Weichang an and 5-fluorouracil suppresses colorectal cancer in a mouse model

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1 Submit Mnuscript: Help Desk: DOI: /wjg.v21.i World J Gstroenterol 215 Jnury 28; 21(4): ISSN (print) ISSN (online) 215 Bishideng Publishing Group Inc. All rights reserved. Bsic Study ORIGINAL ARTICLE Weichng n nd 5-fluorourcil suppresses colorectl cncer in mouse model Li To, Jin-Kun Yng, Ying Gu, Xin Zhou, Ai-Gung Zho, Jin Zheng, Ying-Jie Zhu Li To, Jin-Kun Yng, Ying Gu, Ai-Gung Zho, Jin Zheng, Ying-Jie Zhu, Deprtment of Oncology, Longhu Hospitl, Shnghi University of Trditionl Chinese Medicine, Shnghi 232, Chin Xin Zhou, Deprtment of Phrmcy, Longhu Hospitl, Shnghi University of Trditionl Chinese Medicine, Shnghi 232, Chin Author contributions: To L nd Yng JK designed the reserch; To L, Gu Y nd Zho AG performed the reserch; Zhu YJ nd Zheng J nlyzed the dt; nd To L, Yng JK nd GuY, Zheng J wrote the pper; Zhou X performed preprtion of the Chinese medicine nd qulity control. Supported by Nturl Science Reserch Fund of Longhu Hospitl Affilited to Shnghi University of Trditionl Chinese Medicine; Budgetry Scientific Reserch Project of the Eduction Commission of Shnghi The impct of Weichng n decoction on β-ctenin/mmp7 signling pthwy in nude mice with heptic metstsis from colorectl cncer: study on the moleculr mechnism, No. 211JW33; Young Tlent Scientific Reserch Project of Shnghi Municipl Commission of Helth nd Fmily Plnning Efficcy Evlution of combined therpy with TCM nd western medicine for the tretment of heptic metstsis from unresectble colorectl cncer, No. 2134y141. Open-Access: This rticle is n open-ccess rticle which ws selected by n in-house editor nd fully peer-reviewed by externl reviewers. It is distributed in ccordnce with the Cretive Commons Attribution Non Commercil (CC BY-NC 4.) license, which permits others to distribute, remix, dpt, build upon this work non-commercilly, nd license their derivtive works on different terms, provided the originl work is properly cited nd the use is non-commercil. See: licenses/by-nc/4./ Correspondence to: Jin-kun Yng, Chief physicin, professor, Deprtment of Oncology, Longhu Hospitl, Shnghi University of Trditionl Chinese Medicine, No. 725, South Wnping Rod, Shnghi 232, Chin. yngjinkun131229@163.com Telephone: Fx: Received: My 6, 214 Peer-review strted: My 11, 214 First decision: June 1, 214 Revised: July 17, 214 Accepted: August 13, 214 Article in press: August 28, 214 Published online: Jnury 28, 215 Abstrct AIM: To exmine the effect of Weichng n (WCA) nd 5-fluorourcil (5-fu) on colorectl tumor nd heptic metstsis in mouse model. METHODS: Quntittive determintion of hesperidin, the effective component in WCA decoction, ws performed using HPLC. In vitro cytotoxicity of WCA ws determined by treting HCT-116 cells with WCA diluents or serum extrcted from rts tht received WCA by orl gvge for 1 wk nd MTT ssys. Forty-eight nude mice received cecum implnttion with tumor blocks subcutneously mplified from humn colon cncer cell line HCT-116. Mice were rndomly divided into four tretment groups: control (CON), WCA, 5-FU nd combintion (WCA + 5-FU). Pthologicl exmintion of tumors consisted of tissue sectioning nd hemtoxylin nd eosin stining. Tumor weight nd size were mesured, nd the number of metsttic lesions ws counted. Serum crcinoembryonic ntigen (CEA) level ws determined by ELISA. The expression levels of tumor genesis nd metstsisrelted proteins β-ctenin nd mtrix metlloproteinse (MMP)-7 were mesured by rel-time quntittive reverse trnscriptse polymerse chin rection (RT- PCR), immunohistochemistry nd immunoblotting. Cell frctiontion ws used to investigte intrcellulr distribution of β-ctenin. RESULTS: Prenchyml tumors were plpble in the bdomens of nude mice 2 wk post-implnttion nd orthotopic tumor formtion rte ws 1% in ll groups. 5-FU tretment lone significntly decresed tumor weight compred to the CON group (1.23 ±.284 g vs 1.84 ±.649 g, P <.1). WCA tretment lone reduced the rte of metstsis (5% vs 1%, 1125 Jnury 28, 215 Volume 21 Issue 4

