Tylophorine, a phenanthraindolizidine alkaloid isolated from Tylophora indica exerts ...

16 Pages·2013·2.98 MB·English

Saraswatietal.MolecularCancer2013,12:82
http://www.molecular-cancer.com/content/12/1/82
RESEARCH Open Access
Tylophorine, a phenanthraindolizidine alkaloid
Tylophora indica
isolated from exerts
antiangiogenic and antitumor activity by
targeting vascular endothelial growth factor
–
receptor 2 mediated angiogenesis
Sarita Saraswati1*, Pawan K Kanaujia2, Shakti Kumar3, Ranjeet Kumar4 and Abdulqader A Alhaider5
Abstract
Background: Anti-angiogenesis targeting VEGFR2 has been considered as an important strategy for cancer therapy.
Tylophorine is known to possess anti-inflammatoryand antitumor activity, but itsroles intumor angiogenesis, the
key step involved in tumor growth and metastasis, and theinvolved molecular mechanism is still unknown.
Therefore, we examined itsanti-angiogenic effects and mechanisms in vitro and in vivo.
Methods: We used tylophorine and analyzedits inhibitory effects on human umbilical vein endothelial cells
(HUVEC) invitro and Ehrlich ascites carcinoma (EAC)tumor in vivo.
Results: Tylophorine significantly inhibited a series of VEGF-induced angiogenesisprocesses including proliferation,
migration, and tube formation of endothelial cells. Besides, it directly inhibited VEGFR2 tyrosine kinase activity and
itsdownstream signaling pathways including Akt, Erk and ROS in endothelial cells. UsingHUVECswedemonstrated
that tylophorineinhibited VEGF-stimulatedinflammatory responses including IL-6, IL-8, TNF-α,IFN-γ,MMP-2 and NO
secretion. Tylophorine significantly inhibited neovascularization insponge implant angiogenesis assay and also
inhibited tumor angiogenesis and tumor growth in vivo. Molecular docking simulation indicated that tylophorine
could form hydrogen bonds and aromatic interactions within the ATP-binding region of the VEGFR2 kinase unit.
Conclusion: Tylophorine exerts anti-angiogenesiseffectsvia VEGFR2 signaling pathway thus, may be a viable drug
candidate inanti-angiogenesis and anti-cancer therapies.
Keywords: Tylophorine, VEGFR2, Antiangiogenesis,Microvessel density, Molecular docking
Background VEGF-C and VEGF-D, as numerous splice variant
Angiogenesis, the formation of new blood vessels by isoforms [4]. VEGF exerts its biological actions on the
sprouting from pre-existing endothelium [1], one of the endothelialcellsismediatedbytwotypesofreceptortyro-
characteristic of malignant neoplasia development [2]. sinekinases(RTKs),namelyVEGFR1(Flt-1)andVEGFR2
Angiogenesis blockade has been shown to be an effective (KDR/Flk-1)withhighaffinities[4].VEGFR2playsanim-
strategy in inhibiting tumor growth and metastasis [3]. A portant role in mediating the mitogenesis and permeabil-
major pro-angiogenic cytokine is vascular endothelial ityofendothelialcells.AutophosphorylationofTyr1175on
growth factor (VEGF) which comprises several isotypes, VEGFR2 is crucial for endothelial cell proliferation, and
includingVEGF-A(vascularpermeabilityfactor),VEGF-B, leads to the activation of downstreamsignaling events in-
cluding Src family kinase [5], focal adhesion kinase (FAK)
[6,7], phosphoinisitide 3 kinase/AKT kinase, Mammalian
*Correspondence:[email protected]
1CamelBiomedicalResearchUnit,CollegeofPharmacyandMedicine,King targetofrapamycin(mTOR)[8],proteinkinaseC/protein
SaudUniversity,Riyadh,KingdomofSaudiArabia kinased D, mitogen extracelluar kinase/ extracellular
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Saraswatietal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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signal related kinase (ERK) that subsequently promote and in vivo mouse model. Our results demonstrate that
proliferation,migration,andtubeformationofendothelial tylophorine significantly inhibited VEGF-stimulated endo-
cells in pre-existing vasculature. Recently many studies thelial cell proliferation, migration and tube formation
showed the important role of VEGFR2 in potential drug invitro.Tylophorineinhibitedneovascularizationinsponge
discoveryandmolecularmechanismresearch[9].Consid- implant angiogenesis assay in vivo and further attenuated
ering anti-angiogenesis therapy is to target endothelial tumor associated angiogenesis. Furthermore, mechanistic-
cells that support tumor growth rather than cancer cells ally, tylophorine suppressed VEGFR2-mediated signaling
themselves,VEGFR2hasbecomeanimportanttherapeutic pathway. Meanwhile, the structure-based interaction be-
targetforcanceranti-angiogenesistherapy[10-13]. tween tylophorine and VEGFR2 was found to be stable
TheNationalInstitutesofHealth(NIH)websiteprovides conformation based on in-silico analysis which revealed
a basic summary of anti-angiogenic drugs that were or are thathydrogenbondandaromaticinteractionswereformed.
