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CHAPTER 2

CHAPTER 2 Proteomic analysis of PAP and proangiogenic activity

Cellular Signalling 25 (2013) 277–294 34

2. INTRODUCTION

ngiogenesis, or neovascularization, is a complex process leading to

formation of new blood vessels from the pre-existing vascular

network of the tissue. Angiogenesis plays a central role in various

physiological and pathological conditions [Szekanecz et al., 2010]. Numerous

inducers of angiogenesis have been identified, including members of the

VEGF and of the FGF families [Carmeliet, 2005]. These angiogenic growth

factors induce a complex “proangiogenic phenotype” in endothelial cells that

recapitulates several aspects of the in vivo angiogenesis process. VEGF-A

exerts proangiogenic effects by binding to the endothelial cell specific

tyrosine-kinase receptor VEGFR2 leading to autophosphorylation of the

receptor. A study has shown that inhibition of p38MAPK activity abrogated

VEGF induced vascular permeability in vivo and in vitro, suggesting the

involvement of p38MAPK in the control of vascular permeability [Issbrücker

et.al 2003]. The c-Jun NH2-terminal kinase (JNK), is a subgroup of MAP

kinase stimulated by mitogens, inflammatory cytokines and inducers of cell

differentiation. However, the cross-talk between ERK and JNK pathways in

response to angiogenic factors (apparently identical to that derived from

tumors) has not been well documented under pathological conditions. Finally,

Ang1 and Ang2 have been shown to be required for the formation of mature

blood vessels, as demonstrated by mouse knockout studies [Thurston, 2003].

Understanding the basic mechanisms will therefore help in controlling and/or

inhibiting angiogenesis. The redundancy in angiogenic factor expression

suggests that inhibition of VEGF bioactivity alone might not be a sufficient

approach for antiangiogenic therapy. In our previous study, novel approaches

for targeted therapy were developed using the angiopoietin/Tie-2 system. The

effect of sTie-2 treatment alone or in combination with sFlt-1 was used to

sequester angiopoietins in the murine ascites carcinoma model [D'Souza et al

2010, Ramachandra et al.,2009]. Besides anti-angiogenic cocktails,

combining anti-angiogenic and vascular disruption strategies can lead to a

very efficient treatment against tumor examples like the enhancement of

radiation therapy by ZD6126 (AstraZeneca) in KHT sarcomamodel [Landuyt

A



et al.,2001] and the combination of Avastin with Fluorouracil, Irinotecan and

Leucovorin in metastatic colorectal cancer [Hurwitz et.al,2004]. Despite the

fact that these approaches put forward an innovative idea for successful

cancer treatment, at present there are a number of problems in clinical trials

on humans that require very attentive studies and critical interpretations.

Therefore, angiogenic profiling is a very important tool that helps deciding on

the most appropriate combination of different therapies for each patient.

Different novel proangiogenic molecules have been isolated and shown to

trigger angiogenesis. Zhang et al., 2009 genetic studies observed that Slit3 is

a novel angiogenic factor. In another report Stabile et al., 2007 identified

Drm/gremlin as a novel proangiogenic factor expressed by endothelium. In

one study Ho et al., 2004 showed Del-1, a novel angiogenic role in ischemia.

The authors Hong et al., 2005 identified MCP-1 as an angiogenic

chemokines. Hu et al., 2007 identified brain derived neurotrophic factor as a

novel angiogenic protein in multiple myeloma and Dardik et al. showed novel

proangiogenic effect of Factor XIII. Understanding the complete mechanisms

of angiogenesis/vasculogenesis, including the knowledge of the involved

angiogenic factors, may provide new insights and possible approaches for the

treatment of cancer patients and perhaps even prognosis. To date, several

purification procedures have been reported for angiogenesis factor extracted

from cells and extracellular form. The starting materials for these purification

procedures have been lysates of tumor cells [Fenselau et al.,1981, Weiss et

al.,1979 and Folkman et al., 1971], an extract of retinal cell [D'Amore et al.,

1981], Synovium tissue [Weiler et al.,2007], and wound fluid [Banda et

al.,1982]. The hyperplastic synovial pannus in rheumatoid arthritis resembles

a solid tumor in certain ways, especially in its intrinsic cellular proliferation, its

invasive properties and the association of angiogenesis with the development

of this highly vascularized tissue [Pufe et al., 2003]. Rheumatoid arthritis (RA)

synovial fluids (SF) were shown to induce morphological changes in human

endothelial cells, with formation of tube-like structure and induction of

angiogenesis in an in vitro assay [Thairu et al., 2011]. Thus, it is well founded

to consider SF as a source of novel proteins involved in the action of

angiogenesis. Although such reports indicate that angiogenesis factor in SFs



from RA (apparently identical from tumors) plays an important role in the

ability to induce angiogenesis, the mechanism by with these factors regulates

the processes is yet to be understood. In this study we explore the molecular

mechanisms underlying the proangiogenic activity of novel proangiogenic

protein (PAP). In this study we have purified and characterized a novel

proangiogenic protein (PAP) from SF of RA patients, with an apparent

molecular weight of 67 kDa, and matrix-assisted laser desorption/ ionization

time-of-flight mass spectrometry (MALDI-TOF-MS) and nano-ESI-MS/MS

were conducted for peptide profile. N-terminal amino acid sequence showed

no identity to sequence currently available. The angiogenic activity of PAP

was revealed in both in vivo chicken chorioallantoic membrane (CAM) and rat

corneal micropocket bioassays.

In vitro, PAP enhanced angiogenic properties of HUVEC, with an increased

cell proliferation and capacity to establish capillary like structures. A

competitive enzyme-linked immunosorbent assay (ELISA) confirmed the

presence ofmicrogramlevels of PAP in the cytosolic extract of tumor cells and

synovial fluids of arthritic patients. This finding is in accordance with the

observation of the presence of PAP in tumor cells by Western blot and

Immunofluorescence, and in clinical specimens of different grades of breast

cancer biopsy samples. Promoter reporter gene assay indicates that PAP

induces VEGF and Flt-1 gene expression. PAP induces NFκB–DNA binding

transcriptional programs in promoting tumorigenesis including cell migration.

Consistent with a proangiogenic role, PAP induced angiogenesis is mediated

through pathways involving VEGF and MAP kinases. On the other hand, the

genetic and molecular events underlying the structural and functional

differences between normal and tumor vasculature are constantly being

revealed, suggesting the possibility of producing more specific and clinically

meaningful mAbs. In vitro and in vivo functional analysis indicated that anti-

PAP-mAb reduced neoangiogenesis. These data provide new insights into the

mechanism underlying the proangiogenic activity of PAP and the anti-PAP-

mAb has therapeutic potential for antiangiogenic therapy of cancer.

CHAPTER 2 Materials & Methods


2.1. MATERIALS

2.1.1. RECRUITMENT OF PATIENTS

Recruitment of patients diagnosed with RA, aged between 38 and 67 years

(10 numbers), was made as per the guidelines and protocol of the Institutional

review board and Department of Pathology, J.S.S. Hospital, Mysore, India.

Informed consent was obtained from all the patients. Synovial fluid (SF)

samples were collected in sterile tubes, centrifuged at 10,000 g for 10 min at

4 °C. The cell free supernatant from all 10 patient s was pooled. Human breast

lesion tissue samples were collected with informed consent, from either

diagnostic biopsies or upon surgery from the Department of Pathology, J.S.S.

Hospital, Mysore, India. Based on clinical investigation they were classified as

invasive ductal carcinoma of the breast

2.1.2. ANIMALS

Swiss albino mice (6–8 weeks old), Balb/c mice (6–8 weeks old), Wistar rats

(4–6 months old) and New Zealand white rabbit (3 months old) were obtained

from the central animal facility, Department of Zoology, University of Mysore,

and Mysore, India. All the animal experiments were approved by the

Institutional animal ethics committee, University of Mysore, Mysore and

studies were conducted according to guidelines of the committee for purpose

of control and supervision of experiments on animals (CPCSEA), Government

of India, India.

2.1.3. REAGENTS

Sephadex G-100 was purchased from Pharmacia fine chemicals, Uppsala,

Sweeden. Protein molecular weight marker for SDS-PAGE was procured

from MBI Fermentas, Hanover, MD, USA. Periodic Acid Schiff’s staining

(PAS) for glycoprotein, Sodium meta-periodate, Freund’s complete and

incomplete adjuvant, ethylene glycol tetra acetic acid (EGTA), Ammonium

bicarbonate, Iadoacetamide (IAA), Dithio-thretol (DTT), Formic acid,

Nitrocellulose and PVDF membrane from Sigma Aldrich St.Louis, USA.

CHAPTER 2 Materials & Methods


Sequencing grade Trypsin obtained from Promega, USA. Zip tips from

Millipore, India. Protein A agarose, secondary antibody (goat anti-rabbit IgG,

ALP/HRP tagged), FITC tagged goat anti-rabbit IgG, colouring reagent 5-

bromo-4chloro-3-indolyl-phosphate (BCIP)/ 4-nitroblue tetrazolium (NBT)

were obtained from Bangalore Genei, Bangalore, India. Electro

chemiluminescence (ECL) kit was obtained from Upstate biotechnology, New

York, USA. All the other chemical/reagents were of the highest grade, which

are commercially available.

CHAPTER 2 Proteomic Studies of PAP


2.2. METHODS

2.2.1. PROTEOMIC STUDIES OF PAP

2.2.1.1. ISOLATION AND PURIFICATION OF PAP

We have earlier isolated calcium and membrane-binding proteins (CaMBPs)

from Ehrlich ascites tumor (EAT) cells. Further we have shown that CaMBPs

are substrate for protein kinase C and phosphorylated CaMBPs could trigger

the generation of ROS in tumor cells. In this study we hypothesize that

secreted CaMBPs may have a role not only in tumor growth but also in

inflammatory disease like RA. Hence by using calcium membrane affinity

binding technique [Sharma et al., 1993] we attempted to purify CaMBPs from

SF of RA patients. In brief inside-out vesicles prepared from RBC (3–4 mg

vesicle protein/ml of SF) were mixed with SF and incubated at 37 °C for 20

min in the presence of 1 mM calcium containing buffer (2 mM NaCl/5 mM

KCl/0.5 mM EGTA/2 mM Tris pH 7.4). Suspensions were washed twice using

this buffer in order to eliminate unspecifically bound proteins. The specifically

bound proteins were released from membrane by including 1 mM EGTA

minus calcium containing buffer by centrifugation at 28,000g for 30 min at 4

°C. The supernatant containing CaMBPs was dialyzed and further purified by

size exclusion chromatography using Sephadex G-100. The apparent mass of

the purified CaMBPs was determined by SDS-PAGE [described in section

2.2.1.2.2] and silver stained [described in section 2.2.1.2.3] protein showed

that the fraction contained a homogeneous protein (PAP) with a molecular

mass of approximately 67 kDa. The purified fraction was assayed for

formation of tube like structures using HUVECs. Accordingly 67 kDa protein

was excised from SDS-PAGE subjected to Mass spectroscopy (MS)

identification [described in section 2.2.1.4].

2.2.1.2 PROTEIN ANALYSIS Protein concentration was determined by the method of Lowry et al. [1951]

using bovine serum albumin as standard.



