Bioanalytical strategies for the quantification of xenobiotics in biological fluids and tissues 7

Chapter 7 Detection of endorepellin in ovarian tumor serum and plasma samples by atomic force microscopic imaging study: Insights to early detection of ovarian tumor... For the first tim

Chapter 7 Detection of endorepellin in ovarian tumor serum and plasma samples by atomic force microscopic imaging study: Insights to early detection of ovarian tumor

7.1 Preface to Chapter 7

Recent advances in angiogenesis research and vascular biology have led to the discovery of a powerful angiogenesis protein inhibitor named endorepellin Endorepellin is found to be in higher concentration in normal healthy humans and lower concentration in cancer patients hence it has been identified as a potential cancer biomarker and therapeutic drug For the first time, an attempt was made to apply the atomic force microscopic study on tumor and control serum samples to compare the levels of endorepellin expression in both tumor and control samples To identify and understand the biological activities and chemistry involved, computational modelling of the protein was done

Conventional proteomics were done in this study in a bid to differentiate endorepellin expression in the tumour and control samples Plasma (n=2) and serum (n=2) from healthy human and cancer patient were used Through the Bradford assay,

it was apparent that the total protein concentration for both healthy and cancerous samples was similar and fall within a range of 69 mg mL-1 to 75 mg mL-1 Protein profiling was done using one-dimensional polyacrylamide gel electrophoresis (1D SDS-PAGE) and LG3 was successfully found to be less expressed in cancerous plasma and serum than in healthy samples That was further proved by AFM imaging study on tumor and control serum and plasma samples

7.1 Introduction

Angiogenesis is the development of new blood capillaries and is widely involved in several physiologic and pathologic processes such as invasive tumour growth [1] In particular, it was first hypothesized by Folkman that tumour-growth is angiogenesis dependant in 1971 [2] However, it was only after the discovery of the first angiogenesis inhibitor and the purification of the first angiogenesis protein in the mid 1980s that resulted in the widespread acceptance of the concept The discovery of

an efficacious angiogenesis protein inhibitor named endorepellin in recent times; signify an exciting potential breakthrough in the detection of tumour cancers and its subsequent cancer therapies [3]

Endorepellin (85-kDa) is the C-terminal domain of a large modular protein called Perlecan (~470kDa) which is composing of five structural domains Perlecan is

a basement membrane heparan sulphate proteoglycan that is involved extensively in vascular growth and tumour angiogenesis Endorepellin consists of three laminin-like globular domains (LG1-LG3) and is found to interact solely with the α2β1 integrin, a receptor for collagen I, in platelets and endothelial cells

Being one of the key receptors of endothelial cells, α2β1 provides vital support for vascular endothelial growth factor (VEGF) signalling, endothelial cell migration, and tumour angiogenesis [4] Therefore, by binding to α2β1 integrin, endorepellin causes disorder to the cell’s cycloskeleton and adhesion properties [5] On the whole,

endorepellin was shown to inhibit three major steps in angiogenesis namely adhesion, migration and morphogenesis Research studies had shown that systematic delivery of human recombinant endorepellin to tumour xenograft mice causes a considerable

down-regulation of the tumour angiogenic network [6] Apart from down-regulating pro-angiogenic proteins, endorepellin can also attach to endostatin (another matrix-derived inhibitor of angiogenesis that had been tested in clinical trials) and work against its anti-angiogenic effects [3] The superior anti-angiogenic abilities of endorepellin further strengthen the belief that it will serve as a better biomarker and therapeutic drug

In particular, the last laminin-like globular domain, LG3 (~26kDa) is found to acquire most of the biological activities and thus has most of the anti-angiogenesis ability LG3 can interact and be released by partial proteolysis during physiologic and pathologic processes such as tissue remodelling and cancer growth [7] This is proven

by the fact that LG3 fragments were found in the urine of patients with end-stage renal failure and chronic allograft nephropathy, and in the amniotic fluid of pregnant woman [8-10] More importantly, it was shown for the first time that circulating LG3 levels in human breast cancer plasma was significantly lower than the LG3 levels in healthy human plasma, indicating endorepellin, more specifically, LG3 as a potential biomarker for cancer detection, progression and invasion [11]

