Research Article Moscatilin Inhibits Lung Cancer Cell...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 765894 11 pageshttpdxdoiorg1011552013765894

Research ArticleMoscatilin Inhibits Lung Cancer Cell Motility and Invasion viaSuppression of Endogenous Reactive Oxygen Species

Akkarawut Kowitdamrong1 Pithi Chanvorachote12

Boonchoo Sritularak3 and Varisa Pongrakhananon12

1 Department of Pharmacology and Physiology Faculty of Pharmaceutical Sciences Chulalongkorn UniversityBangkok 10330 Thailand

2 Cell-Based Drug and Health Product Development Research Unit Faculty of Pharmaceutical Sciences Chulalongkorn UniversityBangkok 10330 Thailand

3Department of Pharmacognosy and Pharmaceutical Botany Faculty of Pharmaceutical Sciences Chulalongkorn UniversityBangkok 10330 Thailand

Correspondence should be addressed to Pithi Chanvorachote pithi chanyahoocom andVarisa Pongrakhananon varisappharmchulaacth

Received 15 February 2013 Accepted 2 April 2013

Academic Editor David J Yang

Copyright copy 2013 Akkarawut Kowitdamrong et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Lung cancer is the leading cause of death among cancer patients worldwide andmost of them have died frommetastasis Migrationand invasion are prerequisite processes associated with high metastasis potential in cancers Moscatilin a bibenzyl derivativeisolated from theThai orchidDendrobium pulchellum has been shown to have anticancer effect against numerous cancer cell linesHowever little is known regarding the effect of moscatilin on cancer cell migration and invasion The present study demonstratesthat nontoxic concentrations of moscatilin were able to inhibit human nonsmall cell lung cancer H23 cell migration and invasionThe inhibitory effect of moscatilin was associated with an attenuation of endogenous reactive oxygen species (ROS) in whichhydroxyl radical (OH∙) was identified as a dominant species in the suppression of filopodia formation Western blot analysisalso revealed that moscatilin downregulated activated focal adhesion kinase (phosphorylated FAK Tyr 397) and activated ATP-dependent tyrosine kinase (phosphorylated Akt Ser 473) whereas their parental counterparts were not detectable changed Inconclusion our results indicate the novel molecular basis of moscalitin-inhibiting lung cancer cell motility and invasion anddemonstrate a promising antimetastatic potential of such an agent for lung cancer therapy

1 Introduction

Lung cancer incidences have continued to increase world-wide [1] More than 90 of lung cancer patients have diedfrommetastasis because of late diagnosis after metastasis wasestablished [2] Treatment of metastasis lung cancer oftenfails due to the acquisition of chemotherapeutic resistanceand the fact that cancer metastasis is still remaining even ifthe tumor is removed by a surgery [3] Recently a numberof researches have been conducted to explore potentialagents for treatment of cancer metastasis Cancer metastasisconsists of multistep events facilitating the establishmentof secondary tumor in which migration and invasion play

critical steps during metastasis involving the elongation offilopodia cell contraction and gliding and cell protrusionand reattachment to extracellular matrix [4 5]

Aberrant generation of cellular ROS was tightly asso-ciated with several metastasis cancers such as lung andcolon cancers [6 7] Reactive oxygen species (ROS) suchas superoxide anion (O

2

minus∙) hydrogen peroxide (H2

O2

)and hydroxyl radical (OH∙) served as important regulatorsof various physiological pathways during cancer metastasisincluding angiogenesis cancer motility and invasiveness [68] Scientific evidence also showed that cancer migrationand invasion are obviously regulated by reactive oxygenspecies (ROS) [6 8] It was demonstrated that treatment with

2 BioMed Research International

antioxidant such as ascorbic acid caused a reduction in cancermotility and invasion [9] and conversely the addition ofexogenous ROS enhances these activities [10]

Moscatilin (441015840-dihydroxy-33101584051015840-trimethoxybibenzyl)is a bibenzyl derivative extracted from Thai orchid Dendro-bium pulchellum (Orchidaceae) which is known as ldquoUeangchang naordquo inThai (Figure 1)Moscatilinwas reported to havevarious pharmacological properties such as antiinflamma-tory [11] antioxidant [12] and antiplatelet aggregation [13]Recently it has shown anticancer activity against many kindsof cancers for example induction of cell cycle G2-M arrest[14 15] and cell apoptosis involving its antioxidant effect [1215] In present study we investigated that the administrationof moscatilin attenuates migration and invasion in lungcancer cells Its negative regulator is associated with theability of compound to suppress endogenous ROS generationand consequently inhibits FAK and Akt activation-mediatingcancer motility and invasiveness Our finding reveals thenovel mechanism of moscatilin on the regulation of cancermigration and invasion which could be an advantage indevelopment of this compound for cancer therapy

2 Material and Methods

21 Cells and Reagents Human lung adenocarcinoma H23cells were obtained from American Type Culture Collec-tion (ATCC Manassas VA USA) and cultured in RPMI-1640 medium containing 10 fetal bovine serum 2mML-glutamine 100 IUmL penicillin and 100 120583gmL strepto-mycin (Gibco MD USA) in 37∘C with 5 CO

2

-humidifiedincubator Moscatilin was isolated from Thai orchid Den-drobium pulchellum as previously described [16] Moscatilinwas dissolved in DMSO and deionized water for the indi-cated working concentrations The amount of DMSO inthe final solution was less than 01 which showed nocytotoxic in H23 cells The 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazoliumbromide (MTT)Hoechst33342 propid-ium iodide (PI) phalloidin tetramethylrhodamine B isoth-iocyanate ribonuclease A bovine serum albumin (BSA)and dimethylsulfoxide (DMSO) were purchased from SigmaChemical Inc (St Louis MO USA) Annexin V ApoptosisDetection Kit was obtained from BD Biosciences (WoburnMA USA) Antibodies for phosphorylated Akt (S473) Aktphosphorylated FAK (Y397) FAK phosphorylated ERK(Thr202Tyr204) ERK Cdc42 120573-actin and peroxidase-conjugated secondary antibodies were obtained from CellSignaling (Denvers MA USA)

22 Cell Viability Assay Cells viability was determined byMTT assay as previously described [17] Initially cells wereseeded at a density of 104 cellswell onto 96-well plateovernight After that they were treated with various concen-trations ofmoscatilin for 24 hThemediumwas then replacedwith MTT solution (50mgmL in PBS) and incubated at37∘C for 4 h To solubilize formazan product the mediumwas replaced with 100 120583L DMSO and an intensity readingof the formazan product was measured at 550 nm usinga microplate reader (Anthros Durham NC USA) Cell

OMe

OMe

OH

MeO

HO

Figure 1 Chemical structure of moscatilin

viability was expressed as the percentage calculated fromabsorbance of MTT-treated cells relative to control cells

23 Apoptosis Assay Cells were seeded at a density of 104cellswell onto 96-well plate and incubated overnight for cellattachment After the indicated treatments cells were washedand incubated with 10 120583gmL Hoechst33342 and 5 120583gmLpropidium iodide (PI) for 30min Additionally apoptoticcells were determined using Annexin-V Staining Assay Afterthe indicated treatments cells were washed and subsequentlystained with 100120583L of 1x binding buffer containing 5120583Lof Annexin V-FITC for 15min at room temperature in thedark Nuclei condensation and DNA fragmentation of apop-totic cells Annexin-V-positive cells and PI-positive necroticcells were visualized and scored by fluorescence microscopy(Olympus IX51 with DP70) as previously described [17]

24 DNA Content Analysis Cells were seeded at a density of3 times 105 cellswell onto 6-well plate and incubated overnightfor cell attachment After the indicated treatments cells weretrypsinized and fixed in 70 absolute ethanol at minus20∘Covernight After washing with PBS cells were incubated inpropidium iodide solution containing 01Triton-X 1120583gmLRNase and 1mgmL propidium iodide at room temperaturefor 30min DNA content was analyzed using flow cytometry(FACSort Becton Dickinson Rutherford NJ USA) as previ-ously described [17]