2.2.1.2.1 PREPARATION OF PROTEIN SAMPLES AND MOLECULAR WEIGHT IDENTIFICATION

Protein samples were mixed with equal volume of 2x sample buffer (4% SDS,

40% glycol in 0.5 M Tris-HCl buffer pH 6.8 with β mercaptoethanol and

bromophenol blue (40 μg/ml) kept in boiling water bath for 5-8 min. The

samples were then cooled to room temperature and loaded on to the

polyacrylamide gel.

2.2.1.2.2 MOLECULAR WEIGHT DETERMINATION BY SODIUM DO-DECYLSULPHATE

POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE)

SDS-PAGE was carried out according to the method of Laemmli, 1970 under

both reducing and non-reducing conditions. Slab gel of 1mm thickness was

prepared in gel chambers. A gel chamber consisted of two glass plates (8x8

cm) spaced to 1 mm thickness using three vacuum greased spacers, two on

either side and one at the bottom. The glass plates were clamped together.

The gel chamber was filled with a separation gel phase and a staking gel

phase as follows. Resolving gel (12.5%) was prepared by mixing 2.1 ml of

monomeric acrylamide solution (30% acrylamide and 0.8% N-N-bisacrylamide

made upto 100 ml in distilled water), 1.25 ml separation gel buffer (1.5 M Tris-

HCl, pH 8.8) 50 μl 10% sodium dodecyl sulfate (SDS), 50 μl 10% ammonium

per sulphate (APS) and 1.65 ml distilled water. The mixture was deaerated

and 10 μl of N,N,N',N'- tetramethylethylene diamine (TEMED) was added.

The contents were poured into a vertical slab gel plate to form 1 mm thick gel

slab. Stacking gel (4.5%) was prepared by mixing 0.75 ml monomer

acrylamide solution, 1.25 ml stacking gel buffer (0.5 M Tris-HCl pH 6.8), 50 μl

10% SDS, 50 μl 10% APS and 2.95 ml distilled water. The mixture was de-

aerated and 10 μl TEMED was added and mixed. The contents were poured

over resolving gel, plastic comb was inserted into the stacking phase to make

slots for loading the samples. The comb was removed after the gel had

polymerized and the slots were cleaned. The spacer at the bottom was

removed and the glass chamber now containing the polymerized resolving

and stacking gel was mounted on the electrophoretic apparatus.The

electrophoresis chambers were filled with the running buffer (12.4 mM Tris-



HCl, 192 mM glycine, 0.1% SDS, pH 8.3) and electrophoresed at 100 V for

3 h.

2.2.1.2.3. SILVER STAINING After electrophoresis the gels were silver stained by the following protocol.

a. Gels were placed in fixative solution (50% v/v ethanol 12% acetic acid,

50 μl formaldehyde) for 30 min.

b. Gels were washed with 50% ethanol thrice for 20 min each.

c. Gels were pretreated with 0.02% sodium thiosulphate for 5 min, washed in

distilled water thrice for 5 min each.

d. Gels were soaked in 0.2% silver nitrate, 75 μl formaldehyde solution for

40-45 min and thoroughly washed with distilled water.

e. Gels were developed in developing solution (6% sodium carbonate, 50 μl

formaldehyde) and reaction was terminated using acetic acid.

f. Gels were transferred to 50% ethanol and stored at 4 oC.

2.2.1.3 MALDI-TOF ANALYSIS OF INTACT PROTEIN Molecular mass of PAP protein was determined using MALDI-TOF MS on a

Kompact SEQ, Kratos Analytical, Manchester, UK. 1 μl of matrix solution (α-

cyano-4-hydroxycinnamic acid powder (Sigma Aldrich Chemicals Pvt. Ltd.,

Bangalore, India) in acetonitrile:water (1:1v/v) with 0.1% (v/v) trifluoroacetic

acid to obtain a saturated solution) followed by 1 μl of sample (1–5 pmol/ml)

and allowed to dry. Ions were generated by irradiation with nitrogen pulse

generated laser. Positive ions were accelerated, detected in the reflection and

linear mode.

2.2.1.4 IN-GEL ENZYMATIC DIGESTION OF PROTEIN

A purified protein band was excised from SDS-PAGE (12.5%) [described in

section 2.2.1.2.2] and was subjected to in-gel digestion with trypsin. Gel

pieces were washed with 50 mM and 25 mM NH4HCO3 solution and

acetonitrile, reduced with 50 μl of 10 mM DTT in 100 mM NH4HCO3 and



alkylated with 50 μl of 55 mM iodoacetamide in 100 mM NH4HCO3. Gel pieces

were washed once with 100 mM NH4HCO3, and twice with acetonitrile.

Enzymatic digestion was carried out by incubating the dried gel pieces with

trypsin (Procine trypsin, Promega, USA; 20 μl of 20 ng/μl stock solution in 50

mM ammonium bicarbonate) at 37 °C overnight. Subseq uently, 10 μl of 50mM

NH4HCO3 was added, and peptides were extracted by adding 30 μl of 50%

ACN/2% formic acid. After extraction of the peptides, the sample volume was

reduced using SPD 111V speed-vac system (Savant Instruments, Holbrook,

NY, USA). Peptide mixtures from tryptic digestions were desalted and

concentrated using C18 zip tips (Millipore Billerica, MA, USA). The resulting

reaction mixture was analyzed by MALDI-TOF-MS. The mass spectra were

acquired by scanning m/z range from 10,000 to 70,000. MALDI-TOF-MS

analyses were performed using an Ultra flex TOF/TOF mass spectrometer

(Bruker Daltonics, Bremen, Germany) in reflectron (positive ion) mode, using

a 90 ns time delay, and a 25 kV accelerating voltage in the positive ion mode.

Identical conditions were maintained while analyzing samples in the negative

ion mode. The system utilizes a 50 Hz pulsed nitrogen laser, emitting at 337

nm. The ion source and the flight tube were kept at a pressure of about 7×107

mbar by turbo molecular pumps. The sample was prepared by mixing an

equal volume of peptide solution and a saturated solution of matrix (α-cyano-

4-hydroxycinnamic acid) in 1:1 (vol/vol) acetonitrile: water mixture. A standard

peptide mixture was used for external calibration. Database searches were

performed using the Mascot software 1.8 (Matrix Science, Oxford, UK) with

carboxyamidomethylation of cysteines as fixed modifications and methionine

oxidations as variable modifications searching the NCBI database. In all

searches, 1 missed tryptic cleavage was accepted and a mass tolerance of

±0.6 Da was set for both precursor ion and fragment ion mass. The probability

MOWSE score was used in the searches by MASCOT. For amino acid

sequencing, the peptide mixtures were analyzed by MS/MS (tandem mass

spectrometry) using a Q-q-TOF hybrid system equipped with a nanospray ion

source.



2.2.1.5 DETERMINATION OF N-TERMINAL AMINO ACID SEQUENCE

Purified PAP protein (100 μg) was resolved on 15% SDS-PAGE [described in

section 2.2.1.2.2] under reducing conditions and then electro-blotted onto

PVDF membrane (Millipore). The transfer membrane was first washed with

methanol and equilibrated with transfer buffer (39 mM Glycine, 48 mM Tris–

HCl, 20% methanol, and 0.37% SDS). Filter papers (Whatman Ltd.,

Maidstone, U.K., 3MM)were soaked with transfer buffer, and a sandwich was

assembled from 7 sheets of paper, the membrane, the gel (rinsed with

transfer buffer), and another 7 sheets of paper. Electroblotting was performed

in a semi-dry blot chamber (Pharmacia) at 130 mA/cm2 40 min. The blotted

membrane was washed in double-distilled water for 5 min, stained with

Ponceau S stain for 5 min and destained by washing thoroughly with double-

distilled water, air dried and stored at 4 °C. N-te rminal amino acid sequence

was obtained by automated Edman degradation followed by HPLC and UV

detection using PROCISE protein sequencing system of Applied Biosystems

(850 Lincoln Centre Drive, CA, USA).

2.2.1.5 PERIODIC ACID SCHIFF'S (PAS) STAINING FOR GLYCOPROTEIN

PAS staining was done according to the method of Trivedi et al.,1982. After

electrophoresis [described in section 2.2.1.2.2] the gel was placed in fixative

(7.5% acetic acid), rocked gently for 60 min and then the contents were

drained.

a. To oxidize the oligosaccharides, the gel was treated with 1% periodic acid

for 45 min at 4 oC.

b. To remove excess of periodic acid, the gel was treated with 0.5% sodium

meta bisulphate in 0.1 N HCl at room temperature.

c. The gel was stained with Schiff’s reagent by submerging the gel in solution

and allowed for pink colour to develop in dark at 4 oC.

d. The gel was destained in 10% acetic acid.

CHAPTER 2 Antibody production for PAP


2.2.2. PRODUCTION OF POLYCLONAL ANTIBODY FOR PURIFIED PAP

The purified PAP obtained from gel filtration chromatography was used as an

antigen for raising polyclonal antibodies in New Zealand white male rabbit

(weighing 3 - 4 kg). Approximately 100 μg protein was emulsified in Freund’s

complete adjuvant and administered to rabbit at different sites subcutaneously

(s.c). Subsequent booster injections were given with antigen (50 μg)

emulsified in incomplete adjuvant. After each booster rabbit was

anaesthetized and 15 ml blood was collected from the ear vein using 21-

guage needle into a sterile tube. After collection, blood was allowed to clot for

30 min at 37 oC. Serum was separated from the clot and remaining insoluble

materials removed by centrifugation at 10,000g for 10 min at 4 oC. Pre-

immune serum was also separated from blood, which was collected before

immunisation.

2.2.2.1 DETERMINATION OF ANTIBODY TITER FOR PAP ANTISERUM Flat bottom 96 well ELISA plates were coated with 100 µl of purified PAP at a

concentration of 100 ng/ml in coating buffer ( 50 mM sodium carbonate buffer,

pH 9.6) and incubated over night at 4 oC. The plate was washed with washing

buffer (50 mM PBS containing 1% tween-20, pH 7.4) thrice and blocked with

blocking buffer (0.05 PBS containing 0.1% tween-20 + 0.4% BSA) for 2 h at

37 oC. Serially diluted 100 µl of PAP specific rabbit hyper immune serum (1:0

to 1:50,000 in 0.05 M PBS pH 7.4) was added and incubated for 2 h at 37 oC.

Plates were washed with washing buffer thrice and incubated with 100 µl of

secondary antibody (1:2500) conjugated to ALP and incubated for 1 h at

37 oC. After washing thrice with washing buffer 100 µl of substrate chromogen

(pNPP 5 mg/15 ml of 50 mM PBS, pH 7.4) was added and incubated at 37 oC.

Reaction was terminated with 50 µl of 0.1 N NaOH and absorbance was read

at 405 nm using Medispec ELISA reader.



2.2.2.2 PURIFICATION OF POLYCLONAL ANTIBODIES FROM ANTISERA

2.2.2.2.1 AMMONIUM SULPHATE PRECIPITATION

The immunoglobulin from antiserum was purified by ammonium sulphate

precipitation method [Heide et al., 1973].

a. To 10 ml of rabbit antiserum, ammonium sulphate (40% precipitation) was

gradually added pinch by pinch with constant stirring at ice-cold

temperature. Suspension was left overnight at 4 oC with occasional

stirring.

b. The mixture was centrifuged at 3,000 rpm for 30 min at 4 oC, supernatant

was discarded.

c. To the precipitate 3 ml of 40% ammonium sulphate was added and the

precipitate was suspended uniformly, washed thrice (3,000 rpm, 30 min at

4 oC).

d. Precipitate was reconstituted in a small volume of 0.01 M sodium

phosphate buffer pH 7.2 and it was dialyzed against same buffer for 24 h

with frequent change of buffer.

e. Precipitate, which appears in this step, was removed by centrifugation and

the supernatant was dialyzed against water before the final dialysis

against borate buffered saline (0.05 M, pH 8.4). Serum and purified

antibodies were subjected to SDS-PAGE.

