8-bromo-7-methoxychrysin inhibits properties of liver cancer stem cells via downregulation of β-catenin

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1 Online Submissions: doi:1.3748/wjg.v19.i World J Gstroenterol 213 November 21; 19(43): ISSN (print) ISSN (online) 213 Bishideng Publishing Group Co., Limited. All rights reserved. ORIGINAL ARTICLE 8-bromo-7-methoxychrysin inhibits properties of liver cncer stem cells vi downregultion of β-ctenin Mei-Fng Qun, Li-Hong Xio, Zhi-Hong Liu, Hui Guo, Ki-Qun Ren, Fei Liu, Jin-Guo Co, Xi-Yun Deng Mei-Fng Qun, Li-Hong Xio, Ki-Qun Ren, Fei Liu, Jin- Guo Co, Xi-Yun Deng, Medicl College, Hunn Norml University, Chngsh 4113, Hunn Province, Chin Zhi-Hong Liu, Hui Guo, Deprtment of pthology, Hunn Provincil Tumor Hospitl, Chngsh 4113, Hunn Province, Chin Author contributions: Qun MF nd Xio LH contributed eqully to this work; Qun MF, Xio LH, Liu ZH, Guo H, Ren KQ nd Liu F performed the mjority of experiments; Co JG designed the study nd wrote the mnuscript; Deng XY prticipted in the study nd helped to drft the mnuscript. Supported by Ntionl Nturl Science Foundtion of Chin, No ; nd Scientific Reserch Fund of Hunn Norml University, No Correspondence to: Jin-Guo Co, Professor, Medicl College, Hunn Norml University, Yuelu District, Chngsh 4113, Hunn Province, Chin. cojinguo25@126.com Telephone: Fx: Received: My 22, 213 Revised: September 13, 213 Accepted: September 16, 213 Published online: November 21, 213 Abstrct AIM: To evlute whether 8-bromo-7-methoxychrysin (BrMC), synthetic nlogue of chrysin, inhibits the properties of cncer stem cells derived from the humn liver cncer MHCC97 cell line nd to determine the potentil mechnisms. METHODS: CD133 + cells were sorted from the MHCC97 cell line by mgnetic ctivted cell sorting, nd mplified in stem cell-conditioned medium to obtin the enriched CD133 + sphere forming cells (SFCs). The stem cell properties of CD133 + SFCs were vlidted by the tumorsphere formtion ssy in vitro nd the xenogrft nude mouse model in vivo, nd termed liver cncer stem cells (LCSCs). The effects of BrMC on LC- SCs in vitro were evluted by MTT ssy, tumorsphere formtion ssy nd trnswell chmber ssy. The effects of BrMC on LCSCs in vivo were determined using primry nd secondry xenogrft model in Blb/c-nu mice. Expressions of the stem cell mrkers, epithelilmesenchyml trnsition (EMT) mrkers nd β-ctenin protein were nlyzed by western blotting or immunohistochemicl nlysis. RESULTS: CD133 + SFCs exhibited stem-like cell properties of tumorsphere formtion nd tumorigenesis cpcity in contrst to the prentl MHCC97 cells. We found tht BrMC preferentilly inhibited prolifertion nd self-renewl of LCSCs (P <.5). Furthermore, BrMC significntly suppressed EMT nd invsion of LCSCs. Moreover, BrMC could efficciously eliminte LCSCs in vivo. Interestingly, we showed tht BrMC decresed the expression of β-ctenin in LCSCs. Silencing of β-ctenin by smll interfering RNA could synergize the inhibition of self-renewl of LCSCs induced by BrMC, while Wnt3 tretment ntgonized the inhibitory effects of BrMC. CONCLUSION: BrMC cn inhibit the functions nd chrcteristics of LCSCs derived from the liver cncer MHCC97 cell line through downregultion of β-ctenin expression. 213 Bishideng Publishing Group Co., Limited. All rights reserved. Key words: Liver cncer; Cncer stem cell; 8-bromo- 7-methoxychrysin; Self-renewl; β-ctenin Core tip: We successfully obtined liver cncer stem cells (LCSCs) from the liver cncer MHCC97 cell line by employing the combintion of mgnetic ctivted cell sorting nd tumorsphere culture. We showed for the first time tht 8-bromo-7-methoxychrysin (BrMC), synthetic nlogue of chrysin, could preferentilly inhibit prolifertion nd self-renewl, suppress epithelilmesenchyml trnsition nd invsion of LCSCs, nd further erdicte LCSCs in vivo. The results of this study support the use of BrMC for liver cncer chemopreven- 768 November 21, 213 Volume 19 Issue 43

