Research Article Plasmid Transfer of Plasminogen K1-5 ...downloads.hindawi.com/journals/bmri/2014/656527.pdf · Research Article Plasmid Transfer of Plasminogen K1-5 Reduces Subcutaneous

Research ArticlePlasmid Transfer of Plasminogen K1-5 Reduces SubcutaneousHepatoma Growth by Affecting Inflammatory Factors

Lea A Koch1 Volker Schmitz2 Christian P Strassburg1 and Esther Raskopf1

1 Department of Inner Medicine I University of Hospital Bonn Sigmund-Freud-Straszlige 25 53127 Bonn Germany2 Krankenhaus Marienworth Muhlenstrasse 39 55543 Bad Kreuznach Germany

Correspondence should be addressed to Esther Raskopf estherraskopfukbuni-bonnde

Received 17 January 2014 Revised 9 April 2014 Accepted 10 April 2014 Published 8 May 2014

Academic Editor Guillermo Mazzolini

Copyright copy 2014 Lea A Koch 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

There is evidence that plasminogen K1-5 (PlgK1-5) directly affects tumour cells and inflammationTherefore we analysed if PlgK1-5has immediate effects on hepatoma cells and inflammatory factors in vitro and in vivo In vitro effects of plasmid encoding PlgK1-5(pK1-5) on Hepa129 Hepa1-6 and HuH7 cell viability apoptosis and proliferation as well as VEGF and TNF-alpha expression andSTAT3-phosphorylation were investigated In vivo tumour growth proliferation vessel density and effects on vascular endothelialgrowth factor (VEGF) and tumour necrosis factor alpha (TNF-alpha) expression were examined following treatment with pK1-5In vivo pK1-5 halved cell viability cell death was increased by up to 15 compared to the corresponding controls Proliferationwas not affected VEGF TNF-alpha and STAT3-phosphorylation were affected following treatment with pK1-5 In vivo ten daysafter treatment initiation pK1-5 reduced subcutaneous tumour growth by 32 and mitosis by up to 77 compared to the controlsVessel density was reduced by 50 TNF-alpha levels in tumour and liver tissue were increased whereas VEGF levels in tumoursand livers were reduced after pK1-5 treatment Taken together plasmid gene transfer of PlgK1-5 inhibits hepatoma (cell) growth notonly by reducing vessel density but also by inducing apoptosis inhibiting proliferation and triggering inflammation

1 Introduction

Hepatocellular carcinomas (HCC) continue to pose a chal-lenge in establishing effective therapies for a broad collectiveof patients Novel therapeutic approaches such as sorafenibhave been shown to inhibit HCC growth and prolong patientsurvival [1] But in the light of possible escape mechanismswith monotarget treatments a combination of antitumoralmechanisms may be preferable [2ndash4]

Angiostatin (PlgK1-4) and its derivate plasminogen K1-5 (PlgK1-5) are powerful angiostatic factors which inhibitendothelial cell functions in vitro and in vivo [5ndash8] Howeverthere has been mounting evidence that antitumor effects ofangiostatin and its derivates go beyond effects on endothelialcells Davidson et al showed that kringle 5 alone induced notonly apoptosis in endothelial cells but also in tumour cells aswell [9] This goes in line with a study by our workgroup alsoshowing direct effects on hepatoma cells [10]

Additionally there is evidence that antiendothelial andantitumoral effects of PlgK1-5 are complemented by a modu-lation of inflammatory pathways

Albini et al showed that angiostatin induces an upreg-ulation of IL-12 in macrophages and concomitantly thatthe angiostatic properties are partly dependent on the IL-12 signal cascade [11] IL-12 is a proinflammatory cytokinesupporting our theory that antitumor effects of angiostatinand its derivates are both angiostatic and proinflammatoryIn line with this Perri et al found that kringle 5 (K5)supported the recruitment of neutrophils and NKT-cells [12]whereas Mauceri and coworkers demonstrated that tumourproduction of angiostatin was heightened after exposure toTNF-alpha [13]

It has been shown that heightened TNF-alpha levelscorrelate with elevated vascular endothelial growth factor(VEGF) levels [14 15] whereas angiostatin has an antagonis-

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 656527 14 pageshttpdxdoiorg1011552014656527

2 BioMed Research International

tic effect on VEGF [16 17] However to our knowledge thishas not been shown for PlgK1-5

Many studies used an adenoviral vector approach toinvestigate the antitumoral effects of PlgK1-5 [8 10 18 19]Yet adenovirus based therapies are prone to cause vehementallergic reactions in follow-up treatments [20]

Therefore we analysed effects of plasmid vectormediatedgene transfer of PlgK1-5 on hepatomas in vitro and in vivoSince angiostatic effects are well documented we placedour focus on other factors We examined proapoptoticantiproliferative and proinflammatory pathways in vitro andin vivo Additionally we wanted to further distinguish thelink between inflammation and angiogenesis

2 Materials and Methods

21 Animals and Cell Lines Eight-week-old male C3H micewere supplied by Charles River (Sulzfeld Germany) and keptin the local central animal facility of the University HospitalBonn The mice were housed under standard conditionsand had free access to water and food Animal procedureswere performed in accordance with approved protocolsand followed recommendations for proper care and use oflaboratory animals

The hepatocyte derived cellular carcinoma cell lineHuH7 and the mouse hepatoma cell line Hepa1-6 (bothobtained from American Type Culture Collection (ATCCRockvilleMD)) were cultivated inDulbeccorsquosmodified Eaglemedium (DMEM) supplemented with 10 heat-inactivatedfetal bovine serum (FBS)

Hepa129 cells (Mouse Hepatoma 129 obtained fromNCI-Frederick Cancer Research and Development Centre(DCT Tumour Repository)) were maintained in RPMI1640supplemented with 10 FBS 200mM glutamine

22 Synthesis and Amplification of pCMVTNT Vectors Frag-ments encoding plasminogen K1-5 (PlgK1-5 amino acids 1-546) were generated and validated as described previously[8] PlgK1-5 was then subcloned into the eukaryotic expres-sion vector pCMVTNT (Promega Mannheim Germany)according to the manufacturerrsquos protocol correct insertionwas determined through enzymatic restriction digestion andgel electrophoresis of pCMVTNT-K1-5 (pK1-5) on a 1agarose gel pCMVTNTwithout transgene (pMock)was usedas a vector control

pK1-5 and pMock were amplified using the E coli K12strain DH10B and plasmid vectors were isolated using theNucleoBond Xtra Maxi Plus EF system (Macherey-NagelDuren Germany) DNA content and purity were determinedby measuring the OD260280 ratio

23 Plasmid Transfection In Vitro and In Vivo In vitro 106cells (HuH7 Hepa1-6 Hepa129) were seeded on 10 cm platesand cultured in antibiotic-free medium One day later cellswere transfectedwith either pK1-5 or pMock (119899=4per group)using Lipofectamine 2000 (Invitrogen Karlsruhe Germany)according to the manufacturerrsquos protocol Cells treated withplain culture medium were used as negative controls

For establishment of subcutaneous tumours 106 Hepa129cells in 150120583L plain RPMI were injected subcutaneously intothe paramedian hind of male C3Hmice [8] Reaching ameantumour diameter of 150 cm3 treatment was started In a two-day intervalmicewere treatedwith intraperitoneal injectionswith NaCl (150120583L NaClmouse 119899 = 5) DOTAP (Carl RothKarlsruhe Germany) alone (100120583L NaCl with 50 120583L DOTAPper animal 119899 = 6) pMock (50120583g pMock in 100 120583L NaCl and50 120583L DOTAP 119899 = 6) or pK1-5 (50120583g pK1-5 in 100 120583L NaClwith 50120583L DOTAP 119899 = 5)

24 Analysis of K1-5 Protein Expression In Vitro and InVivo To determine K1-5 expression in vitro cells weretransfected as described above Two days later the cellswere harvested in 100120583L PBS with protease inhibitors(Complete Roche Diagnostics Mannheim Germany) Foranalysis of K1-5 expression in vivo liver and tumour tissues(50mm3) were harvested after termination of the in vivoexperiments Tumour and liver tissues were homogenisedin 500120583L PBS and protease inhibitors (Complete) using thePrecellys system (Peqlab Heidelberg Germany) Cells andtissue homogenisates were then lysed by repeated freeze-thaw cycles Protein concentrations of tissue and cells weredetermined using the DC protein assay (Biorad MunichGermany) according to the manufacturerrsquos protocol

50120583g protein was used for SDS-polyacrylamide gelelectrophoresis Proteins were transferred on PVDF mem-brane (Biorad) Detection of K1-5 was done using an anti-angiostatin antibody (A1101 Sigma-Aldrich Hamburg Ger-many) and a corresponding secondary antibody (rabbit anti-goat Santa Cruz Heidelberg Germany) GAPDH was usedas a control (sc-25778 Santa Cruz) Chemiluminescent signalwas developed using AceGlow (Peqlab) and the Chemismartsystem with ChemiCapt software (Peqlab)

25 Determination of Tumour Growth In Vivo and SampleCollection Tumour growth was determined every secondday bymeasuringmaximum length andwidth using a caliperTumour volumes were calculated as 119881 = length times (width)2 times052

Ten days after treatment initiation mice were sacrificedand tumours and livers were explanted Blood was collectedTumour liver and blood samples were harvested and con-served at minus80∘C after freezing in liquid nitrogen Tumoursamples were embedded in Tissue Tek (Sakura FinetekHeppenheim Germany) and cryopreserved for immunohis-tochemistry

