RESEARCH ARTICLE Open Access

Butein inhibits metastatic behavior inmouse melanoma cells through VEGFexpression and translation-dependentsignaling pathway regulationYu-Wei Lai1,2, Shih-Wei Wang3, Chien-Hsin Chang4, Shih-Chia Liu5, Yu-Jen Chen6,7, Chih-Wen Chi6, Li-Pin Chiu1,2,Shiou-Sheng Chen1,2, Allen W. Chiu1,2 and Ching-Hu Chung3*

Abstract

Background: Melanoma is an aggressive skin cancer and a predominant cause of skin cancer-related deaths. Aprevious study has demonstrated the ability of butein to inhibit tumor proliferation and invasion. However, theanti-metastatic mechanisms and in vivo effects of butein have not been fully elucidated.

Methods: MTT cell viability assays were used to evaluate the antitumor effects of butein in vitro. Cytotoxiceffects of butein were measured by lactate dehydrogenase assay. Anti-migratory effects of butein wereevaluated by two-dimensional scratch and transwell migration assays. Signaling transduction and VEGF-releasing assayswere measured by Western blotting and ELISA. We also conducted an experimental analysis of the metastatic potentialof tumor cells injected into the tail vein of C57BL/6 mice.

Results: We first demonstrated the effect of butein on cell viability at non-cytotoxic concentrations (1, 3, and 10 μM).In vitro, butein was found to inhibit the migration of B16F10 cells in a concentration-dependent manner usingtranswell and scratch assays. Butein had a dose-dependent effect on focal adhesion kinase, Akt, and ERKphosphorylation in B16F10 cells. Butein efficiently inhibited the mTOR/p70S6K translational inhibition machinery anddecreased the production of VEGF in B16F10 cells. Furthermore, the in vivo antitumor effects of butein weredemonstrated using a pulmonary metastasis model.

Conclusion: The results of the present study indicate the potential utility of butein in the treatment of melanoma.

Keywords: Melanoma, Butein, Mammalian target of rapamycin, Metastasis, Vascular endothelial growth factor, mTOR,VEGF

BackgroundThe incidence of melanoma of the skin has rapidlyincreased in recent years and is currently the mostcommon fatal form of skin cancer [1]. New Zealand hasthe highest incidence of invasive melanoma worldwidewith an age-standardized rate of 40.2/100,000 [2]. Asianpopulations, including China, India, Japan, and Singapore,are reported to have relatively lower incidence rates(approximately 1/100,000). Despite being a relatively rare

cancer, melanoma is associated with substantial morbidityand mortality. This notorious aggressiveness of melanomais associated with its metastatic propensity, which canoccur even from shallow primary tumors [3]. Accordingto the clinical evidence, the survival rate in patients withmultiple site of metastasis disease is less than 5 % [4].Despite significant progress in understanding the biologyof melanoma, new targets and immune-modulatory ther-apies (such as vermurafenib and ipilimumab) have dem-onstrated efficacy in improving the overall survival inmelanoma patients [5, 6]. However, the benefit of thesetherapies is variable, and tumor metastasis still occurs inmajority of the melanoma patients.

* Correspondence: chchung@mmc.edu.tw3Department of Medicine, Mackay Medical College, No. 46, Sec. 3,Jhong-Jheng Rd., Sanzhi Dist., Taipei, TaiwanFull list of author information is available at the end of the article

© 2015 Lai et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Lai et al. BMC Complementary and Alternative Medicine (2015) 15:445 DOI 10.1186/s12906-015-0970-3

