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Introduction

Role of hCG in chemo attraction, angiogenesis and invasion 39

Angiogenesis

Blood vessels are a complex network of tubes that carry oxygenated blood and

nutrients. The fundamental biological mechanism by which new blood vessels grow

is called angiogenesis. It is a physiological process in which new blood vessels are

formed from pre-existing vessels (Dewitt, 2005; John, 2008).

A developing child in a mother's womb must create a vast network of arteries, veins

and capillaries. A process called vasculogenesis creates the primary network of

vascular endothelial cells. Later on, angiogenesis remodels this network into the small

new blood vessels or capillaries that constitute the child's circulatory system.

Angiogenesis in adults is a relatively rare event, playing an important role in various

physiological conditions like wound repair. Female reproductive organs (the ovary,

the uterus and the placenta) go through repeated cycles of growth and remodelling

under the influence of hormones (Carmeliet and Jain, 2000). Apart from these normal

conditions, angiogenesis is also observed to occur in diseased states, being associated

with inflammation and tumorogenesis.

Both normal cells and tumor cells require oxygen; for adequate supply, cells must be

located within 100-200 μm of blood vessels (Figure 3.1). As solid tumors grow and

breach this limit, the recruitment of new blood vessels by vasculogenesis and

angiogenesis becomes a necessity (Figure 3.2). Besides fulfilling the tumor’s oxygen

and nutritional needs, newly-sprouted vessels also carry transformed cells to distal

sites during the process of metastasis. Enhanced vascularisation can also be used to

advantage in the delivery of effective concentrations of chemotherapeutic drugs into

the core of the tumor (Carmeliet and Jain, 2000).

Introduction


Figure 3.1

Figure 3.1: Oxygen concentration gradient in tissues.

Introduction


Figure 3.2

Figure 3.2: The figure depicts schematically the generation of a new capillary sprout from a

pre-existing vessel towards the hypoxic region of a tumor, such as a post capillary venule (on

the left). Numbers refer to various, often overlapping stages of the process. Angiogenic factors

secreted by the tumor cells activate the vascular endothelium lining the vessel and pericytes

(white) retract (1). Proteases degrade the basement membrane beneath endothelial cells (2),

which subsequently become less adherent (3). The αvβ3 and αvβ5 integrins are important at

these stages. Vascular permeability is also increased, allowing fibrin deposition into the tissue

(4). Endothelial cells migrate toward the angiogenic stimulus (5) and enter the cell cycle (6).

Circulating endothelial precursors may also become incorporated to the growing vessel sprout

(7). New vessels mature and become established when periendothelial structures are formed

(8) but this process is often defective in tumors. Abundant VEGF is secreted by hypoxic

regions of the tumor (shown dark). VEGF is involved in most of the steps shown, while Ang-

1 may function in concert with VEGF to stimulate vessel sprout invasion. Ang-2, which

becomes up regulated in endothelial cells of angiogenic capillary sprouts may disrupt the

interactions between endothelial cells and pericytes, thus sensitizing the endothelium to the

mitogenic and chemotactic signals secreted by the tumor.

(Veikkola and Alitalo, 1999)

Introduction


Tumor angiogenesis

The process of angiogenesis probably constitutes the most important component of

tumor growth and metastasis; the development of anti-angiogenesis strategies is

therefore an area of intense research (Folkmann, 1971). In the initial stages of

malignancy, there exists a balance between the proliferation and destruction of

neoplastic cells. When the primary tumor reaches a certain size, the local

concentration of oxygen decreases, causing cells to produce angiogenic factors. The

accompanying tissue destruction leads to the production of anti-angiogenic

substances. The average life of anti-angiogenic factors is greater than that of

angiogeneic factors, so initial tumor growth and metastasis are controlled. The

formation of new vessels takes place when the balance is altered in favour of pro-

angiogenic activity. Pro-angiogenic factors include several molecules released by

parenchymal or inflammatory cells in response to mechanical factors, metabolic

factors (hypoxia, acidosis) or the host immune response.

Tumor angiogenesis involves both blood vessels and lymphatic vessels. Differences

are observed between normal and tumor vasculature. Normal vasculature is arranged

in a hierarchy of evenly spaced, well-differentiated arteries, arterioles, capillaries,

venules and veins, whereas tumor vasculature is unevenly distributed and chaotic

(Figure 3.3). Tumour vessels are dilated and serpentine, branched irregularly and

excessively, have an irregular diameter and form arterio-venous shunts (Warren,

1979). Blood flow through tumours does not follow a constant, unidirectional path.