2.2.2.2.2 PROTEIN A AGAROSE COLUMN CHROMATOGRAPHY

The ammonium sulphate precipitated IgG was further purified by protein A

agarose column chromatography according to the manufacturers protocol

(Bangalore Genei, India). In brief, 10 bed volume of 1X equilibration buffer

(0.05 M PBS, pH 7.4) was passed through the protein A agarose column

provided by the manufacturer. Then equal volume of ammonium sulphate

precipitated antibodies and equilibration buffer was mixed and centrifuged at

10,000 rpm for clarity. Equilibrated clear fluid was loaded onto the column at

the rate of <0.25ml/min. Twenty five bed volume of the equilibration buffer

was passed through the column and unbound protein fractions were collected



at the rate of 1.5 ml/6 min until absorbance at 280 nm reaches zero. Finally

five bed volume of elution buffer (0.05 M citrate buffer, pH 3.0) was passed

and fractions were collected at a flow rate of 1.5 ml/5 min in tubes containing

25 μl of neutralization buffer (1.5 M Tris-base, pH 10.0) and absorbance was

read at 280 nm. The bound fractions were pooled and dialyzed overnight

against PBS (0.02 M, pH 7.2) and stored at 4 oC until further use.

2.2.2.2.3. SDS-PAGE FOR PURIFIED ANTIBODIES

Purity of the protein A agarose affinity purified antibodies was carried out by

SDS-PAGE under non reducing condition as described in the section

2.2.1.2.2.

2.2.2.2.4. IMMUNODIFFUSION Immunodiffusion was performed by Ouchterlony double diffusion (ODD)

method. Agarose (0.65%) was dissolved in PBS containing sodium azide and

poured into petridishes. Wells were punched in the agarose and peripheral

wells were charged with antigen, pre-immune serum, BSA or vehicle (PBS)

respectively. The central well contained the antibody (100 μg). Precipitin band

was allowed to develop at 37 oC for 24-48 h in humidified chamber.

2.2.2.2.5. WESTERN BLOT ANALYSIS

a. The purified PAP (80 μg) was resolved on SDS-PAGE and blot transfer

was carried at a constant voltage 50 V for 3 h at 4 °C.

b. The membrane was washed with TBS-T (20 mM Tris-HCl pH 8.0, 150 mM

NaCl, 0.1% tween-20) and non specific sites were blocked with TBS-T

containing 3% BSA.

c. The blot was treated with primary antibody (antiserum, 1:100 dilution) for

90 min and washed before being treated with the secondary antibody

(anti-rabbit IgG, ALP-linked, 1:2500 dilution) for another 60 min.

d. After washing, the membrane was developed with the colouring reagent

5-bromo-4-chloro-3-indolyl-phosphate(BCIP)/4-nitrobluetertrazolium (NBT)

at 27 oC for 4 min.



The reaction was terminated by washing the membrane with distilled water for

10 min.

2.2.3. GENERATION OF MONOCLONAL ANTIBODIES FOR PAP

The PAP monoclonal antibodies were produced by the fusion of PAP primed

B cell (splenocytes) with myeloma cells by hybridoma technology with

standard HAT selection as described previously [Bhavani et al.,1989 and

described in section 3.2.1] The supernatant from single clone was collected

as a source of anti-PAP-mAb and purified using protein-A agarose column

according to the manufacturer's protocol (Bangalore Genei, India). A

competitive ELISA was developed using this antibody [described in Section

2.2.6.3]. The specificity of the antibody was also checked by Western blot

analysis [described in Section 2.2.2.2.5].

2.2.4. IN VIVO PERMEABILITY ASSAY (MILES ASSAY)

In order to verify that the protein purified from SF induces permeability, we

performed the Miles permeability assay according to the method described

previously [Zebrowski et al.,1999]. In brief, mice were anesthetized with 1%

pentobarbital buffer (i.p). Evan's blue dye (0.2 ml/mouse) was administered

via tail vein. Thirty minutes later, mice were injected intradermally with

different concentrations of PAP (250 ng, 500 ng and 1 μg) orwith VEGF (10

ng, 20 ng, and 40 ng) as a positive control or saline (as vehicle control).

Subsequently subdermis was harvested and Evan's blue concentration was

quantified by reading the absorbance at 610 nm.

CHAPTER 2 Proangiogenic Activities of PAP


2.2.5. CHARACTERIZATION OF PROANGIOGENIC ACTIVITIES OF PAP

2.2.5.1. CELL PROLIFERATION ASSAY

Cell proliferation assay was carried as described previously [Lingaraju et al.,

2008 and Zeng et al.,2001]. Briefly, low-passage primary HUVECs were

seeded at 10,000 cells/well on 12 well plates in EGM-2medium (Cambrex

Biosciences, NJ, USA). After 24 h cells were replaced with serum free basal

EGM-2 medium without any cell growth supplement, before stimulation. Cells

were stimulated with the indicated concentration of PAP (1 ng, 3 ng, 10 ng/ml)

or VEGF (10 ng/ml) or treated with neutralizing monoclonal anti-PAP

antibodies (1 μg/ml). After 72 h treatment, 1µCi/well [3H]-thymidine was added

to the media, the cells were incubated for additional 24 h. After this time

period, the cells were washed thrice with cold PBS and incubated in 10% TCA

overnight at 4 oC. Scintillation fluid was added to all of the samples and the

radioactivity was counted in a liquid scintialltion counter. The cell viability was

plotted as percent of control.

2.2.5.2. TUBE FORMATION ASSAY

Tube formation assay of HUVECs was performed as described previously

[Ramachandra et al.,2009]. Briefly, a 96 well plate was coated with 50 μl of

matrigel (BD biosciences, Bedford, MA). HUVECs (5×103 cells per well) were

seeded in complete EGM-2 media. Subsequently media were aspirated and

basal EGM medium lacking growth factors was added. After 12 h cells were

treated with VEGF (10 ng/ml) or PAP (10 ng, 30 ng, 50 ng, 100 ng, 500 ng

and 1 μg/ml) at the indicated concentrations or treated with neutralizing

monoclonal anti-PAP antibodies (1 μg/ml). In these experiments the VEGF

that is normally part of the EGM-2 formulation was omitted. Quantitation of

angiogenesis was evaluated by objective measurement of HUVEC network

formation, i.e. the formation of tube like structures and their intervening

spaces, as the total empty area in a given culture. Tube length measurements

were performed using ImageJ 1.44 (National institute of Health, USA).



2.2.5.3. SHELL LESS CHICK CHORIOALLANTOIC MEMBRANE (CAM)

To check the vascular effect of PAP, shell less CAM was performed as

described previously [Olfa et al.,2005]. Briefly, chick embryos from 3-day-old

eggs were crack out of their shells onto cling film hammocks (7 cm diameter

and 4 cm high). Egg preparations was covered with sterile petridish and

transferred to humified incubator at 37 °C. On day 5 at 37 °C, impregnated

filter disks (2 mm) with PBS or PAP (10 ng/embryo) or VEGF (10 ng/embryo).

To check the effect of monoclonal antibody on PAP-induced angiogenesis,

neutralizing monoclonal anti-PAP antibody (1 μg/embryo) was administrated

topically on the CAM. After 72 h of incubation blood vessels were

photographed with a digital camera at 10× magnification. Quantification of

angiogenesis was carried out in digitized images by measuring the total blood

vessels length using ImageJ 1.44 (National institute of Health, USA).

Measures were performed by three experiments in a circle, centered on filter

disk that represents 50% of the total CAM surface.

2.2.5.4. CORNEAL MICROPOCKET ASSAY

Neovascularization in vivo was examined by implantation of test substances

{1 μg/pellet of VEGF (a positive control) and 1 μg/pellet of PAP protein or

treated with neutralizing monoclonal anti-PAP antibodies (1 μg/pellet)}

formulated in Hydron pellets (Sigma Co., St Louis, MO, USA) into rat corneas

[Ramachandra et al., 2009 and Hasan et al.,2004]. Female Wistar rats were

anesthetized by intravenous injection of sodium pentobarbital, and the rat

corneas were anesthetized with 0.5% proparacaine hydrochloride ophthalmic

solution, followed by implantation of Hydron pellet with test substances into

micropockets made in the normal avascular corneal stroma 1 to 1.5 mm from

the corneal limbus. The pellet sizes and the distance between the pellet and

the limbus for each group were similar. The cornea was covered with

gentamicin ophthalmic ointment once a day and cornea was photographed on

day 7 to inspect the growth of new blood vessels. The number of blood

vessels and length of the vessels were quantified. The area of corneal



neovascularization was determined using the formula: C/12×3.1416

[r2−(r−L)2],where C = clock hour, L = vessels length from the limbus (30°) and

r=2.5 mm (i.e. the measured radius of the rat cornea).

2.2.5.5. IN VIVO CAM ASSAY

In vivo CAM assay was performed as described previously [Borges et al.,

2003]. Briefly, fertilized Giri Raja chicken eggs were incubated at 37 °C at

constant humidity and on day 3 of incubation a square window was opened in

the egg shell after removal of 2– 3 ml of albumin so as to detach the

developing CAM from the shell. The eggs were resealed and return back to

the incubator. Plastic rings were placed on top of the growing CAM at the 10th

day of incubation under sterile conditions. The rings were then adsorbed with

5×106 cell suspension of B16F10 or with VEGF (10 ng/ml) or PAP (10 ng/ml),

used as negative and positive control, respectively. On day 17, the eggs were

opened and the tumors formed were excised from the CAM, weighed and the

tumor size determined. The tumors were processed for

immunohistochemistry. The sections were stained with hematoxylin and

eosin.

2.2.5.5.1. HEMATOXYLIN & EOSIN (H&E) STAINING

Histopathological studies were carried out in order to verify the MVD. Paraffin

sections were made using following protocol.

Reagent preparations:

a. 10% formalin solution: Formaldehyde solution (100 ml) and distilled

water (900 ml) was mixed well and used as a fixative reagent.

b. Mayer’s egg albumin: Egg white 50 ml and glycerol 50 ml were mixed

well and filtered through coarse filter paper and added a crystal of

thymol as preservative.



2.2.5.5.2. PREPARATION OF PARAFFIN EMBEDDED TISSUE SECTIONS

a. Fixation: Biopsy specimens of the primary lesions from each patient/test

sample was fixed in 10% formalin and embedded in paraffin.

b. Embedment: Tissues were processed as below:

i. Alcohol 80% 1-2 hrs

ii. Alcohol 95% -2 changes 1-2 hrs

iii. Alcohol absolute -3 changes 1-2 hrs

iv. Xylene -2 changes 1-2 hrs

v. Melted paraffin 1-2 hrs

vi. Embedded in paraffin and cooled quickly.

c. Preparation of slides: Paraffin blocks were secured, sections were taken

of about 5-micron thickness using microtome. Sections were fixed to the

slides using Mayer’s egg albumin with mild heat treatment. These slides

were used for the different staining purposes. Appropriate positive and

negative controls were included with each set of stains. One set of

sections were stained with H & E, microvessel counts were derived by

averaging the number of vessels with clearly defined lumens or linear

shaped seen in ten high power field (HPF) of high density areas and low

density areas. Final MVD is the mean score obtained from the areas

counted using Leitz Diaplan bright field microscope, Germany, attached

to CCD camera.

