SKLB1002, a Novel Potent Inhibitor of VEGF Receptor 2 ... · SKLB1002, a Novel Potent Inhibitor of...

Cancer Therapy: Preclinical

SKLB1002, a Novel Potent Inhibitor of VEGF Receptor 2 Signaling,Inhibits Angiogenesis and Tumor Growth In Vivo

Shuang Zhang1, Zhixing Cao1, Hongwei Tian1, Guobo Shen1, Yongping Ma1, Huanzhang Xie1, Yalin Liu1,Chengjian Zhao1, Senyi Deng1, Yang Yang1, Renlin Zheng1, Weiwei Li1, Na Zhang1, Shengyong Liu1,Wei Wang1, Lixia Dai1, Shuai Shi1, Lin Cheng1, Youli Pan1, Shan Feng1, Xia Zhao2, Hongxin Deng1,Shengyong Yang1, and Yuquan Wei1

AbstractPurpose: VEGF receptor 2 (VEGFR2) inhibitors, as efficient antiangiogenesis agents, have been applied

in the cancer treatment. However, currently most of these anticancer drugs suffer some adverse effects.

Discovery of novel VEGFR2 inhibitors as anticancer drug candidates is still needed.

Experimental Design: In this investigation, we adopted a restricted de novo design method to design

VEGFR2 inhibitors. We selected the most potent compound SKLB1002 and analyzed its inhibitory effects

on human umbilical vein endothelial cells (HUVEC) in vitro. Tumor xenografts in zebrafish and athymic

mice were used to examine the in vivo activity of SKLB1002.

Results: The use of the restricted de novo design method indeed led to a new potent VEGFR2 inhibitor,

SKLB1002, which could significantly inhibit HUVEC proliferation, migration, invasion, and tube forma-

tion. Western blot analysis was conducted, which indicated that SKLB1002 inhibited VEGF-induced

phosphorylation of VEGFR2 kinase and the downstream protein kinases including extracellular signal-

regulated kinase, focal adhesion kinase, and Src. In vivo zebrafish model experiments showed that

SKLB1002 remarkably blocked the formation of intersegmental vessels in zebrafish embryos. It was

further found to inhibit a new microvasculature in zebrafish embryos induced by inoculated tumor cells.

Finally, compared with the solvent control, administration of 100 mg/kg/d SKLB1002 reached more than

60% inhibition against human tumor xenografts in athymic mice. The antiangiogenic effect was indicated

by CD31 immunohistochemical staining and alginate-encapsulated tumor cell assay.

Conclusions: Our findings suggest that SKLB1002 inhibits angiogenesis and may be a potential drug

candidate in anticancer therapy. Clin Cancer Res; 17(13); 4439–50. �2011 AACR.

Introduction

Angiogenesis is a complex process including endothelialcell proliferation, migration, basement membrane degen-eration, and new tube formation. It is required for a varietyof physiologic processes such as development and repro-duction. However, angiogenesis also plays important rolesin some disease states, typically cancer (1–4). The newblood vessels grow and infiltrate into the tumor, providing

it with essential nutrients and oxygen, and a route fortumor metastasis (5, 6). Thus, antiangiogenesis has beenthought as one of the most important anticancer therapies.Compared with chemotherapy directed at cancer cells,which often rapidly mutate and acquire "drug resistance"to treatment, the antiangiogenic therapy is obviouslyadvantageous.

Numerous growth factors and cytokines are involvedduring tumor neovascularization. Of all the known angio-genic molecules, VEGF is the key mediator that promotesangiogenesis (7–9). VEGF exerts its biological effects bybinding to and activating its receptors. Among them, VEGFreceptor 2 (VEGFR2) is a major receptor transducing VEGF-induced signaling in endothelial cells. Activation ofVEGFR2 leads to phosphorylation of specific downstreamsignal transduction mediators, including extracellular sig-nal-regulated kinases (ERK), protein kinase C, focal adhe-sion kinase (FAK), phosphoinositide 3-kinase, and AKT.Signaling from VEGFR2 is necessary for the execution ofVEGF-stimulated proliferation, migration, and sprouting ofcultured endothelial cells in vitro and angiogenesis in tumor(10–13). Therefore, VEGFR2 has been recognized as themost important target for the antiangiogenesis therapy of

Authors' Affiliations: 1State Key Laboratory of Biotherapy and CancerCenter, West China Hospital, West China Medical School; and 2Depart-ment of Gynecology and Obstetrics, West China Second Hospital,Sichuan University, Chengdu, Sichuan, People's Republic of China

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

S. Zhang and Z. Cao contributed equally to this work.

Corresponding Author: Hongxin Deng or Shengyong Yang, State KeyLaboratory of Biotherapy and Cancer Center, West China Hospital, WestChina Medical School, Sichuan University, Gaopeng Street, Keyuan Road4, Chengdu, Sichuan, PR China. Phone: 86-288-516-4063; Fax: 86-288-516-4060; E-mail: [email protected] or [email protected]

doi: 10.1158/1078-0432.CCR-10-3109

�2011 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 4439

Cancer Research. on February 27, 2020. © 2011 American Association forclincancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 27, 2011; DOI: 10.1158/1078-0432.CCR-10-3109

http://clincancerres.aacrjournals.org/

cancer. Currently, there have beenmany reports of VEGFR2inhibitors, including 2 marketed drugs, namely, vandeta-nib and sunitinib (14–16). However, most of theseVEGFR2 inhibitors suffer some adverse effects in clinicaluse or clinical trials, which prompts that discovery of moreVEGFR2 inhibitors as anticancer drugs is still needed atpresent.

In an effort to discover more potent VEGFR2 inhibitors,we adopted a restricted de novo design strategy for VEGFR2inhibitors; a detailed description about this strategy can befound in our previous study (17). By using this strategy, wedesigned a series of inhibitors derived from quinazoline.SKLB1002 (Fig. 1A) was selected from these compoundsand displayed potent and specific inhibition of VEGFR2tyrosine kinase activity in vitro. The purpose here was tofurther examine its antiangiogenesis potency in vitro and invivo. Our results clearly indicated that SKLB1002 inhibitedVEGF-induced VEGFR2 phosphorylation and activation ofvarious downstream signaling substrates that were respon-sible for endothelial cell viability. According to this mole-cular mechanism, SKLB1002 significantly inhibitedendothelial cell proliferation, migration, invasion, andtube formation. By using xenograft models in zebrafishand athymic mouse, we determined that SKLB1002 pro-longed tyrosine kinase inhibition and antagonized tumorangiogenesis but caused few toxic effects in the host. Takentogether, our data suggest that SKLB1002 functions as anovel VEGFR2 inhibitor and suppresses angiogenesis dur-ing tumor growth.

Materials and Methods

Cell linesHuman colon cancer SW620 cells, hepatic cancer

HepG2 cells, and melanoma B16-F10 cells were obtainedfrom American Type Culture Collection. The normalhuman liver cell line L-02 was obtained from the Cell

Bank of the Chinese Academy of Science (Shanghai,China). They were cultured in Dulbecco’s modifiedEagle’s medium supplemented with 10% FBS. Humanumbilical vein endothelial cells (HUVEC) were isolatedfrom human umbilical cord veins by a standard proce-dure, as previously described, and grown in EBM-2 med-ium with SingleQuots (Lonza) containing VEGF andother growth factors (18). HUVECs at passages 3 to 8were used for all experiments.