Microscopically, the endothelium has several openings (fenestrate endothelial,

vesicles, transcellular holes), demonstrate widened intercellular junctions, with the

basement membrane either discontinuous or absent.

http://www.nature.com/bjc/journal/v100/n6/full/6604929a.html#bib35



Introduction


Figure 3.3

Figure 3.3: Tumours contain regions of hypoxia and necrosis because their vasculature cannot

supply oxygen and other vital nutrients to all the cells. Whereas normal vasculature (a) is

hierarchically organized, with vessels that are sufficiently close to ensure adequate nutrient

and oxygen supply to all cells, tumour vessels (b) are chaotic, dilated, tortuous and are often

far apart and have sluggish blood flow. As a consequence, areas of hypoxia and necrosis often

develop distant from blood vessels. In addition to these regions of chronic (or diffusion-

limited) hypoxia, areas of acute (or perfusion-limited) hypoxia can develop in tumours as a

result of the temporary closure or reduced flow in certain vessels.

(Brown and Wilson, 2004)

Angiogenic factors can modulate the expression of cell adhesion molecules and other

surface markers on tumor vascular endothelium. For example, vascular endothelial

growth factor (VEGF) and tumor necrosis factor-α (TNF-α) up-regulate, while the

fibroblast growth factor (FGF) and transforming growth factor-β1 (TGF-β1) down-

regulate adhesion molecules. Modulation can be influenced both by tumor type and

surrounding stromal cells (Gohongi et al., 1999). Some of the factors that regulate

tumor angiogenesis and their modes of action are described in Table 3.1.

Factor Role in Tumor Neovascularisation

VEGF Secreted by many tumor cells in vitro

Introduction



Highly up regulated in most human cancers

Expression correlates with intratumoral micro vessel density and

poor prognosis in cancer patients

Inhibition decreases tumor vessel density and tumor growth

FGF Inhibition suppresses generation of tumor vessels in vitro and in

vivo and tumor growth in vivo

Important for maintenance vs. induction of tumor angiogenesis

Synergizes with VEGF to promote angiogenesis in vitro and in

vivo

Induces VEGF expression in tumor cells and VEGF receptor

expression in endothelial cells

Heparinase Stimulates invasion and vascular sprouting of endothelial cells

Releases bFGF from extracellular matrix

mRNA and protein are enriched in metastatic tumor cell lines

and human tumors vs. normal tissues

Over expression renders nonmetastatic cell lines metastatic in

vivo and increases tumor neovascularization

Ang 2 Induced in endothelial cells of pre-existing vessels co-opted by a

tumor, leading to vessel regression

Induced in endothelial cells of newly formed vessels of tumor,

leading to vessel plasticity and VEGF-mediated growth

IL-8 Mitogenic and chemotactic for HUVECs in vitro

Stimulates angiogenesis in vivo

mRNA is up regulated in neoplastic vs. normal tissues in vivo;

expression correlates with extent of neovascularisation

Over expression increases invasiveness, tumorigenicity,

neovascularization, and metastatic potential of tumor cells

Mediates stimulation of MMP-2 gene transcription

Introduction



MMP-2 Directly modulates melanoma cell adhesion and spreading on

extracellular matrix

Mediates tumor growth and neovascularization in CAM

Michael and Herman. American j of physiology

Matrix metalloproteases (MMPs) are a family of Zn-dependent endopeptidase (Gros

and Lapier, 1962). MMPs mediate ECM degradation which leads to cancer invasion

and metastasis (Liotta, 1990). They also affect signalling pathways that modulate the

biology of the cell and may be crucial in disrupting the balance between growth and

anti-growth signals. MMP-9, MMP-14 and MMP-2 proteolytically activate TGF-β1

(Mu. et al., 2002; Yu and Stamenkoric, 2000). Expression of MMP-3 in mammary

epithelium can stimulate a cascade of events leading to the cleavage of E-cadherin, a

characteristic of which results in epithelial-mesenchymal transition (EMT) (Lochter,

1997; Radisky, 2005). MMPs also interfere with the induction of apoptosis in

malignant cells, further contributing to tumor burden; MMP-7 cleaves Fas ligand on

deoxyrubicin-treated tumor cells (Mitsiades, 2001). MMP-9 regulates the

bioavailability of VEGF and so can influence the process of angiogenesis. MMPs also

impact on lymphogenesis (Nakamura et al., 2004). Upmodulated expression of MMP-

1, 2 and MMP-3 (Islekal et al., 2007) is related to lymphatic invasion and metastasis.