Surgery is the most important treatment for malignantmelanoma. However, late stage and metastatic melan-oma is known to confer a very poor prognosis, with nostandard of care currently established. Drug therapiesfor melanoma, including chemotherapy and targetedtherapy, are currently under development. The patho-genesis of melanoma is complex and involves a numberof signaling pathways related to tumor cell growth andmetastasis [7]. Up-regulation of the mitogen activatedprotein kinase (MAPK) signaling pathway is common inmany human cancers, including melanoma [8]. Thephosphatidylinositol 3-kinase (PI3K)/Akt signaling path-way represents another key mediator of these processes.PI3K can activate Akt and subsequently phosphorylateseveral target proteins, including mammalian target ofrapamycin (mTOR). Following mTOR activation, cellgrowth promotion and cell cycle progression may occurbecause of mTOR downstream p70S6 kinase (p70S6K)and eukaryotic initiation factor (eIF) 4E-binding protein1 (4E-BP1) phosphorylation [9, 10]. The MAPK andPI3K/Akt/mTOR signaling pathways are frequently acti-vated and dysregulated in melanoma, and both representpromising targets for melanoma treatment. Agents thattarget the MAPK signaling pathway (AS/RAF/MEK/ERK), such as vemurafenib (a BRAF inhibitor) and tra-metinib (a MEK1/2 inhibitor), have increased survival inpatients with metastatic melanoma [11]. Inhibition ofthe Akt/mTOR signaling pathway has also been shownto abolish invasion and metastasis of highly-metastaticmelanoma cells [12]. MAPK and Akt/mTOR signalingpathway inhibitors may represent promising targets forthe inhibition of melanoma cell invasion and metastasis.Butein (3,4,2′,4′ -tetrahydroxychalcone) is a flavonoid

and chalcone derivative [13]. Flavonoids are phenolicagents used in traditional Chinese and Tibetan herbalmedicines, which can be extracted from numerous planttissues, including the stem bark of cashews (Semecarpusanacardium), the heartwood of Dalbergia odorifera, andCaragana jubata and Rhus verniciflua stokes. Butein hasbeen found to have anti-oxidant, anti-inflammatory, andanti-restenosis activities, which may be beneficial in dis-ease treatment [14–16]. Recent studies have reportedthat butein exhibits anti-angiogenesis, anti-proliferation,and apoptotic effects against numerous cancer cell lines[17–19]. In our previous study, we demonstrated thatbutein significantly inhibited invasion of SK-Hep-1 cells,a hepatoma cell line, via Akt/mTOR/p70S6K blockade[20]. However, the effects of butein on tumor metastasisare yet to be determined. Thus, we hypothesized thatbutein has a similar inhibitory effect on Akt/mTOR/p70S6K signaling pathway and the proliferation and me-tastasis of melanoma cells. In the present study, weaimed to elucidate the mechanisms underlying the anti-metastatic effect of butein. We found that butein

diminished the PI3K/Akt/mTOR signaling pathway inthe mouse melanoma cell line, B16F10. In addition,butein demonstrated potent anti-metastatic effects in amodel of lung metastasis.

MethodsMaterials2',3,4,4'-Tetrahydroxychalcone (butein) was purchased fromExtrasynthese Corporation (Genay, France). Dulbecco'sModified Eagle Medium (DMEM), fetal bovine serum(FBS), Antibiotic-Antimycotic and trypsin-EDTA werepurchased from Gibco (Grand Island, NY). Toluidineblue O, RNase A and 3-[4,5-dimethylthiahiazo-2-y1]-2,4-diphenytetrazolium bromide (MTT) were purchasedfrom Sigma Chemical Corporation (St Louis, MO, U.S.A.).Lactate dehydrogenase (LDH) assay reagents werepurchased from Promega Corporation (Madison, WI).Antibodies for phospho-Akt (Ser473), phospho-mTOR(Ser2448) were purchased from Epitomics (Burlin-game, CA). Antibodies for phospho-ERK1/2 (Thr202/Tyr204), phospho-p70S6K (Thr389) and α-tubulinwere purchased from Cell Signaling Technologies(Boston, MA).

Cell cultureMouse melanoma cells (B16F10) were obtained fromthe American Type Culture Collection. B16F10 cellswere cultured at 37 °C in HEPES-buffered Dulbecco’smodified Eagle’s medium (DMEM), containing 10 % fetalbovine serum (Gibco) supplemented with NaHCO3, glu-tamax I (Gibco), 100 IU/mL penicillin G (sodium salt),100 μg/mL streptomycin and 0.25 μg/mL amphotericin B(antibiotic-antimitotic solution, Gibco).