25 Migration Determination Migration was determined bywound healing and Boyden chamber assay as previouslydescribed [6] For wound healing assay cells were seededat a density of 2 times 105 cellswell onto 24-well plate Afterthe cell monolayer was formed a micropipette tip wasused to scratch cell attachment to generate wound spaceThe cells were then washed with PBS and replaced withRPMI medium containing the indicated concentration ofmoscatilinThe progress of cell migration into the woundwasphotographed by inverted microscope (Olympus IX51 withDP70) at indicated time of incubation The average woundspace was calculated from the random field of view andrepresented the relative cell migration Relative cell migrationwas calculated by dividing the change of wound space of

BioMed Research International 3

sample by that of the control cells in each experiment In caseof Boyden chamber assay cells were seeded at a density of 5 times104 cellswell onto upper 24-transwell plate of the transwellfilter (8-120583M pore) in-serum-free medium and incubatedwith various concentrations of moscatilin RPMI mediumcontaining 10 FBS was added at lower chamber Followingthe incubation the nonmigrate cells in the upperside mem-brane were removed by cotton-swab wiping and cells thatmigrated to the underside of themembranewere stainedwith10 120583gmL Hoechst33342 for 10min visualized and scoredunder a fluorescencemicroscope (Olympus IX51 with DP70)

26 Invasion Assay The invasion assay was carried out using24-transwell chambers as previously described [6] whichwere coated with 50 120583L of 05matrigel on the upper surfaceof chamber overnight at 37∘C in a humidified incubatorFollowing the incubation cells were seeded at a density of 5times 104 cellswell onto upper chambers in serum-free mediumcontaining various concentrations of moscatilin and RPMImediumcontaining 10FBSwas added to the lower chamberAfter the indicated time noninvaded cells in the uppersideof membrane were removed by cotton-swab wiping Invadedcells in the underside of membrane were fixed with coldabsolute methanol for 10min and stained with 10 120583gmLHoechst33342 for 10min Cells were then visualized andscored under a fluorescence microscope (Olympus IX51 withDP70)

27 Cell Morphology Characterization Cell morphology wasinvestigated by phalloidin-rhodamine and sulforhodamineB staining assay as described in [18] Cells were seededat a density of 104 cellswell onto 96-well plate overnightCells were treated with various concentrations of moscatilinfor 24 h Cells were then washed with PBS fixed with 4paraformaldehyde in PBS for 10min at 37∘C permeabilizedwith 01 Triton-X100 in PBS for 4min and blocked with02 BSA for 30min Cells were then incubated with either1 100 phalloidin-rhodamine in PBS or 04 sulforhodamineB in 1 acetic acid for 15min rinsed 3 times with PBSand mounted with 50 glycerol Cell morphology was thenimaged by fluorescence (Olympus IX51 with DP70) Filopo-dia protrusion was represented in comparison with controlcells

28 Reactive Oxygen Species Detection Intracellular ROSwere determined using specific ROS detection probeincluding dichlorofluorescein diacetate (DCFH

2

-DAROS probe) hydroxyphenyl fluorescein (HPF specificOH∙ probe) amplex red (specific H

2

O2

probe) anddihydroethidium (DHE specific O

2

∙minus probe) as previouslydescribed in [17] After the indicated treatments cells wereincubated with either 100 120583Mof DCFH

2

-DA 100 120583MofHPF10mM of amplex red or 100 120583M of DHE for 30min at 37∘Cafter which they were washed and immediately analyzed forfluorescence intensity using a microplate reader

29 Western Blotting Cells were seeded at a density of 3 times105 cellswell onto 6-well plates overnight After specific treat-ment cells were washed twice with cold PBS and incubatedwith lysis buffer containing 20mM Tris-HCl (pH 75) 1Triton X-100 150mM sodium chloride 10 glycerol 1mMsodium orthovanadate 50mM sodium fluoride 100mMphenylmethylsulfonyl fluoride and protease inhibitor cock-tail (Roche Molecular Biochemicals) for 40min on ice Celllysates were collected and the protein content was deter-mined using the BCAprotein assay kit (Pierce BiotechnologyRockford IL USA) Equal amounts of protein from eachsample (60 120583g) were denatured by heating at 95∘C for 5minwith Laemmli loading buffer and subsequently loaded ontoa 10 SDS-polyacrylamide gel for electrophoresis Afterseparation proteins were transferred onto 045120583M nitro-cellulose membranes (Bio-Rad) The transferred membraneswere blocked in 5 nonfat dry milk in TBST (25mM Tris-HCl (pH 75) 125mM NaCl 005 Tween 20) for 1 h afterwhich it was incubated with a specific primary antibodyovernight at 4∘C Membranes were washed three times withTBST for 10min and incubated with Horseradish peroxidase(HRP)-conjugated anti-rabbit or anti-mouse IgG for 2 hat room temperature After three times of washing withTBST the immune complexes were detected by enhancementwith a chemiluminescent substrate (Supersignal West PicoPierce Rockfore IL USA) and quantified using analystPCdensitometry software (Bio-Rad)

210 Statistical Analysis All results from four or more inde-pendent experiments were presented as the mean plusmn standarddeviation (SD) Statistical differences between the meanswere analyzed using one-way ANOVA with Turkey post hoctest at a significance level of 119875 lt 005 using SPSS version 190

3 Results

31 Cytotoxicity of Moscatilin to H23 Cells To investigatethe inhibitory effect of moscatilin on cancer migration andinvasion prerequisite information regarding its cytotoxicityis crucial Human lung H23 cells were treated with variousconcentrations of moscatilin (0ndash5120583M) for 0ndash48 h and cellviability was examined by MTT assay Figures 2(a) and2(b) show that a significant cytotoxic effect of moscatilinwas appeared at the concentration of 5120583M at 24 h withapproximately 70 of cells remaining viable while the con-centrations of such a substance less than 1120583M show nontoxiceffect in both dose and time studies Hoechst33342PI assayalso confirmed that apoptosis and necrotic cell death werenot found significantly in response to 0-1 120583M moscatilinwhereas apoptotic nuclei were appeared in the cells treatedwith 5 120583M of moscatilin similar with the data obtained fromAnnexin-V staining assay (Figure 2(c)) Consistent with theabove findings DNA content analysis revealed that treatmentwith 0-1120583M moscatilin caused no detectable change in thepercentage of cells in each phase of cell cycle compared withnontreated control (Figure 2(d)) This result suggests thatlower doses of moscatilin (0-1120583M) caused neither toxic norproliferative effects on lung cancer H23 cells


32 Effect of Moscatilin on H23 Cells Migration The negativeregulatory role of moscatilin on lung cancer migration wasinvestigated by wound healing and Boyden chamber assaysFigures 3(a) and 3(c) show that treatment of the cells withnontoxic doses of moscatilin (0-1 120583M) inhibited migrationof the cells across the wound space in a dose-dependentmanner of which approximately 075- and 055-fold ofrelative migration level were found in cells treated with 05and 1 120583M respectively compared with nontreated controlcells In addition moscatilin also causes antimigrative effectin a time-dependent study (Figures 3(b) and 3(c)) Boydenchamber assay supported our finding that the migrating cellson the lower side of membrane were decreased gradually indose- and time-dependent manners (Figures 3(d) 3(e) and3(f)) These results suggest the promising role of moscatilinin regulation of lung cancer migration

33 Effect of Moscatilin on H23 Cells Invasion and FilopodiaFormation To further investigate the effect of moscatilinin lung cancer cell invasion H23 cells were treated withnontoxic concentrations of moscatilin (0-1 120583M) for varioustimes (0ndash48 h) and invaded cells were examined by transwellinvasion assay Figures 4(a) and 4(c) show that nontoxicconcentrations of moscatilin retarded a number of invadedcells in a dose-dependent fashion in which approximately06- and 05-fold of relative invaded cells were deservedin response to 05 and 1 120583M of moscatilin respectivelyFurthermore moscatilin was able to impede invaded cells inthe time-dependent study (Figures 4(b) and 4(c))