2 tion or chemotherpy. Qun MF, Xio LH, Liu ZH, Guo H, Ren KQ, Liu F, Co JG, Deng XY. 8-bromo-7-methoxychrysin inhibits properties of liver cncer stem cells vi downregultion of β-ctenin. World J Gstroenterol 213; 19(43): Avilble from: URL: DOI: INTRODUCTION Humn liver cncer is the fifth most common cncer in the world nd the third leding cuse of cncer-relted deth [1,2]. Although surgery, liver trnsplnttion or chemotherpy offers the possibility of prolonged survivl for liver cncer ptients, mortlity still remins high, lrgely due to recurrence nd drug-resistnce [3,4]. According to the cncer stem cell hypothesis, this is thought to be due to the survivl of popultion of chemoresistnt cells within the tumor, the cncer stem cells (CSCs) in liver cncer, tht re ble to regenerte the tumor following chemotherpy [5]. However, most currently vilble therpeutic pproches, including chemotherpy nd rdiotherpy, lck the bility to effectively kill these CSCs, which my eventully led to the disese relpse nd metstsis [6,7]. A number of previous studies hve suggested tht CD133, originlly identified s hemtopoietic stem cell mrker, could be used to isolte liver cncer stem cells (LCSCs) from humn liver cncer cell lines, xenogrft tumors nd primry liver cncer specimens [8-13]. These CD133 + liver cncer cells possess mny stem cell properties, including extensive prolifertion, self-renewl, nd differentition into the bulk of cncer cells. Thus, this minor subpopultion of CD133 + LCSCs my contribute to the high recurrence rte of liver cncer. Therefore, the identifiction of compound tht cn trget LCSCs is one of the min steps in improving overll survivl of liver cncer ptients. More recently, number of studies hve found tht severl dietry compounds cn directly or indirectly inhibit cncer stem cell self-renewl pthwys [14]. For exmple, nturl flvonoid, genistein nd synthetic derivtive of didzein, N-t-boc-didzein, hve been reported to possess inhibitory ctivity ginst prostte nd epithelil ovrin CSCs, respectively [15,16]. Chrysin (5,7-dihydroxyflvone), nturlly widely distributed flvonoid, hs been shown to possess promising effects on the inhibition of prolifertion nd induction of poptosis in vriety of cncer cells [17]. 8-bromo-7-methoxychrysin (BrMC) is synthetic derivtive of chrysin, nd our previous study demonstrted the effect of BrMC in inhibiting prolifertion nd induction of poptosis in colon, gstric nd liver cncer cells ws stronger thn tht of chrysin [18-21]. In this study, we investigted the inhibitory effects of BrMC on the chrcteristics of LCSCs. We showed for the first time tht BrMC ws ble to inhibit cncer stem cell-like properties of LCSCs nd eliminte LCSCs in vivo. We lso found tht BrMC significntly decresed β-ctenin expression in LCSCs nd knockdown of β-ctenin expression could synergize the inhibition of self-renewl of LCSCs induced by BrMC. Together, our results indicted tht the downregultion of β-ctenin expression ppered to contribute to the inhibitory effects of BrMC on the properties of LCSCs. MATERIALS AND METHODS Cell culture nd regents The humn liver cncer MHCC97 cell line ws purchsed from Fuxing Biotechnology Co., Ltd. (Shnghi, Chin). MHCC97 cells were mintined in DMEM supplemented with 1% fetl bovine serum, 1 U/mL penicillin nd 1 μg/ml streptomycin (Invitrogen Life Technologies, Crlsbd, CA, United Sttes) in n incubtor contining 5% CO2 t 37. Wnt3-conditioned medium ws prepred s described by Willert et l [22]. BrMC ws synthesized s described previously [18]. MTT ws purchsed from Sigm (St. Louis, MO, United Sttes). Fetl bovine serum ws from Hngzhou Sijiqing Biologicl Engineering Mterils Co., Ltd. (Hngzhou, Chin). Trypsin nd DMSO were from Amersco Compny (Solon, OH, United Sttes). Antibodies used in this study were s follows: rbbit polyclonl ntibodies ginst ZO-1 (Abcm, Cmbridge, MA, United Sttes), mouse monoclonl ntibodies ginst N-cdherin (Upstte Co., Lke Plcid, NY, United Sttes), Vimentin (Neo Mrkers, Fremont, CA, United Sttes), E-cdherin (BD Trnsduction Lbs, Lexington, KY, United Sttes), β-ctenin nd CD44 (Cell Signling Technology Inc., Dnvers, MA, United Sttes), (Sigm Chemicl Co., St Louis, MO, United Sttes), nd horserdish peroxidse-conjugted got ntimouse secondry ntibody (Snt Cruz Biotechnology, Snt Cruz, CA, United Sttes). Cell sorting nd flow cytometry Cell sorting ws performed on MHCC97 cells using the cell surfce mrker CD133 + with mgnetic ctivted cell sorting (MACS) seprtion columns (Miltenyi Biotec, Bergisch Gldbch, Germny) ccording to the mnufcturer s protocol. Cells were trypsinized nd wshed with PBS, nd suspended in Phosphte buffered sline (PBS) contining.5% Bovine Serum Albumin (BSA). 1 μl Fc receptor (FCR) Blocking Regent (nti-cd133 ntibody) nd 1 μl CD133-conjugted MicroBeds (AC133, Cell Isoltion Kit, Miltenyi Biotec) per 1 8 cells were subsequently dded to the smple nd incubted in prllel for 3 min on ice. After wshing the cells, CD133 positive nd negtive frctions were ech isolted through MACS seprtion columns. The qulity of sorting ws controlled by flow cytometry nlysis for CD133 expression using PE-conjugted nti-humn CD133 ntibody nd isotype control mouse IgG2b-PE (Biolegend, Sn Diego, CA, United Sttes). The single cell suspension ws cultured in stem cell-conditioned medium (DMEM/F November 21, 213 Volume 19 Issue 43

