Research ArticleThe Antitumor Effect of Gekko Sulfated Glycopeptide byInhibiting bFGF-Induced Lymphangiogenesis

Xiu-Li Ding1 Ya-Nan Man2 Jian Hao1 Cui-Hong Zhu1

Chang Liu1 Xue Yang1 and Xiong-Zhi Wu1

1Zhong-Shan-Men In-Patient Department Tianjin Medical University Cancer Institute and Hospital National Clinical ResearchCenter for Cancer Key Laboratory of Cancer Prevention andTherapy Huan-Hu-Xi Road He-Xi District Tianjin 300060 China2Department of Radiotherapy The Second Affiliated Hospital of Tianjin Medical University Ping-Jiang RoadHe-Xi District Tianjin 300060 China

Correspondence should be addressed to Xiong-Zhi Wu wuxiongzhi163com

Received 6 December 2015 Revised 16 February 2016 Accepted 20 March 2016

Academic Editor Kallesh D Jayappa

Copyright copy 2016 Xiu-Li Ding et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Objective To study the antilymphangiogenesis effect of Gekko Sulfated Glycopeptide (GSPP) on human lymphatic endothelial cells(hLECs) Methods MTS was conducted to confirm the antiproliferation effect of GSPP on hLECs flow cytometry was employedto detect hLECs cycle distribution the antimigration effect of GSPP on hLECs was investigated by wound healing experiment andtranswell experiment tube formation assaywas used to examine its inhibitory effect on the lymphangiogenesis western blottingwasconducted to detect the expression of extracellular signal-regulated kinase12 (Erk12) and p-Erk12 after GSPP and basic fibroblastgrowth factor (bFGF) treatment Nude mice models were established to investigate the antitumor effect of GSPP in vivo Decreasedlymphangiogenesis caused by GSPP in vivo was verified by immunohistochemical staining Results In vitro GSPP (10 120583gmL100 120583gmL) significantly inhibited bFGF-induced hLECs proliferation migration and tube-like structure formation (119875 lt 005) andantagonized the phosphorylation activation of Erk12 induced by bFGF In vivo GSPP treatment (200mgkgd) not only inhibitedthe growth of colon carcinoma but also inhibited the tumor lymphangiogenesis Conclusion GSPP possesses the antitumor abilityby inhibiting bFGF-inducing lymphangiogenesis in vitro and in vivo which may further inhibit tumor lymphatic metastasis

1 Introduction

There are two vital ways for cancer cells to disseminate theblood pathway leading to the invasion of distant organs suchas liver brain bone or lung and the lymphatic pathway lead-ing to the invasion of the lymph nodes draining the organswhere the tumor evolves [1] For a long time the study ontumor metastasis has been centered on tumor angiogenesisand hematogenous metastasis [2] In the recent years tumorlymphangiogenesis catches the eyes of researchers [3] Oneof the most popular views on cancerous lymphangiogenesisis that tumor forms new lymphatic vessels on the basis of theexisting ones Studies have demonstrated that tumor-inducedlymphangiogenesis plays a significant role in tumor cellstraffic and lymph node metastasis [4ndash6] Further lymphaticmetastasis is amajor and early step during tumor progression

The presence of lymphatic metastasis is a key determinant ofcancer staging treatment and prognosis [7 8] Thus anti-lymphangiogenesis is a new target for cancer therapy How-ever there are no effective antilymphangiogenesis drugs inclinic until now

It was reported that vascular endothelial growth factor-C3(VEGF-C3) and VEGF-D were identified as the stimulatorsof the proliferation of lymphatic endothelial cells Recentstudies showed that basic fibroblast growth factor (bFGF)wasanother effective stimulator for lymphangiogenesis and couldregulate lymphatic endothelial cell proliferation migrationand tube formation as well [9ndash11] bFGF is a heparin depen-dent growth factor and can activate the intracellular signaltransduction pathways only in the form of bFGF-heparin-FGFR terpolymers structure [12] bFGF is bound to heparansulfate (HS) in the extracellular matrix (ECM) and is released

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 7396392 9 pageshttpdxdoiorg10115520167396392

2 BioMed Research International

in an active form when the ECM-HS is degraded by hep-aranase expressed in normal and malignant cells [13 14]

Wepreviously isolated a novel polysaccharideGekko Sul-fated Glycopeptide (GSPP) from Gekko swinhonis Guntherand confirmed it as a homogeneous sulfated polysaccharide-protein complex with O-glycopeptide linkages The molecu-lar weight of GSPPwas estimated to be over 2000 kDa [15] Itsdirect effects on the proliferation differentiation and migra-tion of hepatoma cells have been studied [15ndash17] Our furtherstudy showed that GSPP had a similar structure with heparinand competed with heparin to disturb the bFGF-heparin-FGFR terpolymers forming further blocking bFGFrsquos biologi-cal effect GSPP could inhibit tumor angiogenesis by reducingbFGF production inhibiting the release of bFGF from theextracellular matrix and disturbing the binding of bFGF toits low affinity receptor By inhibiting bFGF-induced angio-genesis GSPP significantly inhibited the growth of nudemicexenografted tumors [12]

In this study whether GSPP could inhibit the lymphan-giogenesis goaded our interests Here we investigated thepotent antilymphangiogenesis ability of GSPP in vitro andin vivo and found that GSPP significantly inhibited bFGF-induced cell proliferation migration and tube formationin hLECs And GSPP demonstrated an excellent antitumoreffect through inhibiting lymphangiogenesis in vivo

2 Materials and Methods

21 Cell Lines hLECs were purchased from CHI ScientificInc (Jiangsu China) The certificate analysis sheet suppliedby CHI Scientific Inc for each vial of cells indicated thatmorethan 95of the cells were hLECs (CD31 and podoplanin dou-ble positive)Thiswas determined by FluorescenceActivatingCell Sorter (FACS) Cells were cultured in EGM-2 mediaaccording to the supplierrsquos instructions (CHI Scientific IncJiangsu China) Cells before 6 generations were used in thisstudy

22 Antibodies and Reagents GSPP used in this study wasprepared in advance which is the same batchwith that in pre-vious study [15] The dried powder is stored in minus80∘C MTStest kit was purchased from Promega Corporation (MadisonWisconsin USA) Fibronectin from human plasma waspurchased from Sigma (St Louis MO USA) Recombinanthuman FGF-basic (154 aa) was purchased from PeproTechCorporation (Rocky Hill NJ USA) Antibodies againstphospho-Erk12 (p4442 MAPK lot 9101S) and total-Erk12(p4442 MAPK 137F5 lot 4695) were obtained from CellSignalingTechnology (DanversMAUSA)Antibody againstLYVE-1 (lot 33504-1) was bought from Abcam Corpora-tion (Cambridge MA USA) Antibody against 120573-actin (lotA2228) was bought from Sigma Corporation (St Louis MOUSA)

23 hLECs Proliferation Assay 96-well plate was precoatedwith fibronectin for 20 minutes at 37∘C in 5 humidifiedCO2 100 120583L hLECs suspension (1 times 105 cellsmL) was seeded

into each well of a 96-well plate After 24 h the mediumwas discarded and replaced with drug-containing medium

6 groups were set up as GSPP 10 120583gmL GSPP 100 120583gmLbFGF 10 ngmL GSPP 10 120583gmL with bFGF 10 ngmL GSPP100 120583gmL with bFGF 10 ngmL and negative control groupAfter cells were exposed to the drugs for indicated times (01 2 3 4 5 and 6 d) 20120583L MTS solution reagent was addedinto each well and incubated at 37∘C for 1ndash4 h and then theOD value was measured with a Microplate reader (iMarkBio-Rad) at 490 nmThe media were not changed during thetreatment period

24 Cell Cycle Detection A flow cytometry (BD BiosciencesFranklin Lakes NJ USA) was used to evaluate cell cycle dis-tribution hLECs (5 times 105 cells2mL) were seeded into 6-wellplates and treated with GSPP (10 120583gmL 100 120583gmL) alone orcombined with bFGF 10 ngmL for 48 h Cells were collectedandwashed twice in cold PBS fixed in 70methanol (minus20∘C)overnight Then cells were washed with PBS twice again andincubated with RNase (20120583gmL) in 37∘C for 1 h Propidiumiodide (50120583gmL) was added before being detected by flowcytometry system

25 Wound Healing Experiment hLECs were seeded in 24-well plates at the density of 1 times 105 cellsmL After cell attach-ment hLECs were starved with serum-free EBM-2 for 24 hA linear wound about 1mm in width was made by scratchingthe monolayer cell culture with a pipette tip after cellconfluency Then EBM-2 with different concentrations ofGSPP (10 120583gmL GSPP 100 120583gmL) andor bFGF (10 ngmL)with 15 FBS were added After 0 and 6 h the photographsof wound healing width of hLECs were observed and takenunder an invert microscope The migration width was mea-sured by the Photoshop software The migration ratio wascalculated as the migration width of experiment groupthemigration width of control group

26 Transwell Experiment After being starved with serum-free medium for 24 h hLECs (5 times 104 cells) in EBM-2media with different concentrations of GSPP (10 120583gmLGSPP 100120583gmL) were added to the upper chambers ofthe transwell insert (BD Biosciences Bedford MA) EBM-2containing bFGF was added to the lower chamber to inducecell migration After being incubated for 12 h at 37∘C cells onthe top surface of the membranes were wiped off with cottonballs and the cells that migrated on the underside of insertswere fixed with methanol and stained with crystal violet Fivedifferent digital images were taken per well and the numbersof migrated cells were counted and calculated

27 Tube Formation Assay Matrigel was thawed at 4∘Covernight 96-well plate and 100 120583L pipette tips were also keptat 4∘C overnight and both the plate and tips were placed onice during the entire experiment process 30 uL Matrigel wasloaded in each well of the 96-well plates and the plate wasincubated at 37∘C in a tissue culture incubator for 30minto allow the matrix to polymerize Trypsinized LECs wereadjusted to the appropriate cell density (15 times 104 cellswell)with different concentration of GSPP and bFGF as describedin the proliferation assay 100 120583L hLECs suspension wasadded on top of the gel in the 96-well plateThe plate was then

BioMed Research International 3

incubated at 37∘C in a tissue culture incubator and the forma-tion of the capillary-like tubes was observed after 4 h ThenhLECs were observed under inverted microscope and 9 pho-tographs (times40) were taken per hole The numbers of matrixform of closed irregular polygon were recorded and calcu-lated

28 Western Blot Experiment For western blot analysis ofErk and p-Erk protein expression confluent cultures ofhLECs in 6-hole pate were homogenized in lysis bufferThe protein concentrations were determined using the BCAProtein Quantitation Kit Equal amounts of lysate proteinwere subjected to 10 SDS-polyacrylamide gel electrophore-sis The proteins were transferred onto PVDF membranesfor immunoblot analysis Blocking was performed with 5nonfat dry milk in 01 Tween 20 in TBS followed byimmunoblotting with a polyclonal goat anti-human Erk anti-body p-Erk antibody and 120573-actin antibody Specific bindingwas detected by the ECL plus Western Blotting DetectionSystem

29 In Vivo Nude Mice Model This study was approved bythe Tianjin Cancer Institutional Animal ethics Committee(number 2014044) Animal care and experimental proce-dures followed the Tianjin Medical University guidelinesfor the care and use of laboratory animals 4ndash6-week-oldmale BALBc mice were purchased from LianHe LiHuacooperation To establish a heterotopic colon carcinoma nudemicemodel 25times 106HT-29 cells in 200 120583L PBSwere injectedinto the right flank of BALBc mice When tumors grew to10mmdiameter size tumors were cut off and cut into 1mm times1mm chips and then transplanted to other 24 BALBc micewhich were divided into four groups of 8 mice each groupMice were treated daily with an intraperitoneal injectionof either 01mL GSPP (20 200mgkgday in PBS) or PBS(control) for 21 daysThe length andwidth of the tumors weremeasured with a caliper every 2-3 days All mice were sacri-ficed 24 days after tumor inoculation and the tumors wereexcised and weighted and the tumor volumes were calculatedusing the standard formula 119881 = 11988611988722 (119886 lengths of thetumors 119887 widths of the tumors)

210 Immunohistochemistry Staining Tumor specimenswereimmediately removed from sacrificed mice and preparedfor immunohistological examination Tumors were fixed in10 (vv) neutral buffered formalin overnight embedded inparaffin and sectioned to a 5 120583m thickness Tumor sectionswere deparaffinized via immersion in xylene dehydrated ina graded series of ethanol and washed with distilled waterThereafter tumor sections were boiled in 10mM sodiumcitrate buffer (pH = 60) for 10min and cooled at room tem-perature To inhibit endogenous peroxidase activity tumorsections were incubated with methanol containing 1 (vv)hydrogen peroxide for 10min Tumor sections were thenblocked with 1 BSA and then incubated overnight withanti-LYVE-1 antibody tumor sections were probed withperoxidase-conjugated secondary antibodies and incubatedwith DAB until the desired stain intensity developed Aftercounterstaining with Harris hematoxylin tumor sections

were examined under an inverted microscope (E100 NikonJapan) To analyze immunohistochemical signals within thespecimens all tumor sections were digitized under a times40magnification and images were captured

211 Statistical Analysis The data were expressed as themean plusmn SD from triplicate experiments and were analyzedusing SPSS software (Version 200 Chicago USA) One-wayANOVA and two-way ANOVA were applied to analyze thesignificance of groupswith one factor and two factors respec-tively 119875 value lt005 was considered statistically significantAll of the experiments were repeated at least three times

3 Results

31 Morphological Characteristics of Lymphatic EndothelialCells InVitroCulture After 3ndash5 h of primary cell culture cellswere able to grow adhering to the wall After 1 d endothelialcells spread forming groups and after 1 week cells grewdensely and formed a single layer with the characteristicof ldquopebblesrdquo Inverted microscopy observation lymphaticendothelial cells were irregular ovoid with big nucleus andthere were many small vacuoles in the cytoplasm (Fig-ure 1(a))

32 GSPP Inhibited bFGF-Induced Proliferation of hLECsMTS assay was performed to investigate the antiproliferationeffect of GSPP After hLECs were exposed to GSPP (0 10 and100 120583gmL) andor bFGF (10 ngmL) for 0 1 2 3 4 5 or 6 dODvalueswere detectedThe results demonstrated that bFGFpromoted the proliferation of hLECs significantly (119875 lt 005)while GSPP alone did not inhibit proliferation of hLECs (119875 gt005) TrypanBlue staining showed that the viability of hLECswas not affected by GSPP (results were not shown) But GSPPcould abrogate bFGF-induced proliferation of hLECs signifi-cantly in a dose- and time-dependent manner (119875 lt 005 Fig-ure 1(b)) In the cell cycle assay bFGF significantly promotedhLECs into proliferation cycle with a high proportion of Sand G2 phase cells while GSPP alone has no effect on the cellcycle distribution of hLECsWhen combinedwith 100 120583gmLGSPP cell proliferative activity was blocked distinctly withan increasing proportion of G1 phase cells compared withbFGF alone group (Figure 1(d))

33 bFGF-Induced p-Erk Was Downregulated by GSPP inhLECs Erk12 signal pathway is reported to be involvedin cell growth migration and angiogenesis The promotingeffect of bFGF on vessel cell proliferation and migrationmay be partly associated with an increased level of Erkphosphorylation [18 19] To explore the possible mechanismof GSPP in inhibiting bFGF-induced lymphangiogenesis Erkand p-Erk protein expressions were detected by westernblotting Results showed that bFGF significantly increased theexpression of p-Erk in hLECs andGSPP decreased the bFGF-induced p-Erk significantly No significant difference of theexpression of total Erk was observed among each group(Figure 1(c))

4 BioMed Research International

(a)

05

10

15

20

OD

val

ue

2 3 6541Time (day)

ControlbFGF 10ngmLGSPP 10120583gmL

GSPP 100120583gmLGSPP 10120583gmL + bFGF 10ngmLGSPP 100120583gmL + bFGF 10ngmL

(b)

+ minus minus +

minus + minus +

minus

minus

minus minus minus + minus

+

minus

+

p-Erk

Erk

120573-actin10ngmL bFGF10120583gmL GSPP100120583gmL GSPP

(c)

(A) (B) (C)

(D) (E) (F)

930

4823

0

300

600

900

1200

Num

ber

0 8020 40 60 100

731

17297

0

400

800

1600

1200

30 60 90 1200

894

4661

0

600

1200

1800

2400

20 40 60 80 1000

946

3717

0100200300400500

Num

ber

1000 60 8020 40

Channels (FL2-A-PI-area)

722

23438

0

600

1200

1800

2400

20 40 60 80 1000

Channels (FL2-A-PI-area)

850

6783

02004006008001000

20 40 60 80 1000

Channels (FL2-A-PI-area)G1SG2

bFGF 10ngmLGSPP 10120583gmL

GSPP 100120583gmL

+ minus minus +

minus + minus +

minus

minus

minus minus minus + minus

+

minus

+

0

50

100

150(

of p

er p

hase

)

(d)

Figure 1 GSPP inhibited bFGF-induced proliferation of hLECs and Erk phosphorylation (a)Morphological characteristics of hLECs in vitroculture hLECs were compressed ovoid short fusiform or polygon and formed a single layer with the characteristic of ldquopebblesrdquo (b) hLECsgrowth curves (1 times 105 cellsmL) were incubated with different concentration of GSPP (0 10 and 100120583gmL) andor bFGF (10 ngmL) for 01 2 3 4 5 and 6 d then cell proliferation was quantified by MTS assay and cell growth curve was made (c) The changes of Erk and p-Erkprotein expression level of hLECs cultured in 6-well plate were incubated with different concentration of GSPP (0 10 and 100120583gmL) andorbFGF (10 ngmL) Erk and p-Erk protein levels were monitored by western blot analysis of whole-cell lysates (d) Cell cycle analysis Lefthistogram of cell cycle distribution Right statistical analysis of cell cycle percentage After exposure to GSPP (0 10 and 100120583gmL) andorbFGF (10 ngmL) for 48 h cell cycle distribution was determined by propidium iodide labeling (A) Control (B) bFGF 10 ngmL (C) GSPP10120583gmL (D) GSPP 100 120583gmL (E) GSPP 10 120583gmL + bFGF 10 ngmL and (F) GSPP 100 120583gmL + bFGF 10 ngmL Data were presented asmean plusmn SD of three independent experiments hLECs human lymphatic endothelial cells bFGF basic fibroblast growth factor GSPP GekkoSulfated Glycopeptide

BioMed Research International 5

(A) (B) (A) (B)

(C) (D) (C) (D)

(E) (F) (E) (F)

6h0h

(a)

00

05

10

15

20

The m

igra

tion

ratio

bFGF 10ngmLGSPP 10120583gmL

GSPP 100120583gmL

+ minus minus +

minus + minus +

minus

minus

minus minus minus + minus

+

minus

+

(b)

(A) (B) (C)

(D) (E) (F)

(c)

0

50

100

150

Num

ber o

f mig

rate

d ce

lls

bFGF 10ngmLGSPP 10120583gmL

GSPP 100120583gmL

+ minus minus +

minus + minus +

minus

minus

minus minus minus + minus

+

minus

+

(d)

Figure 2 GSPP inhibits bFGF-induced migration of hLECs (a) Wound healing assay Left representative images of injury width in woundhealing assay (times40) Right quantification of the migration ratio of hLECs compared to control hLECs (1 times 105 cellswell) were seeded in24-well plates and wounds were generated after cell confluence After hLECs were treated with different concentrations of GSPP (0 10 and100120583gmL) andor bFGF (10 ngmL) for 0 h and 6 h the photos were taken and the injury width was measured The migration ratio wascalculated as the migration width of experiment groupthe migration width of control group (b) Transwell assays Left representative imagesof migrated LECs in transwell assay (times40) Right quantification of migrated LECs compared to control hLECs (5 times 104 cellswell) in EBM-2with different concentrations of GSPP (0 10 and 100 120583gmL) were added to the upper chamber of the transwell insert EBM-2 containingbFGF (10 ngmL) or not was added to the lower chamber to induce cell migration After 12 h at 37∘C cells on the top surface of themembraneswere wiped off with cotton balls and the cells that migrated on the underside of inserts were fixed with methanol and stained with crystalviolet Five different digital images were taken per well and the number of migrated cells was counted (A) Control (B) bFGF 10 ngmL (C)GSPP 10120583gmL (D)GSPP 100120583gmL (E) GSPP 10 120583gmL+ bFGF 10 ngmL and (F) GSPP 100 120583gmL+ bFGF 10 ngmL Data were presentedas mean plusmn SD of three independent experiments ◻119875 lt 005 versus control group and 998810119875 lt 005 versus bFGF-single use group

34 GSPP Inhibited bFGF-Induced Migration of hLECsWound healing experiment and transwell experiment wereconducted to investigate the antimigration effect of GSPP Itwas showed that bFGF significantly upregulated the migra-tion distance of hLECs (119875 lt 005) which was antagonized byGSPP significantly (119875 lt 005) at 6 h (Figure 2(a)) The sameresults were obtained in transwell experimentThe number ofmigrated cells in bFGF-treated group increased significantlycompared to the negative control cells (119875 lt 005) No sig-nificant difference of the cell numbers was observed between

the GSPP-treated group and negative control (119875 gt 005)However concomitant treatment with 10 120583gmL 100 120583gmLGSPP and bFGF inhibited the migration of hLECs comparedwith the bFGF-single use group (119875 lt 005) (Figure 2(b))

35 GSPP Abrogated bFGF-Induced hLECs Lymphangiogen-esis In Vitro Tube-like formation assay was conducted toexamine the inhibitory effect of GSPP on lymphangiogenesishLECs were added on top of the gel in the 96-well plate incu-bated at 37∘C in a tissue culture incubator and the formation

6 BioMed Research International

(A) (B) (C)

(D) (E) (F)

(a)

bFGF 10ngmLGSPP 10120583gmL

GSPP 100120583gmL

+ minus minus +

minus + minus +

minus

minus

minus minus minus + minus

+

minus

+

0

20

40

60

The n

umbe

r of t

ubes

(b)