Since filopodia has been shown to play an essentialrole in cell motility and invasion by protrusion at the edgeof motile cells for attachment and gliding [5] we furtherclarified whether the antimigrative and antiinvasive effectsof moscatilin were related to the presence of filopodia H23cells were treated with nontoxic concentrations of moscatilin(0-1 120583M) for 24 h and filopodia of cells was identified byphalloidin-rhodamine and sulforhodamine B staining assaysFigure 4(d) shows that upon migration motile cells exhib-ited filopodia protrusions accumulating at the cellular edgein which these filopodia were dramatically decreased inresponse tomoscatilin treatmentsThe above finding suggeststhatmoscatilin inhibits filopodia formation and subsequenceimpedes lung cancer cell migration and invasion

34 Moscatilin Attenuates Cell Motility through ROS-Dependent Mechanism It has been well documented thatendogenous ROS namely superoxide anion hydrogenperoxide and hydroxyl radical are continuously producedinside the living cells [19] Substantial studies have indicatedthe regulatory role of such specific ROS in cell behaviorsincluding migration and invasion [6 8] and most evidenceindicated that these specific ROS play distinguishable rolesin cell motility In order to provide the precise mechanismof moscatilin in the regulation of cell migration cells weretreated with various concentrations of moscatilin andcellular ROS were investigated by using DCFH

2

-DA specificROS detection probe As expected moscatilin caused agradual decrease of endogenous ROS level in dose- and

time-dependent manners (Figure 5(a)) In order to identifythe specific ROS involved in our tested conditions cells weretreated with moscatilin (0-1 120583M) for 3 h and incubatedwith specific ROS detection probes hydroxyphenylfluorescein (HPF) amplex red and dihydroethidium(DHE) Interestingly moscatilin shows an antioxidant effectby which the level of OH∙ is substantially decreased inresponse to moscatilin treatment (Figure 5(b)) In additionno change was observed regarding the level of O

2

∙minus andH2

O2

in comparison with nontreated control cells (Figures5(c) and 5(d)) suggesting that endogenous OH∙ is a targetedspecies eliminated by moscatilin To confirm the anti-OH∙effect of moscatilin cells were treated with specific OH∙generator (ferrous sulfate) in the presence of moscatilin for3 h and ROS levels were identified usingDCFH

2

-DA-specificROS detection probes Figure 5(e) clearly demonstrates thatan extensive increase in ROS level mediated by ferroussulfate was in turn suppressed gradually by moscatilin in adose-dependent fashion This novel finding indicated thatmoscatilin shows a potent antioxidant against endogenousROS and OH∙ is the most affected species

Parallel study was conducted to investigate the relevanceof antioxidant effect of moscatilin on cancer migration andcells were preincubated with OH∙ generator in the presenceor absence of moscatilin treatment Wound healing assayshows that ferrous sulfate treatment significantly enhancedthemigration of cells and the addition of moscatilin was ableto abolish such an effect (Figure 5(f)) These findings suggestthat antimigrative effect of moscatilin was associated with itsability to suppress endogenous OH∙

35 Effect of Moscatilin on the FAK Signaling in H23 CellsHaving shown that moscatilin suppressed the migration ofthe cells via hydroxyl radical attenuation we further providedthe possible underlying mechanisms involving migratoryregulating proteins Focal adhesion kinase (FAK) ATP-dependent tyrosine kinase (Akt) p4442 mitogen-activatedprotein kinase (ERK12) and cell division cycle 42 (Cdc42)were reported to implicate cell motility in several studies[5 20 21] therefore the expression and activated levelof the proteins were investigated Cells were treated withmoscatilin for 24 h and the expression levels of these proteinsincluding activated FAK (phosphorylated FAK Tyr 397)FAK activated Akt (phosphorylated Akt Ser 473) Aktactivated ERK12 (phosphorylated ERK12 Thr202Tyr204)ERK12 and Cdc42 were determined by Western blottingFigure 6 shows that treatment with moscatilin caused asubstantially downregulation of activated FAK and activatedAkt as compared to nontreated control In addition activatedERK12 ERK12 and Cdc42 are not affected by moscatilinThese results suggest thatmoscatilin attenuated the activationof migrating-related proteins FAK and Akt in accordancewith the ability of such agent on lowering endogenous ROS

4 Discussion

Cancer metastasis is a complex multistep process wherebycancer cell migration and invasion are crucial in determining


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Figure 2 Cytotoxicity of moscatilin on human lung H23 cells (a) Cells were treated with various concentrations of moscatilin (0ndash5 120583M) for24 h (b) Cells were treatedwithmoscatilin (0-1 120583M) for various times (0ndash72 h) Cytotoxicity was determined by 3-(45-dimethyl-thiazol-2-yl)-25-diphenyl tetrazolium bromide (MTT) assay (c) After indicated treatment for 24 h mode of cell death was examined by Hoechst33342PIcostaining assay and Annexin-V staining assay (d) Cellular apoptosis was determined by DNA content analysis using flow cytometry Datarepresent the means plusmn SD (119899 = 4) lowast119875 lt 005 versus nontreated control cells

the capability of cancer to metastasize Cancer migration ischaracterized by the movement of cancer to other placeswhich initiates by the dynamic change of cytoskeletonincluding protrusion of cell membrane and actin-myosincontraction [4] Even though the invasion of the cancer cellswas shown to share certain molecular mechanisms with cellmigration invasion is more focused on the ability of cancer

to disrupt basement membrane and extracellular matrix bysecreting the proteolytic enzyme to destruct the meshworkof basement membrane prior to migration through sur-rounding tissue [4] Most of metastasis cancer cells exhibitthese aggressive behaviors which limit the effectiveness ofcancer therapy and result in high mortality rate of lungcancer patients [2] Many studies have been conducted in


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Figure 3 Effects of moscatilin on H23 cell migration (a) Confluent monolayer of H23 cells was wounded using a 1mm width tip andincubated with nontoxic dose of moscatilin (0-1120583M) for 24 h Wound space was analyzed and represented as migration level relatively tothe change of those in nontreated cells Data represent the means plusmn SD (119899 = 4) lowast119875 lt 005 versus nontreated control cells (b) Confluentmonolayer of H23 cells was wounded using a 1mm width tip and incubated with moscatilin (1 120583M) or without for various times (12ndash48 h)Wound spacewas analyzed and represented asmigration level relatively to the change of those in nontreated cells lowast119875 lt 005 versus nontreatedcontrol cells (c) After indicated treatment migrating cells in the denuded zone were photographed (d) H23 cell migration was examined bytranswell assay for 24 h Data were plotted as an average number of cells in each field and represented the means plusmn SD (119899 = 4) lowast119875 lt 005versus nontreated control cells (e) Cells were treated with moscatilin (1120583M) or without for various times (12ndash48 h) on transwell Data wereplotted as an average number of cells in each field and represented the means plusmn SD (119899 = 4) lowast119875 lt 005 versus nontreated control cells (f)Migratory cells at the basolateral side ofmembranewere stainedwithHoechst33342 for 30min and visualized under fluorescencemicroscopy


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Figure 4 Effects of moscatilin on H23 cell invasion (a) H23 cells were treated with various nontoxic doses of moscatilin (0-1 120583M) for 24 h(b) H23 cells were treated with moscatilin (1120583M) or left untreated as control for various times (12ndash48 h) Cell invasion was evaluated usingtranswell coated with matrigel as described under Section 2 Invaded cells across the membrane were stained with Hoechst33342 for 30minData were plotted as an average number of cells in each field and represented the means plusmn SD (119899 = 4) lowast119875 lt 005 versus nontreated controlcells (c) Invading cells were stained with Hoechst33342 and visualized under fluorescence microscopy (d) Effect of moscatilin on filopodiaformation and cell morphology After being treated with nontoxic dose of moscatilin for 24 h cells were stained with either phalloidin orsulforhodamine B and visualized under fluorescence microscope Filopodia was indicated by arrow

the past decade to explore biological agents that have anability to inhibit cancer metastasis According to numerousresearches moscatilin a major constituent of Dendrobiumpulchellum is one such interested in its antimutagenic activityagainst several cancer types [14 15] We also providedfurther evidence supporting the promising role of this naturalcompound for treatment of metastasis cancers Our findingsshow that nontoxic doses of moscatilin were able to inhibitlung cancer cell migration and invasion (Figures 3 and 4)Our work also reported herein for the first time that such aninhibitory effect was involved with the potential of moscatilinto attenuate endogenous ROS of which OH∙ was identified tobe an affected species