26 Determination of Cell Viability One day prior totransfection 104 cells were seeded onto 96-well-platesand then cultivated in antibiotic-free medium 100 120583L 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT) was added to the cells 48 hours after transfectionCells were then incubated for 45 minutes Supernatants wereremoved and MTT was solubilized with 100 120583L dimethylsulfoxide (DMSO) Optical density was measured colorimet-rically at 560 nm (GloMax Multi ELISA reader PromegaMannheim Germany)

BioMed Research International 3

To test for effects on tumour cell proliferation cells werelabelled using 5-bromo-21015840-deoxyuridine (BrdU) in accor-dance with the manufacturerrsquos protocol (Cell ProliferationELISA Roche Diagnostics Mannheim Germany) 24 hoursafter transfection Another 24 hours later cells were fixedand BrdU incorporation was determined using a specificantibodyOptical densitywas determined at 450 nm (GloMaxMulti ELISA reader)

Apoptosis was determined by quantifying cytoplasmichistone-associated DNA fragments The Cell Death Detec-tion kit (Roche Diagnostics) was applied following the man-ufacturerrsquos protocol 48 hours after transfection cells werelysed and histone-associated DNA was determined usingspecific antibodies and ELISA technique Optical density wasdetermined at 405 nm (GloMax Multi ELISA reader)

27 Cell-Based Phospho-STAT3 Immunoassay For analysisof (phosphorylated) STAT3 104 cells were seeded on ablack 96-well tissue culture plate with clear bottom Thecells were transfected as described above Two days aftertransfection STAT3 phosphorylation was quantified with acell-based STAT3 Immunoassay (RampD Systems WiesbadenMannheim Germany) as described in the manufacturerrsquosprotocol STAT3 and phosphorylated STAT3 (pSTAT3) weredetected with two different specific antibodies and corre-sponding fluorescent secondary antibodies

Fluorescence was measured with the GloMax MultiELISA reader (Promega) For STAT3 fluorescence was mea-sured at 360 nm (excitation) and 450 nm (extinction) and at540 nm (excitation) and 600 nm (extinction) for pSTAT3

28 Analysis of VEGF and TNF-Alpha Levels In Vitro andIn Vivo Cells were transfected and harvested as describedin the passage about the detection of K1-5 protein Tumourand liver tissues were homogenised as described above Cellsand tissue homogenisates were then lysed by repeated freeze-thaw cycles Protein concentrations of tissue and cells weredetermined using the DC protein assay (Biorad MunichGermany) according to the manufacturerrsquos protocol

VEGF in HuH7 was quantified with the human VEGFDuoSet ELISA kit (RampD Systems Wiesbaden Germany)VEGF in Hepa1-6 Hepa129 and liver and tumour tissuesamples was analysed with the murine VEGF DuoSet ELISAkit (RampD Systems) as instructed VEGF levels in cell super-natants were correlated with the cell viability as establishedin the MTT assay

TNF-alpha in HuH7 was analysed with the humanTNF-alpha DuoSet ELISA kit (RampD Systems) TNF-alphain Hepa1-6 Hepa129 and tumour and liver tissues wasquantified with the murine TNF-alpha DuoSet ELISA kit(RampD Systems)

29 Histological Sections and Staining Cryopreserved tum-our samples were standard HE stained

Immunostaining against CD31 was done with mono-clonal rat anti-mouse CD31 (Cedarlane Ontario Canada)

and Ki67 staining with rat anti-mouse Ki67 (DakoCytoma-tion Hamburg Germany) according to a previous publica-tion [10]

For quantification of CD31 positive blood vessels andKi67 positive mitotic cells microscopic fields of vision (=high power fields hpf) were evaluated

Sectionswere stained against VEGFwith goat anti-mouseVEGF (RampD Systems) For staining against TNF-alpha goatanti-mouse TNF-alpha (Abcam Cambridge UK) was used

Sections were incubated with corresponding secondaryantibodies (DakoCytomation) Detection was done usingstreptavidin and AEC substrate (DakoCytomation) Sectionswere counterstained with hematoxyline

210 Statistical Analysis Data are presented as means withSEM Statistically significant differences between experimen-tal groups were shown by using a nonparametric two-tailedtest (Mann-Whitney test) for unpaired samples Values of119875 lt005 were considered to be significant

3 Results

31 PlgK1-5 Is Expressed In Vitro and In Vivo First weanalysed PlgK1-5 protein expression following transfectionwith pK1-5 in vitro and in vivo

Using western blot technique PlgK1-5 was detected inall three analysed hepatoma cell lines transfected with pK1-5 whereas in the pMock and medium control PlgK1-5 wasbarely detected This was also the case for subcutaneoustumours and corresponding livers PlgK1-5 was strongerexpressed in animals receiving pK1-5 In the control groupsbands were only slightly visible (Figure 1)

32 pK1-5 Reduces Cell Viability by Increasing Cellular Deathbut Has Little Impact on Cell Proliferation After showingPlgK1-5 expression after pK1-5 treatment we examinedwhether pK1-5 affects overall cell viability cell proliferationand cell death in hepatoma cell lines

In an MTT assay pK1-5 reduced cell viability in all threeexamined cell lines HuH7 treated with pK1-5 were 1091(119899 = 4 119875 = 00286) less viable than pMock treated cellsCompared to the medium control viability was reduced by4786 (119899 = 4 119875 = 00286) In pK1-5 treated Hepa1-6 viabilitywas reduced by 736 (119899 = 4 ns) compared to pMock andby 5158 compared to the medium control (119899 = 4 119875 =00286) When treated with pK1-5 Hepa129 showed 496less viability than pMock treated cells (119899 = 4 ns) Viabilitywas decreased by 3537 when compared to the mediumcontrol (119899 = 4 119875 = 00286 Figure 2(a))

To ascertain whether reduced cell viability was due todecreased cell proliferation we examined effects of pK1-5 onBrdU incorporation in HuH7 Hepa1-6 and Hepa129 Whentreated with pK1-5 proliferation of HuH7 was reduced by904 compared to pMock treated cells (119899 = 4 ns) andby 1403 compared to the medium control (119899 = 4 119875 =00286) In Hepa1-6 pK1-5 treated cells showed 1021 lessproliferation than pMock treated cells (119899= 4 ns) but showed


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Figure 1 Protein expression of PlgK1-5 in vitro and in vivo Cells were transfected with pMock or pK1-5 and incubated for 48 hours Cells wereharvested and protein was isolated Tumour and liver tissue fragments from mice treated with NaCl DOTAP alone pMock or pK1-5 werehomogenised using the Precellys system and protein was isolated Whole protein content was determined and 50120583g protein was subjectedto SDS-PAGE Following semidry blotting PlgK1-5 was detected using an angiostatin-specific antibody GAPDH was used as a control CtrlNaCl or plain medium control Dotap DOTAP control Mock pMock K1-5 pK1-5

no effect compared to the medium control (119899 = 4 119875 = 00286Figure 2(b))

To analyse if diminished cell viability is due to increasedapoptosis we applied a cell death detection assay HuH7treated with pK1-5 showed 1833 less apoptotic cells thanHuH7 treated with pMock (119899 = 4 ns) but 125 higher ratesof apoptosis compared to the medium control (119899 = 4 ns)When treated with pK1-5 apoptosis inHepa1-6 was increasedby 2575 compared to pMock treated cells (119899 = 4 ns) andby 1562 compared to the medium control (119899 = 4 ns) InHepa129 apoptosis was reduced by 3746 in pK1-5 treatedcells compared to the pMock group (119899 = 4 ns) but increased26-fold compared to the medium control (119899 = 4 119875 = 00286Figure 2(c))

33 pK1-5 Increases VEGF Expression and Secretion in Hep-atoma Cells In Vitro To test whether pK1-5 affects VEGFexpression a VEGF ELISA was performed

In detail VEGF expression in pK1-5 treated HuH7 wasraised by 1297 compared to pMock treated HuH7 (119899 =4 ns) and 19-fold compared to the medium control (119899= 4 119875 = 00286) In Hepa1-6 VEGF levels in the pK1-5group were elevated compared to pMock treated cells by1159 (119899 = 4 ns) but slightly lowered by 893 compared tomedium treated Hepa1-6 (119899 = 4 ns) In Hepa129 treatment

with pK1-5 increasedVEGF expression by 1712with regardsto the pMock group (119899 = 4 ns) and by 1313 compared tothe medium control (119899 = 4 ns Figure 3(a))

Correlated with cell viability pK1-5 reduced VEGF secre-tion in HuH7 by 955 compared to pMock treated cells(119899 = 4 ns) but increased secretion by 2065 comparedto medium treated HuH7 (119899 = 4 ns) In Hepa1-6 pK1-5appeared to have no significant effect on VEGF secretionVEGF levels in supernatant of pK1-5 treated cells wereincreased by 573 (119899 = 4 ns) compared to the mediumcontrol but decreased by 209 compared with pMock (119899 =4 ns)

Hepa129 treated with pK1-5 secreted 3042 more VEGFthan Hepa129 treated with pMock (119899 = 4 ns) but expressed326 less than the medium control (119899 = 4 119875 = 00286Figure 3(b))

34 TNF-Alpha Is Increased in pK1-5-Treated Hepatoma CellsSince VEGF was affected by pK1-5 we wanted to know if thiswas due to TNF-alpha

In all three investigated cell lines TNF-alpha was elevatedfollowing treatment with pK1-5 (Figure 4) In HuH7 cellstreated with pK1-5 (119899 = 4) expressed 4454more TNF-alphathan the medium control (119899 = 4 119875 = 00286) and 946morethan cells treated with pMock (119899 = 4 ns)


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Figure 2 K1-5 and cell viability 104 hepatoma cells were seeded on 96-well plates transfected with Lipofectamine and pCMVTNT-vectors ortreated with plain medium and incubated for twenty four hours 119899 = 4 lowast119875 lt 005 (plain mediumpK1-5)

