World Journal of Gastroenterology

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1 ISSN (print) ISSN (online) World Journal of Gastroenterology World J Gastroenterol 2017 Novemer 14; 23(42): Pulished y Baishideng Pulishing Group In

2 S Contents Weekly Volume 23 Numer 42 Novemer 14, 2017 EDITORIAL 7495 Current and emerging pharmaologial therapy for non-aloholi fatty liver disease Eshraghian A REVIEW 7505 Intestinal epithelium, intraepithelial lymphoytes and the gut miroiota - Key players in the pathogenesis of elia disease Cukrowska B, Sowińska A, Bierła JB, Czarnowska E, Ryak A, Grzyowska-Chleowzyk U ORIGINAL ARTICLE Basi Study 7519 Pregnane X reeptor and onstitutive androstane reeptor modulate differently CYP3A-mediated metaolism in early- and late-stage holestasis Gaia D, Dalla Pozza A, Alertoni L, Lazzari R, Zigiotto G, Carrara M, Baldo V, Baldovin T, Floreani A, De Martin S 7531 Indution of preoious intestinal maturation in T-ell defiient athymi neonatal rats Arévalo Sureda E, Gidlund C, Weström B, Prykhodko O 7541 Nulear heat shok protein 110 expression is assoiated with poor prognosis and hyperthermohemotherapy resistane in gastri aner patients with peritoneal metastasis Kimura A, Ogata K, Altan B, Yokoori T, Mohiki E, Yanai M, Kogure N, Yanoma T, Suzuki M, Bai T, Kuwano H 7551 Comined treatment of panreati aner xenograft with Y-ITGA6B4-mediated radioimmunotherapy and PI3K/mTOR inhiitor Aung W, Tsuji AB, Sudo H, Sugyo A, Ukai Y, Kouda K, Kurosawa Y, Furukawa T, Saga T, Higashi T 7563 Effets of Hemp seed soft apsule on oloni ion transport in rats Lu XF, Jia MD, Zhang SS, Zhao LQ 7572 Novel D-galatosamine-indued ynomolgus monkey model of aute liver failure Feng L, Cai L, He GL, Weng J, Li Y, Pan MX, Jiang ZS, Peng Q, Gao Y 7584 Diversity of aterial latase genes in intestinal ontents of mie with antiiotis-indued diarrhea Long CX, He L, Guo YF, Liu YW, Xiao NQ, Tan ZJ WJG Novemer 14, 2017 Volume 23 Issue 42

3 Contents World Journal of Gastroenterology Volume 23 Numer 42 Novemer 14, Potential rat model of anxiety-like gastri hypersensitivity indued y sequential stress Jing FC, Zhang J, Feng C, Nian YY, Wang JH, Hu H, Yang BD, Sun XM, Zheng JY, Yin XR Retrospetive Cohort Study 7609 Post-olonosopy oloretal aner rate in the era of high-definition olonosopy Iwatate M, Kitagawa T, Katayama Y, Tokutomi N, Ban S, Hattori S, Hasuike N, Sano W, Sano Y, Tamano M 7618 Right- and left-sided oloretal aners respond differently to traditional Chinese mediine Liu SS, Shi Q, Li HJ, Yang W, Han SS, Zong SQ, Li W, Hou FG Oservational Study 7626 Hepatitis B virus outreah to immigrant population in Greater Boston Area: Key to improving hepatitis B knowledge ng Djoufak R, Cheon SSY, Mohamed A, Faye F, Diouf K, Colvin R, Morrill J, Duffy-Keane AM, Perumalswami P, Jourdain G, Fuso DN 7635 Preditors of healthare-seeking ehavior among Chinese patients with irritale owel syndrome Fan W, Xu D, Chang M, Zhu L, Fei G, Li X, Fang X SYSTEMATIC REVIEWS 7644 Vainations in immunosuppressive-dependent pediatri inflammatory owel disease Nguyen HT, Minar P, Jakson K, Fulkerson PC WJG II Novemer 14, 2017 Volume 23 Issue 42

4 Contents World Journal of Gastroenterology Volume 23 Numer 42 Novemer 14, 2017 ABOUT COVER Editorial oard memer of World Journal of Gastroenterology, Zhao-Shan Niu, MD, Assoiate Professor, Laoratory of Miromorphology, Shool of Basi Mediine, Medial Department of Qingdao University, Qingdao , Shandong Provine, China AIMS AND SCOPE World Journal of Gastroenterology (World J Gastroenterol, WJG, print ISSN , online ISSN , DOI: ) is a peer-reviewed open aess journal. WJG was estalished on Otoer 1, It is pulished weekly on the 7 th, 14 th, 21 st, and 28 th eah month. The WJG Editorial Board onsists of 1375 experts in gastroenterology and hepatology from 68 ountries. The primary task of WJG is to rapidly pulish high-quality original artiles, reviews, and ommentaries in the fields of gastroenterology, hepatology, gastrointestinal endosopy, gastrointestinal surgery, hepatoiliary surgery, gastrointestinal onology, gastrointestinal radiation onology, gastrointestinal imaging, gastrointestinal interventional therapy, gastrointestinal infetious diseases, gastrointestinal pharmaology, gastrointestinal pathophysiology, gastrointestinal pathology, evidene-ased mediine in gastroenterology, panreatology, gastrointestinal laoratory mediine, gastrointestinal moleular iology, gastrointestinal immunology, gastrointestinal miroiology, gastrointestinal genetis, gastrointestinal translational mediine, gastrointestinal diagnostis, and gastrointestinal therapeutis. WJG is dediated to eome an influential and prestigious journal in gastroenterology and hepatology, to promote the development of aove disiplines, and to improve the diagnosti and therapeuti skill and expertise of liniians. INDEXING/ABSTRACTING World Journal of Gastroenterology (WJG) is now indexed in Current Contents /Clinial Mediine, Siene Citation Index Expanded (also known as SiSearh ), Journal Citation Reports, Index Medius, MEDLINE, PuMed, PuMed Central and Diretory of Open Aess Journals. The 2017 edition of Journal Citation Reports ites the 2016 impat fator for WJG as (5-year impat fator: 3.176), ranking WJG as 29 th among 79 journals in gastroenterology and hepatology (quartile in ategory Q2). FLYLEAF I-IX Editorial Board EDITORS FOR THIS ISSUE Responsile Assistant Editor: Xiang Li Responsile Eletroni Editor: Yu-Jie Ma Proofing Editor-in-Chief: Lian-Sheng Ma Responsile Siene Editor: Ze-Mao Gong Proofing Editorial Offie Diretor: Jin-Lei Wang NAME OF JOURNAL World Journal of Gastroenterology ISSN ISSN (print) ISSN (online) LAUNCH DATE Otoer 1, 1995 FREQUENCY Weekly EDITORS-IN-CHIEF Damian Garia-Olmo, MD, PhD, Dotor, Professor, Surgeon, Department of Surgery, Universidad Autonoma de Madrid; Department of General Surgery, Fundaion Jimenez Diaz University Hospital, Madrid 28040, Spain Stephen C Strom, PhD, Professor, Department of Laoratory Mediine, Division of Pathology, Karolinska Institutet, Stokholm , Sweden Andrzej S Tarnawski, MD, PhD, DS (Med), Professor of Mediine, Chief Gastroenterology, VA Long Beah Health Care System, University of California, Irvine, CA, 51 E. Seventh Str., Long Beah, CA 822, United States EDITORIAL BOARD MEMBERS All editorial oard memers resoures online at EDITORIAL OFFICE Jin-Lei Wang, Diretor Yuan Qi, Vie Diretor Ze-Mao Gong, Vie Diretor World Journal of Gastroenterology Baishideng Pulishing Group In 71 Stoneridge Drive, Suite 501, Pleasanton, CA 94588, USA Telephone: Fax: editorialoffie@wjgnet.om Help Desk: PUBLISHER Baishideng Pulishing Group In 71 Stoneridge Drive, Suite 501, Pleasanton, CA 94588, USA Telephone: Fax: pgoffie@wjgnet.om Help Desk: PUBLICATION DATE Novemer 14, 2017 COPYRIGHT 2017 Baishideng Pulishing Group In. Artiles pulished y this Open-Aess journal are distriuted under the terms of the Creative Commons Attriution Nonommerial Liense, whih permits use, distriution, and reprodution in any medium, provided the original work is properly ited, the use is non ommerial and is otherwise in ompliane with the liense. SPECIAL STATEMENT All artiles pulished in journals owned y the Baishideng Pulishing Group (BPG) represent the views and opinions of their authors, and not the views, opinions or poliies of the BPG, exept where otherwise expliitly indiated. INSTRUCTIONS TO AUTHORS Full instrutions are availale online at wjgnet.om/pg/gerinfo/204 ONLINE SUBMISSION WJG III Novemer 14, 2017 Volume 23 Issue 42

