tht Prx1 my meite the ASK1/p38 signlling pthwy. In ition, the effet of oxitive stress on Prx1 n poptosis in orl prenerous lesions ws lso etermine. Unerstning the moleulr mehnisms of Prx1 involve in the initition n progression to mlignny my enefit methos for the prognosis n tretment of orl prenerous lesions. Mterils n methos Experimentl nimls. A totl of 50 wil type C57BL/6 mie (Vitl River Lortory Animl Tehnology Co., Lt., Shenzhen, Chin) n 50 Prx1 knokout mie, whih h een previously estlishe (24), ge 6 8 weeks ol, were use in the present stuy. All the nimls were kept in orne with institutionl guielines in speifi pthogen free units t 24±2 C room temperture with 40 60% humiity, in 14 y light/10 y rk yle with freely essile wter foo. The experimentl protool for the present stuy ws pprove y the lol Ethil Committee for Animl Use. The experimentl mie were rnomly ivie into six groups tht unerwent vrious tretments s follows: Wil type ontrol (n=10), tretment with vehile (istille wter); wil type 4NQO group (n=20), tretment with 50 µg/ml 4NQO (Sigm Alrih, St. Louis, MO, USA) every y; wil type 4NQO + H 2 O 2 group (n=20), tretment with 50 µg/ml 4NQO every y n 3% H 2 O 2 smere on tongue muos three times week; Prx1 knokout ontrol group (n=10), tretment with vehile (istille wter); Prx1 knokout 4NQO group (n=20), tretment with 50 µg/ml 4NQO every y; n Prx1 knokout 4NQO + H 2 O 2 group (n=20), tretment with 50 µg/ml 4NQO every y n 3% H 2 O 2 smere on tongue muos three times week. All these tretments lste for 16 weeks. The mie were euthnize n the tongues were resete n immeitely store in liqui nitrogen for future moleulr/ellulr nlysis, or in formlin for the preprtion of prffin emee tissue loks. Terminl eoxynuleotiyl trnsferse UTP nik en leling (TUNEL) ssy. Apoptosis ws exmine using In Situ Cell Deth Detetion kit, POD (Rohe Dignostis, Mnnheim, Germny), oring to the mnufturer's protool. The prffin emee tissues were ke t 65 C for 1 h, e wxe using xylene n grully ehyrte with 100, 95, 90, 80 n 70% ethnol. The speimens were wshe twie with phosphte uffere sline (PBS) for 5 min eh wsh, trete with proteinse K solution (10 mm Tris HCl with 20 µg/ml proteinse K; Merk Millipore, Drmstt, Germny), inute t 37 C for 15 min, n wshe twie with PBS for 5 min eh wsh. Dry speimens were trete with 50 µl TUNEL retion mixture (ilution, 1:5), overe with over slip, hyrte in light free onitions n inute t 37 C for 60 min. The speimens were susequently wshe three times with PBS for 5 min eh wsh, n ry speimens were trete with 50 µl onverter POD, overe with over slip, hyrte in light free onitions, inute t 37 C for 60 min, n wshe three times in PBS for 5 min eh wsh. Finlly, the speimens were sujete to inution with freshly prepre 3,3' iminoenziine (DAB) solution for 10 min, hemtoxylin stining, soking twie in nhyrous ethnol for 5 min n xylene for 2 min n mounting with neutrl gum. Immunohistohemil stining. The prffin emee mouse tongue speimens (4 µm) were e prffinize n hyrte using grient lohol, n rinse with PBS. Antigen retrievl for Prx1, ASK1, phosphor ASK1 n p38 ws onute with itrte uffer (ph=6.0) in mirowve oven, n for phosphor p38 with n EDTA uffer. Susequently, the setions were loke with 3% H 2 O 2 t room temperture for 15 min to remove the enogenous peroxise n inute in 10% got serum (Beijing Zhongshn