2 P <.5). Combintion tretment of WCA + 5-FU ws the most effective, reducing the tumor weight (1.14 ±.464 g vs 1.84 ±.649 g, P <.1) nd size ( ± mm 3 vs ± mm 3, P <.5), the rte of metstses (4% vs 1%, P <.1), nd serum CEA levels ( ± μg/l vs 43.4 ± μg/l, P <.5). Expression of β-ctenin nd MMP-7 ws decresed in drug-treted groups compred to controls, s detected using reltime quntittive RT-PCR, immunohistochemistry nd immunoblotting, respectively. Cell frctiontion ssys reveled tht nucler trnsloction of β-ctenin ws reduced by WCA nd/or 5-FU tretments. CONCLUSION: Combintion of WCA with 5-FU significntly inhibited colon tumor growth nd heptic metstses. Decresed expression of β-ctenin nd MMP-7 my be importnt. Key words: Colorectl cncer; Heptic metstsis; Weichng n formul; Orthotopic trnsplnt nude mouse model; Chemotherpeutics 5-fluorourcil The Author(s) 215. Published by Bishideng Publishing Group Inc. All rights reserved. Core tip: In this study, the nti-colon cncer ctivity of Weichng n (WCA), Chinese herbl medicine, ws ssessed in n orthotopic trnsplnttion nude mouse model. Combintion of WCA with 5-fluorourcil inhibited orthotopic tumor growth nd heptic metstses. Decresed expression of β-ctenin nd metlloproteinse-7, which re crucil proteins modulting tumor ggression, my be importnt for the nti-tumor properties of WCA. To L, Yng JK, Gu Y, Zhou X, Zho AG, Zheng J, Zhu YJ. Weichng n nd 5-fluorourcil suppresses colorectl cncer in mouse model. World J Gstroenterol 215; 21(4): Avilble from: URL: full/v21/i4/1125.htm DOI: i INTRODUCTION Colorectl cncer is the third most commonly dignosed cncer nd the third most common cuse of cncer-relted deth worldwide. Approximtely one million new cses of colorectl cncer re dignosed in both men nd women round the world ech yer [1]. It is estimted tht 5%-6% of ptients dignosed with colorectl cncer will develop heptic metstses during the course of their disese [2-4], nd unfortuntely, 8%-9% of these metstses will be unresectble [5-7]. While metsttic lesions my be found throughout the body following locoregionl tretments, the most frequent site of colorectl cncer metstsis is the liver [8]. The 5-yer survivl rte of ptients with heptic metstses nd not undergoing surgicl tretment is low [3,9]. Ptients with unresectble heptic metstses require multidisciplinry tretments, including surgery, chemotherpy, liver-trgeting therpies, nd trditionl Chinese medicine (TCM). TCM hs been used extensively in Chin for tretment of vrious diseses, including cncer. TCMs hve number of benefits, including decresing the negtive side effects ssocited with chemotherpy nd rdiotherpy, improving ptients immune function, nd enhncing the effects of conventionl cncer tretments [1-12]. Weichng n (WCA) is trditionl Chinese formul prescribed by prctitioners of TCM, bsed on clinicl experiences nd phrmceuticl screening. The principl elements comprising WCA re invigorting spleen herbs, wheres het-clering nd detoxicting herbs, hrd lump-resolving herbs nd blood stsis removing herbs re djuvnt components. Previous clinicl studies hve indicted tht ptients with gstric crcinom benefit from WCA tretment [13,14]. In ddition, WCA hs been shown to increse the 5-yer survivl rte nd reduce the 1- nd 2-yer metsttic rtes in ptients with colorectl cncer [15]. However, whether WCA is effective in colorectl cncer with heptic metstsis is still uncler. Mny studies hve shown tht the Wnt/β-ctenin signling pthwy regultes tumor cell invsion nd metstsis. In orl squmous cell crcinom cells, the ccumultion of β-ctenin in the cytoplsm induced T-cell fctor/lymphoid enhncing fctor trnscriptionl ctivity, nd incresed mtrix metlloproteinse (MMP)-7 expression, thereby inducing the conversion of epithelil cells to mesenchyml cells, s well s enhncing invsion nd metstsis [16]. The Wnt/β-ctenin pthwy lso plys criticl role in colorectl cncer development [17]. We hypothesized tht WCA my ffect the Wnt/β-ctenin pthwy. To elucidte the efficcy of WCA in inhibiting colorectl cncer cell growth nd heptic metstsis nd underlying mechnisms, we dministrted WCA combined with 5-fluorourcil (5-FU) in n orthotopic trnsplnt nude mouse model grfted with HCT-116 humn colon cncer cells. The pthologicl phenotype, tumor genesis, metsttic lesions, crcinoembryonic ntigen (CEA) levels nd β-ctenin/ MMP-7 expression were detected. MATERIALS AND METHODS Drugs WCA ws provided by the Dispensry of TCM, Longhu Hospitl, Shnghi University of Trditionl Chinese Medicine, Shnghi, Chin. The composition of WCA includes Pseudostellri heterophyll (Miq.) Px, Atrctylodes mcrocephl koidz., Pori cocos 1126 Jnury 28, 215 Volume 21 Issue 4

3 (Schw.) Wolf, Glycyrrhiz urlensis Fisch., Srgentodox cunet, nd Prunell vulgris L. The preprtion of the WCA decoction hs been described previously [13,14]. The concentrtion of hesperidin (Ntionl Institutes for Food nd Drug Control, Beijing, Chin) in the WCA formul ws determined by HPLC (Agilent 11; Plo Alto, CA, United Sttes) using the following conditions: Agilent 11 C18 column (25 mm 4.6 mm, 5 μm); mobile phse, cetonitrile:wter 8% HAC (18:82); wvelength, 284 nm; flow rte, 1 ml/min; nd column temperture, FU ws purchsed from Shnghi Xudong Hipu Phrmceuticl Co. Ltd. (Shnghi, Chin). In the present study, 5-FU ws used t concentrtion of 2.5 mg/ml. Animls Mle BALB/C -nu/nu mice, ged 4-6 wk nd weighing 18-2 g, were purchsed from the Shnghi SLAC Lbortory Animl Co. Ltd. [Shnghi, Chin; certifiction NO. SCXK (Shnghi) 212-2]. Mice were housed under specific pthogen-free conditions nd were given free ccess to food nd wter. All niml experiments were performed in ccordnce with the Guidelines for the Cre nd Use of Lbortory Animls, Ministry of Science nd Technology, Chin. This study ws pproved by the Animl Cre nd Scientific Committee of the Shnghi University of trditionl Chinese medicine. Cell culture The humn colon cncer cell line HCT-116 ws purchsed from the Cell Bnk of the Chinese Acdemy of Sciences (Shnghi, Chin). Cells were cultured in McCoy s 5A modified medium (Gibco, Crlsbd, CA, United Sttes) contining 1% het-inctivted fetl bovine serum nd supplemented with penicillin (1 U/ml) nd streptomycin (1 g/ml). Cells were mintined t 37 nd 5% CO2. MTT ssy During the logrithmic growth phse, HCT-116 cells were conventionlly digested, fter which the cells were seeded into 96-well plte t density of cells/ml. The control group ws set, nd the cells were cultured t 37 in humidified tmosphere of 5% CO2 in ir. The medium ws discrded when cells hd reched 7%-8% confluence. Medium contining, 3%, 6% nd 9% WCA decoction or 5%, 1% nd 2% of serum extrcted from rts tht received WCA by orl gvge (OG) (2 ml/d for 1 wk) ws dded to ech well (1 μl/well). Ech condition ws tested in qudruplicte, nd the cells were treted for, 24, 48 nd 72 h. After tretment, 2 μl MTT regent (Sigm, St Louis, MO, United Sttes) ws dded to ech well for 4 h. The culture medium ws discrded, nd 15 μl dimethyl sulfoxide (Sigm) ws dded. Formzn ws dissolved by gentle gittion for 1 min. Opticl density (OD) for ech well ws determined using plte reder (Synergy2; Bio-tek, Winooski, VT, United Sttes) t wvelength of 49 nm. All experiments were performed three times. Estblishment of the colon cncer trnsplnt nude mouse model nd drug dministrtion HCT-116 cells (2 1 7 ) were subcutneously injected into the right xill of nude mice. After 3 wk, tumors were excised nd cut into 1-2 mm 3 pieces using sterile techniques. Tumor blocks were trnsplnted into the cecum of 48 nude mice by purse-string suture (Figure 1A nd 1B). Drug dministrtion strted 7 d fter tumor implnttion. The 48 nude mice were rndomly divided into four tretment groups (12 mice/group). Mice in the control (CON) group received sline by OG (.5 ml/d) nd by intrperitonel (IP) injection (.4 ml/time, once/ week). Mice in the WCA group received WCA by OG (.5 ml/d) nd sline by IP injection (.4 ml/time, once/week). Mice in the 5-FU group were given sline by OG (.5 ml/d) nd 5-FU by IP injection (5 mg/kg per time, once/week). Mice in the WCA + 5-FU group received WCA by OG (.5 ml/d) nd 5-FU by IP injection (5 mg/kg per time, once/ week). All tretments lsted for 7 wk. Twentyfour hours fter the finl drug dministrtion, lprotomy ws performed nd blood smples were collected from ech niml. Tumors (both orthotopic nd metsttic) nd tumor-djcent tissues were collected, nd conventionl pthologicl exmintion, immunohistochemistry (IHC), rel-time polymerse chin rection (PCR), nd immunoblotting ssys were performed. Tumor mesurement nd observtion Weights of the orthotopic tumors were mesured using n electronic pltform blnce. The width () nd length (b) of ech orthotopic tumor were mesured with cliper, nd tumor size ws clculted ccording to the following formul: tumor size (mm 3 ) = π 2 b/6. Inhibition of tumor growth cused by drug tretment ws estimted bsed on the following formul: inhibition rte of tumor (%) = (1 - men tumor weight in tretment group/men tumor weight in CON group) 1%. Pthologicl exmintion Orthotopic tumors, tumor-djcent tissues, nd metsttic tumors were embedded in prffin nd cut into sections. These sections were then stined with hemtoxylin nd eosin (HE; Sigm) nd visulized under BH2 opticl microscope (Olympus, Tokyo, Jpn). Determintion of serum CEA level Blood smples obtined from mice nd were nturlly 1127 Jnury 28, 215 Volume 21 Issue 4