3 medium supplemented with 1 B27, 2 ng/ml EGF, 2 ng/ml bfgf,.4% BSA, 4 μg/ml Insulin, 1 U/mL penicillin nd 1 μg/ml streptomycin; Invitrogen) for the following ssys. Tumorsphere culture Single-cell suspensions were suspended t density of 2 cells/ml in stem cell-conditioned medium nd seeded into ultrlow ttchment 24-well pltes (Corning, NY, United Sttes). When the dimeter of the spheroid reched 5 μm, suspension cultures were pssged every 6 d. Colonies were counted t 1 different views under microscope (Nikon, Jpn). The volume of the spheroid ws estimted using V = (4/3) π R 3. Experiments were repeted 3 times with dupliction in ech experiment. Western blotting nlysis The procedures for preprtion of whole cell lystes nd western blotting nlysis hve been previously described [23]. Mouse nti-humn β-ctenin, N-cdherin, vimentin, E-cdherin, ZO-1, CD133, CD44 nd ntibodies were used s primry ntibodies. Signls were visulized using chemiluminescent substrte (ECL; Amershm, Arlington Heights, IL, United Sttes). ws used s n internl control. Imges were scnned, followed by densitometry nlysis with UN-SCAN-IT softwre (Silk Scientific Inc., Orem, UT, United Sttes). MTT ssy CD133 + sphere-forming cells (SFCs) or prentl MHCC97 cells were seeded in 96-well plte pre-coted with.6% grose t density of 5 cells/well s described previously [24]. One dy fter plting, vrious concentrtions of BrMC (.1,.3 1., 3. or 1. μmol/l) were dded to ech well nd the culture continued for 48 h. After removl of the medium, cells were incubted with 5 mg/ml of MTT for 4 h. Cells were then extrcted with cidic isopropnol nd the bsorbnce t 57 nm (A57) ws mesured by mens of n enzyme-lbeling instrument (EXL-8 type). The reltive cell prolifertion inhibition rte = (verge A57 of the experimentl group/verge A57 of the control group) 1%. Mtrigel invsion ssy The invsion bility of tumor cells ws exmined in vitro using trnswell chmber system with 8. μm pore polycrbonte filter inserts (Corning Coster, Cmbridge, MA, United Sttes). The lower side of the filter ws coted with 1 μl geltin (1 mg/ml), nd the upper side ws coted with 1 μl of Mtrigel. Prentl MHCC97 cells or LCSCs (2 1 3 ) were plced in the upper prt of the filter. 1% fetl bovine serum ws dded in the lower prt of the chmber s chemicl ttrctnt. The chmber ws then incubted t 37 for 48 h. Cells tht could not invde through the filter were removed with cotton swb. The cells in the lower prt of the chmber were fixed with methnol nd stined with crystl violet. The invsiveness of tumor cells ws determined by counting the totl number of cells on the lower side of the filter t 1 mgnifiction. In the drug-intervention experiment, cells were pretreted with different concentrtions of BrMC for 24 h prior to the trnswell chmber ssy. In vivo tumorigenicity experiments Pthogen-free Blb/c-nu mice ged 5-6 wk were purchsed from Shnghi Lbortory Animl Center (Shnghi, Chin). All niml studies were performed in ccordnce with the stndrd protocols pproved by the Ethicl Committee of Hunn Norml University nd the Committee of Experimentl Animl Feeding nd Mngement. Mice were rndomly divided into 3 groups (4 mice/group) nd mintined under stndrd conditions, ccording to the stndrd protocols. Cells were suspended in serum free-dmem/mtrigel (BD Biosciences, Sn Jose, CA, United Sttes) mixture (1:1 volume). Ech recipient Blb/c-nu mouse ws inoculted subcutneously with vrious numbers of CD133 + SFCs (2 1 3, nd cells) in one flnk nd prentl MHCC97 cells (1 1 4, nd ) in the other. Tumorigenicity experiments were terminted 2 mo fter cell inocultion. Tumor size were mesured with cliper, nd the volume ws clculted using V (mm 3 ) = L W 2.5. Hrvested tumors were imged nd weighed immeditely. Specimens from tumor tissue smples were fixed in 1% neutrl buffered formlin, processed in prffin blocks, nd sectioned. The sections were stined with hemtoxylin nd eosin (HE) nd exmined for the histopthology. For BrMC tretment studies, LCSCs per mouse were injected subcutneously. Two weeks fter inocultion, nimls were rndomly divided into 4 groups. One group underwent dily gstric lvge with refined olive oil s control, nd the other 3 groups were treted with 12.5, 25 or 5 mg/kg BrMC. After 2 d of tretment, living cells from the primry tumors were dissocited nd injected into 3 groups of mice (4 mice per group). Ech mouse ws implnted with cells from the control group nd from the 5 mg/kg BrMC treted group in ech flnk. The growth of tumors ws monitored, nd tumor volumes were mesured every 3 d. Animls were humnely scrificed when the lrger of the two tumors reched 5 mm 3. Immunohistochemicl exmintion For immunohistochemicl nlysis of CD44 nd CD133, tissues of the LCSCs derived-tumors in the nude mouse xenogrft model were performed with formlin-fixed, prffin-embedded sectioning s previously described by Moinfr et l [25]. After incubtion with 1% non-ft dry milk in PBS (ph 7.4), the sections were then rected with mouse nti-cd44 monoclonl ntibody (1:25, Cell Signling Technology Inc.) or mouse nti-cd133 monoclonl ntibody (1:2, Abzoom, Dlls, TX, United Sttes) for 1 h t room temperture followed by incubtion with the secondry biotinylted ntibody for 3 min. After wshing, sections were subsequently incubted with streptvidin-peroxidse for 3 min. Finlly, the results were vi November 21, 213 Volume 19 Issue 43