Jinqio Biotehnology Co., Lt., Beijing, Chin) s loking solution t 37 C for 30 min. The speimens were inute with the following primry ntioies: Polylonl rit nti Prx1 (ilution, 1:5,000; #41906; Am, Cmrige, MA, USA), polylonl rit nti ASK1 (ilution, 1:200; #s 1425R; Bioss, In., Beijing, Chin), monolonl rit nti phosphor ASK1 (ilution, 1:400; #GTX50229; GeneTex, In., Irvine, CA, USA), p38 (ilution, 1:800; #s-0637r; Bioss, In.) n phosphor p38 (ilution, 1:200; #4631; Cell Signling Tehnology, In., Dnvers, MA, USA) t 4 C overnight. The speimens were inute with iotinylte seonry IgG ntioy (from the MxVision HRP Polymer nti Mouse IHC kit; Fuzhou Mixin Bioteh Co., Lt., Fuzhou, Chin) t 37 C for 30 min, n then visulize using DAB stining for 2 5 min. The speimens were sujete to Myer's hemtoxylin stining, ehyrtion n mounting. For the negtive ontrol, PBS ws use in ple of primry ntioy. Heptoellulr rinom tissue n smll intestine tissue were use s the positive ontrols for Prx1 n p38, respetively, while rest rinom tissue ws use s the positive ontrol for ASK1, phosphor ASK1 n phosphor p38. For evluting the poptosis level n the expression of phosphor p38, the ells with positive stining were

3 ONCOLOGY LETTERS 12: , etermine y ounting the stine ells using Imge Pro Plus version 7.0 (Mei Cyernetis, In., Rokville, MD, USA). In totl, ~1,000 ells were ounte for eh tumor speimen. In orer to evlute the expression of Prx1, ASK1, p38 n phosphor ASK1, the stine ells from three to five representtive mirosope fiels were ounte for eh speimen (mgnifition, x200) n the men optil ensity (MOD) ws lulte for eh mouse tongue tissue using Imge Pro Plus version 7.0 softwre s follows: MOD = integrte option ensity / re. Reverse trnsription quntittive polymerse hin retion (RT qpcr). Totl RNA ws extrte from mouse tongue tissues using TRIzol Regent (Invitrogen ; Thermo Fisher Sientifi, In., Wlthm, MA, USA), oring to the mnufturer's protool. DNA ws synthesize y reverse trnsriing 2 µg RNA with the High Cpity DNA Reverse Trnsription kit (Applie Biosystems ; Thermo Fisher Sientifi, In.). In totl, 1 µl liquots of DNA were use s the templtes for qpcr. Sequenes for ll trget gene primers were synthesize y Sngon Bioteh (Shnghi, Chin) s follows: Prx1, forwr: 5' AAT GCA AAA ATT GGG TAT CCT GC 3' n reverse 5' CGT GGG ACA CAC AAA AGT AAA GT 3'; ASK1, forwr: 5' AAG TCC CAA CCC ATA GAA ATT CCT 3' n reverse 5' AGC CAG TCG GTA AGT TCA GAA TCT T 3'; p38, forwr 5' GAG CTG AAG ATT CTG GAT TTT GG 3' n reverse 5' TAG CCA CGT AGC CGG TCA TT 3'; glyerlehye 3 phosphte ehyrogense (GAPDH), forwr 5' AGG TCG GTG TGA ACG GAT TTG 3' n reverse 5' TGT AGA CCA TGA GTT GAG GTC A 3'. The yling onitions for RT PCR were s follows: 25 C for 10 min, 37 C for 120 min n 85 C for 5 min. The UltrSYBR Mixture (With ROX) (ComWin Bioteh Co., Lt., Beijing, Chin) ws use for qpcr, n the yling onitions were s follows: 95 C for 10 min, 95 C for 15 se n 60 C for 15 se for 40 yles. For t nlysis, the 2 ΔΔCq metho (25) ws use for the normliztion of the genes of interest ginst GAPDH. The experiments were onute three times. Sttistil nlysis. Sttistilly signifint ifferenes were nlyze y χ 2, two tile Stuent's t test n Kruskl Wllis one wy nlysis of vrine test. Bonferroni ws use s post ho test. SPSS version 17.0 softwre (SPSS, In., Chigo, IL, USA) ws use for