4 A B C D E F G H I J Figure 1 Pthology of primry colon cncer tissues (HE stining) nd gross ntomy of nude mice implnted with humn colon cncer HCT-116 cells. A, B: tumor pieces were trnsplnted into the cecum of nude mice by purse-string suture; C, E, G nd I: HE stining of colon cncer tissues from mice in the CON, WCA, 5-FU nd WCA + 5-FU groups (mgnifiction 1); D, F, H nd J: Gross ntomy of mice in the CON, WCA, 5-FU nd WCA + 5-FU groups. Blck rrows indicte orthotopic tumors, nd white rrows indicte the bdominl wll tumor. WCA: Weichng n; 5-FU: 5-fluorourcil; CON: Control Jnury 28, 215 Volume 21 Issue 4

5 cogulted for 2 min fter collection. Smples were centrifuged t 1 g for 1 min, nd the superntnt ws collected nd stored t -2. Serum CEA levels were determined using CEA ELISA kit (Bio-Swmp, Wuhn, Chin) ccording to the mnufcturer s instructions. Absorbnce ws mesured t 45 nm on n MK3 microplte reder (Thermo Fisher Scientific, Wlthm, MA, United Sttes). Rel-time PCR Totl RNA ws extrcted with Trizol (Invitrogen, Crlsbd, CA, United Sttes), nd the concentrtion nd purity of RNA were determined using UV spectrophotometer. Totl RNA ws reverse-trnscribed into cdna with reverse trnscriptse regents (Shnghi Jierdun Biotech Co. Ltd., Shnghi, Chin) ccording to the mnufcturer s protocol. An ABI Step One Plus Rel-Time-PCR System (Applied Biosystems, Foster City, CA, United Sttes) ws used with SYBR Green Mster Mix (ABI) nd primers (Invitrogen, Shnghi, Chin) for mesurement of β-ctenin nd MMP-7. Primers were: β-ctenin sense (5 -GGT TTC CCA TTG GTT CAC-3 ) nd ntisense (5 -CAT AAA TCC CGC CTA ACG-3 ); nd MMP-7 sense (5 -GAA CAG GCT CAG GAC TAT CTC-3 ) nd ntisense (5 -ACA TCT GGC ACT CCA CAT C-3 ). GAPDH ws used s reference to obtin the reltive fold chnge for trget genes using the comprtive Ct method. Primer sequences for GAPDH were: sense (5 -CGG AGT CAA CGG ATT TGG TCG TAT-3 ) nd ntisense (5 -AGC CTT CTC CAT GGT GGT GAA GAC-3 ). Reltive β-ctenin nd MMP-7 expression were estimted using the 2 - CT method. IHC We performed IHC on orthotopic nd metsttic tumors to determine protein expression levels of both β-ctenin nd MMP-7. The following primry ntibodies were used: rbbit monoclonl ntiβ-ctenin ntibody (diluted 1:2, rbbit IgG1; b7989, Abcm, Cmbridge, United Kingdom) nd nti-mmp-7 ntibody (diluted 1:2, rbbit IgG1; b444, Abcm, Cmbridge, United Kingdom). The secondry ntibody used ws biotinylted got nti-rbbit IgG (Beyotime Institute of Biotechnology, Shnghi, Chin). Sections were visulized with diminobenzidine (DAB; Shnghi Jierdun Biotech Co. Ltd., Shnghi, Chin) nd counterstined with HE. IHC imges were cptured with digitl cmer (Nikon, Tokyo, Jpn) nd nlyzed using the IMS imging processing system (Shnghi Jierdun Biotech). Positively stined regions were counted nd nlyzed. Cell frctiontion Cells were frctionted into cytosolic nd nucler frctions, with little modifiction [18]. Cells from both orthotopic tumor tissues nd heptic metsttic cncer tissues were collected, wshed with PBS nd centrifuged t 5 g. Pelleted cells were resuspended in ice-cold.1% NP4-PBS nd lysed by pipetting up nd down severl times. A portion of the cell suspension ws kept s whole cell lyste. The cell lystes were centrifuged t 14 g for 1 min nd the superntnts were collected s cytosolic frction, while the pellets (nuclei) were wshed twice with ice-cold.1% NP4-PBS. The hrvested pellets were resuspended in Lemmli smple buffer (Bio-Rd), sonicted for 3 s, nd collected s nucler frctions. Equivlent proportions of two frctions were nlyzed by SDS-PAGE nd immunoblotting. The purity of the frctions ws ssessed by detecting specific subcellulr mrker proteins such s GAPDH s cytosolic protein nd H3 s nucler protein. Immunoblotting Totl protein ws extrcted from both orthotopic tumor tissues nd heptic metsttic cncer tissues, nd protein concentrtion ws determined using the BCA protein ssy kit (Thermo Fisher Scientific). Immunoblotting ws performed with the following primry ntibodies: rbbit monoclonl nti-β-ctenin ntibody (diluted 1:1, rbbit IgG1; b7989, Abcm, Cmbridge, MA, United Sttes) nd nti- MMP-7 ntibody (diluted 1:3, rbbit IgG1; b444, Abcm, Cmbridge, MA, United Sttes). Anti-rbbit horserdish-peroxidse-conjugted secondry ntibody ws used t dilution of 1:2. Detection of GAPDH [diluted 1:15; Cell Signling Technology (CST), Beverly, MA, United Sttes] nd H3 (diluted 1:1, #4499S, CST) served s internl loding controls. All blots were scnned with the Lbwork imging processing system. Protein bnd pixels were clculted using Gel-pro Anlyzer 4. (Medi Cybernetics, Rockville, MD, United Sttes). Sttisticl nlysis Continuous dt re expressed s men ± stndrd devition (SD) nd comprison of the mens mong four groups ws performed using one-wy nlysis of vrince (ANOVA). If Levene s test reveled homogeneity of vrince, multiple comprisons were performed using Fisher s lest significnt difference test. If the Levene s test reveled heterogeneity of vrince, the Welch nd Brown-Forsythe tests were used. In this cse, multiple comprisons were performed using Dunnett s T3 nd Dunnett s C tests. Dt with smll smple size re expressed s men ± SD nd comprisons mong four groups were performed using Kruskl-Wllis tests. Nemenyi tests were further used to perform multiple comprisons. Ctegoricl dt were nlyzed using χ 2 tests. R 3.1 softwre ws used to perform Nemenyi tests nd other sttisticl nlyses were performed using 1129 Jnury 28, 215 Volume 21 Issue 4