5 Sumit a Manusript: DOI: /wjg.v23.i World J Gastroenterol 2017 Novemer 14; 23(42): ISSN (print) ISSN (online) Basi Study ORIGINAL ARTICLE Comined treatment of panreati aner xenograft with Y-ITGA6B4-mediated radioimmunotherapy and PI3K/ mtor inhiitor Winn Aung, Atsushi B Tsuji, Hitomi Sudo, Aya Sugyo, Yoshinori Ukai, Katsushi Kouda, Yoshikazu Kurosawa, Takako Furukawa, Tsuneo Saga, Tatsuya Higashi Winn Aung, Atsushi B Tsuji, Hitomi Sudo, Aya Sugyo, Tatsuya Higashi, Department of Moleular Imaging and Theranostis, National Institute of Radiologial Sienes, National Institutes for Quantum and Radiologial Siene and Tehnology (QST-NIRS), Chia , Japan Yoshinori Ukai, Katsushi Kouda, Perseus Proteomis In., Tokyo , Japan Yoshikazu Kurosawa, Innovation Center for Advaned Mediine, Fujita Health University, Toyoake, Aihi , Japan Takako Furukawa, Department of Radiologial and Medial Laoratory Sienes, Nagoya University Graduate Shool of Mediine, Nagoya , Japan Tsuneo Saga, Department of Diagnosti Radiology, Kyoto University Hospital, Kyoto , Japan ORCID Numer: Winn Aung ( ); Atsushi B Tsuji ( X); Hitomi Sudo ( ); Aya Sugyo ( ); Yoshinori Ukai ( ); Katsushi Kouda ( ); Yoshikazu Kurosawa ( ); Takako Furukawa ( ); Tsuneo Saga ( ); Tatsuya Higashi ( ). Author ontriutions: Aung W designed the researh, performed the majority of experiments, analyzed the data and wrote the manusript; Ukai K, Kouda K and Kurozawa Y provided the anti-integrin α6β4 antiody; Sudo H perfomed the antiody radiolaeling; Sugyo A partiipated in the animal experiments; Tsuji AB, Higashi T, Furukawa T and Tsuneo S oordinated the researh and helped for the manusript preparation; all authors revised and endorsed the final draft. Supported y (partially) a Grant-in-Aid for Sientifi Researh (C) from the Ministry of Eduation, Culture, Sports, Siene and Tehnology, Japan, No. 17K10460 to Aung W. Institutional animal are and use ommittee statement: All proedures involving animals were reviewed and approved y the Institutional Animal Care and Use Committee of National Institute of Radiologial Sienes. Conflit-of-interest statement: The authors delare no potential onflits of interest relevant to this artile. Data sharing statement: All relevant data were presented in the manusript. Further information is availale from the orresponding author at winn.aung@qst.go.jp. Open-Aess: This artile is an open-aess artile whih was seleted y an in-house editor and fully peer-reviewed y external reviewers. It is distriuted in aordane with the Creative Commons Attriution Non Commerial (CC BY-NC 4.0) liense, whih permits others to distriute, remix, adapt, uild upon this work non-ommerially, and liense their derivative works on different terms, provided the original work is properly ited and the use is non-ommerial. See: lienses/y-n/4.0/ Manusript soure: Unsoliited manusript Correspondene to: Winn Aung, MBBS, PhD, Senior Researher, Department of Moleular Imaging and Theranostis, National Institute of Radiologial Sienes, National Institutes for Quantum and Radiologial Siene and Tehnology, Anagawa, Inage-ku, Chia , Japan. winn.aung@qst.go.jp Telephone: Fax: Reeived: June 23, 2017 Peer-review started: June 26, 2017 First deision: july 13, 2017 Revised: july 31, 2017 Aepted: Septemer 9, 2017 WJG Novemer 14, 2017 Volume 23 Issue 42