119875 lt 005 (pMockpK1-5) Opticaldensity of medium treated cells was set as 100 Data are shown as mean percentage plusmn SEM (a) Supernatants were removed and MTTwas added and incubated Supernatants were removed MTT was solubilized with DMSO and OD was measured at 560 nm (b) Cells werelabeled with BrdU incubated for 24 hours and fixed with a specific antibody and optical density was measured at 560 nm (c) Cytoplasmatichistone-associated DNA fragments were quantified with the Cell Death Detection kit (Roche diagnostics) and optical density measured at405 nm

Hepa1-6 treated with pK1-5 (119899 = 4) produced 311 moreTNF-alpha than the medium control (119899 = 4 ns) but 858less than cells treated with pMock (119899 = 4 ns)

In pK1-5 treated Hepa129 (119899 = 4) TNF-alpha wasincreased by 1731 when compared to medium treated cells(119899 = 4 ns) When compared to pMock pK1-5 treatedHepa129 expressed 535 more TNF-alpha (119899 = 4 ns)

35 STAT3 and pSTAT3 Expression Is Modulated by pK1-5Signal transducer and activator of transcription 3 (STAT3)is an acute-phase response factor that is activated throughphosphorylation To detect possible effects of pK1-5 on cell

growth and further elucidate pK1-5 effects on inflammationwe analysed STAT3 phosphorylation We found that bothtotal STAT3 expression and phosphorylation were altered bytreatment with pK1-5 in all tested cell lines

In HuH7 cells treated with pK1-5 expressed 7645less total STAT3 In comparison with pMock treated cellspK1-5 increased total STAT3 by 1495 Levels of pSTAT3were reduced by 6102 compared to the medium controlCompared with pMock pK1-5 increased pSTAT3 by 2853(Figure 5(a))

When treated with pK1-5 Hepa1-6 produced 3351 lesstotal STAT3Compared to pMock pK1-5 reduced total STAT3


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Figure 3 VEGF expression after pK1-5 treatment in vitro Cells were transfected and incubated for 48 hours Cells and supernatants wereharvested and cells were solubilized in PBScomplete and lysed with repeated freeze-thaw cycles Protein levels were analysed and VEGFlevels were quantified with human (HuH7) or murine (Hepa1-6 Hepa129) VEGF ELISAs VEGF levels in supernatants were correlated toOD acquired by MTT assays (a) VEGF levels in cell lysates Data are shown as mean pg VEGF100120583g protein plusmn SEM (b) VEGF levels insupernatants MTT correlated Data are shown as mean pg VEGFmL cell supernatant plusmn SEM 119899 = 4 lowast119875 lt 005 (compared to plain medium)119875 lt 005 (compared to pMock)

levels by 3173 pK1-5 treated Hepa1-6 showed 6873 lesspSTAT3 in comparison to the medium control Compared topMock pK1-5 reduced pSTAT3 levels by 433 (Figure 5(b))

Hepa129 treated with pK1-5 showed increased totalSTAT3 (by 3877) and phosphorylation of STAT3 (by2481) compared to the medium control When comparedwith the pMock control total STAT3 levels were reduced by6451 and pSTAT3 was decreased by 8096 lower whentreated with pK1-5 (Figure 5(c))

36 Subcutaneous Tumour Growth Is Strongly Reduced in pK1-5 TreatedMice After showing that pK1-5 affects tumour cellsin vitro we wanted to investigate whether these effects couldbe transferred in a subcutaneous tumour model

Ten days after treatment initiation tumour growth wasreduced by 2236 in pK1-5 treated mice when compared topMock treated animals Compared to the DOTAP controltumour growth of pK1-5 treated animals was reduced by3444 and by 3244 in comparison to NaCl treated mice(Figure 6(a))

In order to differentiate whether inhibitory effects ofpK1-5 on tumour growth were due to increased cell deathandor reduced proliferation we analysed tumour sectionsfor necrosis and mitosis

pK1-5 (119899 = 4) reduced necrotic areas by 37 plusmn 2364compared to pMock (119899 = 4 ns) and by 22 plusmn 816compared to DOTAP controls (119899 = 4 ns) Necrotic areasof sections of pK1-5 treated animals were reduced by 82 plusmn245 compared to NaCl (119899 = 4 119875 = 00286) as determinedby HE staining (data not shown)

Ki67 staining revealed that pK1-5 reduced mitosis by684 compared to pMock treated animals and by 7349 inthe DOTAP control Mitotic cells in pK1-5 treated mice weredecreased by 7737 compared with NaCl controls (Figures6(b) and 6(c))

37 pK1-5 Affects VEGF Expression In Vivo VEGF levels invitro had been elevated following treatment with pK1-5 Inorder to examine pK1-5 effects on VEGF levels in vivo weperformed a VEGF ELISA with tumour and liver tissues

VEGF levels in tumour samples of pK1-5 treated ani-mals were reduced by 1267 in comparison to the NaClcontrol but heightened when compared to the DOTAPcontrol (+1808) and pMock control (+1263 Figure 7(a))This was confirmed by immunohistochemistry (Figure 7(c))VEGF levels in liver samples were not altered in the pK1-5 group compared to pMock and DOTAP treated mice butreduced by 17 compared to the NaCl control (Figure 7(b))

38 TNF-Alpha Expression In Vivo Is Altered by pK1-5Following treatment with pK1-5 intratumoural TNF-alphalevels were increased by 182 in comparison to the NaClcontrol In the DOTAP and the pMock control TNF-alphaconcentrations were decreased compared to the NaCl controlby 1969 and by 2798 respectively TNF-alpha levels inthe pK1-5 group showed a significant increase comparedto the DOTAP (+4718) and the pMock group (+6413Figure 8(a))

Mice treated with pK1-5 showed altered TNF-alphaexpression in livers as well When compared with pMock


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treated mice pK1-5 increased TNF-alpha levels by 1022Compared to DOTAP pK1-5 treated mice showed 1141higher TNF-alpha Comparing NaCl treated mice with pK1-5 treated animals pK1-5 increased TNF-alpha expression inlivers by 575 (Figure 8(b))

Elevated intratumoural TNF-alpha levels were supportedby TNF-alpha staining in tumour sections Intensity ofstaining indicating presence of TNF-alpha was noticeablyincreased in sections of pK1-5 treated mice compared to thecontrols (Figure 8(c))

39 pK1-5 Strongly Reduces Vessel Density in Subcuta-neous Hepatomas Angiostatin and its derivate PlgK1-5 havealready been shown in various studies to be potent angiostaticfactors [8 21] The focus of this study was therefore not onantiangiogenesis Nevertheless we performed CD31 stainingof tumour sections to demonstrate the angiostatic potency ofpK1-5

CD31 staining showed that intratumoural microvesselswere reduced by 5483 in pK1-5 treated mice when com-pared to the NaCl control In comparison with DOTAPtreated mice pK1-5 treated animals had 4062 less intratu-moural vessels and showed 3481 less vessels than pMocktreated mice (Figure 9)

4 Discussion

To this day hepatocellular carcinomas (HCC) belong to themost frequent malignant neoplastic diseases worldwide [2223] Yet despite of new developments therapeutic options are

still rare and prognosis of patients in advanced stages remainspoor [24]

In our opinion it is achievable that molecular treatmentstrategies should not only aim at one specific moleculemdashwhich could lead to escape mechanisms [2]mdashbut ratherinhibit multiple targets This is mainly accomplished bycombining therapies but a single adjuvant with for exampleangiostatic and inflammatory properties would be preferable

The angiostatic potency of angiostatin and its derivatePlgK1-5 have been shown in various studies [25ndash27] Butbeyond that recent data showed that angiostatin and PlgK1-5 not only affected endothelial cells but tumour cells as well[9 10 28]

Adenoviral gene therapies are viewed critically because ofrestrictions on follow-up treatments due to their high allergicpotential [20 29] and hydrodynamic injections practised insomemousemodels are potentially fatal in humans Previousstudies already demonstrated that a plasmid based genetransfer of angiostatin and its derivates is effective [30ndash32]For this reason we applied a eukaryotic plasmid encodingPlgK1-5

In this study we demonstrated that pK1-5 stronglyreduced cell viability in three different human and murinehepatoma cell lines On further examination we showed thatreduced viability was caused by increased cell death throughapoptosis This confirms the antitumoral potential of PlgK1-5 as was already demonstrated in previous studies by ourworkgroup adenoviral mediated gene transfer of PlgK1-5reduced the expression of ICAM on murine (Hepa129) andhuman (HuH7) hepatoma cells which led to heightenedapoptosis [10] This is also in accordance with prior findingsby Davidson et al Kringle 5 alone increased apoptosis inD54 human glioma tumour cells [9] Induction of apoptosiscould be due to the inhibition of mitochondrial proteins aswas shown by a study by Lee et al Angiostatin downregu-lated BCL-2 protein resulting in the activation of apoptoticpathways in humanA2058melanoma and BxPC-3 pancreatictumour cell lines [28]This supports our hypothesis that pK1-5 activates apoptotic pathways in hepatoma cells

A previous study showed that antiendothelial and anti-tumoral effects of plasminogen kringle 5 are supported byproinflammatory mediators such as VCAM-1 and E-selectin[12] Therefore we analysed if TNF-alpha and VEGF whichlink inflammation and angiogenesis were also affected bypK1-5 treatment