6 A mau DAD1 A, Sig = 283, 4 Ref = 36, 1 (CPG17.D) min B mau DAD1 A, Sig = 283, 4 Ref = 36, 1 (CPG16.D) min C Cell vibility (OD vlue) Control 3% WCA 6% WCA 9% WCA D Cell vibility (OD vlue) Control 5% serum 1% serum 2% serum Figure 2 HPLC fingerprinting of hesperidin (A) nd Weichng n (B); HCT-116 cells were treted with different concentrtions of Weichng n (C) nd different concentrtions of Weichng n-contining serum (D) in vitro. Prolifertion of cells ws detected using the MTT ssy. Cell vibility ws expressed s opticl density (OD) vlue (men ± SD). P <.5 vs cell vibility of the control group t the sme time point. WCA: Weichng n. SPSS version 18. (SPSS Inc., Chicgo, IL, United Sttes). P <.5 were considered significnt. RESULTS Determintion of WCA concentrtion nd ex vivo effects on HCT-116 cells To normlize the concentrtion of WCA, the concentrtion of hesperidin in WCA ws determined by HPLC nd stndrd curve (y = x ; correltion coefficient r =.999) ws obtined with the pek re s the Y xis nd concentrtion of control hesperidin s the X xis. HPLC fingerprinting of control hesperidin ws performed t min (Figure 2A), nd HPLC fingerprinting of WCA ws done t min (Figure 2B). Three btches of WCA hd similr concentrtions of hesperidin. The humn colon cncer cell line HCT-116 ws treted with, 3%, 6% nd 9% WCA decoction filtrte or with 5%, 1% nd 2% of serum extrcted from rts tht received WCA by OG (2 ml/d for 1 wk). As shown in Figure 2C nd 2D, 72 h fter tretment, WCA exerted significnt inhibition on HCT-116 cell prolifertion (P <.5). 113 Jnury 28, 215 Volume 21 Issue 4