Cell viability assayB16F10 cells were sub-cultured onto 24-well plates andstarved with serum-free DMEM for 48 hours. Then,melanoma cells were grown in DMEM, included 10 %FBS in absence or presence of various concentrations ofbutein (1, 3, 10 μM) for 48 hours. Cells were incubatedwith MTT (0.5 mg/mL) for 4 hours. Formazan crystal(purple) was lysed by dimethyl sulfoxide (DMSO) andabsorbance was measured at 550 nm with ELISA-reader(Thermo, U.S.A.).

Lactate Dehydrogenase AssayB16F10 cells were seeded onto 48-well plates in adensity of 2 × 104 cells/well and starvation for48 hours with serum-free DMEM and incubated withor without vehicle and indicated concentrations ofbutein (1, 3 and 10 μM) in DMEM with 10 % FBS.The percentage of LDH release was calculated fromthe ratio of LDH activity in the medium to LDH ac-tivity in the cell lysate.

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Migration assayMigration assay of B16F10 cells was measured byCoaster Transwells (polycarbonate filter, 8 μm pore size)which were coated with 0.2 % gelatin. B16F10 cells(5x104cell/well) were treated with vehicle or variousconcentrations of butein (1, 3, 10 μM), and then cellswere seeded into the upper chamber. The bottom cham-ber was added DMEM medium supplemented with 10 %FBS. After incubation at 37 °C for 16 hr, all non-migrantcells were removed from the upper face of the Transwellmembrane with a cotton swab, and the B16F10 cells thathad transmigrated through the micropore and that werestill bound to the membrane were fixed with 4 % paraf-ormadehyde in PBS solution and stained with 0.5 %toluidine blue in 4 % paraformadehyde. Migration wasquantified by counting the number of stained cells onthe membrane under a light microscope (Nikon, Japan)at a magnification of 200× in 3 random fields, and thenphotographed. All determinations were obtained by rep-lication in at least three independent experiments.

Two-dimensional Migration Scratch AssaySub-cultured B16F10 cells were seeded into 6-wellplates. B16F10 cells were starved with serum-freeDMEM for 48 hours. Part of the dish was denuded byscratching along a straight line with a 200 μl pipet tipand incubated with or without vehicle and indicatedconcentrations of butein (1, 3 and 10 μM) in DMEMwith 10 % FBS. After 16 hours of incubation at 37 °C,images of the wounded area were captured immediatelyby microscope (Nikon, Japan).

Western blottingAfter cells pretreated with or without various concentra-tions of butein (1, 3, 10 μM) and vehicle in serum-freeDMEM for 2 hours, cells were harvested and lysed in alysis buffer containing 15 mM Tris–HCl, 50 mM NaCl,5 mM EGTA (ethyleneglycoltetraacetic acid), 1 % TritonX-100, 1 % sodium deoxycholate, 0.1 % sodium dodecylsulfate (SDS), 1 mM NaF, 1 mM phenylmethylsulfonylfluoride (PMSF), 1 mM Na3VO4, 10 μg/mL aprotinin,and 10 μg/mL leupeptin, pH 7.4. The protein samplesseparated on 10 % SDS-PAGE were then transferred toPVDF membranes by electro-blotting. The membraneswere blocked with 0.25 % (wt/vol) gelatin at roomtemperature 1 hour. Afterward, immune-detection forphosphorylated (activated) FAK, Akt, ERK, mTor,p70S6K, eIF4E and α-tubulin were accomplished withantibody in 0.25 % (wt/vol) gelatin. Blots were washedfor at least 30 mins in wash buffer (Tris/phosphate/sa-line/Tween). After incubation with each antibody, theimmunoreactive band was detected by an EnhancedChemiluminiscence (ECL) Western blotting detectionsystem.