General computational methodsA restricted de novo design method was used to con-

struct new molecules. In this approach, quinazoline wastaken as a general support-nog. Various fragments wereallowed to grow at position 4 of quinazoline if theirchemical structures and physicochemical properties con-formed to the requirements of the hydrophobic pocket.Molecular docking experiments and scoring-rankingoperations were carried out on the created molecules,which were suitable for assessing how the molecules fitthe kinase active site. The de novo design and dockingexperiments were carried out by using AutoLudi andLigandFit in Discovery studio 2.5, respectively. The scor-ing functions adopted here were Ludi Energy Estimate1 and Chemscore.

Kinase inhibition assaysKinase inhibition was measured by the use of radio-

metric assays conducted by Kinase Profiler service (Milli-pore). Briefly, in the presence or absence of SKLB1002,VGFR2 (5–10 mU) was incubated in 25-mL reactionsolution containing 8 mmol/L 3-(N-morpholino)propa-nesulfonic acid (MOPS), pH 7.0, 0.2 mmol/L EDTA, 0.33mg/mL myelin basic protein, 10 mmol/L Mg acetate, andg-[33P]ATP. After incubation for 40 minutes at roomtemperature, the reaction was stopped and 10 mL of thereaction solution was then spotted onto a P30 filtermatand washed 3 times for 5 minutes in 75 mmol/L phos-phoric acid and once in methanol prior to scintillationcounting.

Preparation of SKLB1002For all in vitro assays and zebrafish studies, SKLB1002

was prepared initially as a 20 mmol/L stock solution indimethylsulfoxide (DMSO). Stock solution was diluted inthe relevant assay media, and 0.1% DMSO served as avehicle control. For studies in athymic mice, SKLB1002 wassuspended in 35% (v/v) polyethylene glycol solution con-taining 5% (v/v) DMSO and dosed at 0.1 mL/10 g of bodyweight.

Proliferation assayCell proliferation was measured using MTT assay as

previously described (19). Various cells includingHUVECs,L-02, B16-F10, HepG2, and SW620 were treated withindicated concentrations of SKLB1002 for 24 hours. Van-detanib and sunitinib (Sigma) served as positive controls.Each assay was replicated 3 times.

Translational Relevance

Inhibition of VEGF signaling is a promising thera-peutic approach for tumors by targeting tumor-inducedangiogenesis. This may be accomplished by abrogatingthe kinase activity of VEGF receptor 2 (VEGFR2), whichplays a critical role in mediating VEGF-induced signal-ing in endothelial cells. In the current study, a restrictedde novo design method was used to construct newmolecules that targeted VEGFR2, which indeed led toa new potent and specific VEGFR2 inhibitor, SKLB1002,which showed dose-dependent inhibitory activity inhuman umbilical vein endothelial cells and humantumor xenografts in athymic mice with limited toxicity.Moreover, the current study provided the potentialtechnique for high-throughput antiangiogenic drug dis-covery, which was shown for the first time by usingcomputer-aided drug design and tumor xenograftmodel in a whole vertebrate system.

Zhang et al.

Clin Cancer Res; 17(13) July 1, 2011 Clinical Cancer Research4440




Wound healing assayMonolayer HUVECs were wounded by scratching with

pipette tips and washed with PBS. Fresh EGM2 mediumcontaining vehicle or different concentrations of SKLB1002was added to the scratched monolayers. Images were takenby an OLYMPUS digital camera after 24 hours. Themigrated cells were quantified by manual counting, andthe percentage of inhibition was expressed using untreatedcells at 100%. Vandetanib and sunitinib served as positivecontrols.

Transwell invasion assayInvasion assay was done as described previously, with

some modifications (20). Briefly, the filter of the Transwellplate (Millipore) was coated with 50 mL Matrigel (BDBiosciences). After Matrigel polymerization, the bottomchambers were filled with EGM-2 medium containingvarious growth factors and the top chambers were seededwith 100 mL EBM-2 medium (without growth factors) andHUVECs (2 � 104 cells per well). The top chamber con-tained vehicle or various concentrations of SKLB1002.Cells were allowed to migrate for 24 hours. Nonmigratedcells were scraped with a cotton swab, and migrated cellswere fixed with 100% methanol and stained with 0.05%

crystal violet. The cells were quantified bymanual countingand photographed under a light microscope. The percen-tage of migrated cells inhibited by SKLB1002 was expressedon the basis of vehicle control wells, and vandetanib andsunitinib served as positive controls.

Tube formation assayThe tube formation assay was conducted as described

previously (21). After polymerization at 37�C for 30 min-utes, HUVECs suspended in EBM-2 medium were seededonto the Matrigel. They were then treated with SKLB1002,vandetanib, sunitinib, or vehicle. After 6 hours, cells werephotographed with a digital camera attached to an invertedmicroscope.

Western blot analysisTo determine the effects of SKLB1002 on VEGFR2-

dependent signaling cascade, subconfluent HUVECs wereserum-starved overnight and incubated with SKLB1002 for90 minutes, followed by 50 ng/mL VEGF165 treatmentfor 10 minutes. Cells were lysed with buffer containing1% Triton X-100, 1% deoxycholate, and proteinaseinhibitor cocktail (Sigma). Protein concentrations weredetermined using a modified Lowry protein assay kit

NA C

B

N

N

N

SS

O

O

Phe918

Thr916

Phe918(A)

Gly922(A)

Cys919(A)

Thr916(A)

Key

Ligand bond Nonligand residues involved inhydrophobic contact(s)

Corresponding atoms involved inhydrophobic contact(s)

Nonligand bondHydrogen bond andits length

Leu840(A)

Leu1035(A)

Phe1047(A)

Ala866(A)

CB

CAN

2.92

N7

3.01

C8C5

C10

S11

S13

C12N16

N15

C21C14

His 53

CO

CB

CA

N

CG2 OG1

N9

C

O

C20

O19

C2

C3

C1C6

C18

C4

O17SG

Cys919

Figure 1. A, chemical structure of SKLB1002. B, SKLB1002 is docked into the active site of VEGFR2, showing interactions between SKLB1002 and VEGFR2by using the in silico model. C, a 2-dimensional interaction map of SKLB1002 and VEGFR2.

SKLB1002 Inhibits Angiogenesis and Tumor Growth

www.aacrjournals.org Clin Cancer Res; 17(13) July 1, 2011 4441




(ThermoScientific) and equalized before loading. Fortymicrograms of cellular protein from each sample wasapplied to 8% to 12% SDS-PAGE gels and probed withspecific antibodies (Cell Signaling Technology) includingphospho-VEGFR2 (p-VEGFR2; Tyr1175), VEGFR2, phos-pho-Erk1/2 (p-Erk1/2; Thr202/Tyr204), Erk1/2, phospho-FAK (p-FAK; Tyr925), FAK, phospho-Src (p-Src; Tyr416),Src. Blots were developed with horseradish peroxidase(HRP)-conjugated secondary antibodies and chemilumi-nescent substrate on Kodak X-ray films. To rule out thepossibility that SKLB1002 killed tumor cells directly,VEGFR2 expression was detected in various cells includingHUVECs, SW620, HepG2, B16-F10, and L-02.