7 Tble 1 Weight nd size of orthotopic tumors in nude mice nd tumor inhibition rtes resulting from drug tretments Group n Weight (g) Size (mm 3 ) Tumor inhibition CON ± ± WCA ± ± % 5-FU ±.284 b ± % WCA + 5-FU ±.464 b ± % P <.5 vs CON; b P <.1 vs CON. WCA: Weichng n; 5-FU: 5-fluorourcil; CON: Control. Mouse survivl nd pthogenic mnifesttions fter tumor implnttion One mouse in ech group died within 1 wk of orthotopic trnsplnt surgery; in ech instnce, deth my hve been cused by the surgicl procedure itself. Additionlly, one mouse died 12 d fter surgery in the WCA group, nd one 1 d fter surgery in the WCA + 5-FU group. Therefore, 42 mice survived the entire length of the procedure nd drug dministrtion. Prenchyml tumors were plpble in the bdomens of mice 2 wk post-surgery. Furthermore, the body weights of mice were reduced by 5 wk fter surgery. At 7 wk post-surgery, mrsmus nd loss of ppetite occurred. Additionlly, feces ppered soft nd red in color; mice lso displyed skin csting nd slow movement nd ctivity. Effect of WCA on orthotopic tumor growth nd heptic metstsis All mice tht underwent orthotopic trnsplnttion surgery developed tumors. HE stining nd gross ntomy of tumors re shown in Figure 1. The CON group showed obvious typi of orthotopic cncer cells; lrge cncerous cells displying kryokinesis could lso be seen. Irregulrly rrnged tumorgint cells were lso present. Blood vessels were bundnt, nd there ws little connective tissue found in the interstitium. Monocyte infiltrtion nd necrosis were pprent in the tumor interstitium (Figure 1A). The WCA, 5-FU nd WCA + 5FU groups showed reduced typi of orthotopic cncer cells nd the presence of lrge cncerous cells. Irregulrly rrnged tumor-gint cells were lso seen. Blood vessels were bundnt in the interstitium. Incresed monocyte infiltrtion ws observed in the tumor interstitium, nd enlrged necrotic res were pprent. A significnt increse in the cvity-like structure could lso be observed (Figure 1B, 1C nd 1E). One-wy ANOVA showed significnt differences in the men tumor weight mong the four groups (P =.1). Moreover, the men tumor weight in the CON group ws significntly different from the men tumor weight in both the 5-FU (P =.5) nd WCA + 5-FU groups (P =.3). Tretment of mice with WCA, 5-FU nd WCA + 5-FU inhibited tumor growth by 19.87%, % nd 36.82%, respectively (Tble 1). The verge size of orthotopic tumors between the CON nd the WCA + 5-FU groups ws significntly different (P =.31); however, no other significnt differences were identified (Tble 1). Compred with the CON group, mice in the WCA nd the WCA + 5-FU groups hd fewer liver, bdominl nd intestinl metstses (Figure 3). Heptic metsttic tumors ppered s nodules with mssive typi in mice belonging to the CON group. Cncer cells were lrge, nd little connective tissue ws observed in the interstitium. Infiltrtion nd necrotic ltertion of monocytes were found in the tumor interstitium. There were heptic tissue necrosis nd lymphocyte infiltrtion t the boundry of the tumor interstitium (figure 3A). As for heptic metsttic tumors in mice in the WCA group, no pprent boundry between the metstses ws evident. There were obvious heptic tissue necrosis nd infiltrtion of both lymphocytes nd monocytes (Figure 3B). In heptic metsttic tumors in mice from the 5-FU nd WCA + 5FU groups, cncer cells were irregulrly rrnged nd n increse in the interstitium ws observed. Mssive bleeding hs lso occurred (Figure 3C nd 3D). The gross metsttic rtes in the WCA nd the WCA + 5-FU groups were significntly lower thn in the CON group (P =.12 nd.4, respectively). However, there ws no significnt difference in heptic metstses mong ny of the drug-treted groups (Tble 2). Effect of WCA on serum CEA level There ws no significnt difference in serum CEA levels mong the CON (43.4 ± μg/l), WCA ( ± μg/l), nd 5-FU ( ± μg/l) groups (P =.122). However, serum CEA levels in the WCA + 5-FU group ( ± μg/l) were significntly lower thn tht in the CON group (P =.23). Effect of WCA on β-ctenin nd MMP-7 mrna expression in orthotopic tumors nd liver metstses To begin to investigte the moleculr mechnisms underlying metstsis in our model, we mesured mrna expression of β-ctenin, Wnt pthwy regultor of cell-cell dhesion, by rel-time RT-PCR. One-wy ANOVA reveled significnt differences in β-ctenin expression in orthotopic tumors mong the four groups (P =.1). Specificlly, mice treted with WCA, 5-FU nd WCA + 5-FU showed decresed β-ctenin mrna expression in orthotopic tumors compred to control treted mice (P =,.21 nd.6, respectively; Tble 3). In liver metstses, s smple size ws smll (3-5 per group), Kruskl-Wllis nonprmetric tests were performed. The results showed tht there ws no significnt difference in β-ctenin mrna expression levels in the CON, WCA, 5-FU nd WCA + 5-FU tretment groups (P =.155; Tble 3) Jnury 28, 215 Volume 21 Issue 4

8 A E B F C G D H Figure 3 Heptic metstses of colon cncer in the orthotopic trnsplnt nude mouse model. A-D: HE stining of heptic metstses tissues from mice in the CON, WCA, 5-FU nd WCA + 5-FU groups (mgnifiction 2); E-H: Gross ntomy of mice in the CON, WCA, 5-FU nd WCA + 5-FU groups. The blck rrow indictes orthotopic tumor, the long white rrow indictes the heptic metsttic tumor, nd the short white rrow indictes the metstsis of colon cncer to the intestinl wll. WCA: Weichng n; 5-FU: 5-fluorourcil; CON: Control Jnury 28, 215 Volume 21 Issue 4

9 Tble 2 Cncer metstses in nude mice implnted with tumors formed from HCT-116 cell injection Group mice survived, n Heptic metstses Abdominl wll metstses Mesenteric metstses Splenic metstsis Renl metstsis Gross metsttic rte Heptic metstsis rte CON % 45.45% WCA % 3% 5-FU % 36.36% WCA + 5-FU % b 3% P <.5 vs CON; b P <.1 vs CON. WCA: Weichng n; 5-FU: 5-fluorourcil; CON: Control. Tble 3 β-ctenin nd mtrix metlloproteinse-7 mrna levels in orthotopic nd heptic metsttic tumors in the trnsplnt model Group β-ctenin MMP-7 Orthotopic tumors Heptic metsttic tumors Orthotopic tumors Heptic metsttic tumors n reltive expression n reltive expression 1 n reltive expression n reltive expression 2 CON ± ± ± ±.593 WCA ±.173 b ± ± ±.25 b 5-FU ± ± ± ±.21 WCA + 5-FU ±.325 b ± ± 1.11 b 3.44 ±.19 1 β-ctenin mrna expression levels in heptic metsttic tumors were presented s men ± SD nd nonprmetric tests (Kruskl-Wllis tests) were performed, P =.155; 2 Mtrix metlloproteinse-7 (MMP-7) mrna expression levels in heptic metsttic tumors were presented s men ± SD nd nonprmetric tests (Kruskl-Wllis tests) were performed, P =.12. P <.5 vs CON; b P <.1 vs CON. WCA: Weichng n; 5-FU: 5-fluorourcil; CON: Control. MMP-7 is n importnt trget gene in the Wnt/ β-ctenin signling pthwy nd the min member of the MMP fmily, nd it ws lso exmined in our model. The Welch nd Brown-Forsythe tests showed significnt differences in MMP-7 mrna expression in orthotopic tumors mong the four groups (P =.1). Moreover, MMP-7 trnscript levels were significntly decresed in both the WCA nd WCA + 5-FU tretment groups (P =.38 nd.3, respectively; Tble 3). In liver metstses, MMP-7 mrna ws significntly decresed (P =.12, Kruskl-Wllis tests; Tble 3) nd Nemenyi tests reveled tht both WCA nd WCA + 5-FU tretments decresed MMP-7 mrna expression compred to the CON group (P =.1 nd.13, respectively; Tble 3). Effect of WCA on β-ctenin nd MMP-7 protein expression in orthotopic tumors nd liver metstses We mesured protein expression of β-ctenin nd MMP-7 in both orthotopic tumors nd liver metstses by IHC nd immunoblotting. Figure 4A nd 4B show integrted OD of β-ctenin nd MMP-7 protein detected by IHC in orthotopic tumors nd liver metstses, respectively. As shown in Figure 4C nd 4E, in both orthotopic tumors nd liver metstses, β-ctenin ws expressed t high levels nd ws locted in both the nucleus nd cytoplsm in the CON group, nd totl expression of β-ctenin ws reduced in the WCA/5-FU/WCA + 5-FU groups compred with the CON group. As shown in Figure 4D nd 4F, MMP-7 ws highly expressed (brown stining) in the cytoplsm of orthotopic nd liver metsttic tumors from the CON group. We lso observed some membrne stining nd some stining of the tumor interstitium. Expression of MMP-7 ws reduced in the WCA, 5-FU nd WCA + 5-FU groups compred with the CON group. The structure of the tumor tissue ws lso destroyed by mssive necrotic res. One-wy ANOVA nlysis of IHC stining showed significnt difference in β-ctenin protein expression in orthotopic tumors mong the four groups (P =.12, 1 or 11 per group). Furthermore, β-ctenin protein ws decresed in ll tretment groups compred with CON (WCA, P =.3; 5-FU, P =.33; WCA + 5-FU, P =.8; Figure 4A). Levene s test reveled heterogeneity of vrince in MMP-7 protein levels in the orthotopic tumors, nd the Brown-Forsythe test showed significnt differences in MMP-7 protein expression in the orthotopic tumors mong the four groups (P =.2, 1 or 11 per group). Pirwise comprisons showed no significnt difference between groups (ll P >.5). However, decresing trend ws observed in both the WCA group (776.3 ± 124.3) nd the WCA + 5-FU group (97.3 ± ) compred with the CON group ( ± ; Figure 4A). β-ctenin protein expression in heptic metstses detected using IHC ws significntly different mong the four groups [CON group ( ± ); WCA group ( ± 17.39); 5-FU group ( ± 86.83); nd WCA + 5-FU group (838. ± ); P =.25 using the Kruskl-Wllis test, Figure 4B, 3-5 per group]. Pirwise comprisons using Nemenyi tests reveled tht the WCA + 5-FU tretment decresed β-ctenin expression compred to the CON group (P =.2) Jnury 28, 215 Volume 21 Issue 4