stillcurrentlyunderclinicalinvestigations(http://www.can- Taken together our results suggest that tylophorine could
cer.gov/clinicaltrials/developments/antiangio-table). These be used as a potential anti-angiogenesis agent that targets
include monoclonal antibodies targeting VEGF ligands or VEGF/VEGFR2 signaling pathways and inhibits tumor in-
VEGFRs [14], soluble receptors that sequester ligands [15] ducedangiogenesis.
and small molecule inhibitors that inhibit kinase activity
[16]. Three drugs developed for their anti-angiogenic ac- Results
tions, bevacizumab (Avastin®), sunitinib malate (Sutent®, Tylophorineinhibitedcellviabilityinendothelialcells
SU11248) and sorafenib (Nexavar®, BAY 43-9006), have Angiogenesisisprimarilyinitiatedbygrowthfactorsthere-
beenapprovedbytheUnitedStatesFoodandDrugAdmin- fore we tested whether tylophorine decreases VEGF-
istrationfortreatmentofpatientswithspecifictypesofcan- mediated HUVEC viability and proliferation. We found
cer—all three inhibit VEGF signaling by blocking VEGF that when HUVECs were cultured in normal cell culture
ligand or VEGFR [17]. However, serious side effects, such medium (ECGM supplemented with 20% FBS) in absence
as hypertension, bleeding and gastrointestinal perforation, of VEGF, tylophorine inhibited cell viabilityin a dose- and
have been associated with currently available anti-VEGF time-dependentmanner.Significantcellviabilityinhibitory
agents, limiting their chronic use [17]. Hence, there is an effect of tylophorine was observed in HUVECs at concen-
urgent need to find a molecule that can be more specific trations more than 10 μM (Figure 1B). As shown in
and less toxic for the treatment of cancer, particularly Figure 1C, the proliferation of endothelial cells stimulated
agents that exhibit activity against drug-resistant strains, by VEGF was markedly decreased after tylophorine treat-
completelysterilizetheinfection,orshortenthedurationof mentrangingfrom2.5to20μMatdifferenttimeintervals
drugtherapyandthuspromotedrugcompliance. of 24 and 48 h indicating extracellular VEGF acted as a
Tylophorine (Figure 1A) and its analogs are strong attractant for endothelial cells proliferation.
phenanthroindolizidine alkaloids, isolated from Tylophora TylophorinealoneinhibitedthegrowthofHUVECindose
indica(Asclepiadaceae)[18].Tylophoraindicahasbeenin- dependent manner (Additional file 1: Figure S1A). As
cluded as an official drug in the Bengal pharmacoepia of detected by BrdU incorporation assay (Figure 1D), DNA
1884 [19]. The leaves of this plant have been used for the synthesis of HUVECs was also significantly inhibited by
treatmentofasthmaaswell asbronchitis,rheumatism and tylophorineinadose-dependentmanner.Tofurtherexam-
dysentery in India. These alkaloids possesses anticancer ine whether tylophorine would result in toxic effects of
[20-24], anti-inflammatory [19,25], anti-ameobicidal [26] HUVEC, LDH cytotoxic assay was carried out. As shown
and anti-viral [27] activity. Several key metabolic enzymes, in Figure 1E, Tylophorine caused minute toxicity on
including thymidylate synthase [28] and dihydrofolate re- HUVECs.