4 sulized fter 15-min incubtion with diminobenzidine. RNA interference A control non-specific smll interfering RNA (sirna) (5 -GACTTCATAAGGCGCATGC-3 ) nd β-ctenin sirna (5 -AGCUGAUAUUGAUGGACAGTT-3 ) were synthesized by Shnghi Sngon Biotech Co., Ltd. (Shnghi, Chin). Trnsfection of sirna ws crried out with Lipofectmine 2 (Invitrogen Life Technologies) ccording to the procedure recommended by the mnufcturer. Twenty-four hours fter trnsfection, the cells were treted with DMSO (control) or BrMC t the indicted concentrtions for 24 h. The cells were then collected nd processed for western blotting nd the tumorsphere formtion ssy. RESULTS Isoltion nd chrcteriztion of LCSCs derived from MHCC97 cell line CD133 hs previously been clssified s CSC mrker in liver cncer. Therefore, we first isolted the CD133 + subpopultion from MHCC97 cells by MACS. Following sorting, we exmined the expression of CD133 by flow cytometry. As shown in Figure 1A, the sorted CD133 + cells showed high purity of 57.29% ± 4.61%, s compred with purity of 1.2% ±.65% for CD133 - counterprts nd 7.21% ± 1.34% for non-sorted MHCC97 cells. To estblish long-term cultures enriched in stem cells from sorted CD133 +, we performed the tumorsphere ssy by culturing the cells in stem cellconditioned medium. Within 6 d of culture, we obtined liver cncer spheroids both in CD133 + cells nd prentl MHCC97 cells (Figure 1B). As shown in Tble 1, the CD133 + subpopultion exhibited 2.7- nd 2.5-fold enhncement in tumorsphere formtion mount nd size, respectively, compred with tht of prentl cells, wheres, CD133 - counterprts could not grow s spheroids in the nondherent nd serum-free conditions. To further confirm the stem cell properties nd functions of the CD133 + SFCs, we evluted their self-renewl cpcity nd tumorigenic potentil. First, we mesured the cpcity of single cells obtined from these CD133 + dissocited spheres to form secondry tumorspheres. Within 9 d of culture, we obtined new LCSC spheroids of growing undifferentited CD133 + cells (Figure 1C). These suggest n in vitro self-renewing cpcity of CD133 + SFCs. In ddition, CD133 + SFCs lso expressed n enhnced level of stem cell mrkers, CD133 nd CD44, compred with their prentl cells (Figure 1D). Next, we evluted the tumorigenic potentil of CD133 + SFCs. We investigted the bility of CD133 + SFCs nd prentl cells to give rise to tumors in Blb/c-nu mice. As mny s prentl cells were needed to initite stble tumor formtion 39 d fter injection, while, in contrst, s few s CD133 + SFCs were sufficient to generte visible tumors only 23 d post-injection (Tble 2). These dt indicte tht CD133 + SFCs, nmely LCSCs, re more tumorigenic thn their prentl cells in vivo. Additionlly, HE stining ws performed nd reveled similr histologicl chrcteristics in tumor xenogrfts derived from CD133 + SFCs nd their prentl cells (Figure 1E). BrMC inhibits prolifertion nd self-renewl of LCSCs derived from MHCC97 cell line CSCs possess the property of limitless prolifertive potentil. A number of previous studies hve demonstrted tht some nturlly-occurring polyphenol compounds such s genistein preferentilly inhibit prolifertion of pncretic cncer stem cells [26]. In this study, we thus evluted the nti-prolifertive effects of BrMC on LCSCs derived from MHCC97 cell line by MTT ssy. As shown in Figure 2A, when cells were treted with different concentrtions of BrMC for 48 h, BrMC preferentilly inhibited prolifertion of LCSCs in dose-dependent mnner, with the IC5 round.5 μmol/l for LCSCs nd 17.9 μmol/l for prentl MHCC97 cells. In order to evlute whether BrMC could suppress the self-renewl of LCSCs derived from the MHCC97 cell line in vitro, we treted the primry tumorspheres with vrying concentrtions of BrMC nd then removed the drug nd cultured them for nother pssge to form the secondry spheres. Results showed tht BrMC tretment resulted in decrese both in tumorsphere number nd size of LCSCs. Furthermore, significnt decrese in the number nd size of the secondry tumorspheres indicted reduced self-renewl cpcity of these LCSCs by BrMC tretment (Figure 2B nd C). BrMC inhibits Epithelil-mesenchyml trnsition nd invsion of LCSCs derived from MHCC97 cell line Epithelil-mesenchyml trnsition (EMT) is n importnt process during metstsis of LCSCs. Therefore, we sought to exmine whether morphologicl chnges existed between LCSCs nd prentl MHCC97 cells cultured dherently in vitro. As observed in Figure 3A, LCSCs exhibited spindle-like shpe, while prentl MHCC97 cells displyed cobble-stone-like phenotype. However, tretment with.1 μmol/l BrMC suppressed EMT in LCSCs s morphologicl chnges from spindle-like shpe to cobble-stone-like ppernce were displyed. Moreover, similr results were further confirmed by western blotting using specific ntibodies ginst EMT-reltive mrkers. Figure 3B shows tht LCSCs expressed higher vimentin nd N-cdherin protein levels, which re typiclly ssocited with mesenchyml cells, nd lower expression of epithelium-ssocited E-cdherin nd ZO-1 proteins. However, BrMC induced the upregultion of epithelil mrkers E-cdherin nd ZO-1 nd the downregultion of mesenchyml mrkers N-cdherin nd vimentin fter tretment for 24 h of LCSCs derived from MHCC97 cell line. Since EMT hs been identified s being ssocited with incresed cncer cell invsion, we next evluted the effect of BrMC on cell invsion of LCSCs in vitro using trnswell chmber coted with Mtrigel brrier. As 7683 November 21, 213 Volume 19 Issue 43

5 A 852 Control 52 PC 639 R2: 1.3% 378 R2: 7.19% Cell counts CD CD R2: 54.71% 687 R2: 1.83% CD133-PE B MHCC97 CD133 + MHCC97 C Dy 1 Dy 3 Dy 6 Dy November 21, 213 Volume 19 Issue 43

6 D CD44 PC CD133 + PC CD133 + CD133 E CD133 + PC Figure 1 Isoltion nd chrcteriztion of liver cncer stem cells derived from the MHCC97 cell line. A: Flow cytometry nlysis of CD133 expression following sorting. CD133 + cells from MHCC97 cells formed liver cncer spheroids in stem cell-conditioned medium (2 mgnifiction); B: Anchorge-dependent growth of MHCC97 cells, tumor spheroid formed by CD133 + cells, tumor spheroid formed by prentl MHCC97 cells; C: Secondry tumorspheres formed by single cells from dissocited primry liver spheroids (4 mgnifiction); D: Expression of stem cell surfce mrkers CD44 nd CD133 in CD133 + sphere-forming cells (SFCs) nd prentl cells; E: Hemtoxylin-eosin stining reveled similr histologicl chrcteristics in tumor xenogrfts derived from CD133 + SFCs nd their prentl cells (1 mgnifiction). Tble 1 Tumorsphere formtion bility of CD133 + cells derived from the MHCC97 cell line (men ± SD, n = 3) Cell line Spheroid number/2 1 3 cells Volume of spheroid (μm 3 ) Prentl cells CD133 + cells Prentl cells CD133 + cells MHCC97 61 ± ± ± ± 45 P <.5 vs prentl MHCC97 cells. shown in Figure 3C nd 3D, BrMC significntly reduced the invsiveness cpcity of LCSCs in dose-dependent mnner. These results demonstrted tht BrMC possesses inhibitory effects on EMT nd invsion in LCSCs derived from MHCC97 cell line. BrMC elimintes LCSCs derived from MHCC97 cell line in vivo In order to evlute whether BrMC could trget LCSCs in vivo, we utilized the xenogrft model of LCSCs from MHCC 97 cells in Blb/c-nu mice. Two weeks fter cell inocultion with LCSCs resuspended in Mtrigel, nimls underwent dily gstric lvge with vrious concentrtions of BrMC. After 2 d of tretment, tumors in 25 nd 5 mg/kg BrMC-treted mice were less thn 5% of the size of those in refined olive oil control nimls (Figure 4A nd B). Immunohistochemicl nlysis of CD44 nd CD133 in LCSC-derived tumors reveled tht the LCSC mrkers CD44 nd CD133 were minly expressed on the cell surfce of the cncer cells, nd tht the tumors derived from CD133 + SFCs showed significntly higher CD44 nd CD133 positive rtes thn tht of tumors derived from prentl cells (Figure 4C). Furthermore, BrMC tretment cn significntly decrese the CD44 nd CD133 expression frequency of the tumors derived from LCSCs (Figure 4D). To further confirm the results, we investigted the growth of secondry tumors in Blb/c-nu mice inoculted with tumor cells dissocited from primry tumor xenogrfts. In order to void possible vritions due to heterogeneity, ech recipient mouse ws inoculted with cells obtined from 5 mg/kg BrMC-treted tumors nd nother cells obtined from control tumors in two opposite sides. Interestingly, we found tht tumor cells from control nimls exhibited rpid tumor re-growth, reching finl tumor volume of mm 3. However, the tumor cells from 5 mg/kg BrMC November 21, 213 Volume 19 Issue 43