6 Aung W et al. Radioimmunotherapy and BEZ235 for panreati aner Artile in press: Septemer 9, 2017 Pulished online: Novemer 14, 2017 Astrat AIM To investigate the therapeuti effet of omined integrin α6β4-targeted radioimmunotherapy (RIT) and PI3K/mTOR inhiitor BEZ235 in a panreati aner model. METHODS Phosphorylation of Akt, mtor, the downstream effetors eukaryoti initiation fator 4E inding protein 1 (4EBP1) and S6 riosomal protein (S6) were evaluated in BxPC-3 human panreati aner ells treated with Yttrium- ( Y) laeled anti-integrin α6β4 antiody (ITGA6B4) and BEZ235 y western lotting. The ytotoxi effet of BEZ235 was investigated using a olony formation assay. Therapeuti effiay enhanement y oral BEZ235 administration was assessed using mie earing BxPC-3 xenograft tumors. Tumor volume measurements and immunohistohemial analyses (ell proliferation marker Ki-67, DNA damage marker p-h2ax and p-4ebp1 staining) of tumors were performed for evaluation of omined treatment with Y-ITGA6B4 plus BEZ235, or eah arm alone. Key words: Radioimmunotherapy; Panreati aner; anti-integrin α6β4 antiody; Yttrium-; NVP-BEZ235 The Author(s) Pulished y Baishideng Pulishing Group In. All rights reserved. Core tip: We examined whether the therapeuti effet of Y-laeled anti-α6β4 integrin antiody (ITGA6B4)- mediated radioimmunotherapy (RIT) is improved y dual PI3K/mTOR inhiitor BEZ235 in the treatment of panreati aner xenograft. There is no report aout the omined therapeuti effets of RIT and BEZ235 in aner treatment, though BEZ235 has een tested for its antianer and potential radiosensitizing effet. Our studies (in vitro/in vivo ) and results suggest for the first time that it is possile to improve the therapeuti effiay y omining Y-ITGA6B4-RIT and BEZ235 and this omination an e a potential enouraging treatment modality in the future. Aung W, Tsuji AB, Sudo H, Sugyo A, Ukai Y, Kouda K, Kurosawa Y, Furukawa T, Saga T, Higashi T. Comined treatment of panreati aner xenograft with Y-ITGA6B4- mediated radioimmunotherapy and PI3K/mTOR inhiitor. World J Gastroenterol 2017; 23(42): Availale from: URL: DOI: RESULTS We found that phosphorylation of Akt (p-akt), 4EBP1 (p-4ebp1) and S6 (p-s6) was inhiited y BEZ235. Colony formation in BxPC-3 ells was additively suppressed y the omination of Y-ITGA6B4 and BEZ235. Pretreatment with BEZ235 efore Y-ITGA6B4 exposure resulted in signifiant redution of ells plating effiieny (PE) (0.54 ± 0.11 vs 2.81 ± 0.14 with 185 kbq/ml Y-ITGA6B4 exposure, P < 0.01; 0.39 ± 0.08 vs 1.88 ± 0.09 with 370 kbq/ml Y-ITGA6B4 exposure, P < 0.01) when ells per dish were plated. In vivo, the omined treatment with Y-ITGA6B4 plus BEZ235 enhaned the inhiition of tumor growth and statistially signifiant differenes of relative tumor volume were oserved for 27 d after the treatment start date when ompared with the Y-ITGA6B4 single injetion treatment (1.03 ± 0.38 vs 1.5 ± 0.15 at Day 27, P < 0.05), and for 41 d when ompared with the BEZ235 treatment alone (1.8 ± 0.7 vs 3.14 ± 1.19 at Day 41, P < 0.05). Tumors from treatment groups showed redution in volumes, dereased Ki-67-positive ells, inreased p-h2ax-positive ells and dereased p-4ebp1 expression. CONCLUSION The therapeuti effiay of Y-ITGA6B4-RIT an e improved y omining with dual PI3K and mtor inhiitor, BEZ235, in a panreati aner model suggesting potential linial appliation. INTRODUCTION Panreati aner is one of the most diffiult malignant diseases to ure [1]. Its treatment options are limited inluding surgery, adjuvant hemotherapy, and radiation therapy and have not given enouraging outomes so far. A possile solution might emerge from the use of targeted therapy suh as radioimmunotherapy (RIT) [2]. RIT involves a seletive internal radiation therapy using ytotoxi radionulides onjugated to tumor-direted antiodies [3]. We reently reported the results otained from a prelinial study of RIT using a novel monolonal anti-integrin α6β4 antiody (ITGA6B4) laeled with eta-emitter Yttrium- ( Y) ( Y-ITGA6B4). Aording to our study, although Y-ITGA6B4 showed signifiant anti-tumor effets, the myelotoxiity aused y an overdose was a major hallenge to overome [4]. To ounterat this prolem, the feasiility of reduing the radiation dose y omining other therapeuti modalities and retaining the same or etter therapeuti effet is important. Thus, omining RIT with other hemotherapeuti andidates is one of the options to redue the radiation dose to normal organs espeially one marrow. The phosphatidylinositol-3-kinase (PI3K)/Akt/ mammalian target of rapamyin (mtor) pathway is a ritial intraellular signaling pathway involved in regulating ell metaolism and survival, ell yle WJG Novemer 14, 2017 Volume 23 Issue 42

7 Aung W et al. Radioimmunotherapy and BEZ235 for panreati aner progression, proliferation, adhesion, and migration, partiularly during aner progression, metastasis, and radioresistane [5-10]. Moreover, this pathway is frequently aerrant and ativated in aner ells. Several downstream targets inluding the serine/ threonine kinase Akt that ativates mtor are ativated y PI3K. Furthermore, ativation of this pathway is known to derease sensitivity to hemotherapeutis as well as to irradiation (IR) [11,12], resulting in a limited treatment outome. The PI3K/Akt and mtor signaling pathways are also frequently dysregulated in panreati dutal adenoarinoma (PDAC) [13]. Meanwhile, BEZ235 (also known as NVP-BEZ235, Datolisi) is a potent dual pan-lass I PI3K and mtor inhiitor that suppresses PI3K and mtor kinase ativity and has een tested in prelinial studies for many aners to demonstrate remarkale antianer effets [14]. This orally administrale inhiitor is the first PI3K/mTOR dual inhiitor to undergo linial trials [15,16], and already shown promising ytostati results in reast aner treatment [17]. Cao et al [18] reported that aute oral dosing with BEZ235 strongly suppressed the phosphorylation of protein kinase B (PKB)/Akt in primary human panreati aners grown as orthotopi xenografts. They also showed the inhiition of downstream Thr37/46 eukaryoti initiation fator 4E inding protein 1 (4EBP1) and Ser235/236 S6 riosomal protein (S6), onsistent with the effets of BEZ235 as a dual PI3K/ mtor inhiitor [18]. Matsushima et al [19] reported that the phosphorylation of Akt (p-akt), 4EBP1 (p-4ebp1), and S6 (p-s6) was inhiited in BEZ235-treated MBT-2 murine ladder aner ells. Similarly, Kuger et al [20] reported that inhiiting the PI3K/Akt/mTOR pathway with BEZ235 aused dephosphorylation of the transription and translation regulators 4EBP1 and S6. Although several studies have demonstrated that BEZ235 ould e a potential radiosensitizer to external radiation therapy [21-29], there are urrently no reports evaluating BEZ235 as a suitale drug to improve the therapeuti effet of RIT. The present study aimed to investigate whether the omination of RIT with moleular targeting BEZ235 therapy ould enhane the therapeuti effiay. MATERIALS AND METHODS Cell ulture and drug preparation Human panreati aner ell line BxPC-3 was purhased from Amerian Type Culture Colletion (Manassas, VA, United States) and ultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrih, St. Louis, MO, United States) supplemented with 10% fetal ovine serum (FBS, Nihirei Biosienes, Tokyo, Japan), 100 U/mL peniillin G sodium, and 100 mg/ml streptomyin sulfate (Invitrogen, Carlsad, CA, United States) at 37 in a humidified atmosphere ontaining 5% CO2. NVP-BEZ235 was purhased from Sellek Chemials (Houston, TX, United States), dissolved in dimethyl sulfoxide (Sigma-Aldrih) and the 2.5 mmol/l stok was stored at -20. For in vivo treatment, it was mixed with the vehile NMP/polyethylene glyol 300 (10/, v/v). Antiody radiolaeling Human anti-α6β4 monolonal antiody (IgG1) was laeled with eta-emitter Y, as previously reported [30]. Briefly, the antiody solution and a helating agent, N-[(R)-2-amino-3-(p-isothioyanato-phenyl)propyl]- trans-(s,s)-ylohexane-1,2-diamine-n,n,n,n,n -p entaaeti aid (CHX-A -DTPA) (Maroylis, Dallas, TX, United States) were mixed at a molar ratio of 1:2.5 and inuated overnight at 37. The onjugation ratio of DTPA and the antiody was estimated to e 1.3 alulated from the ratio (of 111 In-DTPAantiody to 111 In-DTPA) determined y isoeletri fousing. Unonjugated DTPA was removed using a Sephadex G-50 olumn eluted with 0.1 mol/l sodium aetate uffer (GE Healthare, Little Chalfont, United Kingdom). Afterward, the DTPA-onjugated antiody (71.2 μg in 0.1 mol/l sodium aetate uffer, ph 6.0) was inuated with a mixture of Y-hloride (74 MBq, Ekert & Ziegler Radiopharma GmH, Berlin, Germany) and 1 mol/l sodium aetate uffer (ph 6.0) for 30 min at room temperature (RT). The radiolaeled antiody was purified using a Sephadex G-50 olumn (730 g for 2 min). The radiohemial purity as determined y TLC was > 95%. The radiohemial yield was approximately 80%, and the speifi ativity was approximately 1500 kbq/μg. Western lot analysis Western lotting was performed to analyze the proteins of interest from ultured ells. Caner ells were ultured and treated with medium ontaining 0.1 μmol/l BEZ235 or DMSO (vehile) for 1 h. The medium was then disarded and ells were exposed to medium ontaining Y-ITGA6B4 (indiated doses 185 and 370 kbq/ml) in the presene and asene of BEZ235 treatment. At 18 h after inuation, whole-ell lysates were prepared using radioimmunopreipitation assay uffer (Wako Pure Chemial Industries, Osaka, Japan) with protease inhiitor oktail. Total protein onentration was measured using the NanoDrop One Spetrophotometer (Thermo Fisher Sientifi, Wilmington, DE, United States). Protein samples (45 μg) were separated on a 4%-20% polyarylamide gel (ATTO Corporation, Tokyo, Japan) and transferred to an Immoilon-P memrane (Millipore, Billeria, MA, United States). The following antiodies: antihuman phospho-akt (Ser473) (D9E) monolonal antiody, anti-human phospho-4ebp1 (Thr37/46) (236B4) monolonal antiody, anti-human phosphomtor (Ser2448) (D9C2) monolonal antiody, antihuman phospho-s6 Riosomal protein (Ser235/236) WJG Novemer 14, 2017 Volume 23 Issue 42