Figure 3 GSPP inhibits bFGF-induced lymphangiogenesis in vitro In vitro tube formation assay hLECs (15 times 104 cellswell) were seeded inMatrigel-coated 96-well plates and treated with different concentration of GSPP (0 10 and 100120583gmL) andor bFGF (10 ngmL) for 4 h andthe tube-like structure formation was observed (a) Representative images of tube formation (times40) (b) Quantification of inhibitory ratios oftube branches (A) Control (B) bFGF 10 ngmL (C) GSPP 10 120583gmL (D) GSPP 100120583gmL (E) GSPP 10 120583gmL + bFGF 10 ngmL and (F)GSPP 100 120583gmL + bFGF 10 ngmL Data were presented as mean plusmn SD of three independent experiments ◻119875 lt 005 versus control groupand 998810119875 lt 005 versus bFGF-single use group

of the capillary-like tubes was observed after 4 h There wasno difference in the number of tube-like structures formationbetween GSPP treatment group and the negative controlgroup in hLECs (119875 gt 005) The number of tube-likestructures treated with bFGF group was much more than thenegative control group (119875 lt 005) However GSPP signif-icantly attenuated bFGF-induced tube formation in a dose-dependent manner with simultaneous incubation with GSPPand bFGF (Figures 3(a) and 3(b))

36 GSPP Inhibited Tumor Growth and Lymphangiogenesis inHT-29 Colon Carcinoma Nude Mice Model The antitumoractivity of GSPP was investigated in HT-29 colon carcinomaxenograft model using BALBc nude mice Growth of thetumors was significantly inhibited in the mice treated withGSPP compared with the growth of tumors in control mice(Figure 4(a)) In control group (intraperitoneal injectionwithPBS) tumors grew rapidly and reached an average volume of45903 plusmn 2892mm3 (mean plusmn SD) by day 24 after being trans-plantedwithHT-29 tumor blocks while the sizes of tumors in20 and 200mgkgday GSPP-treated groups were only 34825plusmn 622mm3 and 25518 plusmn 6072mm3 respectively (759 and559 decrease) (119875 lt 001 Figure 4(b)) The tumor weightof the control group was 028 plusmn 003 g whereas the weightsof GSPP-treated groups decreased to 022 plusmn 004 g and 014plusmn 003 g respectively (119875 lt 001 Figure 4(c)) To evaluate theadverse effects of GSPP wemeasured the weights and visceralindex of the mice and found that there was no significantdifference between the control and GSPP-treated groups(Figure 4(d)) Tumor lymphangiogenesis was analyzedusing immunohistochemical staining with LYVE-1 antibodyResults showed that 200mgkgday GSPP markedly reducedtumor microvessel density in the tissue sections comparedwith the control group (Figure 4(e)) These results indicatedthat GSPP efficiently inhibits tumor growth in carcinoma

animal model Suppression of tumor growth due to GSPPcould be caused by inhibition of lymphangiogenesis

4 Discussion

Metastasis is a key cause for the failure of tumor treatmentand patient death The lymph node metastasis is the firststep of the tumor dissemination and is also the main signof poor prognosis of tumor [20ndash22] More and more studiesshowed that the tumor-induced lymphangiogenesis played anextremely important role in cancer cells spreading to somelocal lymph nodes and distant metastasis [23ndash25] Studieson antitumor lymphangiogenesis have gradually become aresearch hotspot and bFGF as an important factor to pro-mote lymphangiogenesis has been researched intensively

Gekko swinhonis Gunther was a traditional Chinesemedicine which has been used as an anticancer drug intraditionalChinesemedicine for hundreds of years especiallyin hepatoma [26] Soaking in alcohol is very useful forpharmaceutical ingredients dissolving out and is also themost common method of extraction Oral administrationand external usewere themainmethods of administration Inour previous study we isolated GSPP from Gekko swinhonisGunther and confirmed that GSPP could induce hepatomacell differentiation inhibit cell proliferation and migration invitro and inhibit hepatic carcinoma growth in vivo [12 1517] In this study we verified another mechanism of GSPPrsquosantitumor effects by inhibiting tumor lymphangiogenesisResults showed that GSPP significantly inhibited bFGF-induced human hLECs proliferation migration and tube-like structure formation in vitro Moreover GSPP treatment(200mgkgd) not only inhibited the growth of breast carci-noma but also inhibited the lymphangiogenesis in vivo Thedosage was determined by previous studies which is a safedose with an appropriate tumor inhibition rate [12] Through

BioMed Research International 7

Con

trol

GSP

P (m

gkg

)20

020

(a)

0

200

400

600

The v

olum

e of t

umor

s (m

m3)

10 14 18 22 246Days after tumor-bearing

Control

GSPP 200mgkgGSPP 20mgkg

(b)

00

01

02

03

The w

eigh

t of t

umor

s (g)

20 2000GSPP (mgkgd)

lowast

lowast

(c)

0

8

16

24

The w

eigh

t of m

ice (

g)

20 2000GSPP (mgkgd)

(d)

Control GSPP 200mgkgGSPP 20mgkg

(e)

Figure 4 GSPP inhibited colon carcinoma HT-29 xenograft growth and lymphangiogenesis in vivo Male nunu nude mice were inoculatedsubcutaneously with colon carcinoma HT-29 cells Three days after inoculation mice were treated with GSPP (20 or 200mgkg) or PBSevery day for 21 days via intraperitoneal injection The lengths and widths of tumors were measured individually every 3 days At the endof the experiment the implanted tumors were sectioned (a) Effect of GSPP on tumor volume Left image of excised tumors Right tumorgrowth curves (b) Effect of GSPP on tumor weight (c) Effect of GSPP onmouse weight Mice were weighed at the end of the experiment (d)Tumor lymphatic microvessel density The implanted tumors were sectioned and stained against LYNE-1 antibody Tumor lymphatic vesselsare shown as LYNE-1 positive (yellow color) Data were presented as mean plusmn SD of three independent experiments lowast119875 lt 005 versus controlgroup

this study we further uncovered the mechanisms of GSPPrsquosantitumor effects besides inhibiting cancer cells prolifer-ation and migration inducing tumor cell differentiationand inhibiting cancer angiogenesis and cancer-associatedfibroblast growth (unpublished) showing that GSPP is apromising antitumor drug in future cancer treatment

bFGF as an important prolymphangiogenesis factorgenerated by tumor cells can significantly promote lym-phatic vessel endothelial cell proliferation and migration and

promote tumor lymphangiogenesis by a variety of ways [27]The most classic way on which bFGF promotes tumor lym-phangiogenesis is through VEGF-A VEGF-C and VEGF-Dwhich are known prolymphangiogenesis factors to promotetumor lymphatic vessel grow [23 28] bFGF is a heparindependent growth factor which means that it can have effectonly in the form of bFGF-heparin-FGFR terpolymers struc-ture to further activate the intracellular signal transductionpathways GSPP is a kind of polysaccharide sulfate which

8 BioMed Research International

is similar to the structure of heparin active site both ofwhich contain a sulfuric acid base Previous study showedthat GSPP works by three distinct mechanisms (a) blockingthe bFGF production (b) inhibiting the release of bFGF fromthe extracellular matrix and (c) directly binding to bFGFand competitively inhibiting the binding of bFGF to its lowaffinity receptor heparinHS [12] In this study we found thatGSPP alone has no effect on the viability growth of hLECsAnd it does not inhibit the migration and tube formation ofhLECs whichmay be due to the absence of bFGF bFGF playsan important role in the growth migration and lymphan-giogenesis of hLECs With the addition of exogenous bFGFwhich simulates the environment in the body GSPP signifi-cantly inhibited bFGF-induced cell proliferation migrationand tube formation This means that GSPP works throughdirectly binding to bFGF and competitively inhibiting thebinding of bFGF to its low affinity receptor heparinHS

Extracellular signal-regulated kinase12 (Erk12) is a pro-tein kinase separated and identified in the early 1990s and itssignal transduction is involved in cell growth developmentand differentiation [19] Studies found that the promotingeffect of bFGF on endothelial cell proliferation andmigrationof part is associated with an increased level of Erk phos-phorylation [18 29] To explore the possible mechanism ofGSPPrsquos inhibiting effect of bFGF-induced lymphangiogene-sis western blot was performed We examined the Erk andp-Erk expression level changes in lymphatic endothelial cellsafter being exposed to GSPP for a certain time Our resultsshowed that bFGF significantly increased the expression ofp-Erk in hLECs However cotreatment with GSPP and bFGFdecreased the expression of p-Erk in hLECs compared withthe bFGF-single use group This further proved that GSPPcould bind to bFGF and competitively inhibit the bindingof bFGF to its low affinity receptor thus blocking the bFGF-induced phosphorylation of Erk12 in hLECs

5 Conclusions

Our study showed that inhibiting bFGF-induced lymphan-giogenesis was one of GSPPrsquos anticancer mechanisms GSPPas an effective bFGF-targeted inhibitor can be a notableantilymphatic metastasis drug in future cancer treatment

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Xiu-Li Ding and Ya-Nan Man contributed equally to thiswork

Acknowledgments

This study was supported by major scientific and technicalproject of Tianjin Science and Technology Commission(05YFGDSF02600) andNationalNatural Science Foundationof China (no 81173376 and no 81473441)

References

[1] J Robert ldquoBiology of cancermetastasisrdquoBulletin du Cancer vol100 no 4 pp 333ndash342 2013

[2] R Bicknell ldquoVascular targeting and the inhibition of angiogene-sisrdquoAnnals of Oncology vol 5 supplement 4 pp S45ndashS50 1994

[3] D Ribatti ldquoHistory of research on angiogenesisrdquo ChemicalImmunology and Allergy vol 99 pp 1ndash14 2014

[4] R Shayan M G Achen and S A Stacker ldquoLymphatic vesselsin cancer metastasis bridging the gapsrdquo Carcinogenesis vol 27no 9 pp 1729ndash1738 2006

[5] J P Sleeman and W Thiele ldquoTumor metastasis and the lym-phatic vasculaturerdquo International Journal of Cancer vol 125 no12 pp 2747ndash2756 2009

[6] R L Ferris M T Lotze S P L Leong D S B Hoon and D LMorton ldquoLymphatics lymph nodes and the immune systembarriers and gateways for cancer spreadrdquo Clinical and Experi-mental Metastasis vol 29 no 7 pp 729ndash736 2012

[7] S S Sundar and T S Ganesan ldquoRole of lymphangiogenesis incancerrdquo Journal of Clinical Oncology vol 25 no 27 pp 4298ndash4307 2007

[8] M G Achen B K McColl and S A Stacker ldquoFocus on lym-phangiogenesis in tumor metastasisrdquo Cancer Cell vol 7 no 2pp 121ndash127 2005

[9] R H Adams and K Alitalo ldquoMolecular regulation of angio-genesis and lymphangiogenesisrdquoNature Reviews Molecular CellBiology vol 8 no 6 pp 464ndash478 2007

[10] P M Linares and J P Gisbert ldquoRole of growth factors inthe development of lymphangiogenesis driven by inflammatorybowel disease a reviewrdquo Inflammatory Bowel Diseases vol 17no 8 pp 1814ndash1821 2011

[11] L K Chang G Garcia-Cardena F Farnebo et al ldquoDose-dependent response of FGF-2 for lymphangiogenesisrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 101 no 32 pp 11658ndash11663 2004

[12] S-X Zhang C Zhu Y Ba et al ldquoGekko-sulfated glycopeptideinhibits tumor angiogenesis by targeting basic fibroblast growthfactorrdquo The Journal of Biological Chemistry vol 287 no 16 pp13206ndash13215 2012

[13] M Benezra R Ishai-Michaeli S A Ben-Sasson and I Vlo-davsky ldquoStructure-activity relationships of heparin-mimickingcompounds in induction of bFGF release from extracellularmatrix and inhibition of smooth muscle cell proliferation andheparanase activityrdquo Journal of Cellular Physiology vol 192 no3 pp 276ndash285 2002

[14] M Fannon K E Forsten and M A Nugent ldquoPotentiation andinhibition of bFGF binding by heparin a model for regulationof cellular responserdquo Biochemistry vol 39 no 6 pp 1434ndash14452000

[15] D ChenW-J Yao X-L Zhang et al ldquoEffects of Gekko sulfatedpolysaccharide-protein complex on human hepatoma SMMC-7721 cells inhibition of proliferation and migrationrdquo Journal ofEthnopharmacology vol 127 no 3 pp 702ndash708 2010

[16] X Wu D Chen and G-R Xie ldquoEffects of Gekko sulfatedpolysaccharide on the proliferation and differentiation of hep-atic cancer cell linerdquoCell Biology International vol 30 no 8 pp659ndash664 2006

[17] X-Z Wu D Chen and X-Q Han ldquoAnti-migration effects ofGekko sulfated glycopeptide on human hepatoma SMMC-7721cellsrdquoMolecules vol 16 no 6 pp 4958ndash4970 2011

BioMed Research International 9

[18] L Yang J Zhang C Wang et al ldquoInteraction between 8-hydroxyquinoline ruthenium(II) complexes and basic fibrob-last growth factors (bFGF) inhibiting angiogenesis and tumorgrowth through ERK and AKT signaling pathwaysrdquo Metal-lomics vol 6 no 3 pp 518ndash531 2014

[19] K Balmanno and S J Cook ldquoTumour cell survival signallingby the ERK12 pathwayrdquo Cell Death and Differentiation vol 16no 3 pp 368ndash377 2009

[20] N E Tobler andMDetmar ldquoTumor and lymph node lymphan-giogenesismdashimpact on cancer metastasisrdquo Journal of LeukocyteBiology vol 80 no 4 pp 691ndash696 2006

[21] Y Cao ldquoOpinion emerging mechanisms of tumour lymphan-giogenesis and lymphatic metastasisrdquo Nature Reviews Cancervol 5 no 9 pp 735ndash743 2005

[22] K Alitalo T Tammela and T V Petrova ldquoLymphangiogenesisin development and human diseaserdquo Nature vol 438 no 7070pp 946ndash953 2005

[23] M Skobe T Hawighorst D G Jackson et al ldquoInduction oftumor lymphangiogenesis by VEGF-C promotes breast cancermetastasisrdquo Nature Medicine vol 7 no 2 pp 192ndash198 2001

[24] SA Stacker CCaesarM E Baldwin et al ldquoVEGF-Dpromotesthe metastatic spread of tumor cells via the lymphaticsrdquo NatureMedicine vol 7 no 2 pp 186ndash191 2001

[25] M S Pepper ldquoLymphangiogenesis and tumor metastasis mythor realityrdquo Clinical Cancer Research vol 7 no 3 pp 462ndash4682001

[26] C L Yang and Y J Qi Animal Drug of Chinese Tradi-tional Medicine Ancient Books Press of Chinese TraditionalMedicine Beijing China 1st edition 2001

[27] R Cao A Eriksson H Kubo K Alitalo Y Cao and JThyberg ldquoComparative evaluation of FGF-2- VEGF-A- andVEGF-C-induced angiogenesis lymphangiogenesis vascularfenestrations and permeabilityrdquo Circulation Research vol 94pp 664ndash670 2004

[28] M J Karkkainen R E Ferrell E C Lawrence et al ldquoMissensemutations interfere with VEGFR-3 signalling in primary lym-phoedemardquo Nature Genetics vol 25 no 2 pp 153ndash159 2000

[29] S Guo L Yu Y Cheng et al ldquoPDGFR120573 triggered by bFGF pro-motes the proliferation andmigration of endothelial progenitorcells via p-ERK signallingrdquo Cell Biology International vol 36no 10 pp 945ndash950 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 BioMed Research International

in an active form when the ECM-HS is degraded by hep-aranase expressed in normal and malignant cells [13 14]

Wepreviously isolated a novel polysaccharideGekko Sul-fated Glycopeptide (GSPP) from Gekko swinhonis Guntherand confirmed it as a homogeneous sulfated polysaccharide-protein complex with O-glycopeptide linkages The molecu-lar weight of GSPPwas estimated to be over 2000 kDa [15] Itsdirect effects on the proliferation differentiation and migra-tion of hepatoma cells have been studied [15ndash17] Our furtherstudy showed that GSPP had a similar structure with heparinand competed with heparin to disturb the bFGF-heparin-FGFR terpolymers forming further blocking bFGFrsquos biologi-cal effect GSPP could inhibit tumor angiogenesis by reducingbFGF production inhibiting the release of bFGF from theextracellular matrix and disturbing the binding of bFGF toits low affinity receptor By inhibiting bFGF-induced angio-genesis GSPP significantly inhibited the growth of nudemicexenografted tumors [12]

In this study whether GSPP could inhibit the lymphan-giogenesis goaded our interests Here we investigated thepotent antilymphangiogenesis ability of GSPP in vitro andin vivo and found that GSPP significantly inhibited bFGF-induced cell proliferation migration and tube formationin hLECs And GSPP demonstrated an excellent antitumoreffect through inhibiting lymphangiogenesis in vivo

2 Materials and Methods

21 Cell Lines hLECs were purchased from CHI ScientificInc (Jiangsu China) The certificate analysis sheet suppliedby CHI Scientific Inc for each vial of cells indicated thatmorethan 95of the cells were hLECs (CD31 and podoplanin dou-ble positive)Thiswas determined by FluorescenceActivatingCell Sorter (FACS) Cells were cultured in EGM-2 mediaaccording to the supplierrsquos instructions (CHI Scientific IncJiangsu China) Cells before 6 generations were used in thisstudy

22 Antibodies and Reagents GSPP used in this study wasprepared in advance which is the same batchwith that in pre-vious study [15] The dried powder is stored in minus80∘C MTStest kit was purchased from Promega Corporation (MadisonWisconsin USA) Fibronectin from human plasma waspurchased from Sigma (St Louis MO USA) Recombinanthuman FGF-basic (154 aa) was purchased from PeproTechCorporation (Rocky Hill NJ USA) Antibodies againstphospho-Erk12 (p4442 MAPK lot 9101S) and total-Erk12(p4442 MAPK 137F5 lot 4695) were obtained from CellSignalingTechnology (DanversMAUSA)Antibody againstLYVE-1 (lot 33504-1) was bought from Abcam Corpora-tion (Cambridge MA USA) Antibody against 120573-actin (lotA2228) was bought from Sigma Corporation (St Louis MOUSA)

23 hLECs Proliferation Assay 96-well plate was precoatedwith fibronectin for 20 minutes at 37∘C in 5 humidifiedCO2 100 120583L hLECs suspension (1 times 105 cellsmL) was seeded

into each well of a 96-well plate After 24 h the mediumwas discarded and replaced with drug-containing medium

6 groups were set up as GSPP 10 120583gmL GSPP 100 120583gmLbFGF 10 ngmL GSPP 10 120583gmL with bFGF 10 ngmL GSPP100 120583gmL with bFGF 10 ngmL and negative control groupAfter cells were exposed to the drugs for indicated times (01 2 3 4 5 and 6 d) 20120583L MTS solution reagent was addedinto each well and incubated at 37∘C for 1ndash4 h and then theOD value was measured with a Microplate reader (iMarkBio-Rad) at 490 nmThe media were not changed during thetreatment period

24 Cell Cycle Detection A flow cytometry (BD BiosciencesFranklin Lakes NJ USA) was used to evaluate cell cycle dis-tribution hLECs (5 times 105 cells2mL) were seeded into 6-wellplates and treated with GSPP (10 120583gmL 100 120583gmL) alone orcombined with bFGF 10 ngmL for 48 h Cells were collectedandwashed twice in cold PBS fixed in 70methanol (minus20∘C)overnight Then cells were washed with PBS twice again andincubated with RNase (20120583gmL) in 37∘C for 1 h Propidiumiodide (50120583gmL) was added before being detected by flowcytometry system

25 Wound Healing Experiment hLECs were seeded in 24-well plates at the density of 1 times 105 cellsmL After cell attach-ment hLECs were starved with serum-free EBM-2 for 24 hA linear wound about 1mm in width was made by scratchingthe monolayer cell culture with a pipette tip after cellconfluency Then EBM-2 with different concentrations ofGSPP (10 120583gmL GSPP 100 120583gmL) andor bFGF (10 ngmL)with 15 FBS were added After 0 and 6 h the photographsof wound healing width of hLECs were observed and takenunder an invert microscope The migration width was mea-sured by the Photoshop software The migration ratio wascalculated as the migration width of experiment groupthemigration width of control group

26 Transwell Experiment After being starved with serum-free medium for 24 h hLECs (5 times 104 cells) in EBM-2media with different concentrations of GSPP (10 120583gmLGSPP 100120583gmL) were added to the upper chambers ofthe transwell insert (BD Biosciences Bedford MA) EBM-2containing bFGF was added to the lower chamber to inducecell migration After being incubated for 12 h at 37∘C cells onthe top surface of the membranes were wiped off with cottonballs and the cells that migrated on the underside of insertswere fixed with methanol and stained with crystal violet Fivedifferent digital images were taken per well and the numbersof migrated cells were counted and calculated

27 Tube Formation Assay Matrigel was thawed at 4∘Covernight 96-well plate and 100 120583L pipette tips were also keptat 4∘C overnight and both the plate and tips were placed onice during the entire experiment process 30 uL Matrigel wasloaded in each well of the 96-well plates and the plate wasincubated at 37∘C in a tissue culture incubator for 30minto allow the matrix to polymerize Trypsinized LECs wereadjusted to the appropriate cell density (15 times 104 cellswell)with different concentration of GSPP and bFGF as describedin the proliferation assay 100 120583L hLECs suspension wasadded on top of the gel in the 96-well plateThe plate was then

BioMed Research International 3

incubated at 37∘C in a tissue culture incubator and the forma-tion of the capillary-like tubes was observed after 4 h ThenhLECs were observed under inverted microscope and 9 pho-tographs (times40) were taken per hole The numbers of matrixform of closed irregular polygon were recorded and calcu-lated

28 Western Blot Experiment For western blot analysis ofErk and p-Erk protein expression confluent cultures ofhLECs in 6-hole pate were homogenized in lysis bufferThe protein concentrations were determined using the BCAProtein Quantitation Kit Equal amounts of lysate proteinwere subjected to 10 SDS-polyacrylamide gel electrophore-sis The proteins were transferred onto PVDF membranesfor immunoblot analysis Blocking was performed with 5nonfat dry milk in 01 Tween 20 in TBS followed byimmunoblotting with a polyclonal goat anti-human Erk anti-body p-Erk antibody and 120573-actin antibody Specific bindingwas detected by the ECL plus Western Blotting DetectionSystem