ductase have been reported as biological targets of
tylophorine alkaloids [29]. Tylophorine derivatives also in- TylophorineinhibitedVEGF-inducedendothelialcell
hibits activator protein-1–mediated, CRE-mediated, and migrationandinvasionandtubeformationofHUVECs
nuclear factor kappaB (NF-κB)-mediated transcription Cell migration is an essential step in angiogenesis [33];
[30,31]. Tylophorine arrests the cells at G1 phase in therefore we investigated the effects of tylophorine on
HepG2,HONE-1,andNUGC-3carcinomacellsanddown the chemotactic motility of the endothelial cells by using
regulates cyclin A2 expression [32]. Preliminary studies wound-healing (Figure 2A) assay. The results showed
illustrate the potential of tylophorine as a new class of that tylophorine significantly inhibited VEGF-induced
anticancer drugs. However, the molecular mechanism re- HUVECs migration in a dose-dependent manner ran-
sponsible of its inhibitory effects on cancer cell growth is ging from 2.5 μM to 20 μM. Directional motility and
largely unknown. In this study, we evaluated for the first matrixdegradationarecrucialforangiogenesissprouting
time how tylophorine inhibits tumor angiogenesis by therefore, we next examined the effect of tylophorine on
targetingkeysignalingpathwaysonhumanendothelialcells the invasion ability of HUVECs using the Boyden
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Figure1EffectoftylophorineoncellproliferationinHUVECs.(A)Chemicalstructure(B)Undernormalculturecondition.HUVECswere
culturedinECGMcontaining20%FBS,thencells(5×104cells/well)weretreatedwithDMSO(0.1%)orvariousconcentrationsoftylophorinefor
24,48and72h.CellviabilitywasdeterminedbyMTTassay.CellsreceivingonlyDMSO(0.1%)servedasavehiclecontrol.Datawereexpressed
aspercentagesofthevehiclecontrol(100%)asmean±SEM,n=6wells.**p<0.01;***p<0.001versuscontrolgroup.(C)UnderVEGF-stimulated
conditionHUVECs(5×104cells/well)werestarvedwithECGMsupplementedwith0.5%FBSfor24h,andthentreatedwithorwithoutVEGF(10ng/
mL)andDMSO(0.1%)orvariousconcentrationsoftylophorineforanother24and48h.Datawereexpressedaspercentagesofthevehiclecontrol(100%)
asmean±SEM,n=6wells.(D)EffectsoftylophorineonDNAsynthesiswasexaminedbyBrdUcellproliferationenzymelinkedimmunosorbent
assay.Datawereexpressedaspercentagesofthevehiclecontrol(100%)asmean±SEM,n=6wells.*p<0.05;**p<0.01;***p<0.001versus
controlgroup.(E)TylophorineadministrationdidnotresultinLDHreleasefromendothelialcellsasstudiedwithLDHcytotoxicityassaykit
indicatingthattylophorineposedlittlecytotoxicityeffectsuponHUVECs.Datawereexpressedaspercentagesofthevehiclecontrol(100%)
asmean±SEM,n=6wells.
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Figure2TylophorineinhibitedVEGF-inducedendothelialcellmigrationandinvasionandtubeformationofHUVECs.(A)Effectof
tylophorineonVEGF-inducedcellmotility(wound-healingassay).ConfluentHUVECmonolayerson0.1%gelatin-coatedsix-wellplateswere
scratchwounded.Thecellsweretreatedwithvariousconcentrationsoftylophorinewith0.5%FBSand10ng/mLVEGFfor16h.Representative
fieldswerephotographed,×100magnification.GraphshowsthequantitativeeffectoftylophorineonVEGF-inducedHUVECmotility.Data
werepresentedasmean±SEM,n=6wells.##p<0.01VEGF-treatedgroupversusnoVEGF-treatedgroup;**p<0.01;***p<0.001versusVEGF-
stimulatedgroup.(B)EffectoftylophorineonVEGF-inducedinvasionofHUVECthroughMatrigelin24h.Representativefieldswere
photographed,×100magnification.GraphshowsthequantitativeeffectoftylophorineonVEGF-inducedHUVECinvasion.Datawerepresented
asmeans±SEM,n=6wells.##p<0.01VEGF-treatedgroupversusnoVEGF-treatedgroup;**p<0.01;***p<0.001comparedwithVEGF-
stimulatedgroup.(C)EffectoftylophorineonVEGF-inducedcapillary-liketubeformationofHUVECthroughMatrigelin24h.Representativefields
werephotographed,×100magnification.GraphshowsthequantitativeeffectoftylophorineonVEGF-inducedHUVECtubeformation.Datawere
presentedasmean±SEM,n=6wells.##p<0.01VEGF-treatedgroupversusnoVEGF-treatedgroup;**p<0.01;***p<0.001versusVEGF-stimulatedgroup.