The role of ROS in cancer behavior has been welldescribed including the regulation of cell motility and inva-siveness [6 10] Recently specific ROS O

2

∙minus and H2

O2

wereshown to exhibit a negative regulatory effect on cell migration

and invasion whereas OH∙ encourages such activities [6]Previously moscatilin was reported to have antioxidant effect[12] and we further found that this substance reducedendogenous OH∙ and thus inhibited migratory action of thecells (Figure 5) Consistent with previous study we found thatthe addition of ferrous sulfate promoted cancer cell motilitywhich can be conversed by treatment with moscatilin

Emerging evidence showed that several signalingmolecules such as focal adhesion kinase (FAK) Aktphosphatidylinositol-3-kinase (PI3 K) and p4442 Mitogen-activated protein kinases (ERK12) play enhancing roles inmotility of cells [22 23] Recently focal adhesion kinas (FAK)pathway has gained increasing attention as migratory-relatedproteins [23] During cell motility FAK accumulated atthe site of integrin and the phosphorylated form of FAKwas shown to serve as binding site for Src [20] FAK-Srccomplexes enhance actin polymerization and filopodia


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Figure 5 Effect of moscatilin on endogenous reactive oxygen species (ROS) generation H23 cells were treated with various nontoxic doses ofmoscatilin (0-1120583M) for various times (0ndash3 h) (a) Endogenous cellular ROS levels were determined by dichlorofluorescein diacetate (DCFH

2

-DA) probe Values are mean plusmn SD (119899 = 4) lowast119875 lt 005 versus nontreated control cells of each time point (b) After the indicated treatmentfor 3 h cells were incubated with hydroxyphenyl fluorescein (HPF) probe Hydroxyl radical level was detected using fluorescence microplatereader lowast119875 lt 005 versus nontreated control cells (c) Hydrogen peroxide level was examined using amplex red probe lowast119875 lt 005 versusnontreated control cells (d) Superoxide anion level was detected by dihydroethidium (DHE) probe lowast119875 lt 005 versus nontreated controlcells (e) Cells were pretreated with 50120583M of ferrous sulfate (FeSO

4

) for 30min prior to moscatilin treatments (0-1120583M) for 3 h EndogenousROS level were determined by using dichlorofluorescein diacetate (DCFH

2

-DA) probe Values are mean plusmn SD (119899 = 4) lowast119875 lt 005 versusnontreated control cells

119875 lt 005 versus ferrous sulfate treated cells (f) Confluent monolayer of H23 cells was wounded using a 1mmwidthtip and treated with moscatilin (1120583M) in the presence or absence of 50120583M of ferrous sulfate (FeSO

4

) for 24 h Wound space was analyzedand represented as migration level relatively to the change of those in nontreated cells lowast119875 lt 005 versus nontreated control cells

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versus ferrous sulfate treated cells


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Cdc42

120573-actin

0 025 05 1Moscatilin (120583M)

(a)

Cdc42p-FAKFAKp-Erk12Erk12

p-AktAkt

14

12

1

08

06

04

02

0

Relat

ive p

rote

in le

vel

0 025 05 1Moscatilin (120583M)

lowast

lowastlowast

lowast lowastlowast

(b)

FAK FAK

Akt

Cdc42

ERK12

Cell migration and invasion

P

ROS

Moscatilin

Moscatilin

Moscatilin

(c)

Figure 6 Effect of moscatilin on migratory-related proteins (a) H23 cells was treated with various nontoxic doses of moscatilin (0-1 120583M)for 24 h and analyzed for protein expression by using western blot analysis as described under Section 2 Cells were collected and analyzedfor phosphorylated FAK (Tyr 397) FAK proteins phosphorylated Akt (Ser 473) Akt phosphorylated-Erk12 (Thr202Tyr204) Erk12 andCdc42 proteins Blots were reprobed with 120573-actin to confirm equal loading (b)The immunoblot signals were quantified by densitometry andmean data from four independent experiments were presented Values are means of samples plusmn SD lowast119875 lt 005 versus nontreated control cells(c) A schematic diagram summarizes the inhibitory effect of moscatilin on lung cancer cell migration and invasion Moscatilin suppressesROS production and consequently attenuates the activation of FAK and Akt in H23 cells

formation through Cdc42-dependent mechanism [5 20]In addition Akt and ERK signaling were implicated incancer migration and invasion of which the suppressionof either their expressions or activity by silencing plasmidor specific inhibitor was able to attenuate these activities[21] Accumulative studies have demonstrated that thesementioned proteins function independently from each other

[24] and some evidence showed the linkage of them oncell motility [25] FAK activation was shown to mediateAkt phosphorylation which resulted in cell movementAccording to this report the reduction of Akt activationmight be a consequent event as downstream effector inresponse to moscatilin-attenuating FAK phosphorylationEven ERK and Cdc42 were indicated to potentiate cells to


migrate and invade [5 23] this study demonstrated thatmoscatilin impeded migratory activity of H23 cells via ERK-and Cdc42-independent mechanisms These results providea mechanistic insight into the mechanism of moscatilin oncancer cell migration and invasion through the suppressionof endogenous ROS and FAK and Akt activation

5 Conclusion

In conclusion we reported a novel finding on moscatilin-suppressing migratory behavior of lung cancer cells andits molecular mechanism The migrative-inhibitory effectof moscatilin was through an attenuation of endogenousOH∙ (Figure 6(c)) In addition moscatilin reduced FAK andAkt activation which at least in part is responsible forits antimigratory effects Since cell motility and invasionwere critical implicated in cancer metastasis this studythus provides information and highlights potential of thisnatural-based compound for clinical use to overcome cancermetastasis

Abbreviations

Akt ATP-dependent tyrosine kinaseCdc42 Cell division cycle 42DCFH

2

-DA Dichlorofluorescein-diacetateDHE DihydroethidiumERK12 Extracellular signal-regulated kinaseFAK Focal-adhesion kinaseHPF Hydroxyphenyl fluoresceinH2

O2

Hydrogen peroxideMTT 3-(45-Dimethylthiazol-2-yl)-25-

diphenyltetrazoliumbromide

OH∙ Hydroxyl radicalO2

∙minus Superoxidep-Akt Phosphorylated AktPBS Phosphate-buffered salinep-ERK12 Phosphorylated-ERK12p-FAK Phosphorylated-FAKPI Propidium iodideROS Reactive oxygen speciesTBST Tris-buffered saline with 01 Tween

Acknowledgments

This work was supported by the 90th Anniversary of Chu-lalongkorn University Fund and RatchadaphisaksomphotEndowment Fund

References

[1] R Siegel A Jemal E Ward and D Naishadham ldquoCancerstatistic 2012rdquo CA A Cancer Journal for Clinicians vol 62 no 1pp 11ndash29 2012

[2] M R Ray and D M Jablons ldquoLung cancer metastasis novelbiological mechanisms and impact on clinical practicerdquo inHallmarks of Metastasis V Keshamouni D Arenberg and G

Kalemkerian Eds pp 29ndash46 Springer New York NY USA2009

[3] M Shanker D Willcutts J A Roth and R Ramesh ldquoDrugresistance in lung cancerrdquo Lung Cancer vol 1 pp 23ndash36 2010

[4] I J Fidler ldquoThe organ microenvironment and cancer metasta-sisrdquo Differentiation vol 70 no 9-10 pp 498ndash505 2002