10 A 3 B 25 Integrted opticl density β-ctenin MMP-7 Integrted opticl density β-ctenin MMP-7 CON WCA 5-FU WCA + 5-FU CON WCA 5-FU WCA + 5-FU C β-ctenin D MMP-7 Heptic metsttic tumors Orthotopic tumors E F Figure 4 immunohistochemistry detection of β-ctenin nd mtrix metlloproteinse-7 in orthotopic tumors nd heptic metsttic tumors. A, B: Integrted opticl density (OD) of β-ctenin nd mtrix metlloproteinse (MMP)-7 protein expressed in orthotopic tumors (A) or heptic metsttic tumors (B) from mice in the CON, WCA, 5-FU nd WCA + 5-FU groups. Dt re presented s men ± SD nd P <.5 vs corresponding CON group; C: IHC stining for β-ctenin in orthotopic tumors (mgnifiction 2); D: IHC stining for MMP-7 in orthotopic tumors (mgnifiction 2); E: IHC stining for β-ctenin in heptic metsttic tumors (mgnifiction 2); F: IHC stining for MMP-7 in heptic metsttic tumors (mgnifiction 2). WCA: Weichng n; 5-FU: 5-fluorourcil; CON: Control. MMP-7 expression in heptic metstses ws not significntly different mong the groups (P =.113 using Kruskl-Wllis test, 3-5 per group), while trend towrd drug-medited decrese in MMP-7 protein expression ws observed in heptic metstses by IHC (179. ± , ± , 126. ± nd 1353.± in the CON, WCA, 5-FU nd WCA + 5-FU groups nd men rnk 11.4, 5.67, 4.75 nd 9., respectively). Figure 5A nd B show gry scle scnning of β-ctenin nd MMP-7 protein detected by western blot in orthotopic tumors nd liver metstses (3 per group), respectively. β-ctenin expression ws significntly different mong the four groups in both orthotopic nd heptic metsttic tumors (P =.43 nd P =.41 by Kruskl-Wllis test, respectively). Pirwise comprisons using Nemenyi tests showed decresed expression of β-ctenin in the 5-FU nd WCA + 5-FU groups compred with the CON group (P =.23 nd P =.43, respectively) in orthotopic tumors, while in heptic metsttic tumors, β-ctenin expression in the WCA + 5-FU group ws decresed compred with the CON group (P =.12) Jnury 28, 215 Volume 21 Issue 4