8 Aung W et al. Radioimmunotherapy and BEZ235 for panreati aner A Y-ITGA6B4 + BEZ235 Day B Y-ITGA6B4 Day C BEZ235 Day D Vehile Day Y-ITGA6B4 Inj BEZ235 administration Vehile administration IHC Figure 1 Experimental treatment sheme. Group A: Y-ITGA6B4 + BEZ235; Group B: Y-ITGA6B4; Group C: BEZ235; Group D: Vehile. polylonal antiody, and anti-human GAPDH monolonal antiody were purhased from Cell Signaling tehnology (Danvers, MA, United States). Anti-human Akt1 (C-20) polylonal antiody was purhased from Santa Cruz Biotehnology (Dallas, TX, United States). These were used as primary antiodies. Horseradish peroxidase (HRP)-linked anti-rait IgG antiody purhased from GE Healthare (Little Chalfont, United Kingdom) was used as the seondary antiody. Immunoreative ands were visualized using the Enhaned Chemiluminesene Plus western lotting detetion system (GE Healthare). Colony formation assay Cells (10, 5, ells/dish) were plated in tripliate onto 60-mm dishes. After overnight inuation, exponentially growing ells were treated with the medium ontaining 0.1 μmol/l μmol BEZ235 or DMSO (vehile) for 1 h. The medium was then disarded and adherent ells were exposed to medium ontaining Y-ITGA6B4 (indiated doses 185 and 370 kbq/ml) in the presene and asene of BEZ235 treatment for 24 h. The medium was then replaed with drug-free medium and the ells were ultured for 7 d for olony formation. At the indiated time point, ells were fixed and stained with Gentian violet and the grown olonies (lusters of > 50 ells) were ounted. Plating effiienies (PE) were determined as (numer of olonies ounted/numer of ell inoulated) 100. Mouse panreati tumor xenograft model All animal experiments were performed in aordane with the animal experimentation protool approved y the Animal Care and Use Committee of National Institute of Radiologial Sienes. Nude mie (7-wkold female BALB/A Jl-nu/nu mie) were otained ommerially from CLEA, Shizuoka, Japan. They were housed in a restrited aess room and alimatized to standard laoratory onditions (23, 12 h/12 h light/dark, 50% humidity, free aess to food and water). Suutaneous tumors were generated y injeting a suspension of BxPC-3 ells in 100 µl RPMI medium mixed with BD Matrigel matrix (BD Biosienes, Bedford, MA, United States) into the right thigh of nude mie. In vivo tumor treatment study When the suutaneous tumors in mie reahed approximately 10 mm at the longest diameter, the xenograft tumor-earing mie were randomly assigned to 4 groups (n = 10 for eah group) for the treatment study. Experimental treatment was performed aording to the shown sheme (Figure 1). Group a reeived daily oral administration of BEZ mg/kg, 5 d/wk for a 6-wk shedule with a single administration of Y-ITGA6B4 (2.8 MBq) following 1 h of the seond BEZ235 dose, group reeived a single administration of Y-ITGA6B4 (2.8 MBq), group reeived daily oral WJG Novemer 14, 2017 Volume 23 Issue 42