[5] P K Mattila and P Lappalainen ldquoFilopodia Molecular archi-tecture and cellular functionsrdquo Nature Reviews Molecular CellBiology vol 9 no 6 pp 446ndash454 2008

[6] S Luanpitpong S J Talbott Y Rojanasakul et al ldquoRegulationof lung cancer cell migration and invasion by reactive oxygenspecies and caveolin-1rdquo Journal of Biological Chemistry vol 285no 50 pp 38832ndash38840 2010

[7] Y Lei K Huang C Gao et al ldquoProteomics Identification ofITGB3 as a key regulator in reactive oxygen species inducedmigration and invasion of colorectal cancer cellsrdquoMolecular ampCellular Proteomics vol 10 no 10 pp 1ndash16 2011

[8] W Y Hung K H Huang C W Wu et al ldquoMitochondrialdysfunction promotes cellmigration via reactive oxygen speciesenhanced beta5-integrin expression inhuman gastric cancerSC-M1 cellsrdquo Biochimica et Biophysica Acta vol 1820 no 7 pp1102ndash1110 2012

[9] D Z Wei J Y Yang J W Liu and W Y Tong ldquoInhibition ofliver cancer cell proliferation and migration by a combinationof (-)-epigallocatechin-3-gallate and ascorbic acidrdquo Journal ofChemotherapy vol 15 no 6 pp 591ndash595 2003

[10] P Storz ldquoReactive oxygen species in tumor progressionrdquo Fron-tiers in Bioscience vol 10 no 2 pp 1881ndash1896 2005

[11] Y N Liu S L Pan C Y Peng et al ldquoMoscatilin repressedlipopolysaccaride-induced HIF-1 alpha accumulation and NF-kappaB activation in murine RAW264 7 cellsrdquo Shock vol 33no 1 pp 70ndash75 2009

[12] X Zhang J K Xu JWang et al ldquoBioactive bibenzyl derivativesand fluorenones from Dendrobium nobilerdquo Journal of NaturalProducts vol 70 no 1 pp 24ndash28 2007

[13] C C Chen L G Wu F N Ko and C M Teng ldquoAntiplateletaggregation principles of Dendrobium loddigesiirdquo Journal ofNatural Products vol 57 no 9 pp 1271ndash1274 1994

[14] C K Ho and C C Chen ldquoMoscatilin from the orchiddendrobrium ioddigesii is a potential anticancer agentrdquo CancerInvestigation vol 21 no 5 pp 729ndash736 2003

[15] T H Chen S L Pan J H Guh et al ldquoMoscatilin inducesapoptosis in human colorectal cancer cells a crucial role of c-Jun NH2-terminal protein kinase activation caused by tubulindepolymerization and DNA damagerdquo Clinical Cancer Researchvol 14 no 13 pp 4250ndash4258 2008

[16] P Chanvorachote A Kowitdamrong T Ruanghirun et alldquoAnti-metastatic activities of bibenzyls from Dendrobium pul-chellumrdquoNatural Product Communications vol 8 no 1 pp 115ndash118 2012

[17] P Chanvorachote and V Pongrakhananon ldquoOuabain downreg-ulates Mcl-1 and sensitizes lung cancer cellsto TRAIL-inducedapoptosisrdquo American Journal of Physiology-Cell Physiology vol304 no 3 pp 263ndash272 2013

[18] F Wang M Yamauchi M Muramatsu T Osawa R Tsuchidaand M Shibuya ldquoRACK1 regulates VEGFFlt1-mediated cellmigration via activation of a PI3KAkt pathwayrdquo Journal ofBiological Chemistry vol 286 no 11 pp 9097ndash9106 2011

[19] H C Lee P H Yin C Y Lu C W Chi and Y H WeildquoIncrease of mitochondria andmitochondrial DNA in responseto oxidative stress in human cellsrdquoBiochemical Journal vol 348no 2 pp 425ndash432 2000


[20] S K Mitra and D D Schlaepfer ldquoIntegrin-regulated FAK-Srcsignaling in normal and cancer cellsrdquo Current Opinion in CellBiology vol 18 no 5 pp 516ndash523 2006

[21] H Si C Peng J Li et al ldquoRNAi-mediated knockdown ofERK12 inhibits cell proliferation and invasion and increaseschemosensitivity to cisplatin in human osteosarcoma U2-OScells in vitrordquo International Journal of Oncology vol 40 no 4pp 1291ndash1297 2012

[22] S Shukla G T MacLennan D J Hartman P Fu M I Resnickand S Gupta ldquoActivation of PI3K-Akt signaling pathway pro-motes prostate cancer cell invasionrdquo International Journal ofCancer vol 121 no 7 pp 1424ndash1432 2007

[23] S Teranishi K Kimura and T Nishida ldquoRole of formation ofan ERK-FAK-paxillin complex in migration of human cornealepithelial cells during wound closure in vitrordquo InvestigativeOphthalmology and Visual Science vol 50 no 12 pp 5646ndash5652 2009

[24] S B Peng V Peek Y Zhai et al ldquoAkt activation but notextracellular signal-regulated kinase activation is required forSDF-1120572CXCR4-mediated migration of epitheloid carcinomacellsrdquo Molecular Cancer Research vol 3 no 4 pp 227ndash2362005

[25] H Hayashi Y Tsuchiya K Nakayama T Satoh and E NishidaldquoDown-regulation of the PI3-kinaseAkt pathway by ERKMAPkinase in growth factor signalingrdquo Genes to Cells vol 13 no 9pp 941ndash947 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


antioxidant such as ascorbic acid caused a reduction in cancermotility and invasion [9] and conversely the addition ofexogenous ROS enhances these activities [10]

Moscatilin (441015840-dihydroxy-33101584051015840-trimethoxybibenzyl)is a bibenzyl derivative extracted from Thai orchid Dendro-bium pulchellum (Orchidaceae) which is known as ldquoUeangchang naordquo inThai (Figure 1)Moscatilinwas reported to havevarious pharmacological properties such as antiinflamma-tory [11] antioxidant [12] and antiplatelet aggregation [13]Recently it has shown anticancer activity against many kindsof cancers for example induction of cell cycle G2-M arrest[14 15] and cell apoptosis involving its antioxidant effect [1215] In present study we investigated that the administrationof moscatilin attenuates migration and invasion in lungcancer cells Its negative regulator is associated with theability of compound to suppress endogenous ROS generationand consequently inhibits FAK and Akt activation-mediatingcancer motility and invasiveness Our finding reveals thenovel mechanism of moscatilin on the regulation of cancermigration and invasion which could be an advantage indevelopment of this compound for cancer therapy

2 Material and Methods

21 Cells and Reagents Human lung adenocarcinoma H23cells were obtained from American Type Culture Collec-tion (ATCC Manassas VA USA) and cultured in RPMI-1640 medium containing 10 fetal bovine serum 2mML-glutamine 100 IUmL penicillin and 100 120583gmL strepto-mycin (Gibco MD USA) in 37∘C with 5 CO

2

-humidifiedincubator Moscatilin was isolated from Thai orchid Den-drobium pulchellum as previously described [16] Moscatilinwas dissolved in DMSO and deionized water for the indi-cated working concentrations The amount of DMSO inthe final solution was less than 01 which showed nocytotoxic in H23 cells The 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazoliumbromide (MTT)Hoechst33342 propid-ium iodide (PI) phalloidin tetramethylrhodamine B isoth-iocyanate ribonuclease A bovine serum albumin (BSA)and dimethylsulfoxide (DMSO) were purchased from SigmaChemical Inc (St Louis MO USA) Annexin V ApoptosisDetection Kit was obtained from BD Biosciences (WoburnMA USA) Antibodies for phosphorylated Akt (S473) Aktphosphorylated FAK (Y397) FAK phosphorylated ERK(Thr202Tyr204) ERK Cdc42 120573-actin and peroxidase-conjugated secondary antibodies were obtained from CellSignaling (Denvers MA USA)