11 A Reltive expression mount β-ctenin MMP-7 B Reltive expression mount β-ctenin MMP CON WCA 5-FU WCA + 5-FU CON WCA 5-FU WCA + 5-FU C β-ctenin GAPDH D MMP-7 GAPDH E β-ctenin F MMP-7 GAPDH GAPDH Figure 5 Western blot nlysis of β-ctenin nd mtrix metlloproteinse-7 protein expression in orthotopic nd heptic metsttic tumors. A, B: Quntifiction of β-ctenin nd mtrix metlloproteinse (MMP)-7 expression in orthotopic tumors (A) nd heptic metsttic tumors (B). Dt were presented s men ± SD nd P <.5 vs corresponding CON group; C: β-ctenin expression in orthotopic tumors; D: MMP-7 expression in orthotopic tumors; E: β-ctenin expression in heptic metsttic tumors; F: MMP-7 expression in heptic metsttic tumors. Lnes 1-3, CON group; 4-6, WCA group; 7-9, 5-FU group; nd 1-12, WCA + 5-FU group. WCA: Weichng n; 5-FU: 5-fluorourcil; CON: Control. Using Kruskl-Wllis tests, MMP-7 showed difference in orthotopic tumors mong the four groups (P =.33) but not in heptic metsttic tumors (P =.69). Pirwise comprisons using Nemenyi tests showed decresed expression of MMP-7 in the WCA + 5-FU group compred with the CON group (P =.1) in orthotopic tumors. Effect of WCA on intrcellulr distribution of β-ctenin protein To clrify whether the intrcellulr distribution of β-ctenin ws ffected by WCA, we performed cell frctiontion nd detected β-ctenin in the cytosolic nd nucler frctions in orthotopic tumors nd heptic metsttic tumors, respectively. The immunoblotting imges nd quntittive digrms re shown in Figure 6. In orthotopic tumors (Figure 6A), Kruskl-Wllis tests showed significnt cytosolic nd nucler β-ctenin expression (P =.19 nd P =.33, respectively). Pirwise comprisons using Nemenyi tests reveled tht WCA + 5-FU tretment decresed cytosolic β-ctenin expression compred to the CON group (P <.1), while WCA nd WCA + 5-FU decresed nucler β-ctenin expression compred to the CON group (P =.43 nd P =.12, respectively). In heptic metsttic tumors (Figure 6B), Kruskl- Wllis tests reveled tht there ws significnt difference in cytosolic (P =.16) or nucler (P =.31) β-ctenin expression mong groups, nd Nemenyi tests showed tht WCA + 5-FU tretment significntly decresed cytosolic (P <.1) nd nucler (P =.1) β-ctenin. These dt showed tht WCA/5-FU tretments not only depressed β-ctenin expression but lso inhibited nucler trnsloction both in orthotopic tumors nd heptic metsttic tumors. DISCUSSION Colorectl cncer most frequently metstsizes to the liver; n event tht is the primry cuse of deth in ptients with this disese [8,19]. Those with unresectble colorectl heptic metstses hve medin survivl of 6.9 mo nd 5-yer survivl rte close to zero [2,21]. Therefore, it is criticl to understnd better the mechnisms underlying the process of metstsis development from colorectl cncer to the liver, in order to prolong ptient survivl nd improve qulity of life. Quntittive determintion of WCA ws conducted using HPLC detection of hesperidin, which is citrus flvonoid known to be ctive ginst some oxidtive-stress-medited diseses. Hesperidin found in ornge peel is flvnone glycoside consisting of the flvone hesperidin bound to the discchride rutinose. The sugr group mkes hesperidin more wter-soluble thn hesperitin; nother compound in ornge peel [22]. Hesperidin my modertely 1135 Jnury 28, 215 Volume 21 Issue 4

12 A Reltive expression of β-ctenin Cytosolic frction Nucler frction B Reltive expression of β-ctenin Cytosolic frction Nucler frction CON WCA 5-FU WCA + 5-FU CON WCA 5-FU WCA + 5-FU C Cytosolic frction Nucler frction Orthotopic tumors β-ctenin GAPDH β-ctenin H Heptic metsttic tumors β-ctenin GAPDH β-ctenin H3 Figure 6 Cell frctiontion nd western blot nlysis of β-ctenin protein expression in both orthotopic tumors nd heptic metsttic tumors. A, B: Quntifiction of β-ctenin expression in cytosolic frction nd nucler frction in orthotopic tumors (A) nd in heptic metsttic tumors (B). Dt were presented s men ± SD nd P <.5 vs corresponding CON group; C: β-ctenin expression in cytosolic frction nd nucler frction in orthotopic tumors nd heptic metsttic tumors. Lnes 1-3, CON group; 4-6, WCA group; 7-9, 5-FU group; nd 1-12, WCA + 5-FU group. WCA: Weichng n; 5-FU: 5-fluorourcil; CON: Control. stimulte the gstrointestinl trct, promote the secretion of digestive enzymes, remove intestinl pneumtosis, nd invigorte the stomch to relieve phlegm. Exogenous hesperidin hs been shown to influence wide vriety of biologicl functions, such s inducing poptosis nd suppressing prolifertion of humn cncer cells [23], s well s inhibiting tumor development in vrious tissues, including colon cncer [24,25]. In WCA, hesperidin coopertes with other components to exert spleen invigorting, het clering, detoxicting nd hrd lump resolving effects. Hesperidin is stble nd controllble component, nd HPLC detection of hesperidin fcilittes the monitoring of WCA concentrtion. In our current study, orthotopic colon tumors grown in 1% of the mice. Importntly, tumors displyed pthogenic chrcteristics similr to those observed in clinicl cses of dvnced colorectl cncer [26]. The men weights of the orthotopic tumors in the 5-FU nd WCA + 5-FU groups were significntly lower thn in the CON group (both P <.1). Additionlly, the men size of orthotopic tumors in the WCA + 5-FU group ws significntly smller thn in the CON group (P <.5). Tretment with WCA, 5-FU, or their combintion resulted in orthotopic tumor inhibition of 19.9%, 33.3% nd 36.8%, respectively, compred with controls. We detected heptic metstses in third of the mice used in the study. Overll, the occurrence of heptic metstsis in the CON group ws 45.45% nd 36.36% in the 5-FU group, 3% in the WCA group, nd 3% in the WCA + 5-FU group. Although the percentge of heptic metstsis ws lower in the three tretment groups compred with the CON group, sttisticl significnce ws not reched. This should be followed up by dditionl studies with lrger smple sizes to determine if the drugmedited decrese in metsttic rte is significnt. We lso found significntly lower gross metsttic rte in the WCA group (5%) nd in the WCA + 5-FU group (4%) compred with the CON group (1%). Upon tretment by OG, WCA is primrily bsorbed by the intestine, resulting in high concentrtion of WCA in the liver. This likely reduces heptic metstsis of colon cncer. Furthermore, dministrtion of WCA combined with bdominl 5-FU chemotherpy reduces peritonel metstsis of the tumor cells. Previous reserch hs estblished tht deregulted Wnt signling is involved in the development of tumors nd closely correltes with the invsiveness nd metsttic potentil of colorectl cncer [27]. β-ctenin is key downstrem meditor of the Wnt signling pthwy [28,29]. Nucler ccumultion of β-ctenin t the invsive front of tumors nd within blood vessels is strongly ssocited with 1136 Jnury 28, 215 Volume 21 Issue 4