29 In Vivo Nude Mice Model This study was approved bythe Tianjin Cancer Institutional Animal ethics Committee(number 2014044) Animal care and experimental proce-dures followed the Tianjin Medical University guidelinesfor the care and use of laboratory animals 4ndash6-week-oldmale BALBc mice were purchased from LianHe LiHuacooperation To establish a heterotopic colon carcinoma nudemicemodel 25times 106HT-29 cells in 200 120583L PBSwere injectedinto the right flank of BALBc mice When tumors grew to10mmdiameter size tumors were cut off and cut into 1mm times1mm chips and then transplanted to other 24 BALBc micewhich were divided into four groups of 8 mice each groupMice were treated daily with an intraperitoneal injectionof either 01mL GSPP (20 200mgkgday in PBS) or PBS(control) for 21 daysThe length andwidth of the tumors weremeasured with a caliper every 2-3 days All mice were sacri-ficed 24 days after tumor inoculation and the tumors wereexcised and weighted and the tumor volumes were calculatedusing the standard formula 119881 = 11988611988722 (119886 lengths of thetumors 119887 widths of the tumors)

210 Immunohistochemistry Staining Tumor specimenswereimmediately removed from sacrificed mice and preparedfor immunohistological examination Tumors were fixed in10 (vv) neutral buffered formalin overnight embedded inparaffin and sectioned to a 5 120583m thickness Tumor sectionswere deparaffinized via immersion in xylene dehydrated ina graded series of ethanol and washed with distilled waterThereafter tumor sections were boiled in 10mM sodiumcitrate buffer (pH = 60) for 10min and cooled at room tem-perature To inhibit endogenous peroxidase activity tumorsections were incubated with methanol containing 1 (vv)hydrogen peroxide for 10min Tumor sections were thenblocked with 1 BSA and then incubated overnight withanti-LYVE-1 antibody tumor sections were probed withperoxidase-conjugated secondary antibodies and incubatedwith DAB until the desired stain intensity developed Aftercounterstaining with Harris hematoxylin tumor sections

were examined under an inverted microscope (E100 NikonJapan) To analyze immunohistochemical signals within thespecimens all tumor sections were digitized under a times40magnification and images were captured

211 Statistical Analysis The data were expressed as themean plusmn SD from triplicate experiments and were analyzedusing SPSS software (Version 200 Chicago USA) One-wayANOVA and two-way ANOVA were applied to analyze thesignificance of groupswith one factor and two factors respec-tively 119875 value lt005 was considered statistically significantAll of the experiments were repeated at least three times

3 Results

31 Morphological Characteristics of Lymphatic EndothelialCells InVitroCulture After 3ndash5 h of primary cell culture cellswere able to grow adhering to the wall After 1 d endothelialcells spread forming groups and after 1 week cells grewdensely and formed a single layer with the characteristicof ldquopebblesrdquo Inverted microscopy observation lymphaticendothelial cells were irregular ovoid with big nucleus andthere were many small vacuoles in the cytoplasm (Fig-ure 1(a))

32 GSPP Inhibited bFGF-Induced Proliferation of hLECsMTS assay was performed to investigate the antiproliferationeffect of GSPP After hLECs were exposed to GSPP (0 10 and100 120583gmL) andor bFGF (10 ngmL) for 0 1 2 3 4 5 or 6 dODvalueswere detectedThe results demonstrated that bFGFpromoted the proliferation of hLECs significantly (119875 lt 005)while GSPP alone did not inhibit proliferation of hLECs (119875 gt005) TrypanBlue staining showed that the viability of hLECswas not affected by GSPP (results were not shown) But GSPPcould abrogate bFGF-induced proliferation of hLECs signifi-cantly in a dose- and time-dependent manner (119875 lt 005 Fig-ure 1(b)) In the cell cycle assay bFGF significantly promotedhLECs into proliferation cycle with a high proportion of Sand G2 phase cells while GSPP alone has no effect on the cellcycle distribution of hLECsWhen combinedwith 100 120583gmLGSPP cell proliferative activity was blocked distinctly withan increasing proportion of G1 phase cells compared withbFGF alone group (Figure 1(d))

33 bFGF-Induced p-Erk Was Downregulated by GSPP inhLECs Erk12 signal pathway is reported to be involvedin cell growth migration and angiogenesis The promotingeffect of bFGF on vessel cell proliferation and migrationmay be partly associated with an increased level of Erkphosphorylation [18 19] To explore the possible mechanismof GSPP in inhibiting bFGF-induced lymphangiogenesis Erkand p-Erk protein expressions were detected by westernblotting Results showed that bFGF significantly increased theexpression of p-Erk in hLECs andGSPP decreased the bFGF-induced p-Erk significantly No significant difference of theexpression of total Erk was observed among each group(Figure 1(c))

4 BioMed Research International

(a)

05

10

15

20

OD

val

ue

2 3 6541Time (day)

ControlbFGF 10ngmLGSPP 10120583gmL

GSPP 100120583gmLGSPP 10120583gmL + bFGF 10ngmLGSPP 100120583gmL + bFGF 10ngmL

(b)

+ minus minus +

minus + minus +

minus

minus

minus minus minus + minus

+

minus

+

p-Erk

Erk

120573-actin10ngmL bFGF10120583gmL GSPP100120583gmL GSPP

(c)

(A) (B) (C)

(D) (E) (F)

930

4823

0

300

600

900

1200

Num

ber

0 8020 40 60 100

731

17297

0

400

800

1600

1200

30 60 90 1200

894

4661

0

600

1200

1800

2400

20 40 60 80 1000

946

3717

0100200300400500

Num

ber

1000 60 8020 40

Channels (FL2-A-PI-area)

722

23438

0

600

1200

1800

2400

20 40 60 80 1000

Channels (FL2-A-PI-area)

850

6783

02004006008001000

20 40 60 80 1000

Channels (FL2-A-PI-area)G1SG2

bFGF 10ngmLGSPP 10120583gmL

GSPP 100120583gmL

+ minus minus +

minus + minus +

minus

minus

minus minus minus + minus

+

minus

+

0

50

100

150(

of p

er p

hase

)

(d)

Figure 1 GSPP inhibited bFGF-induced proliferation of hLECs and Erk phosphorylation (a)Morphological characteristics of hLECs in vitroculture hLECs were compressed ovoid short fusiform or polygon and formed a single layer with the characteristic of ldquopebblesrdquo (b) hLECsgrowth curves (1 times 105 cellsmL) were incubated with different concentration of GSPP (0 10 and 100120583gmL) andor bFGF (10 ngmL) for 01 2 3 4 5 and 6 d then cell proliferation was quantified by MTS assay and cell growth curve was made (c) The changes of Erk and p-Erkprotein expression level of hLECs cultured in 6-well plate were incubated with different concentration of GSPP (0 10 and 100120583gmL) andorbFGF (10 ngmL) Erk and p-Erk protein levels were monitored by western blot analysis of whole-cell lysates (d) Cell cycle analysis Lefthistogram of cell cycle distribution Right statistical analysis of cell cycle percentage After exposure to GSPP (0 10 and 100120583gmL) andorbFGF (10 ngmL) for 48 h cell cycle distribution was determined by propidium iodide labeling (A) Control (B) bFGF 10 ngmL (C) GSPP10120583gmL (D) GSPP 100 120583gmL (E) GSPP 10 120583gmL + bFGF 10 ngmL and (F) GSPP 100 120583gmL + bFGF 10 ngmL Data were presented asmean plusmn SD of three independent experiments hLECs human lymphatic endothelial cells bFGF basic fibroblast growth factor GSPP GekkoSulfated Glycopeptide

BioMed Research International 5

(A) (B) (A) (B)

(C) (D) (C) (D)

(E) (F) (E) (F)

6h0h

(a)

00

05

10

15

20

The m

igra

tion

ratio

bFGF 10ngmLGSPP 10120583gmL

GSPP 100120583gmL

+ minus minus +

minus + minus +

minus

minus

minus minus minus + minus

+

minus

+

(b)

(A) (B) (C)

(D) (E) (F)

(c)

0

50

100

150

Num

ber o

f mig

rate

d ce

lls

bFGF 10ngmLGSPP 10120583gmL

GSPP 100120583gmL

+ minus minus +

minus + minus +

minus

minus

minus minus minus + minus

+

minus

+

(d)

Figure 2 GSPP inhibits bFGF-induced migration of hLECs (a) Wound healing assay Left representative images of injury width in woundhealing assay (times40) Right quantification of the migration ratio of hLECs compared to control hLECs (1 times 105 cellswell) were seeded in24-well plates and wounds were generated after cell confluence After hLECs were treated with different concentrations of GSPP (0 10 and100120583gmL) andor bFGF (10 ngmL) for 0 h and 6 h the photos were taken and the injury width was measured The migration ratio wascalculated as the migration width of experiment groupthe migration width of control group (b) Transwell assays Left representative imagesof migrated LECs in transwell assay (times40) Right quantification of migrated LECs compared to control hLECs (5 times 104 cellswell) in EBM-2with different concentrations of GSPP (0 10 and 100 120583gmL) were added to the upper chamber of the transwell insert EBM-2 containingbFGF (10 ngmL) or not was added to the lower chamber to induce cell migration After 12 h at 37∘C cells on the top surface of themembraneswere wiped off with cotton balls and the cells that migrated on the underside of inserts were fixed with methanol and stained with crystalviolet Five different digital images were taken per well and the number of migrated cells was counted (A) Control (B) bFGF 10 ngmL (C)GSPP 10120583gmL (D)GSPP 100120583gmL (E) GSPP 10 120583gmL+ bFGF 10 ngmL and (F) GSPP 100 120583gmL+ bFGF 10 ngmL Data were presentedas mean plusmn SD of three independent experiments ◻119875 lt 005 versus control group and 998810119875 lt 005 versus bFGF-single use group

34 GSPP Inhibited bFGF-Induced Migration of hLECsWound healing experiment and transwell experiment wereconducted to investigate the antimigration effect of GSPP Itwas showed that bFGF significantly upregulated the migra-tion distance of hLECs (119875 lt 005) which was antagonized byGSPP significantly (119875 lt 005) at 6 h (Figure 2(a)) The sameresults were obtained in transwell experimentThe number ofmigrated cells in bFGF-treated group increased significantlycompared to the negative control cells (119875 lt 005) No sig-nificant difference of the cell numbers was observed between

the GSPP-treated group and negative control (119875 gt 005)However concomitant treatment with 10 120583gmL 100 120583gmLGSPP and bFGF inhibited the migration of hLECs comparedwith the bFGF-single use group (119875 lt 005) (Figure 2(b))

35 GSPP Abrogated bFGF-Induced hLECs Lymphangiogen-esis In Vitro Tube-like formation assay was conducted toexamine the inhibitory effect of GSPP on lymphangiogenesishLECs were added on top of the gel in the 96-well plate incu-bated at 37∘C in a tissue culture incubator and the formation

6 BioMed Research International

(A) (B) (C)

(D) (E) (F)

(a)

bFGF 10ngmLGSPP 10120583gmL

GSPP 100120583gmL

+ minus minus +

minus + minus +

minus

minus

minus minus minus + minus

+

minus

+

0

20

40

60

The n

umbe

r of t

ubes

(b)

Figure 3 GSPP inhibits bFGF-induced lymphangiogenesis in vitro In vitro tube formation assay hLECs (15 times 104 cellswell) were seeded inMatrigel-coated 96-well plates and treated with different concentration of GSPP (0 10 and 100120583gmL) andor bFGF (10 ngmL) for 4 h andthe tube-like structure formation was observed (a) Representative images of tube formation (times40) (b) Quantification of inhibitory ratios oftube branches (A) Control (B) bFGF 10 ngmL (C) GSPP 10 120583gmL (D) GSPP 100120583gmL (E) GSPP 10 120583gmL + bFGF 10 ngmL and (F)GSPP 100 120583gmL + bFGF 10 ngmL Data were presented as mean plusmn SD of three independent experiments ◻119875 lt 005 versus control groupand 998810119875 lt 005 versus bFGF-single use group

of the capillary-like tubes was observed after 4 h There wasno difference in the number of tube-like structures formationbetween GSPP treatment group and the negative controlgroup in hLECs (119875 gt 005) The number of tube-likestructures treated with bFGF group was much more than thenegative control group (119875 lt 005) However GSPP signif-icantly attenuated bFGF-induced tube formation in a dose-dependent manner with simultaneous incubation with GSPPand bFGF (Figures 3(a) and 3(b))

36 GSPP Inhibited Tumor Growth and Lymphangiogenesis inHT-29 Colon Carcinoma Nude Mice Model The antitumoractivity of GSPP was investigated in HT-29 colon carcinomaxenograft model using BALBc nude mice Growth of thetumors was significantly inhibited in the mice treated withGSPP compared with the growth of tumors in control mice(Figure 4(a)) In control group (intraperitoneal injectionwithPBS) tumors grew rapidly and reached an average volume of45903 plusmn 2892mm3 (mean plusmn SD) by day 24 after being trans-plantedwithHT-29 tumor blocks while the sizes of tumors in20 and 200mgkgday GSPP-treated groups were only 34825plusmn 622mm3 and 25518 plusmn 6072mm3 respectively (759 and559 decrease) (119875 lt 001 Figure 4(b)) The tumor weightof the control group was 028 plusmn 003 g whereas the weightsof GSPP-treated groups decreased to 022 plusmn 004 g and 014plusmn 003 g respectively (119875 lt 001 Figure 4(c)) To evaluate theadverse effects of GSPP wemeasured the weights and visceralindex of the mice and found that there was no significantdifference between the control and GSPP-treated groups(Figure 4(d)) Tumor lymphangiogenesis was analyzedusing immunohistochemical staining with LYVE-1 antibodyResults showed that 200mgkgday GSPP markedly reducedtumor microvessel density in the tissue sections comparedwith the control group (Figure 4(e)) These results indicatedthat GSPP efficiently inhibits tumor growth in carcinoma

animal model Suppression of tumor growth due to GSPPcould be caused by inhibition of lymphangiogenesis

4 Discussion

Metastasis is a key cause for the failure of tumor treatmentand patient death The lymph node metastasis is the firststep of the tumor dissemination and is also the main signof poor prognosis of tumor [20ndash22] More and more studiesshowed that the tumor-induced lymphangiogenesis played anextremely important role in cancer cells spreading to somelocal lymph nodes and distant metastasis [23ndash25] Studieson antitumor lymphangiogenesis have gradually become aresearch hotspot and bFGF as an important factor to pro-mote lymphangiogenesis has been researched intensively

Gekko swinhonis Gunther was a traditional Chinesemedicine which has been used as an anticancer drug intraditionalChinesemedicine for hundreds of years especiallyin hepatoma [26] Soaking in alcohol is very useful forpharmaceutical ingredients dissolving out and is also themost common method of extraction Oral administrationand external usewere themainmethods of administration Inour previous study we isolated GSPP from Gekko swinhonisGunther and confirmed that GSPP could induce hepatomacell differentiation inhibit cell proliferation and migration invitro and inhibit hepatic carcinoma growth in vivo [12 1517] In this study we verified another mechanism of GSPPrsquosantitumor effects by inhibiting tumor lymphangiogenesisResults showed that GSPP significantly inhibited bFGF-induced human hLECs proliferation migration and tube-like structure formation in vitro Moreover GSPP treatment(200mgkgd) not only inhibited the growth of breast carci-noma but also inhibited the lymphangiogenesis in vivo Thedosage was determined by previous studies which is a safedose with an appropriate tumor inhibition rate [12] Through

BioMed Research International 7

Con

trol

GSP

P (m

gkg

)20

020

(a)

0

200

400

600

The v

olum

e of t

umor

s (m

m3)

10 14 18 22 246Days after tumor-bearing

Control

GSPP 200mgkgGSPP 20mgkg

(b)

00

01

02

03

The w

eigh

t of t

umor

s (g)

20 2000GSPP (mgkgd)

lowast

lowast

(c)

0

8

16

24

The w

eigh

t of m

ice (

g)

20 2000GSPP (mgkgd)

(d)

Control GSPP 200mgkgGSPP 20mgkg

(e)

Figure 4 GSPP inhibited colon carcinoma HT-29 xenograft growth and lymphangiogenesis in vivo Male nunu nude mice were inoculatedsubcutaneously with colon carcinoma HT-29 cells Three days after inoculation mice were treated with GSPP (20 or 200mgkg) or PBSevery day for 21 days via intraperitoneal injection The lengths and widths of tumors were measured individually every 3 days At the endof the experiment the implanted tumors were sectioned (a) Effect of GSPP on tumor volume Left image of excised tumors Right tumorgrowth curves (b) Effect of GSPP on tumor weight (c) Effect of GSPP onmouse weight Mice were weighed at the end of the experiment (d)Tumor lymphatic microvessel density The implanted tumors were sectioned and stained against LYNE-1 antibody Tumor lymphatic vesselsare shown as LYNE-1 positive (yellow color) Data were presented as mean plusmn SD of three independent experiments lowast119875 lt 005 versus controlgroup

this study we further uncovered the mechanisms of GSPPrsquosantitumor effects besides inhibiting cancer cells prolifer-ation and migration inducing tumor cell differentiationand inhibiting cancer angiogenesis and cancer-associatedfibroblast growth (unpublished) showing that GSPP is apromising antitumor drug in future cancer treatment

bFGF as an important prolymphangiogenesis factorgenerated by tumor cells can significantly promote lym-phatic vessel endothelial cell proliferation and migration and

promote tumor lymphangiogenesis by a variety of ways [27]The most classic way on which bFGF promotes tumor lym-phangiogenesis is through VEGF-A VEGF-C and VEGF-Dwhich are known prolymphangiogenesis factors to promotetumor lymphatic vessel grow [23 28] bFGF is a heparindependent growth factor which means that it can have effectonly in the form of bFGF-heparin-FGFR terpolymers struc-ture to further activate the intracellular signal transductionpathways GSPP is a kind of polysaccharide sulfate which

8 BioMed Research International

is similar to the structure of heparin active site both ofwhich contain a sulfuric acid base Previous study showedthat GSPP works by three distinct mechanisms (a) blockingthe bFGF production (b) inhibiting the release of bFGF fromthe extracellular matrix and (c) directly binding to bFGFand competitively inhibiting the binding of bFGF to its lowaffinity receptor heparinHS [12] In this study we found thatGSPP alone has no effect on the viability growth of hLECsAnd it does not inhibit the migration and tube formation ofhLECs whichmay be due to the absence of bFGF bFGF playsan important role in the growth migration and lymphan-giogenesis of hLECs With the addition of exogenous bFGFwhich simulates the environment in the body GSPP signifi-cantly inhibited bFGF-induced cell proliferation migrationand tube formation This means that GSPP works throughdirectly binding to bFGF and competitively inhibiting thebinding of bFGF to its low affinity receptor heparinHS

Extracellular signal-regulated kinase12 (Erk12) is a pro-tein kinase separated and identified in the early 1990s and itssignal transduction is involved in cell growth developmentand differentiation [19] Studies found that the promotingeffect of bFGF on endothelial cell proliferation andmigrationof part is associated with an increased level of Erk phos-phorylation [18 29] To explore the possible mechanism ofGSPPrsquos inhibiting effect of bFGF-induced lymphangiogene-sis western blot was performed We examined the Erk andp-Erk expression level changes in lymphatic endothelial cellsafter being exposed to GSPP for a certain time Our resultsshowed that bFGF significantly increased the expression ofp-Erk in hLECs However cotreatment with GSPP and bFGFdecreased the expression of p-Erk in hLECs compared withthe bFGF-single use group This further proved that GSPPcould bind to bFGF and competitively inhibit the bindingof bFGF to its low affinity receptor thus blocking the bFGF-induced phosphorylation of Erk12 in hLECs

5 Conclusions

Our study showed that inhibiting bFGF-induced lymphan-giogenesis was one of GSPPrsquos anticancer mechanisms GSPPas an effective bFGF-targeted inhibitor can be a notableantilymphatic metastasis drug in future cancer treatment

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Xiu-Li Ding and Ya-Nan Man contributed equally to thiswork

Acknowledgments

This study was supported by major scientific and technicalproject of Tianjin Science and Technology Commission(05YFGDSF02600) andNationalNatural Science Foundationof China (no 81173376 and no 81473441)

References

[1] J Robert ldquoBiology of cancermetastasisrdquoBulletin du Cancer vol100 no 4 pp 333ndash342 2013

[2] R Bicknell ldquoVascular targeting and the inhibition of angiogene-sisrdquoAnnals of Oncology vol 5 supplement 4 pp S45ndashS50 1994

[3] D Ribatti ldquoHistory of research on angiogenesisrdquo ChemicalImmunology and Allergy vol 99 pp 1ndash14 2014

[4] R Shayan M G Achen and S A Stacker ldquoLymphatic vesselsin cancer metastasis bridging the gapsrdquo Carcinogenesis vol 27no 9 pp 1729ndash1738 2006

[5] J P Sleeman and W Thiele ldquoTumor metastasis and the lym-phatic vasculaturerdquo International Journal of Cancer vol 125 no12 pp 2747ndash2756 2009

[6] R L Ferris M T Lotze S P L Leong D S B Hoon and D LMorton ldquoLymphatics lymph nodes and the immune systembarriers and gateways for cancer spreadrdquo Clinical and Experi-mental Metastasis vol 29 no 7 pp 729ndash736 2012

[7] S S Sundar and T S Ganesan ldquoRole of lymphangiogenesis incancerrdquo Journal of Clinical Oncology vol 25 no 27 pp 4298ndash4307 2007

[8] M G Achen B K McColl and S A Stacker ldquoFocus on lym-phangiogenesis in tumor metastasisrdquo Cancer Cell vol 7 no 2pp 121ndash127 2005

[9] R H Adams and K Alitalo ldquoMolecular regulation of angio-genesis and lymphangiogenesisrdquoNature Reviews Molecular CellBiology vol 8 no 6 pp 464ndash478 2007

[10] P M Linares and J P Gisbert ldquoRole of growth factors inthe development of lymphangiogenesis driven by inflammatorybowel disease a reviewrdquo Inflammatory Bowel Diseases vol 17no 8 pp 1814ndash1821 2011

[11] L K Chang G Garcia-Cardena F Farnebo et al ldquoDose-dependent response of FGF-2 for lymphangiogenesisrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 101 no 32 pp 11658ndash11663 2004