MMP-11 is the only MMP which expressed in adipose tissue as tumor cells invade the

surroundings. Over-expression of MMP-3, -7 and -14 results in enhanced

carcinogenesis (Egeblad and Werb, 2002). Conversion of TNF-α into the soluble

cytokine form require proteolytic cleavage by ADAM-17 and MMPs (Manicone and

McGuire, 2008). Processing of CXCL8/IL-8 by MMP-9 leads to a 10-fold increase in

chemotactic activity (Van ad steer, 2000).

VEGF is a signal protein that stimulates vasculogenesis and angiogenesis. It is part of

the system that restores the oxygen supply to tissues when blood circulation is

inadequate. Many tumor cell lines secrete VEGF in vitro (Senger et al., 1986.) VEGF

expression is up-regulated by hypoxia (Shweiki et al., 1992), and it serves as a major

http://ajpcell.physiology.org/search?author1=Michael+Papetti&sortspec=date&submit=Submit

http://ajpcell.physiology.org/search?author1=Ira+M.+Herman&sortspec=date&submit=Submit

Introduction


angiogenic factor in normal vascular development (Shalaby et al., 1995; Fong et al.,

1995). VEGF can help in the angiogenic response by increasing microvascular

permeability (Dvorak et al., 1995). VEGF stimulates several endothelial cell responses

in cell culture, including proliferation, migration and survival. As indicated above,

once a tumor grows to a certain size, the cells at the core are too far from existing

blood vessels to receive necessary oxygen and nutrients. Sensors within these

“starved” cells recognize the decrease in oxygen and initiate processes for producing

angiogenic growth factors, most notably VEGF. Both VEGF and its receptor (Flk-1)

are highly expressed in metastatic human colon carcinomas and their associated

endothelial cells; thus, the production of these two proteins correlates the tumor

vascularisation (Takahashi et al., 1995). Elevations in VEGF levels have been

detected in the serum of some cancer patients (Kondo et al., 1994) and a correlation

has been observed between VEGF expression and microvascular density in primary

breast cancer sections (Toi et al., 1996). Increased VEGF expression is closely

associated with increased intratumoral microvessel density and poor prognosis in

breast cancer patients (Toi et al., 1996). Intraperitoneal administration of anti-VEGF

antibody to nude mice implanted with either sarcoma and glioblastoma cells leads to

significant decreases in tumor vessel density and a suppression of tumor cell growth

(Kim et al., 1993). The three dimensional structure of VEGF is very similar to

platelet-derived growth factor, and both growth factors share conserved cysteine

amino acids (Keck et al., 1989). In situ hybridization has identified VEGF mRNA in

hypoxic regions of glioblastoma cells and capillary bundles have been found next to

the VEGF-producing cells (Shweiki et al., 1992). VEGF induces the migration of

monocytes from the periphery and also promotes angiogenesis by causing the

chemotaxis and growth of endothelial cells (Rudolfsson et al., 2004). Inhibition of

VEGF-induced angiogenesis has been shown to inhibit the growth of tumour cells in

vivo (Millauer et al., 1994; Saleh et al., 1996) and the molecule has emerged as an

important target for anti-cancer therapeutics (Gimbrone, 1972).

A growth factor not well-characterized for its role in physiological angiogenesis but

has nevertheless attracted attention in the context of tumor neovascularisation is IL-8.

Initial observations suggested that IL-8 is made by macrophages and mediates

angiogenesis in chronic inflammatory diseases such as psoriasis and rheumatoid

Introduction


arthritis (Koch et al., 1992). IL-8 is a pro-inflammatory CXC cytokine and is

increasingly recognized for its role in the progression and pathogenesis of cancer

(Rubie, 2007). IL-8 synthesis is believed to be a mediator of both tumorigenesis and

metastasis (Inoue et al., 2000). Heightened expression of both IL-8 and its two

receptors (CXCR1 and CXCR2) has been observed on tumours (Murphy et al., 2005).