2.2.5.6. CELL MIGRATION ASSAY

Migration of MDA-MB-231was measured using two assays, transwell

migration assay and wound migration assay. The migration assay was

performed as described previously [Pang et al., 2009]. Briefly, the transwell

(Corning Inc.) was coated with 0.1% gelatin for 30 min in cell incubator. Cells

were allowed to attach to the membrane for 30 min before the addition of

inhibitors. The bottom chambers of the transwell were filled with basal

medium with VEGF (10 ng/ml) or PAP (10 ng/ml) or treated with neutralizing

CHAPTER 2 Molecular Mechanism of PAP


monoclonal anti-PAP antibody (1 μg/ml), and the top chambers were seeded

inactivated 5×104 cell/well MDA-MB-231 (pretreated with mitomycin C, 10

μg/ml) in 100 μl of basal medium. After 16 h of migration, the cells on the top

surface of the membrane (non migrated cells) were scraped with cotton swab

and the cells spreading on the bottom sides of the membrane (invasive cells)

were fixed with cold 4% paraformaldehyde for 30 min. After that, these

migrated cells were stained with hematoxylin. Images were taken using

inverted microscope (Carl Zeiss, Germany) and invasive cells were quantified

by manual counting. Experiments were performed in triplicate.

The wound-migration assay has been previously described and used

with slight modification [Liang et al., 2007]. Briefly, MDA-MB-231 was allowed

to grow into full confluence in six well plates and then incubated with 10 μg/ml

mitomycin C for 2 h to inactivate cell proliferation. After that cells were

wounded by pipette tips and washed with PBS. Basal medium was added into

the well with or without VEGF (10 ng/ml) or PAP (10 ng/ml) or treated with

neutralizing monoclonal anti-PAP antibodies (1 μg/ml). Images were taken at

different time intervals (0, 6, 12, 24 and 48 h) of incubation at 37 °C, 5% CO 2.

Migration of the cells across the sharp wound edge to the cell free region was

quantified by manual counting. Experiments were performed in triplicate.

2.2.6. DETECTION OF PAP IN TUMOR

2.2.6.1. IMMUNOLOCALIZATION OF ANGIOGENIC PROTEIN

Standard immunofluorescence staining of PAP was carried out as previously

described [Lucas et al., 2010]. Briefly, tumor cells (Glioblastoma and BeWo)

were detached from a plastic tissue culture dish with trypsin–EDTA solution

(1×). The cells (1×104) were suspended in DMEM medium and transferred to

six well culture dishes (Nunc, USA) with sterile cover slips and grownup to

semi confluency. The next day wells were rinsed thrice carefully with PBS.

Cells grown on cover slips were fixed for 10 min on ice with 3% formaldehyde

(freshly prepared from paraformaldehyde) in PBS, and permeabilized using

0.01% TritonX-100/PBS for 10 min at room temperature. Cells were quenched

for 30 min in 5% BSA/PBS and washed with PBS. The cells were incubated



for 2 h at room temperature with PAP antibody (1:50 dilution). The cells were

washed and incubated with FITC-conjugated secondary antibody (1:100

dilution) for 2 h in the dark at room temperature and mounting of the

coverslips on glass slides, and analyzed under a fluorescence microscope

(Leitz-Diaplan fluorescence microscope, Leitz, Germany) with an attached

CCD camera.

2.2.6.2. IMMUNOBLOT ANALYSIS OF PAP IN DIFFERENT TUMOR CELLS

We determined the existence of PAP in in vitro cultures. Breast cancer cells

(MCF-7), glioblastoma multiforme (GBM), choriocarcinoma cells (BeWo), triple

negative breast cancer cells (MDA-MB-231), Ehrlich ascites tumor cells (EAT)

and Human Embryonic Kidney 293 cells (HEK 293) were grown overnight to

confluency in 150 cm dishes (Nunc, USA). Cells were washed with PBS,

collected in modified cold radio immune precipitation buffer (RIPA) {50 mM

Tris–HCl pH7.4, 150 mM NaCl, 1% NP-40, 0.25% C24H39O4Na, 1 mM

Na3VO4, 1 mM NaF, 1 mM EDTA, added freshly with protease and

phosphatase inhibitors 1 mM phenylmethylsulfonyl fluoride, 10 μl/mg protease

cocktail (Sigma)}, and homogenized for 15 min on ice. Extracts were clarified

at 10,000g / 30 min/ 4 °C. Western blotting was performed as p reviously

[described in section 2.2.2.2.5]. Briefly, the sample containing equal amounts

of protein was separated on SDS polyacrylamide gel under reducing

conditions and transferred to nitrocellulose membrane (Millipore, Bedford,

MA). The blot was incubated for 2 h at room temperature with PAP primary

antibody (1:1000), followed by incubation for 2 h with horseradish peroxidase-

conjugated secondary antibody (1:2500). PAP protein was detected by ECL

method (Santa Cruz Inc., USA) and analyzed using phosphorimage analyzer

(Fujifilm, FLA5000, Tokyo, Japan).

2.2.6.3. ENZYME LINKED IMMUNOSORBENT ASSAY

We have developed a sensitive and specific quantification indirect ELISA

system for PAP. This assay was performed as previously described

[Shivakumar et al., 2009] with modification.



2.2.6.3.1. ANALYSIS OF PAP LEVEL IN SYNOVIAL FLUID

To generate a standard curve, purified PAP was diluted in coating buffer at

concentrations ranging from10 pg/ml to 100 ng/ml. The diluted PAP protein

standards and aliquots of synovial fluid (100 μl/well) were coated to the 96-

well microtiter ELISA plates (Nunc MaxiSorp™, Nunc, USA) using a coating

buffer (50 mM sodium carbonate buffer, pH 9.6) at 4 °C overnight.

Subsequently, blocked for 2 h with blocking buffer (5% BSA). Affinity purified

anti-PAP antibodies (dilution 1:1000), 100 μl/well were added and incubated

for 2 h at 37 °C followed by incubation with 100 μl of secondary antibody

(1:5000) conjugated to alkaline phosphatase and developed with 100 μl of p-

nitrophenyl phosphate solution. The optical density at 405 nm was measured

in a Medispec ELISA reader. The PAP concentration in the synovial fluid was

calculated based on the standard curve.

2.2.6.3.2. ANALYSIS OF PAP LEVEL IN CYTOSOLIC EXTRACTS OF TUMOR CELLS

Different malignant cell lines (HEK-293, MCF-7, Glioma, MDAMB- 231 and

EAT) were grown to 80% to 90% confluency in six well plates and were serum

deprived for 12 h. After incubation, the media were collected and centrifuged

at 14,000 g/10 min/4 °C to remove any cellular debr is. The centrifuged

conditioned media were used for ELISA. Cytosolic extract was prepared [as

described in section 2.2.6.2]. 100 μl aliquot of each cell lysate was used for

the ELISA, which was performed as outlined above. The PAP concentration in

the cell supernatant samples was calculated based on the standard curve.

2.2.6.3.3. MEASUREMENT OF PAP LEVEL IN CONDITIONED MEDIUM

In the next step, where we investigated for PAP in conditioned supernatant of

tumor cells, within the detection limit of the assay (10 pg/ml) no detectable

amount of PAP was found.



2.2.7. CLINICAL SPECIMENS FOR IMMUNOHISTOCHEMICAL STUDIES

Human breast lesion tissue samples were collected with informed consent,

from either diagnostic biopsies or upon surgery from the Department of

Pathology, J.S.S. Hospital, Mysore, India. Based on clinical investigation they

were classified as invasive ductal carcinoma of the breast. Paraffin embedded

tissue blocks were cut into 5 μm sections and processed for

immunohistopathology as described previously [Lucas et al., 2010]. Briefly,

a. Sections were de-paraffinized and hydrated. Antigen unmasking was

performed using the heat induced epitope retrieval (HIER) method

b. Then paraffin sections were washed thrice with xylene for 5 min.

c. The sections were hydrated with 100% ethanol for 5 min followed by 95%

for 2 min and 80% for 2 min.

d. Then, the sections were rinsed with distilled water and incubated with 3%

H2O2 in PBS to block endogenous peroxidase activity.

e. After 2 min, the sections were rinsed with PBS for 2 min and baked at 450

Hz for 10 min to retrieve the antigen and again hydrated with PBS for 2

min.

f. Sections were incubated with anti-PAP primary antibodyfor 1 hr at RT.

After tapping off the antibody, the slides were dipped for 5 min in PBS.

g. The sections were incubated with secondary antibody with biotinylated

rabbit anti-mouse IgG for 30 min at room temperature.

h. The slides were washed in PBS for 5 min and incubated (100 μl/section)

for 45 min with SS polymer-HRP detection kit was(Biogenex, Hyderabad)

i. After incubation, the slides were washed again with histo buffer and

incubated for 5-7 min in the substrate (100 μl /section, DAB peroxidase

substrate tablet). The sections were washed thrice for 2 min in tap water

and twice in distilled water.

j. Subsequently, the sections were incubated in 2% hematoxylin solution for

7 min and washed again in tap water thrice for 5 min each.



k. The slides were dehydrated successively for 2 min each in 50% ethanol,

70% ethanol, 80% ethanol, 95% ethanol and absolute alcohol. After

xylene wash, the slides were mounted using Entellan mountant solution.

l. Labeled cells were imaged on a Carl Zeiss fluorescence microscope,

AX10.Imager.A2, Germany with an attached CCD camera.

2.2.8. MOLECULAR MECHANISM OF PAP ACTION

2.2.8.1 VEGF LUCIFERASE REPORTER GENE ASSAY

Transient transfection and luciferase assay was performed as described

previously [Buijs et al.,2007]. MCF-7 cells were transfected with 2 μg of

luciferase reporter construct (pVEGF-Luc) and 2 μg of the β-galactosidase

expression vector pRSV-βgal (Promega,USA). After 24 h of transfection, the

cells were serum starved overnight before stimulation with varying doses of

PAP (2 ng, 5 ng, and 10 ng/ml) or VEGF (2 ng, 5 ng, and 10 ng/ml) as a

positive control for 6 h. Cells were washed once with cold PBS and lysed with

reporter lysis buffer. Luciferase (Luc) activity of the cell extract [as described

in section 2.2.6.2] was determined using luciferase assay system

(Promega,USA). β-galactosidease (β-Gal) activity was determined by

measuring hydrolysis of o-nirophenyl- β-D-galactopyranoside using 50µl of

cell extract at 37 oC for 2 h. Absorbance was measured at 405nm.Luciferase

activity was determined by Luminometer using 50µl of cell extract. The

reaction was initiated by adding 100µl of luciferase assay substrate. Relative

Luc activity with respect to control was calculated as Luc (relative light

units/50µl cell extract)/ β-gal activity (405 per 50µl cell extract per 2hr).