Drug studies in zebrafishFLK-1 promoter EGFP transgenic zebrash (FLK-1:EGFP)

was used in all of our experiments. We used 30 embryos perexperimental group in our study, and each experiment wascarried out in 3 independent replicates. Embryos weremaintained in Holtfreter’s solution in a humidified incu-bator at 28�C (22). The bright and consistent fluorescenceof blood vessels in the zebrafish embryos suggested thatthey could provide an ideal tool for testing antiangiogen-esis drugs. Fifteen hours postfertilization (hpf), zebrafishembryos were incubated overnight with 2.5 mmol/LSKLB1002, vandetanib, sunitinib, or vehicle. At 30 hpf,zebrafish were anesthetized with 0.01% tricaine andimaged under a fluorescence microscope (Carl Zeiss Micro-imaging Inc.) equipped with a AxioCamMRc5 digital CCDcamera (Carl Zeiss Microimaging Inc.).

Histone H3 phosphorylation may initiate at differentphases of the cell division in different organisms duringboth mitosis and meiosis (23). In this study, normal cellproliferation in the zebrafish embryos was observed usingan antibody to phosphorylated histone-3 (Millipore).Briefly, zebrafish embryos at 15 hpf were incubated over-night with 2.5 mmol/L SKLB1002 or vehicle. At 24 hpf,zebrafish embryos were fixed in 4% formaldehyde solu-tion for 4 hours at 4�C and permeablized in acetone at�20�C for 10 minutes. Subsequently, zebrafish embryoswere processed for immunofluorescence by using anti-phosphorylated histone-3 antibody (1:1,000) and TRITC-conjugated antibodies (red label; 1:2,000). In this way,the initiation of intersegmental vessels sprouting andnormal cell proliferation in the zebrafish embryos couldbe simultaneously observed using a fluorescence micro-scope.

We then investigated that the implantation of murinetumor cells B16-F10 could trigger an angiogenic responsein zebrafish embryos. It was a suitable model for us tostudy the antiangiogenic effect of SKLB1002. We used redfluorescent dye CM-DiI (Invitrogen Corporation)-labeled tumor cells for easy observation in our zebra-fish/tumor xenograft model, which was inspected with afluorescence microscope provided with a multidimen-sional acquisition software (Carl Zeiss MicroimagingInc.). Briefly, 48 hpf zebrafish embryos were anesthe-tized with 0.01% of tricaine and injected with 300

murine B16-F10 melanoma cells per embryo. Cells wereresuspended in Hank’s balanced salt medium anddirectly injected into zebrafish perivitelline space byusing an air-driven Cell Tram microinjector (MedicalSystem Corp.). Compounds were added into the incu-bating water at a concentration of 2.5 mmol/L 1 day afterinjection of cells. At 5-day postinoculation (dpi), digitalmicrographs were taken using a fluorescence microscopeas described above.

Xenograft mouse modelAnimal studies were conducted in conformity with insti-

tutional guide for the care and use of laboratory animals.All mouse protocols were approved by the Animal Care andUse Committee of Sichuan University (Chengdu, Sichuan,China). Six-week-old female athymic (nu/nu) mice wereobtained from Chinese Academy of Medical Science (Beij-ing, China). SW620 and HepG2 tumors were establishedby s.c. injection of 5� 106 cells. After 10 days, mice bearingtumors around 100 mm3 were selected and randomizedinto treatment groups (6 mice per group). The dosingschedules were SKLB1002 100 mg/kg/d, 50 mg/kg/d, orvehicle once a day intraperitoneally. Tumor length andwidth were determined every 3 days and tumor volume(TV) was calculated using the following formula: TV ¼length � width2 � 0.52. At the end of experiment, micewere sacrificed. Solid tumors were removed and processedfor immunohistochemical analysis and terminal deoxynu-cleotidyl transferase–mediated dUTP nick end labeling(TUNEL) assay.

Immunohistochemistry and alginate-encapsulatedtumor cell assay

To investigate whether SKLB1002 inhibited tumorgrowth by suppressing tumor angiogenesis, detection ofvessel density in tumor tissue was done as described pre-viously (24). Frozen sections of SW620 tumor xenograftswere used to determine vessel density with an anti-CD31antibody (BD Biosciences).

An alginate-encapsulated assay was conducted asdescribed (25). Briefly, alginate beads containing 5 �104 tumor cells per bead were formed and implanted s.c. into both dorsal sides of the athymic mice. Then micewere treated with SKLB1002 at 100 mg/kg, 50 mg/kg, orvehicle once a day intraperitoneally for 12 days. At the endof experiment, 0.1 mL of 2% fluorescein isothiocyanate(FITC)–dextran solution (Sigma) was injected i.v. into thelateral tail vein of mice. Alginate beads were removed andphotographed within 20 minutes after being exposed sur-gically. The uptake of FITC–dextran was measured asdescribed (25).

In situ TUNELCell apoptosis in SW620 xenograft tumors was deter-

mined using a TUNEL assay following the manufacturer’sinstructions (Promega). Three tumors per group were ana-lyzed. The number of TUNEL-positive cells was quantified

Zhang et al.





by fluorescence microscopy, and the apoptotic index in 6random fields per group was counted.

Toxicity evaluationTo investigate potential side effects or toxicity on mice

during the treatment, they were observed continuously forrelevant indexes such as weight loss, diarrhea, anorexia,

skin ulceration, and toxic deaths. The tissues of heart, liver,spleen, lung, and kidney were stained with hematoxylinand eosin (H&E).

Statistical analysisSPSS 11.5 was used for statistical analysis. Data were

analyzed statistically by using 1-way ANOVA followed by

Vandetanib 2.5 mmol/L Sunitinib 2.5 µmol/LSKLB1002 2.5 µmol/LVehicleA

B

120

100

80

Mig

ratio

n ce

ll nu

mbe

r(%

of c

ontr

ol)

60

40

20

00SKLB1002

(µmol/L)1.25 2.5 5 10

****

*

120

100

80

Mig

ratio

n ce

ll nu

mbe

r(%

of c

ontr

ol)

60

40

20

00Vandetanib

(µmol/L)1.25 2.5 5 10

****

*

120

100

80

Mig

ratio

n ce

ll nu

mbe

r(%

of c

ontr

ol)

60

40

20

00Sunitinib

(µmol/L)

100 µm

100 µm

1.25 2.5 5 10

****

**

**

120

100

80

Inva

sive

cel

l num

ber

(% o

f con

trol

)

60

40

20

00SKLB1002

(µmol/L)1.25 2.5 5 10

******

**

120

100

80

Inva

sive

cel

l num

ber

(% o

f con

trol

)

60

40

20

00Vandetanib

(µmol/L)1.25 2.5 5 10

**

**

**

*

120

100

80

Inva

sive

cel

l num

ber

(% o

f con

trol

)

60

40

20

00Sunitinib

(µmol/L)1.25 2.5 5 10

******

**

Figure 2. SKLB1002 and positive control inhibited migration, invasion, and tube formation of HUVECs. A, SKLB1002 inhibited HUVEC migration in woundhealing assay. Cells were wounded with pipette and treated with vehicle or indicated concentrations of compounds. After 24 hours, the migrated cellswere quantified by manual counting. B, SKLB1002 inhibited HUVEC invasion in Transwell assay. A total of 2 � 104 HUVECs were seeded in the top chamberand treated with vehicle or different concentrations of compounds. After 24 hours, the HUVECs that invaded through the membrane were stained andquantified.






the Tukey test. Differences were considered significant ifP < 0.05.