chamber assay. As shown in Figure 2B, a large number incubated with tylophorine at 10 μM (p<0.001). Taken
of cells migrated to the lower side of membrane in the together, tylophorine suppressed VEGF-induced angio-
transwell chamber after stimulation with VEGF. How- genesis in vitro by inhibiting the migration, invasion and
ever, the number of invaded cells were significantly low tubular structureformationofendothelialcells.
in the presence of tylophorine (p<0.001). The matur-
ation of migrated endothelialcellsinto acapillary tube is DifferentialeffectoftylophorineonthebindingofVEGF
a critical step during angiogenesis [34]. Thus, we investi- toitsreceptors
gated its effect on HUVEC tube formation. When Further, we investigated whether tylophorine inhibits
HUVECs were seeded on the growth factor–reduced the binding of VEGF to its receptors, VEGFR1 (Flt-1)
matrigel, robust tubular-like structures were formed in and VEGFR2 (Flk-1/KDR). As shown in Figure 3A,
the presence of VEGF (Figure 2C). Almost 80% destruc- tylophorine decreased the binding of VEGFR2 to
tion of tube network was observed when HUVECs were immobilized VEGF with IC of∼12.29 μM. However,
50
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Figure3TylophorineinhibitsVEGFR2bindingwithVEGFandattenuatedVEGFR2tyrosinekinaseactivity.(A)Effectoftylophorineon
thebindingofVEGFR1(Flt-1)andVEGFR2(KDR/Flk-1)toimmobilizedVEGF.Datawerepresentedasmeans±SEM,n=6.**p<0.01;***p<0.001
comparedwithcontrol.(B)InhibitionofVEGFR2kinaseactivitybytylophorinewasanalyzedusinganinvitroHTScan®VEGFreceptor2kinasekit
(CellSignalingTechnology,Danvers,MA,USA)combinedwithcolorimetricELISAdetectionaccordingtothemanufacturer’sinstructions.The
reactionprocessedwithDMSO(0.1%)servedasavehiclecontrol.Datawereexpressedaspercentagesofthevehiclecontrol.(C)Westernblot
analysesofeffectoftylophorineonphosphorylationofVEGFR2.HUVECswerepre-treatedwithtylophorinefollowedbythestimulationwith
50ng/mLofVEGFfor2min.Datawerepresentedasmeans±SEM,n=6.(D)QuantitativedensitometryofVEGFR2phosphorylationisshown
aspercentage(%)ofcontrol.Datawerepresentedasmeans±SEM,n=6.##P<0.01VEGF-treatedgroupversusnoVEGF-treatedgroup;
**p<0.01;***p<0.001versusVEGF-stimulatedgroup.
tylophorine did affected the binding between VEGF and tyrosine kinase assay. Tylophorine was found to inhibit
VEGFR1(Figure3A)but itdidnotreached tosignificant kinase activity of VEGFR2 (Figure 3B) with an IC of
50
level. Antihuman VEGFR1 antibody (AF321, R&D Sys- ~9.2 μM. SU5416, a known inhibitor of VEGFR2, was
tems) and antihuman VEGFR2 antibody (MAB3572, used as a positive control and showed inhibition of
R&DSystems)wereusedaspositive control forVEGFR1 kinase activity with an IC of 1.5 μM (data not shown),
50
andVEGFR2respectively(datanot shown). asdescribedpreviously[35].
We further examined the effects of tylophorine on
TylophorineattenuatedVEGFR2tyrosinekinaseactivity phosphorylation of VEGFR2 to determine its inhibitory
Previousstudiessuggestedthatblockage ofVEGFR-2ac- effect on VEGFR2-mediated signaling pathways in endo-
tivity could significantly limit tumoral neovascularization thelial cells. We found that VEGFR2 was phosphorylated
process [12]. Therefore, we first investigated whether by the addition of exogenous VEGF to HUVECs
tylophorine decreased P-VEGFR2 levels by inhibiting the (Figure 3C). Pretreatment of cells with tylophorine sig-
kinase activity of VEGFR2 using an ELISA-based nificantly blocked VEGF-induced phosphorylation of
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VEGFR2, without affecting overall VEGFR2 expression either P-AKT (Ser473) or P-ERK1/2. The result showed
levels. Quantitative densitometry of protein phosphoryl- that P-ERK1/2 is enhanced by VEGF treatment while
ation is shown as percentage (%) of vehicle control the expression level of ERK1/2 remains unchanged.