9 Aung W et al. Radioimmunotherapy and BEZ235 for panreati aner administration of BEZ mg/kg, 5 d/wk for a 6-wk shedule, and group d reeived no treatments exept oral vehile administration. Intragastri gavage administration of BEZ235 was arried out with onsious mie, using disposale flexile gavage needles (1 inh length, 1.25 all diameter). Eah quantity of the injeted antiody was adjusted to 20 μg y the addition of intat unlaeled antiody. Aording to previous pharmaokineti studies, mie were injeted with Y-ITGA6B4 at 1 h following BEZ235 administration eause within this time frame, BEZ235 ahieves effetive intra-tumoral onentrations [15]. To oserve the tumor response, tumor volumes of mie (n = 6 for eah group) were measured twie a week throughout the experiment using alipers, and were approximated using the equation: volume (mm 3 ) = [length (mm)] [width (mm)] 2 /2. Relative tumor volume was alulated as the volume on the indiated day divided y the volume on the day treatment egan. Immunohistohemial analysis On Day 1 and Day 3 after Y-ITGA6B4 administration, 2 mie of eah group (n = 2) were euthanized y ervial disloation under anaesthesia (Isoflurane). Tumor tissue speimens were extirpated, fixed in 4% paraformaldehyde, and emedded in paraffin. Tumor speimens otained from untreated mie were used as ontrol. Paraffin-emedded tissue setions were ut (5 μm), rehydrated, and sujeted to antigen retrieval. Ki-67 staining of setions was performed using an antihuman Ki-67 polylonal antiody (Dako Denmark, Glostrup, Denmark), as previously desried [31]. Phospho-Histone H2AX (p-h2ax) and Phospho- 4EBP1 (p-4ebp1) staining were also deteted using anti-human p-h2ax (Ser139) (20E3) monolonal antiody and anti-human p-4ebp1 (Thr37/46) (236B4) monolonal antiody (Cell Signaling tehnology), respetively. To detet the apoptoti tumor ells, terminal deoxynuleotidyl transferase-mediated deoxyuridine triphosphate nik end laeling (TUNEL) staining was performed with an ApopTag Peroxidase In situ Apoptosis Detetion Kit (Millipore Corporation, Temeula, CA, United states). Eah slide was oserved using the Olympus BX43 mirosope system (Olympus, Tokyo, Japan). For quantitative and statistial analysis, we ounted the numer of Ki-67-positive ells oserved in three random fields of view at 200 magnifiation in 2 different tumors setions from eah group (n = 6). Similarly, p-h2ax-positive ells were also ounted. Statistial analysis All results were expressed as mean ± SD. Signifiant differenes etween groups were determined y Student s t-test (Exel, Mirosoft, Redmond, WA, United states). Two-tailed unpaired t-test was used for omparisons of relative tumor volume, Ki-67 positive ells and p-h2ax-positive ells. Two-tailed paired t-test was used for omparisons of PE. P-values < 0.05 were onsidered signifiant. RESULTS BEZ235 downregulates the PI3K/Akt signaling pathway in BxPC-3 panreati aner ells The effets of BEZ235 on the PI3K/Akt signaling pathway were determined y western lotting using BxPC-3 ells. Cells were treated with or without 0.1 μmol/l BEZ235 for 1 h efore exposure to Y-ITGA6B4. Total ell lysates were prepared after 18 h of Y-ITGA6B4 treatment. Akt1 expression and phosphorylation of Akt (p-akt) were inreased at 18 h after exposure to 185 or 370 kbq/ml Y-ITGA6B4. These inreased expression levels were attenuated y pretreatment with 0.1 μmol/l BEZ235. Similarly, phosphorylation of 4EBP1 (p-4ebp1) and S6 (p-s6), whih are Akt and mtor downstream effetors, was inhiited y BEZ235 (Figure 2). No ovious hanges in mtor expression and phosphorylation were noted in our experiments (data not shown). BEZ235 augments the ytotoxi effet exerted y Y-ITGA6B4 treatment In the olony formation assay, 24 h exposure of BxPC-3 ells to Y-ITGA6B4 (185 or 370 kbq/ml) apparently resulted in radiation dose-dependent inhiition of ell survival and proliferation, whih was evidened y the redued size and numer of olonies and y the dereased PE. Moreover, pretreatment with 0.1 μmol/l BEZ235 for 1 h efore Y-ITGA6B4 exposure resulted in signifiant redution of olony formation and PE to a greater degree than in the single-drug treatment. For example, omined treatment with BEZ235 and Y-ITGA6B4 (185 kbq/ml) to ells ( ells/dish) showed.9% redution of PE from that of ontrol while Y-ITGA6B4 treatment alone showed 52.5% redution of PE from that of ontrol. All relevant PE were ompared in Figure 3 (P < 0.01). These results indiate that omination of BEZ235 and RIT markedly exaggerates the ytotoxi ativity. BEZ235 inhiits the growth of BxPC-3 xenografts and potentiates Y-ITGA6B4 mediated RIT The anti-tumor effet of Y-ITGA6B4 with or without BEZ235 was evaluated using a BxPC-3 xenograft tumor model. Tumor growth was signifiantly delayed y Y-ITGA6B4 (2.8 MBq) treatment alone for 58 days following the treatment start date ompared with that in vehile treatment (ontrol). Tumor growth was also signifiantly delayed y BEZ235 (35 mg/kg, 5 d/wk for 6 wk) treatment alone for 23 d ompared with that in ontrol. Comined treatment with Y-ITGA6B4 plus BEZ235 enhaned the inhiition of tumor growth and statistially signifiant differenes were oserved for 27 d after the treatment start date when ompared with the Y-ITGA6B4 single injetion treatment, and WJG Novemer 14, 2017 Volume 23 Issue 42

10 Aung W et al. Radioimmunotherapy and BEZ235 for panreati aner BEZ235 (0.1 μmol/l) Y-ITGA6B4 (kbq/ml) After 1 h (BEZ) + 18 h ( Y-ITGA6B4) AKt1 60 kda p-akt1 60 kda p-4ebp kda p-s6 32 kda GAPDH 36 kda Figure 2 Effet of BEZ235 and Y-ITGA6B4 on PI3K/Akt signaling. Expression levels of seletive proteins assoiated with the PI3K/Akt pathway were determined at 18 h after Y-ITGA6B4 exposure (185 or 370 kbq/ml). Representative western lot analyses are shown. Akt1 expression and Akt phosphorylation (p-akt) likely indued y Y-ITGA6B4 were inhiited upon BEZ235 treatment. Conordantly, phosphorylation of 4EBP1 (p-4ebp1) and S6 (p-s6), oth Akt and mtor downstream effetors, was markedly inhiited y BEZ235 pretreatment. Tale 1 Comparison of the numer of Ki-67 positive ells and p-h2ax-positive ells in tumor setions from xenografted mie treated with the vehile ontrol, BEZ235, Y-ITGA6B4, or Y-ITGA6B4 + BEZ235 Group Ki-67-positive ells p-h2ax-positive ells Day 1 Day 3 Day 1 Day 3 Vehile 95.7 ± ± ± ± 1.2 (ontrol) BEZ ± ± ± ± 2.2 Y-ITGA6B ± ± ± 2.8,e 12.7 ± 4.7,d Y-ITGA6B4 + BEZ 13.0 ± ± 3.6 a,,d 12.3 ± 5.5, 19.3 ± 7.0,d Values are expressed as mean ± SD, n = 6. P < 0.01 vs Vehile, d P < 0.01 vs BEZ235; a P < 0.05 vs Y-ITGA6B4, P < 0.05 vs BEZ235, e P < 0.05 vs BEZ235. for 41 d when ompared with the BEZ235 treatment alone (P < 0.05, Figure 4A). At 27 d after starting the treatment, the omination treatment showed 30.9% or 53.9% further redution of the tumor volume ratio ompared to that attained with RIT alone or BEZ235 alone, respetively. Neither signifiant average ody weight loss (Figure 4B) nor differenes in general onditions of mie were oserved etween the groups throughout the experiment. Immunohistohemial evaluation of the therapeuti effets of omined Y-ITGA6B4 plus BEZ235 treatment or eah arm alone Proportions of ell positive for the ell proliferation marker protein [32] Ki-67 were dereased in tumor setions of mie treated with the omination Y-ITGA6B4 plus BEZ235, or eah arm alone, when ompared with those of the ontrol group. It is wellknown that phosphorylation of the histone variant H2AX (p-h2ax) is a highly speifi and sensitive moleular marker to monitor oth DNA doule-strand reak (DSB) initiation and resolution, espeially during radiation treatment [33]. P-H2AX-positive ells were inreased in tumor setions of mie reeiving the omined treatment or Y-ITGA6B4 treatment alone, when ompared with those in the ontrol group. The quantitative results and statistial analysis were summarized in Tale 1. Immunohistohemial staining with the p-4ebp1 antiody depited dereased expression of p-4ebp1 in the omined treatment or eah arm alone, when ompared with that in the ontrol (Figure 5). On the other hand, TUNEL-positive ells were rare and there was no lear differene in the TUNEL staining patterns among all the groups (Data not shown). DISCUSSION Several previous in vitro and in vivo studies have revealed that BEZ235 ould e a potential radiosensitizer in the treatment of various aners suh as firosaroma as well as hypopharyngeal arinoma [21], prostate arinoma [22,23], non-small ell lung aner (NSCLC) [24], gliolastoma [25], oloretal aner (CRC) [26], and reast aner [34]. Chen et al [26] reported that inativation of the PI3K/Akt pathway y BEZ235 ould enhane radiosensitivity y hindering the PI3K/Akt/mTOR pathway and DNA repair mehanisms. To prevent the radiation indued ativation of PI3K/Akt/mTOR WJG Novemer 14, 2017 Volume 23 Issue 42