Results

Design, synthesis, and evaluation of SKLB1002Seventy-five novel molecules were designed with the

use of the proposed restricted de novo design approach. Atotal of 15 molecules, which were ranked at the top 20%of the created molecules according to values of the Ludienergy estimate 1, were chemically synthesized, followedby in vitro kinase inhibition assays (SupplementaryTable S1). Among all the tested compounds, 6,7-dimethoxy-4-(5-methyl-1,3,4-thiadiazol-2-ylthio)quina-zoline (SKLB1002) was found to be the most potent one.SKLB1002 showed potent inhibition of VEGFR2 kinaseactivity (IC50 ¼ 32 nmol/L), which was similar with thepositive drugs vandetanib (IC50 ¼ 40 nmol/L) and suni-tinib (IC50 ¼ 9 nmol/L).

Figure 1 presents a possible binding mode of SKLB1002in the VEGFR2 active site. The quinazoline ring locates atthe front of the ATP binding pocket, where it is sandwichedbetween 2 loops in the N- and C-lobes of the catalyticdomain. As anticipated, the quinazoline ring of SKLB1002is oriented with its N1 atom hydrogen bonded to theCys919 residue in the hinge region of VEGFR2. The N4atom in thiadiazole moiety of SKLB1002 forms anotherinteraction with gatekeeper residues: an N (thiadiazole)��O–H (Thr916) hydrogen bonding interaction. Further-more, the quinazoline ring forms an important interactionwith hinge region residues: an aromatic-aromatic face-to-

face p–p interaction with the benzene ring in the side chainof Phe918. SKLB1002 also forms hydrophobic interactionwith residues Leu840, Ala866, Phe918, Gly922, Leu1035,and Phe1047 in ATP binding pocket of VEGFR2.

SKLB1002 shows strikingly lower cytotoxicity thanvandetanib and sunitinib

L-02 cell strain was established from normal human liverand considered to have extensive application in cytotoxicityassay (26). To determine the cytotoxicity of SKLB1002 onnormal human cells, we conducted MTT assay in L-02 cellsafter administration with indicated concentrations of com-pounds. As shown in Supplementary Figure S2B,SKLB1002 effectively induces cell growth inhibition of L-02 cells at concentrations over 150 mmol/L. In contrast,vandetanib and sunitinib sufficiently inhibited L-02 cellsonly at concentrations of 40.9 and 30.1 mmol/L, respec-tively.

SKLB1002 inhibits HUVEC proliferation, migration,invasion, and tube formation

To assess the antiangiogenic activity of SKLB1002 in vitro,its inhibitory effects on VEGF-induced proliferation ofendothelial cells were first evaluated by MTT assay.SKLB1002 significantly inhibited endothelial cell prolifera-tion with an IC50 value of 11.9 mmol/L. However, vande-tanib and sunitinib inhibited cell viability at a much lowerconcentration with an IC50 value of 3.8 mmol/L and 1.9mmol/L, respectively (Supplementary Fig. S2A).

Cell migration and invasion are essential for endo-thelial cells in angiogenesis. We carried out wound

C

100 µm

120

100

80

HU

VE

C tu

be fo

rmat

ion

(% o

f con

trol

)

60

40

20

00SKLB1002

(µmol/L)1.25 2.5 5 10

****

****

120

100

80H

UV

EC

tube

form

atio

n(%

of c

ontr

ol)

60

40

20

00Vandetanib

(µmol/L)1.25 2.5 5 10

**

**

**

**

120

100

80

HU

VE

C tu

be fo

rmat

ion

(% o

f con

trol

)

60

40

20

00Sunitinib

(µmol/L)1.25 2.5 5 10

****

****

Figure 2. (Continued) C, SKLB1002 inhibited tube formation of HUVECs. After treated with vehicle or indicated concentrations of compounds for 6 hours,tubular structure in each group was quantified by manual counting. The percentage of inhibition was expressed using vehicle treated cells at 100%. Columns,mean; bars, SD (n ¼ 3; ANOVA; *, P < 0.05; **, P < 0.01 vs. vehicle control).

Zhang et al.





healing assays to investigate the effects of SKLB1002 oncell migration and found that 2.5 mmol/L SKLB1002 obvi-ously inhibited the migration of HUVECs (Fig. 2A). Wealso conducted Transwell invasion assays to evaluate theability of HUVECs to pass through the Matrigel andmembrane barrier of the Transwell in the presence ofvehicle or various concentrations of SKLB1002. Datashowed that SKLB1002 significantly inhibited the inva-sion properties of endothelial cells in a dose-dependentmanner (Fig. 2B).Although angiogenesis is a complex process of several

kinds of cells, tube formation of endothelial cells is one ofthe key steps. HUVECs plated on the surface of Matrigelformed capillary-like structures in the vehicle group within6 hours. However, treatment with SKLB1002 dose depen-dently inhibited the tube formation (Fig. 2C).As shown in Figure 2 and Supplementary Figure S2, we

indicated that SKLB1002 had similar antiangiogenic effi-cacy in HUVEC migration, invasion, and tube formationbut strikingly lower cytotoxicity in comparison with thepositive drugs vandetanib and sunitinib.

SKLB1002 inhibits VEGFR2 signaling pathwayVEGFR2 phosphorylation leads to the activation of

various downstream signaling substrates that are respon-

sible for endothelial cell migration and tube formation.To investigate whether SKLB1002 inhibited VEGFR2 andits downstream signaling, we screened some essentialkinases involved in VEGFR2 signaling pathway. As shownin Figure 3A, 10 mmol/L SKLB1002 significantly sup-pressed the phosphorylation of VEGFR2, ERK, FAK,and Src induced by VEGF, which suggested thatSKLB1002 exerted its antiangiogenic function by directlytargeting VEGFR2 on the surface of endothelial cells andfurther antagonizing VEGFR2-mediated downstream sig-naling cascade. Consistent with previous reports (27, 28),SW620 expressed moderate VEGFR2, which was not suf-ficient to lead to the activation of various downstreamsignaling substrates that were responsible for cell prolif-eration.

SKLB1002 inhibits embryonic angiogenesisin zebrash

Fifteen-hpf zebrafish embryos were incubated overnightwith SKLB1002, vandetanib, sunitinib, or vehicle, zebrafishwere anesthetized with 0.01% tricaine, and a digitalimage of each embryo was captured using the fluorescencemicroscope. Intersegmental blood vessel growth wasgreatly inhibited in the 2.5 mmol/L SKLB1002 group com-pared with vehicle treated and untreated embryos (Fig. 4A).