(Figure 3D). The protein levels were normalized to β- Tylophorine was found to inhibit the phosphorylation of
actin. In addition, previous studies supported that ERK1/2 at the concentration of 20 μM without affecting
phosphorylation of VEGFR2 could subsequently trigger total ERK1/2 expression level (Figure 4A). A recent
multiple downstream signals that induced proliferation study suggests that the AKT/mTOR pathways and
anddifferentiationactivitiesofendothelial cells [36,37]. Hsp90, which are criticalfor angiogenesis,are phosphor-
ylated or activated by VEGFR2 activation in the endo-
TylophorineinhibitedtheactivationofVEGFR2-mediated thelial cells [39]. As shown in Figure 4A, expression
signalingpathways levels of P-AKTand p-mTOR increases by VEGF treat-
Binding of VEGFR2 with VEGF led to the activation of ment. Pretreatment of the HUVECs with tylophorine
various downstream signaling molecules responsible for significantly inhibited the phosphorylation of AKT and
endothelial cell migration, proliferation and survival mTOR, while the total amount of AKT and mTOR re-
[35]. To further delineate the mechanism that underlies mains unchanged. Further, the action of tylophorine on
the anti-angiogenic effects of tylophorine, we screened the phosphorylation of FAK and Src were determined.
some key kinases involved in VEGFR2-mediated signal- The result showed that tylophorine inhibited VEGF-
ing pathway. VEGF induces survival of endothelial cells induced phosphorylation of FAK at the dose of 10 and
(ECs) mainly via the activation of AKT [37], whereas 20 μM and Src at the concentration of 20 μM respect-
activation of ERK1/2 MAPKs is thought to be essential ively (Figure 4A). Tylophorine could evidently inhibit
for VEGF-induced proliferation [38]. To assess the VEGF-stimulated eNOS expression. In addition, both
effect of tylophorine on these pathways, serum-starved the MMP-9 and MMP-2 activities were suppressed with
HUVECs were treated withVEGF for 20 minutes in the tylophorine treatment (Figure 4B). ROS is known to be
presence or absence of tylophorine and cell lysates were downstream signaling after VEGFR2 activation [40],
subjected to immunodetection using antibodies against therefore, we detected the ROS levels by DCFH-DA
Figure4WesternblotanalysisoftheeffectoftylophorineonVEGFR2-mediateddownstreamsignaling.(A)HUVECswerepre-treated
withtylophorinefollowedbythestimulationwith50ng/mLofVEGFfor20min.Datawerepresentedasmeans±SEM,n=6.(B).Effectof
tylophorineonVEGF-inducedMMP-2secretionfromHUVECsafter20hexaminedbyzymography.Datawerepresentedasmeans±SEM,n=6.
(C).EffectoftylophorineonHUVECsintracellularROSlevelasdetectedbyDCFH-DAstainingassay.Datawerepresentedasmeans±SEM,n=6.
##p<0.01VEGF-treatedgroupversusnoVEGF-treatedgroup;**p<0.01;***p<0.001versusVEGF-stimulatedgroup.
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probe. The results showed that the intracellular ROS Tylophorineinhibitedneovascularizationinvivo
levels were significantly reduced after tylophorine ad- To determine whether tylophorine has an effect on
ministration (Figure 4C). Taken together, our result re- angiogenesis in vivo, we performed a sponge implant
vealed that tylophorine inhibited in vitro angiogenesis by angiogenesis assay in Swiss albino mice. Sponge disks
directly targeting VEGFR2 on the surface of endothelial were s.c. implanted into mice and treatment with
cells, and further downregulating VEGFR2-mediated tylophorine or DMSO was continued, once daily, for
signalingpathway. 14 days. Over 14 day experimental period, the weight of
sponge granuloma tissues increased gradually in vehicle-
TylophorineinhibitedVEGF-inducedIL-6,IL-8,TNF-α, control group, whereas in tylophorine treated group
IFN-γ,MMP-2andNO sponge weight was reduced dramatically (Figure 6A).