2.2.8.2 DNA TRANSFECTION AND CAT ASSAY

Transient transfection and CAT assay was performed as described previously

[Jeyaseelan et al.,2001]. Cells were seeded at 2×105 cells in six well tissue

culture plates. Subconfluent MCF-7 cells were transiently transfected with 2

μg of Flt-1 CAT reporter plasmid according to the manufacturer's instructions



(Promega, USA). After 24 h of transfection, the cells were serum starved

overnight before stimulation with varying doses of PAP (2 ng, 5 ng, and 10

ng/ml) for 6 h. The cells were also treated with VEGF (2 ng, 5 ng, and 10

ng/ml) at 37 °C for 6 h as a positive control. pRSV -βgal was co-transfected to

serve as an internal control for transfection efficiency. 48 h after transfection,

cell lysates were prepared using the freeze/thaw method. Endogenous

deacetylases were inactivated by incubation at 65°C for 10 min and the lysate

was clarified by centrifugation. The standard assay is performed according to

the manufacturer's instructions (Promega, USA) by incubating cell extract (in

50 µl- 100 µl) with 3µl of [14C]-chloramphenicol (GE healthcare,0.5 mCi/ml)

and 5µl n-Butyryl CoA and distilled water in a final volume of 125 µl. After

incubation at 37 oC for 4 h, the reaction is terminated by addition of 300 µl of

xylenes, and mixed vigorously by vortexing and centrifuged at top speed in a

micro centrifuge for 3 min. 90% of the xylene phase is removed, and extracted

twice with 100 µl of fresh 0.25 M Tris-HCl (pH 8.0), with complete removal of

the aqueous phase after each extraction. The remaining organic phase is

counted in Beckmen scintillation counter. The CAT activities of the transient

transfection assay were normalized to β-galactosidase activity. Changes in

CAT activity with respect to the control were calculated and the CAT

(Promega, USA) activity was determined by using a Beckman liquid

scintillation counter. All results were normalized by using pRSV-βgal as an

internal control.

2.2.8.3. ELECTROPHORETIC MOBILITY SHIFT ASSAY (EMSA)

Extraction of nuclear proteins and electrophoreticmobility shift assay (EMSA)

were performed as described by us previously [Prasanna et al.,2008]. EAT

cells were grown to 80% to 90% confluency in six well plates and were serum

deprived for 12 h before stimulation. Cells were stimulated with 100 ng/ml

PAP for different time intervals (0, 2, 4, 8, 16, 24, 32 and 48 h). Nuclear

extract prepared (60 µg) were incubated with 40 µl of reaction mixture

containing 100 mM HEPES (pH 7.9),10 mM MgCl2, 25 mM KCL,0.5 mM

EDTA, 4% glycerol, 0.5% NP-40,1 µg of polydeoxyinosinic deoxycytidylic acid



[poly(dI-dC)]. 40 fmoles of γ-[32P] labeled double stranded NF-κB

oligonucleotied (5'AGT TGA GGG GAC TTT CCC AGG C 3') was added to

the reaction mixture last and incubated for 30 min at 25 oC. Two microliters of

10x agarose dye loading buffer (50 % glycerol,0.25 % bromophenol blue, and

0.25 % xylene cyanlo) was added at the end of the incubation. The specificity

of the NF-κB DNA binding was determined in completion reactions in which a

40-fold molar excess of unlabelled NF-κB oligonucleotides was added to the

binding reactions 20 min prior to the addition of radio labeled probes. The

samples were electrophoresed in 4 % native polyacrylamide gel in 0.5 % TBE

at room temperature for 2 h at 200 V.The gel was dried, transferred to

imaging plate (IP) and the image was scanned by Phosphorimage analyzer to

examine the effect of PAP on NFκB–DNA binding activity using specific

oligonucleotides probe for NFκB binding element in the VEGF gene.

2.2.8.4. IMMUNOPRECIPITATION FOLLOWED BY WESTERN BLOT ANALYSIS

HUVEC or MDA-MB-231 cells were seeded in 150 cm dishes, grown to 90%

confluency, and serum starved for 24 h. Cells were preincubated with 2 mM

sodium orthovanadate for 2 h at 37 °C. Then, HUVECs were treated with

VEGF (10 ng/ml) or PAP (10 ng/ml) for different time intervals (0, 5, 15, 30

and 60 min). In other experiments MDA-MB-231 cells were treated with VEGF

(10 ng/ml) for various time points (0, 2, 5, 10, 15, 30, 60 and 120 min). In a

separate experiment prior to addition of VEGF, MDA-MB-231 cells were

preincubated with SB202190 (p38MAPK inhibitor, 10 μM/ml; Sigma-Aldrich)

for 2 h under serum free conditions. Thereafter, cells were incubated with

VEGF (10 ng/ml) for different time intervals (0, 10 and 30 min). Cells were

directly lysed with modified RIPA buffer [as described in section 2.2.6.2] and

incubated with the appropriate primary antibody (anti-VEGFR2 (Santa Cruz

Biotechnologies, USA) or anti-PAP) overnight at 4 °C, followed by addition of

Protein A-sepharose beads (Merck, Germany). The beads were washed 3×

with RIPA lysis buffer lacking detergent and boiled in SDS-PAGE sample

buffer for 5 min. The precipitated proteins were resolved by 10% SDS-PAGE

and transferred to a nylonmembrane [as described in section 2.2.2.2.5]. Blots



containing the proteins immunoprecipitated with anti-Flk1 antibody were

probed with anti-pFlk1 (Santa Cruz Biotechnologies, USA). Blots containing

the proteins immunoprecipitated with the anti-PAP were probed with mouse

anti-pY (Santa Cruz Biotechnologies, USA) antibodies. The signals were

detected using the appropriate HRP-conjugated antibodies followed by

enhance chemiluminescence.

2.2.8.5. IN VITRO KINASE ASSAY

The JNK or ERK activity was measured as described previously [Kralova et

al.,2010]. Briefly, MDA-MB-231 cells were seeded in 150 cm dishes, grown

overnight to 95% confluency, and serum starved for 24 h before the

treatment. Cells were incubated with VEGF (10 ng/ml) for different time

intervals (0, 10 and 30 min) or with PAP (10 ng/ml) for various time periods (0,

1, 2, 4, 8, 16 and 32 min). Samples were prepared [as described in section

2.2.6.2] and followed by direct Western blotting [described in section

2.2.2.2.5]] using their specific antibodies.

2.2.9. STATISTICAL ANALYSIS

Unless stated otherwise, all experiments were performed in triplicates.

Wherever appropriate, data are expressed as the mean ± SD and means

were compared using one-way analysis of variance (ANOVA). Statistical

significance of differences between control, VEGF, PAP and monoclonal

antibody treated cells was determined by Duncan's multiple range test

(DMRT). P < 0.05 is considered statistically significant. All statistical analyses

were performed using SPSS statistical software package version 13.0.

CHAPTER 2 Results: Proteomic studies of PAP


2.3. RESULTS

2.3.1. PROTEOMIC STUDIES OF PAP

CaMBP was purified from synovial fluid of RA patients using membrane

affinity binding technique followed by gel filtration chromatography. CaMBPs

which were specifically bound to inside-out red cell membrane vesicles in the

presence of calcium and released by EGTA are shown in Fig. 2.1(Inset).

Membrane affinity purified protein was fractionated into five peaks by gel

filtration. The bioactivity of eluted fractions was monitored, based on the ability

of individual fractions from the column to elicit formation of tube like structures

by HUVECs in matrigel. The major peak fraction exhibited induction of

capillary like tubes and contained protein with proangiogenic potential.

Moreover, purified CaMBP henceforth called as novel angiogenic protein

(PAP) was analyzed by SDS-PAGE under reducing conditions. The analysis

revealed a monomeric band as is shown in Fig. 2.1, lane 4 (Inset) and the

identified protein was a glycoprotein as revealed by Periodic acid Schiff's

staining Fig. 2.3. Results of the MALDI mass spectrum of the intact protein

revealed one pure peptide with a mass of 67,218.93 Da (Fig. 2.2, Inset).

Peptide ions are analyzed by the data dependent method to the obtained

spectrum (Fig. 2.2) by sequencing of the peptides generated by tryptic

digestion of the purified protein. The MASCOT search results (Table 2)

showed sequence coverage of 29% with maximum identity for human

retinoblastoma binding protein 2-Homo sapiens (Accession: AAB28544.1)

(Fig. 2.2). N-terminal amino acid sequence of the purified glycoprotein as

determined by automated Edman's degradation showed

D-A-A-A-X-E-V-A-A-A as the N-terminal sequence and by comparing its N-

terminal sequence with those available in data bank

(http://blast.ncbi.nlm.nih.gov/) revealed no sequence homology to currently

known proteins.

CHAPTER 2 Results: Antibody Production


2.3.2. PRODUCTION AND PURIFICATION OF POLYCLONAL ANTIBODIES

Antibody titer was determined by ELISA using serially diluted PAP anti-serum

was found to be 1:50,000. Anti-PAP antibodies were precipitated using 40%

ammonium sulphate and subjected to protein A agarose affinity coloumn

chromatography. The homogenity of purified IgG fractions was verified

SDS-PAGE under non-reducing condition where it shown the purified IgG

having molecular mass of 150 kDa (Fig. 2.4 A).

2.3.3. IMMUNODETECTION

The purified PAP was used for the production of polyclonal antibodies in New

Zealand white rabbit. Ouchterlony double diffusion results showed a single

precipitin band upon reactivity of the antigen with anti-PAP IgG. As seen in

the picture, no cross reactivity was obtained with PBS (vehicle) or BSA or pre

immune serum (Fig. 2.4 B). Western blot analysis indicates specific

interaction of purified PAP with affinity purified anti-PAP polyclonal antibodies.

The results indicate specificity of antigen to antibody (Fig. 2.4 C).

2.3.4. IN VIVO PERMEABILITY ENHANCING ACTIVITY OF PAP

The data as is shown in (Fig. 2.5) reveals that PAP is a permeability factor

with a similar permeability activity to VEGF.PAP (250 ng, 500 ng and 1 μg)

increased vascular permeability in a dose-dependent manner (Fig. 2.5 B)

similar to permeability activity of VEGF (10 ng, 20 ng and 40 ng) (Fig. 2.5 A).

Intradermal injection of the vehicle control (saline) (0.1 ml) did not increase

vascular permeability. The experiment was repeated thrice and representative

data is shown in figures.

CHAPTER 2 Results: Proangiogenic activities of PAP


2.3.5. CHARACTERIZATION OF PROANGIOGENIC ACTIVITIES OF PAP

Proangiogenic potential of PAP was revealed by the data presented in

Fig. 2.6. As is shown in the figure, increasing doses of PAP increased

proliferation of HUVECs as determined by [3H]-thymidine incorporation.

HUVEC cell proliferation potential of PAP (10 ng/ml) was comparable to that

induced by VEGF (10 ng/ml). In contrast, the monoclonal antibody treatment

significantly reduced the cell proliferation induced by the PAP (Fig. 2.6) but

not the proliferation induced by the VEGF. Further the effect of PAP on the

formation of functional tubes by HUVECs plated on the matrigel was

evaluated. When compared to the HUVECs without VEGF, cells treated with

VEGF rapidly aligned with one another and formed tube like structures

resembling capillary plexus within 8 h (Fig. 2.7). However addition of

exogenous PAP resulted in a biphasic response. Sprout formation was

optimal at low concentration of PAP (10 ng/ml) and decreased at higher

concentration (1 μg/ml). Correlating with increase of sprouting, the length of

sprouts also tends to increase slightly at low concentration of PAP (10 ng–100

ng/ml) and decreased at high concentration of PAP (500 ng–1 μg/ml). We

also noted an increase in vessel diameter at higher concentration (50 ng/ ml).