The cytokine can have significant autocrine growth-promoting effects (Brew et al.,

2000) and can promote angiogenesis by activating endothelial cells in the tumour

vasculature (Li et al., 2003). The molecule has been shown to confer drug resistance

in some systems (Huang et al., 2010). IL-8 has been shown to be mitogenic and

chemotactic for HUVECs in vitro and also stimulates angiogenesis in the rat cornea

(Koch et al., 1986). IL-8 mRNA is up regulated in neoplastic tissues such as non-

small cell lung cancer (Yuan et al., 2000) and melanoma (Luca et al., 1997) and its

expression correlates with the extent of neovascularization. Overexpression of IL-8 in

non-metastatic, IL-8-negative melanoma cells not only increases their ability to invade

Matrigel-coated filters but also makes them highly tumorigenic and metastatic in nude

mice (Bar-Eli M, 1999). When gastric carcinoma cells producing low amounts of IL-8

were transfected with the IL-8 gene, they produced highly vascular neoplasms

(Kitadai et al., 1999).

hCG and angiogenesis

The female reproductive system undergoes physiological angiogenesis during the

menstrual cycle, folliculogenesis, ovulation and corpus luteum formation,

implantation, and in particular, during placenta formation (Gutman et al., 2008).

Successful implantation, placentation and subsequent gestation require finely-

regulated vascular development and adaptations on both sides of the maternal-fetal

interface. Due to demand for increased blood supply, the vasculature of the uterus and

endometrium undergoes three main adaptative changes: vasodilatation, increased

permeability and development and maturation of new vessels (Torry et al., 2007).

Disturbance in uterine blood supply or vascular remodelling is associated with higher

fetal morbidity and mortality due to miscarriage, pre-eclampsia or intrauterine growth

restriction. The physiological changes in uterine vascular remodelling are regulated by

growth factors such as VEGF, as well as by hormonal factors. hCG acts on several

molecules implicated in angiogenesis such as VEGF and both its receptors VEGFR-1

Introduction


and -2, angiopoietins and their receptor Tie-2, bFGF and placental-derived growth

factor (Reisinger et al., 2007).

Endothelial cells of the uterine vessels have been shown to express the LH/hCG

receptor (Toth et al., 1994). In vivo administration of hCG reduces vascular resistance

in the human uterus (Toth et al., 2001). hCG is now proposed as a new angiogenic

factor (Zygmunt et al., 2002). In an in vitro 3-D model, hCG promotes angiogenesis

by supporting the migration and formation of capillary structures by uterine

endothelial cells. hCG induces an increase in vessel sprouting on endothelial

endometrial cells, with an indirect effect demonstrated via the increase of VEGF

(Berndt et al., 2006) (Figure 3.4). hCG also stimulates proliferation of human

placental microvascular endothelial cells (HPMVEC) in a dose-dependent manner and

stimulates sprout formation in a spheroid angiogenesis assay (Herr et al., 2007). hCG

is known to increase the secretion of MMP2 and MMP9 from cytotrophoblast (Fluhr

et al., 2008).

Figure 3.4: The possible roles of HCG in human embryo invasion. The invading human

embryo locally secretes HCG at high concentration and stimulates recruited or resident

immune cells to produce chemoattractants and then these factors in turn induce embryo

invasion toward endometrial stromal tissue.

Hiroshi Fujiwara1, Molecular Human Reproduction. 2009.

hCG can induce the expression of VEGF transcripts in tumor cells (Mukhopadhyay et

al., 2004). Tumor-associated neo-angiogenesis and the production of VEGF (both by

tumor cells and by tumor-associated macrophages) are accompanied by the heightened

Introduction


secretion of MMPs which aids in both endothelial cell and tumor cell escape, thus

promoting extravasations and metastasis (Bazarbachi et al., 2004). MMP-2 and MMP-

9 are considered potential proteases involved in extracellular matrix remodelling

during trophoblast invasion (Bishop et al., 1998; Librach et al., 1991). Cytokines

expressed in the secretory endometrium such as Leukemia inhibitory factor (LIF), IL-

6 and Insulin Growth Factor Binding Protein 1 (IGFBP-1) can also modulate

trophoblastic invasion; these regulators of trophoblastic MMPs have been shown to be

influenced by hCG (Litch et al., 2001, Figure 3.5). Proteolytic degradation of the

stroma causes the release of sequestered fibroblast growth factor and VEGF, further

promoting tumor growth (Bergers et al., 2000).

Figure 3.5

Figure 3.5: Coordinated effects of hCG on several endometrial functions may lead to

prolongation of endometrial receptivity, increased angiogenesis, modulation of implantation

parameters and tissue remodelling.