We found that TNF-alpha was heightened in all threeexamined hepatoma cell lines indicating a proinflammatorypotential of pK1-5 This goes in line with a study by Perriet al showing that retroviral vector based transduction ofkringle 5 activated proinflammatory pathways resulting indecreased tumour growth in murine mammary adenocar-cinoma (DA3) cells [12] We found that in two out of thethree examined hepatoma cell lines STAT3 expression andSTAT3 phosphorylation as markers for immune responseswere strongly reduced following pK1-5 treatmentThe down-regulation of STAT3 expression corresponds to the reductionof cell viability as shown in the MTT assay Both TNF-alpha and STAT3 are key actors that are regulated throughvarious mechanisms [33ndash35] thus the exact mechanism of


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[RFU

]

Plain mediumpMock

Total STAT3 pSTAT3

pK1-5

(c)

Figure 5 STAT3 phosphorylation 104 HuH7 (a) Hepa1-6 (b) or Hepa129 (c) were seeded on a sterile black 96-well tissue plate withclear bottom transfected with Lipofectamine and pCMVTNT vectors or treated with plain medium and incubated for 48 hours STAT3phosphorylation was quantified according to manufacturerrsquos protocol (STAT3 immunoassay RampD systems) Fluorescence was measuredwith the GloMax Multi (Promega) ELISA reader For STAT3 fluorescence was measured at 360 nm (excitation) and 450 nm (extinction)and at 540 nm (excitation) and 600 nm (extinction) for pSTAT3 Data are presented as relative fluorescence units plusmn SEM 119899 = 4 lowast119875 lt 005(compared to plain medium)

119875 lt 005 (compared to pMock)

pK1-5 effects on either remain to be elucidated Therefore itis also possible that STAT3 expression and phosphorylationare activated through mediators other than pK1-5 Signaltransduction leading to inflammatory responses should befurther investigated this study gives first indications that pK1-5 activates inflammation via an upregulation of TNF-alpha

In previous studies raised TNF-alpha levels correlatedwith heightened VEGF levels [14 15] VEGF is a keymediatorin tumour angiogenesis and for this reason we examinedpK1-5 effects on VEGF expression in vitro evaluating bothinflammatory and angiostatic capacities of pK1-5 mediatedthrough VEGF We found that in cell lysates VEGF levels

were considerably heightened In cell supernatants correlatedto viability VEGF levels were raised in two out of threeexamined hepatoma cell lines

Our findings of increased VEGF levels despite of theangiostatic potency of PlgK1-5 can be explained by theinduction of TNF-alpha Here the angiostatic and proinflam-matory potency of PlgK1-5 counteracts the proangiogeniceffects of VEGF

Additionally previous studies showed that VEGF itselfplays a role in inflammation [36 37] We postulate thatthrough this pathway pK1-5 activates inflammation in hep-atoma cells through an induction of TNF-alpha and increases


3500

3000

2500

2000

1500

1000

500

0

0 2 4 6 8 10

Days after treatment initiation

Mea

n tu

mor

vol

ume (

mm

3)

NaClDOTAP

pMockpK1-5

(a)

400

350

300

250

200

150

100

50

0

∘lowast

lowast

Mito

tic ce

llsh

pf

NaClDOTAP

pMockpK1-5

(b)

NaCl DOTAP

pMock pK1-5

(c)

Figure 6 Antiproliferative and proapoptotic effects of pK1-5 in murine subcutaneous tumours (a) tumour development after treatment withNaCl (119899 = 5) DOTAP (119899 = 6) pMock (119899 = 6) or pK1-5 (119899 = 5) 106 Hepa129 were injected subcutaneously into the paramedian hind of maleC3Hmice Treatment was started when tumours reached amean volume of 150mm3 with subcutaneous injections every second day Tumoursize was measured every second day using a caliper and volumes were calculated as 119881 = length times (width)2 times 052 Data are presented as themean tumour volume in mm3 plusmn SEM (b) Mitosis in Ki67 immunostained sections Tumour sections were immunostained against Ki67 andcounterstained with hematoxyline Mitotic cells were counted per hpf (119899 = 16) Data are shown as mitotic cells plusmn SEM lowastP lt 005 (comparedto NaCl) ∘P lt 005 (compared to DOTAP) P lt 005 (compared to pMock) (c) Exemplary photomicrographs of animals treated with NaClDOTAP pMock pK1-5 Mitotic cells show a brown nuclear staining


140

120

100

80

60

40

20

0

lowast

TumourPg

VEG

F100120583

g pr

otei

n

NaCl DOTAP pMock pK1-5

(a)

Pg V

EGF100120583

g pr

otei

n

NaCl DOTAP pMock

2250

2000

1750

1500

1250

1000

750

500

250

0

Liver

pK1-5

(b)

NaCl DOTAP

pMock pK1-5

(c)

Figure 7 In vivo effects of pK1-5 on VEGF expression Mice with subcutaneous tumours were treated with NaCl (119899 = 5) DOTAP (119899 = 6)pMock (119899 = 6) or pK1-5 (119899 = 5) and sacrificed after ten days Tumours and livers were explanted and homogenized serum was collectedand protein concentrations were evaluated VEGF levels were quantified with a murine VEGF ELISA Data are presented as mean pg VEGFplusmn SEM lowast119875 lt 005 (compared to NaCl) (a) Tumour samples (b) liver samples and (c) tumour sections were stained against VEGF andcounterstained with hematoxyline Exemplary photomicrographs of animals treated with NaCl DOTAP pMock and pK1-5 VEGF is shownby brown staining

this proinflammatory impact via a TNF-alpha triggeredupregulation of VEGF

After showing the proapoptotic and proinflammatoryeffects of pK1-5 in vitro we wanted to validate these resultsin a subcutaneous mouse model

In one of our own studies we already demonstrated thatadenoviralmediated gene transfer of PlgK1-5 led to drasticallyreduced tumour growth and blood vessel density [8] Cor-responding to this we showed in the study presented herethat also plasmid based gene transfer of PlgK1-5 decreased


Tumour24

22

20

18

16

14

12

10

8

6

4

2

0

∘


pg T

NF-

alph

a100120583

g pr

otei

n

(a)

4000

3500

3000

2500

2000

1500

1000

500

0

Liver


TNF-

alph

a100120583

g pr

otei

n

(b)

NaCl DOTAP

pMock pK1-5

(c)

Figure 8 In vivo effects of pK1-5 on TNF-alpha expression Mice with subcutaneous tumours were treated with NaCl (119899 = 5) DOTAP (119899 = 6)pMock (119899 = 6) or pK1-5 (119899 = 5) and sacrificed after ten days Tumours and livers were explanted and homogenized and protein concentrationswere evaluated TNF-alpha levels were quantified with amurine TNF-alpha ELISA Data are presented asmean pg TNF-alpha100 120583g protein∘

119875 lt 005 (compared to DOTAP) 119875 lt 005 (compared to pMock) (a) Tumour samples (b) liver samples and (c) tumour sections were

stained against TNF-alpha and counterstained with hematoxyline Exemplary photomicrographs of animals treated with NaCl DOTAPpMock and pK1-5 TNF-alpha is shown by brown staining

subcutaneous tumour growthThe inhibition of experimentaltumour growth of for example murine adenocarcinoma orhuman glioma cells after the treatment with angiostatin orits derivates for example via retroviral or adenoviral genetransfer had already been described in previous studies goingin line with our findings [7 12 19]

Tumour cell proliferationwas reduced in our setting con-firming previous findings that PlgK1-5 exerts antiproliferativecapacities in hepatoma and other tumour entities [5 9 10]In contrast to that necrosis found in HE stained sectionsof animals treated with PlgK1-5 was decreased comparedto the NaCl control This could be caused by markedly


∘lowast

80

60

40

20

0

Mic

rove

ssels

hpf

NaClDOTAP

pMockpK1-5

(a)

NaCl DOTAP

pMock pK1-5

(b)

Figure 9 Microvessel density in subcutaneous tumours Tumours of killed mice were explanted shock frozen stained against CD31 andcounterstained against hematoxyline Vessels show a red-brownish colour (a)Mean tumour vesselsplusmn SEMhpf 119899= 16 lowast119875 lt 005 (compared toNaCl) ∘119875 lt 005 (compared toDOTAP)

119875 lt 005 (compared to pMock)Microvessels per hpf weremicroscopically counted (b) Exemplaryphotomicrographs of tumour sections from animals treated with NaCl DOTAP pMock and pK1-5

lessened tumour growth and mitosis resulting in a decreaseof apoptotic pressure on the tumour cells

In addition to reduced tumour growth expression ofproangiogenicproinflammatory factors likeVEGF andTNF-alpha was affected by pK1-5 Corresponding to the in vitroexperiments TNF-alpha levels in tumour and liver tissuewere heightened

While we found a correlation between heightened TNF-alpha levels and increased VEGF expression in vitro VEGFwas decreased in tumour and liver tissue This discrepancyof VEGF levels in vitro and in vivo may be caused by theconsiderable reduction of tumour growth and mitotic activecells resulting in a lower number of VEGF secreting cellsFurthermore PlgK1-5 has angiostatic potencies that may


play a role in reducing in vivo VEGF levels Additionallyangiostatin is an antagonist of VEGF [38 39] But so far theexact link between TNF-alpha and VEGF regarding PlgK1-5remains to be found

In this context two different approaches to furtherelucidate pK1-5 and its role in inflammation and reducedtumour progression appear promising One step could beto evaluate the recruitment of inflammatory cells such asT-cell lymphocytes NK-cells and macrophages This couldbe extended with an analysis of cell adhesion moleculesmodulating leukocyte and neutrophil recruitment such asPECAM-1 and ICAM or VCAM First hints that PlgK1-5affects these proinflammatory factors came from a study byour workgroup [10] Another access to a further insight intoinflammatory responses could be the expression of cytokinessuch as IL-12 The role of IL-12 in tumour suppression hasalready been shown by Albini and coworkers who found thatangiostatin-induced upregulation of IL-12 is an importantmechanism behind angiostasis [11] In our study we placedthe focus on inflammation and apoptosis rather than antian-giogenesis Nevertheless we evaluated microvessel densityto demonstrate that plasmid based gene transfer of PlgK1-5exhibits angiostatic effects comparable to an adenoviral basedgene transfer [5 7] As expected we found a strong reductionof microvessel density in animals treated with pK1-5 Thisdemonstrates that antiangiogenic capacities of PlgK1-5 arenot limited to adenoviral gene transfer