ELISAB16F10 cells (105cells/well in 96well) were treated withvarious concentrations of Butein or vehicle for 24 hr,and then the supernatant was collected to examine theamount of vascular endothelial growth factor (VEGF)protein. VEGF mouse ELISA Kit (Novex®, Invitrogen)was used according to the manufacturer’s instructions.

Animals8-10 weeks-old male C57BL/6 mice were used in allstudies. All the experimental protocols regarding animalstudy have been approved by the Institutional AnimalCare and Use Committee at College of Medicine, Tzu-ChiUniversity.

Experimental metastasis analysisMetastasis analysis was performed as previously described[21]. Briefly, B16F10 cells (2 × 106 cells) were slowlyinjected into the lateral tail vein of C57BL/6 mouse to ini-tiate tumor metastasis. Butein (1、3、10 mg/kg) was in-traperitoneally administered to mice every day from3 days before tumor cell injection to 21 days after tumorcell injection (the day sacrified).

Statistical analysisAll values are presented as mean ± SEM. Differences be-tween groups were assessed by one-way ANOVA andNewman–Keuls multiple comparison test where appro-priate. P values less than 0.05 (P < 0.05) were consideredto be significantly different.

ResultsEffects of butein on the inhibition of B16F10 melanomaproliferationThe structure of butein is shown in Fig. 1a. The effectsof butein on melanoma cell proliferation were deter-mined by MTT assay. As shown in Fig. 1b, buteininhibited B16F10 proliferation in a concentration-dependent manner (IC50 = 9.53 μM). To confirm thatthe anti-proliferative effect of butein was not becauseof cytotoxicity, an LDH assay was also performed.Our data indicates that butein does not have cyto-toxic effects on B16F10 cells at a concentration of10 μM (Fig. 1c).

Butein inhibits serum-stimulated cell motilityMigration is a major step in melanoma metastasis.The pre-treatment addition of various concentrationsof butein (1, 3, and 10 μM) to the upper wells of transwellchamber significantly inhibited B16F10s migration in re-sponse to 10 % FBS (Fig. 2a). Furthermore, we producedscrape wounds on B16F10 monolayers to observe the con-tinuous movement of B16F10 cells in closing the gap thatwas denuded. After 16 h of continuous treatment, butein

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(1, 3, and 10 μM) inhibited the serum-stimulated cell mo-tility of melanoma cells (Fig. 3a). Migration responses ofmelanoma cells in both modified Boyden chamber assaysand scrape models were significantly reduced in aconcentration-dependent manner.

Butein inhibits the ERK, focal adhesion kinase (FAK), andPI3K/Akt/mTOR/p70S6K signaling pathways in melanomacellsThe MAPK and PI3K/Akt/mTOR signaling pathwaysare well-known important signaling pathways for melan-oma growth and metastasis. Follow-up experiments weredesigned to determine the effect of butein on signaling-related protein phosphorylation in B16F10 cells. Buteindramatically inhibited Akt and ERK1/2 phosphorylationin a concentration-dependent manner in B16F10 cells.mTOR, an important downstream effector of the PI3K/Akt signaling pathway, is known to regulate translational-related processes via stimulation of p70S6K and 4EBP1phosphorylation. Thus, we evaluated the effects of buteinin the modulation of these signaling pathways. As shownin Fig. 4a, serum (10 % FBS) induced a significant increasein mTOR, p70S6K, 4E-BP1, and eIF4E phosphorylation.In response to the serum, mTOR phosphorylation andmajor downstream targets, including p70S6K, 4EBP, andeIF4E, were significantly attenuated by butein treatmentin a concentration-dependent manner. The FAK-mediatedsignaling cascade has also been shown to be involved inmelanoma migration and mitogenic responses [22]. Ac-cordingly, we further assessed the effect of butein on FAKphosphorylation. We observed that the levels of serum-induced FAK phosphorylation were also inhibited bybutein. These results indicate that butein may inhibitB16F10 growth and migration through the inhibition ofcellular signaling pathways.