[12] S-X Zhang C Zhu Y Ba et al ldquoGekko-sulfated glycopeptideinhibits tumor angiogenesis by targeting basic fibroblast growthfactorrdquo The Journal of Biological Chemistry vol 287 no 16 pp13206ndash13215 2012

[13] M Benezra R Ishai-Michaeli S A Ben-Sasson and I Vlo-davsky ldquoStructure-activity relationships of heparin-mimickingcompounds in induction of bFGF release from extracellularmatrix and inhibition of smooth muscle cell proliferation andheparanase activityrdquo Journal of Cellular Physiology vol 192 no3 pp 276ndash285 2002

[14] M Fannon K E Forsten and M A Nugent ldquoPotentiation andinhibition of bFGF binding by heparin a model for regulationof cellular responserdquo Biochemistry vol 39 no 6 pp 1434ndash14452000

[15] D ChenW-J Yao X-L Zhang et al ldquoEffects of Gekko sulfatedpolysaccharide-protein complex on human hepatoma SMMC-7721 cells inhibition of proliferation and migrationrdquo Journal ofEthnopharmacology vol 127 no 3 pp 702ndash708 2010

[16] X Wu D Chen and G-R Xie ldquoEffects of Gekko sulfatedpolysaccharide on the proliferation and differentiation of hep-atic cancer cell linerdquoCell Biology International vol 30 no 8 pp659ndash664 2006

[17] X-Z Wu D Chen and X-Q Han ldquoAnti-migration effects ofGekko sulfated glycopeptide on human hepatoma SMMC-7721cellsrdquoMolecules vol 16 no 6 pp 4958ndash4970 2011

BioMed Research International 9

[18] L Yang J Zhang C Wang et al ldquoInteraction between 8-hydroxyquinoline ruthenium(II) complexes and basic fibrob-last growth factors (bFGF) inhibiting angiogenesis and tumorgrowth through ERK and AKT signaling pathwaysrdquo Metal-lomics vol 6 no 3 pp 518ndash531 2014

[19] K Balmanno and S J Cook ldquoTumour cell survival signallingby the ERK12 pathwayrdquo Cell Death and Differentiation vol 16no 3 pp 368ndash377 2009

[20] N E Tobler andMDetmar ldquoTumor and lymph node lymphan-giogenesismdashimpact on cancer metastasisrdquo Journal of LeukocyteBiology vol 80 no 4 pp 691ndash696 2006

[21] Y Cao ldquoOpinion emerging mechanisms of tumour lymphan-giogenesis and lymphatic metastasisrdquo Nature Reviews Cancervol 5 no 9 pp 735ndash743 2005

[22] K Alitalo T Tammela and T V Petrova ldquoLymphangiogenesisin development and human diseaserdquo Nature vol 438 no 7070pp 946ndash953 2005

[23] M Skobe T Hawighorst D G Jackson et al ldquoInduction oftumor lymphangiogenesis by VEGF-C promotes breast cancermetastasisrdquo Nature Medicine vol 7 no 2 pp 192ndash198 2001

[24] SA Stacker CCaesarM E Baldwin et al ldquoVEGF-Dpromotesthe metastatic spread of tumor cells via the lymphaticsrdquo NatureMedicine vol 7 no 2 pp 186ndash191 2001

[25] M S Pepper ldquoLymphangiogenesis and tumor metastasis mythor realityrdquo Clinical Cancer Research vol 7 no 3 pp 462ndash4682001

[26] C L Yang and Y J Qi Animal Drug of Chinese Tradi-tional Medicine Ancient Books Press of Chinese TraditionalMedicine Beijing China 1st edition 2001

[27] R Cao A Eriksson H Kubo K Alitalo Y Cao and JThyberg ldquoComparative evaluation of FGF-2- VEGF-A- andVEGF-C-induced angiogenesis lymphangiogenesis vascularfenestrations and permeabilityrdquo Circulation Research vol 94pp 664ndash670 2004

[28] M J Karkkainen R E Ferrell E C Lawrence et al ldquoMissensemutations interfere with VEGFR-3 signalling in primary lym-phoedemardquo Nature Genetics vol 25 no 2 pp 153ndash159 2000

[29] S Guo L Yu Y Cheng et al ldquoPDGFR120573 triggered by bFGF pro-motes the proliferation andmigration of endothelial progenitorcells via p-ERK signallingrdquo Cell Biology International vol 36no 10 pp 945ndash950 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

BioMed Research International 3

incubated at 37∘C in a tissue culture incubator and the forma-tion of the capillary-like tubes was observed after 4 h ThenhLECs were observed under inverted microscope and 9 pho-tographs (times40) were taken per hole The numbers of matrixform of closed irregular polygon were recorded and calcu-lated

28 Western Blot Experiment For western blot analysis ofErk and p-Erk protein expression confluent cultures ofhLECs in 6-hole pate were homogenized in lysis bufferThe protein concentrations were determined using the BCAProtein Quantitation Kit Equal amounts of lysate proteinwere subjected to 10 SDS-polyacrylamide gel electrophore-sis The proteins were transferred onto PVDF membranesfor immunoblot analysis Blocking was performed with 5nonfat dry milk in 01 Tween 20 in TBS followed byimmunoblotting with a polyclonal goat anti-human Erk anti-body p-Erk antibody and 120573-actin antibody Specific bindingwas detected by the ECL plus Western Blotting DetectionSystem

29 In Vivo Nude Mice Model This study was approved bythe Tianjin Cancer Institutional Animal ethics Committee(number 2014044) Animal care and experimental proce-dures followed the Tianjin Medical University guidelinesfor the care and use of laboratory animals 4ndash6-week-oldmale BALBc mice were purchased from LianHe LiHuacooperation To establish a heterotopic colon carcinoma nudemicemodel 25times 106HT-29 cells in 200 120583L PBSwere injectedinto the right flank of BALBc mice When tumors grew to10mmdiameter size tumors were cut off and cut into 1mm times1mm chips and then transplanted to other 24 BALBc micewhich were divided into four groups of 8 mice each groupMice were treated daily with an intraperitoneal injectionof either 01mL GSPP (20 200mgkgday in PBS) or PBS(control) for 21 daysThe length andwidth of the tumors weremeasured with a caliper every 2-3 days All mice were sacri-ficed 24 days after tumor inoculation and the tumors wereexcised and weighted and the tumor volumes were calculatedusing the standard formula 119881 = 11988611988722 (119886 lengths of thetumors 119887 widths of the tumors)

210 Immunohistochemistry Staining Tumor specimenswereimmediately removed from sacrificed mice and preparedfor immunohistological examination Tumors were fixed in10 (vv) neutral buffered formalin overnight embedded inparaffin and sectioned to a 5 120583m thickness Tumor sectionswere deparaffinized via immersion in xylene dehydrated ina graded series of ethanol and washed with distilled waterThereafter tumor sections were boiled in 10mM sodiumcitrate buffer (pH = 60) for 10min and cooled at room tem-perature To inhibit endogenous peroxidase activity tumorsections were incubated with methanol containing 1 (vv)hydrogen peroxide for 10min Tumor sections were thenblocked with 1 BSA and then incubated overnight withanti-LYVE-1 antibody tumor sections were probed withperoxidase-conjugated secondary antibodies and incubatedwith DAB until the desired stain intensity developed Aftercounterstaining with Harris hematoxylin tumor sections

were examined under an inverted microscope (E100 NikonJapan) To analyze immunohistochemical signals within thespecimens all tumor sections were digitized under a times40magnification and images were captured

211 Statistical Analysis The data were expressed as themean plusmn SD from triplicate experiments and were analyzedusing SPSS software (Version 200 Chicago USA) One-wayANOVA and two-way ANOVA were applied to analyze thesignificance of groupswith one factor and two factors respec-tively 119875 value lt005 was considered statistically significantAll of the experiments were repeated at least three times

3 Results

31 Morphological Characteristics of Lymphatic EndothelialCells InVitroCulture After 3ndash5 h of primary cell culture cellswere able to grow adhering to the wall After 1 d endothelialcells spread forming groups and after 1 week cells grewdensely and formed a single layer with the characteristicof ldquopebblesrdquo Inverted microscopy observation lymphaticendothelial cells were irregular ovoid with big nucleus andthere were many small vacuoles in the cytoplasm (Fig-ure 1(a))

32 GSPP Inhibited bFGF-Induced Proliferation of hLECsMTS assay was performed to investigate the antiproliferationeffect of GSPP After hLECs were exposed to GSPP (0 10 and100 120583gmL) andor bFGF (10 ngmL) for 0 1 2 3 4 5 or 6 dODvalueswere detectedThe results demonstrated that bFGFpromoted the proliferation of hLECs significantly (119875 lt 005)while GSPP alone did not inhibit proliferation of hLECs (119875 gt005) TrypanBlue staining showed that the viability of hLECswas not affected by GSPP (results were not shown) But GSPPcould abrogate bFGF-induced proliferation of hLECs signifi-cantly in a dose- and time-dependent manner (119875 lt 005 Fig-ure 1(b)) In the cell cycle assay bFGF significantly promotedhLECs into proliferation cycle with a high proportion of Sand G2 phase cells while GSPP alone has no effect on the cellcycle distribution of hLECsWhen combinedwith 100 120583gmLGSPP cell proliferative activity was blocked distinctly withan increasing proportion of G1 phase cells compared withbFGF alone group (Figure 1(d))

33 bFGF-Induced p-Erk Was Downregulated by GSPP inhLECs Erk12 signal pathway is reported to be involvedin cell growth migration and angiogenesis The promotingeffect of bFGF on vessel cell proliferation and migrationmay be partly associated with an increased level of Erkphosphorylation [18 19] To explore the possible mechanismof GSPP in inhibiting bFGF-induced lymphangiogenesis Erkand p-Erk protein expressions were detected by westernblotting Results showed that bFGF significantly increased theexpression of p-Erk in hLECs andGSPP decreased the bFGF-induced p-Erk significantly No significant difference of theexpression of total Erk was observed among each group(Figure 1(c))

4 BioMed Research International

(a)

05

10

15

20

OD

val

ue

2 3 6541Time (day)

ControlbFGF 10ngmLGSPP 10120583gmL

GSPP 100120583gmLGSPP 10120583gmL + bFGF 10ngmLGSPP 100120583gmL + bFGF 10ngmL

(b)

+ minus minus +

minus + minus +

minus

minus

minus minus minus + minus

+

minus

+

p-Erk

Erk

120573-actin10ngmL bFGF10120583gmL GSPP100120583gmL GSPP

(c)

(A) (B) (C)

(D) (E) (F)

930

4823

0

300

600

900

1200

Num

ber

0 8020 40 60 100

731

17297

0

400

800

1600

1200

30 60 90 1200

894

4661

0

600

1200

1800

2400

20 40 60 80 1000

946

3717

0100200300400500

Num

ber

1000 60 8020 40

Channels (FL2-A-PI-area)

722

23438

0

600

1200

1800

2400

20 40 60 80 1000

Channels (FL2-A-PI-area)

850

6783

02004006008001000

20 40 60 80 1000

Channels (FL2-A-PI-area)G1SG2

bFGF 10ngmLGSPP 10120583gmL

GSPP 100120583gmL

+ minus minus +

minus + minus +

minus

minus

minus minus minus + minus

+

minus

+

0

50

100

150(

of p

er p

hase

)

(d)

Figure 1 GSPP inhibited bFGF-induced proliferation of hLECs and Erk phosphorylation (a)Morphological characteristics of hLECs in vitroculture hLECs were compressed ovoid short fusiform or polygon and formed a single layer with the characteristic of ldquopebblesrdquo (b) hLECsgrowth curves (1 times 105 cellsmL) were incubated with different concentration of GSPP (0 10 and 100120583gmL) andor bFGF (10 ngmL) for 01 2 3 4 5 and 6 d then cell proliferation was quantified by MTS assay and cell growth curve was made (c) The changes of Erk and p-Erkprotein expression level of hLECs cultured in 6-well plate were incubated with different concentration of GSPP (0 10 and 100120583gmL) andorbFGF (10 ngmL) Erk and p-Erk protein levels were monitored by western blot analysis of whole-cell lysates (d) Cell cycle analysis Lefthistogram of cell cycle distribution Right statistical analysis of cell cycle percentage After exposure to GSPP (0 10 and 100120583gmL) andorbFGF (10 ngmL) for 48 h cell cycle distribution was determined by propidium iodide labeling (A) Control (B) bFGF 10 ngmL (C) GSPP10120583gmL (D) GSPP 100 120583gmL (E) GSPP 10 120583gmL + bFGF 10 ngmL and (F) GSPP 100 120583gmL + bFGF 10 ngmL Data were presented asmean plusmn SD of three independent experiments hLECs human lymphatic endothelial cells bFGF basic fibroblast growth factor GSPP GekkoSulfated Glycopeptide

BioMed Research International 5

(A) (B) (A) (B)

(C) (D) (C) (D)

(E) (F) (E) (F)

6h0h

(a)

00

05

10

15

20

The m

igra

tion

ratio

bFGF 10ngmLGSPP 10120583gmL

GSPP 100120583gmL

+ minus minus +

minus + minus +

minus

minus

minus minus minus + minus

+

minus

+

(b)

(A) (B) (C)

(D) (E) (F)

(c)

0

50

100

150

Num

ber o

f mig

rate

d ce

lls

bFGF 10ngmLGSPP 10120583gmL

GSPP 100120583gmL

+ minus minus +

minus + minus +

minus

minus

minus minus minus + minus

+

minus

+

(d)

Figure 2 GSPP inhibits bFGF-induced migration of hLECs (a) Wound healing assay Left representative images of injury width in woundhealing assay (times40) Right quantification of the migration ratio of hLECs compared to control hLECs (1 times 105 cellswell) were seeded in24-well plates and wounds were generated after cell confluence After hLECs were treated with different concentrations of GSPP (0 10 and100120583gmL) andor bFGF (10 ngmL) for 0 h and 6 h the photos were taken and the injury width was measured The migration ratio wascalculated as the migration width of experiment groupthe migration width of control group (b) Transwell assays Left representative imagesof migrated LECs in transwell assay (times40) Right quantification of migrated LECs compared to control hLECs (5 times 104 cellswell) in EBM-2with different concentrations of GSPP (0 10 and 100 120583gmL) were added to the upper chamber of the transwell insert EBM-2 containingbFGF (10 ngmL) or not was added to the lower chamber to induce cell migration After 12 h at 37∘C cells on the top surface of themembraneswere wiped off with cotton balls and the cells that migrated on the underside of inserts were fixed with methanol and stained with crystalviolet Five different digital images were taken per well and the number of migrated cells was counted (A) Control (B) bFGF 10 ngmL (C)GSPP 10120583gmL (D)GSPP 100120583gmL (E) GSPP 10 120583gmL+ bFGF 10 ngmL and (F) GSPP 100 120583gmL+ bFGF 10 ngmL Data were presentedas mean plusmn SD of three independent experiments ◻119875 lt 005 versus control group and 998810119875 lt 005 versus bFGF-single use group

34 GSPP Inhibited bFGF-Induced Migration of hLECsWound healing experiment and transwell experiment wereconducted to investigate the antimigration effect of GSPP Itwas showed that bFGF significantly upregulated the migra-tion distance of hLECs (119875 lt 005) which was antagonized byGSPP significantly (119875 lt 005) at 6 h (Figure 2(a)) The sameresults were obtained in transwell experimentThe number ofmigrated cells in bFGF-treated group increased significantlycompared to the negative control cells (119875 lt 005) No sig-nificant difference of the cell numbers was observed between

the GSPP-treated group and negative control (119875 gt 005)However concomitant treatment with 10 120583gmL 100 120583gmLGSPP and bFGF inhibited the migration of hLECs comparedwith the bFGF-single use group (119875 lt 005) (Figure 2(b))

35 GSPP Abrogated bFGF-Induced hLECs Lymphangiogen-esis In Vitro Tube-like formation assay was conducted toexamine the inhibitory effect of GSPP on lymphangiogenesishLECs were added on top of the gel in the 96-well plate incu-bated at 37∘C in a tissue culture incubator and the formation

6 BioMed Research International

(A) (B) (C)

(D) (E) (F)

(a)

bFGF 10ngmLGSPP 10120583gmL

GSPP 100120583gmL

+ minus minus +

minus + minus +

minus

minus

minus minus minus + minus

+

minus

+

0

20

40

60

The n

umbe

r of t

ubes

(b)

Figure 3 GSPP inhibits bFGF-induced lymphangiogenesis in vitro In vitro tube formation assay hLECs (15 times 104 cellswell) were seeded inMatrigel-coated 96-well plates and treated with different concentration of GSPP (0 10 and 100120583gmL) andor bFGF (10 ngmL) for 4 h andthe tube-like structure formation was observed (a) Representative images of tube formation (times40) (b) Quantification of inhibitory ratios oftube branches (A) Control (B) bFGF 10 ngmL (C) GSPP 10 120583gmL (D) GSPP 100120583gmL (E) GSPP 10 120583gmL + bFGF 10 ngmL and (F)GSPP 100 120583gmL + bFGF 10 ngmL Data were presented as mean plusmn SD of three independent experiments ◻119875 lt 005 versus control groupand 998810119875 lt 005 versus bFGF-single use group

of the capillary-like tubes was observed after 4 h There wasno difference in the number of tube-like structures formationbetween GSPP treatment group and the negative controlgroup in hLECs (119875 gt 005) The number of tube-likestructures treated with bFGF group was much more than thenegative control group (119875 lt 005) However GSPP signif-icantly attenuated bFGF-induced tube formation in a dose-dependent manner with simultaneous incubation with GSPPand bFGF (Figures 3(a) and 3(b))

36 GSPP Inhibited Tumor Growth and Lymphangiogenesis inHT-29 Colon Carcinoma Nude Mice Model The antitumoractivity of GSPP was investigated in HT-29 colon carcinomaxenograft model using BALBc nude mice Growth of thetumors was significantly inhibited in the mice treated withGSPP compared with the growth of tumors in control mice(Figure 4(a)) In control group (intraperitoneal injectionwithPBS) tumors grew rapidly and reached an average volume of45903 plusmn 2892mm3 (mean plusmn SD) by day 24 after being trans-plantedwithHT-29 tumor blocks while the sizes of tumors in20 and 200mgkgday GSPP-treated groups were only 34825plusmn 622mm3 and 25518 plusmn 6072mm3 respectively (759 and559 decrease) (119875 lt 001 Figure 4(b)) The tumor weightof the control group was 028 plusmn 003 g whereas the weightsof GSPP-treated groups decreased to 022 plusmn 004 g and 014plusmn 003 g respectively (119875 lt 001 Figure 4(c)) To evaluate theadverse effects of GSPP wemeasured the weights and visceralindex of the mice and found that there was no significantdifference between the control and GSPP-treated groups(Figure 4(d)) Tumor lymphangiogenesis was analyzedusing immunohistochemical staining with LYVE-1 antibodyResults showed that 200mgkgday GSPP markedly reducedtumor microvessel density in the tissue sections comparedwith the control group (Figure 4(e)) These results indicatedthat GSPP efficiently inhibits tumor growth in carcinoma

animal model Suppression of tumor growth due to GSPPcould be caused by inhibition of lymphangiogenesis

4 Discussion

Metastasis is a key cause for the failure of tumor treatmentand patient death The lymph node metastasis is the firststep of the tumor dissemination and is also the main signof poor prognosis of tumor [20ndash22] More and more studiesshowed that the tumor-induced lymphangiogenesis played anextremely important role in cancer cells spreading to somelocal lymph nodes and distant metastasis [23ndash25] Studieson antitumor lymphangiogenesis have gradually become aresearch hotspot and bFGF as an important factor to pro-mote lymphangiogenesis has been researched intensively

Gekko swinhonis Gunther was a traditional Chinesemedicine which has been used as an anticancer drug intraditionalChinesemedicine for hundreds of years especiallyin hepatoma [26] Soaking in alcohol is very useful forpharmaceutical ingredients dissolving out and is also themost common method of extraction Oral administrationand external usewere themainmethods of administration Inour previous study we isolated GSPP from Gekko swinhonisGunther and confirmed that GSPP could induce hepatomacell differentiation inhibit cell proliferation and migration invitro and inhibit hepatic carcinoma growth in vivo [12 1517] In this study we verified another mechanism of GSPPrsquosantitumor effects by inhibiting tumor lymphangiogenesisResults showed that GSPP significantly inhibited bFGF-induced human hLECs proliferation migration and tube-like structure formation in vitro Moreover GSPP treatment(200mgkgd) not only inhibited the growth of breast carci-noma but also inhibited the lymphangiogenesis in vivo Thedosage was determined by previous studies which is a safedose with an appropriate tumor inhibition rate [12] Through

BioMed Research International 7

Con

trol

GSP

P (m

gkg

)20

020

(a)

0

200

400

600

The v

olum

e of t

umor

s (m

m3)

10 14 18 22 246Days after tumor-bearing

Control

GSPP 200mgkgGSPP 20mgkg

(b)

00

01

02

03

The w

eigh

t of t

umor

s (g)

20 2000GSPP (mgkgd)

lowast

lowast

(c)

0

8

16

24

The w

eigh

t of m

ice (

g)

20 2000GSPP (mgkgd)

(d)

Control GSPP 200mgkgGSPP 20mgkg

(e)

Figure 4 GSPP inhibited colon carcinoma HT-29 xenograft growth and lymphangiogenesis in vivo Male nunu nude mice were inoculatedsubcutaneously with colon carcinoma HT-29 cells Three days after inoculation mice were treated with GSPP (20 or 200mgkg) or PBSevery day for 21 days via intraperitoneal injection The lengths and widths of tumors were measured individually every 3 days At the endof the experiment the implanted tumors were sectioned (a) Effect of GSPP on tumor volume Left image of excised tumors Right tumorgrowth curves (b) Effect of GSPP on tumor weight (c) Effect of GSPP onmouse weight Mice were weighed at the end of the experiment (d)Tumor lymphatic microvessel density The implanted tumors were sectioned and stained against LYNE-1 antibody Tumor lymphatic vesselsare shown as LYNE-1 positive (yellow color) Data were presented as mean plusmn SD of three independent experiments lowast119875 lt 005 versus controlgroup

this study we further uncovered the mechanisms of GSPPrsquosantitumor effects besides inhibiting cancer cells prolifer-ation and migration inducing tumor cell differentiationand inhibiting cancer angiogenesis and cancer-associatedfibroblast growth (unpublished) showing that GSPP is apromising antitumor drug in future cancer treatment

bFGF as an important prolymphangiogenesis factorgenerated by tumor cells can significantly promote lym-phatic vessel endothelial cell proliferation and migration and

promote tumor lymphangiogenesis by a variety of ways [27]The most classic way on which bFGF promotes tumor lym-phangiogenesis is through VEGF-A VEGF-C and VEGF-Dwhich are known prolymphangiogenesis factors to promotetumor lymphatic vessel grow [23 28] bFGF is a heparindependent growth factor which means that it can have effectonly in the form of bFGF-heparin-FGFR terpolymers struc-ture to further activate the intracellular signal transductionpathways GSPP is a kind of polysaccharide sulfate which