Materials and methods


Cell culture

COLO 205 (human colorectal cancer), ChaGo (human lung cancer), JEG-3 (human

choriocarcinoma and LLC (murine lung cancer) were obtained from the American

Type Culture Collection (ATCC). Cell cultures were maintained under standard

conditions as described in Chapter 2. HBMEC (Human Brain Microvascular

Endothelial Cells), kindly gifted by Dr Kwang Sik Kim were cultured in RPMI 1640

(without antibiotics) supplemented with 10% FCS (Gibco), 10% NuSerum IV

(Becton Dickinson), 1% Modified Eagle’s Medium nonessential amino acids (Gibco),

1% vitamins (Gibco), 5 U/ml heparin (Sigma), 1 mM sodium pyruvate (Sigma) and

2mM L-glutamine (Sigma).

Effect of hCG on IL-8, VEGF and MMPs levels

Experimental set up

5 x 105

cells were incubated with medium, 1µg hCG, anti-hCG antiserum (1:500),

hCG + anti-hCG antiserum, non-immune serum (1:500) or hCG + non-immune

serum. After 24 hrs, supernatants were collected for the estimation of IL-8, VEGF and

MMP levels and RNA was isolated from the cells for PCR.

Reverse transcriptase PCR

Total RNA was isolated using a RNA isolation kit (Intron) as described in Chapter 2.

PCR was carried out with the help of a one-step reverse transcriptase-PCR kit

(Qiagen).

Reactions were set as follows:

RNA 1 µg

DNTP mix 1 µl

Buffer 10 µl

Primer (Forward) 10-15 pmol

Primer (Reverse) 10-15 pmol

Enzyme 1 µl

Nuclease free water was used to make up the volume up to 50 µl.



Primer sequences and PCR conditions were as follows:

VEGF (human): Forward 5’-CCATGAACTTTCTGCTGTCTT-3’

Reverse 5’-ATCGCATCAGGGGCACACAAG-3’

VEGF (murine): Forward 5’-CTGTGCAGGCTGCTGTAACG-3’

Reverse 5’-GTTCCCGAAACCCTGAGGAG-3’

Reverse Transcription: 500C for 30 min followed by a hold at 95

0C for 15 min.

Denaturation at 940C for 1 min, Annealing at 55.3

0C for 1 min, Extension at 68

0C for

1 min, Final Extension at 680C for 2 min. Number of cycles: 25.

MMP-2 (human): Forward 5’-GTGCTGAAGGACACACTAAAGAAGA-3’

Reverse 5’ -TTGCCATCCTTCTCAAAGTTGTAGG-3’

MMP-2 (murine): Forward 5’-CACCTACACCAAGAACTTCC-3’

Reverse 5’-AACACAGCCTTCTCCTCCTG-3’

MMP-9 (human): Forward 5’-CACTGTCCACCCCTCAGAGC-3’

Reverse 5’-GCCACTTGTCGGCGATAAGG-3’

MMP-9 (murine): Forward 5’-TTGAGTCCGGCAGACAATCC-3’

Reverse 5’-CCTTATCCACGCGAATGACG-3’

Reverse Transcription: 500C for 30 min followed by a hold 95

0C for 15 min.

Denaturation at 940C for 1 min, Annealing at 58


0C for 1

min, Final Extension at 720C for 10 min. Number of cycles: 35.

IL 8 (human): Forward 5’-AACTTTCAGAGACAGCAGAG-3’

Reverse 5’-TACAACAGACCCACACAATA-3’

KC (murine): Forward 5’-CTTGAAGGTGTTGCCCTCAG-3’

Reverse 5’-TGGGGACACCTTTTAGCATC-3’




0C for 15 min.



0C for 1


-actin (human): Forward 5’-AGATGACCCAGATCATGTTTGAGA-3’

Reverse 5’-CTAAGTCATAGTCCGCCTAGAAGC-3’

-actin (murine): Forward 5’- ATCCGTAAAGACCTCTATGC-3’

Reverse 5’- AACGCAGCTCAGTAACAGTC-3’


0C for 15 min.



0C for 1


Enzyme Linked Immunosorbant Assay (ELISA)

ELISA kits were employed for the estimation of IL-8 (BD PharMingen) and VEGF

(Peprotec) in cell culture supernatants. Briefly, capture antibody (500ng/100µl/well,

diluted in Coating Buffer (Appendix) was dispensed to wells of a 96-well flat bottom

ELISA plates. The plates were sealed and incubated overnight at 4°C. Plates were the

“washed” three times with Wash Buffer (PBS containing 1% BSA + 0.05% Tween-

20). 200µl/well of Blocking Buffer (PBS containing 3% BSA) was then dispensed and

an incubation was then carried out for 2 hrs at RT. Plates were the “washed” six times

with Wash Buffer. 100µl/well of the standards and samples diluted Assay Diluent

(PBS containing 1% BSA) were added to designated wells. The plates were then

covered and incubated at for 2 hrs at RT following which they were “washed”

extensively. 100µl/well of detection antibody (diluted in Assay Diluent) was then

dispensed. The plates were covered and incubated at room temperature for 1 hour

followed by extensive “washes”. 100µl/well of Substrate Solution (Appendix) was

then added to each well. The enzyme reaction was arrested by the addition of 50 µl

Stop Solution (Appendix). Optical Densities were determined at 450 nm.