7 A Reltive inhibition rte (%) Unsorted cells CD133 + sphere-forming cells Concentrtion (μmol/l) B Primry tumorspheres Secondry tumorspheres No. of tumorspheres (per 1 3 cells) ,c Unsorted cells.1% DMSO c CD133 + sphere-forming cells,c c,c No. of tumorspheres (per 1 3 cells) c,c.1% DMSO c Unsorted cells CD133 + sphere-forming cells,c,c c C Control st 2 nd Figure 2 Effects of 8-bromo-7-methoxychrysin on cell prolifertion nd self-renewl. 8-bromo-7-methoxychrysin (BrMC) inhibited prolifertion (A), self-renewl (B nd C) of liver cncer stem cells derived from MHCC97 cell line (men ± SD, n = 3). P <.5 vs unsorted MHCC97 cells treted with corresponding concentrtions of BrMC. c P <.5 vs corresponding.1% DMSO treted group. Tumor sphere morphology is shown s the phse contrst imge (4 mgnifiction). treted mice mostly filed to generte ny tumors up to 33 d fter inocultion (Tble 3). These results suggest tht BrMC ws ble to eliminte LCSCs in primry tumor xenogrfts, thereby inhibiting tumor regrowth in secondry inoculted mice. BrMC inhibits self-renewl in LCSCs through modultion of β-ctenin expression To exmine whether BrMC could regulte expression of stem cell mrkers of LCSCs, we determined the expression of CD44 nd CD133 following BrMC tretment 7686 November 21, 213 Volume 19 Issue 43

8 A PC CD μmol/l BrMC B PC CD133 + Control E-Cd Actin N-Cd Actin Vimentin Actin ZO-1 Actin C PC CD November 21, 213 Volume 19 Issue 43

9 D Invded cell numbers (per cells) c,c Unsorted cells CD133 + sphere-forming cells c,c c,c.1% DMSO Figure 3 8-bromo-7-methoxychrysin inhibition of liver cncer stem cells. 8-bromo-7-methoxychrysin (BrMC) inhibited epithelil-mesenchyml trnsition (EMT, A nd B) nd invsion (C nd D) of liver cncer stem cells derived from the MHCC97 cell line (men ± SD, n = 3). Cell morphologicl chnges ssocited with EMT re shown s the phse contrst imge (2 mgnifiction). P <.5 vs unsorted MHCC97 cells treted with corresponding concentrtions of BrMC, c P <.5 vs corresponding.1% DMSO treted group. by western blotting nlysis. Results showed tht BrMC downregulted CD44 nd CD133 expression in dosedependent mnner (Figure 5A). This ws in ccordnce with our previous immunohistochemicl nlysis in LCSC-derived tumors (Figure 4D). CD44 hs been shown to be downstrem trget of the β-ctenin signling pthwy [27]. Wnt/β-ctenin signling hs been implicted in the mintennce of CSCs of liver cncer [28]. Therefore, we mesured the expression level of stem cell signl molecule β-ctenin in LCSCs nd prentl MHCC97 cells, nd exmined whether β-ctenin ws downregulted by BrMC in LCSCs. Western blotting nlysis showed tht β-ctenin ws highly expressed in LCSCs compred with tht of prentl MHCC97 cells. We lso found tht BrMC (.1,.3, 1. μmol/l) tretment resulted in significnt decrese in β-ctenin expression of LCSCs (Figure 5B). We further determined the role of β-ctenin in the mintennce of self-renewl of LCSCs. Silencing of β-ctenin by sirna trnsfection resulted in less expression of β-ctenin protein, s confirmed by Western blotting (Figure 5C). We lso found tht the downregultion of β-ctenin expression significntly decresed the tumorsphere formtion bility nd inhibited expression of stem cell mrkers of LCSCs (Figure 5D nd 5E). BrMC (.1 μmol/l) plus β-ctenin sirna inhibited β-ctenin expression to greter degree compred to either lone (Figure 6A). Moreover, β-ctenin sirna potentited the BrMC-induced decrese in tumorsphere formtion of LCSCs (Figure 6B). We lso treted LCSCs with Wnt3, lignd known to ctivte the Wnt/β-ctenin pthwy. As expected, Wnt3 induced β-ctenin stbiliztion nd resulted in corresponding up-regultion of β-ctenin in LCSCs (Figure 6C). This upregultion of β-ctenin ttenuted BrMC-induced downregultion of β-ctenin nd stem cell mrkers nd ntgonized BrMC-induced inhibition of self-renewl of LCSCs (Figure 6D, 6E nd 6F). Tken together, these results provide some moleculr evidence suggesting tht the downregultion of β-ctenin expression my contribute to the inhibitory effects of BrMC on LCSCs. DISCUSSION Cncer stem cells re defined s minor popultion of tumorigenic cells tht re cpble of continuous selfrenewl nd differentition, nd undergo unlimited prolifertion, giving rise to new tumors [29,3]. Therefore, finding compound(s) tht re cpble of inhibiting or killing the CSCs is extremely importnt to overcome tumor resistnce, reduce relpse, nd eventully improve overll survivl. Our previous study hs shown tht BrMC possessed promising inhibitory effects on prolifertion nd poptosis of colon, gstric nd liver cncer cells. In the current study, we first successfully isolted nd identified LCSCs from the liver cncer MHCC97 cell line. Further, we showed for the first time tht BrMC could preferentilly inhibit prolifertion nd self-renewl, nd suppress EMT nd invsion of LCSCs. Moreover, BrMC ws ble to erdicte LCSCs in vivo, s ssessed by n in vivo tumorigenicity ssy using primry nd secondry Blb/ c-nu mouse models. Secondly, we found tht the inhibitory effects of BrMC on stem cell function nd properties of LCSCs were medited by inhibition of β-ctenin pthwys: β-ctenin sirna trnsfection nd BrMC were synergistic in inhibiting the self-renewl of LCSCs. Conversely, the inhibition of Wnt3 in LCSCs resulted in n opposite effect. More recently, CD133 hs been used s surfce mrker of CSCs in vrious solid tumors, including liver cncer. However, the function of CD133 is not entirely known yet. Thus, the single phenotypic mrker CD133 is not sufficient to identify LCSCs. Tumorsphere culture my provide n lterntive pproch to identify nd enrich LCSCs. Under non-dherent serum-free conditions in vitro, most tumor cells undergo progrmmed cell deth, 7688 November 21, 213 Volume 19 Issue 43