GAPDH

GAPDH

ERK1/2

p-ERK1/2(Thr202/Tyr204)

Src

p-Src (Tyr416)

FAK

p-FAK (Tyr925)

VEGFR2

VEGF 50 ng/mL

VEGF receptor 2

p-VEGFR2 (Tyr1175)

p-VEGF receptor 2 (Tyr1175)

SKLB1002 (µmol/L) 0A

C

B

D

0 1 5 10

VEGF 50 ng/mL

SKLB1002

VEGFR2

ERKFAK

Cell migration

Src

Vascularpermeability

Cell proliferation

Angiogenesis

HUVEC SW620 HepG2 B16-F10 L-02

bFGF 10 ng/mL

0 0 1 5 10

GAPDH

ERK1/2

p-ERK1/2(Thr202/Tyr204)

Src

p-Src (Tyr416)

FAK

p-FAK (Tyr925)

VEGFR2

p-VEGFR2(Tyr1175)

SKLB1002 (µmol/L)

Figure 3. SKLB1002 inhibited VEGFR2 kinase activity and its downstream signaling molecules. A, SKLB1002 suppressed the phosphorylation ofVEGFR2 induced by VEGF in HUVECs. SKLB1002 also suppressed VEGFR2-mediated protein kinase activation of ERK, FAK, and Src. Blots were developedwith HRP-conjugated secondary antibodies and chemiluminescent substrate on Kodak X-ray films. B, the negative control study was carried out by Westernblot analysis, in which HUVECs were activated by basic fibroblast growth factor (bFGF) and show no response to SKLB1002 in ERK, FAK, and Srcphosphorylation levels. C, to rule out that SKLB1002 killed tumor cells directly, expression of VEGFR2 and 50 ng/mL VEGF-induced p-VEGFR2 wasdetected in various cells. In agreement with previous reports, HepG2, B16-F10, and L-02 did not express VEGFR2 and SW620 expressed moderate VEGFR2,which was not sufficient to lead to the activation of various downstream signaling substrates that were responsible for cell proliferation. D, diagram of signalingpathways for SKLB1002-regulated antiangiogenesis.






We also observed a slight decrease in body length ofzebrafish embryos, as the inhibition of developmentalangiogenesis had moderate effects on the growth of theembryo.

To evaluate whether SKLB1002 affected proliferation ofnormal cells in the zebrafish embryos, we used the anti-body of phosphorylated histone-3 to observe normal cellproliferation in the zebrafish embryos treated withSKLB1002 or vehicle, respectively. Nine hours after drugadministration, zebrafish embryos were processed forimmunofluorescence. We found that SKLB1002 stronglyantagonized the initiation of intersegmental vesselssprouting with no or least impact on normal cell pro-liferation in the zebrafish embryos (SupplementaryFig. S4A).

SKLB1002 inhibits tumor-induced angiogenesis inzebrash

Red fluorescence-labeled B16-F10 melanoma cells wereresuspended in Hank’s balanced salt medium and micro-injected in zebrafish embryos at 48 hpf through the peri-vitelline space between the periderm and the yolk. By5 days after the implantation in zebrafish, endothelial cellshad filled the inner space of the xenografted tumor incontrol group and built up its primary vascular network.However, at the same time, minimal angiogenic effect wasobserved in the embryos treated with 2.5 mmol/LSKLB1002, vandetanib, or sunitinib (Fig. 4B).

SKLB1002 inhibits tumor growth in vivoWe used 2 xenograft tumor models to investigate the

effect of SKLB1002 on tumor growth. Athymic mice bear-ing SW620 or HepG2 xenografts were treated daily for3 weeks with SKLB1002. As shown in Figure 5, we foundthat 100 mg/kg SKLB1002 significantly suppressed TV andcaused more than 60% inhibition of tumor growth com-pared with vehicle-treated mice.

To evaluate the health status of mice treated withSKLB1002, weight ofmice wasmonitored once every 3 daysthroughout the whole experiment. As shown in Supple-mentary Figure S5, no significant differences in weightswere found among the 3 groups. No adverse effects in othergross measures such as skin ulcerations or toxic death wereobserved in SKLB1002 group. Furthermore, toxic patholo-gic changes in liver, lungs, kidneys, spleen, and heart werenot detected by microscopic examination (Fig. 5E). Thesedata determined that the inhibition of tumor growth wasnot attributable to systemic toxicity.

SKLB1002 inhibits tumor angiogenesis in vivoImmunohistochemical anti-CD31 staining of the

tumor tissue from SKLB1002-treated mice showed sig-nificantly decreased microvessel density compared withvehicle groups (Fig. 6B). In addition, inhibition of angio-genesis could also be detected in the alginate-encapsu-lated tumor cell assay. New blood vessels in alginatebeads from SKLB1002-treated mice were apparently

BlankA

C D

B100µm

50 µm

50µm100 µm

Blank

Vandetanib

SKLB1002

Vehicle

Sunitinib

Vandetanib

SKLB1002

Vehicle

Sunitinib

60

40

20

01 2 3

**

**

**** **

**

4 5 1 2 3 4 50

400

800

1200

Leng

th o

f IS

Vs

(µm

)

Leng

th o

f tum

or v

esse

ls

Figure 4. SKLB1002 and positive control inhibited embryonic and tumor-induced angiogenesis. The experiments included 5 independent treatmentgroups: 1, untreated; 2, vehicle treated; 3, SKLB1002 treated; 4, vandetanib treated; and 5, sunitinib treated. A and C, brightfield and fluorescent imagesof 30 hpf zebrafish treated with blank control, vehicle, 2.5 mmol/L SKLB1002, vandetanib, or sunitinib. Intersegmental vessel (ISV) growth was obviouslyinhibited in the 2.5 mmol/L SKLB1002 and positive control groups. Columns, mean; bars, SD (n¼ 30; ANOVA; **, P < 0.01 vs. vehicle control). B and D, B16-F10cells were labeled with red fluorescence for easy observation in the zebrafish/tumor xenograft model. A total of 2.5 mmol/L SKLB1002 greatly inhibitedtumor-induced angiogenesis in 5-dpi zebrafish embryos compared with vehicle-treated and blank embryos. Columns, mean; bars, SD (n ¼ 30; ANOVA;**, P < 0.01 vs. vehicle control).

Zhang et al.





sparse. Besides, FITC–dextran uptake of mice treated withSKLB1002 was significantly decreased when comparedwith control groups (Fig. 6A). These results suggested thattumor angiogenesis was inhibited in mice treated withSKLB1002, which participated in suppression of tumorgrowth.

Reduced neovascular growth induces more apoptosisin vivoWe next analyzed the effect of SKLB1002 on apoptosis

in the SW620 xenograft tumors by TUNEL staining.TUNEL-positive cells were counted only in regions of in-tact tumor in such a way that the central necrosis typi-cally observed in xenografts did not interfere withquantification of apoptotic cells. Representative fieldsfrom each group were shown, which clearly indicatedthe higher rate of apoptosis in mice treated withSKLB1002. The number of apoptotic cells in 6 randomfields from 3 different tumors in each group was counted,and the apoptotic index is shown in Figure 6C.