In summary we found that plasmid vector based genetransfer of PlgK1-5 had marked proapoptotic and proinflam-matory effects on hepatoma cells in vitro and in vivo inaddition to the predescribed effects on endothelial cells Thissupports our theory that pK1-5 not only has direct apoptoticand antiproliferative effects on hepatoma cells but also leadsto inflammation in addition to the known angiostasis

Our findings underline the antitumoral potency of PlgK1-5 on hepatoma cells making it an interesting molecule in thefuture treatment of hepatocellular carcinomas

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported by a grant from theHWamp J HectorStiftung to Esther Raskopf The authors would like to thankSabine Schmitt for her technical support

References

[1] T Zhang X Ding D Wei et al ldquoSorafenib improves thesurvival of patients with advanced hepatocellular carcinoma ameta-analysis of randomized trialsrdquo Anti-Cancer Drugs vol 21no 3 pp 326ndash332 2010

[2] L Rivera M Pandika and G Bergers ldquoEscape mechanismsfrom antiangiogenic therapy an immune cellrsquos perspectiverdquoAdvances in Experimental Medicine and Biology vol 772 pp83ndash99 2014

[3] D A Yardley ldquoDrug resistance and the role of combinationchemotherapy in improving patient outcomesrdquo InternationalJournal of Breast Cancer vol 2013 Article ID 137414 15 pages2013

[4] M Saraswathy and S Gong ldquoDifferent strategies to overcomemultidrug resistance in cancerrdquo Biotechnology Advances vol 31no 8 pp 1397ndash1407 2013

[5] Y Cao A Chen S S A An et al ldquoKringle 5 of plasminogen is anovel inhibitor of endothelial cell growthrdquo Journal of BiologicalChemistry vol 272 no 36 pp 22924ndash22928 1997

[6] Y Cao R W Ji D Davidson et al ldquoKringle domains of humanangiostatin characterization of the anti-proliferative activity onendothelial cellsrdquo Journal of Biological Chemistry vol 271 no46 pp 29461ndash29467 1996

[7] S R Perri J Nalbantoglu B Annabi et al ldquoPlasminogenkringle 5-engineered glioma cells block migration of tumor-associated macrophages and suppress tumor vascularizationand progressionrdquo Cancer Research vol 65 no 18 pp 8359ndash8365 2005

[8] V Schmitz E Raskopf M A Gonzalez-Carmona et alldquoPlasminogen fragment K1-5 improves survival in a murinehepatocellular carcinoma modelrdquo Gut vol 56 no 2 pp 271ndash278 2007

[9] D J Davidson C Haskell S Majest et al ldquoKringle 5 of humanplasminogen induces apoptosis of endothelial and tumor cellsthrough surface-expressed glucose-regulated protein 78rdquo Can-cer Research vol 65 no 11 pp 4663ndash4672 2005

[10] V Schmitz T Sauerbruch and E Raskopf ldquoAnti-tumouraleffects of PlgK1-5 are directly linked to reduced ICAM expres-sion resulting in hepatoma cell apoptosisrdquo International Journalof Colorectal Disease vol 27 no 8 pp 1029ndash1038 2012

[11] A Albini C Brigati A Ventura et al ldquoAngiostatin anti-angiogenesis requires IL-12 the innate immune system as a keytargetrdquo Journal of Translational Medicine vol 7 article 5 2009

[12] S R Perri D Martineau M Francois et al ldquoPlasminogenKringle 5 blocks tumor progression by antiangiogenic andproinflammatory pathwaysrdquo Molecular Cancer Therapeuticsvol 6 no 2 pp 441ndash449 2007

[13] H J Mauceri S Seetharam M A Beckett et al ldquoTumorproduction of angiostatin is enhanced after exposure to TNF-120572rdquo International Journal of Cancer vol 97 no 4 pp 410ndash4152002

[14] Z Xiao Q Liu F Mao J Wu and T Lei ldquoTNF-120572-inducedVEGF and MMP-9 expression promotes hemorrhagic trans-formation in pituitary adenomasrdquo International Journal ofMolecular Sciences vol 12 no 6 pp 4165ndash4179 2011

[15] P Lu L Li G Liu et al ldquoCritical role of TNF-alpha-inducedmacrophage VEGF and iNOS production in the experimen-tal corneal neovascularizationrdquo Investigative Ophthalmology ampVisual Science vol 53 no 7 pp 3516ndash3526 2012

[16] Y-H Chen H-L Wu C-K Chen Y-H Huang B-C Yangand L-W Wu ldquoAngiostatin antagonizes the action of VEGF-Ain human endothelial cells via two distinct pathwaysrdquo Biochem-ical and Biophysical Research Communications vol 310 no 3pp 804ndash810 2003

[17] A Hajitou C Grignet L Devy et al ldquoThe antitumoral effect ofendostatin and angiostatin is associated with a down-regulationof vascular endothelial growth factor expression in tumor cellsrdquoThe FASEB Journal vol 16 no 13 pp 1802ndash1804 2002

[18] V Schmitz E Raskopf M-A Gonzalez-Carmona et al ldquoPlas-minogen derivatives encoding kringles 1-4 and kringles 1-5


exert indirect antiangiogenic and direct antitumoral effects inexperimental lung cancerrdquo Cancer Investigation vol 26 no 5pp 464ndash470 2008

[19] A Galaup C Magnon V Rouffiac et al ldquoFull kringles ofplasminogen (aa 1-566) mediate complete regression of humanMDA-MB-231 breast tumor xenograted in nude micerdquo GeneTherapy vol 12 no 10 pp 831ndash842 2005

[20] N Bessis F J GarciaCozar and M-C Boissier ldquoImmuneresponses to gene therapy vectors influence on vector functionand effector mechanismsrdquo Gene Therapy vol 11 no 1 pp S10ndashS17 2004

[21] R Cao H-L Wu N Veitonmaki et al ldquoSuppression ofangiogenesis and tumor growth by the inhibitor K1-5 generatedby plasmin-mediated proteolysisrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 96 no10 pp 5728ndash5733 1999

[22] J M Llovet A Burroughs and J Bruix ldquoHepatocellularcarcinomardquoThe Lancet vol 362 no 9399 pp 1907ndash1917 2003

[23] C Genco G Cabibbo M Maida et al ldquoTreatment of hep-atocellular carcinoma present and futurerdquo Expert Review ofAnticancer Therapy vol 13 no 4 pp 469ndash479 2013

[24] J M Llovet and J Bruix ldquoMolecular targeted therapies inhepatocellular carcinomardquo Hepatology vol 48 no 4 pp 1312ndash1327 2008

[25] Y-H Chen H-L Wu C Li et al ldquoAnti-angiogenesis mediatedby angiostatin K1-3 K1-4 and K1-45 Involvement of p53 FasLAKT and mRNA deregulationrdquo Thrombosis and Haemostasisvol 95 no 4 pp 668ndash677 2006

[26] J I Weidong-Richard F J Castellino Y Chang et al ldquoChar-acterization of kringle domains of angiostatin as antagonists ofendothelial cell migration an important process in angiogene-sisrdquoThe FASEB Journal vol 12 no 15 pp 1731ndash1738 1998

[27] H A Hanford C A Wong H Kassan et al ldquoAngiostatin45-mediated apoptosis of vascular endothelial cellsrdquo CancerResearch vol 63 no 14 pp 4275ndash4280 2003

[28] T-Y Lee S Muschal E A Pravda J Folkman A Abdollahiand K Javaherian ldquoAngiostatin regulates the expression ofantiangiogenic and proapoptotic pathways via targeted inhibi-tion of mitochondrial proteinsrdquo Blood vol 114 no 9 pp 1987ndash1998 2009

[29] ldquoAssessment of adenoviral vector safety and toxicity report ofthe National Institutes of Health Recombinant DNA AdvisoryCommitteerdquoHuman GeneTherapy vol 13 no 1 pp 3ndash13 2002

[30] S Indraccolo S Minuzzo E Gola et al ldquoGeneration ofexpression plasmids for angiostatin endostatin and TIMP-2for cancer gene therapyrdquo International Journal of BiologicalMarkers vol 14 no 4 pp 251ndash256 1999

[31] X Sun H Qiao H Jiang et al ldquoIntramuscular delivery ofantiangiogenic genes suppresses secondary metastases afterremoval of primary tumorsrdquo Cancer Gene Therapy vol 12 no1 pp 35ndash45 2005

[32] Q-R Chen D Kumar S A Stass andA JMixson ldquoLiposomescomplexed to plasmids encoding angiostatin and endostatininhibit breast cancer in nudemicerdquoCancer Research vol 59 no14 pp 3308ndash3312 1999

[33] J Li Q Yin and HWu ldquoStructural basis of signal transductionin the TNF receptor superfamilyrdquoAdvances in Immunology vol119 pp 135ndash153 2013

[34] S Giraud F Bienvenu S Avril H Gascan D M Heery andO Coqueret ldquoFunctional interaction of STAT3 transcriptionfactor with the coactivator NcoASRC1ardquo Journal of BiologicalChemistry vol 277 no 10 pp 8004ndash8011 2002

[35] S Aznar P F Valeron S V Del Rincon L F Perez RPerona and J C Lacal ldquoSimultaneous tyrosine and serinephosphorylation of STAT3 transcription factor is involved inRhoA GTPase oncogenic transformationrdquoMolecular Biology ofthe Cell vol 12 no 10 pp 3282ndash3294 2001