13 heptic metstsis nd my be useful predictor of heptic metstsis in colorectl cncer [3-32]. MMP-7 is trnscribed in response to ctive Wnt-β-ctenin signling. Once trnslted into functionl protein, MMP-7 ssists in the degrdtion of extrcellulr mtrix, which is criticl event in tumor invsion nd metstsis. IHC expression of MMP-7 hs been shown to correlte with Dukes clssifiction in colorectl cncer specimens, nd introduction of MMP-7 into colorectl cncer cells mrkedly upregultes their in vivo invsive nd metsttic potentil [33]. Furthermore, MMP-7 promotes heptic metstsis in colorectl cncer [34]. Reserch hs lso shown tht elevted MMP-7 expression is relted to decresed survivl, nd it is considered s predictor of disese recurrence nd heptic metstsis [35]. The present study showed tht dministrtion of WCA significntly reduced the expression of β-ctenin nd MMP-7 mrna in orthotopic tumors nd in heptic metsttic tumors in mice. Similrly, WCA, lone nd in combintion with 5-FU, reduced β-ctenin nd MMP-7 protein expression in orthotopic nd metsttic tumors. Some results detected using immunoblotting, IHC nd rel-time PCR did not show significnt differences. The orthotopic trnsplnt model used in this study is reltively nturl nude mouse model of heptic metstses, nd the metstsis rte is not very high. Only 3-5 nimls in ech group developed liver metstses, which is not sttisticlly powerful. In future study, we will increse the smple size nd this problem my be resolved. CEA is often expressed in colorectl cncer nd medites the metstsis of these cncerous cells from the colon to the liver [36]. CEA binds to receptors on the surfce of Kupffer cells nd promotes the relese of interleukin (IL)-1, IL-6, nd tumor necrosis fctor-α [37]. In turn, these molecules increse the expression of intercellulr dhesion molecule-1 nd vsculr cell dhesion molecule-1 by endothelil cells, thereby reinforcing the dhesion between the tumor cells nd endothelil cells. Recent reports hve shown tht mesurement of serum CEA levels, in combintion with CA199 nd CA125, cn serve s n importnt predictor of heptic metstsis of colorectl cncer [36]. Moreover, serum CEA levels in combintion with imging techniques my ccurtely predict tumor recurrence following rdicl surgery of colorectl heptic metstses [38]. Importntly, our dt showed tht dministrtion of WCA nd 5-FU effectively reduced serum CEA levels in the orthotopic trnsplnt nude mouse model, which my be ssocited with the decresed rte of heptic metstsis tht we observed. Pthologicl nlysis reveled tht the degree of typi in both orthotopic tumors nd heptic metstses ws significntly lower in the WCA group compred with the CON group. The difference between the CON group nd the WCA + 5-FU group ws even more striking. Considering the reltively low rte of dverse effects of TCM, the combintion of WCA with 5-FU my present good strtegy in colon cncer tretment. Limittions of this study included the reltively smll number of nimls in ech group, uncler exct components in WCA, nd indequte ssessment of the immunologicl effects of WCA. In ddition, different tretment strtegies need to be investigted to optimize the effect of WCA. Furthermore, how WCA ffects the ngiogenesis of metsttic tumor, tumor microenvironment nd immunologicl response requires further reserch. In summry, combintion of the TCM WCA with 5-FU inhibited colon tumor growth nd heptic metstsis in n orthotopic trnsplnt nude mouse model. Decresed expression of β-ctenin nd MMP-7 my be importnt for the nti-tumor properties of WCA nd 5-FU. COMMENTS Bckground It is estimted tht 5%-6% of ptients dignosed with colorectl cncer will develop heptic metstses nd the most frequent site of metstsis is the liver. Eighty to ninety percent of these metstses will be unresectble nd require multidisciplinry tretments, including chemotherpy, liver-trgeting therpies nd trditionl Chinese medicine (TCM). TCM hs number of benefits, including decresing the negtive side effects ssocited with chemotherpy nd rdiotherpy, improving ptients immune function, nd enhncing the effects of conventionl cncer tretments. Reserch frontiers A previous clinicl study hs indicted tht ptients with gstric crcinom benefit from Weichng n (WCA) tretment. In ddition, WCA hs been shown to increse the 5-yer survivl rte nd reduce the 1- nd 2-yer metsttic rtes in ptients with colorectl cncer. Innovtions nd brekthroughs We demonstrted tht combintion of WCA with 5-fluorourcil (5-FU) suppressed colon tumor growth nd heptic metstses, reducing the tumor weight nd size, the rte of metstses nd serum crcinoembryonic ntigen levels. The uthors lso confirmed tht expression levels of β-ctenin nd mtrix metlloproteinse (mmp)-7 re involved in the process of metstsis from colorectl cncer to the liver, s well s the inhibitory effect of WCA nd 5-FU. Applictions This study provided new therpeutic strtegy for colorectl cncer with heptic metstsis nd my gretly contribute to prolong ptient survivl nd improve their qulity of life. Terminology Orthotopic trnsplnt nude mouse model: HCT-116 cells were subcutneously injected into the right xill of nude mice. After 3 wk, tumors were excised nd cut into 1-2-mm 3 pieces using sterile techniques. Tumor blocks were trnsplnted into the cecum of nude mice by purse-string suture. Peer review The uthors nlyzed the effect of the TCM wc on colorectl crcinom in cliniclly interesting orthotopic model. their mjor finding ws reduction in tumor weight nd size s well s the number of metstses when wc ws combined with 5-fu. they lso speculte tht decresed expression of β-ctenin nd mmp-7 my be involved in the nti-tumorigenic properties of wc. the design, technicl performnce nd some of the conclusions mde in this study re comprehensible. the uthors ddress cliniclly very relevnt topic. the mnuscript is overll written in good English Jnury 28, 215 Volume 21 Issue 4

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15 35 Fng YJ, Lu ZH, Wng GQ, Pn ZZ, Zhou ZW, Yun JP, Zhng MF, Wn DS. Elevted expressions of MMP7, TROP2, nd survivin re ssocited with survivl, disese recurrence, nd liver metstsis of colon cncer. Int J Colorectl Dis 29; 24: [PMID: DOI: 1.17/s z] 36 Zhng D, Yu M, Xu T, Xiong B. Predictive vlue of serum CEA, CA19-9 nd CA125 in dignosis of colorectl liver metstsis in Chinese popultion. Heptogstroenterology 213; 6: [PMID: DOI: /hge121125] 37 Gngopdhyy A, Lzure DA, Thoms P. Adhesion of colorectl crcinom cells to the endothelium is medited by cytokines from CEA stimulted Kupffer cells. Clin Exp Metstsis 1998; 16: [PMID: ] 38 Verberne CJ, Wiggers T, Vermeulen KM, de Jong KP. Detection of recurrences during follow-up fter liver surgery for colorectl metstses: both crcinoembryonic ntigen (CEA) nd imging re importnt. Ann Surg Oncol 213; 2: [PMID: DOI: /s ] P- Reviewer: Cre F, Linnebcher M, Mrtinez-Zorzno VS S- Editor: M YJ L- Editor: Kerr C E- Editor: Zhng DN 1139 Jnury 28, 215 Volume 21 Issue 4

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