Apart from its anti-angeogenis activity and its ability to reduce tumour to a manageable size or inhibit tumour growth, other intrinsic characteristics of endorepellin also justify its selection for this study Being a protein-based inhibitor, it does not induce resistance and the toxicity is low It is able to work in low concentration (i.e nM) and it may also exert an anti-adhesive action on certain tumour cells It shows better anti-angiogenic properties and therefore a greater potential as a tumour biomarker and therapeutic drug Endorepellin is found to be in higher concentration in normal healthy humans and lower concentration in cancer patients

The malignant transformation of a normal epithelial cell is generally thought

to be caused by genetic alterations or mutations that disrupt the regulation of proliferation and apoptosis, in turn leading to an altered protein expression and modification [12] Alterations in protein levels can be detected not only in the cancer cells, but also in the blood and other body fluids into which these proteins are secreted This can therefore aid in the identification of a normal cell transforming into

a cancerous state Hence analysis of those body fluids by proteomic studies for quantification of endorepellin will lead to the clue about the state of the tumour cells

Electrophoresis is the separation of macromolecules in an electrically charged field For this, a support medium such as polyacrylamide or agarose is required Gel electrophoresis is a simple way to separate proteins prior to downstream detection or analysis PAGE is most commonly used to separate proteins in a sample based on their molecular weight (or length of polypeptide chain) However, the general electrophoresis methods cannot be used to separate proteins according to molecular weight alone because the mobility of a substance in the gel is influenced by both charge and size In order to overcome this, the proteins undergoing electrophoresis are treated with SDS, an anionic detergent, so that proteins have a uniform charge The method of SDS-PAGE that is currently being used, involving the use of a Tris-glycine running buffer to carry out electrophoresis, was first described by Laemmli and is better known as the Laemmli method [13]

Atomic force microscopy is an imaging technique which permits the investigation of molecules in their native physiological buffer condition without subjecting the sample to harsh treatments such as drying, crystallizing or vaporizing in

sample This feature made this technique highly suitable for topographical imaging of biological samples [14] AFM can provide nanometer-resolution images of living cells

in gaseous and liquid environments

In an AFM, a sharp stylus (approximately tenths of a nanometer) attached to the end of a cantilever is approached to the surface As a consequence, a force appears between the tip and surface that can be attractive or repulsive causing the cantilever to bend When this bending is controlled with a feedback algorithm, it is possible to obtain a topographic map by scanning the surface in a plane perpendicular to the tip

By using this technique individual protein molecules in aqueous solutions can be imaged directly at sub molecular resolution If suitable antibody reagents were available, this technology could be used to detect the presence of a specific protein by identifying its protein-antibody complex

In this present study, the structure and environment of the LG3 domain in the endorepellin was identified using homology modelling and the presence of endorepellin in plasma and serum samples was established by conducting conventional proteomic studies i.e SDS-PAGE for the quantification, separation and

identification of LG3 domain Further, the expression of endorepellin in tumour

plasma and serum samples was detected by Atomic force microscopic imaging studies and compared with control plasma and serum samples

7.2 Experimental

7.2.1 Chemicals and reagents

Acetic acid, Acetone, Formic acid, Tris base (Merck); Bovine serum albumin (BSA) standards, Bradford dye, bromophenol blue, dithiothreitol (DTT), glycerol, glycine, (Sigma-Aldrich); ammonium persulphate, 30% bis/Acrylamide, Precision

Plus protein all blue standards, SDS, N,N,N',N'-Tetramethylethylenediamine (TEMED) (Bio-Rad Laboratories, Hercules, CA, USA); Silver Stain plus kit (Bio-Rad laboratories), mouse monoclonal [a74] to heparan sulfate proteoglycan 2 antibody (Anti-HSPG2) (Abcam, Cambridge, UK),

7.2.2 Water and solutions

Autoclaved water, 1x phosphate-buffered saline (PBS); 5x SDS/Glycine electrophoresis buffer (15.1 g tris base, 72 g glycine and 5 g SDS); Silver stain fixative solution (40% methanol, 10% acetic acid (v/v); Silver stain stop solution (5% acetic acid, 95% water); 2x loading buffer (0.313 M Tris-HCl pH 6.8 at 25oC, 10% SDS, 0.05% bromophenol blue, 50% glycerol, and 0.5 M DTT); upper Tris solution (0.5 M Tris [pH 6.8], 0.4% SDS); lower Tris solution (1.6 M Tris [pH8.8], 0.4% SDS); rehydration buffer (7 M urea, 2 M thiourea, 100 mM DTT, 4% CHAPS, 0.5% carrier ampholytes pH 4–7, 0.01% Bromophenol blue (BPB) and 40 mM Tris)