8 BioMed Research International

is similar to the structure of heparin active site both ofwhich contain a sulfuric acid base Previous study showedthat GSPP works by three distinct mechanisms (a) blockingthe bFGF production (b) inhibiting the release of bFGF fromthe extracellular matrix and (c) directly binding to bFGFand competitively inhibiting the binding of bFGF to its lowaffinity receptor heparinHS [12] In this study we found thatGSPP alone has no effect on the viability growth of hLECsAnd it does not inhibit the migration and tube formation ofhLECs whichmay be due to the absence of bFGF bFGF playsan important role in the growth migration and lymphan-giogenesis of hLECs With the addition of exogenous bFGFwhich simulates the environment in the body GSPP signifi-cantly inhibited bFGF-induced cell proliferation migrationand tube formation This means that GSPP works throughdirectly binding to bFGF and competitively inhibiting thebinding of bFGF to its low affinity receptor heparinHS

Extracellular signal-regulated kinase12 (Erk12) is a pro-tein kinase separated and identified in the early 1990s and itssignal transduction is involved in cell growth developmentand differentiation [19] Studies found that the promotingeffect of bFGF on endothelial cell proliferation andmigrationof part is associated with an increased level of Erk phos-phorylation [18 29] To explore the possible mechanism ofGSPPrsquos inhibiting effect of bFGF-induced lymphangiogene-sis western blot was performed We examined the Erk andp-Erk expression level changes in lymphatic endothelial cellsafter being exposed to GSPP for a certain time Our resultsshowed that bFGF significantly increased the expression ofp-Erk in hLECs However cotreatment with GSPP and bFGFdecreased the expression of p-Erk in hLECs compared withthe bFGF-single use group This further proved that GSPPcould bind to bFGF and competitively inhibit the bindingof bFGF to its low affinity receptor thus blocking the bFGF-induced phosphorylation of Erk12 in hLECs

5 Conclusions

Our study showed that inhibiting bFGF-induced lymphan-giogenesis was one of GSPPrsquos anticancer mechanisms GSPPas an effective bFGF-targeted inhibitor can be a notableantilymphatic metastasis drug in future cancer treatment

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Xiu-Li Ding and Ya-Nan Man contributed equally to thiswork

Acknowledgments

This study was supported by major scientific and technicalproject of Tianjin Science and Technology Commission(05YFGDSF02600) andNationalNatural Science Foundationof China (no 81173376 and no 81473441)

References

[1] J Robert ldquoBiology of cancermetastasisrdquoBulletin du Cancer vol100 no 4 pp 333ndash342 2013

[2] R Bicknell ldquoVascular targeting and the inhibition of angiogene-sisrdquoAnnals of Oncology vol 5 supplement 4 pp S45ndashS50 1994

[3] D Ribatti ldquoHistory of research on angiogenesisrdquo ChemicalImmunology and Allergy vol 99 pp 1ndash14 2014

[4] R Shayan M G Achen and S A Stacker ldquoLymphatic vesselsin cancer metastasis bridging the gapsrdquo Carcinogenesis vol 27no 9 pp 1729ndash1738 2006

[5] J P Sleeman and W Thiele ldquoTumor metastasis and the lym-phatic vasculaturerdquo International Journal of Cancer vol 125 no12 pp 2747ndash2756 2009

[6] R L Ferris M T Lotze S P L Leong D S B Hoon and D LMorton ldquoLymphatics lymph nodes and the immune systembarriers and gateways for cancer spreadrdquo Clinical and Experi-mental Metastasis vol 29 no 7 pp 729ndash736 2012

[7] S S Sundar and T S Ganesan ldquoRole of lymphangiogenesis incancerrdquo Journal of Clinical Oncology vol 25 no 27 pp 4298ndash4307 2007

[8] M G Achen B K McColl and S A Stacker ldquoFocus on lym-phangiogenesis in tumor metastasisrdquo Cancer Cell vol 7 no 2pp 121ndash127 2005

[9] R H Adams and K Alitalo ldquoMolecular regulation of angio-genesis and lymphangiogenesisrdquoNature Reviews Molecular CellBiology vol 8 no 6 pp 464ndash478 2007

[10] P M Linares and J P Gisbert ldquoRole of growth factors inthe development of lymphangiogenesis driven by inflammatorybowel disease a reviewrdquo Inflammatory Bowel Diseases vol 17no 8 pp 1814ndash1821 2011

[11] L K Chang G Garcia-Cardena F Farnebo et al ldquoDose-dependent response of FGF-2 for lymphangiogenesisrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 101 no 32 pp 11658ndash11663 2004

[12] S-X Zhang C Zhu Y Ba et al ldquoGekko-sulfated glycopeptideinhibits tumor angiogenesis by targeting basic fibroblast growthfactorrdquo The Journal of Biological Chemistry vol 287 no 16 pp13206ndash13215 2012

[13] M Benezra R Ishai-Michaeli S A Ben-Sasson and I Vlo-davsky ldquoStructure-activity relationships of heparin-mimickingcompounds in induction of bFGF release from extracellularmatrix and inhibition of smooth muscle cell proliferation andheparanase activityrdquo Journal of Cellular Physiology vol 192 no3 pp 276ndash285 2002

[14] M Fannon K E Forsten and M A Nugent ldquoPotentiation andinhibition of bFGF binding by heparin a model for regulationof cellular responserdquo Biochemistry vol 39 no 6 pp 1434ndash14452000

[15] D ChenW-J Yao X-L Zhang et al ldquoEffects of Gekko sulfatedpolysaccharide-protein complex on human hepatoma SMMC-7721 cells inhibition of proliferation and migrationrdquo Journal ofEthnopharmacology vol 127 no 3 pp 702ndash708 2010

[16] X Wu D Chen and G-R Xie ldquoEffects of Gekko sulfatedpolysaccharide on the proliferation and differentiation of hep-atic cancer cell linerdquoCell Biology International vol 30 no 8 pp659ndash664 2006

[17] X-Z Wu D Chen and X-Q Han ldquoAnti-migration effects ofGekko sulfated glycopeptide on human hepatoma SMMC-7721cellsrdquoMolecules vol 16 no 6 pp 4958ndash4970 2011

BioMed Research International 9

[18] L Yang J Zhang C Wang et al ldquoInteraction between 8-hydroxyquinoline ruthenium(II) complexes and basic fibrob-last growth factors (bFGF) inhibiting angiogenesis and tumorgrowth through ERK and AKT signaling pathwaysrdquo Metal-lomics vol 6 no 3 pp 518ndash531 2014

[19] K Balmanno and S J Cook ldquoTumour cell survival signallingby the ERK12 pathwayrdquo Cell Death and Differentiation vol 16no 3 pp 368ndash377 2009

[20] N E Tobler andMDetmar ldquoTumor and lymph node lymphan-giogenesismdashimpact on cancer metastasisrdquo Journal of LeukocyteBiology vol 80 no 4 pp 691ndash696 2006

[21] Y Cao ldquoOpinion emerging mechanisms of tumour lymphan-giogenesis and lymphatic metastasisrdquo Nature Reviews Cancervol 5 no 9 pp 735ndash743 2005

[22] K Alitalo T Tammela and T V Petrova ldquoLymphangiogenesisin development and human diseaserdquo Nature vol 438 no 7070pp 946ndash953 2005

[23] M Skobe T Hawighorst D G Jackson et al ldquoInduction oftumor lymphangiogenesis by VEGF-C promotes breast cancermetastasisrdquo Nature Medicine vol 7 no 2 pp 192ndash198 2001

[24] SA Stacker CCaesarM E Baldwin et al ldquoVEGF-Dpromotesthe metastatic spread of tumor cells via the lymphaticsrdquo NatureMedicine vol 7 no 2 pp 186ndash191 2001

[25] M S Pepper ldquoLymphangiogenesis and tumor metastasis mythor realityrdquo Clinical Cancer Research vol 7 no 3 pp 462ndash4682001

[26] C L Yang and Y J Qi Animal Drug of Chinese Tradi-tional Medicine Ancient Books Press of Chinese TraditionalMedicine Beijing China 1st edition 2001

[27] R Cao A Eriksson H Kubo K Alitalo Y Cao and JThyberg ldquoComparative evaluation of FGF-2- VEGF-A- andVEGF-C-induced angiogenesis lymphangiogenesis vascularfenestrations and permeabilityrdquo Circulation Research vol 94pp 664ndash670 2004

[28] M J Karkkainen R E Ferrell E C Lawrence et al ldquoMissensemutations interfere with VEGFR-3 signalling in primary lym-phoedemardquo Nature Genetics vol 25 no 2 pp 153ndash159 2000

[29] S Guo L Yu Y Cheng et al ldquoPDGFR120573 triggered by bFGF pro-motes the proliferation andmigration of endothelial progenitorcells via p-ERK signallingrdquo Cell Biology International vol 36no 10 pp 945ndash950 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 BioMed Research International

(a)

05

10

15

20

OD

val

ue

2 3 6541Time (day)

ControlbFGF 10ngmLGSPP 10120583gmL

GSPP 100120583gmLGSPP 10120583gmL + bFGF 10ngmLGSPP 100120583gmL + bFGF 10ngmL

(b)

+ minus minus +

minus + minus +

minus

minus

minus minus minus + minus

+

minus

+

p-Erk

Erk

120573-actin10ngmL bFGF10120583gmL GSPP100120583gmL GSPP

(c)

(A) (B) (C)

(D) (E) (F)

930

4823

0

300

600

900

1200

Num

ber

0 8020 40 60 100

731

17297

0

400

800

1600

1200

30 60 90 1200

894

4661

0

600

1200

1800

2400

20 40 60 80 1000

946

3717

0100200300400500

Num

ber

1000 60 8020 40

Channels (FL2-A-PI-area)

722

23438

0

600

1200

1800

2400

20 40 60 80 1000

Channels (FL2-A-PI-area)

850

6783

02004006008001000

20 40 60 80 1000

Channels (FL2-A-PI-area)G1SG2

bFGF 10ngmLGSPP 10120583gmL

GSPP 100120583gmL

+ minus minus +

minus + minus +

minus

minus

minus minus minus + minus

+

minus

+

0

50

100

150(

of p

er p

hase

)

(d)

Figure 1 GSPP inhibited bFGF-induced proliferation of hLECs and Erk phosphorylation (a)Morphological characteristics of hLECs in vitroculture hLECs were compressed ovoid short fusiform or polygon and formed a single layer with the characteristic of ldquopebblesrdquo (b) hLECsgrowth curves (1 times 105 cellsmL) were incubated with different concentration of GSPP (0 10 and 100120583gmL) andor bFGF (10 ngmL) for 01 2 3 4 5 and 6 d then cell proliferation was quantified by MTS assay and cell growth curve was made (c) The changes of Erk and p-Erkprotein expression level of hLECs cultured in 6-well plate were incubated with different concentration of GSPP (0 10 and 100120583gmL) andorbFGF (10 ngmL) Erk and p-Erk protein levels were monitored by western blot analysis of whole-cell lysates (d) Cell cycle analysis Lefthistogram of cell cycle distribution Right statistical analysis of cell cycle percentage After exposure to GSPP (0 10 and 100120583gmL) andorbFGF (10 ngmL) for 48 h cell cycle distribution was determined by propidium iodide labeling (A) Control (B) bFGF 10 ngmL (C) GSPP10120583gmL (D) GSPP 100 120583gmL (E) GSPP 10 120583gmL + bFGF 10 ngmL and (F) GSPP 100 120583gmL + bFGF 10 ngmL Data were presented asmean plusmn SD of three independent experiments hLECs human lymphatic endothelial cells bFGF basic fibroblast growth factor GSPP GekkoSulfated Glycopeptide

BioMed Research International 5

(A) (B) (A) (B)

(C) (D) (C) (D)

(E) (F) (E) (F)

6h0h

(a)

00

05

10

15

20

The m

igra

tion

ratio

bFGF 10ngmLGSPP 10120583gmL

GSPP 100120583gmL

+ minus minus +

minus + minus +

minus

minus

minus minus minus + minus

+

minus

+

(b)

(A) (B) (C)

(D) (E) (F)

(c)

0

50

100

150

Num

ber o

f mig

rate

d ce

lls

bFGF 10ngmLGSPP 10120583gmL

GSPP 100120583gmL

+ minus minus +

minus + minus +

minus

minus

minus minus minus + minus

+

minus

+

(d)

Figure 2 GSPP inhibits bFGF-induced migration of hLECs (a) Wound healing assay Left representative images of injury width in woundhealing assay (times40) Right quantification of the migration ratio of hLECs compared to control hLECs (1 times 105 cellswell) were seeded in24-well plates and wounds were generated after cell confluence After hLECs were treated with different concentrations of GSPP (0 10 and100120583gmL) andor bFGF (10 ngmL) for 0 h and 6 h the photos were taken and the injury width was measured The migration ratio wascalculated as the migration width of experiment groupthe migration width of control group (b) Transwell assays Left representative imagesof migrated LECs in transwell assay (times40) Right quantification of migrated LECs compared to control hLECs (5 times 104 cellswell) in EBM-2with different concentrations of GSPP (0 10 and 100 120583gmL) were added to the upper chamber of the transwell insert EBM-2 containingbFGF (10 ngmL) or not was added to the lower chamber to induce cell migration After 12 h at 37∘C cells on the top surface of themembraneswere wiped off with cotton balls and the cells that migrated on the underside of inserts were fixed with methanol and stained with crystalviolet Five different digital images were taken per well and the number of migrated cells was counted (A) Control (B) bFGF 10 ngmL (C)GSPP 10120583gmL (D)GSPP 100120583gmL (E) GSPP 10 120583gmL+ bFGF 10 ngmL and (F) GSPP 100 120583gmL+ bFGF 10 ngmL Data were presentedas mean plusmn SD of three independent experiments ◻119875 lt 005 versus control group and 998810119875 lt 005 versus bFGF-single use group

34 GSPP Inhibited bFGF-Induced Migration of hLECsWound healing experiment and transwell experiment wereconducted to investigate the antimigration effect of GSPP Itwas showed that bFGF significantly upregulated the migra-tion distance of hLECs (119875 lt 005) which was antagonized byGSPP significantly (119875 lt 005) at 6 h (Figure 2(a)) The sameresults were obtained in transwell experimentThe number ofmigrated cells in bFGF-treated group increased significantlycompared to the negative control cells (119875 lt 005) No sig-nificant difference of the cell numbers was observed between

the GSPP-treated group and negative control (119875 gt 005)However concomitant treatment with 10 120583gmL 100 120583gmLGSPP and bFGF inhibited the migration of hLECs comparedwith the bFGF-single use group (119875 lt 005) (Figure 2(b))

35 GSPP Abrogated bFGF-Induced hLECs Lymphangiogen-esis In Vitro Tube-like formation assay was conducted toexamine the inhibitory effect of GSPP on lymphangiogenesishLECs were added on top of the gel in the 96-well plate incu-bated at 37∘C in a tissue culture incubator and the formation

6 BioMed Research International

(A) (B) (C)

(D) (E) (F)

(a)

bFGF 10ngmLGSPP 10120583gmL

GSPP 100120583gmL

+ minus minus +

minus + minus +

minus

minus

minus minus minus + minus

+

minus

+

0

20

40

60

The n

umbe

r of t

ubes

(b)

Figure 3 GSPP inhibits bFGF-induced lymphangiogenesis in vitro In vitro tube formation assay hLECs (15 times 104 cellswell) were seeded inMatrigel-coated 96-well plates and treated with different concentration of GSPP (0 10 and 100120583gmL) andor bFGF (10 ngmL) for 4 h andthe tube-like structure formation was observed (a) Representative images of tube formation (times40) (b) Quantification of inhibitory ratios oftube branches (A) Control (B) bFGF 10 ngmL (C) GSPP 10 120583gmL (D) GSPP 100120583gmL (E) GSPP 10 120583gmL + bFGF 10 ngmL and (F)GSPP 100 120583gmL + bFGF 10 ngmL Data were presented as mean plusmn SD of three independent experiments ◻119875 lt 005 versus control groupand 998810119875 lt 005 versus bFGF-single use group

of the capillary-like tubes was observed after 4 h There wasno difference in the number of tube-like structures formationbetween GSPP treatment group and the negative controlgroup in hLECs (119875 gt 005) The number of tube-likestructures treated with bFGF group was much more than thenegative control group (119875 lt 005) However GSPP signif-icantly attenuated bFGF-induced tube formation in a dose-dependent manner with simultaneous incubation with GSPPand bFGF (Figures 3(a) and 3(b))

36 GSPP Inhibited Tumor Growth and Lymphangiogenesis inHT-29 Colon Carcinoma Nude Mice Model The antitumoractivity of GSPP was investigated in HT-29 colon carcinomaxenograft model using BALBc nude mice Growth of thetumors was significantly inhibited in the mice treated withGSPP compared with the growth of tumors in control mice(Figure 4(a)) In control group (intraperitoneal injectionwithPBS) tumors grew rapidly and reached an average volume of45903 plusmn 2892mm3 (mean plusmn SD) by day 24 after being trans-plantedwithHT-29 tumor blocks while the sizes of tumors in20 and 200mgkgday GSPP-treated groups were only 34825plusmn 622mm3 and 25518 plusmn 6072mm3 respectively (759 and559 decrease) (119875 lt 001 Figure 4(b)) The tumor weightof the control group was 028 plusmn 003 g whereas the weightsof GSPP-treated groups decreased to 022 plusmn 004 g and 014plusmn 003 g respectively (119875 lt 001 Figure 4(c)) To evaluate theadverse effects of GSPP wemeasured the weights and visceralindex of the mice and found that there was no significantdifference between the control and GSPP-treated groups(Figure 4(d)) Tumor lymphangiogenesis was analyzedusing immunohistochemical staining with LYVE-1 antibodyResults showed that 200mgkgday GSPP markedly reducedtumor microvessel density in the tissue sections comparedwith the control group (Figure 4(e)) These results indicatedthat GSPP efficiently inhibits tumor growth in carcinoma

animal model Suppression of tumor growth due to GSPPcould be caused by inhibition of lymphangiogenesis

4 Discussion

Metastasis is a key cause for the failure of tumor treatmentand patient death The lymph node metastasis is the firststep of the tumor dissemination and is also the main signof poor prognosis of tumor [20ndash22] More and more studiesshowed that the tumor-induced lymphangiogenesis played anextremely important role in cancer cells spreading to somelocal lymph nodes and distant metastasis [23ndash25] Studieson antitumor lymphangiogenesis have gradually become aresearch hotspot and bFGF as an important factor to pro-mote lymphangiogenesis has been researched intensively

Gekko swinhonis Gunther was a traditional Chinesemedicine which has been used as an anticancer drug intraditionalChinesemedicine for hundreds of years especiallyin hepatoma [26] Soaking in alcohol is very useful forpharmaceutical ingredients dissolving out and is also themost common method of extraction Oral administrationand external usewere themainmethods of administration Inour previous study we isolated GSPP from Gekko swinhonisGunther and confirmed that GSPP could induce hepatomacell differentiation inhibit cell proliferation and migration invitro and inhibit hepatic carcinoma growth in vivo [12 1517] In this study we verified another mechanism of GSPPrsquosantitumor effects by inhibiting tumor lymphangiogenesisResults showed that GSPP significantly inhibited bFGF-induced human hLECs proliferation migration and tube-like structure formation in vitro Moreover GSPP treatment(200mgkgd) not only inhibited the growth of breast carci-noma but also inhibited the lymphangiogenesis in vivo Thedosage was determined by previous studies which is a safedose with an appropriate tumor inhibition rate [12] Through

BioMed Research International 7

Con

trol

GSP

P (m

gkg

)20

020

(a)

0

200

400

600

The v

olum

e of t

umor

s (m

m3)

10 14 18 22 246Days after tumor-bearing

Control

GSPP 200mgkgGSPP 20mgkg

(b)

00

01

02

03

The w

eigh

t of t

umor

s (g)

20 2000GSPP (mgkgd)

lowast

lowast

(c)

0

8

16

24

The w

eigh

t of m

ice (

g)

20 2000GSPP (mgkgd)

(d)

Control GSPP 200mgkgGSPP 20mgkg

(e)

Figure 4 GSPP inhibited colon carcinoma HT-29 xenograft growth and lymphangiogenesis in vivo Male nunu nude mice were inoculatedsubcutaneously with colon carcinoma HT-29 cells Three days after inoculation mice were treated with GSPP (20 or 200mgkg) or PBSevery day for 21 days via intraperitoneal injection The lengths and widths of tumors were measured individually every 3 days At the endof the experiment the implanted tumors were sectioned (a) Effect of GSPP on tumor volume Left image of excised tumors Right tumorgrowth curves (b) Effect of GSPP on tumor weight (c) Effect of GSPP onmouse weight Mice were weighed at the end of the experiment (d)Tumor lymphatic microvessel density The implanted tumors were sectioned and stained against LYNE-1 antibody Tumor lymphatic vesselsare shown as LYNE-1 positive (yellow color) Data were presented as mean plusmn SD of three independent experiments lowast119875 lt 005 versus controlgroup

this study we further uncovered the mechanisms of GSPPrsquosantitumor effects besides inhibiting cancer cells prolifer-ation and migration inducing tumor cell differentiationand inhibiting cancer angiogenesis and cancer-associatedfibroblast growth (unpublished) showing that GSPP is apromising antitumor drug in future cancer treatment

bFGF as an important prolymphangiogenesis factorgenerated by tumor cells can significantly promote lym-phatic vessel endothelial cell proliferation and migration and

promote tumor lymphangiogenesis by a variety of ways [27]The most classic way on which bFGF promotes tumor lym-phangiogenesis is through VEGF-A VEGF-C and VEGF-Dwhich are known prolymphangiogenesis factors to promotetumor lymphatic vessel grow [23 28] bFGF is a heparindependent growth factor which means that it can have effectonly in the form of bFGF-heparin-FGFR terpolymers struc-ture to further activate the intracellular signal transductionpathways GSPP is a kind of polysaccharide sulfate which