nlysis. P<0.05 ws onsiere to inite sttistilly signifint ifferene. P<0.017 ws onsiere to inite sttistilly signifint ifferene in the Bonferroni test. Results Tongue prenerous lesion moel estlishe in Prx1 knokout mie. 4NQO ws use to inue the evelopment of tongue prenerous lesions in Prx1 knokout n wil type mie. No tongue prenerous lesions were oserve in the ontrol mie t the en of the 16th week, while in Prx1 knokout n wil type mie trete with 4NQO or 4NQO + H 2 O 2 the tongues of the mie exhiite white, thik, rough n visile white pthes s well s surfe toughness. Histologil oservtion revele epithelil ysplsi with vrying egrees n OSCC on the tongues, initing tht the moel of tongue Figure 1. Iniene n type of mouse tongue prenerous lesions in six experimentl mouse moels use in the present stuy. Control groups, n=10; other groups, n=20. Prx1, peroxireoxin 1; 4NQO, 4 nitroquinoline 1 oxie; OSCC, orl squmous ell rinom. prenerous lesions in Prx1 knokout mie ws suessfully estlishe. There ws signifint erese in the egree of moerte or severe epithelil ysplsi (P=0.016), n mil epithelil ysplsi ws lerly elevte (P=0.011), in Prx1 knokout mie trete with 4NQO + H 2 O 2 ompre with wil type mie trete with 4NQO + H 2 O 2 (Fig. 1; Tle I). The pplition of 3% H 2 O 2 lone (3 times/week) i not inue epithelil ysplsi of tongue muos over 16 weeks (t not shown). These results inite tht Prx1 n H 2 O 2 ply oorintion role in promoting the progression of tongue prenerous lesions. Prx1 is over expresse in tongue prenerous lesions. The expression of Prx1 ws nlyze y RT qpcr n immunohistohemil stining. The mrna expression of Prx1 ws inrese in the wil type 4NQO group ompre with the wil type ontrol group (P=0.046). The mrna expression level of Prx1 ws lso inrese in the wil type 4NQO + H 2 O 2 group ompre with the wil type ontrol group (P=0.009). There ws no sttistilly signifint ifferene in mrna expression etween the wil type 4NQO n 4NQO + H 2 O 2 groups (Fig. 2A). The protein expression levels of Prx1 were inrese in the 4NQO n 4NQO + H 2 O 2 groups ompre with mie from the wil type ontrol group (P=0.035 n P=0.024, respetively). The expression of Prx1 in the 4NQO + H 2 O 2 group ws inrese ompre with the 4NQO group, ut this ws not sttistilly signifint (P=0.847; Fig. 2B). These results inite tht Prx1 my e importnt in promoting ell prolifertion in orl prenerous lesions. Prx1 knokout inreses ell poptosis in tongue prenerous lesions. The poptoti rte in the wil type 4NQO group ws elevte ompre with the wil type ontrol group (P<0.001). The poptoti rte in the wil type 4NQO + H 2 O 2 group ws erese ompre with the 4NQO group (P=0.004). An inrese poptoti rte in the Prx1 knokout 4NQO n Prx1 knokout 4NQO + H 2 O 2 groups ws oserve ompre with the wil type 4NQO (P=0.009) n wil type

4 416 ZHANG et l: ANTI-APOPTOTIC ROLE OF PEROXIREDOXIN 1 IN ORAL PRECANCEROUS LESION MOUSE MODELS Tle I. Iniene n type of mouse tongue prenerous lesions in six experimentl mouse moels. Norml Mil Moerte severe Group n muosl, n (%) ysplsi, n (%) ysplsi, n (%) OSCC, n (%) Totl 100 Wil type ontrol (100) 0 (0) 0 (0) 0 (0) Prx1 knokout ontrol (100) 0 (0) 0 (0) 0 (0) Wil type 4NQO 20 0 (0) 6 (30) 14 (70) 0 (0) Prx1 knokout 4NQO 20 0 (0) 7 (35) 12 (50) 1 (5) Wil type 4NQO + H 2 O (0) 1 (5) 18 (90) 1 (5) Prx1 knokout 4NQO + H 2 O (0) 9 (45) 10 (50) 