[36] Y B Shaik-Dasthagirisaheb G Varvara G Murmura et alldquoVascular endothelial growth factor (VEGF) mast cells andinflammationrdquo International Journal of Immunopathology andPharmacology vol 26 no 2 pp 327ndash335 2013

[37] M Azimi-Nezhad M G Stathopoulou A Bonnefond et alldquoAssociations of vascular endothelial growth factor (VEGF)with adhesion and inflammation molecules in a healthy pop-ulationrdquo Cytokine vol 61 no 2 pp 602ndash607 2013

[38] A W Y Chung Y N Hsiang L A Matzke B M McManusC Van Breemen and E B Okon ldquoReduced expression ofvascular endothelial growth factor paralleled with the increasedangiostatin expression resulting from the upregulated activitiesof matrix metalloproteinase-2 and -9 in human type 2 diabeticarterial vasculaturerdquoCirculation Research vol 99 no 2 pp 140ndash148 2006

[39] H Yang Z Xu P M Iuvone and H E GrossniklausldquoAngiostatin decreases cell migration and vascular endotheliumgrowth factor (VEGF) to pigment epithelium derived factor(PEDF) RNA ratio in vitro and in a murine ocular melanomamodelrdquoMolecular Vision vol 12 pp 511ndash517 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


tic effect on VEGF [16 17] However to our knowledge thishas not been shown for PlgK1-5

Many studies used an adenoviral vector approach toinvestigate the antitumoral effects of PlgK1-5 [8 10 18 19]Yet adenovirus based therapies are prone to cause vehementallergic reactions in follow-up treatments [20]

Therefore we analysed effects of plasmid vectormediatedgene transfer of PlgK1-5 on hepatomas in vitro and in vivoSince angiostatic effects are well documented we placedour focus on other factors We examined proapoptoticantiproliferative and proinflammatory pathways in vitro andin vivo Additionally we wanted to further distinguish thelink between inflammation and angiogenesis

2 Materials and Methods

21 Animals and Cell Lines Eight-week-old male C3H micewere supplied by Charles River (Sulzfeld Germany) and keptin the local central animal facility of the University HospitalBonn The mice were housed under standard conditionsand had free access to water and food Animal procedureswere performed in accordance with approved protocolsand followed recommendations for proper care and use oflaboratory animals

The hepatocyte derived cellular carcinoma cell lineHuH7 and the mouse hepatoma cell line Hepa1-6 (bothobtained from American Type Culture Collection (ATCCRockvilleMD)) were cultivated inDulbeccorsquosmodified Eaglemedium (DMEM) supplemented with 10 heat-inactivatedfetal bovine serum (FBS)

Hepa129 cells (Mouse Hepatoma 129 obtained fromNCI-Frederick Cancer Research and Development Centre(DCT Tumour Repository)) were maintained in RPMI1640supplemented with 10 FBS 200mM glutamine

22 Synthesis and Amplification of pCMVTNT Vectors Frag-ments encoding plasminogen K1-5 (PlgK1-5 amino acids 1-546) were generated and validated as described previously[8] PlgK1-5 was then subcloned into the eukaryotic expres-sion vector pCMVTNT (Promega Mannheim Germany)according to the manufacturerrsquos protocol correct insertionwas determined through enzymatic restriction digestion andgel electrophoresis of pCMVTNT-K1-5 (pK1-5) on a 1agarose gel pCMVTNTwithout transgene (pMock)was usedas a vector control

pK1-5 and pMock were amplified using the E coli K12strain DH10B and plasmid vectors were isolated using theNucleoBond Xtra Maxi Plus EF system (Macherey-NagelDuren Germany) DNA content and purity were determinedby measuring the OD260280 ratio

23 Plasmid Transfection In Vitro and In Vivo In vitro 106cells (HuH7 Hepa1-6 Hepa129) were seeded on 10 cm platesand cultured in antibiotic-free medium One day later cellswere transfectedwith either pK1-5 or pMock (119899=4per group)using Lipofectamine 2000 (Invitrogen Karlsruhe Germany)according to the manufacturerrsquos protocol Cells treated withplain culture medium were used as negative controls

For establishment of subcutaneous tumours 106 Hepa129cells in 150120583L plain RPMI were injected subcutaneously intothe paramedian hind of male C3Hmice [8] Reaching ameantumour diameter of 150 cm3 treatment was started In a two-day intervalmicewere treatedwith intraperitoneal injectionswith NaCl (150120583L NaClmouse 119899 = 5) DOTAP (Carl RothKarlsruhe Germany) alone (100120583L NaCl with 50 120583L DOTAPper animal 119899 = 6) pMock (50120583g pMock in 100 120583L NaCl and50 120583L DOTAP 119899 = 6) or pK1-5 (50120583g pK1-5 in 100 120583L NaClwith 50120583L DOTAP 119899 = 5)

24 Analysis of K1-5 Protein Expression In Vitro and InVivo To determine K1-5 expression in vitro cells weretransfected as described above Two days later the cellswere harvested in 100120583L PBS with protease inhibitors(Complete Roche Diagnostics Mannheim Germany) Foranalysis of K1-5 expression in vivo liver and tumour tissues(50mm3) were harvested after termination of the in vivoexperiments Tumour and liver tissues were homogenisedin 500120583L PBS and protease inhibitors (Complete) using thePrecellys system (Peqlab Heidelberg Germany) Cells andtissue homogenisates were then lysed by repeated freeze-thaw cycles Protein concentrations of tissue and cells weredetermined using the DC protein assay (Biorad MunichGermany) according to the manufacturerrsquos protocol

50120583g protein was used for SDS-polyacrylamide gelelectrophoresis Proteins were transferred on PVDF mem-brane (Biorad) Detection of K1-5 was done using an anti-angiostatin antibody (A1101 Sigma-Aldrich Hamburg Ger-many) and a corresponding secondary antibody (rabbit anti-goat Santa Cruz Heidelberg Germany) GAPDH was usedas a control (sc-25778 Santa Cruz) Chemiluminescent signalwas developed using AceGlow (Peqlab) and the Chemismartsystem with ChemiCapt software (Peqlab)

25 Determination of Tumour Growth In Vivo and SampleCollection Tumour growth was determined every secondday bymeasuringmaximum length andwidth using a caliperTumour volumes were calculated as 119881 = length times (width)2 times052

Ten days after treatment initiation mice were sacrificedand tumours and livers were explanted Blood was collectedTumour liver and blood samples were harvested and con-served at minus80∘C after freezing in liquid nitrogen Tumoursamples were embedded in Tissue Tek (Sakura FinetekHeppenheim Germany) and cryopreserved for immunohis-tochemistry

26 Determination of Cell Viability One day prior totransfection 104 cells were seeded onto 96-well-platesand then cultivated in antibiotic-free medium 100 120583L 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT) was added to the cells 48 hours after transfectionCells were then incubated for 45 minutes Supernatants wereremoved and MTT was solubilized with 100 120583L dimethylsulfoxide (DMSO) Optical density was measured colorimet-rically at 560 nm (GloMax Multi ELISA reader PromegaMannheim Germany)
















3 Results







HuH7


GAPDH

Hepa129

K1-5K1-5K1-5

Hepa1-6

PlgK1-5

(a)

Dotap

Liver Tumour


GAPDH

K1-5 K1-5 K1-5 K1-5

PlgK1-5

(b)












07

06

05

04

03

02

01

00HuH7

NaCl

lowast lowast

lowast

lowastlowast

lowast

Hepa129Hepa1-6

Relat

ive u

nits

pMockpK1-5

(a)

110

100

90

80

70

60

50

40

30

20

10

0

()

HuH7

NaCl

Hepa129Hepa1-6

pMockpK1-5

(b)

500

450

400

350

300

200

250

150

125

100

75

50

25

0

()

HuH7

NaCl

Hepa129

lowast

lowast

Hepa1-6

pMockpK1-5

(c)










225

200

175

150

125

100

75

50

25

0

Pg V

EGF100120583

g pr

otei

n

Plain mediumpMock

HuH7 Hepa129

lowastlowast

lowast

Hepa1-6

pK1-5

(a)

lowast

lowast

800

700

600

250

200

150

100

50

0

Pg V

EGF

mL

cell

supe

rnat

ant

Plain mediumpMock

HuH7 Hepa129Hepa1-6

pK1-5

(b)














280260240220200180160140120

40

20

0

lowast

lowast

Plain mediumpMock

HuH7 Hepa129Hepa1-6

pK1-5

TNF-

alph

a100120583

g pr

otei

n






4 Discussion










lowast

lowast lowast

12000

10000

8000

6000

4000

2000

0

Relat

ive fl

uore

scen

ce u

nits

[RFU

]

Plain mediumpMock

Total STAT3 pSTAT3

pK1-5

(a)

20000

18000

16000

14000

12000

10000

8000

6000

4000

2000

0

lowast

Relat

ive fl

uore

scen

ce u

nits

[RFU

]

Plain mediumpMock

Total STAT3 pSTAT3

pK1-5

(b)

2500

2250

2000

1750

1500

1250

1000

750

500

250

0

lowast

lowast

Relat

ive fl

uore

scen

ce u

nits

[RFU

]

Plain mediumpMock

Total STAT3 pSTAT3

pK1-5

(c)









3500

3000

2500

2000

1500

1000

500

0

0 2 4 6 8 10


Mea

n tu

mor

vol

ume (

mm

3)

NaClDOTAP

pMockpK1-5

(a)

400

350

300

250

200

150

100

50

0

∘lowast

lowast

Mito

tic ce

llsh

pf

NaClDOTAP

pMockpK1-5

(b)