22 Cell Viability Assay Cells viability was determined byMTT assay as previously described [17] Initially cells wereseeded at a density of 104 cellswell onto 96-well plateovernight After that they were treated with various concen-trations ofmoscatilin for 24 hThemediumwas then replacedwith MTT solution (50mgmL in PBS) and incubated at37∘C for 4 h To solubilize formazan product the mediumwas replaced with 100 120583L DMSO and an intensity readingof the formazan product was measured at 550 nm usinga microplate reader (Anthros Durham NC USA) Cell

OMe

OMe

OH

MeO

HO

Figure 1 Chemical structure of moscatilin

viability was expressed as the percentage calculated fromabsorbance of MTT-treated cells relative to control cells

23 Apoptosis Assay Cells were seeded at a density of 104cellswell onto 96-well plate and incubated overnight for cellattachment After the indicated treatments cells were washedand incubated with 10 120583gmL Hoechst33342 and 5 120583gmLpropidium iodide (PI) for 30min Additionally apoptoticcells were determined using Annexin-V Staining Assay Afterthe indicated treatments cells were washed and subsequentlystained with 100120583L of 1x binding buffer containing 5120583Lof Annexin V-FITC for 15min at room temperature in thedark Nuclei condensation and DNA fragmentation of apop-totic cells Annexin-V-positive cells and PI-positive necroticcells were visualized and scored by fluorescence microscopy(Olympus IX51 with DP70) as previously described [17]

24 DNA Content Analysis Cells were seeded at a density of3 times 105 cellswell onto 6-well plate and incubated overnightfor cell attachment After the indicated treatments cells weretrypsinized and fixed in 70 absolute ethanol at minus20∘Covernight After washing with PBS cells were incubated inpropidium iodide solution containing 01Triton-X 1120583gmLRNase and 1mgmL propidium iodide at room temperaturefor 30min DNA content was analyzed using flow cytometry(FACSort Becton Dickinson Rutherford NJ USA) as previ-ously described [17]

25 Migration Determination Migration was determined bywound healing and Boyden chamber assay as previouslydescribed [6] For wound healing assay cells were seededat a density of 2 times 105 cellswell onto 24-well plate Afterthe cell monolayer was formed a micropipette tip wasused to scratch cell attachment to generate wound spaceThe cells were then washed with PBS and replaced withRPMI medium containing the indicated concentration ofmoscatilinThe progress of cell migration into the woundwasphotographed by inverted microscope (Olympus IX51 withDP70) at indicated time of incubation The average woundspace was calculated from the random field of view andrepresented the relative cell migration Relative cell migrationwas calculated by dividing the change of wound space of






2


2

O2


2


2




3 Results







2



2

∙minus andH2

O2


2




4 Discussion



lowast

lowast

140120100

80604020

00 025 05 1 5

Cel

l via

bilit

y (

)

100

80

60

40

20

00 025 05 1 5



lowast

100

80

60

40

20

00

0

025 02505

05

1

1


0 025 05 1 5Moscatilin (120583M)


Cell

dea

th (

)140120100

80604020

0

Cel

l via

bilit

y (

)

12 24 48 72Time (h)

0 120583M025 120583M

05 120583M1 120583M

ApoptosisNecrosis

Ann

exin

V-p

ositi

ve ce

lls (

) 200

0

Cou

nts


200

0

Cou

nts

0

0


200

0

Cou

nts


0

200

0

Cou

nts


PIH

oech

st

(a) (b)

(c) (d)





0 025 05 1Moscatilin (120583M)


0h

12h

24h

48h

0 025 05 1Moscatilin (120583M)

12h

24h

48h

(c)

(f)

1120583M

14

12

1

08

06

04

02

0

Rela

tive m

igra

tion

Rela

tive m

igra

tion

lowast

lowast

lowast

lowast

lowast

0 025 05 10

1

2

3

4

5

12 24 48Time (h)

0 120583M

1120583M0 120583M

(a) (b)1412

108060402

0

Rela

tive m

igra

tion

Rela

tive m

igra

tion

lowast

lowast

lowast

lowast

lowast

0 025 05 1

12 24 48Time (h)

10

8

6

4

2

0

(d)

(e)




Relat

ive i

nvas

ion

14

12

1

08

06

04

02

0

lowastlowast

0 025 05 1Moscatilin (120583M)

(a)

Relat

ive i

nvas

ion

lowast

lowast

12 24 48Time (h)

0 120583M

10

8

6

4

2

0

1120583M (b)

0 025 05 1Moscatilin (120583M)

12h

24h

48h

(c)

SRB

Phal

loid

in

0 025 05 1Moscatilin (120583M)

(d)




2

∙minus and H2

O2





Relat

ive R

OS

leve

l

1 2 30

1

2

3

4

5

Time (h)

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

12

0 120583M025 120583M

05 120583M1120583M

(a)

14

12

1

08

06

04

02

0

lowastlowast

lowast

0 025 05 1Moscatilin (120583M)

Rela

tive O

H∙

leve

l

(b)

0 025 05 1Moscatilin (120583M)

14

12

1

08

06

04

02

0

Rela

tive H

2O2

leve

l

(c)

14

12

1

08

06

04

02

00 025 05 1


Rela

tive O

2∙minus

leve

l

(d)

Relat

ive R

OS

leve

l

0

1

2

3

4

5

025 05 1

lowast

minus

minus minus

50 50 50 50

lowast lowast

lowast


(e)

0

15

05

1

lowast

lowast



Rela

tive m

igra

tion

(f)


2


4


2



4


119875 lt 005



(kD

a)

125-

125-

60-

60-

44-42-

44-42-

21-

43-

p-FAK (y397)

FAK

p-Akt (S473)

Akt

p-Erk12

Erk

Cdc42

120573-actin

0 025 05 1Moscatilin (120583M)

(a)


p-AktAkt

14

12

1

08

06

04

02

0

Relat

ive p

rote

in le

vel

0 025 05 1Moscatilin (120583M)

lowast

lowastlowast

lowast lowastlowast

(b)

FAK FAK

Akt

Cdc42

ERK12


P

ROS

Moscatilin

Moscatilin

Moscatilin

(c)






5 Conclusion


Abbreviations


2


O2





Acknowledgments


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















2


2

O2


2


2




3 Results







2



2

∙minus andH2

O2


2




4 Discussion



lowast

lowast

140120100

80604020

00 025 05 1 5

Cel

l via

bilit

y (

)

100

80

60

40

20

00 025 05 1 5



lowast

100

80

60

40

20

00

0

025 02505

05

1

1


0 025 05 1 5Moscatilin (120583M)


Cell

dea

th (

)140120100

80604020

0

Cel

l via

bilit

y (

)

12 24 48 72Time (h)

0 120583M025 120583M

05 120583M1 120583M

ApoptosisNecrosis

Ann

exin

V-p

ositi

ve ce

lls (

) 200

0

Cou

nts


200

0

Cou

nts

0

0


200

0

Cou

nts


0

200

0

Cou

nts


PIH

oech

st

(a) (b)

(c) (d)





0 025 05 1Moscatilin (120583M)


0h

12h

24h

48h

0 025 05 1Moscatilin (120583M)

12h

24h

48h

(c)

(f)

1120583M

14

12

1

08

06

04

02

0

Rela

tive m

igra

tion

Rela

tive m

igra

tion

lowast

lowast

lowast

lowast

lowast

0 025 05 10

1

2

3

4

5

12 24 48Time (h)

0 120583M

1120583M0 120583M

(a) (b)1412

108060402

0

Rela

tive m

igra

tion

Rela

tive m

igra

tion

lowast

lowast

lowast

lowast

lowast

0 025 05 1

12 24 48Time (h)

10

8

6

4

2

0

(d)

(e)




Relat

ive i

nvas

ion

14

12

1

08

06

04

02

0

lowastlowast

0 025 05 1Moscatilin (120583M)

(a)

Relat

ive i

nvas

ion

lowast

lowast

12 24 48Time (h)

0 120583M

10

8

6

4

2

0

1120583M (b)

0 025 05 1Moscatilin (120583M)