Butein inhibits VEGF releaseThe expression and production of VEGF is regulatedby downstream transcript factors of the PI3K/Akt/mTOR signaling pathway. Therefore, we examined theeffect of butein on VEGF release in B16F10 cells.After 24-h treatment with various concentrations ofbutein, supernatants were collected for analysis. VEGFrelease was found to be suppressed by butein in aconcentration-dependent manner in B16F10 cells(Fig. 5).

Butein inhibits in vivo pulmonary metastasis inB16F10 cellsAs the migration activity of B16F10 cells is associatedwith tumor metastasis, we investigated the in vivo effectof butein in a murine pulmonary metastasis model. Sin-gle intravenous bolus injections of 2 × 106 B16F10 cellswere used to induce lung metastases. Butein (1, 3, or10 mg/kg) was intraperitoneally administered to micefrom 3 days before tumor cell injection. Twenty-onedays later, the mice were euthanized, and their lungswere removed. Mice treated with butein were found tohave a significant dose-dependent reduction in lung me-tastases (Fig. 6a). The quantitative of lung metastasis

Fig. 1 The structure of butein and its effect on melanoma cellsviability and cytotoxicity. The structure of butein is shown in a.B16F10 cells were seeded onto plates for 24 hr and starved foranother 24 hr with serum-free DMEM. b Quiescent cells werecultivated with DMEM containing 10 % FBS in the absence orpresence of various concentrations of butein. After 48 h, cell viabilitywas detected by the MTT assay. c Cytotoxicity was detected bymanufacture’s protocol and finally recorded absorbance at 490 nm.Control was taken as basal condition of LDH release. The results arepresented as percentages of inhibition (mean ± SEM, n = 4). AllP values were obtained from comparisons between control andindicated concentration-treated cells. **, P < 0.01; ***, P < 0.001

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colony was shown in Fig. 6b. Butein significantly attenu-ated pulmonary metastasis of B16F10 cells in vivo.

DiscussionMetastasis is a deadly stage in cancer progression andthe also is an important prerequisite for cancer me-tastasis [23, 24]. There remains a lack of promising

therapeutic agents for the prevention of melanoma in-vasion and metastasis. The results of the presentstudy demonstrated that a naturally occurring chalconederivative, butein, significantly inhibited melanoma prolif-eration and migration in a concentration-dependent man-ner (1, 3, and 10 μM) without significant cytotoxicity(Fig. 1b, c). We previously reported that butein has

Fig. 2 Effect of butein on serum-stimulated B16F10 cell migration. a B16F10 cells (2 × 104) cultured in serum-free DMEM were pretreated with orwithout butein for 30 minutes, and placed in the upper chamber of a Transwell containing with 0.2 % gelatin-coated filter membrane. DMEMwith 10 % FBS was added to the lower chamber. After removal of non-migrated cells and fixation, cells that migrated to the underside filtermembrane were stained and quantified by phase-contrast light microscope under a high-power field (HPF; magnification, 100×). Three fields perfilter were counted at 100× magnification. The quantitative of migratory cell was shown in b. Data are expressed as mean ± S.E.M. of fiveindependent experiments. *p < 0.05; **p < 0.01 compare with the control (Veh) group

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anti-metastatic activity by reducing MMP-9/uPA pro-teolytic and Akt/mTOR/p70S6K translational activity[20]. In the present study, butein treatment for 16 hwas found to inhibit cell migration at concentrationsranging from 1 to 10 μM. Under similar conditions,

butein was found to induce substantial amounts ofcell death at a concentration of 10 μM only. Thesedata indicate that the migration inhibitory effect ofbutein is not because of cytotoxicity. To the best ofour knowledge the present study is the first to

Fig. 3 Effect of butein on serum-stimulated B16F10 cell motility. a B16F10 cells (5 × 104) were seeded onto 6-well plates for 24 hr and starved foranother 24 hr with serum-free DMEM. Cells were subjected to injury by scratching with a plastic pipette tip (200 μl). The cells were then treatedfor 24 hours with or without serum or butein. The quantitative of migratory cell was shown in b. Data are presented as percentages of control(mean ± S.E.M., n = 3). **, P < 0.01 and ***,P < 0.001 versus control group