7.2.3 Hardware and equipment

P-2, P-10, P-20, P-100, P-200 and P-1000 pipettes (eppendorf); 96 well microtiter plate (Tecan Asia); 0.75 mm spacer plates, short glass plates, gel casting stand and combs (Bio-Rad Laboratories); GS-800 calibrated densitometer, UltraRocker Rocking Platform (Bio-Rad Laboratories); Pχ2 programmable thermal cycler (Thermo Hybraid, Middlesex, TW, USA); bench top microcentrifuge for 0.5- and 1.5 ml polypropylene tubes (Sanyo Gallenkamp PLC, Loughborough, UK); PowerWaveX Select Microplate Spectrophotometer (BioTek, Winooski, VT, USA);

pH meter, Weighing Balance (Sartorius)

7.2.4 Computer software

The PyMOL molecular graphics system (DeLano Scientific, Palo Alto, CA, USA), Accerlys Discovery Studio 2.0 client (Accerlys Inc, San Diego, CA, USA), KC4 (BioTek, Winooski, VT, USA); PDQuest version 7.2 software package (Bio-Rad Laboratories,) GwyddionTM 2.29 (Czech republic)

7.2.5 Atomic force microscopy

The imaging of endorepellin expression was performed using NanoMan AFM system (Veeco metrology group, USA) which allows contact and tap mode image, multichannel data acquisition, and operates under ambient laboratory conditions, in vacuum, or in solution The system equipped with calibrated silicon nitride AFM cantilever (OTR8- 35) with force constant of 0.57 N/m, tip size of 15 nm and resonant frequency 300 kHz (Veeco)

7.2.6 Preparation of plasma and serum samples

Plasma (n=2) and serum (n=2) samples from a healthy being and ovarian cancer patient were each obtained from the Department of Obstetrics & Gynaecology, National University Hospital, Singapore The fluids were centrifuged at 15,000 rpm for 10 min at 4˚C The supernatants were then divided into aliquots of 1 mL, snap

frozen in liquid nitrogen, and stored at -80˚C until analysis

7.3 Methodology

7.3.1 Homology modelling

Homology Modelling is fundamentally made up of two principles Firstly, the structure of a protein is distinctly identified by its amino acid sequence [15] This implies that the sequence information alone is sufficient to obtain the protein structure Secondly, the structure is more highly conserved than the sequence,

suggesting that the structure is more stable and changes less significantly during evolution [16] As such, similar sequences are assumed, and later proven, to fold into practically identical structures and that distantly related sequences will still adopt similar structures [17] It is with these principles that allow modelling of an unknown target based on the sequence similarity with other homologous proteins that have known crystal structures These homologous proteins are referred as templates in this chapter

The search for templates was first carried out using the PSI-BLAST and BLASTp server at NCBI Unfortunately, the search did not return with substantial results, thus another technique called the fold recognition was also employed This technique specifically searches for similar secondary structures (such as the folding of alpha helix or beta sheets etc) in addition to searching for similar sequences Through LOMETS, an automatic mail server for protein secondary structure prediction, templates with the highest identities match were obtained (Table 7.1) The templates were named as according to their Protein Data Bank (PDB) number

Table 7.1

Top 2 templates results obtained through secondary structure prediction

1dyk Laminin alpha 2

chain LG 4-5 domain

House mouse

2.00 Å 42.90% 25.00%

1pz7

Modulation of agrin function by alternative splicing and Ca2+ binding

Chicken 1.42 Å 41.60% 21.50%

From Table 7.1, it is evident that murine laminin α2LG4-5 domain has the

highest percentage of identity (25%) when matched with the endorepellin LG3 sequence The low similarity and identity of all the templates are typical of homology modelling among LG domains [18] However, the low resolution of 1dyk posed as an obstacle to creating a good homology model With respect to this, the templates 1dyk and 1pz7 were superimposed onto each other in an attempt to resolve the resolution problem while not comprising much on the identity percentages The newly generated multiple sequence alignment was then aligned with the target LG3 sequence (Figure 7.1), thereby creating the basis of the final LG3 homology model The overall sequence identity obtained is 22.6% with the model having a resolution of 1.68Å