12h

24h

48h

(c)

SRB

Phal

loid

in

0 025 05 1Moscatilin (120583M)

(d)




2

∙minus and H2

O2





Relat

ive R

OS

leve

l

1 2 30

1

2

3

4

5

Time (h)

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

12

0 120583M025 120583M

05 120583M1120583M

(a)

14

12

1

08

06

04

02

0

lowastlowast

lowast

0 025 05 1Moscatilin (120583M)

Rela

tive O

H∙

leve

l

(b)

0 025 05 1Moscatilin (120583M)

14

12

1

08

06

04

02

0

Rela

tive H

2O2

leve

l

(c)

14

12

1

08

06

04

02

00 025 05 1


Rela

tive O

2∙minus

leve

l

(d)

Relat

ive R

OS

leve

l

0

1

2

3

4

5

025 05 1

lowast

minus

minus minus

50 50 50 50

lowast lowast

lowast


(e)

0

15

05

1

lowast

lowast



Rela

tive m

igra

tion

(f)


2


4


2



4


119875 lt 005



(kD

a)

125-

125-

60-

60-

44-42-

44-42-

21-

43-

p-FAK (y397)

FAK

p-Akt (S473)

Akt

p-Erk12

Erk

Cdc42

120573-actin

0 025 05 1Moscatilin (120583M)

(a)


p-AktAkt

14

12

1

08

06

04

02

0

Relat

ive p

rote

in le

vel

0 025 05 1Moscatilin (120583M)

lowast

lowastlowast

lowast lowastlowast

(b)

FAK FAK

Akt

Cdc42

ERK12


P

ROS

Moscatilin

Moscatilin

Moscatilin

(c)






5 Conclusion


Abbreviations


2


O2





Acknowledgments


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















2



2

∙minus andH2

O2


2




4 Discussion



lowast

lowast

140120100

80604020

00 025 05 1 5

Cel

l via

bilit

y (

)

100

80

60

40

20

00 025 05 1 5



lowast

100

80

60

40

20

00

0

025 02505

05

1

1


0 025 05 1 5Moscatilin (120583M)


Cell

dea

th (

)140120100

80604020

0

Cel

l via

bilit

y (

)

12 24 48 72Time (h)

0 120583M025 120583M

05 120583M1 120583M

ApoptosisNecrosis

Ann

exin

V-p

ositi

ve ce

lls (

) 200

0

Cou

nts


200

0

Cou

nts

0

0


200

0

Cou

nts


0

200

0

Cou

nts


PIH

oech

st

(a) (b)

(c) (d)





0 025 05 1Moscatilin (120583M)


0h

12h

24h

48h

0 025 05 1Moscatilin (120583M)

12h

24h

48h

(c)

(f)

1120583M

14

12

1

08

06

04

02

0

Rela

tive m

igra

tion

Rela

tive m

igra

tion

lowast

lowast

lowast

lowast

lowast

0 025 05 10

1

2

3

4

5

12 24 48Time (h)

0 120583M

1120583M0 120583M

(a) (b)1412

108060402

0

Rela

tive m

igra

tion

Rela

tive m

igra

tion

lowast

lowast

lowast

lowast

lowast

0 025 05 1

12 24 48Time (h)

10

8

6

4

2

0

(d)

(e)




Relat

ive i

nvas

ion

14

12

1

08

06

04

02

0

lowastlowast

0 025 05 1Moscatilin (120583M)

(a)

Relat

ive i

nvas

ion

lowast

lowast

12 24 48Time (h)

0 120583M

10

8

6

4

2

0

1120583M (b)

0 025 05 1Moscatilin (120583M)

12h

24h

48h

(c)

SRB

Phal

loid

in

0 025 05 1Moscatilin (120583M)

(d)




2

∙minus and H2

O2





Relat

ive R

OS

leve

l

1 2 30

1

2

3

4

5

Time (h)

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

12

0 120583M025 120583M

05 120583M1120583M

(a)

14

12

1

08

06

04

02

0

lowastlowast

lowast

0 025 05 1Moscatilin (120583M)

Rela

tive O

H∙

leve

l

(b)

0 025 05 1Moscatilin (120583M)

14

12

1

08

06

04

02

0

Rela

tive H

2O2

leve

l

(c)

14

12

1

08

06

04

02

00 025 05 1


Rela

tive O

2∙minus

leve

l

(d)

Relat

ive R

OS

leve

l

0

1

2

3

4

5

025 05 1

lowast

minus

minus minus

50 50 50 50

lowast lowast

lowast


(e)

0

15

05

1

lowast

lowast



Rela

tive m

igra

tion

(f)


2


4


2



4


119875 lt 005



(kD

a)

125-

125-

60-

60-

44-42-

44-42-

21-

43-

p-FAK (y397)

FAK

p-Akt (S473)

Akt

p-Erk12

Erk

Cdc42

120573-actin

0 025 05 1Moscatilin (120583M)

(a)


p-AktAkt

14

12

1

08

06

04

02

0

Relat

ive p

rote

in le

vel

0 025 05 1Moscatilin (120583M)

lowast

lowastlowast

lowast lowastlowast

(b)

FAK FAK

Akt

Cdc42

ERK12


P

ROS

Moscatilin

Moscatilin

Moscatilin

(c)






5 Conclusion


Abbreviations


2


O2





Acknowledgments


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowast

lowast

140120100

80604020

00 025 05 1 5

Cel

l via

bilit

y (

)

100

80

60

40

20

00 025 05 1 5



lowast

100

80

60

40

20

00

0

025 02505

05

1

1


0 025 05 1 5Moscatilin (120583M)


Cell

dea

th (

)140120100

80604020

0

Cel

l via

bilit

y (

)

12 24 48 72Time (h)

0 120583M025 120583M

05 120583M1 120583M

ApoptosisNecrosis

Ann

exin

V-p

ositi

ve ce

lls (

) 200

0

Cou

nts


200

0

Cou

nts

0

0


200

0

Cou

nts


0

200

0

Cou

nts


PIH

oech

st

(a) (b)

(c) (d)





0 025 05 1Moscatilin (120583M)


0h

12h

24h

48h

0 025 05 1Moscatilin (120583M)

12h

24h

48h

(c)

(f)

1120583M

14

12

1

08

06

04

02

0

Rela

tive m

igra

tion

Rela

tive m

igra

tion

lowast

lowast

lowast

lowast

lowast

0 025 05 10

1

2

3

4

5

12 24 48Time (h)

0 120583M

1120583M0 120583M

(a) (b)1412

108060402

0

Rela

tive m

igra

tion

Rela

tive m

igra

tion

lowast

lowast

lowast

lowast

lowast

0 025 05 1

12 24 48Time (h)

10

8

6

4

2

0

(d)

(e)




Relat

ive i

nvas

ion

14

12

1

08

06

04

02

0

lowastlowast

0 025 05 1Moscatilin (120583M)

(a)

Relat

ive i

nvas

ion

lowast

lowast

12 24 48Time (h)

0 120583M

10

8

6

4

2

0

1120583M (b)

0 025 05 1Moscatilin (120583M)

12h

24h

48h

(c)

SRB

Phal

loid

in

0 025 05 1Moscatilin (120583M)

(d)




2

∙minus and H2

O2





Relat

ive R

OS

leve

l

1 2 30

1

2

3

4

5

Time (h)

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

12

0 120583M025 120583M

05 120583M1120583M

(a)

14

12

1

08

06

04

02

0

lowastlowast

lowast

0 025 05 1Moscatilin (120583M)

Rela

tive O

H∙

leve

l

(b)

0 025 05 1Moscatilin (120583M)

14

12

1

08

06

04

02

0

Rela

tive H

2O2

leve

l

(c)

14

12

1

08

06

04

02

00 025 05 1


Rela

tive O

2∙minus

leve

l

(d)

Relat

ive R

OS

leve

l

0

1

2

3

4

5

025 05 1

lowast

minus

minus minus

50 50 50 50

lowast lowast

lowast


(e)