NaCl DOTAP

pMock pK1-5

(c)



140

120

100

80

60

40

20

0

lowast

TumourPg

VEG

F100120583

g pr

otei

n


(a)

Pg V

EGF100120583

g pr

otei

n

NaCl DOTAP pMock

2250

2000

1750

1500

1250

1000

750

500

250

0

Liver

pK1-5

(b)

NaCl DOTAP

pMock pK1-5

(c)






Tumour24

22

20

18

16

14

12

10

8

6

4

2

0

∘


pg T

NF-

alph

a100120583

g pr

otei

n

(a)

4000

3500

3000

2500

2000

1500

1000

500

0

Liver


TNF-

alph

a100120583

g pr

otei

n

(b)

NaCl DOTAP

pMock pK1-5

(c)







∘lowast

80

60

40

20

0

Mic

rove

ssels

hpf

NaClDOTAP

pMockpK1-5

(a)

NaCl DOTAP

pMock pK1-5

(b)













Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































3 Results







HuH7


GAPDH

Hepa129

K1-5K1-5K1-5

Hepa1-6

PlgK1-5

(a)

Dotap

Liver Tumour


GAPDH

K1-5 K1-5 K1-5 K1-5

PlgK1-5

(b)












07

06

05

04

03

02

01

00HuH7

NaCl

lowast lowast

lowast

lowastlowast

lowast

Hepa129Hepa1-6

Relat

ive u

nits

pMockpK1-5

(a)

110

100

90

80

70

60

50

40

30

20

10

0

()

HuH7

NaCl

Hepa129Hepa1-6

pMockpK1-5

(b)

500

450

400

350

300

200

250

150

125

100

75

50

25

0

()

HuH7

NaCl

Hepa129

lowast

lowast

Hepa1-6

pMockpK1-5

(c)










225

200

175

150

125

100

75

50

25

0

Pg V

EGF100120583

g pr

otei

n

Plain mediumpMock

HuH7 Hepa129

lowastlowast

lowast

Hepa1-6

pK1-5

(a)

lowast

lowast

800

700

600

250

200

150

100

50

0

Pg V

EGF

mL

cell

supe

rnat

ant

Plain mediumpMock

HuH7 Hepa129Hepa1-6

pK1-5

(b)














280260240220200180160140120

40

20

0

lowast

lowast

Plain mediumpMock

HuH7 Hepa129Hepa1-6

pK1-5

TNF-

alph

a100120583

g pr

otei

n






4 Discussion










lowast

lowast lowast

12000

10000

8000

6000

4000

2000

0

Relat

ive fl

uore

scen

ce u

nits

[RFU

]

Plain mediumpMock

Total STAT3 pSTAT3

pK1-5

(a)

20000

18000

16000

14000

12000

10000

8000

6000

4000

2000

0

lowast

Relat

ive fl

uore

scen

ce u

nits

[RFU

]

Plain mediumpMock

Total STAT3 pSTAT3

pK1-5

(b)

2500

2250

2000

1750

1500

1250

1000

750

500

250

0

lowast

lowast

Relat

ive fl

uore

scen

ce u

nits

[RFU

]

Plain mediumpMock

Total STAT3 pSTAT3

pK1-5

(c)









3500

3000

2500

2000

1500

1000

500

0

0 2 4 6 8 10


Mea

n tu

mor

vol

ume (

mm

3)

NaClDOTAP

pMockpK1-5

(a)

400

350

300

250

200

150

100

50

0

∘lowast

lowast

Mito

tic ce

llsh

pf

NaClDOTAP

pMockpK1-5

(b)

NaCl DOTAP

pMock pK1-5

(c)



140

120

100

80

60

40

20

0

lowast

TumourPg

VEG

F100120583

g pr

otei

n


(a)

Pg V

EGF100120583

g pr

otei

n

NaCl DOTAP pMock

2250

2000

1750

1500

1250

1000

750

500

250

0

Liver

pK1-5

(b)

NaCl DOTAP

pMock pK1-5

(c)






Tumour24

22

20

18

16

14

12

10

8

6

4

2

0

∘


pg T

NF-

alph

a100120583

g pr

otei

n

(a)

4000

3500

3000

2500

2000

1500

1000

500

0

Liver


TNF-

alph

a100120583

g pr

otei

n

(b)

NaCl DOTAP

pMock pK1-5

(c)







∘lowast

80

60

40

20

0

Mic

rove

ssels

hpf

NaClDOTAP

pMockpK1-5

(a)

NaCl DOTAP

pMock pK1-5

(b)













Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HuH7


GAPDH

Hepa129

K1-5K1-5K1-5

Hepa1-6

PlgK1-5

(a)

Dotap

Liver Tumour


GAPDH

K1-5 K1-5 K1-5 K1-5

PlgK1-5

(b)












07

06

05

04

03

02

01

00HuH7

NaCl

lowast lowast

lowast

lowastlowast

lowast

Hepa129Hepa1-6

Relat

ive u

nits

pMockpK1-5

(a)

110

100

90

80

70

60

50

40

30

20

10

0

()

HuH7

NaCl

Hepa129Hepa1-6

pMockpK1-5

(b)

500

450

400

350

300

200

250

150

125

100

75

50

25

0

()

HuH7

NaCl

Hepa129

lowast

lowast

Hepa1-6

pMockpK1-5

(c)










225

200

175

150

125

100

75

50

25

0

Pg V

EGF100120583

g pr

otei

n

Plain mediumpMock

HuH7 Hepa129

lowastlowast

lowast

Hepa1-6

pK1-5

(a)

lowast

lowast

800

700

600

250

200

150

100

50

0

Pg V

EGF

mL

cell

supe

rnat

ant

Plain mediumpMock

HuH7 Hepa129Hepa1-6

pK1-5

(b)














280260240220200180160140120

40

20

0

lowast

lowast

Plain mediumpMock

HuH7 Hepa129Hepa1-6

pK1-5

TNF-

alph

a100120583

g pr

otei

n






4 Discussion










lowast

lowast lowast

12000

10000

8000

6000

4000

2000

0

Relat

ive fl

uore

scen

ce u

nits

[RFU

]

Plain mediumpMock

Total STAT3 pSTAT3

pK1-5

(a)

20000

18000

16000

14000

12000

10000

8000

6000

4000

2000

0

lowast

Relat

ive fl

uore

scen

ce u

nits

[RFU

]

Plain mediumpMock

Total STAT3 pSTAT3

pK1-5

(b)

2500

2250

2000

1750

1500

1250

1000

750

500

250

0

lowast

lowast

Relat

ive fl

uore

scen

ce u

nits

[RFU

]

Plain mediumpMock

Total STAT3 pSTAT3

pK1-5

(c)









3500

3000

2500

2000

1500

1000

500

0

0 2 4 6 8 10


Mea

n tu

mor

vol

ume (

mm

3)

NaClDOTAP

pMockpK1-5

(a)

400

350

300

250

200

150

100

50

0

∘lowast

lowast

Mito

tic ce

llsh

pf

NaClDOTAP

pMockpK1-5

(b)

NaCl DOTAP

pMock pK1-5

(c)



140

120

100

80

60

40

20

0

lowast

TumourPg

VEG

F100120583

g pr

otei

n


(a)

Pg V

EGF100120583

g pr

otei

n

NaCl DOTAP pMock

2250

2000

1750

1500

1250

1000

750

500

250

0

Liver

pK1-5

(b)

NaCl DOTAP

pMock pK1-5

(c)






Tumour24

22

20

18

16

14

12

10

8

6

4

2

0

∘


pg T

NF-

alph

a100120583

g pr

otei

n

(a)

4000

3500

3000

2500

2000

1500

1000

500

0

Liver


TNF-

alph

a100120583

g pr

otei

n

(b)

NaCl DOTAP

pMock pK1-5

(c)







∘lowast

80

60

40

20

0

Mic

rove

ssels

hpf

NaClDOTAP

pMockpK1-5

(a)

NaCl DOTAP

pMock pK1-5

(b)













Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















07

06

05

04

03

02

01

00HuH7

NaCl

lowast lowast

lowast

lowastlowast

lowast

Hepa129Hepa1-6

Relat

ive u

nits

pMockpK1-5

(a)

110

100

90

80

70

60

50

40

30

20

10

0

()

HuH7

NaCl

Hepa129Hepa1-6

pMockpK1-5

(b)

500

450

400

350

300

200

250

150

125

100

75

50

25

0

()

HuH7

NaCl

Hepa129

lowast

lowast

Hepa1-6

pMockpK1-5

(c)










225

200

175

150

125

100

75

50

25

0

Pg V

EGF100120583

g pr

otei

n

Plain mediumpMock

HuH7 Hepa129

lowastlowast

lowast

Hepa1-6

pK1-5

(a)

lowast

lowast

800

700

600

250

200

150

100

50

0

Pg V

EGF

mL

cell

supe

rnat

ant

Plain mediumpMock

HuH7 Hepa129Hepa1-6

pK1-5

(b)














280260240220200180160140120

40

20

0

lowast

lowast

Plain mediumpMock

HuH7 Hepa129Hepa1-6

pK1-5

TNF-

alph

a100120583

g pr

otei

n






4 Discussion










lowast

lowast lowast

12000

10000

8000

6000

4000

2000

0

Relat

ive fl

uore

scen

ce u

nits

[RFU

]

Plain mediumpMock

Total STAT3 pSTAT3

pK1-5

(a)

20000

18000

16000

14000

12000

10000

8000

6000

4000

2000

0

lowast

Relat

ive fl

uore

scen

ce u

nits

[RFU

]

Plain mediumpMock

Total STAT3 pSTAT3

pK1-5

(b)