1 (5) P<0.05 vs. wil type 4NQO + H 2 O 2 group. OSCC, orl squmous ell rinom; Prx1, peroxireoxin 1; 4NQO, 4 nitroquinoline 1 oxie. B A Figure 2. Prx1 is over expresse in tongue prenerous lesions. (A) mrna level of Prx1 in wil type mie etermine y reverse trnsription polymerse hin retion. (B) Positive expression of Prx1 in mouse tongue premlignnt lesions in the () wil type ontrol group, () wil type 4NQO group n () wil type 4NQO + H 2 O 2 group (mgnifition, x200). () Prx1 MOD vlue. * 0.01<P<0.05; ** 0.001<P<0.01. Prx1, peroxireoxin 1; 4NQO, 4 nitroquinoline 1 oxie; MOD, men optil ensity. 4NQO + H 2 O 2 groups (P=0.024), respetively. These results inite tht Prx1 inhiits poptosis in tongue prenerous lesions (Fig. 3A n B). Prx1 knokout results in the ownregultion of ASK1. In orer to evlute the effet of Prx1 on the tivtion of ASK1 in tongue prenerous lesions, the expression of totl ASK1 n phosphor ASK1 ws oserve in Prx1 knokout n wil type mie. The present results emonstrte tht the mrna expression level of ASK1 ws inrese in wil type 4NQO n wil type 4NQO + H 2 O 2 groups ompre with the wil type ontrol group (P=0.001 n P=0.002, respetively; Fig. 4A). A sttistilly signifint ifferene in the mrna expression level of ASK1 etween wil type 4NQO n 4NQO + H 2 O 2 groups ws oserve. The mrna expression level of ASK1 ws inrese in Prx1 knokout ontrol group ompre with wil type ontrol group (P=0.003; Fig. 4B). The mrna expression level of ASK1 in Prx1 knokout 4NQO n Prx1 knokout 4NQO + H 2 O 2 groups ws inrese ompre with the wil type group, lthough this ws not sttistilly signifint (P=0.704 n P=0.24, respetively; Fig. 4B n ). Immunohistohemil nlysis revele tht there ws no sttistilly signifint ifferene in protein expression of ASK1 etween ny groups (Fig. 4C). Compre with the wil type 4NQO group, the expression of phosphor ASK1 ws erese in the Prx1 knokout 4NQO group (P=0.022). A similr expression pttern ws oserve in wil type n Prx1 knokout 4NQO + H 2 O 2 groups (P=0.001). There ws no signifint ifferene in the phosphoryltion of ASK1 in the wil type 4NQO n wil type 4NQO + H 2 O 2 groups ompre with the wil type ontrol group (P=0.481 n P=0.104), suggesting tht phosphor ASK1 hs positive ssoition with Prx1 expression (Fig. 4D). Prx1 knokout suppresses the expression of p38. In orer to evlute the effet of Prx1 on the tivtion of p38 MAPK in tongue prenerous lesions, the expression of totl p38 n

5 ONCOLOGY LETTERS 12: , A B e f Figure 3. Prx1 knokout inreses ell poptosis in tongue prenerous lesions. (A) Terminl eoxynuleotiyl trnsferse UTP nik en leling ssy in () wil type ontrol group, () wil type 4NQO group, () wil type 4NQO + H 2 O 2 group, () Prx1 knokout ontrol group, (e) Prx1 knokout 4NQO group n (f) Prx1 knokout 4NQO + H 2 O 2 group (mgnifition, x400). (B) Prx1 negtively regulte the poptoti rte of ells in mouse tongue premlignnt lesions. * 0.01<P<0.05; ** 0.000<P<0.01; *** P= Prx1, peroxireoxin 1; 4NQO, 4 nitroquinoline 1 oxie. A B C D e f e f g g Figure 4. Prx1 knokout les to ownregultion of ASK1. (A) mrna expression level of ASK1 ws elevte in mouse tongue premlignnt lesions, s etermine y RT qpcr. (B) RT qpcr etermintion of the reltive expression of ASK1 mrna in Prx1 knokout mie () ontrol group, () 4NQO group n () 4NQO + H 2 O 2 group. (C) Prx1 h no ler ssoition with ASK1 in mouse tongue premlignnt lesions in the () wil type ontrol group, () wil type 4NQO group, () wil type 4NQO + H 2 O 2 group, () Prx1 knokout ontrol group, (e) Prx1 knokout 4NQO group n (f) Prx1 knokout 4NQO + H 2 O 2 group (mgnifition, x200). (g) ASK1 MOD vlue. (D) Prx1 positively regulte the tivtion of phosphor ASK1 in mouse tongue premlignnt lesions in the () wil type ontrol group, () wil type 4NQO group, () wil type 4NQO + H 2 O 2 group, () Prx1 knokout ontrol group, (e) Prx1 knokout 4NQO group n (f) Prx1 knokout 4NQO + H 2 O 2 group (mgnifition, x200). (g) Phosphor ASK1 MOD vlue. * 0.01<P<0.05; ** 0.000<P<0.01. Prx1, peroxireoxin 1; 4NQO, 4 nitroquinoline 1 oxie; ASK 1, poptosis signl regulting kinse 1; MOD, men optil ensity; RT qpcr, reverse trnsription quntittive polymerse hin retion.

6 418 ZHANG et l: ANTI-APOPTOTIC ROLE OF PEROXIREDOXIN 1 IN ORAL PRECANCEROUS LESION MOUSE MODELS A B C D e f e f g g Figure 5. Prx1 knokout suppresses the expression of p38. (A) mrna expression level of p38 ws elevte in mouse tongue premlignnt lesions, s etermine y RT qpcr. (B) RT qpcr etermine the reltive expression level of p38 mrna in Prx1 knokout mie () ontrol group, () 4NQO group n () 4NQO + H 2 O 2 group. (C) Prx1 positively regulte the tivtion of p38 in mouse tongue premlignnt lesions in the () wil type ontrol group, () wil type 4NQO group, () wil type 4NQO + H 2 O 2 group, () Prx1 knokout ontrol group, (e) Prx1 knokout 4NQO group n (f) Prx1 knokout 4NQO + H 2 O 2 group (mgnifition, x200). (g) p38 MOD vlue. (D) Prx1 positively regulte the tivtion of phosphor p38 in mouse tongue premlignnt lesions in the () wil type ontrol group, () wil type 4NQO group, () wil type 4NQO + H 2 O 2 group, () Prx1 knokout ontrol group, (e) Prx1 knokout 4NQO group n (f) Prx1 knokout 4NQO + H 2 O 2 group (mgnifition, x400). (g) Nuler positive rte of phosphor p38. * 0.01<P<0.05; ** 0.000<P<0.01. Prx1, peroxireoxin 1; 4NQO, 4 nitroquinoline 1 oxie; MOD, men optil ensity; RT qpcr, reverse trnsription quntittive polymerse hin retion. phosphor p38 ws etete in Prx1 knokout n wil type mie. The mrna expression level of p38 ws inrese in the wil type 4NQO n 4NQO + H 2 O 2 groups ompre with the wil type ontrol group (P=0.021 n P=0.001, respetively). The ifferene in mrna expression levels of p38 etween wil type 4NQO n 4NQO + H 2 O 2 groups ws not sttistilly signifint (P=0.401; Fig. 5A). The mrna expression level of p38 ws erese in the Prx1 knokout 4NQO group ompre with the wil type 4NQO group (P=0.006). The mrna expression of p38 ws erese in the Prx1 knokout 4NQO + H 2 O 2 group, lthough no sttistilly signifint ifferene ws oserve with the wil type 4NQO + H 2 O 2 group (P=0.649; Fig. 5B). The protein expression of p38 ws lerly inrese in the wil type 4NQO + H 2 O 2 group ompre with the wil type ontrol group (P=0.002; Fig. 5C). The level of phosphor p38 ws erese in the Prx1 knokout 4NQO group ompre with the wil type 4NQO group (P=0.022). The sme expression pttern ws oserve in Prx1 knokout n wil type 4NQO + H 2 O 2 groups (P=0.001). The expression level of phosphor p38 ws not signifintly ifferent in the wil type 4NQO n 4NQO + H 2 O 2 groups ompre with the wil type ontrol groups (P=0.606 n P=0.333, respetively), initing tht phosphor p38 h ler positive ssoition with the expression of Prx1 (Fig. 5D). Disussion OLK is the most ommon orl prenerous lesion, whih my unergo rinomtous hnge to OSCC (26). At present, little is known onerning the pthogenesis of OLK. Previous stuies hve emonstrte tht ell poptosis is suppresse y Prx1 vi the ASK1 meite signling pthwy in humn emryoni kiney 293 n ervil ner HeL ells (23). In previous stuy y the present uthors, n inrese level of poptosis ws oserve in OLK tissues, n Prx1 knokown signifintly enhne the level of poptosis in ysplsti orl kertinoyte

7 ONCOLOGY LETTERS 12: , ells (t not shown). However, even though there re numerous in vitro stuies onerning Prx1 n ell poptosis, there re few in vivo stuies. The present stuy hs for the first time, to the est of our knowlege, esigne rinogeni experiments in vivo to oserve the effet of Prx1 on ell poptosis uring the initition n progression to mlignny of orl muos. In orer to onfirm the role of Prx1 in orl prenerous lesions in vivo, the present stuy estlishe tongue prenerous lesion mouse moels in Prx1 knokout mie n investigte whether Prx1 suppresses poptosis inue y oxitive stress. The present stuy eluite the possile moleulr mehnism uring the pthogenesis n evelopment of orl prenerous lesions. In eukryoti ells, four MAPK signl trnsution pthwys, inluing extrellulr signl regulte kinse (ERK) 1/2, JNK, p38 n ERK5, hve een ientifie. ERK1/2, JNK n p38 pthwys re typil MAPK signl trnsution pthwys. Furthermore, JNK n p38 signling pthwys re ssoite with ell poptosis (27 29). ASK1 is known s propoptoti, stress tivte signling moleule, n is n uiquitously expresse serine theronine protein kinse tht funtions s MAPK kinse to tivte JNK n p38 MAPK signling ses (30). Prx1 is the most unnt n uiquitously istriute memer of the mmmlin Prx fmily. It hs een implite in regulting ell prolifertion, ifferentition n poptosis (31,32). ASK1 interts with Prx1 in the presene of H 2 O 2 inue stress n is negtively regulte y Prx1 (23). Nkgw et l (33) hve emonstrte tht the tivtion of JNK n p38 is ttenute n heptorinogenesis is inrese in ASK1 efiient mie. Yn et l (34) hve ientifie tht ASK1 tivte y rseni trioxie in leukemi ells my ply n ntipoptoti role, n Prk et l (35) hve emonstrte tht Billus nthris inues the poptosis of tivte mrophges y inhiiting the p38 MAPK pthwy. In the present stuy, the poptoti rte of ells inrese n the expression of phosphor ASK1 n phosphor p38 ws ownregulte in tongue prenerous lesions of Prx1 knokout mie. These results emonstrte tht poptosis suppression y Prx1 my e ssoite with the phosphoryltion of ASK1 n p38, n tht Prx1 hs positive regultory role in the phosphoryltion of ASK1 n p38. In ition, the present stuy lso etete the trnsription level of Prx1, ASK1 n p38 ompre with tht in norml epithelium, n the expression of Prx1, ASK1 n p38 ws lerly inrese in prenerous lesions ompre with norml epithelium. When Prx1 ws knoke own, the ASK1 trnsription level ws signifintly inrese in the ontrol group, initing tht Prx1 lerly inhiits the trnsription of ASK1 in norml muos. By ontrst, knokown of Prx1 resulte in signifint ownregultion of p38 t trnsriptionl level in the prenerous lesions, suggesting tht Prx1 lso positively regultes p38 in prenerous lesions. Overll, Prx1 suppresses oxitive stress inue poptosis in tongue prenerous lesions y positively regulting ASK1 n p38 expression t moleulr level. In the present stuy, in the Prx1 knokout 4NQO + H 2 O 2 mie, the egree of moerte to severe epithelil ysplsi ws signifintly reue n mil epithelil ysplsi ws lerly elevte ompre with wil type 4NQO + H 2 O 2 mie. This suggests tht Prx1 enhnes ell prolifertion uring the pthogenesis of orl prenerous lesions. Therefore, when orl prenerous lesions re ffete y oxitive stress, Prx1 is importnt in inhiiting oxitive mge n poptosis of ells, n promotes the progression of tongue prenerous lesions. Lee et l (36) hve lso emonstrte tht Prx1 knokout results in the erese of ell prolifertion, n Prx1 is ssoite with tumor size, miro vssl invsion n Emonson tumor gre (37). In ition, mirorna 510 iretly ins to the 3' untrnslte region of Prx1 n loks its protein expression, leing to suppression in the migrtion of humn rest ner ells (38). In the present stuy, the pplition of H 2 O 2 lone s n oxitive stressor h no ovious effet on lesion evelopment. However, more severe lesions were oserve in mie from the wil type 4NQO + H 2 O 2 group ompre with mie from the wil type 4NQO group, initing tht H 2 O 2 pplition ouple with 4NQO hs positive effet on promoting the evelopment n progression of lesions. H 2 O 2 is known s the most ommon memer of ROS n inues poptosis in vrious types of mlignnes (39,40). However, in the present stuy, ompre with 4NQO inue tongue prenerous lesions, ell poptosis ws moertely reue in mie from the 4NQO + H 2 O 2 group. A similr pttern ws oserve in Prx1 knokout mie. Previous stuies hve revele tht 4NQO tretment les to the formtion of H 2 O 2 superoxie n hyroxyl rils, thus resulting in the proution of sustntil mount of 8 OHG in DNA n oxitive mge in norml humn firolsts (41). Tng et l (42) hve emonstrte tht H 2 O 2 preonitioning t low onentrtions my protet rt pheohromoytom PC12 ells from poptosis inue y H 2 O 2. In ition, oxint preonitioning protets humn proximl tuulr ells ginst lethl oxint injury (43). In the present stuy, 3% H 2 O 2 ws pplie to mouse tongue muos three times week uring the evelopment of 4NQO inue mouse tongue prenerous lesions for 16 weeks. The 3% H 2 O 2 ws revele to e mil stimulus ompre with 50 µg/ml 4NQO. The 3% H 2 O 2 tretment my llevite poptosis inue y susequent 4NQO exposure in tongue muos epitheli of the mie. These t inite tht H 2 O 2 t low onentrtion my inhiit poptosis. A previous stuy lso revele tht H 2 O 2 t low onentrtion promotes ell prolifertion (44). A low ose of H 2 O 2 ws le to reverse DHM inue ell poptosis of humn heptoellulr rinom (44). This my inite tht the lne etween ROS proution n vrious ntioxints is vitlly importnt for ner ell growth. The present rinogeni in vivo experiments were use to oserve the effet of Prx1 on ell poptosis uring the evelopment n progression to mlignny of mouse tongue muos. The present stuy onlues tht Prx1 my suppress oxitive stress inue poptosis vi the ASK1/p38 signling pthwy in mouse tongue prenerous lesions, n H 2 O 2 n 4NQO ply oorintion role in promoting the progression of tongue muos prenerous lesions. In ition, H 2 O 2 t low onentrtion level my inhiit poptosis. The present finings provie novel insights into Prx1 funtion n the mehnisms of OLK pthogenesis. Aknowlegements The present stuy ws fune y the Ntionl Nturl Siene Fountion of Chin, Beijing, Chin (grnt nos n

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