7.3.2 Protein quantification, sample preparation, separation and identification

of LG3

7.3.2.1 Total protein quantification by the Bradford assay

The Bradford method is a colorimetric assay technique used to determine protein concentration in a sample It uses the Coomassie Brilliant Blue G-250 dye, which has a maximum absorbance at 595 nm when bound to proteins The dye binds primarily to lysine and arginine residues on the protein, where it becomes ionised and its maximum absorbance increases The increase in absorbance at 595 nm is thus proportional to the amount of protein present

As the Bradford assay is only linear over a short range between 100 and 1500

μg mL-1

, samples were diluted with a 100 factor before quantification begins Therefore, 5 µl of each samples were added with 495 µl of 1x PBS Next, 250 µl of the Bradford (Coomassie Brilliant Blue G-250) dye were added to 5 µl of each standards and samples in a 96-well microtiter plate Duplicate standards and samples

were prepared and analysed at 595 nm using a micro plate spectrophotometer in each run so that the average protein concentration could be calculated, thus taking into account any intra-assay error The results were then computed with the KC4 program The average protein concentration found in plasma and serum samples are 74.942 and 71.569 mg mL-1 for healthy sample and 74.11 and 70.886 mg mL-1for cancer sample respectively

7.3.2.2 Acetone precipitation

The purpose of protein precipitation is to concentrate samples that have low protein concentration, and to remove substances that may interfere with protein separation by SDS-PAGE e.g nucleic acids, lipids, polysaccharides and salts Acetone denatures the proteins and causes more hydrophobic areas of the protein to

be exposed This causes the proteins to clump together and form a solid precipitate

As a sample calculation, 133.436 (≈133.4) µL of healthy plasma and 141.072 (≈141.1) µL of cancer serum were needed in order to precipitate 100 µg of proteins

Following which, five volumes of ice-cold acetone (667 µL for plasma and 705 µL for serum) was added into the respective samples and mixed thoroughly, before incubating overnight at -20 °C

7.3.2.3 SDS-PAGE

7.3.2.3.1 Assembly of apparatus before casting the polyacrylamide gels

To prepare for casting the polyacrylamide gels, the 0.75 mm spacer and short plates were cleaned with 70% ethanol The cleaned 0.75 mm spacer and short plates were then inserted into the casting frame, placed on the gasket and held together by the casting stand

7.3.2.3.2 Preparation of SDS-PAGE

SDS-PAGE gels with 2 different acrylamide concentrations (10 and 12%) were used in this study The resolving gel mixture was prepared by mixing the reagents listed in Table 7.2 The mixture was then loaded into the space between the glass plates, and the resolving gel was left to polymerize at room temperature for 45 minutes Once it had polymerized, the stacking solution (also prepared by mixing the reagents listed in Table 7.2) was loaded above the resolving gel to the top of the short glass plate The 10-well Bio-Rad Laboratories comb was cleaned with 70% ethanol and inserted into the stacking solution The stacking solution was then left to polymerise for at least 4 hours at room temperature or left overnight at 4 °C

7.3.2.3.3 Running SDS-PAGE

Following acetone precipitation overnight, the plasma and serum samples were thawed to solution before centrifugation at 13000 RPM was applied for 5 minutes The supernatant was then removed and the pellet was air-dried for 1 minute, after which, the pellet was re-solubilised in 50 µL of 1x PBS or rehydration buffer and liquated into 5 aliquots of 10 µL each 5 µL of re-dissolved sample were used in each SDS-PAGE run

The 5 µl of re-dissolved sample was mixed with 5 µl of 2x loading buffer (0.313 M Tris-HCl, pH 6.8, 10% SDS, 0.05% bromophenol blue (BPB), 50% glycerol, 0.5 M DTT) such that the resulting sample had a protein concentration of 1 μg/μl The samples were then heated at 99o