10 A Tumor volumes (mm 3 ) BrMC (mg/kg) Tumor weight (g) ,c,c Dys post tretment BrMC tretment (mg/kg) B Control C PC SFCs CD133 CD November 21, 213 Volume 19 Issue 43

11 D Control CD133 CD44 Figure 4 8-bromo-7-methoxychrysin eliminted liver cncer stem cells derived from MHCC97 cell line in vivo. Effects of 8-bromo-7-methoxychrysin (BrMC) on growth of primry nd secondry tumor xenogrfts derived from liver cncer stem cells (LCSCs) (A nd B, men ± SD, n = 12). P <.5 vs refined olive oil tretment model, c P <.5 vs tretment with 12.5 mg/kg BrMC. Immunohistochemicl nlysis of CD44 nd CD133 in LCSC-derived tumors before nd fter BrMC tretment (C nd D). Tble 2 Tumorigenicity of CD133 + sphere forming cell derived from MHCC97 cells in Blb/c-nu mice Cell type No. inoculted cells Tumor incidence 1 Ltency (d) 2 Prentl cells / / /4 39 CD133 + SFCs / / /4 8 1 Number of tumors detected/number of injections; 2 Approximte number of dys from tumor cell injection to ppernce of tumor. SFC: Sphere forming cells. wheres the rre CSCs divide to generte multicellulr 3-dimensionl spheres [31,32]. This ssy is powerful tool to enrich CSCs nd further ssess the functionl properties of the isolted CSCs. By employing combintion of this technique nd MACS bsed on the CD133 + surfce mrker, we hve successfully obtined the puttive LC- SCs, nmely CD133 + SFCs, from the MHCC97 cell line. We demonstrted tht these CD133 + SFCs possess stemlike properties, including self-renewl, initition of tumor growth in mice t very low cell numbers nd higher expression level of stem cell mrker compred with their prentl cells. These dt indicted tht the method which we used to isolte nd indentify LCSCs from liver cncer cell lines my be fster, more economic nd effective, compred with methods bsed on two or more surfce mrkers. Tble 3 Effects of 8-bromo-7-methoxychrysin on growth of secondry tumors in Blb/c-nu mice inoculted with tumor cells obtined from primry xenogrfts Time (d) Tumor incidence 1 Volume (mm 3 ) Control BrMC-treted group Control BrMC-treted group 1 /12 / /12 / /12 /12 28 ± /12 / ± /12 3/ ± ± /12 3/ ± ± /8 2/8 351 ± ± /8 2/8 593 ± ± /4 1/4 264 ± /4 1/4 387 ± /4 1/4 567 ± Number of tumors detected/number of injections. BrMC: 8-bromo-7- methoxychrysin The Wnt/β-ctenin pthwy is one of the key pthwys tht modultes stem cell self-renewl [14]. For exmple, overexpression of β-ctenin enhnced self-renewl preferentilly nd medited rdition resistnce of Sc1 + progenitors in n immortlized mmmry glnd cell line [33]. Hllett et l [34] reported tht phrmcologicl inhibitors of Wnt/β-ctenin signling could inhibit the vibility nd or self-renewl of brest tumor-inititing cells, nd trget brest tumor-inititing cells in Her2/Neu mouse model of brest cncer. Consistent with these previous reports, we found tht downregultion of β-ctenin 769 November 21, 213 Volume 19 Issue 43

12 A Control CD44 CD133 B β-ctenin PC CD133 + Control C β-ctenin CD133 + Control sirna β-ctenin sirna D CD133 + Control sirna β-ctenin sirna No. of tumorspheres (per cells) CD133 + cells Control sirna β-ctenin sirna 7691 November 21, 213 Volume 19 Issue 43