8 BioMed Research International

is similar to the structure of heparin active site both ofwhich contain a sulfuric acid base Previous study showedthat GSPP works by three distinct mechanisms (a) blockingthe bFGF production (b) inhibiting the release of bFGF fromthe extracellular matrix and (c) directly binding to bFGFand competitively inhibiting the binding of bFGF to its lowaffinity receptor heparinHS [12] In this study we found thatGSPP alone has no effect on the viability growth of hLECsAnd it does not inhibit the migration and tube formation ofhLECs whichmay be due to the absence of bFGF bFGF playsan important role in the growth migration and lymphan-giogenesis of hLECs With the addition of exogenous bFGFwhich simulates the environment in the body GSPP signifi-cantly inhibited bFGF-induced cell proliferation migrationand tube formation This means that GSPP works throughdirectly binding to bFGF and competitively inhibiting thebinding of bFGF to its low affinity receptor heparinHS

Extracellular signal-regulated kinase12 (Erk12) is a pro-tein kinase separated and identified in the early 1990s and itssignal transduction is involved in cell growth developmentand differentiation [19] Studies found that the promotingeffect of bFGF on endothelial cell proliferation andmigrationof part is associated with an increased level of Erk phos-phorylation [18 29] To explore the possible mechanism ofGSPPrsquos inhibiting effect of bFGF-induced lymphangiogene-sis western blot was performed We examined the Erk andp-Erk expression level changes in lymphatic endothelial cellsafter being exposed to GSPP for a certain time Our resultsshowed that bFGF significantly increased the expression ofp-Erk in hLECs However cotreatment with GSPP and bFGFdecreased the expression of p-Erk in hLECs compared withthe bFGF-single use group This further proved that GSPPcould bind to bFGF and competitively inhibit the bindingof bFGF to its low affinity receptor thus blocking the bFGF-induced phosphorylation of Erk12 in hLECs

5 Conclusions

Our study showed that inhibiting bFGF-induced lymphan-giogenesis was one of GSPPrsquos anticancer mechanisms GSPPas an effective bFGF-targeted inhibitor can be a notableantilymphatic metastasis drug in future cancer treatment

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Xiu-Li Ding and Ya-Nan Man contributed equally to thiswork

Acknowledgments

This study was supported by major scientific and technicalproject of Tianjin Science and Technology Commission(05YFGDSF02600) andNationalNatural Science Foundationof China (no 81173376 and no 81473441)

References

[1] J Robert ldquoBiology of cancermetastasisrdquoBulletin du Cancer vol100 no 4 pp 333ndash342 2013

[2] R Bicknell ldquoVascular targeting and the inhibition of angiogene-sisrdquoAnnals of Oncology vol 5 supplement 4 pp S45ndashS50 1994

[3] D Ribatti ldquoHistory of research on angiogenesisrdquo ChemicalImmunology and Allergy vol 99 pp 1ndash14 2014

[4] R Shayan M G Achen and S A Stacker ldquoLymphatic vesselsin cancer metastasis bridging the gapsrdquo Carcinogenesis vol 27no 9 pp 1729ndash1738 2006

[5] J P Sleeman and W Thiele ldquoTumor metastasis and the lym-phatic vasculaturerdquo International Journal of Cancer vol 125 no12 pp 2747ndash2756 2009

[6] R L Ferris M T Lotze S P L Leong D S B Hoon and D LMorton ldquoLymphatics lymph nodes and the immune systembarriers and gateways for cancer spreadrdquo Clinical and Experi-mental Metastasis vol 29 no 7 pp 729ndash736 2012

[7] S S Sundar and T S Ganesan ldquoRole of lymphangiogenesis incancerrdquo Journal of Clinical Oncology vol 25 no 27 pp 4298ndash4307 2007

[8] M G Achen B K McColl and S A Stacker ldquoFocus on lym-phangiogenesis in tumor metastasisrdquo Cancer Cell vol 7 no 2pp 121ndash127 2005

[9] R H Adams and K Alitalo ldquoMolecular regulation of angio-genesis and lymphangiogenesisrdquoNature Reviews Molecular CellBiology vol 8 no 6 pp 464ndash478 2007

[10] P M Linares and J P Gisbert ldquoRole of growth factors inthe development of lymphangiogenesis driven by inflammatorybowel disease a reviewrdquo Inflammatory Bowel Diseases vol 17no 8 pp 1814ndash1821 2011

[11] L K Chang G Garcia-Cardena F Farnebo et al ldquoDose-dependent response of FGF-2 for lymphangiogenesisrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 101 no 32 pp 11658ndash11663 2004

[12] S-X Zhang C Zhu Y Ba et al ldquoGekko-sulfated glycopeptideinhibits tumor angiogenesis by targeting basic fibroblast growthfactorrdquo The Journal of Biological Chemistry vol 287 no 16 pp13206ndash13215 2012

[13] M Benezra R Ishai-Michaeli S A Ben-Sasson and I Vlo-davsky ldquoStructure-activity relationships of heparin-mimickingcompounds in induction of bFGF release from extracellularmatrix and inhibition of smooth muscle cell proliferation andheparanase activityrdquo Journal of Cellular Physiology vol 192 no3 pp 276ndash285 2002

[14] M Fannon K E Forsten and M A Nugent ldquoPotentiation andinhibition of bFGF binding by heparin a model for regulationof cellular responserdquo Biochemistry vol 39 no 6 pp 1434ndash14452000

[15] D ChenW-J Yao X-L Zhang et al ldquoEffects of Gekko sulfatedpolysaccharide-protein complex on human hepatoma SMMC-7721 cells inhibition of proliferation and migrationrdquo Journal ofEthnopharmacology vol 127 no 3 pp 702ndash708 2010

[16] X Wu D Chen and G-R Xie ldquoEffects of Gekko sulfatedpolysaccharide on the proliferation and differentiation of hep-atic cancer cell linerdquoCell Biology International vol 30 no 8 pp659ndash664 2006

[17] X-Z Wu D Chen and X-Q Han ldquoAnti-migration effects ofGekko sulfated glycopeptide on human hepatoma SMMC-7721cellsrdquoMolecules vol 16 no 6 pp 4958ndash4970 2011

BioMed Research International 9

[18] L Yang J Zhang C Wang et al ldquoInteraction between 8-hydroxyquinoline ruthenium(II) complexes and basic fibrob-last growth factors (bFGF) inhibiting angiogenesis and tumorgrowth through ERK and AKT signaling pathwaysrdquo Metal-lomics vol 6 no 3 pp 518ndash531 2014

[19] K Balmanno and S J Cook ldquoTumour cell survival signallingby the ERK12 pathwayrdquo Cell Death and Differentiation vol 16no 3 pp 368ndash377 2009

[20] N E Tobler andMDetmar ldquoTumor and lymph node lymphan-giogenesismdashimpact on cancer metastasisrdquo Journal of LeukocyteBiology vol 80 no 4 pp 691ndash696 2006

[21] Y Cao ldquoOpinion emerging mechanisms of tumour lymphan-giogenesis and lymphatic metastasisrdquo Nature Reviews Cancervol 5 no 9 pp 735ndash743 2005

[22] K Alitalo T Tammela and T V Petrova ldquoLymphangiogenesisin development and human diseaserdquo Nature vol 438 no 7070pp 946ndash953 2005

[23] M Skobe T Hawighorst D G Jackson et al ldquoInduction oftumor lymphangiogenesis by VEGF-C promotes breast cancermetastasisrdquo Nature Medicine vol 7 no 2 pp 192ndash198 2001

[24] SA Stacker CCaesarM E Baldwin et al ldquoVEGF-Dpromotesthe metastatic spread of tumor cells via the lymphaticsrdquo NatureMedicine vol 7 no 2 pp 186ndash191 2001

[25] M S Pepper ldquoLymphangiogenesis and tumor metastasis mythor realityrdquo Clinical Cancer Research vol 7 no 3 pp 462ndash4682001

[26] C L Yang and Y J Qi Animal Drug of Chinese Tradi-tional Medicine Ancient Books Press of Chinese TraditionalMedicine Beijing China 1st edition 2001

[27] R Cao A Eriksson H Kubo K Alitalo Y Cao and JThyberg ldquoComparative evaluation of FGF-2- VEGF-A- andVEGF-C-induced angiogenesis lymphangiogenesis vascularfenestrations and permeabilityrdquo Circulation Research vol 94pp 664ndash670 2004

[28] M J Karkkainen R E Ferrell E C Lawrence et al ldquoMissensemutations interfere with VEGFR-3 signalling in primary lym-phoedemardquo Nature Genetics vol 25 no 2 pp 153ndash159 2000

[29] S Guo L Yu Y Cheng et al ldquoPDGFR120573 triggered by bFGF pro-motes the proliferation andmigration of endothelial progenitorcells via p-ERK signallingrdquo Cell Biology International vol 36no 10 pp 945ndash950 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

BioMed Research International 5

(A) (B) (A) (B)

(C) (D) (C) (D)

(E) (F) (E) (F)

6h0h

(a)

00

05

10

15

20

The m

igra

tion

ratio

bFGF 10ngmLGSPP 10120583gmL

GSPP 100120583gmL

+ minus minus +

minus + minus +

minus

minus

minus minus minus + minus

+

minus

+

(b)

(A) (B) (C)

(D) (E) (F)

(c)

0

50

100

150

Num

ber o

f mig

rate

d ce

lls

bFGF 10ngmLGSPP 10120583gmL

GSPP 100120583gmL

+ minus minus +

minus + minus +

minus

minus

minus minus minus + minus

+

minus

+

(d)

Figure 2 GSPP inhibits bFGF-induced migration of hLECs (a) Wound healing assay Left representative images of injury width in woundhealing assay (times40) Right quantification of the migration ratio of hLECs compared to control hLECs (1 times 105 cellswell) were seeded in24-well plates and wounds were generated after cell confluence After hLECs were treated with different concentrations of GSPP (0 10 and100120583gmL) andor bFGF (10 ngmL) for 0 h and 6 h the photos were taken and the injury width was measured The migration ratio wascalculated as the migration width of experiment groupthe migration width of control group (b) Transwell assays Left representative imagesof migrated LECs in transwell assay (times40) Right quantification of migrated LECs compared to control hLECs (5 times 104 cellswell) in EBM-2with different concentrations of GSPP (0 10 and 100 120583gmL) were added to the upper chamber of the transwell insert EBM-2 containingbFGF (10 ngmL) or not was added to the lower chamber to induce cell migration After 12 h at 37∘C cells on the top surface of themembraneswere wiped off with cotton balls and the cells that migrated on the underside of inserts were fixed with methanol and stained with crystalviolet Five different digital images were taken per well and the number of migrated cells was counted (A) Control (B) bFGF 10 ngmL (C)GSPP 10120583gmL (D)GSPP 100120583gmL (E) GSPP 10 120583gmL+ bFGF 10 ngmL and (F) GSPP 100 120583gmL+ bFGF 10 ngmL Data were presentedas mean plusmn SD of three independent experiments ◻119875 lt 005 versus control group and 998810119875 lt 005 versus bFGF-single use group

34 GSPP Inhibited bFGF-Induced Migration of hLECsWound healing experiment and transwell experiment wereconducted to investigate the antimigration effect of GSPP Itwas showed that bFGF significantly upregulated the migra-tion distance of hLECs (119875 lt 005) which was antagonized byGSPP significantly (119875 lt 005) at 6 h (Figure 2(a)) The sameresults were obtained in transwell experimentThe number ofmigrated cells in bFGF-treated group increased significantlycompared to the negative control cells (119875 lt 005) No sig-nificant difference of the cell numbers was observed between

the GSPP-treated group and negative control (119875 gt 005)However concomitant treatment with 10 120583gmL 100 120583gmLGSPP and bFGF inhibited the migration of hLECs comparedwith the bFGF-single use group (119875 lt 005) (Figure 2(b))

35 GSPP Abrogated bFGF-Induced hLECs Lymphangiogen-esis In Vitro Tube-like formation assay was conducted toexamine the inhibitory effect of GSPP on lymphangiogenesishLECs were added on top of the gel in the 96-well plate incu-bated at 37∘C in a tissue culture incubator and the formation

6 BioMed Research International

(A) (B) (C)

(D) (E) (F)

(a)

bFGF 10ngmLGSPP 10120583gmL

GSPP 100120583gmL

+ minus minus +

minus + minus +

minus

minus

minus minus minus + minus

+

minus

+

0

20

40

60

The n

umbe

r of t

ubes

(b)

Figure 3 GSPP inhibits bFGF-induced lymphangiogenesis in vitro In vitro tube formation assay hLECs (15 times 104 cellswell) were seeded inMatrigel-coated 96-well plates and treated with different concentration of GSPP (0 10 and 100120583gmL) andor bFGF (10 ngmL) for 4 h andthe tube-like structure formation was observed (a) Representative images of tube formation (times40) (b) Quantification of inhibitory ratios oftube branches (A) Control (B) bFGF 10 ngmL (C) GSPP 10 120583gmL (D) GSPP 100120583gmL (E) GSPP 10 120583gmL + bFGF 10 ngmL and (F)GSPP 100 120583gmL + bFGF 10 ngmL Data were presented as mean plusmn SD of three independent experiments ◻119875 lt 005 versus control groupand 998810119875 lt 005 versus bFGF-single use group

of the capillary-like tubes was observed after 4 h There wasno difference in the number of tube-like structures formationbetween GSPP treatment group and the negative controlgroup in hLECs (119875 gt 005) The number of tube-likestructures treated with bFGF group was much more than thenegative control group (119875 lt 005) However GSPP signif-icantly attenuated bFGF-induced tube formation in a dose-dependent manner with simultaneous incubation with GSPPand bFGF (Figures 3(a) and 3(b))

36 GSPP Inhibited Tumor Growth and Lymphangiogenesis inHT-29 Colon Carcinoma Nude Mice Model The antitumoractivity of GSPP was investigated in HT-29 colon carcinomaxenograft model using BALBc nude mice Growth of thetumors was significantly inhibited in the mice treated withGSPP compared with the growth of tumors in control mice(Figure 4(a)) In control group (intraperitoneal injectionwithPBS) tumors grew rapidly and reached an average volume of45903 plusmn 2892mm3 (mean plusmn SD) by day 24 after being trans-plantedwithHT-29 tumor blocks while the sizes of tumors in20 and 200mgkgday GSPP-treated groups were only 34825plusmn 622mm3 and 25518 plusmn 6072mm3 respectively (759 and559 decrease) (119875 lt 001 Figure 4(b)) The tumor weightof the control group was 028 plusmn 003 g whereas the weightsof GSPP-treated groups decreased to 022 plusmn 004 g and 014plusmn 003 g respectively (119875 lt 001 Figure 4(c)) To evaluate theadverse effects of GSPP wemeasured the weights and visceralindex of the mice and found that there was no significantdifference between the control and GSPP-treated groups(Figure 4(d)) Tumor lymphangiogenesis was analyzedusing immunohistochemical staining with LYVE-1 antibodyResults showed that 200mgkgday GSPP markedly reducedtumor microvessel density in the tissue sections comparedwith the control group (Figure 4(e)) These results indicatedthat GSPP efficiently inhibits tumor growth in carcinoma

animal model Suppression of tumor growth due to GSPPcould be caused by inhibition of lymphangiogenesis

4 Discussion

Metastasis is a key cause for the failure of tumor treatmentand patient death The lymph node metastasis is the firststep of the tumor dissemination and is also the main signof poor prognosis of tumor [20ndash22] More and more studiesshowed that the tumor-induced lymphangiogenesis played anextremely important role in cancer cells spreading to somelocal lymph nodes and distant metastasis [23ndash25] Studieson antitumor lymphangiogenesis have gradually become aresearch hotspot and bFGF as an important factor to pro-mote lymphangiogenesis has been researched intensively

Gekko swinhonis Gunther was a traditional Chinesemedicine which has been used as an anticancer drug intraditionalChinesemedicine for hundreds of years especiallyin hepatoma [26] Soaking in alcohol is very useful forpharmaceutical ingredients dissolving out and is also themost common method of extraction Oral administrationand external usewere themainmethods of administration Inour previous study we isolated GSPP from Gekko swinhonisGunther and confirmed that GSPP could induce hepatomacell differentiation inhibit cell proliferation and migration invitro and inhibit hepatic carcinoma growth in vivo [12 1517] In this study we verified another mechanism of GSPPrsquosantitumor effects by inhibiting tumor lymphangiogenesisResults showed that GSPP significantly inhibited bFGF-induced human hLECs proliferation migration and tube-like structure formation in vitro Moreover GSPP treatment(200mgkgd) not only inhibited the growth of breast carci-noma but also inhibited the lymphangiogenesis in vivo Thedosage was determined by previous studies which is a safedose with an appropriate tumor inhibition rate [12] Through

BioMed Research International 7

Con

trol

GSP

P (m

gkg

)20

020

(a)

0

200

400

600

The v

olum

e of t

umor

s (m

m3)

10 14 18 22 246Days after tumor-bearing

Control

GSPP 200mgkgGSPP 20mgkg

(b)

00

01

02

03

The w

eigh

t of t

umor

s (g)

20 2000GSPP (mgkgd)

lowast

lowast

(c)

0

8

16

24

The w

eigh

t of m

ice (

g)

20 2000GSPP (mgkgd)

(d)

Control GSPP 200mgkgGSPP 20mgkg

(e)

Figure 4 GSPP inhibited colon carcinoma HT-29 xenograft growth and lymphangiogenesis in vivo Male nunu nude mice were inoculatedsubcutaneously with colon carcinoma HT-29 cells Three days after inoculation mice were treated with GSPP (20 or 200mgkg) or PBSevery day for 21 days via intraperitoneal injection The lengths and widths of tumors were measured individually every 3 days At the endof the experiment the implanted tumors were sectioned (a) Effect of GSPP on tumor volume Left image of excised tumors Right tumorgrowth curves (b) Effect of GSPP on tumor weight (c) Effect of GSPP onmouse weight Mice were weighed at the end of the experiment (d)Tumor lymphatic microvessel density The implanted tumors were sectioned and stained against LYNE-1 antibody Tumor lymphatic vesselsare shown as LYNE-1 positive (yellow color) Data were presented as mean plusmn SD of three independent experiments lowast119875 lt 005 versus controlgroup

this study we further uncovered the mechanisms of GSPPrsquosantitumor effects besides inhibiting cancer cells prolifer-ation and migration inducing tumor cell differentiationand inhibiting cancer angiogenesis and cancer-associatedfibroblast growth (unpublished) showing that GSPP is apromising antitumor drug in future cancer treatment

bFGF as an important prolymphangiogenesis factorgenerated by tumor cells can significantly promote lym-phatic vessel endothelial cell proliferation and migration and

promote tumor lymphangiogenesis by a variety of ways [27]The most classic way on which bFGF promotes tumor lym-phangiogenesis is through VEGF-A VEGF-C and VEGF-Dwhich are known prolymphangiogenesis factors to promotetumor lymphatic vessel grow [23 28] bFGF is a heparindependent growth factor which means that it can have effectonly in the form of bFGF-heparin-FGFR terpolymers struc-ture to further activate the intracellular signal transductionpathways GSPP is a kind of polysaccharide sulfate which

8 BioMed Research International

is similar to the structure of heparin active site both ofwhich contain a sulfuric acid base Previous study showedthat GSPP works by three distinct mechanisms (a) blockingthe bFGF production (b) inhibiting the release of bFGF fromthe extracellular matrix and (c) directly binding to bFGFand competitively inhibiting the binding of bFGF to its lowaffinity receptor heparinHS [12] In this study we found thatGSPP alone has no effect on the viability growth of hLECsAnd it does not inhibit the migration and tube formation ofhLECs whichmay be due to the absence of bFGF bFGF playsan important role in the growth migration and lymphan-giogenesis of hLECs With the addition of exogenous bFGFwhich simulates the environment in the body GSPP signifi-cantly inhibited bFGF-induced cell proliferation migrationand tube formation This means that GSPP works throughdirectly binding to bFGF and competitively inhibiting thebinding of bFGF to its low affinity receptor heparinHS

Extracellular signal-regulated kinase12 (Erk12) is a pro-tein kinase separated and identified in the early 1990s and itssignal transduction is involved in cell growth developmentand differentiation [19] Studies found that the promotingeffect of bFGF on endothelial cell proliferation andmigrationof part is associated with an increased level of Erk phos-phorylation [18 29] To explore the possible mechanism ofGSPPrsquos inhibiting effect of bFGF-induced lymphangiogene-sis western blot was performed We examined the Erk andp-Erk expression level changes in lymphatic endothelial cellsafter being exposed to GSPP for a certain time Our resultsshowed that bFGF significantly increased the expression ofp-Erk in hLECs However cotreatment with GSPP and bFGFdecreased the expression of p-Erk in hLECs compared withthe bFGF-single use group This further proved that GSPPcould bind to bFGF and competitively inhibit the bindingof bFGF to its low affinity receptor thus blocking the bFGF-induced phosphorylation of Erk12 in hLECs

5 Conclusions

Our study showed that inhibiting bFGF-induced lymphan-giogenesis was one of GSPPrsquos anticancer mechanisms GSPPas an effective bFGF-targeted inhibitor can be a notableantilymphatic metastasis drug in future cancer treatment

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Xiu-Li Ding and Ya-Nan Man contributed equally to thiswork

Acknowledgments

This study was supported by major scientific and technicalproject of Tianjin Science and Technology Commission(05YFGDSF02600) andNationalNatural Science Foundationof China (no 81173376 and no 81473441)

References

[1] J Robert ldquoBiology of cancermetastasisrdquoBulletin du Cancer vol100 no 4 pp 333ndash342 2013

[2] R Bicknell ldquoVascular targeting and the inhibition of angiogene-sisrdquoAnnals of Oncology vol 5 supplement 4 pp S45ndashS50 1994