This effect of PAP was abolished by a monoclonal anti-PAP antibody (1

μg/ml) (Fig. 2.7). Further confirmation of proangiogenic role of PAP came from

the data on chick chorioallantoic membrane (CAM) assay. Upon analysis of

the CAM of 6-day-old chick embryos, the spontaneous angiogenesis in CAM

was clearly observed after 72 h. As illustrated in Fig. 2.8 topical application of

PAP showed similar effect as VEGF (10 ng/embryo). In contrast, topical

application of anti-PAP-mAb inhibited the spontaneous angiogenesis. The

PAP induced a pronounced angiogenic response in this model. Quantification

of angiogenesis clearly shows that anti-PAP-mAb treatment significantly

reduced angiogenesis. Corneal micropocket assay is considered as one of

the gold standards, for in vivo evaluation of angio-stimulatory or angio-

inhibitory activity. On this basis, we investigated the proangiogenic activity of

PAP in corneal micropocket assay. As shown in Fig. 2.9, cornea treated

with PAP showed extensive angiogenesis similar to that induced by VEGF

CHAPTER 2 Results: Proangiogenic activities of PAP


when compared with vehicle treated control animals. In contrast, pellet

containing PAP and anti-PAP-mAb had markedly reduced neovascularization.

Quantitative comparison showed that all parameters relevant for measuring

the extent of corneal neovascularization were significantly higher in PAP-

treated when compared with control animals. Quantification of angiogenesis

clearly shows that anti-PAP-mAb treatment significantly reduced

angiogenesis. The maximum vessel lengths were increased by 80% when

compared with controls, and circumference of neovascularization in PAP

treated rat corneas was increased by 85% (bar graph).While the above data

are in a non tumor context we verified the proangiogenic activity of PAP in a

xenograft model. On day 17 of incubation, macroscopic observation showed

that the plastic ring containing cells with PAP was surrounded by numerous

allantoic vessels that developed and surrounded the implant, and tumor size

was measured (Fig. 2.10). The angiogenic response was comparable to that

induced by VEGF, a well known angiogenic cytokine. On the contrary, few

blood vessels were recognizable around the plastic ring containing medium

with cells. At the microscopic level, in H & E stained sections , an augmented

MVD count was noticed in the PAP treated cells in contrast to that of

untreated cells (bar graph).

2.3.7. PAP ENHANCES IN VITRO CELL MIGRATION AND ECM INVASION

To assess the effects of PAP on breast cancer cell migration and ECM

(extracellular matrix) invasion, MDA-MB-231 cells were evaluated using

established in vitro assay systems. In the wound healing assay, migration of

the cells across the sharp wound edge to the cell-free region was assessed.

As shown in Fig. 2.11 migration of cells increased in a time dependent

manner. Cells completely migrated after 16 h of exposure to PAP. Similar

result was obtained in transwell assay (Fig. 2.12), and there was significant

increase in ECM-invasion in time dependent manner. PAP or VEGF untreated

cells were used as the control, where no migration was observed.

Interestingly, the wound healing-accelerating effect of PAP treatment was

blocked by anti-PAP–mAb (Fig.2.11). To further determine whether PAP

CHAPTER 2 Results: PAP in Tumor


stimulated migration of breast cancer cells depended on MAPK activation, we

investigated the effect of p38MAPK inhibitor SB202190 on cell migration using

transwell migration assay. Treatment of cells with 10 ng PAP increased the

migration of MDA-MB-231 cells when compared with control cells and anti-

PAP-mAb treated cells (Fig. 2.12). Pretreatment with SB202190 not only

eliminated PAP-stimulated migration, but also reduced cell migration in the

absence of PAP treatment. These results indicate that the activation of MAPK

is essential for both basal and PAP-stimulated breast cancer cell migration.

2.3.8. DETECTION OF PAP IN TUMOR

2.3.8.1. LOCALIZATION OF PAP IN TUMOR CELLS

To detect the intracellular localization of PAP, we used the anti-PAP antibody.

Cells grown on cover slides were fixed, incubated with anti- PAP antibody,

incubated further with FITC-conjugated IgG secondary antibody, and

analyzed under fluorescence microscope (Leitz-Diaplan fluorescence

microscope, Leitz, Germany) with an attached CCD camera. Fig. 2.13A

showed that PAP is localized in cytoplasm.

2.3.8.2. IMMUNOBLOT ANALYSIS

The above preliminary observation had shown that PAP is a potent

proangiogenic molecule. On this basis we investigated the possible presence

of PAP in tumor cells. We carried out Western blot of the cell lysates derived

from tumor cells. Interestingly PAP was identified in several tumor cell lines

(Fig. 2.13B). Although PAP was detected in HEK-293 cells, a strong

expression was evident in Glioblastoma, MCF-7, MDA-MB-231, BeWo and

EAT cell lines.

2.3.8.3. ELISA

Indirect PAP ELISA assay was developed as a direct test of this possibility to

measure the PAP levels in synovial fluid. The synovial fluids from 10 different

CHAPTER 2 Results: Molecular Mechanism of PAP


patients with RA were examined for the presence of PAP. Levels of PAP were

significantly higher in synovial fluid (Fig. 2.13 C). The cellular distribution of

PAP was unknown at this time; however based on the immunolocalization

studies, it appears that the protein may be present in cytosol. So indirect

ELISA was performed to detect the concentration of PAP in cell lysates.

Consistent with the above result PAP levels in tumor cell lysate (Fig. 2.13 D)

were also significantly higher when compared to lysates from normal cells.

2.3.8.3. EXPRESSION OF PAP IN HUMAN BREAST CANCER SPECIMENS

The in vitro data prompted to examine human clinical specimens, and

mammary ductal carcinoma tissues were collected with informed consent,

from either diagnostic biopsies or upon surgery. The levels of expression of

PAP were determined by immunohistochemical analysis. The result revealed

that PAP was present in human mammary ductal carcinoma specimens of all

20 patients (Fig. 2.13 E).

2.3.9. MOLECULAR MECHANISM OF PAP

2.3.9.1. EFFECT OF PAP ON VEGF GENE EXPRESSION

The effect of PAP on VEGF transcriptional activity was monitored using VEGF

gene promoter luciferase reporter gene assay. The cells were transiently

transfected with VEGF-luciferase reporter construct (pVEGF-Luc). After 24 h

of transfection, cells were either stimulated with increasing concentration of

PAP (2 ng, 5 ng and 10 ng/ml) or VEGF, which served as a positive control.

The cell lysate was used to measure luciferase activity. The data

demonstrated that PAP stimulates VEGF gene expression in a dose

dependent manner (Fig. 2.14 A).

2.3.9.2. EFFECT OF PAP ON FLT-1 PROMOTER CAT REPORTER ACTIVITY

Based on the results obtained with regulation of VEGF gene expression by

PAP, it was expected that PAP could stimulate the transcriptional activation of



the Flt-1 gene in MCF-7 cells. Flt-1 promoter-CAT reporter gene was

transiently transfected into MCF-7 cells, then analyzed the promoter activity in

the transfected MCF-7 cells. The CAT activity was increased about by 3–4-

fold higher by PAP or VEGF (Fig. 2.14 B). By contrast, the CAT activity was

not changed in control cells. The effect of PAP was examined at 2 ng, 5 ng

and 10 ng/ml on the Flt-1 promoter activity, and it was observed a 4-fold

higher stimulation at 10 ng/ml PAP.

2.3.9.3. EMSA

To examine the effect of PAP on NFκB binding activity to VEGF gene

promoter,MCF-7 cells were treated with 100 ng/ml of PAP for different time

intervals. Results in Fig. 2.14 C show that PAP induced NFκB binding to the

promoter region of VEGF gene in a time dependent manner (0 h to 48 h). The

data indicate that there is increase of NFκB DNA binding in PAP treated cells

as compared to untreated cells. Specificity of binding was also confirmed by

incubating the nuclear extract with 40-fold molar excess of unlabeled

oligonucleotide, and the data showed that there is a complete displacement of

NFκB specific band.

2.3.9.4. EFFECT OF PAP ON VEGFR2 PHOSPHORYLATION

First assayed the kinetics of phosphorylation of tyrosine within VEGFR2 in

response to PAP. HUVECs were treated with VEGF (10 ng/ml) or PAP (10

ng/ml) for increasing time intervals varying from 0 to 60 min. Thereafter, the

phosphorylation of VEGFR2 was verified by immunoprecipitation of VEGFR2

followed by Western blot analysis with anti-pFlk1 antibody. VEGFR2 was

tyrosine phosphorylated in VEGF treated samples. In contrast, VEGFR2 is not

phosphorylated by PAP treated samples (Fig. 2.15 A).



2.3.9.5. TYROSINE PHOSPHORYLATION OF PAP IN TUMOR CELLS INDUCED BY

VEGF

Instantly tested whether VEGF affects the phosphorylation of PAP. Cell lysate

from a time kinetics study of control and VEGF treated MDA- MB-231 cells

were immunoprecipitated with anti-PAP antibody followed by Western blot

analysis using anti-pY antibody (Fig. 2.15 B). Interestingly PAP was tyrosine

phosphorylated in VEGF treated cells, in a time-dependent manner. PAP was

phosphorylated to maximum extent at 30 min. There was subsequent

dephosphorylation of PAP.

2.3.9.6. INHIBITION OF P38MAPK PHOSPHORYLATION CAN REDUCE PAP

PHOSPHORYLATION IN MDA-MB-231

To determine whether PAP-stimulated angiogenic activities are MAPK-

dependent, MAPK activity was blocked by treating the cells with SB202190, a

p38 MAPK inhibitor, and examined p38MAPK activities after stimulation with

VEGF. Cleared cell lysate of control and VEGF treated cells were

immunoprecipitated with anti-PAP antibody followed by Western blot analysis

using anti-pY antibody (Fig. 2.16 A). The results showed that pretreatment

with 10 μM/ml SB202190 inhibited VEGF induced phosphorylation of PAP as

compared with control cells. Western blot with anti-pY revealed that PAP has

not been phosphorylated in the presence of SB202190. This result suggests

that VEGF regulates PAP phosphorylation through MAPK activation.

2.3.9.7. PAP-INDUCED JNK KINASE ACTIVITY BUT NOT BY ERK

To examine the effect of PAP on activity of the MAPK pathway components

like ERK and JNK we treated cells with PAP prior to assay for MAPK

activation. Our results showed PAP-induced phosphorylation and activation of

JNK or ERK. Accordingly, cells were treated with PAP (10 ng/ml) for 0, 1, 2, 4,

8, 16 and 32 min or VEGF (10 ng/ml) for 0, 10 and 30 min and lysed. The

lysates containing an equal amount of protein were resolved by SDS-PAGE



and phosphorylated ERK or JNK was detected by Western blot analysis using

anti-pERK or anti-pJNK antibodies. The data demonstrated that PAP induces

JNK phosphorylation in 8 and 16 min (Fig. 2.16 C). No effect was detected in

phosphorylation of ERK (Fig. 2.16 B). The blot was reprobed with anti-ERK or

anti-JNK antibody as loading control. These data suggested that PAP is

activating MAPK cascade reactions through JNK.

CHAPTER 2 Results: Purification of PAP


Figure 2.1 | Purification of PAP from SF from RA patients. Partially

purified PAP was chromatographed on Sephadex G-100 gel column.

Fractions of 1.5 ml at a flow rate of 15 ml/h were collected. Protein elution

profile was monitored spectrophotometrically at 280 nm. Partially purified PAP

resolved into five peaks. The bioactivity of eluted fractions was monitored,

based on the ability of individual fractions from the column to elicit formation

of tube like structures by HUVECs in matrigel (Inset). The major peak fraction

(*) exhibited induction of capillary like tubes and contained protein with

proangiogenic potential. (Inset) Silver stained SDS-PAGE profile of SF PAP

isolated from membrane affinity binding technique. Lane M: Protein molecular

weight marker, Lane 1: Synovial fluid, Lane 2: Membrane bound PAP, Lane 3:

Purified PAP.

CHAPTER 2 Results: MS/MS of PAP


Figure 2.2 | MALDI-TOF MS analysis of PAP. MALDI-TOF-TOFMS

fingerprint obtained by in-gel tryptic digestion of PAP. Numbers in the mass

spectrum give precise m/z values for the detected peptide signals followed by

MS/MS scans of the most abundant ions. Intact mass of PAP is given in the

inset.

Partial amino acid sequence of PAP. Tryptic digested peptides were

sequenced by nano-ESI-MS/MS. Matched sequences of PAP to RBP2 are

highlighted in bold.

CHAPTER 2 Results: PAS staining of PAP


Figure 2.3 | Periodic acid Schiff staining technique for detection of

glycoprotein. SDS-PAGE was carried out in slab gels. After electrophoresis

the gel was placed in fixative (7.5% acetic acid), rocked gently for 60 min. To

oxidize the oligosaccharides the gel was treated with 0.2% periodic acid for 45

min at 4 oC, then gel was washed with distilled water. Finally stained with

Schiff’s reagent by submerging the gel and allowing pink colour to develop in

the dark at 4 oC.

CHAPTER 2 Results: Western blot of PAP


Figure 2.4 | (A) SDS-PAGE profile of purified antibody: Purified fractions of

anti-PAP polyclonal antibody was subjected to SDS-PAGE analysis under

reducing condition. (B) Ouchterlony double diffusion: Immunodiffusion was

performed by Ouchterlony double diffusion method in 0.65% agarose in saline

containing 0.1% sodium azide. Wells were made in the agarose by sucking

out circular portion of the agarose. These wells were charged with antigen

and antibody, precipitin band was allowed to develop at 37 °C for 24 – 48 hrs.

Wells A, B, C and D contains BSA, PAP, PBS and pre immune serum

respectively. The central well contained the antibody. (C) Western blot:

Western blot analysis of PAP showing the strong detection of PAP by affinity

purified antibody. Lane M: Molecular weight marker. Lane 1: PAP.

CHAPTER 2 Results: PAP is a permeability factor


Figure 2.5 | Miles permeability assay: (A) VEGF (10, 20 and 40 ng)

increased vascular permeability (positive control). (B) Varying amounts (250

ng, 500 ng and 1 μg) of purified PAP injected intradermally in to mice

increased vascular permeability in a dose dependent manner. There was no

effect by saline (control) as indicated by the arrows. The intensity of the visible

spots proportional to the amount of vascular leakage of dye from the plasma

into the skin confirmed that of PAP is a permeability inducing factor. Vascular

permeability profile of leaky dye spots and Evan's blue concentration is

quantified by eluting leaky spots and then measured in a spectrophotometer.

Absorbance was read at 610 nm (bar graphs).

CHAPTER 2 Results: PAP induces capillaries


Figure 2.6 | Effect of PAP on HUVEC proliferation. HUVECs were seeded

and processed as described in “Materials and Methods”. HUVECs were

stimulated with different concentrations of PAP (1 ng, 3 ng and 10 ng/ml) or

VEGF (10 ng/ml) as a positive control or anti-PAP-mAb (1 μg/ml). Data shown

is the mean±SD of three independent experiments. a = statistically significant

at P<0.05 when VEGF compared with control, b = statistically significant at

P<0.05 when PAP compared with VEGF and c = statistically significant at

P<0.05 when PAP+mAb compared with PAP alone.

CHAPTER 2 Results: PAP induces capillaries


Figure 2.7 | Effect of PAP on tube formation in matrigel. HUVECs 5×103

cells/well were plated on matrigel and incubated with control medium (serum

free basal medium) containing different concentrations of PAP (10 ng, 30 ng,

50 ng, 100 ng, 500 ng and 1 μg/ml) or VEFG (10 ng/ml) or anti-PAP-mAb (1

μg/ml), for 8 h at 37 °C and photographed using Olym pus inverted microscope

(CK X40; Olympus, New York, NY, USA) (Magnification, ×40). Sprouting is

maximal at low concentration of PAP and the number of intersections started

decreasing as the concentration increased and at 500 ng/ml no intersection

was visible in both the groups. Average sprout length and intersections were

highest at 10 ng/ml of PAP or VEGF and slightly decrease as PAP

concentration increases. Monoclonal antibody anti-PAP-mAb (1 μg/ml)

inhibited tube formation in HUVEC cells, after which the relative intersections

and capillaries per field from each well were calculated.

CHAPTER 2 Results: PAP induces blood vessels


Figure 2.8 | Shell less CAM: Effect of PAP on angiogenesis in CAM

assay. The CAM models were prepared using 6-day old embryos treated as

described in “Materials and Methods”. Filter disks were soaked either in PBS

alone or 10 ng of PAP or 10 ng of VEGF or anti-PAP-mAb (1 μg). After

incubation for 72 h, CAMs were photographed (Leica, Germany). Each group

contained 6 eggs and the experiment was repeated three times. Quantitation

of the total blood vessel length was performed in a circle representing 50% of

the total CAM surface. Data shown is the mean±SD of three independent

experiments. a = statistically significant at P<0.05 when VEGF compared with

control, b = statistically significant at P<0.05 when PAP compared with VEGF

and c = statistically significant at P<0.05 when PAP+mAb compared with PAP

alone.

CHAPTER 2 Results: PAP induces corneal vasculature


Figure 2.9 | Corneal neovascularization induced by PAP. Hydron Polymer pellet containing 1 μg of PAP or 1 μg of VEGF (+ve control) and Hydron polymer with PBS (control) or anti-PAP-mAb (1 μg), was implanted into a surgically created corneal micropocket in one eye of Wistar rats. On day 7, the corneal neovasculature was visualized. In PAP treated animals, abundant new vessels grew from the limbus towards the pellet which was similar to that of VEGF treated animals. Treatment with anti-PAP-mAb (1 μg) inhibited PAP-stimulated angiogenesis. Corneas were photographed at 40× magnification (Leica, Germany). The maximum vessel lengths in PAP treated and VEGF treated animals were 1.2±0.1 mm and 1.4±0.1 mm, respectively. (C) The total vascular areas in PAP and VEGF treated animals were 5.0±0.3 mm2 and 7.0±0.6 mm2 respectively. CAMs treated with PAP+ mAb showed a statistically significant decrease in angiogenesis. No effect to CAMs treated with VEGF+anti-PAP-mAb. Data shown is the mean±SD of three independent experiments. a=statistically significant at P<0.05 when VEGF compared with control, b = statistically significant at P<0.05 when PAP compared with VEGF and c = statistically significant at P<0.05 when PAP+mAb compared with PAP alone.

CHAPTER 2 Results: Xenograft model


Figure 2.10 | Effects of PAP on angiogenesis in xenograft model.

Representative photograph of tumor on CAM at day 17 of incubation treated

with cells alone or VEGF or PAP. The histogram showing the excised tumor

size.

Immunohistochemistry studies of CAM tumors. Microvessel density was

determined by counting the blood vessels (black arrow).The histogram

showing the quantification of microvessel. Data shown is the mean±SD of

three independent experiments. a = statistically significant at P<0.05 when

VEGF compared with control and b = statistically significant at P<0.05 when

PAP compared with VEGF.

CHAPTER 2 Results: PAP enhances cell migration


Figure 2.11 | Effect of PAP on cell migration assay. The wound healing

assays were performed either by using untreated cells (4×105 cells/well), cells

treated with VEGF (10 ng/ml) or PAP (10 ng/ml) or anti-PAP-mAb (1 μg/ml).

Cells treated with mitomycin C (10 μg/ml) for 2 h. Cells were wounded with

sterile pipette tip and treated with or without VEGF (10 ng/ml) or PAP (10

ng/ml) or anti-PAP-mAb (1 μg/ml) in serum free media. In contrast, cells

treated with PAP+mAb inhibited migration, whereas cell migration is not

inhibited in VEGF+anti-PAP-mAb. The histogram showing the migrated cells.

Data shown is the mean±SD of three independent experiments. a =

statistically significant at P<0.05 when VEGF compared with control, b =

statistically significant at P<0.05 when PAP compared with VEGF and c =

statistically significant at P<0.05 when PAP+mAb compared with PAP alone.

CHAPTER 2 Results: PAP induces invasion


Figure 2.12 | PAP induced invasion of MDA-MB-231 cells. Cells were

seeded in the upper chamber of the transwell and the bottom chamber was

filled with serum free medium supplemented with or without VEGF (10 ng/ml)

or PAP (10 ng/ml) or anti-PAP-mAb (1 μg/ml). After about 8 to 10 h, the

invasive MDA-MB-231 cells passed through the membrane and were

quantified by counting the cells that migrated onto the membrane. Data for

migration are presented as number of migrating cells per field (mean±SD of 3

separate experiments performed in triplicates). a = statistically significant at

P<0.05 when VEGF compared with control, b = statistically significant at

P<0.05 when VEGF+SB compared with VEGF, c = statistically significant at

P<0.05 when PAP compared with VEGF, d = statistically significant at P<0.05

when PAP+SB compared with PAP and e = statistically significant at P<0.05

when PAP+mAb compared with PAP.

CHAPTER 2 Results: PAP present in human tumor biopsies


Figure 2.13 | (A) Immunolocalization of PAP in tumor cells. Cells grown on

glass slides and were fixed using paraformaldehyde, incubated with anti-PAP

antibody, followed by further incubation with FITC-conjugated IgG, and

photographed using fluorescence microscope.PAP localized to the cytoplasm.

(B) Western blot analysis of PAP detection in tumor cells.Cytoplasmic

extracts from tumor cells were used for Western blot analysis with PAP

antibody. PAP was detected in highly metastatic tumor cells (lanes 2 to 6) but

not in HEK-293. (C) Expression of PAP in human clinical specimens.

Representative photomicrograph of different grades of biopsy sections of

infiltrating ductal carcinoma stained for PAP. (D) ELISA of PAP protein in SF

samples of RA patients. The PAP protein content of SF from RA patients

was detected by PAP indirect ELISA. (E) ELISA of PAP of tumor cells. The

presence of the PAP was detected in cytosolic extract by the anti-PAP

antibody. The levels of PAP in cell supernatant were undetectable. The data

are presented as the mean±SD of three independent experiments.

CHAPTER 2 Results: PAP induced VEGF expression


Figure 2.14 | (A) VEGF gene promoter luciferase reporter analysis. Transfected cells were either stimulated with VEGF (2 ng, 5 ng, and 10 ng/ml) or different doses of PAP (2 ng, 5 ng, and 10 ng/ml) for 6 h. The cell lysates were used to measure the luciferase activity. (B) Induction of CAT-Flt-1 promoter activity by PAP. Transfected cells were either stimulated with VEGF (2 ng, 5 ng, and 10 ng/ml) or different doses of PAP (2 ng, 5 ng, and 10 ng/ml) for 6 h. CAT activity was corrected for differences in transfection efficiency (C) EMSA. Cells under serum starvation were incubated with PAP (100 ng/ml) for different time intervals , incubated with 32P-labeled NFκB binding region on VEGF promoter site consensus oligonucleotides .Data shown is the mean±SD of three independent experiments. a = statistically significant at P<0.05 when VEGF compared with control and b = statistically significant at P<0.05 when PAP compared with VEGF.