During inflammation VEGFR activation is linked Daily administration of tylophorine into the sponge
to cytokine release, pro-inflammatory molecules and implants caused a marked decrease in angiogenesis as
leukocyte endothelial interactions, which exacerbate the evident by pictorial representation (Figure 6B) and de-
inflammatory response [41]. Therefore, we investigated creased hemoglobin concentration (Figure 6C)in sponge
the effect tylophorine on endothelial cell cytokine re- granuloma tissues. In implants of control group, the
lease. As shown in Figure 5, HUVECs treated for 24 h hemoglobin levels were found to be 3.11±0.17 μg Hb/
withVEGF up-regulated the secretion of IL-6 (Figure 5A), mg wet tissue (n=10); versus 2.21±0.52 μg Hb/mg
IL-8 (Figure 5B), TNF-α (Figure 5C), IFN-γ (Figure 5D) (tylophorine 7.5 mg/kg; n=10) and 1.24±0.19 μg Hb/
and MMP-2 (Figure 5E). HUVECs pretreated with mg wet tissue (tylophorine 15 mg/kg; n=10). The differ-
tylophorine, before the addition of VEGF (10 ng/mL), sig- ence in between control and treated groups were further
nificantly (P<0.001) decreased the cytokine secretionIL-6, confirmed by morphometric analysis of implants that
IL-8,TNF-α,IFN-γandMMP-2inadose-dependentman- the number of blood vessels was markedly lower in the
ner (Figure 5). Further tylophorine significantly inhibited treatedgroupsascomparedtocontrolgroup(Figure6D),
NOlevels(Figure5F,P<0.001)inHUVECat24hincuba- which was confirmed by staining with CD31. It was ob-
tioninadose-dependentmanner. served that tylophorine treatment significantly reduced
Figure5EffectoftylophorineonsecretionofVEGF-inducedcytokines.(A)IL-6,(B)IL-8,(C)TNF-α(D)IFN-γ(E)MMP-2and(F)NO.
Serum-starvedHUVECswerepreincubatedwithDMSO(0.1%)ortylophorine(20μM),beforestimulationwithVEGF(10ng/mL),andsupernatants
wereharvestedafter24hforcytokineassays.IL-6,IL-8,TNF-α,IFN-γandMMP-2weremeasuredbysandwichELISAfollowingthemanufacturer’s
instructions(RandDsystems,USA).Absorbancewasdeterminedusingamicroplatereader(Biorad,USA)at450nm.TheNOlevelsintheHUVECs
weremeasuredwithNitricoxidecolorimetricassaykit(Biovision,USA)followingthemanufacturer’sinstructions.Absorbancewasdetermined
usingamicroplatereader(Biorad,USA)at540nm.Datawerepresentedasmeans±SEM,n=3.##p<0.01VEGF-treatedgroupversusnoVEGF-
treatedgroup;**p<0.01;***p<0.001versusVEGF-stimulatedgroup.
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Figure6Effectoftylophorineonspongeimplantangiogenesisassayinvivo.Sterilecircularspongediscswereinsertedsubcutaneously
intomaleSwissalbinomiceandtreatedwithtylophorinefor14days.Miceweresacrificedandspongewasexcised,(A)weighedand(B)
photographed.Datawerepresentedasmeans±SEM,n=10.**p<0.01;***p<0.001versuscontrolgroup.(C)Excisedspongewashomogenized
inDrabkinReagenttoquantifythehemoglobinlevel.Thecontentofhemoglobinineachimplantisexpressedasg/dl/perwettissue.Datawere
presentedasmeans±SEM,n=10.***p<0.001versuscontrolgroup.(D)Representativehistologicalsections(5μm,stainedwithH&E)of
spongeimplants.Theporesofthespongematrixarefilledwithinflammatorycells,spindle-shapedfibroblasts,bloodvessels.Inthecontrolgroup
fibrovasculartissueisdenserandmorevascularizedcomparedwiththetylophorine-treatedgroupsatthedosesof7.5and15mg/kg,Arrows
showsthebloodvesselsincontrolgroup.s:sponge:Scalebar:100μm.(E)ImmunohistochemicalstainingofspongewithCD31.Sectionsfrom
thespongetissuethatwereeitheruntreatedor-treatedwereincubatedwithanti-CD31overnightat4°CandstainedwithABC-reagent
accordingtothemanufacturersprotocol.Scalebar:100μm.(F)Effectsoftylophorineonmicrovesseldensity(MVD)inspongeimplants.Effectof
tylophorineoncytokinelevels(G)VEGF(H)TNF-α(I)TGF-β.Datawerepresentedasmeans±SEM,n=10.**p<0.01;***p<0.001versuscontrolgroup.
the CD 31 expression as compared to control group inhibitedVEGFlevelinspongeimplanttissues(Figure6G).