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provide evidence of the inhibitory effects of butein onmelanoma metastasis, without showing a significanteffect on cell proliferation. We demonstrated thatbutein exhibited anti-metastatic activity at low con-centrations (1 μM) without inducing significant cell

death in B16F10 cells. Notably, we also found thatbutein significantly attenuated pulmonary metastasisof B16F10 cells in vivo (Fig. 6). We also summarizethe contents and major findings in Fig. 7.Dysregulation of the PI3K/Akt/mTOR signaling path-

way has been observed in various human cancers. Asthe PI3K/Akt/mTOR signaling pathway regulates vari-ous important cellular processes, it has become anmost important therapeutic targets for anti-cancer drugdevelopment [9, 10, 25]. The mTOR signaling pathwayis a major chemotherapy target for the treatment ofcancers, including melanoma [11]. We found thatmTOR phosphorylation, its upstream kinases, PI3K,Akt, and the downstream effector, p70S6K, in B16F10cells (Fig. 4) were all effectively inhibited by butein.The mTOR signaling pathway is also regulated byPI3K/Akt-independent signaling pathways, such asERK, p38 MAPK, and AMPK [26]. FAK has beenshown to be important for melanoma cell proliferation,adhesion, and invasion in vitro and in vivo [27]. FAKinactivation reportedly results in decreased ERK phos-phorylation and VEGF expression [22]. These studieshave demonstrated that FAK plays a critical role in mel-anoma metastasis and may represent an anti-metastatictarget for melanoma treatment. As butein also signifi-cantly inhibited the FAK and ERK signaling pathways,the anti-cancer effect of butein may also be dependenton the inhibition of the FAK and ERK signalingpathways.Activation of mTOR via the PI3K/Akt signaling

pathway is associated with increased translation ofhypoxia-inducible factor (HIF)-1α, which drives theexpression of angiogenic growth factors [28]. Asbutein was found to inhibit mTOR activation, wehypothesize that butein may suppress VEGF gene ex-pression in B16F10 cells. Previous studies have re-ported that aggressive melanoma cell lines expresshigher levels of VEGF and that melanoma patientswith higher VEGF concentrations have a higher rateof relapse [29, 30]. Inhibition of tumor growth hasbeen achieved in different melanoma xenograftmodels through the use of a number of anti-VEGFstrategies [31]. In the present study, VEGF productionwas decreased in B16F10 cells after 24-h treatment withbutein in a concentration-dependent manner (Fig. 5).VEGF production has also been reported to be associatedwith activation of downstream signaling pathways andphenotypic changes, such as increased cell migration andinvasion [32]. Furthermore, VEGF expression has beenreported to have a correlation with apoptosis, and VEGFreduces cell death through an autocrine mechanism[33, 34]. The inhibitory effect of butein on VEGF ex-pression may induce apoptosis of B16F10 cells inaddition to reducing tumor metastasis.

Fig. 4 Effect of butein on PI3K/Akt/mTOR, ERK and FAK pathways.a B16F10 cells were incubated in the absence or presence of butein(1- to 10 μM) for 12 hrs. Then, the cells were harvested and lysed forthe detection of the PI3K/Akt/mTOR, ERK and FAK pathwaysactivation by Western blot. The quantitative densitometry of therelative level of protein phosphorylation (phosphortlation protein/total protein) was performed with Image-Pro Plus and was shown inb. Data are expressed as mean ± S.E.M. of five independent ex-periments. The protein phosphorylation in all treatment cellswere significant lower than control (Veh) group (***p < 0.001 ascompare with control (Veh) group)

Fig. 5 Effects of butein on VEGF production in melanoma cells, B16F10.VEGF protein expression was evaluated by ELISA in conditioned mediumof B16F10 in the presence or absence of butein at the indicatedconcentrations. Data are represented as mean ± SEM (n = 6). *P < 0.05and **P < 0.01 indicates significant differences from the vehicle control

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Surgery remains the major treatment for malignantmelanoma. However, the prognosis of melanoma re-mains poor because of the invasiveness and metastasisof the tumors [35]. Therefore, more efficacious treat-ments for melanoma metastasis are required. Butein re-duced the number of lung metastasis foci in a dose-dependent manner in an in vivo pulmonary metastasismodel, indicating the anti-metastatic and antitumor ac-tivities of butein towards melanoma (Fig. 6).