13 E CD133 + Control sirna β-ctenin sirna F BrMC (.1 μmol/l) CD44 β-ctenin sirna β-ctenin CD133 G CD133 + BrMC (.1 μmol/l) + Control sirna β-ctenin sirna BrMC (.1 μmol/l) + β-ctenin sirna 25 No. of tumorspheres (per cells) ,c sirna Control Control β-ctenin β-ctenin BrMC (.1 μmol/l) Figure 5 β-ctenin sirna synergized the inhibitory effects of 8-bromo-7-methoxychrysin. 8-bromo-7-methoxychrysin (BrMC) downregulted CD44 nd CD133 expression in liver cncer stem cells in concentrtion-dependent mnner (A). β-ctenin ws highly expressed in CD133 + sphere forming cells (SFCs) nd ws downregulted by BrMC tretment (B). β-ctenin sirna decresed the protein level of β-ctenin (C) nd stem cell mrkers (E), nd significntly inhibited self-renewl cpcity (D) of CD133 + SFCs (men ± SD, n = 3). P <.5 vs CD133 + SFCs or control sirna trnsfected CD133 + SFCs. BrMC enhnced β-ctenin sirna induced downregultion of β-ctenin expression (F) nd inhibition of self-renewl cpcity (G) in CD133 + SFCs (men ± SD, n = 3). P <.5 vs CD133 + SFCs derived from the MHCC97 cell line. c P <.5 vs.1 μmol/l BrMC or β-ctenin sirna treted CD133 + SFCs November 21, 213 Volume 19 Issue 43

14 A β-ctenin MHCC97 cells CD133 + SFCs Control wnt3 Control wnt3 B wnt BrMC (.1 μmol/l) β-ctenin C wnt BrMC (.1 μmol/l) CD44 CD133 D No. of tumorspheres (per cells) ,c Wnt BrMC (.1 μmol/l) Figure 6 Wnt3 tretment ntgonized the inhibitory effects of 8-bromo-7-methoxychrysin. Wnt3 tretment resulted in n increse in the expression of β-ctenin in both liver cncer stem cells (LCSCs) nd prentl MHCC97 cells (A) nd ttenuted the effects of 8-bromo-7-methoxychrysin (BrMC) on the expression of β-ctenin (B) nd stem cell mrkers (C), nd self-renewl cpcity (D) of LCSCs derived from the MHCC97 cell line. P <.5 vs CD133 + SFCs, c P <.5 vs.1 μmol/l BrMC or Wnt 3 lone treted group. by BrMC resulted in inhibition of CSC function nd chrcteristics of LCSCs, such s significnt inhibition of prolifertion nd self-renewl, suppression of EMT nd invsiveness, downregultion of the expression of stem cell mrkers of LCSCs, nd further efficcious promotion of the elimintion of LCSCs in vivo. Previous studies hve shown tht ctivted Akt ws ble to phosphorylte Ser9 on GSK3β, which my decrese the ctivity of GSK3β, thereby leding to stbiliztion of β-ctenin in the cytoplsm [35]. Chrysin ws reported to induce poptosis through cspse ctivtion nd Akt inctivtion in leukemi cells [36]. Our previous study lso demonstrted tht 5,7-dihydroxy-8-nitrochrysin, nother synthetic chrysin nlogue, could induce ctivtion nd nucler locliztion of FOXO3, which ws ssocited with reduced levels of Akt phosphoryltion. Therefore, we speculte tht the downregultion of β-ctenin by BrMC probbly occurs vi reduced levels of Akt nd ctivtion of GSK3β, with the consequent degrdtion of β-ctenin. Wnt3 tretment cn induce stbiliztion of β-ctenin, with entry into the nucleus nd subsequent ctivtion of the β-ctenin pthwy. Thus, Wnt3 tretment cn ntgonize the inhibitory effects of BrMC on self-renewl of LCSCs. On the other hnd, Su et l [37] reported tht genistein increses levels of membrne E-cdherin nd E-cdherin-β-ctenin cell dhesion complex, nd eventully ttenutes β-ctenin signling in mmmry epithelil cells. We lso found tht E-cdherin, n epithelil mrker, ws upregulted by BrMC in LCSCs. E-cdherin is known to nchor nd to sequester β-ctenin in the membrne nd prevent its ctivtion. Therefore, we suppose tht this inctivtion of β-ctenin by upregultion of E-cdherin cn lso contribute to the inhibitory effects of LCSCs by BrMC. Interestingly, the inhibition of β-ctenin t the protein level ws not optiml, s tretment of β-ctenin sirna cn further downregulte β-ctenin t the trnscription level nd synergize the inhibition of self-renewl of LC- SCs induced by BrMC. In conclusion, we hve presented supportive evidence for the first time tht BrMC, novel synthetic chrysin nlogue, cn trget LCSCs both in vitro nd in vivo. Furthermore, our study identified the downregultion of β-ctenin expression by BrMC s one of the possible mechnisms for its efficcy. These studies support the use of BrMC for liver cncer chemoprevention or chemotherpy. These findings provide strong rtionle for preclinicl nd subsequent clinicl evlution of BrMC for liver cncer therpy. COMMENTS Bckground Liver cncer is the fifth most common cncer in the world nd the third leding cuse of cncer-relted deth. Recent studies indicted tht cncer stem cells (CSCs) my be responsible for tumor recurrence nd drug-resistnce. Therefore, the identifiction of compound tht cn trget liver CSCs (LCSCs) is one of the min steps in improving overll survivl of liver cncer ptients. Reserch frontiers More recently, number of studies hve found tht some dietry compounds cn directly or indirectly ffect CSC self-renewl pthwys. 8-bromo-7- methoxychrysin (BrMC) is synthetic derivtive of chrysin, nd their previous study hve demonstrted the effect of BrMC on the inhibition of prolifertion 7693 November 21, 213 Volume 19 Issue 43