(Figure 6E). The microvessel density was statistically The inflammatory components of the sponge-induced in-
lowered in tylophorine treated sponge tissue (Figure 6F). flammationweredeterminedbyestimatingthenumbersof
Subsequently, it was sought to correlate this change in the leukocytes in the implant by assaying levels of pro-
vascularization with change in the level of VEGF in the inflammatory cytokines TNF-α. Tylophorine at 15 mg/kg
implants. It was found that tylophorine significantly reduced theTNF-αlevel by41.81%(Figure6H). Asshown
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inFigure6I,therewasacleardecreaseintheTGF-βlevels Figure S2. Quantitatively weights of tumor lumps treated
(38.92 and 59.73% at 7.5 and 15 mg/kg respectively) after with tylophorine were also found smaller (p<0.001) as
tylophorinetreatment. comparedtocontrolgroup(Figure7C).Theaveragetumor
weight in the control group was 8.34±1.85 g; whereas the
Tylophorineinhibitedtumorgrowthinvivo averagetumorweightinthetylophorine-treatedgroupwas
Prompted by the in vitro and in vivo data supporting a foundtobe0.98±0.07g(Figure7D)indicatingthatprolif-
potential antiangiogenic activity of tylophorine, we ex- eration rateof tumorcells inmice wasgreatlyinhibited by
amined the in vivo efficacy of tylophorine on the growth tylophorine.Tofurtherexaminewhethertylophorinecould
of mouse Ehrlich ascites solid tumor, which is highly suppress tumor growth by inhibiting angiogenesis, tumor
dependent on angiogenesis. As compared to control tissues were stained with specific antibodies against CD31,
group treated with vehicle, tylophorine -treated group P-VEGFR2 (Tyr 1175), P-AKT, and P-Erk in Figure 7E.
showed slower growth kinetics of EAC solid tumor CD31 is a widely used endothelial marker for quantifying
(Figure 7A). It was found that treatment with tylophorine angiogenesis by calculating microvessel density (MVD)
significantly led to suppression of EAC solid tumor vol- [42]. Our data showed that the average number of blood
umeswhencomparedwiththe controlgroup.Theaverage vessels in tylophorine treated group is 4.87±0.34 blood
tumor volumeinthecontrolgroupincreasesfrom91.35± vessels/HPF (Figure 7F) as compared with 11.93±2.84
21.64mm3to2139.05±193.09mm3after30days,whereas blood vessels/HPF in the control group (P<0.001).
the average tumor volume in the tylophorine-treated mice SuppressedCD31expressionanddecreasedtumor vol-
increased from 93.28±31.98 mm3 to 213.96±65.61 mm3 ume and tumor weight suggests that tylophorine tar-
(Figure 7A). The body weights of animals corresponded gets endothelial cells (ECs) as well as tumor cells. In
well with the growth of tumors in respective group of ani- addition, tylophorine down-regulated the expressions
mals (Figure 7B). The effect of tylophorine alone on body of P-VEGFR2, P-Akt, and P-Erk (Figure 7E) further
weight of normal mice is depicted in Additional file 2: demonstrating that tylophorine played an important
Figure7EffectoftylophorineontumorgrowthandVEGFR2phosphorylationinEACtumormodel.15×106EACcells/mousewere
injecteds.c.into5–6weekoldSwissalbinomice.Aftersolidtumorsgrewto~100mm3,themicewerei.p.treatedwithtylophorine(7.5mg/kg
bw).Tumorgrowthwasmeasuredwithcaliperseveryfifthdaysusingtheformula:Tumorvolume(mm3)=(width)2×(length)×π/6.Tylophorine
treatmentdecreases(A)tumorvolume(B)bodyweight.Datawerepresentedasmeans±SEM,n=15.(C)Representiveimagesofsolidtumor
lumpshowstylophorinetreatedgroupissignificantlysmallerthanthoseinthecontrolgroup.(D)Thetumortissuewasremovedfrommiceat
30daysaftertreatmentandweighed.(E)Immunohistochemicalstainingoftumortissue(n=6)withantibodiesagainstP-VEGFR2,P-ERK,P-Akt
andCD31.(F)%MVDwasdeterminedbyselectingthebloodvessel(CD31)areaperfieldinselectedvascularizedareasdividedbythewhole
area.Datawerepresentedasmeans±SEM,n=6.***p<0.001versuscontrolgroup.
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role in suppressing angiogenesis at least partly through pocketandinthiswayithasprovidedadirectionfordevel-
VEGFR2signalingpathways. opmentofsmallnaturalinhibitors.