[3] D Ribatti ldquoHistory of research on angiogenesisrdquo ChemicalImmunology and Allergy vol 99 pp 1ndash14 2014

[4] R Shayan M G Achen and S A Stacker ldquoLymphatic vesselsin cancer metastasis bridging the gapsrdquo Carcinogenesis vol 27no 9 pp 1729ndash1738 2006

[5] J P Sleeman and W Thiele ldquoTumor metastasis and the lym-phatic vasculaturerdquo International Journal of Cancer vol 125 no12 pp 2747ndash2756 2009

[6] R L Ferris M T Lotze S P L Leong D S B Hoon and D LMorton ldquoLymphatics lymph nodes and the immune systembarriers and gateways for cancer spreadrdquo Clinical and Experi-mental Metastasis vol 29 no 7 pp 729ndash736 2012

[7] S S Sundar and T S Ganesan ldquoRole of lymphangiogenesis incancerrdquo Journal of Clinical Oncology vol 25 no 27 pp 4298ndash4307 2007

[8] M G Achen B K McColl and S A Stacker ldquoFocus on lym-phangiogenesis in tumor metastasisrdquo Cancer Cell vol 7 no 2pp 121ndash127 2005

[9] R H Adams and K Alitalo ldquoMolecular regulation of angio-genesis and lymphangiogenesisrdquoNature Reviews Molecular CellBiology vol 8 no 6 pp 464ndash478 2007

[10] P M Linares and J P Gisbert ldquoRole of growth factors inthe development of lymphangiogenesis driven by inflammatorybowel disease a reviewrdquo Inflammatory Bowel Diseases vol 17no 8 pp 1814ndash1821 2011

[11] L K Chang G Garcia-Cardena F Farnebo et al ldquoDose-dependent response of FGF-2 for lymphangiogenesisrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 101 no 32 pp 11658ndash11663 2004

[12] S-X Zhang C Zhu Y Ba et al ldquoGekko-sulfated glycopeptideinhibits tumor angiogenesis by targeting basic fibroblast growthfactorrdquo The Journal of Biological Chemistry vol 287 no 16 pp13206ndash13215 2012

[13] M Benezra R Ishai-Michaeli S A Ben-Sasson and I Vlo-davsky ldquoStructure-activity relationships of heparin-mimickingcompounds in induction of bFGF release from extracellularmatrix and inhibition of smooth muscle cell proliferation andheparanase activityrdquo Journal of Cellular Physiology vol 192 no3 pp 276ndash285 2002

[14] M Fannon K E Forsten and M A Nugent ldquoPotentiation andinhibition of bFGF binding by heparin a model for regulationof cellular responserdquo Biochemistry vol 39 no 6 pp 1434ndash14452000

[15] D ChenW-J Yao X-L Zhang et al ldquoEffects of Gekko sulfatedpolysaccharide-protein complex on human hepatoma SMMC-7721 cells inhibition of proliferation and migrationrdquo Journal ofEthnopharmacology vol 127 no 3 pp 702ndash708 2010

[16] X Wu D Chen and G-R Xie ldquoEffects of Gekko sulfatedpolysaccharide on the proliferation and differentiation of hep-atic cancer cell linerdquoCell Biology International vol 30 no 8 pp659ndash664 2006

[17] X-Z Wu D Chen and X-Q Han ldquoAnti-migration effects ofGekko sulfated glycopeptide on human hepatoma SMMC-7721cellsrdquoMolecules vol 16 no 6 pp 4958ndash4970 2011

BioMed Research International 9

[18] L Yang J Zhang C Wang et al ldquoInteraction between 8-hydroxyquinoline ruthenium(II) complexes and basic fibrob-last growth factors (bFGF) inhibiting angiogenesis and tumorgrowth through ERK and AKT signaling pathwaysrdquo Metal-lomics vol 6 no 3 pp 518ndash531 2014

[19] K Balmanno and S J Cook ldquoTumour cell survival signallingby the ERK12 pathwayrdquo Cell Death and Differentiation vol 16no 3 pp 368ndash377 2009

[20] N E Tobler andMDetmar ldquoTumor and lymph node lymphan-giogenesismdashimpact on cancer metastasisrdquo Journal of LeukocyteBiology vol 80 no 4 pp 691ndash696 2006

[21] Y Cao ldquoOpinion emerging mechanisms of tumour lymphan-giogenesis and lymphatic metastasisrdquo Nature Reviews Cancervol 5 no 9 pp 735ndash743 2005

[22] K Alitalo T Tammela and T V Petrova ldquoLymphangiogenesisin development and human diseaserdquo Nature vol 438 no 7070pp 946ndash953 2005

[23] M Skobe T Hawighorst D G Jackson et al ldquoInduction oftumor lymphangiogenesis by VEGF-C promotes breast cancermetastasisrdquo Nature Medicine vol 7 no 2 pp 192ndash198 2001

[24] SA Stacker CCaesarM E Baldwin et al ldquoVEGF-Dpromotesthe metastatic spread of tumor cells via the lymphaticsrdquo NatureMedicine vol 7 no 2 pp 186ndash191 2001

[25] M S Pepper ldquoLymphangiogenesis and tumor metastasis mythor realityrdquo Clinical Cancer Research vol 7 no 3 pp 462ndash4682001

[26] C L Yang and Y J Qi Animal Drug of Chinese Tradi-tional Medicine Ancient Books Press of Chinese TraditionalMedicine Beijing China 1st edition 2001

[27] R Cao A Eriksson H Kubo K Alitalo Y Cao and JThyberg ldquoComparative evaluation of FGF-2- VEGF-A- andVEGF-C-induced angiogenesis lymphangiogenesis vascularfenestrations and permeabilityrdquo Circulation Research vol 94pp 664ndash670 2004

[28] M J Karkkainen R E Ferrell E C Lawrence et al ldquoMissensemutations interfere with VEGFR-3 signalling in primary lym-phoedemardquo Nature Genetics vol 25 no 2 pp 153ndash159 2000

[29] S Guo L Yu Y Cheng et al ldquoPDGFR120573 triggered by bFGF pro-motes the proliferation andmigration of endothelial progenitorcells via p-ERK signallingrdquo Cell Biology International vol 36no 10 pp 945ndash950 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 BioMed Research International

(A) (B) (C)

(D) (E) (F)

(a)

bFGF 10ngmLGSPP 10120583gmL

GSPP 100120583gmL

+ minus minus +

minus + minus +

minus

minus

minus minus minus + minus

+

minus

+

0

20

40

60

The n

umbe

r of t

ubes

(b)

Figure 3 GSPP inhibits bFGF-induced lymphangiogenesis in vitro In vitro tube formation assay hLECs (15 times 104 cellswell) were seeded inMatrigel-coated 96-well plates and treated with different concentration of GSPP (0 10 and 100120583gmL) andor bFGF (10 ngmL) for 4 h andthe tube-like structure formation was observed (a) Representative images of tube formation (times40) (b) Quantification of inhibitory ratios oftube branches (A) Control (B) bFGF 10 ngmL (C) GSPP 10 120583gmL (D) GSPP 100120583gmL (E) GSPP 10 120583gmL + bFGF 10 ngmL and (F)GSPP 100 120583gmL + bFGF 10 ngmL Data were presented as mean plusmn SD of three independent experiments ◻119875 lt 005 versus control groupand 998810119875 lt 005 versus bFGF-single use group

of the capillary-like tubes was observed after 4 h There wasno difference in the number of tube-like structures formationbetween GSPP treatment group and the negative controlgroup in hLECs (119875 gt 005) The number of tube-likestructures treated with bFGF group was much more than thenegative control group (119875 lt 005) However GSPP signif-icantly attenuated bFGF-induced tube formation in a dose-dependent manner with simultaneous incubation with GSPPand bFGF (Figures 3(a) and 3(b))

36 GSPP Inhibited Tumor Growth and Lymphangiogenesis inHT-29 Colon Carcinoma Nude Mice Model The antitumoractivity of GSPP was investigated in HT-29 colon carcinomaxenograft model using BALBc nude mice Growth of thetumors was significantly inhibited in the mice treated withGSPP compared with the growth of tumors in control mice(Figure 4(a)) In control group (intraperitoneal injectionwithPBS) tumors grew rapidly and reached an average volume of45903 plusmn 2892mm3 (mean plusmn SD) by day 24 after being trans-plantedwithHT-29 tumor blocks while the sizes of tumors in20 and 200mgkgday GSPP-treated groups were only 34825plusmn 622mm3 and 25518 plusmn 6072mm3 respectively (759 and559 decrease) (119875 lt 001 Figure 4(b)) The tumor weightof the control group was 028 plusmn 003 g whereas the weightsof GSPP-treated groups decreased to 022 plusmn 004 g and 014plusmn 003 g respectively (119875 lt 001 Figure 4(c)) To evaluate theadverse effects of GSPP wemeasured the weights and visceralindex of the mice and found that there was no significantdifference between the control and GSPP-treated groups(Figure 4(d)) Tumor lymphangiogenesis was analyzedusing immunohistochemical staining with LYVE-1 antibodyResults showed that 200mgkgday GSPP markedly reducedtumor microvessel density in the tissue sections comparedwith the control group (Figure 4(e)) These results indicatedthat GSPP efficiently inhibits tumor growth in carcinoma

animal model Suppression of tumor growth due to GSPPcould be caused by inhibition of lymphangiogenesis

4 Discussion

Metastasis is a key cause for the failure of tumor treatmentand patient death The lymph node metastasis is the firststep of the tumor dissemination and is also the main signof poor prognosis of tumor [20ndash22] More and more studiesshowed that the tumor-induced lymphangiogenesis played anextremely important role in cancer cells spreading to somelocal lymph nodes and distant metastasis [23ndash25] Studieson antitumor lymphangiogenesis have gradually become aresearch hotspot and bFGF as an important factor to pro-mote lymphangiogenesis has been researched intensively

Gekko swinhonis Gunther was a traditional Chinesemedicine which has been used as an anticancer drug intraditionalChinesemedicine for hundreds of years especiallyin hepatoma [26] Soaking in alcohol is very useful forpharmaceutical ingredients dissolving out and is also themost common method of extraction Oral administrationand external usewere themainmethods of administration Inour previous study we isolated GSPP from Gekko swinhonisGunther and confirmed that GSPP could induce hepatomacell differentiation inhibit cell proliferation and migration invitro and inhibit hepatic carcinoma growth in vivo [12 1517] In this study we verified another mechanism of GSPPrsquosantitumor effects by inhibiting tumor lymphangiogenesisResults showed that GSPP significantly inhibited bFGF-induced human hLECs proliferation migration and tube-like structure formation in vitro Moreover GSPP treatment(200mgkgd) not only inhibited the growth of breast carci-noma but also inhibited the lymphangiogenesis in vivo Thedosage was determined by previous studies which is a safedose with an appropriate tumor inhibition rate [12] Through

BioMed Research International 7

Con

trol

GSP

P (m

gkg

)20

020

(a)

0

200

400

600

The v

olum

e of t

umor

s (m

m3)

10 14 18 22 246Days after tumor-bearing

Control

GSPP 200mgkgGSPP 20mgkg

(b)

00

01

02

03

The w

eigh

t of t

umor

s (g)

20 2000GSPP (mgkgd)

lowast

lowast

(c)

0

8

16

24

The w

eigh

t of m

ice (

g)

20 2000GSPP (mgkgd)

(d)

Control GSPP 200mgkgGSPP 20mgkg

(e)

Figure 4 GSPP inhibited colon carcinoma HT-29 xenograft growth and lymphangiogenesis in vivo Male nunu nude mice were inoculatedsubcutaneously with colon carcinoma HT-29 cells Three days after inoculation mice were treated with GSPP (20 or 200mgkg) or PBSevery day for 21 days via intraperitoneal injection The lengths and widths of tumors were measured individually every 3 days At the endof the experiment the implanted tumors were sectioned (a) Effect of GSPP on tumor volume Left image of excised tumors Right tumorgrowth curves (b) Effect of GSPP on tumor weight (c) Effect of GSPP onmouse weight Mice were weighed at the end of the experiment (d)Tumor lymphatic microvessel density The implanted tumors were sectioned and stained against LYNE-1 antibody Tumor lymphatic vesselsare shown as LYNE-1 positive (yellow color) Data were presented as mean plusmn SD of three independent experiments lowast119875 lt 005 versus controlgroup

this study we further uncovered the mechanisms of GSPPrsquosantitumor effects besides inhibiting cancer cells prolifer-ation and migration inducing tumor cell differentiationand inhibiting cancer angiogenesis and cancer-associatedfibroblast growth (unpublished) showing that GSPP is apromising antitumor drug in future cancer treatment

bFGF as an important prolymphangiogenesis factorgenerated by tumor cells can significantly promote lym-phatic vessel endothelial cell proliferation and migration and

promote tumor lymphangiogenesis by a variety of ways [27]The most classic way on which bFGF promotes tumor lym-phangiogenesis is through VEGF-A VEGF-C and VEGF-Dwhich are known prolymphangiogenesis factors to promotetumor lymphatic vessel grow [23 28] bFGF is a heparindependent growth factor which means that it can have effectonly in the form of bFGF-heparin-FGFR terpolymers struc-ture to further activate the intracellular signal transductionpathways GSPP is a kind of polysaccharide sulfate which

8 BioMed Research International

is similar to the structure of heparin active site both ofwhich contain a sulfuric acid base Previous study showedthat GSPP works by three distinct mechanisms (a) blockingthe bFGF production (b) inhibiting the release of bFGF fromthe extracellular matrix and (c) directly binding to bFGFand competitively inhibiting the binding of bFGF to its lowaffinity receptor heparinHS [12] In this study we found thatGSPP alone has no effect on the viability growth of hLECsAnd it does not inhibit the migration and tube formation ofhLECs whichmay be due to the absence of bFGF bFGF playsan important role in the growth migration and lymphan-giogenesis of hLECs With the addition of exogenous bFGFwhich simulates the environment in the body GSPP signifi-cantly inhibited bFGF-induced cell proliferation migrationand tube formation This means that GSPP works throughdirectly binding to bFGF and competitively inhibiting thebinding of bFGF to its low affinity receptor heparinHS

Extracellular signal-regulated kinase12 (Erk12) is a pro-tein kinase separated and identified in the early 1990s and itssignal transduction is involved in cell growth developmentand differentiation [19] Studies found that the promotingeffect of bFGF on endothelial cell proliferation andmigrationof part is associated with an increased level of Erk phos-phorylation [18 29] To explore the possible mechanism ofGSPPrsquos inhibiting effect of bFGF-induced lymphangiogene-sis western blot was performed We examined the Erk andp-Erk expression level changes in lymphatic endothelial cellsafter being exposed to GSPP for a certain time Our resultsshowed that bFGF significantly increased the expression ofp-Erk in hLECs However cotreatment with GSPP and bFGFdecreased the expression of p-Erk in hLECs compared withthe bFGF-single use group This further proved that GSPPcould bind to bFGF and competitively inhibit the bindingof bFGF to its low affinity receptor thus blocking the bFGF-induced phosphorylation of Erk12 in hLECs

5 Conclusions

Our study showed that inhibiting bFGF-induced lymphan-giogenesis was one of GSPPrsquos anticancer mechanisms GSPPas an effective bFGF-targeted inhibitor can be a notableantilymphatic metastasis drug in future cancer treatment

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Xiu-Li Ding and Ya-Nan Man contributed equally to thiswork

Acknowledgments

This study was supported by major scientific and technicalproject of Tianjin Science and Technology Commission(05YFGDSF02600) andNationalNatural Science Foundationof China (no 81173376 and no 81473441)

References

[1] J Robert ldquoBiology of cancermetastasisrdquoBulletin du Cancer vol100 no 4 pp 333ndash342 2013

[2] R Bicknell ldquoVascular targeting and the inhibition of angiogene-sisrdquoAnnals of Oncology vol 5 supplement 4 pp S45ndashS50 1994

[3] D Ribatti ldquoHistory of research on angiogenesisrdquo ChemicalImmunology and Allergy vol 99 pp 1ndash14 2014

[4] R Shayan M G Achen and S A Stacker ldquoLymphatic vesselsin cancer metastasis bridging the gapsrdquo Carcinogenesis vol 27no 9 pp 1729ndash1738 2006

[5] J P Sleeman and W Thiele ldquoTumor metastasis and the lym-phatic vasculaturerdquo International Journal of Cancer vol 125 no12 pp 2747ndash2756 2009

[6] R L Ferris M T Lotze S P L Leong D S B Hoon and D LMorton ldquoLymphatics lymph nodes and the immune systembarriers and gateways for cancer spreadrdquo Clinical and Experi-mental Metastasis vol 29 no 7 pp 729ndash736 2012

[7] S S Sundar and T S Ganesan ldquoRole of lymphangiogenesis incancerrdquo Journal of Clinical Oncology vol 25 no 27 pp 4298ndash4307 2007

[8] M G Achen B K McColl and S A Stacker ldquoFocus on lym-phangiogenesis in tumor metastasisrdquo Cancer Cell vol 7 no 2pp 121ndash127 2005

[9] R H Adams and K Alitalo ldquoMolecular regulation of angio-genesis and lymphangiogenesisrdquoNature Reviews Molecular CellBiology vol 8 no 6 pp 464ndash478 2007

[10] P M Linares and J P Gisbert ldquoRole of growth factors inthe development of lymphangiogenesis driven by inflammatorybowel disease a reviewrdquo Inflammatory Bowel Diseases vol 17no 8 pp 1814ndash1821 2011

[11] L K Chang G Garcia-Cardena F Farnebo et al ldquoDose-dependent response of FGF-2 for lymphangiogenesisrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 101 no 32 pp 11658ndash11663 2004

[12] S-X Zhang C Zhu Y Ba et al ldquoGekko-sulfated glycopeptideinhibits tumor angiogenesis by targeting basic fibroblast growthfactorrdquo The Journal of Biological Chemistry vol 287 no 16 pp13206ndash13215 2012

[13] M Benezra R Ishai-Michaeli S A Ben-Sasson and I Vlo-davsky ldquoStructure-activity relationships of heparin-mimickingcompounds in induction of bFGF release from extracellularmatrix and inhibition of smooth muscle cell proliferation andheparanase activityrdquo Journal of Cellular Physiology vol 192 no3 pp 276ndash285 2002

[14] M Fannon K E Forsten and M A Nugent ldquoPotentiation andinhibition of bFGF binding by heparin a model for regulationof cellular responserdquo Biochemistry vol 39 no 6 pp 1434ndash14452000

[15] D ChenW-J Yao X-L Zhang et al ldquoEffects of Gekko sulfatedpolysaccharide-protein complex on human hepatoma SMMC-7721 cells inhibition of proliferation and migrationrdquo Journal ofEthnopharmacology vol 127 no 3 pp 702ndash708 2010

[16] X Wu D Chen and G-R Xie ldquoEffects of Gekko sulfatedpolysaccharide on the proliferation and differentiation of hep-atic cancer cell linerdquoCell Biology International vol 30 no 8 pp659ndash664 2006

[17] X-Z Wu D Chen and X-Q Han ldquoAnti-migration effects ofGekko sulfated glycopeptide on human hepatoma SMMC-7721cellsrdquoMolecules vol 16 no 6 pp 4958ndash4970 2011

BioMed Research International 9

[18] L Yang J Zhang C Wang et al ldquoInteraction between 8-hydroxyquinoline ruthenium(II) complexes and basic fibrob-last growth factors (bFGF) inhibiting angiogenesis and tumorgrowth through ERK and AKT signaling pathwaysrdquo Metal-lomics vol 6 no 3 pp 518ndash531 2014

[19] K Balmanno and S J Cook ldquoTumour cell survival signallingby the ERK12 pathwayrdquo Cell Death and Differentiation vol 16no 3 pp 368ndash377 2009

[20] N E Tobler andMDetmar ldquoTumor and lymph node lymphan-giogenesismdashimpact on cancer metastasisrdquo Journal of LeukocyteBiology vol 80 no 4 pp 691ndash696 2006

[21] Y Cao ldquoOpinion emerging mechanisms of tumour lymphan-giogenesis and lymphatic metastasisrdquo Nature Reviews Cancervol 5 no 9 pp 735ndash743 2005

[22] K Alitalo T Tammela and T V Petrova ldquoLymphangiogenesisin development and human diseaserdquo Nature vol 438 no 7070pp 946ndash953 2005

[23] M Skobe T Hawighorst D G Jackson et al ldquoInduction oftumor lymphangiogenesis by VEGF-C promotes breast cancermetastasisrdquo Nature Medicine vol 7 no 2 pp 192ndash198 2001

[24] SA Stacker CCaesarM E Baldwin et al ldquoVEGF-Dpromotesthe metastatic spread of tumor cells via the lymphaticsrdquo NatureMedicine vol 7 no 2 pp 186ndash191 2001

[25] M S Pepper ldquoLymphangiogenesis and tumor metastasis mythor realityrdquo Clinical Cancer Research vol 7 no 3 pp 462ndash4682001

[26] C L Yang and Y J Qi Animal Drug of Chinese Tradi-tional Medicine Ancient Books Press of Chinese TraditionalMedicine Beijing China 1st edition 2001

[27] R Cao A Eriksson H Kubo K Alitalo Y Cao and JThyberg ldquoComparative evaluation of FGF-2- VEGF-A- andVEGF-C-induced angiogenesis lymphangiogenesis vascularfenestrations and permeabilityrdquo Circulation Research vol 94pp 664ndash670 2004

[28] M J Karkkainen R E Ferrell E C Lawrence et al ldquoMissensemutations interfere with VEGFR-3 signalling in primary lym-phoedemardquo Nature Genetics vol 25 no 2 pp 153ndash159 2000

[29] S Guo L Yu Y Cheng et al ldquoPDGFR120573 triggered by bFGF pro-motes the proliferation andmigration of endothelial progenitorcells via p-ERK signallingrdquo Cell Biology International vol 36no 10 pp 945ndash950 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

BioMed Research International 7

Con

trol

GSP

P (m

gkg

)20

020

(a)

0

200

400

600

The v

olum

e of t

umor

s (m

m3)

10 14 18 22 246Days after tumor-bearing

Control

GSPP 200mgkgGSPP 20mgkg

(b)

00

01

02

03

The w

eigh

t of t

umor

s (g)