CHAPTER 2 Results: PAP signaling axis


Figure 2.15 | (A) Effect of PAP on VEGFR2 phosphorylation. Whole cell

extract of either HUVECs untreated or HUVECs treated with VEGF (10 ng/ml)

or PAP (10 ng/ml) was prepared. The samples were subjected to

immunoprecipitated (IP) with anti-Flk1 antibody. The immunoprecipitated

proteins were analyzed by Western blot using anti-pFlk1 antibody. Total

amount of Flk1 was used to ensure equal protein loading. (B) Tyrosine

phosphorylation of PAP after VEGF treatment. MDA-MB-231 cells were

stimulated with VEGF (10 ng/ml) for different time intervals. PAP was

immunoprecipitated from whole cell extracts, resolved by SDS-PAGE and

immunoblotted with anti-pY antibodies, followed by anti-PAP as loading

control.

CHAPTER 2 Results: PAP activity mediated through JNK


Figure 2.16 | (A) Inhibition of p38MAPK successively inhibits PAP

phosphorylation. MDA-MB-231 cells were pretreated with SB202190 (10

μM/ml) and then treated with VEGF or VEGF+inhibitor (0 and 30 min). Cell

lysate was immunoprecipitated with anti-PAP antibody followed by

immunoblotting with anti-pY antibodies. (B,C) Induction of JNK and ERK

MAPK pathways by PAP. Total protein extracts of MDA-MB-231 treated with

either VEGF (10 ng/ml) or PAP (10 ng/ml) for various time periods were

analyzed with either anti-pERK, anti-ERK, anti-pJNK, or anti-JNK antibodies.

CHAPTER 2 Results: Summary of Proteome analysis


TABLE 2 SUMMARY OF PROTEINS IDENTIFIED BY PROTEOME ANALYSIS

Match Score Sequence coverage

Retinoblastoma binding protein 2 [Homo sapiens] 95.1 29%

Jumonji, AT rich interactive domain 1A (RBBP2-like), isoform CRA_b [Homo sapiens]

73.6 23%

JARID1A variant protein [Homo sapiens] 73.6 18%

CHAPTER 2 DISCUSSION


2.4. DISCUSSION

Angiogenesis and inflammation are codependent processes [Szekanecz et

al., 2009]. Our results show for the first time the capacity of PAP, to induce

capillary network formation and proliferation phenotype in endothelial cells in

vitro and a potent angiogenic response in vivo. This reflects the previously

unrecognized ability of SF derived protein to trigger a complex intracellular

signaling cascade. Here we report the purification and characterization of a

novel proangiogenic protein (PAP), isolated from synovial fluid of RA patients.

We have explored the molecular mechanisms that underlie the proangiogenic

activity of PAP. The purification to homogeneity of a 67 kDa angiogenic factor

has been achieved with good recoveries of activity. As a preliminary step for

isolation of protein we have adopted membrane bound affinity method which

was described by us previously [Ranjana et al.,1993]. To gain more insight

into the structural and functional relationship, the purified glycoprotein was

subjected to mass spectroscopy and N-terminal analysis. Proteomic analysis

of the protein revealed that 29% sequence coverage with maximum identity

for human retinoblastoma binding protein 2, a critical mediator of cell cycle

progression, is functionally inactivated in the majority of human cancers

[Sharma et al., 2007]. Previous observations by Tanaka M. had shown that

rheumatoid arthritis antigenic protein is similar to retinoblastoma binding

protein 1 (RBP1). Migita K. et al.2001, have shown the role of retinoblastoma

gene product in the regulation of rheumatoid synoviocyte proliferation. N-

terminal sequencing of the 67 kDa protein revealed sequences that do not

match with sequence available in protein or gene databank to date. Thus,

these sequence data are presented for the first time. Experiments have been

initiated to clone and express PAP with degenerate/specific primers derived

from the protein/peptide sequences presented. Only one previous study has

investigated angiogenic factor from synovial fluid resembling that from tumors

[Brown et al., 1980], but it was only a preliminary communication, which we

have taken further. Interestingly we identified PAP in cytosol of different tumor

cells which was evident by immunofluorescence, immunoblot and ELISA

analysis. Clinical data revealed the presence of PAP in ductal breast



carcinoma biopsies. PAP stimulated cell supernatant was collected and tested

for the protein by standardized ELISA but it was below detection level. This

was a surprising result because PAP is a secretary protein found in SF, but in

cancer cells it was present in cytosol which was also confirmed by ELISA,

wherein cytosolic extract was used. However, the role of this protein in both

health and disease especially cancer biology remains to be elucidated. This

study now reports a role of PAP in angiogenesis, a third important pillar in

tumor growth and inflammatory diseases [Zeng et al., 2001].We first

examined its effect on endothelial cell tube formation and proliferation.

Addition of exogenous PAP resulted in a biphasic response. Sprout formation

was optimal at low concentrations of PAP (10 ng/ml) and decreased at higher

concentration (1 μg/ml) correlating with the increase of sprouting, and the

length of sprouts also tended to increase slightly at low concentration and

decreased at higher concentration (Fig. 2.6). We also noted an increase in

vessel diameter at higher concentration of PAP. Interestingly, PAP may not be

required for maintenance of the capillaries once they have formed. At higher

concentration PAP did not stimulate tube formation, but behaved as survival

factor. This data support the angiogenic property of PAP which was very

similar to previously reported data on VEGF [Nakatsu et al., 2003]. PAP also

augmented the rate of thymidine incorporation, endothelial cell proliferation,

tube formation, and decreased number of dead cells is crucial steps in new

blood vessel formation. Our results are consistent with proangiogenic nature

of PAP. The ability of cancer cells to undergo invasion and migration is a

prerequisite for tumor metastasis. In this study, we found that PAP time

dependently stimulated migration of MDA-MB-231 breast cancer cells, which

is a critical step for tumor metastasis. Based on the above functional

evidences, the signaling mechanisms underlying the effect of PAP on

promoting angiogenic activity were investigated. A primary observation in the

present study is that VEGF induced activation of PAP in a time dependent

fashion in breast cancer. In addition, we observed that VEGF enhanced the

PAP phosphorylation. The VEGF receptor KDR, seems to mediate almost all

the observed angiogenic responses to VEGF. In contrast phosphorylation of

VEGFR2 in response to the PAP treatment was studied. We found that the



protein does not phosphorylated VEGFR2. The mechanism by which PAP

affects tube formation, cell proliferation and migration is not known. We began

identifying pathway(s) necessary for proangiogenic property of PAP, using a

panel of pharmacological inhibitors. One such inhibitor, SB202190 targets

p38MAPK, eliminated PAP-stimulated migration. Similar result was seen in

p38MAPK phosphorylation where treatment of cells with SB202190, inhibited

VEGF induced PAP phosphorylation. These data suggest that VEGF

regulates PAP phosphorylation through the p38MAPK pathway. It has been

reported that ERK and JNK pathways play a crucial role in regulation of

angiogenic response of endothelial cells to VEGF-A [Petrova et al., 1999].

These results prompted us to investigate whether PAP induces JNK or ERK

activation. We have shown that PAP induces JNK phosphorylation but not

ERK phosphorylation. A possible explanation is that a crosstalk between the

JNK and p38MAPK pathways, is emerging as an important regulatory

mechanism in cellular response [Wagner et al., 2009]. The present data

indicate that, proinflammatory protein displays angiogenic properties. One can

thus hypothesize that PAP involvement in pathologies could reflect not only

the inflammatory but also the angiogenic status of the patient. In this way, we

suggest that PAP of synovial fluid may constitute a marker for synovial

membrane angiogenesis in rheumatoid arthritis. In the literature, inflammation

and angiogenesis have been frequently linked [Szekanecz et al., 2007].

Common molecular mechanism and signaling pathways have been described

in these two processes and it has been shown that several proinflammatory

cytokines induced during inflammation could promote a suitable

microenvironment for angiogenesis [Harhouri et al., 2010]. The presence of

measurable amounts of PAP in human SF was documented for the first time

in the current study. We have quantified PAP level in SF of RA patients.

Though we could include few patient samples for our study, the result suggest

that the measurement of PAP in joint fluid may prove to be of potential value

in the clinical evaluation of joint disease processes and targeting of PAP can

be efficient angiostatic and inflammatory therapeutic strategy. The number of

functions attributed to being regulated by the transcription factor NFκB is

rapidly increasing. It is involved in the control of a large number of normal



cellular processes such as inflammatory and immune responses,

developmental process, and cell growth. In addition, NFκB is activated in

several pathological conditions like arthritis, inflammation, neurodegenerative

diseases and cancers. IPAPpropriate NFκB activity has been reported in

several cancers [Romieu-Mourez et al., 2001]. To check whether PAP

regulates NFκB–DNA binding, EMSA was performed. The data revealed that

PAP induced NFκB–DNA binding. Activation of NFκB is known to confer

resistance to apoptotic signals [Collart et al., 1990 and Wang et al.,1998].

Enhanced proliferation rates and resistance to apoptosis-inducing signals are

important factors contributing to tumor growth. Similar results were obtained in

the effect of PAP on VEGF gene promoter luciferase reporter analysis and Flt-

1 gene promoter-CAT reporter assay. Effects observed with PAP were similar

to that obtained with VEGF. Our experiments show that PAP alone can

stimulate angiogenesis. So may be, PAP signaling pathway is different from

that of VEGF. Interestingly, signaling pathways of both factors are however

inter-connected since our data show that PAP induces VEGFR1 expression,

VEGF expression, p38MAPKactivation/JNK activation and NFκB signaling

pathways, leading to endothelial cell proliferation, tumor cell migration,

angiogenesis and invasion. These data suggest that there could be a positive

feedback loop between PAP and VEGF which may escalate inflammatory

responses and angiogenesis per se. Evidence of proangiogenic properties of

PAP in vitro led us to investigate whether it could promote neovascularization

in vivo. Our in vivo CAM assays consistently reproduced the results with rat

cornea assays showing that PAP induces neovascularization. Monoclonal

antibodies against PAP are especially valuable for angiogenesis targeted

therapy. To verify whether mAb against PAP had this property, we examined

the in vitro and in vivo effect of the antibody. The antibody inhibited tube

formation, cell proliferation and migration. These result prompted us to

investigate the in vivo effects of the antibody on neovascularization Our

observations were consistent with those for the in vitro experiments. We

confirmed the in vivo antiangiogenic effect of anti-PAP-mAb. Importantly, the

antibody significantly inhibited neovascularization in rat cornea and it could

not promote neovascularization in vivo CAM assay. These findings and those



of the in vitro and in vivo functional analysis provide compelling evidence that

PAP-induced angiogenesis is composed largely of two sequential steps. The

induction of VEGF-A gene expression by PAP and the subsequent VEGF-A

induced angiogenesis. Notably, PAP induced angiogenesis measured as

potent as that induced by VEGF. In conclusion, PAP displays angiogenic

properties and promotes neovascularization both in vitro and in vivo animal

model. Anti- PAP-mAb inhibited angiogenesis. Thus, besides its role as an

angiogenic marker, it sheds new light on its putative role in angiogenesis and

human disease, including cancer.

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