15 nd induction of poptosis in colon, gstric nd liver cncer cells ws stronger thn tht of chrysin. However, the inhibitory effects of BrMC on the chrcteristics of CSCs hve not been reported yet. Innovtions nd brekthroughs The uthors firstly showed tht BrMC, novel synthetic chrysin nlogue, ws ble to inhibit cncer stem cell-like properties of LCSCs nd eliminte LCSCs in vivo. They lso found tht BrMC significntly decresed β-ctenin expression in LCSCs nd knockdown of β-ctenin expression could synergize the inhibition of self-renewl of LCSCs induced by BrMC. The downregultion of β-ctenin expression ppers to contribute to the inhibitory effects of BrMC on the properties of LCSCs. Applictions The present study provided strong evidences for the first time tht BrMC ws ble to trget LCSCs both in vitro nd in vivo. These studies support the use of BrMC for liver cncer chemoprevention or chemotherpy. Terminology Chrysin (5,7-dihydroxyflvone), nturlly wide distributed flvonoid, hs been reported to possess nti-cncer ctivities. BrMC is novel synthetic chrysin nlogue. Peer review This mnuscript concludes tht 8-bromo-7-methoxychrysin cn inhibit the functions nd chrcteristics of liver cncer stem cells derived from liver cncer MHCC97 cell line through downregultion of β-ctenin expression. It is good reserch with necessry informtion. REFERENCES 1 Dudeck O, Ricke J. Advnces in regionl chemotherpy of the liver. Expert Opin Drug Deliv 211; 8: [PMID: DOI: / ] 2 Jeml A, Bry F, Center MM, Ferly J, Wrd E, Formn D. Globl cncer sttistics. CA Cncer J Clin 211; 61: 69-9 [PMID: DOI: /cc.217] 3 Snyl AJ, Yoon SK, Lencioni R. The etiology of heptocellulr crcinom nd consequences for tretment. Oncologist 21; 15 Suppl 4: [PMID: DOI: /theo ncologist.21-s4-14] 4 Villnuev A, Llovet JM. Trgeted therpies for heptocellulr crcinom. Gstroenterology 211; 14: [PMID: DOI: 1.153/j.gstro ] 5 Oishi N, Wng XW. 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J Microbiol Biotechnol 29; 19: [PMID: ] 25 Moinfr F, Okcu M, Tsybrovskyy O, Regitnig P, Lx SF, Weybor W, Rtschek M, Tvssoli FA, Denk H. Androgen receptors frequently re expressed in brest crcinoms: potentil relevnce to new therpeutic strtegies. Cncer 23; 98: [PMID: DOI: 1.12/cncr.11532] 26 Bo B, Wng Z, Ali S, Kong D, Bnerjee S, Ahmd A, Li Y, Azmi AS, Miele L, Srkr FH. Over-expression of FoxM1 leds to epithelil-mesenchyml trnsition nd cncer stem cell phenotype in pncretic cncer cells. J Cell Biochem 211; 112: [PMID: DOI: 1.12/jcb.2315] 27 Li J, Zhou BP. Activtion of β-ctenin nd Akt pthwys by Twist re criticl for the mintennce of EMT ssocited cncer stem cell-like chrcters. BMC Cncer 211; 11: 49 [PMID: DOI: / ] 7694 November 21, 213 Volume 19 Issue 43

16 28 Yng W, Yn HX, Chen L, Liu Q, He YQ, Yu LX, Zhng SH, Hung DD, Tng L, Kong XN, Chen C, Liu SQ, Wu MC, Wng HY. Wnt/bet-ctenin signling contributes to ctivtion of norml nd tumorigenic liver progenitor cells. Cncer Res 28; 68: [PMID: DOI: / CAN ] 29 Zhou BB, Zhng H, Dmelin M, Geles KG, Grindley JC, Dirks PB. Tumour-inititing cells: chllenges nd opportunities for nticncer drug discovery. Nt Rev Drug Discov 29; 8: [PMID: DOI: 1.138/nrd2137] 3 Curtin JC, Lorenzi MV. Drug discovery pproches to trget Wnt signling in cncer stem cells. Oncotrget 21; 1: [PMID: ] 31 Singh SK, Hwkins C, Clrke ID, Squire JA, Byni J, Hide T, Henkelmn RM, Cusimno MD, Dirks PB. Identifiction of humn brin tumour inititing cells. Nture 24; 432: [PMID: DOI: 1.138/nture3128] 32 Ponti D, Cost A, Zffroni N, Prtesi G, Petrngolini G, Cordini D, Pilotti S, Pierotti MA, Didone MG. Isoltion nd in vitro propgtion of tumorigenic brest cncer cells with stem/progenitor cell properties. Cncer Res 25; 65: [PMID: DOI: / CAN-5-626] 33 Chen MS, Woodwrd WA, Behbod F, Peddibhotl S, Alfro MP, Buchholz TA, Rosen JM. Wnt/bet-ctenin medites rdition resistnce of Sc1+ progenitors in n immortlized mmmry glnd cell line. J Cell Sci 27; 12: [PMID: DOI: /jcs.3348] 34 Hllett RM, Kondrtyev MK, Gicomelli AO, Nixon AM, Girgis-Gbrdo A, Iliev D, Hssell JA. Smll molecule ntgonists of the Wnt/β-ctenin signling pthwy trget brest tumor-inititing cells in Her2/Neu mouse model of brest cncer. PLoS One 212; 7: e33976 [PMID: DOI: /journl.pone.33976PONE-D ] 35 Cohen P, Frme S. The renissnce of GSK3. Nt Rev Mol Cell Biol 21; 2: [PMID: DOI: 1.138/359675] 36 Woo KJ, Jeong YJ, Prk JW, Kwon TK. Chrysin-induced poptosis is medited through cspse ctivtion nd Akt inctivtion in U937 leukemi cells. Biochem Biophys Res Commun 24; 325: [PMID: DOI: 1.116/j.bbrc ] 37 Su Y, Simmen RC. Soy isoflvone genistein upregultes epithelil dhesion molecule E-cdherin expression nd ttenutes bet-ctenin signling in mmmry epithelil cells. Crcinogenesis 29; 3: [PMID: DOI: 1.193/crcin/bgn279] P- Reviewer: Cong WM S- Editor: Wen LL L- Editor: Cnt MR E- Editor: M S 7695 November 21, 213 Volume 19 Issue 43

17 Published by Bishideng Publishing Group Co., Limited Flt C, 23/F., Lucky Plz, Lockhrt Rod, Wn Chi, Hong Kong, Chin Fx: Telephone: E-mil: I S S N Bishideng Publishing Group Co., Limited. All rights reserved.