20 2000GSPP (mgkgd)

lowast

lowast

(c)

0

8

16

24

The w

eigh

t of m

ice (

g)

20 2000GSPP (mgkgd)

(d)

Control GSPP 200mgkgGSPP 20mgkg

(e)

Figure 4 GSPP inhibited colon carcinoma HT-29 xenograft growth and lymphangiogenesis in vivo Male nunu nude mice were inoculatedsubcutaneously with colon carcinoma HT-29 cells Three days after inoculation mice were treated with GSPP (20 or 200mgkg) or PBSevery day for 21 days via intraperitoneal injection The lengths and widths of tumors were measured individually every 3 days At the endof the experiment the implanted tumors were sectioned (a) Effect of GSPP on tumor volume Left image of excised tumors Right tumorgrowth curves (b) Effect of GSPP on tumor weight (c) Effect of GSPP onmouse weight Mice were weighed at the end of the experiment (d)Tumor lymphatic microvessel density The implanted tumors were sectioned and stained against LYNE-1 antibody Tumor lymphatic vesselsare shown as LYNE-1 positive (yellow color) Data were presented as mean plusmn SD of three independent experiments lowast119875 lt 005 versus controlgroup

this study we further uncovered the mechanisms of GSPPrsquosantitumor effects besides inhibiting cancer cells prolifer-ation and migration inducing tumor cell differentiationand inhibiting cancer angiogenesis and cancer-associatedfibroblast growth (unpublished) showing that GSPP is apromising antitumor drug in future cancer treatment

bFGF as an important prolymphangiogenesis factorgenerated by tumor cells can significantly promote lym-phatic vessel endothelial cell proliferation and migration and

promote tumor lymphangiogenesis by a variety of ways [27]The most classic way on which bFGF promotes tumor lym-phangiogenesis is through VEGF-A VEGF-C and VEGF-Dwhich are known prolymphangiogenesis factors to promotetumor lymphatic vessel grow [23 28] bFGF is a heparindependent growth factor which means that it can have effectonly in the form of bFGF-heparin-FGFR terpolymers struc-ture to further activate the intracellular signal transductionpathways GSPP is a kind of polysaccharide sulfate which

8 BioMed Research International

is similar to the structure of heparin active site both ofwhich contain a sulfuric acid base Previous study showedthat GSPP works by three distinct mechanisms (a) blockingthe bFGF production (b) inhibiting the release of bFGF fromthe extracellular matrix and (c) directly binding to bFGFand competitively inhibiting the binding of bFGF to its lowaffinity receptor heparinHS [12] In this study we found thatGSPP alone has no effect on the viability growth of hLECsAnd it does not inhibit the migration and tube formation ofhLECs whichmay be due to the absence of bFGF bFGF playsan important role in the growth migration and lymphan-giogenesis of hLECs With the addition of exogenous bFGFwhich simulates the environment in the body GSPP signifi-cantly inhibited bFGF-induced cell proliferation migrationand tube formation This means that GSPP works throughdirectly binding to bFGF and competitively inhibiting thebinding of bFGF to its low affinity receptor heparinHS

Extracellular signal-regulated kinase12 (Erk12) is a pro-tein kinase separated and identified in the early 1990s and itssignal transduction is involved in cell growth developmentand differentiation [19] Studies found that the promotingeffect of bFGF on endothelial cell proliferation andmigrationof part is associated with an increased level of Erk phos-phorylation [18 29] To explore the possible mechanism ofGSPPrsquos inhibiting effect of bFGF-induced lymphangiogene-sis western blot was performed We examined the Erk andp-Erk expression level changes in lymphatic endothelial cellsafter being exposed to GSPP for a certain time Our resultsshowed that bFGF significantly increased the expression ofp-Erk in hLECs However cotreatment with GSPP and bFGFdecreased the expression of p-Erk in hLECs compared withthe bFGF-single use group This further proved that GSPPcould bind to bFGF and competitively inhibit the bindingof bFGF to its low affinity receptor thus blocking the bFGF-induced phosphorylation of Erk12 in hLECs

5 Conclusions

Our study showed that inhibiting bFGF-induced lymphan-giogenesis was one of GSPPrsquos anticancer mechanisms GSPPas an effective bFGF-targeted inhibitor can be a notableantilymphatic metastasis drug in future cancer treatment

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Xiu-Li Ding and Ya-Nan Man contributed equally to thiswork

Acknowledgments

This study was supported by major scientific and technicalproject of Tianjin Science and Technology Commission(05YFGDSF02600) andNationalNatural Science Foundationof China (no 81173376 and no 81473441)

References

[1] J Robert ldquoBiology of cancermetastasisrdquoBulletin du Cancer vol100 no 4 pp 333ndash342 2013

[2] R Bicknell ldquoVascular targeting and the inhibition of angiogene-sisrdquoAnnals of Oncology vol 5 supplement 4 pp S45ndashS50 1994

[3] D Ribatti ldquoHistory of research on angiogenesisrdquo ChemicalImmunology and Allergy vol 99 pp 1ndash14 2014

[4] R Shayan M G Achen and S A Stacker ldquoLymphatic vesselsin cancer metastasis bridging the gapsrdquo Carcinogenesis vol 27no 9 pp 1729ndash1738 2006

[5] J P Sleeman and W Thiele ldquoTumor metastasis and the lym-phatic vasculaturerdquo International Journal of Cancer vol 125 no12 pp 2747ndash2756 2009

[6] R L Ferris M T Lotze S P L Leong D S B Hoon and D LMorton ldquoLymphatics lymph nodes and the immune systembarriers and gateways for cancer spreadrdquo Clinical and Experi-mental Metastasis vol 29 no 7 pp 729ndash736 2012

[7] S S Sundar and T S Ganesan ldquoRole of lymphangiogenesis incancerrdquo Journal of Clinical Oncology vol 25 no 27 pp 4298ndash4307 2007

[8] M G Achen B K McColl and S A Stacker ldquoFocus on lym-phangiogenesis in tumor metastasisrdquo Cancer Cell vol 7 no 2pp 121ndash127 2005

[9] R H Adams and K Alitalo ldquoMolecular regulation of angio-genesis and lymphangiogenesisrdquoNature Reviews Molecular CellBiology vol 8 no 6 pp 464ndash478 2007

[10] P M Linares and J P Gisbert ldquoRole of growth factors inthe development of lymphangiogenesis driven by inflammatorybowel disease a reviewrdquo Inflammatory Bowel Diseases vol 17no 8 pp 1814ndash1821 2011

[11] L K Chang G Garcia-Cardena F Farnebo et al ldquoDose-dependent response of FGF-2 for lymphangiogenesisrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 101 no 32 pp 11658ndash11663 2004

[12] S-X Zhang C Zhu Y Ba et al ldquoGekko-sulfated glycopeptideinhibits tumor angiogenesis by targeting basic fibroblast growthfactorrdquo The Journal of Biological Chemistry vol 287 no 16 pp13206ndash13215 2012

[13] M Benezra R Ishai-Michaeli S A Ben-Sasson and I Vlo-davsky ldquoStructure-activity relationships of heparin-mimickingcompounds in induction of bFGF release from extracellularmatrix and inhibition of smooth muscle cell proliferation andheparanase activityrdquo Journal of Cellular Physiology vol 192 no3 pp 276ndash285 2002

[14] M Fannon K E Forsten and M A Nugent ldquoPotentiation andinhibition of bFGF binding by heparin a model for regulationof cellular responserdquo Biochemistry vol 39 no 6 pp 1434ndash14452000

[15] D ChenW-J Yao X-L Zhang et al ldquoEffects of Gekko sulfatedpolysaccharide-protein complex on human hepatoma SMMC-7721 cells inhibition of proliferation and migrationrdquo Journal ofEthnopharmacology vol 127 no 3 pp 702ndash708 2010

[16] X Wu D Chen and G-R Xie ldquoEffects of Gekko sulfatedpolysaccharide on the proliferation and differentiation of hep-atic cancer cell linerdquoCell Biology International vol 30 no 8 pp659ndash664 2006

[17] X-Z Wu D Chen and X-Q Han ldquoAnti-migration effects ofGekko sulfated glycopeptide on human hepatoma SMMC-7721cellsrdquoMolecules vol 16 no 6 pp 4958ndash4970 2011

BioMed Research International 9

[18] L Yang J Zhang C Wang et al ldquoInteraction between 8-hydroxyquinoline ruthenium(II) complexes and basic fibrob-last growth factors (bFGF) inhibiting angiogenesis and tumorgrowth through ERK and AKT signaling pathwaysrdquo Metal-lomics vol 6 no 3 pp 518ndash531 2014

[19] K Balmanno and S J Cook ldquoTumour cell survival signallingby the ERK12 pathwayrdquo Cell Death and Differentiation vol 16no 3 pp 368ndash377 2009

[20] N E Tobler andMDetmar ldquoTumor and lymph node lymphan-giogenesismdashimpact on cancer metastasisrdquo Journal of LeukocyteBiology vol 80 no 4 pp 691ndash696 2006

[21] Y Cao ldquoOpinion emerging mechanisms of tumour lymphan-giogenesis and lymphatic metastasisrdquo Nature Reviews Cancervol 5 no 9 pp 735ndash743 2005

[22] K Alitalo T Tammela and T V Petrova ldquoLymphangiogenesisin development and human diseaserdquo Nature vol 438 no 7070pp 946ndash953 2005

[23] M Skobe T Hawighorst D G Jackson et al ldquoInduction oftumor lymphangiogenesis by VEGF-C promotes breast cancermetastasisrdquo Nature Medicine vol 7 no 2 pp 192ndash198 2001

[24] SA Stacker CCaesarM E Baldwin et al ldquoVEGF-Dpromotesthe metastatic spread of tumor cells via the lymphaticsrdquo NatureMedicine vol 7 no 2 pp 186ndash191 2001

[25] M S Pepper ldquoLymphangiogenesis and tumor metastasis mythor realityrdquo Clinical Cancer Research vol 7 no 3 pp 462ndash4682001

[26] C L Yang and Y J Qi Animal Drug of Chinese Tradi-tional Medicine Ancient Books Press of Chinese TraditionalMedicine Beijing China 1st edition 2001

[27] R Cao A Eriksson H Kubo K Alitalo Y Cao and JThyberg ldquoComparative evaluation of FGF-2- VEGF-A- andVEGF-C-induced angiogenesis lymphangiogenesis vascularfenestrations and permeabilityrdquo Circulation Research vol 94pp 664ndash670 2004

[28] M J Karkkainen R E Ferrell E C Lawrence et al ldquoMissensemutations interfere with VEGFR-3 signalling in primary lym-phoedemardquo Nature Genetics vol 25 no 2 pp 153ndash159 2000

[29] S Guo L Yu Y Cheng et al ldquoPDGFR120573 triggered by bFGF pro-motes the proliferation andmigration of endothelial progenitorcells via p-ERK signallingrdquo Cell Biology International vol 36no 10 pp 945ndash950 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 BioMed Research International

is similar to the structure of heparin active site both ofwhich contain a sulfuric acid base Previous study showedthat GSPP works by three distinct mechanisms (a) blockingthe bFGF production (b) inhibiting the release of bFGF fromthe extracellular matrix and (c) directly binding to bFGFand competitively inhibiting the binding of bFGF to its lowaffinity receptor heparinHS [12] In this study we found thatGSPP alone has no effect on the viability growth of hLECsAnd it does not inhibit the migration and tube formation ofhLECs whichmay be due to the absence of bFGF bFGF playsan important role in the growth migration and lymphan-giogenesis of hLECs With the addition of exogenous bFGFwhich simulates the environment in the body GSPP signifi-cantly inhibited bFGF-induced cell proliferation migrationand tube formation This means that GSPP works throughdirectly binding to bFGF and competitively inhibiting thebinding of bFGF to its low affinity receptor heparinHS

Extracellular signal-regulated kinase12 (Erk12) is a pro-tein kinase separated and identified in the early 1990s and itssignal transduction is involved in cell growth developmentand differentiation [19] Studies found that the promotingeffect of bFGF on endothelial cell proliferation andmigrationof part is associated with an increased level of Erk phos-phorylation [18 29] To explore the possible mechanism ofGSPPrsquos inhibiting effect of bFGF-induced lymphangiogene-sis western blot was performed We examined the Erk andp-Erk expression level changes in lymphatic endothelial cellsafter being exposed to GSPP for a certain time Our resultsshowed that bFGF significantly increased the expression ofp-Erk in hLECs However cotreatment with GSPP and bFGFdecreased the expression of p-Erk in hLECs compared withthe bFGF-single use group This further proved that GSPPcould bind to bFGF and competitively inhibit the bindingof bFGF to its low affinity receptor thus blocking the bFGF-induced phosphorylation of Erk12 in hLECs

5 Conclusions

Our study showed that inhibiting bFGF-induced lymphan-giogenesis was one of GSPPrsquos anticancer mechanisms GSPPas an effective bFGF-targeted inhibitor can be a notableantilymphatic metastasis drug in future cancer treatment

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Xiu-Li Ding and Ya-Nan Man contributed equally to thiswork

Acknowledgments

This study was supported by major scientific and technicalproject of Tianjin Science and Technology Commission(05YFGDSF02600) andNationalNatural Science Foundationof China (no 81173376 and no 81473441)

References

[1] J Robert ldquoBiology of cancermetastasisrdquoBulletin du Cancer vol100 no 4 pp 333ndash342 2013

[2] R Bicknell ldquoVascular targeting and the inhibition of angiogene-sisrdquoAnnals of Oncology vol 5 supplement 4 pp S45ndashS50 1994

[3] D Ribatti ldquoHistory of research on angiogenesisrdquo ChemicalImmunology and Allergy vol 99 pp 1ndash14 2014

[4] R Shayan M G Achen and S A Stacker ldquoLymphatic vesselsin cancer metastasis bridging the gapsrdquo Carcinogenesis vol 27no 9 pp 1729ndash1738 2006

[5] J P Sleeman and W Thiele ldquoTumor metastasis and the lym-phatic vasculaturerdquo International Journal of Cancer vol 125 no12 pp 2747ndash2756 2009

[6] R L Ferris M T Lotze S P L Leong D S B Hoon and D LMorton ldquoLymphatics lymph nodes and the immune systembarriers and gateways for cancer spreadrdquo Clinical and Experi-mental Metastasis vol 29 no 7 pp 729ndash736 2012

[7] S S Sundar and T S Ganesan ldquoRole of lymphangiogenesis incancerrdquo Journal of Clinical Oncology vol 25 no 27 pp 4298ndash4307 2007

[8] M G Achen B K McColl and S A Stacker ldquoFocus on lym-phangiogenesis in tumor metastasisrdquo Cancer Cell vol 7 no 2pp 121ndash127 2005

[9] R H Adams and K Alitalo ldquoMolecular regulation of angio-genesis and lymphangiogenesisrdquoNature Reviews Molecular CellBiology vol 8 no 6 pp 464ndash478 2007

[10] P M Linares and J P Gisbert ldquoRole of growth factors inthe development of lymphangiogenesis driven by inflammatorybowel disease a reviewrdquo Inflammatory Bowel Diseases vol 17no 8 pp 1814ndash1821 2011

[11] L K Chang G Garcia-Cardena F Farnebo et al ldquoDose-dependent response of FGF-2 for lymphangiogenesisrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 101 no 32 pp 11658ndash11663 2004

[12] S-X Zhang C Zhu Y Ba et al ldquoGekko-sulfated glycopeptideinhibits tumor angiogenesis by targeting basic fibroblast growthfactorrdquo The Journal of Biological Chemistry vol 287 no 16 pp13206ndash13215 2012

[13] M Benezra R Ishai-Michaeli S A Ben-Sasson and I Vlo-davsky ldquoStructure-activity relationships of heparin-mimickingcompounds in induction of bFGF release from extracellularmatrix and inhibition of smooth muscle cell proliferation andheparanase activityrdquo Journal of Cellular Physiology vol 192 no3 pp 276ndash285 2002

[14] M Fannon K E Forsten and M A Nugent ldquoPotentiation andinhibition of bFGF binding by heparin a model for regulationof cellular responserdquo Biochemistry vol 39 no 6 pp 1434ndash14452000

[15] D ChenW-J Yao X-L Zhang et al ldquoEffects of Gekko sulfatedpolysaccharide-protein complex on human hepatoma SMMC-7721 cells inhibition of proliferation and migrationrdquo Journal ofEthnopharmacology vol 127 no 3 pp 702ndash708 2010

[16] X Wu D Chen and G-R Xie ldquoEffects of Gekko sulfatedpolysaccharide on the proliferation and differentiation of hep-atic cancer cell linerdquoCell Biology International vol 30 no 8 pp659ndash664 2006

[17] X-Z Wu D Chen and X-Q Han ldquoAnti-migration effects ofGekko sulfated glycopeptide on human hepatoma SMMC-7721cellsrdquoMolecules vol 16 no 6 pp 4958ndash4970 2011

BioMed Research International 9

[18] L Yang J Zhang C Wang et al ldquoInteraction between 8-hydroxyquinoline ruthenium(II) complexes and basic fibrob-last growth factors (bFGF) inhibiting angiogenesis and tumorgrowth through ERK and AKT signaling pathwaysrdquo Metal-lomics vol 6 no 3 pp 518ndash531 2014

[19] K Balmanno and S J Cook ldquoTumour cell survival signallingby the ERK12 pathwayrdquo Cell Death and Differentiation vol 16no 3 pp 368ndash377 2009

[20] N E Tobler andMDetmar ldquoTumor and lymph node lymphan-giogenesismdashimpact on cancer metastasisrdquo Journal of LeukocyteBiology vol 80 no 4 pp 691ndash696 2006

[21] Y Cao ldquoOpinion emerging mechanisms of tumour lymphan-giogenesis and lymphatic metastasisrdquo Nature Reviews Cancervol 5 no 9 pp 735ndash743 2005

[22] K Alitalo T Tammela and T V Petrova ldquoLymphangiogenesisin development and human diseaserdquo Nature vol 438 no 7070pp 946ndash953 2005

[23] M Skobe T Hawighorst D G Jackson et al ldquoInduction oftumor lymphangiogenesis by VEGF-C promotes breast cancermetastasisrdquo Nature Medicine vol 7 no 2 pp 192ndash198 2001

[24] SA Stacker CCaesarM E Baldwin et al ldquoVEGF-Dpromotesthe metastatic spread of tumor cells via the lymphaticsrdquo NatureMedicine vol 7 no 2 pp 186ndash191 2001

[25] M S Pepper ldquoLymphangiogenesis and tumor metastasis mythor realityrdquo Clinical Cancer Research vol 7 no 3 pp 462ndash4682001

[26] C L Yang and Y J Qi Animal Drug of Chinese Tradi-tional Medicine Ancient Books Press of Chinese TraditionalMedicine Beijing China 1st edition 2001

[27] R Cao A Eriksson H Kubo K Alitalo Y Cao and JThyberg ldquoComparative evaluation of FGF-2- VEGF-A- andVEGF-C-induced angiogenesis lymphangiogenesis vascularfenestrations and permeabilityrdquo Circulation Research vol 94pp 664ndash670 2004

[28] M J Karkkainen R E Ferrell E C Lawrence et al ldquoMissensemutations interfere with VEGFR-3 signalling in primary lym-phoedemardquo Nature Genetics vol 25 no 2 pp 153ndash159 2000

[29] S Guo L Yu Y Cheng et al ldquoPDGFR120573 triggered by bFGF pro-motes the proliferation andmigration of endothelial progenitorcells via p-ERK signallingrdquo Cell Biology International vol 36no 10 pp 945ndash950 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

BioMed Research International 9

[18] L Yang J Zhang C Wang et al ldquoInteraction between 8-hydroxyquinoline ruthenium(II) complexes and basic fibrob-last growth factors (bFGF) inhibiting angiogenesis and tumorgrowth through ERK and AKT signaling pathwaysrdquo Metal-lomics vol 6 no 3 pp 518ndash531 2014

[19] K Balmanno and S J Cook ldquoTumour cell survival signallingby the ERK12 pathwayrdquo Cell Death and Differentiation vol 16no 3 pp 368ndash377 2009

[20] N E Tobler andMDetmar ldquoTumor and lymph node lymphan-giogenesismdashimpact on cancer metastasisrdquo Journal of LeukocyteBiology vol 80 no 4 pp 691ndash696 2006

[21] Y Cao ldquoOpinion emerging mechanisms of tumour lymphan-giogenesis and lymphatic metastasisrdquo Nature Reviews Cancervol 5 no 9 pp 735ndash743 2005

[22] K Alitalo T Tammela and T V Petrova ldquoLymphangiogenesisin development and human diseaserdquo Nature vol 438 no 7070pp 946ndash953 2005

[23] M Skobe T Hawighorst D G Jackson et al ldquoInduction oftumor lymphangiogenesis by VEGF-C promotes breast cancermetastasisrdquo Nature Medicine vol 7 no 2 pp 192ndash198 2001

[24] SA Stacker CCaesarM E Baldwin et al ldquoVEGF-Dpromotesthe metastatic spread of tumor cells via the lymphaticsrdquo NatureMedicine vol 7 no 2 pp 186ndash191 2001

[25] M S Pepper ldquoLymphangiogenesis and tumor metastasis mythor realityrdquo Clinical Cancer Research vol 7 no 3 pp 462ndash4682001

[26] C L Yang and Y J Qi Animal Drug of Chinese Tradi-tional Medicine Ancient Books Press of Chinese TraditionalMedicine Beijing China 1st edition 2001

[27] R Cao A Eriksson H Kubo K Alitalo Y Cao and JThyberg ldquoComparative evaluation of FGF-2- VEGF-A- andVEGF-C-induced angiogenesis lymphangiogenesis vascularfenestrations and permeabilityrdquo Circulation Research vol 94pp 664ndash670 2004

[28] M J Karkkainen R E Ferrell E C Lawrence et al ldquoMissensemutations interfere with VEGFR-3 signalling in primary lym-phoedemardquo Nature Genetics vol 25 no 2 pp 153ndash159 2000

[29] S Guo L Yu Y Cheng et al ldquoPDGFR120573 triggered by bFGF pro-motes the proliferation andmigration of endothelial progenitorcells via p-ERK signallingrdquo Cell Biology International vol 36no 10 pp 945ndash950 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom