Melatonin, a novel selective ATF-6 inhibitor, induces human hepatoma cell apoptosis through COX-2 downregulation

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1 Submit Mnusript: Help Desk: DOI: /wjg.v23.i6.986 World J Gstroenterol 2017 Februry 14; 23(6): ISSN (print) ISSN (online) 2017 Bishideng Publishing Group In. All rights reserved. ORIGINAL ARTICLE Bsi Study Meltonin, novel seletive ATF-6 inhibitor, indues humn heptom ell poptosis through COX-2 downregultion Li-Ji Bu, Hn-Qing Yu, Lu-Lu Fn, Xio-Qiu Li, Fng Wng, Ji-To Liu, Fei Zhong, Cong-Jun Zhng, Wei Wei, Hu Wng, Guo-Ping Sun Li-Ji Bu, Hn-Qing Yu, Lu-Lu Fn, Xio-Qiu Li, Fng Wng, Ji-To Liu, Fei Zhong, Cong-Jun Zhng, Hu Wng, Guo- Ping Sun, Deprtment of Onology, the First Affilited Hospitl of Anhui Medil University, Hefei , Anhui Provine, Chin Ji-To Liu, Deprtment of Phrmy, the First Affilited Hospitl of Anhui Medil University, Hefei , Anhui Provine, Chin Wei Wei, Institute of Clinil Phrmology, Anhui Medil University, Hefei , Anhui Provine, Chin Hu Wng, Institute for Liver Diseses of Anhui Medil University, Hefei , Anhui Provine, Chin Author ontributions: Bu LJ, Yu HQ nd Fn LL ontributed eqully to this work; Bu LJ, Yu HQ nd Fn LL performed selet experiments nd nlyzed dt; Li XQ nd Wng F performed selet experiments; Liu JT nd Zhong F performed selet histopthologil experiments; Zhng CJ nd Wei W red the mnusript nd gve importnt intelletul suggestions; Wng H nd Sun GP prepred the mnusript, nd designed nd supervised the projet. Supported by grnts from the Ntionl Nturl Siene Foundtion of Chin, No nd No Institutionl review bord sttement: The study ws rried out in ordne with protool pproved by the Ethis Committee of Anhui Medil University (Anhui, Chin). Conflit-of-interest sttement: No onflits of interest exist for ny of the uthors. Open-Aess: This rtile is n open-ess rtile whih ws seleted by n in-house editor nd fully peer-reviewed by externl reviewers. It is distributed in ordne with the Cretive Commons Attribution Non Commeril (CC BY-NC 4.0) liense, whih permits others to distribute, remix, dpt, build upon this work non-ommerilly, nd liense their derivtive works on different terms, provided the originl work is properly ited nd the use is non-ommeril. See: org/lienses/by-n/4.0/ Mnusript soure: Unsoliited mnusript Correspondene to: Dr. Guo-Ping Sun, Deprtment of Onology, the First Affilited Hospitl of Anhui Medil University, No. 218 Jixi Rod, Hefei , Anhui Provine, Chin. sunguoping@hmu.edu.n Telephone: Fx: Reeived: August 31, 2016 Peer-review strted: August 31, 2016 First deision: September 28, 2016 Revised: Otober 11, 2016 Aepted: Otober 27, 2016 Artile in press: Otober 27, 2016 Published online: Februry 14, 2017 Abstrt AIM To lrify the mehnisms involved in the ritil endoplsmi retiulum (ER) stress inititing unfolded protein response pthwy modified by meltonin. METHODS Heptom ells, HepG2, were ultured in vitro. Flow ytometry nd TUNEL ssy were used to mesure HepG2 ell poptosis. Western blotting nd quntittive reverse trnsription-polymerse hin retion methods were used to determine the protein nd messenger RNA levels of ER stress nd poptosis relted genes expression, respetively. Tissue mirorry onstrution from ptients ws verified by immunohistohemil nlysis. RESULTS In the present study, we first identified tht meltonin 986 Februry 14, 2017 Volume 23 Issue 6

2 seletively bloked tivting trnsription ftor 6 (ATF-6) nd then inhibited ylooxygense-2 (COX-2) expression, leding to enhned liver ner ell poptosis under ER stress ondition. Drmtilly inresed CCAAT-enhner-binding protein homologous protein level, suppressed COX-2 nd deresed Bl-2/ Bx rtio by meltonin or ATF-6 sirna ontributed the enhned HepG2 ell poptosis under tunimyin (n ER stress induer) stimultion. In linil heptoellulr rinom ptients, the lose reltionship between ATF-6 nd COX-2 ws further onfirmed. CONCLUSION These findings indite tht meltonin s novel seletive ATF-6 inhibitor n sensitize humn heptom ells to ER stress induing poptosis. Key words: Meltonin; Endoplsmi retiulum stress; Ativting trnsription ftor 6; Cylooxygense-2; Heptoellulr rinom The Author(s) Published by Bishideng Publishing Group In. All rights reserved. Core tip: Endoplsmi retiulum (ER) stress plys n importnt role in tumor growth nd resistne to tretment. Our previous studies hve lredy shown tht meltonin sensitizes the humn heptoellulr rinom ell to ER stress-indued poptosis nd ttenutes ER stress-indued doxorubiin resistne. In this study, we first identified tht meltonin seletively bloked ER stress downstrem tivting trnsription ftor 6 (ATF-6) nd then inhibited ylooxygense-2 expression, leding to enhned liver ner ell poptosis under ER stress ondition. Our findings indite tht meltonin s novel seletive ATF-6 inhibitor n sensitize humn heptom ells to ER stress induing poptosis. Bu LJ, Yu HQ, Fn LL, Li XQ, Wng F, Liu JT, Zhong F, Zhng CJ, Wei W, Wng H, Sun GP. Meltonin, novel seletive ATF-6 inhibitor, indues humn heptom ell poptosis through COX-2 downregultion. World J Gstroenterol 2017; 23(6): Avilble from: URL: v23/i6/986.htm DOI: tht the liver ner ell hs muh greter tolerne towrds number of ellulr stress onditions, suh s endoplsmi retiulum (ER) stress, hypoxi, nutrient deprivtion nd so on [3,4]. Thus, pprohes to overoming this superior tolerne hve been emerging s potentil drug trgets for the tretment of HCC [5]. All types of stress, inluding onogeneti stress tht interferes with ER funtion, use umultion of unfolded proteins in the ER lumen, referred to s ER stress, nd tivte homeostti signling network known s the unfolded protein response (UPR) [6,7]. The three min ER trnsmembrne sensors tht eliit the UPR re protein kinse RNA-like ER kinse (PERK), inositol-requiring enzyme-1 (IRE-1), nd tivting trnsription ftor 6 (ATF-6). In generl, ER stress initited by IRE-1, PERK, nd ATF-6 nd tivted in solid tumors is ruil for tumor growth nd ggressiveness, s well s for miroenvironment remodeling or drug resistne [3,4]. Meltonin (N-etyl-5-methoxytryptmine), whih is produed in the humn pinel glnd during the night phse of the light-drk yle, plys importnt roles in physiologil nd phrmologil funtions, suh s irdin rhythms nd ntioxidnts. More interestingly to us, however, is the ft tht meltonin exerts ntiner effets through interply with ER stress [8-10]. Our previous study demonstrted tht meltonin ould sensitize the humn HCC ell line HepG2 to ER stress-indued poptosis vi the inhibition of ylooxygense-2 (COX-2) [11]. Furthermore, we found tht meltonin ttenutes ER stress-indued resistne to doxorubiin through reversing tunimyin-indued ER stress [12]. However, the mehnisms involved in the ritil UPR pthwy modified by meltonin must still be lrified. In the present study, we first identified tht meltonin n seletively blok ATF-6 nd then inhibit COX-2 expression, leding to enhned liver ner ell poptosis. In linil HCC ptients, the lose reltionship between ATF-6 nd COX-2 ws further onfirmed. Our study explored the more detiled mehnisms of meltonin enhning ER stress-indued poptosis in humn heptom ells vi inhibition of COX-2 by seletively trgeting ATF-6. INTRODUCTION Heptoellulr rinom (HCC) ws the fifth most frequently dignosed ner worldwide nd the third use of ner-relted deth, responsible for pproximtely humn deths nnully worldwide [1,2]. Chin ounts for the mjority of totl HCC inidene in the world [1]. Most ptients with HCC re dignosed t lte stge, when there is no hne of surgil therpy. Unfortuntely, there re only few effetive hemotherpy gents for this type of mlignnt tumor. One of the mjor resons for untretble HCC is MATERIALS AND METHODS Regents Meltonin (M5250) nd tunimyin (T7765) were obtined from Sigm Chemil (St. Louis, MO, United Sttes). DMEM ws purhsed from Gibo-BRL Life Tehnologies (Grnd Islnd, NY, United Sttes). Anti- COX-2 (b179800), nti-bx (b32503) nd nti- CCAAT/enhner-binding protein homologous protein (CHOP) (b11419) were obtined from Abm (Cmbridge, MA, United Sttes). Anti-ATF-6 (BS6476), nti-perk (BS2156) nd nti-bl-2 (BS3711) were obtined from Bioworld Tehnology In. (St Louis 987 Februry 14, 2017 Volume 23 Issue 6

3 Prk, MN, United Sttes). The terminl deoxynuleotidyltrnsferse-medited dutp-biotin nik end lbeling (TUNEL) system ws purhsed from Rohe (Indinpolis, IN, United Sttes). The nnexin V-FITC kit ws obtined from Shnghi Bestbio (Shnghi, Chin). The reverse trnsription kit (A3500) nd TRIzol ( ) were obtined from Promeg In. (Mdison, WI, United Sttes). The SYBR Green qpcr kit ( ) ws obtined from Invitrogen Life Tehnologies (Grnd Islnd, NY, United Sttes). HCC speimens Tissue smples were obtined from 100 ptients with HCC. These 100 ptients underwent surgery t the First Affilited Hospitl of Anhui Medil University between 2001 nd Ptients with HCC who hd epted hemotherpy or rdition therpy before surgery were exluded from the study. The pthohistologil dignosis of the speimens ws onsistent with HCC in ordne with the World Helth Orgniztion Guidelines. A totl of 85 HCC ptients were mle, nd 15 HCC ptients were femle. The medin ge of the HCC ptient popultion ws 50.7 yers, rnging from 18 to 84 yers. These 100 HCC ptients were stged ording to UICC s follows: 3 HCC ptients were Stge Ⅰ (3%), 73 HCC ptients were Stge Ⅱ (73%), 9 HCC ptients were Stge Ⅲ (9%), nd 15 ptients were Stge Ⅳ (15%). Tumors were pthologilly grded ording to WHO guidelines: 4 HCC ptients were well-differentited, 91 HCC ptients were modertely differentited, nd 5 HCC ptients were poorly differentited. All the linil speimens were olleted from ptients fter obtining written informed onsent. The study ws rried out in ordne with protool pproved by the Ethis Committee of Anhui Medil University (Anhui, Chin). Tissue mirorry onstrution Formlin-fixed prffin-embedded speimens were obtined from the rhives of the Deprtment of Pthology t the First Affilited Hospitl of Anhui Medil University. Hemtoxylin nd eosin-stined tissue setions were reviewed for identifition of the trget re for tissue mirorry onstrution. Three to five representtive 1-mm ores were obtined from eh smple nd inserted in grid pttern into new reipient prffin blok using mnul tissue rryer (Hengti Instruments, Lioning, Chin). Immunohistohemil nlysis Tissue mirorry setions were deprffinized, nd endogenous peroxidse tivity ws bloked with 3% hydrogen peroxide in methnol for 10 min nd heted in 0.01 mol/l sodium itrte buffer (ph 6.0) for 10 min for ntigen retrievl. Subsequently, the setions were inubted with primry ntibody in moist hmber for 1 h t mbient temperture. Then, the setions were wshed in phosphte buffered sline (PBS) (ph 7.2), inubted with biotinylted seondry ntibody nd then inubted with peroxidse-onjugted streptvidin. To observe positive binding of the ntigen, the setions were inubted with diminobenzidine solution nd then ounterstined with hemtoxylin. Next, the setions were viewed under mirosope nd sored on the bsis of stining intensity nd the perentge of stined ells reltive to the bkground: > 10% of tumor ells stined ws onsidered positive stining. Cell ulture The humn heptom ell line HepG2 ws obtined from the Shnghi Cell Bnk (Chinese Ademy of Sienes, Shnghi, Chin). The ells were ultured in high gluose Dulbeo s modified Egle s medium (DMEM) ontining 10% het-intivted fetl bovine serum, 100 U/mL peniillin, nd 100 μg/ml streptomyin. Cell ulture ws rried out t 37 in humidified 5% CO2 tmosphere. Flow ytometry After tretment, ells were dethed from the 6-well pltes by using 0.2% trypsin, hrvested, wshed twie with PBS, nd entrifuged twie t 300 g for 5 min t 4. Then, the superntnt ws disrded, nd the pellet ws resuspended in 400 μl Annexin V binding buffer t 20 for t lest 12 h. Cells were subsequently treted in PBS with RNse A for 30 min t room temperture nd stined with propidium iodide (PI). Flow ytometri nlysis ws performed using n EPICS XL-MCL model ounter (Bekmn Coulter, Fullerton, CA, United Sttes). A totl of 1 ells/ml were nlyzed for eh smple, nd the experiment ws repeted t lest three times. TUNEL ssy Cells were ultured on overslips in 6-well pltes overnight. After tretment with vrious onentrtions of the indited ompounds for eh time period, the overslips were wshed twie with old PBS nd fixed in 4% prformldehyde solution for 1 h t room temperture. Apoptoti ells were deteted by the TUNEL ssy (TUNEL System Kit; Rohe, Bsel, Switzerlnd), whih ws performed ording to the mnufturer s instrutions. The TUNEL ssy results were quntittively nlyzed through the biologil imge nlysis system from the Nikon ECLIPSE 80i biology mirosope, Nikon Digitl Cmer DXM 1200F, ACT-1 version 2.63 softwre (Tokyo, Jpn). Western blotting After drug tretment for the indited time periods nd onentrtions, ells were lysed in RIPA lysis buffer [50 mmol/l Tris-HCl, (ph 7.4), 150 mmol/l NCl, 10 mmol/l phenylmethylsulfonyl fluoride (PMSF), 1 mmol/l ethylene dimine tetreti id (EDTA), 0.1% sodium dodeyl sulfte (SDS), 1% 988 Februry 14, 2017 Volume 23 Issue 6

4 Tble 1 Expression of ylooxygense-2 ws more likely to be ssoited with the expression of tivting trnsription ftor 6 thn with inositol-requiring enzyme-1 nd protein kinse RNA-like endoplsmi retiulum kinse in heptoellulr rinom ptients COX-2 AFP-6 IRE-1 PERK - + r P vlue - + r P vlue - + r P vlue Negtive Positive ATF-6: Ativting trnsription ftor 6; COX-2: Cylooxygense-2; IRE: Inositol-requiring enzyme; PERK: Protein kinse RNA-like endoplsmi retiulum kinse. Triton X-100 nd 1% sodium deoxyholte] for min on ie. Protein onentrtions were determined by the Lowry protein ssy. Lystes were inubted with 2 Lemmli smple buffer (Bio-Rd, Herules, CA, United Sttes) nd heted for 10 min t 95. The proteins were resolved by SDS-polyrylmide gel eletrophoresis (SDS-PAGE), then trnsferred to polyvinylidene fluoride membrnes (Millipore, Bedford, MA, United Sttes) nd inubted with bloking buffer [Tris-buffered sline/tween 20/5% nonft dry milk] overnight t 4. Immunoblots were inubted with the indited primry ntibody followed by the pproprite horserdish peroxidse-onjugted seondry ntibody nd visulized with enhned hemiluminesene (Piere, Rokford, IL, United Sttes) using hydrogen peroxide nd luminol s substrte with Kodk X-AR film. Autordiogrphs were snned using GS-700 Imging Densitometer (Bio-Rd). Quntittive reverse trnsription polymerse hin retion Totl RNA ws extrted from HepG2 ells using the Trizol regent, nd 1 mg RNA ws reverse trnsribed to DNA using the Reverse Trnsription System A3500 (Ferments, Burlington, Cnd). To determine the quntity of mrna, the DNA ws mplified by rel-time PCR with SYBR Green PCR mster mix kit (Invitrogen), nd the housekeeping gene GAPDH ws used s the internl ontrol. The SYBR Green ssys were performed in triplite on 7500 reltime instrument (Applied Biosystems In, Foster City, CA, United Sttes). The primers to detet mrna were 5 -CTGTATCCCGCCCTGCTGGTG-3 nd 5 -ACTTGCGTTG ATGGTGGCTGTCTT-3 for COX-2 nd 5 -AGAAGGCTGG GGCTCATTTG-3 nd 5 -AGGGGCCATCCACAGTCTTC-3 for GADPH. All smples were normlized to internl ontrols, nd fold-hnges were lulted by reltive quntifition. The onditions for quntittive reverse trnsription polymerse hin retion (qrt-pcr) were s follows: 5 min t 94, nd then 50 yles of 94 for 30 s, 59 for 30 s, nd 72 for 1 min. Sttistil nlysis Three or more seprte experiments were performed for eh experiment. Sttistil nlysis ws performed by Student s t-test or ANOVA. Dt re presented s the men ± SD. Signifine ws noted t P < RESULTS COX-2 is the induible form of ylooxygense nd is frequently highly expressed in tumor tissues, inluding liver ner, plying n importnt role in tumor development. To determine whih UPR pthwy ws ssoited with COX-2 expression under ER stress in HCC ptients, we nlyzed the prffin-embedded, formlin-fixed HCC speimens by immunohistohemil stining. For ll 100 speimens, COX-2, ATF-6, IRE-1 nd PERK were stined in the nulei nd/or ytoplsm of tumor ells. We observed tht the expression of COX-2 ws more likely to be ssoited with the expression of ATF-6 thn with IRE-1 nd PERK, whih rehed sttistil signifine (P = 0.011) (Tble 1). These dt suggest the existene of lose reltionship between ATF-6 nd COX-2 (Figure 1). There is limited informtion bout the effets of meltonin under ER stress onditions nd the impt of meltonin on these three UPR pthwys. To ddress this, we next evluted the effets of meltonin on the UPR pthwys in vitro. To mimi the ER stress ondition, HepG2 ells were first pretreted with tunimyin for 8 h; then, the ells were treted with meltonin t four different onentrtions (10-3, 10-5, 10-7 nd 10-9 mmol/l) for nother 24 h. The expression of ll three UPR pthwys ws evluted by western blotting method. As illustrted in Figure 2A, nd onsistent with previous reports, the downstrem signling moleules ATF-6, IRE-1 nd PERK were deteted fter ER stress tivtor tunimyin tretment. Meltonin t onentrtions between 10-7 to 10-3 mmol/l mrkedly inhibited ATF-6 expression. However, only high-onentrtion meltonin (10-3 mmol/l) slightly deresed the expression of IRE-1. Menwhile, meltonin hd no effet on the expression of PERK. These results indite meltonin prominently ffets the ATF-6 pthwy under ER stress ondition. To investigte the underlying mehnisms of whih pthwy is ssoited with the expression of COX-2 under the ondition of ER stress, we used RNA interferene to knokdown the mrna in ll three UPR pthwys nd then observed the hnges in COX-2 levels by western blotting. First, eh UPR sirna hs 989 Februry 14, 2017 Volume 23 Issue 6

5 A B C 50 μm 50 μm D 50 μm 50 μm Figure 1 Reltionship between ylooxygense-2 nd unfolded protein response pthwys. Expression of unfolded protein response (UPR) pthwys nd COX-2 in heptoellulr rinom (HCC) (streptvidin-peroxidse 400). A: Expression of COX-2 in HCC; B: Expression of ATF-6 in HCC; C: Expression of IRE-1 in HCC; D: Expression of PERK in HCC. COX-2: Cylooxygense-2. three ndidte sequenes, nd we hose the most effetive ones by western blot stining (Figure 3A-C). As shown in Figure 4A nd B, the COX-2 protein expression is downregulted fter bloking the ATF-6 mrna under ER stress ondition. In ddition, the mrna level of COX-2 ws lso deresed by si-atf-6 fter ER stress tivtor tunimyin pre-tretment. Thus, we onluded tht COX-2 n be regulted by the ATF-6 pthwy under ER stress. To further determine whether bloking the ATF-6 pthwy influenes ER stress-indued poptosis, FACS nlysis nd TUNEL stining were performed using HepG2 ells. As shown in Figure 5, tunimyin slightly but signifintly indued tumor ell poptosis to round 10%-20%. Interestingly, the perentge of poptoti ells mrkedly inresed to round 30% when ombined with si-atf-6. This result suggests tht bloking ATF-6 pthwy under ER stress n further ggrvte liver tumor ell poptosis. As hs lredy been found, meltonin likely ts s n ER stress inhibitor by seletively bloking the ATF-6 pthwy; thus, we next sked whether meltonin lso n ggrvte liver tumor ell poptosis in mnner similr to si-atf-6 fter ER stress tivtion. To further determine whether meltonin influenes ER stressindued poptosis, FACS nlysis nd TUNEL stining were performed in HepG2 ells in vitro. Similr to the results from si-atf-6, nd s shown in Figure 6, tretment with meltonin for 24 h fter pretretment with tunimyin led to n obvious inrese in poptoti tumor ells. The morphologil hnges inditive of poptosis were lso ssessed by TUNEL stining, s shown in Figures 5 nd 6. Tretment with ATF-6-siRNA nd meltonin resulted in drmti inrese in the number of poptoti HepG2 ells. This lso suggested tht meltonin n effetively downregulte ATF-6 nd led to n inresed number of HepG2 ells, whih supports the results of FACS. Further mehnism study, shown in Figure 7, showed tht meltonin or si-atf-6 inhibits COX-2 expression while inresing CHOP nd the Bx/Bl-2 rtio to indue ner ell poptosis. These dt suggest tht COX-2 expression my be diretly involved in the dpttion of humn heptom ells to ER stressindued poptosis. Bsed on the dt of the reltionship between COX-2 nd UPR pthwys, we onluded tht meltonin n obviously knokdown ATF-6 nd redue poptosis under ER stress by downregulting COX-2. CHOP, lso lled GADD153, is one of the primry effetors of ER stress-medited ell poptosis. As shown in Figure 7A, the expression of CHOP ws mrkedly inresed in the presene of meltonin nd ATF-6 sirna. Similrly, the levels of the nti-poptosis ftor, Bl-2, were deresed, nd the levels of propoptosis ftor, Bx, were inresed when ells were exposed to meltonin or ATF-6 sirna. The Bl-2/Bx rtio lso deresed (Figure 7B). These dt indite 990 Februry 14, 2017 Volume 23 Issue 6

6 A Meltonin mol/l 10-7 mol/l 10-5 mol/l 10-3 mol/l ATF-6 IRE-1 PERK β-tin B 3.0 ATF-6 IRE-1 PERK Fold hnges b b 0.0 TM TM + MT (10-9 mol/l) TM + MT (10-7 mol/l) TM + MT (10-5 mol/l) TM + MT (10-3 mol/l) Figure 2 Seletion of the proper meltonin onentrtion. The effets of meltonin (MT) on the three unfolded protein response (UPR) pthwys of HepG2 ells indued by tunimyin (TM). HepG2 ells were exposed to different onentrtions of meltonin (10-9, 10-7, 10-5 nd 10-3 mol/l) for 24 h. A: Equl protein mounts of ell lystes were subjeted to western blot ssy using nti-atf-6, nti-ire-1 nd nti-perk. β-tin in the sme HepG2 ells extrt ws used s n internl referene; B: Optil density reding vlues of speifi proteins re represented s fold differenes reltive to the loding ontrol protein, β-tin. P < 0.01 vs negtive ontrol (NC); b P < 0.05 vs positive ontrol (TM); P < 0.01 vs positive ontrol (TM). ATF-6: Ativting trnsription ftor 6; IRE: Inositol-requiring enzyme; PERK: Protein kinse RNA-like endoplsmi retiulum kinse. tht inhibition of COX-2 with meltonin by knoking down ATF-6 mrna inreses the number of poptoti ells by upregulting the expression of CHOP. In onlusion, the results of this study indite tht under tunimyin-indued ER stress, meltonin n inhibit the expression COX-2 by downregulting one of the UPR pthwys, ATF-6, whih inreses the poptosis of HepG2 ells vi CHOP nd Bl-2/Bx pthwy. DISCUSSION ER stress produed by tumor ells exposed to intrinsi nd externl ftors often uses tumor growth nd resistne to tretment [4,5]. Trgeting ER stress signling in ner is potentil therpeuti method. Our previous studies nd other lb s dt show tht meltonin exerts its ntiner tions through modultion of the ER stress response [8,9,11,12]. However, there is limited informtion foused on the effet of meltonin under ER stress onditions nd the impt of meltonin on the UPR pthwys. In the present study, we obtined evidene suggesting tht meltonin n seletively trget ATF-6 signl, n importnt pthwy of UPR response initited by ER stress. We found tht meltonin indued heptom ell poptosis through inhibiting the ATF-6 pthwy. Our studies demonstrted tht downregultion of ATF-6 ontributes to the inresed suseptibility of liver ner ells to meltonin tretment under ER stress ondition. HCC is one of the most ommon heptobiliry mlignnt tumors, using inresed ner mortlities worldwide. As the fifth most ommon ner in the world, HCC is lwys ssoited with poor prognosis; the 5-yer survivl rte is less thn 17% [2]. Hepti resetion nd liver trnsplnttion offers tretment to only 20% beuse most ptients re dignosed t lte stge [13]. To eliminte the erly stges of HCC, lol bltion, surgil resetion, or liver trnsplnttion ws pplied to the linil tretment of HCC. Ptients who suffer lte stge HCC lwys present with distnt metstsis nd liver dysfuntions, nd the tumor size no longer llows surgil mngement. Thus, there re few effetive tretments for HCC ptients to dte. 991 Februry 14, 2017 Volume 23 Issue 6

7 A si-atf-6 - Mok NC si-atf-6-1 si-atf-6-2 si-atf-6-3 ATF-6 β-tin B si-ire-1 - Mok NC si-ire-1-1 si-ire-1-2 si-ire-1-3 IRE-1 β-tin C si-perk - Mok NC si-perk-1 si-perk-2 si-perk-3 PERK β-tin Figure 3 Seletion of the most effetive sequenes of eh unfolded protein response pthwys sirna (A-C). All of the ndidte sequenes of the three unfolded protein response (UPR) pthwys were exmined by RNA interferene, nd the expression of UPR proteins ws evluted by western blotting to selet the most effetive one to interfere with the UPR pthwy. ATF-6: Ativting trnsription ftor 6; NC: Negtive ontrol; IRE: Inositol-requiring enzyme; PERK: Protein kinse RNA-like endoplsmi retiulum kinses. A mjor obstle tht we hve yet to overome is hemotherpy resistne nd we still need to lrify the underlying mehnisms. Among the omplex mehnisms involved in HCC development nd progress, ER stress indution ssoited with COX-2 is emerging s very importnt ontributor. COX-2 is well-known induible form of ylooxygense onsidered s good drug trget, whih is frequently elevted in vriety kinds of ner tissues inluding HCC [14,15]. There is inresing evidene tht shows COX-2 indution s losely ssoited with ER stress [16,17]. One reserh group observed tht COX-2 nd the eif2α- ATF-4 pthwy of ER stress were indued by hevy metl dmium in both kidney tissues nd ultured ells [16]. This finding indites the ER stress eif2α- ATF-4 pthwy medites COX-2 overexpression. In the present study, we found tht the expression of COX-2 ws more likely to be ssoited with the expression of ATF-6 but not IRE1 nd PERK. An in vitro ell experiment in whih the ATF-6 pthwy ws bloked using si-atf-6 showed signifint repression of COX-2 expression. These dt suggest the existene of lose reltionship between ATF-6 nd COX-2. Thus, it is rtionl to serh for speifi inhibitors of ATF-6 s potentil ndidtes for use s new therpeuti gents for HCC. As HCC is resistnt to systemi hemotherpy, identifition of new intervention or trgeted therpy is urgently needed for ptients [13]. One of the mehnisms by whih liver ner ells gin resistne to hemotherpy is ER stress [3,4,6]. The ER stress response is proess tht n be tivted by number of ellulr stress onditions, suh s hypoxi, nutrient deprivtion, ltertions in glyosyltion sttus, nd disturbnes of lium flux [3,4,6]. Those onditions lwys use imblnes in intrellulr homeostsis. ER stress plys n importnt role in post-trnsltionl modifitions [3,4,6]. The ER responds to stress onditions by tivting rnge of stress response signling pthwys, whih is referred to s the UPR. The UPR is fundmentlly ytoprotetive response, but exessive or prolonged tivtion of the UPR n result in poptosis. In the present study, single tunimyinindued ER stress only slightly indued HepG2 ell poptosis, wheres ombintion with si-atf-6 strongly inresed the perentge of HepG2 ells undergoing poptosis. This finding suggests tht trgeting the ATF-6 pthwy in HepG2 ells enhnes sensitivity to the poptosis induer. Meltonin is minly sereted by the humn pinel glnd, nd it hs been deteted in mny other tissues or s being sereted by other orgns. Meltonin is highly lipophili moleule tht n esily ross ell membrnes to reh subellulr omprtments, inluding mitohondri, where it exists in high onentrtions [18]. Meltonin is ble to prevent oxidtive 992 Februry 14, 2017 Volume 23 Issue 6

8 A si-rna NC si-atf-6 si-ire-1 si-perk COX-2 β-tin B 3.0 Fold hnges NC + si-ire-1 + si-perk C 6 5 Reltive COX-2 mrna levels NC + si-ire-1 + si-perk Figure 4 Reltionship between tivting trnsription ftor 6 nd ylooxygense-2 under endoplsmi retiulum stress. The mrna of the unfolded protein response (UPR) pthwys ws interfered by ATF-6, IRE-1 nd PERK sirna fter pretretment by tunimyin (TM) for 8 h. A: Equl protein mounts of ell lystes were subjeted to western blot ssy using nti-cox-2. β-tin in the sme HepG2 ell extrt ws used s n internl referene. B: Optil density reding vlues of speifi proteins re represented s fold-differenes reltive to the loding ontrol protein, β-tin. P < 0.01 vs the negtive ontrol. RNA ws hrvested nd gene expression exmined by qrt-pcr in the sme ondition bove. C: The qrt-pcr fold-hnges were normlized using the expression of housekeeping gene (GAPDH) nd vs those obtined from untreted HepG2 ells. P < 0.01 vs the negtive ontrol; P < 0.01 vs the untreted HepG2 ells. ATF-6: Ativting trnsription ftor 6; NC: Negtive ontrol; COX-2: Cylooxygense-2; IRE: Inositol-requiring enzyme; PERK: Protein kinse RNA-like endoplsmi retiulum kinse. stress through both its free rdil svenging effet nd by diretly inresing ntioxidnt tivity [19-25], nd different studies hve demonstrted its protetive role ginst oxidtive dmge indued by drugs, toxins, nd different diseses [26]. In ddition, meltonin lso ts upon omplex funtions through speifi nuler nd plsm membrne reeptors [27,28]. Meltonin MT1 nd MT2 reeptors re G protein oupled reeptors expressed in vrious prts of the entrl nervous system nd in peripherl orgns, whih medite intrellulr effets depending on the hnges in intrellulr yli nuleotides (AMP, GMP) nd lium levels, tivtion of ertin protein kinse C subtypes, intrellulr loliztion of steroid hormone reeptors nd regultion of G protein signling proteins. Altertions in meltonin reeptor expression nd the following bnorml signling pthwy, s well s hnges in endogenous meltonin prodution, ontribute to the pthophysiology of vrious diseses, inluding sleep disorders, depression nd Alzheimer s disese [27,28]. Interestingly, experimentl nd linil studies 993 Februry 14, 2017 Volume 23 Issue 6

9 A 0.365% A1 3.74% 2.23% A2 8.19% B PI 94.5% 1.56% A3 1.36% 8.59% 87.2% 5.02% A4 2.38% 20.0% Apoptosis rtio (%) NC b, 86.3% 3.56% 64.7% 10.2% C C1 Annexin V C2 D μm 100 μm C3 C4 Perent of poptoti ells (%) b b, 0 + NC 100 μm 100 μm Figure 5 Effets of tivting trnsription ftor 6 sirna on ell poptosis in HepG2 ells under endoplsmi retiulum stress. A: HepG2 ells were trnsfeted with ATF-6 sirna for 24 h fter pretretment by tunimyin (TM) for 8 h. Apoptoti ells were determined by FACS, nd the dt re expressed s the men ± SD. A1, HepG2 ells; A2, HepG2 ells treted by TM only; A3, HepG2 ells treted by ombintion of TM nd ATF-6 sirna negtive ontrol; A4, HepG2 ells treted by ATF-6 sirna nd meltonin; B: Dt re presented s the men ± SD for the independent experiments ( P < 0.05 vs untreted HepG2 ells; b P < 0.01 vs untreted HepG2 ells; P < 0.01 vs HepG2 ells treted with TM nd ATF-6 sirna negtive ontrol); C: Cell morphology nd perentge of poptoti ells ws exmined by TUNEL stining. C1, HepG2 ells; C2, HepG2 ells treted by TM only; C3, HepG2 ells treted by ombintion of TM nd ATF-6 sirna negtive ontrol; C4, HepG2 ells treted by ATF-6 sirna nd meltonin; D: Dt re presented s the men ± SD for the independent experiments ( b P < 0.01 vs untreted HepG2 ells; P < 0.01 vs HepG2 ells treted with TM nd ATF-6 sirna negtive ontrol). ATF-6: Ativting trnsription ftor 6; NC: Negtive ontrol. reommend n inrese in the wreness of meltonin s therpeuti gent in ners inluding gstrointestinl trt ner [10,29,30]. Our previous studies nd other lbs findings suggest tht meltonin exerts its ntiner tion through suppressing COX-2 nd ttenuting ER stress-indued drug resistne [11,12,31,32]. A reent study lso showed tht meltonin inhibits the expression of prongiogeni proteins HIF-1α nd VEGF in onditions of normoxi nd hypoxi using the HepG2 ell line [33]. ER stress indued by heptitis B virus X (HBx) protein enhnes COX-2 expression vi tivting trnsription ftor 4 (ATF-4). Further experiment showed tht ATF-4 binding to the COX-2 promoter plys ritil role in HBx-medited COX-2 indution [34]. In ddition, meltonin enhnes ntitumor funtion through upregultion of the pro-poptoti protein BimBim expression nd downregultion of COX-2 expression in tunimyin-treted brest rinom MDA-MB-231 ells [35]. 994 Februry 14, 2017 Volume 23 Issue 6

10 A PI 0.740% 94.3% A1 4.09% 0.865% 2.18% 85.7% A2 9.64% 2.50% B Apoptosis rtio (%) b, b, 4.34% A3 37.7% 6.43% A4 19.4% 0 + MT (10-5 mol/l) 32.4% 25.5% 61.7% 12.4% Annexin V C C1 C2 100 μm 100 μm D Perent of poptoti ells (%) b, b, C3 C4 0 + MT (10-5 mol/l) 100 μm 100 μm Figure 6 Comprison between meltonin nd tivting trnsription ftor 6 sirna trnsfetion on ell poptosis in HepG2 ells under endoplsmi retiulum stress. A: HepG2 ells were exposed to meltonin (10-5 mmol/l) for 24 h fter pretretment by tunimyin (TM) for 8 h or were trnsfeted with ATF-6 sirna for 6 h fter pretretment by tunimyin TM for 8 h. Apoptoti ells were determined by FACS, nd the dt re expressed s the men ± SD. A1, HepG2 ells; A2, HepG2 ells treted by TM only; A3, HepG2 ells treted by ombintion of TM nd meltonin (10-5 mmol/l); A4, HepG2 ells treted by ATF-6 sirna nd meltonin; B: Dt re presented s the men ± SD for the independent experiments ( P < 0.05 vs untreted HepG2 ells; b P < 0.01 vs untreted HepG2 ells; P < 0.01 HepG2 ells treted by TM nd meltonin vs HepG2 ells treted with TM nd ATF-6 sirna negtive ontrol); C: Cell morphology nd perentge of poptoti ells were exmined by TUNEL stining. C1, HepG2 ells; C2, HepG2 ells treted by TM only; C3, HepG2 ells treted by ombintion of TM nd meltonin (10-5 mmol/l); C4, HepG2 ells treted by ATF-6 sirna nd meltonin; D: Dt re presented s the men ± SD for the independent experiments ( P < 0.05 vs untreted HepG2 ells; b P < 0.01 vs untreted HepG2 ells; P < 0.01 HepG2 ells treted by TM nd meltonin vs HepG2 ells treted with TM nd ATF-6 sirna negtive ontrol). ATF-6: Ativting trnsription ftor 6; MT: Meltonin. The present study further explored the new mehnism of speifi effet of meltonin on ATF-6, one of the UPR responses. We found tht meltonin seletively inhibited the ATF-6 expression (Figure 1). As it is well-known tht meltonin hs n inhibitory effet on COX-2 tivity [36], it is esy to speulte nd understnd tht si-atf-6 hs similr COX-2 suppression s meltonin (Figure 4). Furthermore, we onfirmed tht downregultion of ATF-6 by meltonin ontributes to the inresed suseptibility of liver ner ells to meltonin tretment under ER stress ondition (Figure 6). Drmtilly inresed CHOP level led to suppressed COX-2 nd deresed Bl-2/Bx rtio by meltonin, nd ATF-6 sirna ontributed to the enhned HepG2 ell poptosis under ER stress stimultion (Figure 7). These findings indite tht meltonin, s seletive ATF-6 inhibitor, n sensitize humn heptom ells to ER stress-indued poptosis. In summry, our study provides the new mehnism by whih meltonin downregultes COX Februry 14, 2017 Volume 23 Issue 6

11 A MT (10-5 mol/l) si-atf-6 b d COX-2 CHOP Bl-2 BAX β-tin B 6 COX-2 CHOP 5 4 Bl-2/Bx Fold hnges 4 2 b Fold hnges 3 2 b MT (10-5 mol/l) 0 + MT (10-5 mol/l) Figure 7 Pthwy by whih meltonin sensitized HepG2 to endoplsmi retiulum stress-indued poptosis. A:, HepG2 ells; b, HepG2 ells treted by tunimyin (TM) only;, HepG2 ells treted by TM, meltonin (10-5 mmol/l); d: HepG2 ells treted by TM nd ATF-6 sirna. Equl protein mounts of ell lystes were subjeted to western blot ssy using nti-cox-2, nti-chop, nti-bl-2, nd nti-bx. β-tin in the sme HepG2 ell extrt ws used s n internl referene; B: Optil density reding vlues of speifi proteins re represented s fold-differenes reltive to the loding ontrol protein, β-tin. P < 0.01, CHOP vs the Bl-2 nd Bx expression, b P < 0.01, vs the untreted HepG2 ells. ATF-6: Ativting trnsription ftor 6; COX-2: Cylooxygense-2; CHOP: CCAATenhner-binding protein homologous protein; MT: Meltonin. expression nd sensitizes poptosis by seletively trgeting ATF-6 in humn HCC ells under ER stress. Our results rise the possibility tht meltonin my be promising pproh in trgeting ER stress-indued poptosis s therpeuti strtegy for the tretment of HCC nd other ners. We lso identified tht of the three UPR pthwys, ATF-6 ws positively ssoited with COX-2 in HCC ptient smples. Therefore, if there re ny gents tht n knokdown ATF-6, like meltonin, they n be used to tret HCC. However, we still need to investigte whether this effet of meltonin in ER stress-indued tumor poptosis in vitro will lso work well in vivo. Beuse of the low toxiity nd well-doumented onostti effets of meltonin, we believe meltonin hs promising future in the tretment of HCC. COMMENTS Bkground Heptoellulr rinom (HCC) is frequently dignosed ner in Chin nd hs high lethlity rte. Yet, there re few effetive hemotherpy gents for this mlignnt tumor. One of the mjor resons of untretble HCC is tht the liver ner ell hs greter tolerne towrds number of ellulr stress onditions, espeilly endoplsmi retiulum (ER) stress. The published works by other uthors hve shown tht meltonin n sensitize the humn HCC ell to ER stress-indued poptosis nd tht meltonin ttenutes ER stressindued resistne to doxorubiin. However, the preise mehnisms involved in the ritil unfolded protein response (UPR) pthwy modified by meltonin still need to be investigted. Reserh frontiers To the best knowledge of the uthors, this is the first study to identify meltonin s seletive ATF-6 bloker, thereby inhibiting COX-2 nd leding to enhned ell poptosis in liver ner. 996 Februry 14, 2017 Volume 23 Issue 6

12 Innovtions nd brekthroughs This is the first study investigting the preise mehnisms of the ritil UPR pthwys modified by meltonin. Applitions The promising findings presented in the urrent report suggest tht meltonin, s novel seletive ATF-6 inhibitor, n sensitize humn heptom ells to ER stress induing poptosis, whih my be onsidered s therpeuti strtegy for the tretment of HCC nd other ners. Terminology Meltonin plys importnt roles in humn physiologil nd phrmologil funtions, suh s irdin rhythms nd ntioxidnts. Interestingly, meltonin exerts ntiner effets through interply with ER stress. The published studies show tht meltonin sensitizes the humn HCC ell to ER stress-indued poptosis. Furthermore, meltonin lso ttenutes ER stress-indued resistne to doxorubiin through reversing tunimyin-indued ER stress. In the urrent study, the uthors first demonstrted tht meltonin seletively bloks ATF-6 nd then inhibits COX-2 expression, leding to enhned liver ner ell poptosis. In linil HCC ptients, the lose reltionship between ATF-6 nd COX-2 ws further onfirmed. The results presented rise the possibility tht meltonin my be used in the tretment of HCC nd other ners. Peer-review In generl, the enlosed set of dt re very interesting. The rtile will be interesting for reders of this journl. REFERENCES 1 Chen W, Zheng R, Bde PD, Zhng S, Zeng H, Bry F, Jeml A, Yu XQ, He J. Cner sttistis in Chin, CA Cner J Clin 2016; 66: [PMID: DOI: /.21338] 2 Siegel RL, Miller KD, Jeml A. Cner sttistis, CA Cner J Clin 2016; 66: 7-30 [PMID: DOI: /.21332] 3 Binet F, Spieh P. ER Stress nd Angiogenesis. Cell Metb 2015; 22: [PMID: DOI: /j.met ] 4 Ydv RK, Che SW, Kim HR, Che HJ. Endoplsmi retiulum stress nd ner. J Cner Prev 2014; 19: [PMID: DOI: /JCP ] 5 Chevet E, Hetz C, Smli A. Endoplsmi retiulum stresstivted ell reprogrmming in onogenesis. Cner Disov 2015; 5: [PMID: DOI: / CD ] 6 Hirmtsu N, Ching WC, Kurt TD, Sigurdson CJ, Lin JH. Multiple Mehnisms of Unfolded Protein Response-Indued Cell Deth. Am J Pthol 2015; 185: [PMID: DOI: /j.jpth ] 7 Kto H, Nishitoh H. Stress responses from the endoplsmi retiulum in ner. Front Onol 2015; 5: 93 [PMID: DOI: /fon ] 8 Fernández A, Ordóñez R, Reiter RJ, González-Gllego J, Muriz JL. Meltonin nd endoplsmi retiulum stress: reltion to utophgy nd poptosis. J Pinel Res 2015; 59: [PMID: DOI: /jpi.12264] 9 Wu SM, Lin WY, Shen CC, Pn HC, Keh-Bin W, Chen YC, Jn YJ, Li DW, Tng SC, Tien HR, Chiu CS, Tsi TC, Li YL, Sheu ML. Meltonin set out to ER stress signling thwrts epithelil mesenhyml trnsition nd peritonel dissemintion vi lpinmedited C/EBPβ nd NFκB levge. J Pinel Res 2016; 60: [PMID: DOI: /jpi.12295] 10 Xin Z, Jing S, Jing P, Yn X, Fn C, Di S, Wu G, Yng Y, Reiter RJ, Ji G. Meltonin s tretment for gstrointestinl ner: review. J Pinel Res 2015; 58: [PMID: DOI: /jpi.12227] 11 Zh L, Fn L, Sun G, Wng H, M T, Zhong F, Wei W. Meltonin sensitizes humn heptom ells to endoplsmi retiulum stressindued poptosis. J Pinel Res 2012; 52: [PMID: DOI: /j X x] 12 Fn L, Sun G, M T, Zhong F, Lei Y, Li X, Wei W. Meltonin reverses tunimyin-indued endoplsmi retiulum stress in humn heptoellulr rinom ells nd improves ytotoxi response to doxorubiin by inresing CHOP nd deresing survivin. J Pinel Res 2013; 55: [PMID: DOI: /jpi.12061] 13 Bruix J, Reig M, Shermn M. Evidene-Bsed Dignosis, Stging, nd Tretment of Ptients With Heptoellulr Crinom. Gstroenterology 2016; 150: [PMID: DOI: /j.gstro ] 14 Breinig M, Shirmher P, Kern MA. Cylooxygense-2 (COX- 2)-- therpeuti trget in liver ner? Curr Phrm Des 2007; 13: [PMID: ] 15 Misr S, Shrm K. COX-2 signling nd ner: new plyers in old ren. Curr Drug Trgets 2014; 15: [PMID: ] 16 Luo B, Lin Y, Jing S, Hung L, Yo H, Zhung Q, Zho R, Liu H, He C, Lin Z. Endoplsmi retiulum stress eif2α-atf4 pthwymedited ylooxygense-2 indution regultes dmium-indued utophgy in kidney. Cell Deth Dis 2016; 7: e2251 [PMID: DOI: /ddis ] 17 Hung JH, Su IJ, Lei HY, Wng HC, Lin WC, Chng WT, Hung W, Chng WC, Chng YS, Chen CC, Li MD. Endoplsmi retiulum stress stimultes the expression of ylooxygense-2 through tivtion of NF-kppB nd pp38 mitogen-tivted protein kinse. J Biol Chem 2004; 279: [PMID: DOI: /jb.M ] 18 Prdies G, Petrosillo G, Prdies V, Reiter RJ, Ruggiero FM. Meltonin, rdiolipin nd mitohondril bioenergetis in helth nd disese. J Pinel Res 2010; 48: [PMID: DOI: /j X x] 19 Glno A, Tn DX, Reiter RJ. Meltonin s nturl lly ginst oxidtive stress: physiohemil exmintion. J Pinel Res 2011; 51: 1-16 [PMID: DOI: /j X x] 20 Tomás-Zpio C, Coto-Montes A.A proposed mehnism to explin the stimultory effet of meltonin on ntioxidtive enzymes. J Pinel Res 2005; 39: [PMID: DOI: /j X x] 21 Kleszzyński K, Zillikens D, Fisher TW. Meltonin enhnes mitohondril ATP synthesis, redues retive oxygen speies formtion, nd medites trnslotion of the nuler erythroid 2-relted ftor 2 resulting in tivtion of phse-2 ntioxidnt enzymes (γ-gcs, HO-1, NQO1) in ultrviolet rdition-treted norml humn epiderml kertinoytes (NHEK). J Pinel Res 2016; 61: [PMID: DOI: /jpi.12338] 22 Kleszzyński K, Tukj S, Kruse N, Zillikens D, Fisher TW. Meltonin prevents ultrviolet rdition-indued ltertions in plsm membrne potentil nd intrellulr ph in humn kertinoytes. J Pinel Res 2013; 54: [PMID: DOI: /j X x] 23 Fisher TW, Kleszzyński K, Hrdkop LH, Kruse N, Zillikens D. Meltonin enhnes ntioxidtive enzyme gene expression (CAT, GPx, SOD), prevents their UVR-indued depletion, nd protets ginst the formtion of DNA dmge (8-hydroxy-2 - deoxygunosine) in ex vivo humn skin. J Pinel Res 2013; 54: [PMID: DOI: /jpi.12018] 24 Fisher TW, Sholz G, Knöll B, Hipler UC, Elsner P. Meltonin suppresses retive oxygen speies indued by UV irrdition in leukoytes. J Pinel Res 2004; 37: [PMID: DOI: /j X x] 25 Slominski A, Pisrhik A, Zbytek B, Tobin DJ, Kuser S, Wortsmn J. Funtionl tivity of serotoninergi nd meltoninergi systems expressed in the skin. J Cell Physiol 2003; 196: [PMID: DOI: /jp.10287] 26 Grí JJ, López-Pingrrón L, Almeid-Souz P, Tres A, Esudero P, Grí-Gil FA, Tn DX, Reiter RJ, Rmírez JM, Bernl-Pérez M. Protetive effets of meltonin in reduing oxidtive stress nd in preserving the fluidity of biologil membrnes: review. J Pinel Res 2014; 56: [PMID: DOI: /jpi.12128] 27 Pndi-Peruml SR, Trkht I, Srinivsn V, Spene DW, Mestroni GJ, Zispel N, Crdinli DP. Physiologil effets of meltonin: 997 Februry 14, 2017 Volume 23 Issue 6

13 role of meltonin reeptors nd signl trnsdution pthwys. Prog Neurobiol 2008; 85: [PMID: DOI: /j. pneurobio ] 28 Emet M, Ozn H, Ozel L, Yyl M, Hlii Z, Himuftuoglu A. A Review of Meltonin, Its Reeptors nd Drugs. Eursin J Med 2016; 48: [PMID: DOI: /eursinjmed ] 29 Mills E, Wu P, Seely D, Guytt G. Meltonin in the tretment of ner: systemti review of rndomized ontrolled trils nd met-nlysis. J Pinel Res 2005; 39: [PMID: DOI: /j X x] 30 Reiter RJ, Rosles-Corrl SA, Mnhester LC, Liu X, Tn DX. Meltonin in the biliry trt nd liver: helth implitions. Curr Phrm Des 2014; 20: [PMID: ] 31 Crbjo-Pesdor S, Steinmetz C, Kshyp A, Lorenz S, Muriz JL, Heise M, Glle PR, González-Gllego J, Strnd S. Meltonin indues trnsriptionl regultion of Bim by FoxO3 in HepG2 ells. Br J Cner 2013; 108: [PMID: DOI: /bj ] 32 Crbjo-Pesdor S, Grí-Plomo A, Mrtín-Renedo J, Piv M, González-Gllego J, Muriz JL. Meltonin modultion of intrellulr signling pthwys in heptorinom HepG2 ell line: role of the MT1 reeptor. J Pinel Res 2011; 51: [PMID: DOI: /j X x] 33 Colombo J, Miel JM, Ferreir LC, DA Silv RF, Zuri DA. Effets of meltonin on HIF-1α nd VEGF expression nd on the invsive properties of heptorinom ells. Onol Lett 2016; 12: [PMID: DOI: /ol ] 34 Cho HK, Cheong KJ, Kim HY, Cheong J. Endoplsmi retiulum stress indued by heptitis B virus X protein enhnes ylo-oxygense 2 expression vi tivting trnsription ftor 4. Biohem J 2011; 435: [PMID: DOI: /BJ ] 35 Woo SM, Min KJ, Kwon TK. Meltonin-medited Bim up-regultion nd ylooxygense-2 (COX-2) down-regultion enhnes tunimyin-indued poptosis in MDA-MB-231 ells. J Pinel Res 2015; 58: [PMID: DOI: /jpi.12217] 36 Apriio-Soto M, Alrón-de-l-Lstr C, Cárdeno A, Sánhez- Fidlgo S, Snhez-Hidlgo M. Meltonin modultes mirosoml PGE synthse 1 nd NF-E2-relted ftor-2-regulted ntioxidnt enzyme expression in LPS-indued murine peritonel mrophges. Br J Phrmol 2014; 171: [PMID: DOI: /bph.12428] P- Reviewer: Kleszzynski K, Zvodnik IB S- Editor: Yu J L- Editor: Filipodi E- Editor: Liu WX 998 Februry 14, 2017 Volume 23 Issue 6

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