CHAPTER 2shodhganga.inflibnet.ac.in/bitstream/10603/36544/6/chapter 2.pdf · CHAPTER 2 Proteomic...
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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
CHAPTER 2 Proteomic analysis of PAP and proangiogenic activity
Cellular Signalling 25 (2013) 277–294 35
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
CHAPTER 2 Proteomic analysis of PAP and proangiogenic activity
Cellular Signalling 25 (2013) 277–294 36
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
Cellular Signalling 25 (2013) 277–294 37
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
Cellular Signalling 25 (2013) 277–294 38
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
Cellular Signalling 25 (2013) 277–294 39
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.
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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-
CHAPTER 2 Proteomic Studies of PAP
Cellular Signalling 25 (2013) 277–294 41
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
CHAPTER 2 Proteomic Studies of PAP
Cellular Signalling 25 (2013) 277–294 42
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.
CHAPTER 2 Proteomic Studies of PAP
Cellular Signalling 25 (2013) 277–294 43
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
Cellular Signalling 25 (2013) 277–294 44
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.
CHAPTER 2 Antibody production for PAP
Cellular Signalling 25 (2013) 277–294 45
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
CHAPTER 2 Antibody production for PAP
Cellular Signalling 25 (2013) 277–294 46
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.
CHAPTER 2 Antibody production for PAP
Cellular Signalling 25 (2013) 277–294 47
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
Cellular Signalling 25 (2013) 277–294 48
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).
CHAPTER 2 Proangiogenic Activities of PAP
Cellular Signalling 25 (2013) 277–294 49
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
CHAPTER 2 Proangiogenic Activities of PAP
Cellular Signalling 25 (2013) 277–294 50
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.
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Cellular Signalling 25 (2013) 277–294 51
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
Cellular Signalling 25 (2013) 277–294 52
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
CHAPTER 2 Molecular Mechanism of PAP
Cellular Signalling 25 (2013) 277–294 53
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.
CHAPTER 2 Molecular Mechanism of PAP
Cellular Signalling 25 (2013) 277–294 54
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.
CHAPTER 2 Molecular Mechanism of PAP
Cellular Signalling 25 (2013) 277–294 55
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.
CHAPTER 2 Molecular Mechanism of PAP
Cellular Signalling 25 (2013) 277–294 56
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
CHAPTER 2 Molecular Mechanism of PAP
Cellular Signalling 25 (2013) 277–294 57
(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
CHAPTER 2 Molecular Mechanism of PAP
Cellular Signalling 25 (2013) 277–294 58
[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
CHAPTER 2 Molecular Mechanism of PAP
Cellular Signalling 25 (2013) 277–294 59
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
Cellular Signalling 25 (2013) 277–294 60
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
Cellular Signalling 25 (2013) 277–294 61
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
Cellular Signalling 25 (2013) 277–294 62
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
Cellular Signalling 25 (2013) 277–294 63
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
Cellular Signalling 25 (2013) 277–294 64
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
Cellular Signalling 25 (2013) 277–294 65
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
CHAPTER 2 Results: Molecular Mechanism of PAP
Cellular Signalling 25 (2013) 277–294 66
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).
CHAPTER 2 Results: Molecular Mechanism of PAP
Cellular Signalling 25 (2013) 277–294 67
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
CHAPTER 2 Results: Molecular Mechanism of PAP
Cellular Signalling 25 (2013) 277–294 68
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
Cellular Signalling 25 (2013) 277–294 69
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
Cellular Signalling 25 (2013) 277–294 70
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
Cellular Signalling 25 (2013) 277–294 71
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
Cellular Signalling 25 (2013) 277–294 72
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
Cellular Signalling 25 (2013) 277–294 73
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
Cellular Signalling 25 (2013) 277–294 74
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
Cellular Signalling 25 (2013) 277–294 75
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
Cellular Signalling 25 (2013) 277–294 76
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
Cellular Signalling 25 (2013) 277–294 77
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
Cellular Signalling 25 (2013) 277–294 78
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
Cellular Signalling 25 (2013) 277–294 79
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
Cellular Signalling 25 (2013) 277–294 80
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
Cellular Signalling 25 (2013) 277–294 81
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
Cellular Signalling 25 (2013) 277–294 82
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
Cellular Signalling 25 (2013) 277–294 83
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
Cellular Signalling 25 (2013) 277–294 84
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
Cellular Signalling 25 (2013) 277–294 85
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
Cellular Signalling 25 (2013) 277–294 86
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
CHAPTER 2 DISCUSSION
Cellular Signalling 25 (2013) 277–294 87
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
CHAPTER 2 DISCUSSION
Cellular Signalling 25 (2013) 277–294 88
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
CHAPTER 2 DISCUSSION
Cellular Signalling 25 (2013) 277–294 89
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
CHAPTER 2 DISCUSSION
Cellular Signalling 25 (2013) 277–294 90
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.