C for 7 minutes in a thermo-cycler, and then centrifuged at 13,000 RPM for 10 minutes 5 µl of protein standard (Precision plus protein All blue® standards), control (2 x loading buffer) and samples were loaded into the wells once the SDS-PAGE gels had polymerized A constant voltage

of 100V was applied and electrophoresis was run Enhanced resolution of low molecular weight (MW) protein bands was obtained by preparing 12% resolving gel and increasing the final sample loading volume to 15 µl

Table 7.2

Composition of mini-size (0.75mm thick) SDS-PAGE gel

4x Lower Tris (1.6 M Tris, pH 8.8, 0.4% SDS) AA: Acrylamide/Bis Solution

4x Upper Tris (0.5 M Tris, pH 6.8, 0.4% SDS) APS: Ammonium persulphate

7.3.2.3.4 Gel staining

The proteins that have been separated on gels can be made visible by staining them with dyes or metals A number of different protein stains exist like the Coomassie Blue stain, Ruby fluorescent stain and Silver stain Each type of stain has its own characteristics and limitations with regard to the sensitivity of detection and the types of proteins that stain best[19] In this study, silver staining was chosen as the method for staining the gels

Silver Stain Plus kits were used according to manufacturer’s instructions

After electrophoresis, gels were taken out from the glass plates and placed with care into plastic containers, which had already been cleaned with concentrated nitric acid and deionised distilled water The gels were fixed in approximately 50-100 ml of fixative solution containing 40% methanol and 10% acetic acid (v/v) overnight on an

UltraRocker Rocking Platform at room temperature This was followed by rinsing and washing the gels in deionised distilled water for 30 min Gels were then stained in developer solution until desired staining intensity was reached, and placed in 5% acetic acid for 30 minutes to stop the reaction The gels were then scanned using a GS-800 calibrated densitometer and analyzed using the PDQuest version 7.2 software programs

7.3.3 AFM Imaging

7.3.3.1 Preparation of sample, substrate and antibody for imaging

The ovarian tumor and control samples were centrifuged at 15,000 RPM for

10 min at 4 °C The supernatants were then divided into aliquots of 1 ml, snap frozen

in liquid nitrogen, and stored at -80°C until analysis A stock concentration (1µg mL1

-) of anti-HSPG2 antibody in phosphate buffer was prepared The buffer used was prepared by dissolving one tablet of phosphate-buffered saline (PBS) in 200 mL deionized water The obtained solution consisted of a 10 mM phosphate buffer, 2.7

mM potassium chloride, and 137 mM sodium chloride The pH of the solution at 25˚C is 7.2 The stock solution can be pipette in small aliquots (1 to 5 mL) into eppendorf tubes and flash frozen in liquid nitrogen prior to storage at −80°C

Two important criteria for the substrate preparation in this AFM study are (1) the antibody affinity to the substrate must be adequately strong so that they can be immobilized on surface without sacrificing their bio reactivity and (2) the substrate should be smooth enough so that proteins can easily be identified from AFM topographies Mica surface is the most commonly used substrate for protein adsorption because it is hydrophilic and atomically flat

The first step was to immobilize the antibody on the substrate The stock solution of anti-HSPG2 was diluted 2 and 4 fold into Milli Q water Twenty microliters of the Anti-HSPG2 was drawn off with a pipette and deposited on the freshly cleaved mica substrate Then the antibody covered mica substrate was incubated for approximately 20 sec before rinsing in excess Milli Q water to remove any weakly adsorbed antibody and residual salt deposits After washing, the substrate was dried in a stream of dry N2 gas (1 bar pressure at a distance of several cm) Rinsing can be achieved by running up to 5 ml of Milli Q water across the mica sample while it is tilted at a 30 to 45° angle The AFM imaging was done immediately after evaporation of the solvent to prevent contaminations

Subsequently, the target protein in the sample was applied to the antibody adsorbed mica substrate and allowed to dry under a flow of N2 gas Immediately after solvent evaporation, the antigen-antibody layer washed three times with water to remove any residual deposits or loosely adsorbed proteins The samples were then allowed to dry completely and then incubated in a humid chamber at room temperature for a specified period (60 min, 30 min) of time The amount of antibody binding depends on several factors, including degree of washing with water, concentration of antibody, and other incubation conditions The conditions of binding with surface adsorbed protein are summarized in Table 7.3

In the control experiments, Anti-HSPG2 was used with the same incubation times and at the same concentrations as of tumor samples