Discussion
Tylophorineprolongsthesurvivaloftumorbearingmice
Thepresentstudydemonstratedthattylophorineexhibited
The tumor bearing mice administered with DMSO or
anti-angiogenic activities in vivo and suppressed key steps
tylophorine (7.5 mg/kg bw) for 30 days were observed
involvedinangiogenesisincludingproliferation(Figure1C),
andthedaysofsurvivalwererecorded.Withtylophorine
migration (Figure 2A), invasion (Figure 2B), tubulogenesis
treatment, the survival of tumor bearing mice signifi-
(Figure 2C) and expression of pro-MMP2 (Figure 4B) as
cantly increasedfrom 35.2±1.29daysto70.3±3.28 days
detectedbygelatinzymographyinendothelialcells.Bydir-
(Figure8)asobtainedbyKaplanMeier’ssurvivalanalysis
ectly blocking VEGFR2 phosphorylation and activation,
(Figure 8).
tylophorineinhibitedVEGFR2kinaseactivity(Figure3B)as
well as suppressed VEGFR2 signaling pathway (Figure 4A)
TylophorinelocatedattheATP-bindingsitesofVEGFR2 in vitro. Supporting evidences concerning in vivo anti-
kinasedomain angiogenesis effectsof tylophorine then came from sponge
Wenextanalyzedthebindingpatternbetweentylophorine implant angiogenesis model and Ehrlich ascites carcinoma
and VEGFR2 kinase domain to further understand how tumor model. Tylophorine significantly inhibited blood
tylophorine exerted anti-angiogenesis effects via VEGFR2 vessels formation in sponge implant assay (Figure 6) and
anditssignalingpathways.Whenmoleculardockingsimu- significantly suppressed tumor growth accompanied by
lationbetweentylophorineligandandVEGFR2proteinwas reduction in microvessel density (MVD) in tumor tissues
analyzed, it was found that tylophorine has bound at (Figure7).
slightly different location toward N-terminal domain from Our study provides a novel and mechanistic insights
original bound ligand 42Q with-7.00 Kcal/mol binding af- into the mechanism by which tylophorine affects the
finityintheATPbindingpocket(Figure9A).Therearefive multiple facets of vascular endothelial angiogenic signal-
amino acids i.e., Lys868, Leu870, His879, Leu882 and ing through VEGFR1 and VEGFR2. Phosphorylated
Leu912 are actively involved in the binding of tylophorine. Tyr1175 of VEGFR2 mediates activation of the mitogen-
His879 is an active amino acid of the ATP binding pocket activated protein kinase/ERK cascade and was shown to
has participated in hydrogen bond with tylophorine. Rest contribute to cell proliferation in endothelial cells
amino acids are hydrophobic in nature and have made [38-40]. Src family kinase is substantially involved in
strong π-π bonds with the ligand. Therefore hydrophobic VEGF-induced angiogenesis in vitro and in vivo [43-45].
interaction is more dominant than hydrogen and Other signaling molecules that have been involved in
electrostatic interaction in tylophorine-VEGFR2 complex VEGF-induced migration throughVEGFR2 include FAK
(Figure 9B). When structure of tylophorine was inspected, and its substrate paxillin, which are participated in focal
ithasfoundthatitscorestructurehasmadeupwiththree adhesion during cell migration [6,46]. By interacting
fused benzene rings which are also hydrophobic nature between FAK and Src, a dual kinase complex FAK-Src
suggesting,itmaybereasonfordominancyofhydrophobic forms, and is activated by multiple integrin-regulated
interaction. Such binding pattern of tylophorine within linkages [47]. Recent studies show that inhibition of
VEGFR2mayprohibitthebindingoftheATPatitsbinding ERK, phosphoinositide 3-kinase, PDT1/Akt and FAK
downstream of VEGFR2 has emerged as a target for an-
ticancer therapy [48]. AKT/mTOR/ribosomal protein S6
kinase (p70S6K) signaling has also been identified as a
novel, functional mediator in angiogenesis [48,49].
VEGFR1 plays a positive role in promoting tumor
angiogenesis by cross-talks among epithelial cells and
other cell types because VEGFR1 is expressed not only
endothelial cells but also on macrophage lineage cells
and tumor epithelial cells [50]. VEGFR1 is a kinase-
impaired RTK, and may signal in the context of a
receptorheterodimer [51].
Our studies indicated that tylophorine interfered with
the binding of VEGFR2 and reduced the autophospho-
rylation of VEGFR2 whereas; tylophorine did not affect
Figure8Kaplan-Meiersurvivalcurvefortylophorinetreated
the VEGF binding to VEGFR1. We also found that
EACmiceincomparisontoEACcontrolgroup(n=15). a half-maximum inhibitory concentration 9.2 μM