2500

2250

2000

1750

1500

1250

1000

750

500

250

0

lowast

lowast

Relat

ive fl

uore

scen

ce u

nits

[RFU

]

Plain mediumpMock

Total STAT3 pSTAT3

pK1-5

(c)









3500

3000

2500

2000

1500

1000

500

0

0 2 4 6 8 10


Mea

n tu

mor

vol

ume (

mm

3)

NaClDOTAP

pMockpK1-5

(a)

400

350

300

250

200

150

100

50

0

∘lowast

lowast

Mito

tic ce

llsh

pf

NaClDOTAP

pMockpK1-5

(b)

NaCl DOTAP

pMock pK1-5

(c)



140

120

100

80

60

40

20

0

lowast

TumourPg

VEG

F100120583

g pr

otei

n


(a)

Pg V

EGF100120583

g pr

otei

n

NaCl DOTAP pMock

2250

2000

1750

1500

1250

1000

750

500

250

0

Liver

pK1-5

(b)

NaCl DOTAP

pMock pK1-5

(c)






Tumour24

22

20

18

16

14

12

10

8

6

4

2

0

∘


pg T

NF-

alph

a100120583

g pr

otei

n

(a)

4000

3500

3000

2500

2000

1500

1000

500

0

Liver


TNF-

alph

a100120583

g pr

otei

n

(b)

NaCl DOTAP

pMock pK1-5

(c)







∘lowast

80

60

40

20

0

Mic

rove

ssels

hpf

NaClDOTAP

pMockpK1-5

(a)

NaCl DOTAP

pMock pK1-5

(b)













Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















225

200

175

150

125

100

75

50

25

0

Pg V

EGF100120583

g pr

otei

n

Plain mediumpMock

HuH7 Hepa129

lowastlowast

lowast

Hepa1-6

pK1-5

(a)

lowast

lowast

800

700

600

250

200

150

100

50

0

Pg V

EGF

mL

cell

supe

rnat

ant

Plain mediumpMock

HuH7 Hepa129Hepa1-6

pK1-5

(b)














280260240220200180160140120

40

20

0

lowast

lowast

Plain mediumpMock

HuH7 Hepa129Hepa1-6

pK1-5

TNF-

alph

a100120583

g pr

otei

n






4 Discussion










lowast

lowast lowast

12000

10000

8000

6000

4000

2000

0

Relat

ive fl

uore

scen

ce u

nits

[RFU

]

Plain mediumpMock

Total STAT3 pSTAT3

pK1-5

(a)

20000

18000

16000

14000

12000

10000

8000

6000

4000

2000

0

lowast

Relat

ive fl

uore

scen

ce u

nits

[RFU

]

Plain mediumpMock

Total STAT3 pSTAT3

pK1-5

(b)

2500

2250

2000

1750

1500

1250

1000

750

500

250

0

lowast

lowast

Relat

ive fl

uore

scen

ce u

nits

[RFU

]

Plain mediumpMock

Total STAT3 pSTAT3

pK1-5

(c)









3500

3000

2500

2000

1500

1000

500

0

0 2 4 6 8 10


Mea

n tu

mor

vol

ume (

mm

3)

NaClDOTAP

pMockpK1-5

(a)

400

350

300

250

200

150

100

50

0

∘lowast

lowast

Mito

tic ce

llsh

pf

NaClDOTAP

pMockpK1-5

(b)

NaCl DOTAP

pMock pK1-5

(c)



140

120

100

80

60

40

20

0

lowast

TumourPg

VEG

F100120583

g pr

otei

n


(a)

Pg V

EGF100120583

g pr

otei

n

NaCl DOTAP pMock

2250

2000

1750

1500

1250

1000

750

500

250

0

Liver

pK1-5

(b)

NaCl DOTAP

pMock pK1-5

(c)






Tumour24

22

20

18

16

14

12

10

8

6

4

2

0

∘


pg T

NF-

alph

a100120583

g pr

otei

n

(a)

4000

3500

3000

2500

2000

1500

1000

500

0

Liver


TNF-

alph

a100120583

g pr

otei

n

(b)

NaCl DOTAP

pMock pK1-5

(c)







∘lowast

80

60

40

20

0

Mic

rove

ssels

hpf

NaClDOTAP

pMockpK1-5

(a)

NaCl DOTAP

pMock pK1-5

(b)













Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















280260240220200180160140120

40

20

0

lowast

lowast

Plain mediumpMock

HuH7 Hepa129Hepa1-6

pK1-5

TNF-

alph

a100120583

g pr

otei

n






4 Discussion










lowast

lowast lowast

12000

10000

8000

6000

4000

2000

0

Relat

ive fl

uore

scen

ce u

nits

[RFU

]

Plain mediumpMock

Total STAT3 pSTAT3

pK1-5

(a)

20000

18000

16000

14000

12000

10000

8000

6000

4000

2000

0

lowast

Relat

ive fl

uore

scen

ce u

nits

[RFU

]

Plain mediumpMock

Total STAT3 pSTAT3

pK1-5

(b)

2500

2250

2000

1750

1500

1250

1000

750

500

250

0

lowast

lowast

Relat

ive fl

uore

scen

ce u

nits

[RFU

]

Plain mediumpMock

Total STAT3 pSTAT3

pK1-5

(c)









3500

3000

2500

2000

1500

1000

500

0

0 2 4 6 8 10


Mea

n tu

mor

vol

ume (

mm

3)

NaClDOTAP

pMockpK1-5

(a)

400

350

300

250

200

150

100

50

0

∘lowast

lowast

Mito

tic ce

llsh

pf

NaClDOTAP

pMockpK1-5

(b)

NaCl DOTAP

pMock pK1-5

(c)



140

120

100

80

60

40

20

0

lowast

TumourPg

VEG

F100120583

g pr

otei

n


(a)

Pg V

EGF100120583

g pr

otei

n

NaCl DOTAP pMock

2250

2000

1750

1500

1250

1000

750

500

250

0

Liver

pK1-5

(b)

NaCl DOTAP

pMock pK1-5

(c)






Tumour24

22

20

18

16

14

12

10

8

6

4

2

0

∘


pg T

NF-

alph

a100120583

g pr

otei

n

(a)

4000

3500

3000

2500

2000

1500

1000

500

0

Liver


TNF-

alph

a100120583

g pr

otei

n

(b)

NaCl DOTAP

pMock pK1-5

(c)







∘lowast

80

60

40

20

0

Mic

rove

ssels

hpf

NaClDOTAP

pMockpK1-5

(a)

NaCl DOTAP

pMock pK1-5

(b)













Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowast

lowast lowast

12000

10000

8000

6000

4000

2000

0

Relat

ive fl

uore

scen

ce u

nits

[RFU

]

Plain mediumpMock

Total STAT3 pSTAT3

pK1-5

(a)

20000

18000

16000

14000

12000

10000

8000

6000

4000

2000

0

lowast

Relat

ive fl

uore

scen

ce u

nits

[RFU

]

Plain mediumpMock

Total STAT3 pSTAT3

pK1-5

(b)

2500

2250

2000

1750

1500

1250

1000

750

500

250

0

lowast

lowast

Relat

ive fl

uore

scen

ce u

nits

[RFU

]

Plain mediumpMock

Total STAT3 pSTAT3

pK1-5

(c)









3500

3000

2500

2000

1500

1000

500

0

0 2 4 6 8 10


Mea

n tu

mor

vol

ume (

mm

3)

NaClDOTAP

pMockpK1-5

(a)

400

350

300

250

200

150

100

50

0

∘lowast

lowast

Mito

tic ce

llsh

pf

NaClDOTAP

pMockpK1-5

(b)

NaCl DOTAP

pMock pK1-5

(c)



140

120

100

80

60

40

20

0

lowast

TumourPg

VEG

F100120583

g pr

otei

n


(a)

Pg V

EGF100120583

g pr

otei

n

NaCl DOTAP pMock

2250

2000

1750

1500

1250

1000

750

500

250

0

Liver

pK1-5

(b)

NaCl DOTAP

pMock pK1-5

(c)






Tumour24

22

20

18

16

14

12

10

8

6

4

2

0

∘


pg T

NF-

alph

a100120583

g pr

otei

n

(a)

4000

3500

3000

2500

2000

1500

1000

500

0

Liver


TNF-

alph

a100120583

g pr

otei

n

(b)

NaCl DOTAP

pMock pK1-5

(c)







∘lowast

80

60

40

20

0

Mic

rove

ssels

hpf

NaClDOTAP

pMockpK1-5

(a)

NaCl DOTAP

pMock pK1-5

(b)













Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















3500

3000

2500

2000

1500

1000

500

0

0 2 4 6 8 10


Mea

n tu

mor

vol

ume (

mm

3)

NaClDOTAP

pMockpK1-5

(a)

400

350

300

250

200

150

100

50

0

∘lowast

lowast

Mito

tic ce

llsh

pf

NaClDOTAP

pMockpK1-5

(b)

NaCl DOTAP

pMock pK1-5

(c)



140

120

100

80

60

40

20

0

lowast

TumourPg

VEG

F100120583

g pr

otei

n


(a)

Pg V

EGF100120583

g pr

otei

n

NaCl DOTAP pMock

2250

2000

1750

1500

1250

1000

750

500

250

0

Liver

pK1-5

(b)

NaCl DOTAP

pMock pK1-5

(c)






Tumour24

22

20

18

16

14

12

10

8

6

4

2

0

∘


pg T

NF-

alph

a100120583

g pr

otei

n

(a)

4000

3500

3000

2500

2000

1500

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Acknowledgments


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140

120

100

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Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Tumour24

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20

18

16

14

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10

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4000

3500

3000

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1500

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(b)













Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















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60

40

20

0

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rove

ssels

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(b)













Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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