11 Aung W et al. Radioimmunotherapy and BEZ235 for panreati aner Y-ITGA6B4 10 (κbq/ml) 103 ells/dish ells/dish ells/dish BEZ235 (-) BEZ235 (+) 6 8 BEZ (-) BEZ (+) 4 8 BEZ (-) BEZ (+) kbq 185 kbq ells/dish BEZ (-) BEZ (+) ells/dish Plating effiieny ells/dish Plating effiieny Plating effiieny kbq 0 0 kbq 185 kbq 370 kbq 0 0 kbq 185 kbq 370 kbq Figure 3 Cytotoxi effet of BEZ235 that augments the ytotoxi ativity of RIT in BxPC-3 ells. Treatment with Y-ITGA6B4 alone or BEZ235 alone resulted in dereased olony formation aility in ells. Moreover, pretreatment with BEZ235 efore Y-ITGA6B4 exposure indued stronger inhiition of the olony formation aility than that y Y-ITGA6B4 or BEZ235 treatment alone; moreover, plating effiienies (PE) of ells were signifiantly different etween the BEZ235 (+) and (-) onditions (P < 0.01). Data represent mean ± SD, n = 3. signaling, they treated the oloretal aner ells with BEZ235 one hour efore the radiation and found that BEZ235 synergistially inhiited ell viaility. Their [27] findings are in aordane with those of Kuger et al demonstrating that radiosensitivity of gliolastoma ells was enhaned y BEZ235 exposure one hour efore irradiation. Some reent studies desried that BEZ235 ould potently inhiit major DNA damage response kinases, attenuate the repair of irradiationindued DNA damage, and onfer striking tumor [25,29] radiosensitization in gliolastoma. In ontrast to external eam radiation therapy, one of the onvining enefits of RIT is its apaity to strike the primary tumor as well as the systemially metastasizing and residual lesions. However, systemi administration of large amounts of radiolaeled an tiodies may ause one marrow suppression. Small ut adequate quantities of radiolaeled antiody that WJG may ind to targets and produe effiient ytotoxi effets are therefore desirale, and new strategies are onsequently needed to improve the effetiveness of RIT. We speulated that the omination of RIT and BEZ235 may help redue the dose of RIT and generate a greater therapeuti response than eah arm used alone, with less frequent treatment-related toxiity and inreased radiosensitivity. The radiophysial properties of Y (β-partiles with a maximum emitted energy of 2.28 MeV and emission range of 11 mm, half-life 2.7 d) are a relatively preferale and pratial hoie for RIT. In our previous study, we onduted RIT with Y laeled anti-integrin α6β4 antiody ( Y-ITGA6B4) in a [4] pre-linial mouse panreati aner model. As the treatment protool, we used a single administration of Y-ITGA6B4 (3.7 MBq), and doule administrations of Y-ITGA6B4 one-weekly (3.7 MBq 2) in our [4] previous study. We found signifiantly redued tumor 7557 Novemer 14, 2017 Volume 23 Issue 42

12 Aung W et al. Radioimmunotherapy and BEZ235 for panreati aner A 8 B Tumorvolume (ratio) Body weight (gm) a a a a a a a a Day after injetion Day after injetion Y-ITGA6B4 inj: Oral BEZ administration (5 d/wk) Figure 4 Enhanement of mediated radioimmunotherapeuti effets on BxPC-3 xenografts in omination with BEZ235 treatment. A: Tumor volume hanges expressed as the ratio of the volume on the indiated day and the volume on the day treatment egan. Compared with the ontrol vehile administration (light lue squares), single administration of Y-ITGA6B4 (2.8 MBq) alone (red diamonds) or oral administrations of BEZ235 alone (35 mg/kg, 5 d/wk for 6 wk) (lue triangles) resulted in impaired tumor growth. When Y-ITGA6B4 treatment was administered in onjuntion with BEZ235 (orange squares), further redution in tumor volume ratio was oserved indiating enhaned therapeuti effet upon omined treatment. Values represent mean ± SD, a P < 0.05 ( Y-ITGA6B4 + BEZ235 vs Y-ITGA6B4), P < 0.05 (Y-ITGA6B4 + BEZ235 vs BEZ235), P < 0.05 ( Y-ITGA6B4 + BEZ235 vs Vehile, Y-ITGA6B4 vs Vehile); B: Average mouse ody weight did not differ signifiantly among all 4 groups. Vertial orange arrowhead indiates the day of Y-ITGA6B4 injetion. Values represent mean ± SD, n = 6. growth rates in oth groups ompared with those in the untreated ontrol. However, one mouse from the group reeiving the doule administrations showed pale skin and few petehiae during the study, and died on day 22 following the first dose. We onsidered myelotoxiity due to an overdose as the ause of death eause analysis of hematologial parameters at day 27 after starting the RIT indiated dereased RBC, WBC, and platelet ounts in mie treated with doule administrations, whereas only a dereased RBC ount was oserved in mie reeiving a single administration. Based on these previous results [4], we hose a single administration dose of Y-ITGA6B4 (2.8 MBq) for RIT in this study design. Moreover, we deided to inlude daily oral administration of BEZ mg/kg, 5 d/wk over a 6-wk shedule. To the est of our knowledge, this is the first report aout the omined therapeuti effets of RIT and BEZ235 in a prelinial panreati aner model. In the present study, inhiition of the PI3K/Akt/ mtor pathway with BEZ235 was proven y western lotting in BxPC-3 tumor ells upon oserving the downregulation of p-akt, as well as the downstream targets, p-4ebp1 and p-s6. The redution of p-akt, p-4ebp1 and p-s6 suggested that a plausile mehanism of the ytotoxi effet of BEZ235 derived from inhiition of the PI3K/Akt/mTOR pathway. The 4EBP1 protein integrates its funtion at the level of translation regulation [35]. Reently, it has een shown that some kinases phosphorylate 4EBP1 dependent or independent of mtor, indiating that mtor may not e the only kinase that phosphorylates 4EBP1 [36], and that 4EBP1 is regarded as a point of onvergene of various signaling pathways [35]. Phosphorylation of S6 riosomal protein orrelates with an inrease in the translation of mrna transripts enoding proteins involved in ell yle progression as well as riosomal proteins and elongation fators neessary for translation [37]. Upregulation of mrna translation is effetively involved in sustained ell growth and proliferation [37]. The opposite effet was oserved with BEZ235 treatment. The ytotoxi effet of BEZ235 on BxPC-3 ells was evaluated using the olony formation assay. Exposure to Y-ITGA6B4 alone resulted in dereased lonogeniity in ells and BEZ235 pretreatment aused more potent inhiition of olony formation indiated y redued ounts and smaller olonies. Taken together with the results of western lot analysis, BEZ235 might prevent PI3K pathway reativation and further enhane radiation-indued ell killing. Next, we determined the anti-tumor effet of Y-ITGA6B4 with or without BEZ235 treatment in BxPC-3 xenograft tumors through longitudinal measurement of tumor volume and immunohistohemial analyses. We found that a single injetion of Y-ITGA6B4 (2.8 MBq) alone signifiantly delayed tumor growth ompared with that upon vehile administration. Furthermore, omined WJG Novemer 14, 2017 Volume 23 Issue 42