Discussion

We have designed and identified a new small molecule,SKLB1002, as a potent inhibitor of VEGFR2. The com-pound is an ATP-competitive inhibitor of VEGFR2 withIC50 of 32 nmol/L. Our work indicated inhibitory effects ofSKLB1002 on HUVEC migration, invasion, and tube for-mation in a concentration-dependent manner. Notably,the inhibition of VEGF signaling by SKLB1002 occurs atconcentrations below those that show significant directeffects on the normal growth of endothelial cell, as thecompound exhibited an IC50 of 11.9 mmol/L against VEGF-induced HUVECs in theMTT assay. We showed that 2.5 mMSKLB1002 was sufficient to obviously block capillary-likestructure formation and migration in vitro.

VEGFR2 signaling is essential for the functions of vas-cular endothelial cells. Tyr1175 is themajor autophosphor-ylation site within VEGFR2, and its phosphorylationinitiates the downstream signaling events to endothelialcells (29). Phosphorylated Tyr1175 of VEGFR2 mediates

Figure 5. Antitumor effects ofSKLB1002 in vivo and toxicityevaluation. A and C, SW620 orHepG2 tumor–bearing athymicmice were treated as describedwith SKLB1002 at 100 and 50 mg/kg or with vehicle. The treatmentwith SKLB1002 resulted insignificant inhibition of tumorgrowth versus vehicle control (n ¼6; ANOVA; P < 0.01). B and D, theaverage weight of SW620 andHepG2 tumors from vehicle groupwere 2.22 � 0.54 and 1.03 � 0.26g, respectively, whereas that in100 mg/kg SKLB1002-treatedgroups were 0.73 � 0.12 and0.45 � 0.14g (n ¼ 6; ANOVA;**, P < 0.01). E, SKLB1002 did notcause obvious pathologicabnormalities in normal tissues.H&E staining of paraffin-embedded sections of the liver,spleen, kidney, heart,and lung.

2,500A B

C

E

D

Vehicle

Vehicle

**

0

1

2

3

Tum

or w

eigh

t (g)

**

50 mg/kg

50 mg/kg

100 mg/kg

100 mg/kg

2,000

Tum

or v

olum

e (m

m3 )

1,500

1,000

500

010 15 20

Days post–tumor cell inoculation

25 30

1,500Vehicle

Vehicle

**

0.0

0.5

1.0

1.5

Tum

or w

eigh

t (g)

**

50 mg/kg

50 mg/kg

100 mg/kg

Lung

Untreated

SKLB1002

treated

Liver Kidney Heart Spleen

100 mg/kg

1,200

Tum

or v

olum

e (m

m3 )

900

600

300

010 15 20

Days post–tumor cell inoculation

25 30






activation of the MAPK/ERK cascade and proliferation ofendothelial cells. It has also been linked to VEGF-inducedactivation of Src, which regulates vascular permeability andcell migration (30–31). Other signaling molecules thathave been indicated in VEGF-induced migration throughVEGFR2 include FAK and its substrate paxillin, which areinvolved in focal adhesion turnover during cell migration(32–34). In our study, by directly blocking VEGFR2 phos-phorylation, SKLB1002 subsequently blocked the activa-tion of ERK, FAK, and Src signaling pathway and inhibitedcellular activities.

However, endothelial cell models ofmigration, invasion,and tube formation lack the biological complexity of vesselsystem in vertebrate animals (35). To further confirm theability of the antiangiogenic effects of SKLB1002, we usedtumor xenograft models in zebrafish and athymicmouse inour study.

Zebrafish are beginning to be used in the drug discoveryprocess and can be a useful and cost-effective alternative tosome expensive, labor-intensive mammalian models (36).Pathologic angiogenesis has been considered as an impor-tant therapeutic target in several major diseases. Zebrafish

offer a potential tool for antiangiogenic drug developmentin a whole vertebrate system (37–39). In zebrafish, forma-tion of intersegmental vessels is considered to representcapillary sprouting during mammalian developmentwhereas the axial vessels correspond to arteries and veins.Disruption of VEGFR2 signaling impairs intersegmentalsprouting angiogenesis (40). In this assay, we used trans-genic zebrafish with fluorescent blood vessels to facilitateimage analysis and found that angiogenic intersegementalblood vessel growth was inhibited in zebrafish embryostreated with 2.5 mmol/L SKLB1002. Importantly, by detect-ing cells during proliferation in zebrafish embryos treatedby SKLB1002 or vehicle, we determined that SKLB1002 didnot affect normal cell proliferation and its antiangiogenicactivity was not due to broad cytotoxic effects. Recently,some studies have reported that the implantation of mur-ine or human tumor cells could elicit an angiogenicresponse in zebrafish embryos. The tumor xenograft modelin the zebrafish is suitable both for studying the effect ofantiangiogenic compounds and for the identification ofgenes involved in tumor angiogenesis (41, 42). In thisstudy, we used murine B16-F10 melanoma cells to build

3

2

**

**

FIT

C–d

extr

an (

µg/b

ead)

1

0Vehicle

Vehicle

A

B

C

50 mg/kg

SKLB1002 50 mg/kg SKLB1002 100 mg/kg

100 mg/kg

Vehicle 50 mg/kg 100 mg/kg

Vehicle 50 mg/kg 100 mg/kg

120

160

80

****

Ves

sel c

ount

(/m

m2 )

40

0

9

12

6

**

**

Apo

ptot

ic in

dex

(%)

3

0

500 µm

100 µm

50 µm

Figure 6. SKLB1002 inhibited tumor angiogenesis and induced tumor apoptosis in vivo. A, vascularization of alginate implants. Alginate beads containing5 � 104 tumor cells per bead were implanted s.c. into the backs of mice. Mice were then treated with vehicle control or SKLB1002 once a dayintraperitoneally for 12 days. Beads were surgically removed 12 days later, and FITC–dextran was quantified. FITC–dextran uptake of beads from SKLB1002-treated mice showed a significant decrease compared with the vehicle group (n ¼ 6; ANOVA; **, P < 0.01). B, SKLB1002 significantly inhibited tumorvessels in SW620 tumor xenografts. Frozen sections of SW620 tumor tissue were tested by immunohistochemical analysis with anti-CD31 antibody.Representative tumor vasculature from vehicle- or SKLB1002-treated mice was shown. The density of microvessel was calculated in each group(n¼ 5; ANOVA; **,P < 0.01). C, apoptosis wasmeasured on paraffin-embedded SW620 tumor sections by TUNEL staining. The apoptotic index was calculatedby dividing the number of TUNEL-positive cells by the total number of cells. The treatment with SKLB1002 resulted in significantly increased apoptosisin a dose-dependent manner versus vehicle control (n ¼ 6; ANOVA; **, P < 0.01).