There are still some limitations in this study. Accord-ing to the Flaherty et al. study, approximately 50 % ofmelanomas harbor activating BRAF mutations [36].Human melanomas display abnormal activation of theMAPK cascade due to these mutations and may exhibitsensitivity to MEK inhibition. Although the mousemelanoma cell, B16F10, were without mutations in theBRAF and NRAS genes, the MAPK cascade and PI3K/Akt signaling pathway are still the major mediators for

Fig. 6 Effects of butein on experimental lung metastasis. a B16F10 cells (2x106) were injected into the lateral tail vein of C57BL/6 mouse. Twentydays later, the lungs were removed, and surface-visible tumors were counted in the absence and presence of butein treatment. The melanomametastasis area of lung surface was black and background is dark red. White area was quantified using ImageJ software. The quantitative of lungmetastasis colony was shown in b. Corresponding bar graph expressed as percentage of vehicle (n = 8)

Fig. 7 Schematic presentation of butein-mediated signaling transduction modulation

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melanoma metastasis. So far, the best characterized ofmetastasis models is the B16 melanoma model. B16F10cells had an effective metastasis rate of 5 × 10-5 per cellper generation [37]. Beside human melanoma cell trans-planted null mice, this metastasis model in conjunctionwith in vitro modeling and manipulation of tumor cellshave enable investigators to development of agents thatcan be used to prevent or treat overt metastatic disease.

ConclusionWe demonstrated that butein inhibits tumor growth invitro by inhibiting the ERK and PI3K/Akt/mTORsignaling pathways. VEGF production was found to bedecreased following butein treatment. More importantly,butein exhibited anti-tumor activity by in vivo pulmonarymetastasis model. Collectively, the results of the presentstudy indicate that butein has potential anti-cancer prop-erties for the treatment of melanoma. However, clinicaltrials are required to fully validate the potential of buteinin clinical applications.

AbbreviationsPI3K: phosphatidylinositol 3-kinase; mTOR: mammalian target of rapamycin;p70S6K: p70S6 kinase; VEGF: vascular endothelial growth factor.

Competing interestAll authors have no financial or personal relationships with other people ororganizations that could inappropriately influence our work.

Authors’ contributionsConceived and designed the experiments: YL and CC; Performed theexperiments: YL, SW, CC, SL and YC; Analyzed the data: CC, LC and SC;Contributed materials purified/analysis tools: AWC; Wrote the manuscript:YL and CC.

AcknowledgmentsThis study was supported by Mackay Medical College (MMC-1022B14,1032B02, 1041B02 and 1041E01) awarded to Ching-Hu Chung, MackayMemorial Hospital (MMH-103-70, MMH-104-37) and Taipei City Hospital(TPCH-101-016, TPCH-102-006, TPCH-104-010).

Author details1Department of Urology, National Yang-Ming University School of Medicine,Taipei, Taiwan. 2Division of Urology, Taipei City Hospital Renai Branch, Taipei,Taiwan. 3Department of Medicine, Mackay Medical College, No. 46, Sec. 3,Jhong-Jheng Rd., Sanzhi Dist., Taipei, Taiwan. 4Institute of Pharmacology,College of Medicine, National Taiwan University, Taipei, Taiwan. 5Departmentof Orthopaedics, Mackay Memorial Hospital, Taipei, Taiwan. 6Department ofMedical Research, Mackay Memorial Hospital, Taipei, Taiwan. 7Department ofRadiation Oncology, Mackay Memorial Hospital, Taipei, Taiwan.

Received: 14 June 2015 Accepted: 14 December 2015

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