0

15

05

1

lowast

lowast



Rela

tive m

igra

tion

(f)


2


4


2



4


119875 lt 005



(kD

a)

125-

125-

60-

60-

44-42-

44-42-

21-

43-

p-FAK (y397)

FAK

p-Akt (S473)

Akt

p-Erk12

Erk

Cdc42

120573-actin

0 025 05 1Moscatilin (120583M)

(a)


p-AktAkt

14

12

1

08

06

04

02

0

Relat

ive p

rote

in le

vel

0 025 05 1Moscatilin (120583M)

lowast

lowastlowast

lowast lowastlowast

(b)

FAK FAK

Akt

Cdc42

ERK12


P

ROS

Moscatilin

Moscatilin

Moscatilin

(c)






5 Conclusion


Abbreviations


2


O2





Acknowledgments


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 025 05 1Moscatilin (120583M)


0h

12h

24h

48h

0 025 05 1Moscatilin (120583M)

12h

24h

48h

(c)

(f)

1120583M

14

12

1

08

06

04

02

0

Rela

tive m

igra

tion

Rela

tive m

igra

tion

lowast

lowast

lowast

lowast

lowast

0 025 05 10

1

2

3

4

5

12 24 48Time (h)

0 120583M

1120583M0 120583M

(a) (b)1412

108060402

0

Rela

tive m

igra

tion

Rela

tive m

igra

tion

lowast

lowast

lowast

lowast

lowast

0 025 05 1

12 24 48Time (h)

10

8

6

4

2

0

(d)

(e)




Relat

ive i

nvas

ion

14

12

1

08

06

04

02

0

lowastlowast

0 025 05 1Moscatilin (120583M)

(a)

Relat

ive i

nvas

ion

lowast

lowast

12 24 48Time (h)

0 120583M

10

8

6

4

2

0

1120583M (b)

0 025 05 1Moscatilin (120583M)

12h

24h

48h

(c)

SRB

Phal

loid

in

0 025 05 1Moscatilin (120583M)

(d)




2

∙minus and H2

O2





Relat

ive R

OS

leve

l

1 2 30

1

2

3

4

5

Time (h)

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

12

0 120583M025 120583M

05 120583M1120583M

(a)

14

12

1

08

06

04

02

0

lowastlowast

lowast

0 025 05 1Moscatilin (120583M)

Rela

tive O

H∙

leve

l

(b)

0 025 05 1Moscatilin (120583M)

14

12

1

08

06

04

02

0

Rela

tive H

2O2

leve

l

(c)

14

12

1

08

06

04

02

00 025 05 1


Rela

tive O

2∙minus

leve

l

(d)

Relat

ive R

OS

leve

l

0

1

2

3

4

5

025 05 1

lowast

minus

minus minus

50 50 50 50

lowast lowast

lowast


(e)

0

15

05

1

lowast

lowast



Rela

tive m

igra

tion

(f)


2


4


2



4


119875 lt 005



(kD

a)

125-

125-

60-

60-

44-42-

44-42-

21-

43-

p-FAK (y397)

FAK

p-Akt (S473)

Akt

p-Erk12

Erk

Cdc42

120573-actin

0 025 05 1Moscatilin (120583M)

(a)


p-AktAkt

14

12

1

08

06

04

02

0

Relat

ive p

rote

in le

vel

0 025 05 1Moscatilin (120583M)

lowast

lowastlowast

lowast lowastlowast

(b)

FAK FAK

Akt

Cdc42

ERK12


P

ROS

Moscatilin

Moscatilin

Moscatilin

(c)






5 Conclusion


Abbreviations


2


O2





Acknowledgments


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Relat

ive i

nvas

ion

14

12

1

08

06

04

02

0

lowastlowast

0 025 05 1Moscatilin (120583M)

(a)

Relat

ive i

nvas

ion

lowast

lowast

12 24 48Time (h)

0 120583M

10

8

6

4

2

0

1120583M (b)

0 025 05 1Moscatilin (120583M)

12h

24h

48h

(c)

SRB

Phal

loid

in

0 025 05 1Moscatilin (120583M)

(d)




2

∙minus and H2

O2





Relat

ive R

OS

leve

l

1 2 30

1

2

3

4

5

Time (h)

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

12

0 120583M025 120583M

05 120583M1120583M

(a)

14

12

1

08

06

04

02

0

lowastlowast

lowast

0 025 05 1Moscatilin (120583M)

Rela

tive O

H∙

leve

l

(b)

0 025 05 1Moscatilin (120583M)

14

12

1

08

06

04

02

0

Rela

tive H

2O2

leve

l

(c)

14

12

1

08

06

04

02

00 025 05 1


Rela

tive O

2∙minus

leve

l

(d)

Relat

ive R

OS

leve

l

0

1

2

3

4

5

025 05 1

lowast

minus

minus minus

50 50 50 50

lowast lowast

lowast


(e)

0

15

05

1

lowast

lowast



Rela

tive m

igra

tion

(f)


2


4


2



4


119875 lt 005



(kD

a)

125-

125-

60-

60-

44-42-

44-42-

21-

43-

p-FAK (y397)

FAK

p-Akt (S473)

Akt

p-Erk12

Erk

Cdc42

120573-actin

0 025 05 1Moscatilin (120583M)

(a)


p-AktAkt

14

12

1

08

06

04

02

0

Relat

ive p

rote

in le

vel

0 025 05 1Moscatilin (120583M)

lowast

lowastlowast

lowast lowastlowast

(b)

FAK FAK

Akt

Cdc42

ERK12


P

ROS

Moscatilin

Moscatilin

Moscatilin

(c)






5 Conclusion


Abbreviations


2


O2





Acknowledgments


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Relat

ive R

OS

leve

l

1 2 30

1

2

3

4

5

Time (h)

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

12

0 120583M025 120583M

05 120583M1120583M

(a)

14

12

1

08

06

04

02

0

lowastlowast

lowast

0 025 05 1Moscatilin (120583M)

Rela

tive O

H∙

leve

l

(b)

0 025 05 1Moscatilin (120583M)

14

12

1

08

06

04

02

0

Rela

tive H

2O2

leve

l

(c)

14

12

1

08

06

04

02

00 025 05 1


Rela

tive O

2∙minus

leve

l

(d)

Relat

ive R

OS

leve

l

0

1

2

3

4

5

025 05 1

lowast

minus

minus minus

50 50 50 50

lowast lowast

lowast


(e)

0

15

05

1

lowast

lowast



Rela

tive m

igra

tion

(f)


2


4


2



4


119875 lt 005



(kD

a)

125-

125-

60-

60-

44-42-

44-42-

21-

43-

p-FAK (y397)

FAK

p-Akt (S473)

Akt

p-Erk12

Erk

Cdc42

120573-actin

0 025 05 1Moscatilin (120583M)

(a)


p-AktAkt

14

12

1

08

06

04

02

0

Relat

ive p

rote

in le

vel

0 025 05 1Moscatilin (120583M)

lowast

lowastlowast

lowast lowastlowast

(b)

FAK FAK

Akt

Cdc42

ERK12


P

ROS

Moscatilin

Moscatilin

Moscatilin

(c)






5 Conclusion


Abbreviations


2


O2





Acknowledgments


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(kD

a)

125-

125-

60-

60-

44-42-

44-42-

21-

43-

p-FAK (y397)

FAK

p-Akt (S473)

Akt

p-Erk12

Erk

Cdc42

120573-actin

0 025 05 1Moscatilin (120583M)

(a)


p-AktAkt

14

12

1

08

06

04

02

0

Relat

ive p

rote

in le

vel

0 025 05 1Moscatilin (120583M)

lowast

lowastlowast

lowast lowastlowast

(b)

FAK FAK

Akt

Cdc42

ERK12


P

ROS

Moscatilin

Moscatilin

Moscatilin

(c)






5 Conclusion


Abbreviations


2


O2





Acknowledgments


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusion


Abbreviations


2


O2





Acknowledgments


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Moscatilin Inhibits Lung Cancer Cell...

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