13 Aung W et al. Radioimmunotherapy and BEZ235 for panreati aner A Ki-67 p-h2ax p-4ebp1 Vehile ontrol (ontrol) BEZ235 Y-ITGA6B4 2.8 MBq (20 μg) Y-ITGA6B4 2.8 MBq (20 μg) + BEZ m, Magnifiation ( 40) B Vehile ontrol (ontrol) BEZ235 Y-ITGA6B4 2.8 MBq (20 μg) Y-ITGA6B4 2.8 MBq (20 μg) + BEZ235 WJG Novemer 14, 2017 Volume 23 Issue 42

14 Aung W et al. Radioimmunotherapy and BEZ235 for panreati aner Figure 5 Ki-67, p-h2ax, p-4ebp1 immunostaining of tumor setions. A: On Day 1 and; B: On Day 3 after administration of Y-ITGA6B4 alone or omined with BEZ235, intratumoral proliferation was determined y immunostaining for Ki-67 nulear antigen. A marked redution in Ki-67-positive ell numers was oserved in samples from mie treated with Y-ITGA6B4 + BEZ235 as well as with Y-ITGA6B4 alone and BEZ235 alone, ompared with those in the untreated ontrol sample. Meanwhile, inreased p-h2ax-positive ell numers were oserved in samples from mie that reeived Y-ITGA6B4 treatment alone or the omined treatment than those in the ontrol. Immunohistohemial analysis for p-4ebp1 showed that phosphorylation of 4EBP1 was dereased in the treatment groups ompared with the ontrol group. Tumor setion images were aquired at 200 magnifiation and representative images are shown (sale ar, 50 μm). The quantitative and statistial analysis were summarized in Tale 1. treatment with Y-ITGA6B4 plus BEZ235 more strongly and signifiantly inhiited the tumor growth for 27 d when ompared with that in Y-ITGA6B4 treatment alone, and for 41 d when ompared with that in BEZ235 treatment alone (P < 0.05, Figure 4A). To further retain the superior therapeuti effiay of the omined treatment and to impede tumor regrowth, further frationated Y-ITGA6B4 administration with appropriate timings will e required and will e interesting to address in next study. Conerning toxiity, we an judge that there was no enhanement of toxiity during the omined treatment eause ody weight loss, ovious anormal hanges in general onditions and death were not oserved in the mie reeiving treatment (Figure 4B). Previously, Fokas et al [28] has demonstrated that BEZ235 itself auses DNA damage even in nonirradiated ells, as evidened y moderately inreased phosphorylation of the histone variant H2AX, and the enhaned persistene of p-h2ax foi after irradiation, thus attriuting radiosensitivity to head and nek and ladder aner ell lines. In our immunohistohemial analyses, signifiantly inreased DNA damage marker p-h2ax-positive ells were noted in the tumor setions of mie that reeived Y-ITGA6B4 plus BEZ235, or Y-ITGA6B4 treatment alone, when ompared with those of the ontrol group (Figure 4). In ontrast, the proliferation marker nulear protein Ki-67-positive ells were signifiantly redued in treatment groups. Besides, immunohistohemial examination showed dereased phosphorylation of 4EBP1 in the treatment groups. These results suggest that BEZ235 treatment ould potentiate the radioimmunotherapeuti effet of Y-ITGA6B4 in BxPC-3 tumors. We deteted no appreiale indution of apoptoti ells in TUNELstained setions, and the staining patterns among groups were not very different at 1 and 3 d after Y-ITGA6B4 administration (data not shown). This result is in line with the results of our previous RIT study in whih TUNEL assay was onduted at 2 d post-administration [4]. Chen et al [26] have reported that irradiation upregulates the Akt/mTOR signaling pathway inluding the ativation of Akt and mtor, whih were attenuated y BEZ235 pretreatment. Likewise, RIT may also upregulate Akt/mTOR signaling pathway to some extent after administration, ut it seems that this ativated Akt/mTOR signaling pathway is attenuated y BEZ235 pretreatment. The mehanisms underlying the treatment with BEZ235 in omination with RIT for panreati aner still need to e larified with a more detailed study overing evidenes at multiple timespoints during treatment with various dose regimens of oth drugs. In the urrent study, we otained an insight in to the presumptive mehanism of a omination treatment with PI3K/mTOR inhiitors and RIT as well as its therapeuti effets. In onlusion, our findings imply that it is possile to improve the therapeuti effiay y omining Y-ITGA6B4-mediated radioimmunotherapy with the dual PI3K and mtor inhiitor, BEZ235, and this omination is a promising treatment option for future panreati aner therapy, though many hurdles remain to e overome to reah linial use. COMMENTS Bakground Panreati aner is one of the most diffiult malignant diseases to ure. Its treatment options are limited and have not given enouraging outomes so far. A possile solution might emerge from the use of radioimmunotherapy (RIT) and other moleular targeting hemotherapeuti andidates. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamyin (mtor) pathway is frequently dysregulated and ativated in human aners inluding panreati aner. Several studies have revealed that BEZ235 (NVP- BEZ235) an inhiit the PI3K and mtor kinase ativity, ould e a potential radiosensitizer, and has een used in prelinial studies in many aners with exellent results of antianer effets. Researh frontiers The authors have studied the radioimmunotherapeuti effet and toxiity of Yttrium- laeled anti-integrin α6β4 antiody ( Y-ITGA6B4) in a mouse panreati aner model. In the previous study, Y-ITGA6B4 showed antitumor effets ut myelotoxiity aused y an overdose was a major handiap. To overome this ostale, omining RIT with other hemotherapeuti andidates is one of the options for reduing the radiation dose and retaining the therapeuti effet. Innovations and reakthroughs To the est of our knowledge, there was no report aout the therapeuti effets of omined Y-ITGA6B4-RIT and BEZ235 in aner treatment. Thus, The authors examined this effet for the first time in a pre-linial panreati aner xenograft model. Appliations These in vitro and in vivo studies and results suggest that it is possile to improve the therapeuti effiay y oming Y-ITGA6B4-RIT and BEZ235 and this omination an e a potential enouraging treatment option for future panreati aner therapy. Terminology The Y-ITGA6B4-RIT is a speifi treatment option for aner y ytotoxi radionulide (eta-emitter Yttrium-) onjugated to anti-integrin α6β4 monolonoal antiody (ITGA6B4). The PI3K/Akt/mTOR pathway is a WJG Novemer 14, 2017 Volume 23 Issue 42