Zhang et al.





tumor xenograft model in zebrafish embryos and foundthat B16-F10 cell induced the rapid formation of tumormicrovasculature. We found low concentration ofSKLB1002 could remarkably suppress the tumor angiogen-esis in the xenografted tumor mass. Furthermore, in com-bination with computer-aided drug design, tumorxenograft model in zebrafish offers the potential forhigh-throughput antiangiogenic drug discovery in a wholevertebrate system. Briefly, this model can be used to screencompound library built by the restricted de novo designstrategy for antiangiogenic compounds.SW620 is a human carcinoma tumor cell line and highly

tumorigenic in athymic mice (43). In our establishedSW620 tumor xenograft models in athymic mice, tumorgrowth was significantly inhibited by SKLB1002 adminis-tration (100 mg/kg/d) with an inhibitory rate of 67%.Inhibition of angiogenesis was observed both in mousetumor tissue and in an alginate-encapsulated tumor cellassay. Similar to results of a previous report (44), owing toreduced neovascular growth, more apoptotic cells could bevisualized in the tumor tissues treated with SKLB1002 thanin control group by in situ TUNEL assay.Unlike those widely used anticancer drugs that have

adverse effects or severe cytotoxicity to induce cell apop-tosis in modern cancer chemotherapy, various smallmolecule VEGFR tyrosine kinase inhibitors have beenidentified and developed to block tumor angiogenesisand metastasis formation (4, 45). Initially, these agentswere expected to be active without causing toxicities orresistance because of the genetic stability of endothelialcells. However, contrary to initial expectations, VEGFR2inhibitors could cause major side effects in recent clinicalexperiences (46–48). The possible causes of side effectsinduced by VEGFR2 inhibitors are numerous, but themain cause is their poor target selectivity (49, 50). Thus,understanding the molecular mechanisms involved in thetoxicity of angiogenesis inhibition should allow morespecific and more potent inhibitors to be developed.

Following the proposed strategy of designing specifickinase inhibitors, the restricted de novo design methodwas adopted to construct new molecules that targetedVEGFR2, which led to the identification of SKLB1002.The high selectivity of SKLB1002 was evidenced by thelack of activity against a variety of other kinases tested(Supplementary Table S2). SKLB1002 (100 mg/kg/d) didnot affect the body weight of the mice but showedsignificant inhibitory effects on solid tumor growthand tumor angiogenesis. No adverse effects in other grossmeasures such as diarrhea, anorexia, skin ulceration,bleeding, and toxic deaths were observed in SKLB1002-treated group. Furthermore, toxic pathologic changes inliver, lungs, kidneys, spleen, and heart were not found bymicroscopic examination. Thus, we assume thatSKLB1002 may be a novel anticancer agent with limitedtoxicity.

Taken together, our studies indicate that SKLB1002 is apotent and specific inhibitor of tumor angiogenesis bytargeting the VEGFR2 signaling pathways. Further struc-tural modifications of SKLB1002 are still underway.SKLB1002 and its derivates are promising candidates asantiangiogenic and anticancer drugs.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

This study was funded by the National Key Basic Research Program (973Program) of China (2010CB529900) and the National Natural ScienceFoundation of China (30973451).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 3, 2010; revised April 23, 2011; accepted April 28,2011; published OnlineFirst May 27, 2011.

References1. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other

disease. Nat Med 1995;1:27–31.2. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases.

Nature 2000;407:249–57.3. Dvorak HF. Angiogenesis: update 2005. J Thromb Haemost 2005;3:

1835–42.4. Ivy SP, Wick JY, Kaufman BM. An overview of small-molecule

inhibitors of VEGFR signaling. Nat Rev Clin Oncol 2009;6:569–79.5. Bergers G, Benjamin LE. Tumorigenesis and the angiogenic switch.

Nat Rev Cancer 2003;3:401–10.6. Folkman J. Role of angiogenesis in tumor growth and metastasis.

Semin Oncol 2002;29:15–8.7. Carmeliet P. VEGF as a key mediator of angiogenesis in cancer.

Oncology 2005;69:4–10.8. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its

receptors. Nat Med 2003;9:669–76.9. Neufeldaname G, Cohena T, Gengrinovitcha S, Poltorak Z. Vascular

endothelial growth factor (VEGF) and its receptors. FASEB J1999;13:9–22.

10. Holmes K, Roberts OL, Thomas AM, Cross MJ. Vascular endothe-lial growth factor receptor-2:Structure, function, intracellularsignalling and therapeutic inhibition. Cell Signal 2007;19:2003–12.

11. Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L. VEGF receptorsignaling in control of vascular function. Nat Rev Mol Cell Biol2006;7:359–71.

12. Meadows KN, Bryant P, Pumiglia K. Vascular endothelial growthfactor induction of the angiogenic phenotype requires Ras activation.J Biol Chem 2001;276:49289–98.

13. Takahashi T, Ueno H, Shibuya M. VEGF activates protein kinase C-dependent, but Ras-independent Raf-MEK-MAP kinase pathwayfor DNA synthesis in primary endothelial cells. Oncogene 1999;18:2221–30.

14. Ferrara N. Vascular endothelial growth factor as a target for anticancertherapy. Oncologist 2004;9:2–10.

15. Hanrahan EO, Heymach JV. Vascular endothelial growth factor recep-tor tyrosine kinase inhibitors vandetanib (ZD6474) and AZD2171 inlung cancer. Clin Cancer Res 2007;13:s4617–22.






16. Faivre S, Demetri G, Sargent W, Raymond E. Molecular basis forsunitinib efficacy and future clinical development. Nat Rev DrugDiscov 2007;6:734–45.

17. Li WW, Chen JJ, Zheng RL, Zhang WQ, Cao ZX, Yang LL, et al. Takingquinazoline as a general support-nog to design potent and selectivekinase inhibitors: application to FMS-like tyrosine kinase 3. Chem-MedChem 2010;5:513–16.

18. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of humanendothelial cells derived from umbilical veins. Identification bymorphologic and immunologic criteria. J Clin Invest 1973;52:2745–56.

19. Heo DS, Park JG, Hata K, Day R, Herberman RB, Whiteside TL.Evaluation of tetrazolium-based semiautomatic colorimetric assay formeasurement of human antitumor cytotoxicity. Cancer Res 1990;50:3681–90.

20. Timke C, Zieher H, Roth A, Hauser K, Lipson KE, Weber KJ, et al.Combination of vascular endothelial growth factor receptor/platelet-derived growth factor receptor inhibition markedly improves radiationtumor therapy. Clin Cancer Res 2008;14:2210–9.

21. Pang X, Yi Z, Zhang X, Sung B, Qu W, Lian X, et al. Acetyl-11-keto-b-boswellic acid inhibits prostate tumor growth by suppressing vas-cular endothelial growth factor receptor 2-mediated angiogenesis.Cancer Res 2009;69:5893–900.

22. Kimmel CB, BallardWW, Kimmel SR, Ullmann B, Schilling TF. Stages ofembryonic development of the zebrafish. Dev Dyn 1995;203:253–310.

23. Hans F, Dimitrov S. Histone H3 phosphorylation and cell division.Oncogene 2001;20:3021–27.

24. Blezinger P, Wang J, Gondo M, Quezada A, Mehrens D, French M,et al. Systemic inhibition of tumor growth and tumor metastases byintramuscular administration of the endostatin gene. Nat Biotechnol1999;17:343–48.

25. Hoffmann J, Schirner M, Menrad A, Schneider MR. A highly sensitivemodel for quantification of in vivo tumor angiogenesis induced byalginate-encapsulated tumor cells. Cancer Res 1997;57:3847–51.

26. Luo J, Xia Q, Zhang R, Lv C, Zhang W, Wang Y, et al. Treatment ofcancer with a novel dual-targeted conditionally replicative adenovirusarmed with mda-7/IL-24 gene. Clin Cancer Res 2008;14:2450–7.

27. Wang H, Zhang J, Tian J, Qu B, Li T, Chen Y, et al. Using dual-tracerPET to predict the biologic behavior of human colorectal cancer. JNucl Med 2009;50:1857–64.

28. Wang H, Liu B, Tian JH, Xu BX, Guan ZW, Qu BL, et al. Monitoringearly responses to irradiation with dual-tracer micro-PET in dual-tumor bearing mice. World J Gastroenterol 2010;16:5416–23.

29. Liu J, Agarwal S. Mechanical signals activate vascular endothelialgrowth factor receptor-2 to upregulate endothelial cell proliferationduring inflammation. J Immunol 2010;185:1215–21.

30. Takahashi T, Yamaguchi S, Chida K, Shibuya M. A single autopho-sphorylation site on KDR/Flk-1 is essential for VEGF-A-dependentactivation of PLC-g and DNA synthesis in vascular endothelial cells.EMBO J 2001;20:2768–78.

31. Eliceiri BP, Puente XS, Hood JD, Stupack DG, Schlaepfer DD, HuangXZ, et al. Src-mediated coupling of focal adhesion kinase to integrinalpha(v)beta5 in vascular endothelial growth factor signaling. J CellBiol 2002;157:149–60.

32. Holmqvist K, Cross MJ, Rolny C, H€agerkvist R, Rahimi N, MatsumotoT, et al. The adaptor protein shb binds to tyrosine 1175 in vascularendothelial growth factor (VEGF) receptor-2 and regulates VEGF-dependent cellular migration. J Biol Chem 2004;279:22267–75.

33. Abedi H, Zachary I. Vascular endothelial growth factor stimulatestyrosine phosphorylation and recruitment to new focal adhesions of

focal adhesion kinase and paxillin in endothelial cells. J Biol Chem1997;272:15442–51.

34. Avraham HK, Lee TH, Koh Y, Kim TA, Jiang S, Sussman M, et al.Vascular endothelial growth factor regulates focal adhesion assemblyin human brain microvascular endothelial cells through activation ofthe focal adhesion kinase and related adhesion focal tyrosine kinase. JBiol Chem 2003;278:36661–8.

35. Auerbach R, Lewis R, Shinners B, Kubai L, Akhtar N. Angiogenesisassays: a critical overview. Clin Chem 2003;49:32–40.

36. Zon LI, Peterson RT. In vivo drug discovery in the zebrafish. Nat RevDrug Discov 2005;4:35–44.

37. Tran TC, Sneed B, Haider J, Blavo D, White A, Aiyejorun T, et al.Automated, quantitative screening assay for antiangiogenic com-pounds using transgenic zebrafish. Cancer Res 2007;67:11386–92.

38. Wu X, Zhong H, Song J, Damoiseaux R, Yang Z, Lin S. Mycophenolicacid is a potent inhibitor of angiogenesis. Arterioscler Thromb VascBiol 2006;26:2414–16.

39. Cross LM, Cook MA, Lin S, Chen JN, Rubinstein AL. Rapid analysis ofangiogenesis drugs in a live fluorescent zebrafish assay. ArteriosclerThromb Vasc Biol 2003;23:911–12.

40. Habeck H, Odenthal J, Walderich B, Maischein H, Schulte-Merker S.T€ubingen 2000 Screen Consortium. Analysis of a zebrafish VEGFreceptor mutant reveals specific disruption of angiogenesis. Curr Biol2002;12:1405–12.

41. Nicoli S, Ribatti D, Cotelli F, Presta M. Mammalian tumor xenograftsinduce neovascularization in zebrafish embryos. Cancer Res2007;67:2927–31.

42. Stoletov K, Montel V, Lester RD, Gonias SL, Klemke R. High-resolu-tion imaging of the dynamic tumor cell–vascular interface in trans-parent zebrafish. Proc Natl Acad Sci U S A 2007;104:17406–11.

43. Fogh J, Fogh JM, Orfeo T. One hundred and twenty-seven culturedhuman tumor cell lines producing tumors in nude mice. J Natl CancerInst 1977;59:221–26.

44. Rubenstein JL, Kim J, Ozawa T, Zhang M, Westphal M, Deen DF,,et al. Anti-VEGF antibody treatment of glioblastoma prolongssurvival but results in increased vascular cooption. Neoplasia 2000;2:306–14.

45. Liu L, Cao Y, Chen C, Zhang X, McNabola A, Wilkie D, et al. Sorafenibblocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, andinduces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer Res 2006;66:11851–8.

46. Faivre S, Delbaldo C, Vera K, Robert C, Lozahic S, Lassau N, et al.Safety, pharmacokinetic, and antitumor activity of SU11248, a noveloral multitarget tyrosine kinase inhibitor, in patients with cancer. J ClinOncol 2006;24:25–35.

47. Abou-Alfa GK, Schwartz L, Ricci S, Amadori D, Santoro A, Figer A,et al. Phase II study of sorafenib in patients with advanced hepato-cellular carcinoma. J Clin Oncol 2006;24:4293–300.

48. Drevs J, Siegert P, Medinger M, Mross K, Strecker R, Zirrgiebel U,et al. Phase I clinical study of AZD2171, an oral vascular endothelialgrowth factor signaling inhibitor, in patients with advanced solidtumors. J Clin Oncol 2007;25:3045–54.

49. Eskens FA, Verweij J. The clinical toxicity profile of vascular endothe-lial growth factor (VEGF) and vascular endothelial growth factorreceptor (VEGFR) targeting angiogenesis inhibitors; a review. Eur JCancer 2006;42:3127–39.

50. Verheul HM, Pinedo HM. Possible molecular mechanisms involvedin the toxicity of angiogenesis inhibition. Nat Rev Cancer 2007;7:475–85.

Zhang et al.





2011;17:4439-4450. Published OnlineFirst May 27, 2011.Clin Cancer Res Shuang Zhang, Zhixing Cao, Hongwei Tian, et al.

In VivoSignaling, Inhibits Angiogenesis and Tumor Growth SKLB1002, a Novel Potent Inhibitor of VEGF Receptor 2

Updated version

10.1158/1078-0432.CCR-10-3109doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2011/07/01/1078-0432.CCR-10-3109.DC1Access the most recent supplemental material at:

Cited articles

http://clincancerres.aacrjournals.org/content/17/13/4439.full#ref-list-1

This article cites 50 articles, 24 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/17/13/4439.full#related-urls

This article has been cited by 7 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

SubscriptionsReprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://clincancerres.aacrjournals.org/content/17/13/4439To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-10-3109

http://clincancerres.aacrjournals.org/content/suppl/2011/07/01/1078-0432.CCR-10-3109.DC1

http://clincancerres.aacrjournals.org/content/17/13/4439.full#ref-list-1

http://clincancerres.aacrjournals.org/content/17/13/4439.full#related-urls

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/17/13/4439


SKLB1002, a Novel Potent Inhibitor of VEGF Receptor 2 ... · SKLB1002, a Novel Potent Inhibitor of...

Documents

Transcript of SKLB1002, a Novel Potent Inhibitor of VEGF Receptor 2 ... · SKLB1002, a Novel Potent Inhibitor of...