15 Aung W et al. Radioimmunotherapy and BEZ235 for panreati aner ritial intraellular signaling pathway involved in regulating ell metaolism and survival, ell yle progression, proliferation, adhesion, and migration, partiularly during aner progression, metastasis, and radioresistane. Peer-review It s a well-designed study and answers a good sientifi question. REFERENCES 1 Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Reelo M, Parkin DM, Forman D, Bray F. Caner inidene and mortality worldwide: soures, methods and major patterns in GLOBOCAN Int J Caner 2015; 136: E359-E386 [PMID: DOI: /ij.29210] 2 Saah EN, Kadouhe J, Ellison D, Finuane C, Deaudin D, Mather SJ. In vitro and in vivo omparison of DTPA- and DOTAonjugated antiferritin monolonal antiody for imaging and therapy of panreati aner. Nul Med Biol 2007; 34: [PMID: DOI: /j.numedio ] 3 Kawashima H. Radioimmunotherapy: a speifi treatment protool for aner y ytotoxi radioisotopes onjugated to antiodies. 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Aquisition of epithelial-mesenhymal transition and aner stem ell phenotypes is assoiated with ativation of the PI3K/Akt/ mtor pathway in prostate aner radioresistane. Cell Death Dis 2013; 4: e875 [PMID: DOI: /ddis ] 8 Chang L, Graham PH, Hao J, Bui J, Cozzi PJ, Kearsley JH, Li Y. Emerging roles of radioresistane in prostate aner metastasis and radiation therapy. Caner Metastasis Rev 2014; 33: [PMID: DOI: /s ] 9 Bartholomeusz C, Gonzalez-Angulo AM. Targeting the PI3K signaling pathway in aner therapy. Expert Opin Ther Targets 2012; 16: [PMID: DOI: / ] 10 Erahimi S, Hosseini M, Shahidsales S, Maftouh M, Ferns GA, Ghayour-Moarhan M, Hassanian SM, Avan A. Targeting the Akt/PI3K Signaling Pathway as a Potential Therapeuti Strategy for the Treatment of Panreati Caner. Curr Med Chem 2017; 24: [PMID: DOI: / ] 11 Dillon RL, White DE, Muller WJ. The phosphatidyl inositol 3-kinase signaling network: impliations for human reast aner. 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Radiat Onol 2015; 10: 214 [PMID: DOI: /s ] 21 Fokas E, Im JH, Hill S, Yameen S, Stratford M, Beeh J, Hakl W, Maira SM, Bernhard EJ, MKenna WG, Mushel RJ. Dual inhiition of the PI3K/mTOR pathway inreases tumor radiosensitivity y normalizing tumor vasulature. Caner Res 2012; 72: [PMID: DOI: / CAN ] 22 Potiron VA, Aderrahmani R, Giang E, Chiavassa S, Di Tomaso E, Maira SM, Paris F, Supiot S. Radiosensitization of prostate aner ells y the dual PI3K/mTOR inhiitor BEZ235 under normoxi and hypoxi onditions. Radiother Onol 2013; 106: [PMID: DOI: /j.radon ] 23 Zhu W, Fu W, Hu L. NVP-BEZ235, dual phosphatidylinositol 3-kinase/mammalian target of rapamyin inhiitor, prominently enhanes radiosensitivity of prostate aner ell line PC-3. Caner Biother Radiopharm 2013; 28: [PMID: DOI: /r ] 24 Konstantinidou G, Bey EA, Raellino A, Shuster K, Maira MS, Gazdar AF, Amii A, Boothman DA, Saglioni PP. Dual phosphoinositide 3-kinase/mammalian target of rapamyin lokade is an effetive radiosensitizing strategy for the treatment of non-small ell lung aner haroring K-RAS mutations. Caner Res 2009; 69: [PMID: DOI: / CAN ] 25 Gil del Alazar CR, Hardeek MC, Mukherjee B, Tomimatsu N, Gao X, Yan J, Xie XJ, Bahoo R, Li L, Hai AA, Burma S. Inhiition of DNA doule-strand reak repair y the dual PI3K/ mtor inhiitor NVP-BEZ235 as a strategy for radiosensitization of gliolastoma. Clin Caner Res 2014; 20: [PMID: DOI: / CCR ] 26 Chen YH, Wei MF, Wang CW, Lee HW, Pan SL, Gao M, Kuo SH, Cheng AL, Teng CM. Dual phosphoinositide 3-kinase/mammalian target of rapamyin inhiitor is an effetive radiosensitizer for oloretal aner. Caner Lett 2015; 357: [PMID: DOI: /j.anlet ] 27 Kuger S, Graus D, Brendtke R, Günther N, Katzer A, Lutyj P, WJG Novemer 14, 2017 Volume 23 Issue 42

16 Aung W et al. Radioimmunotherapy and BEZ235 for panreati aner Polat B, Chatterjee M, Sukhorukov VL, Flentje M, Djuzenova CS. Radiosensitization of Gliolastoma Cell Lines y the Dual PI3K and mtor Inhiitor NVP-BEZ235 Depends on Drug-Irradiation Shedule. Transl Onol 2013; 6: [PMID: DOI: /tlo.12364] 28 Fokas E, Yoshimura M, Prevo R, Higgins G, Hakl W, Maira SM, Bernhard EJ, MKenna WG, Mushel RJ. NVP-BEZ235 and NVP-BGT226, dual phosphatidylinositol 3-kinase/mammalian target of rapamyin inhiitors, enhane tumor and endothelial ell radiosensitivity. Radiat Onol 2012; 7: 48 [PMID: DOI: / X-7-48] 29 Mukherjee B, Tomimatsu N, Amanherla K, Camaho CV, Pihamoorthy N, Burma S. The dual PI3K/mTOR inhiitor NVP- BEZ235 is a potent inhiitor of ATM- and DNA-PKCs-mediated DNA damage responses. Neoplasia 2012; 14: [PMID: DOI: /neo ] 30 Sogawa C, Tsuji AB, Sudo H, Sugyo A, Yoshida C, Odaka K, Uehara T, Arano Y, Koizumi M, Saga T. C-kit-targeted imaging of gastrointestinal stromal tumor using radiolaeled anti--kit monolonal antiody in a mouse tumor model. Nul Med Biol 2010; 37: [PMID: DOI: / j.numedio ] 31 Sudo H, Tsuji AB, Sugyo A, Ogawa Y, Sagara M, Saga T. ZDHHC8 knokdown enhanes radiosensitivity and suppresses tumor growth in a mesothelioma mouse model. Caner Si 2012; 103: [PMID: DOI: / j x] 32 Sholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown. J Cell Physiol 2000; 182: [PMID: DOI: /(SICI) (200003)182:33.0.CO;2-9] 33 Sak A, Stushke M. Use of γh2ax and other iomarkers of doule-strand reaks during radiotherapy. Semin Radiat Onol 2010; 20: [PMID: DOI: / j.semradon ] 34 Kuger S, Cörek E, Polat B, Kämmerer U, Flentje M, Djuzenova CS. Novel PI3K and mtor Inhiitor NVP-BEZ235 Radiosensitizes Breast Caner Cell Lines under Normoxi and Hypoxi Conditions. Breast Caner (Aukl) 2014; 8: [PMID: DOI: /BCBCR.S13693] 35 She QB, Halilovi E, Ye Q, Zhen W, Shirasawa S, Sasazuki T, Solit DB, Rosen N. 4E-BP1 is a key effetor of the onogeni ativation of the AKT and ERK signaling pathways that integrates their funtion in tumors. Caner Cell 2010; 18: [PMID: DOI: /j.r ] 36 Qin X, Jiang B, Zhang Y. 4E-BP1, a multifator regulated multifuntional protein. Cell Cyle 2016; 15: [PMID: DOI: / ] 37 Peterson RT, Shreier SL. Translation ontrol: onneting mitogens and the riosome. Curr Biol 1998; 8: R248-R250 [PMID: DOI: /S (98) ] P- Reviewer: Roy PK, Wei Dy S- Editor: Gong ZM L- Editor: A E- Editor: Ma YJ WJG Novemer 14, 2017 Volume 23 Issue 42

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