Substrate gel Zymography

Zymography is a technique used to analyze the activity of MMPs. Cell culture

supernatants were electrophoresed on a 10 % SDS polyacrylamide gel containing

0.1% gelatin (Sigma); non-reducing Sample Buffer was employed. 60V were applied

till samples entered the resolving gel after which it was increased to 80V. After

completion of the run, the stacking gel was discarded; the resolving gel was “washed”

with Triton X-100 (2.5 %) on a rocking platform for 30 min at RT. After removal of

the Triton X-100 (save 2-3 ml), Developing Buffer (0.05 mM Tris–HCl pH 8.8, 5M

CaCl2, 0.02 % NaN3) was added and an incubation carried out for 15 min at RT. The

gel was then incubated at 37°C for 24 hr to allow both pro- and active MMPs to digest

the gelatin. Gels were stained by incubation with Coomassie Brilliant Blue R250

(Gibco BRL) for 16 hr and then destained using Destaining Solution (Appendix).

Gelatinylytic activity was visible as clear bands contrasting with the blue background.

Gels were then “washed” with distilled water. A pre-stained protein standard

(Fermantas) was used for estimating molecular mass. Densitometric estimationwere

carried out using the Image J programme.

Effect of hCG on the transmigration of endothelial cells

5 x 104

HBMEC were resuspended in Ex-vivo serum-free medium (BioWhittaker) and

added to uncoated polyethylene terephthalate transwell inserts (pore size: 8 µm pore

size; Becton Dickinson). hCG (1 µg), anti-hCG antiserum (1:500), hCG + anti-hCG

antiserum, non-immune serum (1:500) or hCG + non-immune goat serum were added

to the bottom chamber. After 24 hr incubation, cells in the bottom chamber were

counted (Figure 3.6).

Figure 3.6: Representative diagram for transmigration experiment.

Incubation



Effect of hCG on tumor cell invasion

2x105 cells (COLO 205, ChaGo or LLC) were resuspended in Ex-vivo serum-free

medium and added to transwell inserts previously coated with 50 µg Matrigel (Becton

Dickinson). hCG (40 ng), anti-hCG antiserum (1:10), hCG + anti-hCG antiserum,

non-immune serum (1:10) or hCG + non-immune serum were added to the bottom

chamber. After 48 hr incubation, non-invading cells were removed using a cotton

swab. Filters were excised and incubated in acetone and invading cells were

subsequently visualized upon staining with hematoxylin and eosin.

Effect of hCG on endothelial cell proliferation

5 x 105 HBMEC cells per well were dispensed in a 96-well plate and an incubation

carried out for 16 hrs at 37°C in a 5% CO2 incubator. hCG (1 µg), anti-hCG antiserum

(1:100), non-immune serum (1:100), hCG + anti-hCG antiserum or hCG + non-

immune serum were individually dispensed and a further incubation carried out for 24

hrs. Cells were then “washed” with PBS by repeated centrifugation at 400 g for 5 min

at 4oC. 80 µl of a 5 mg/ml solution of an MTT reagent (3-[4, 5-dimethylthiazol-2-yl]-

2, 5-diphenyltetrazolium bromide; Sigma) was then added, followed by an incubation

for 5 hrs. 50 µl of the Stop Solution (50% DMSO in 20% SDS) was dispensed and

incubation carried out for 1 hr at RT. Cell proliferation/viability was determined

colorimetrically by measuring optical density at 550 nm. A Trypan Blue dye exclusion

assay was employed as an additional assessment of cell death.

Inhibitor analysis

5 x 105

cells were incubated with LY294002 (10 μmol/L; a PI3K pathway inhibitor),

or H89 (10 μmol/L; [2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide

dihydrochloride (H89), a PKA pathway inhibitor) or PD980049 (5µmol/L - (2-Amino-

3-mathoxyphenyl)-4H-1-benzopyran-4-one, a MAPKK pathway inhibitor) or a JNK2

inhibitor (10µmol/L) for 120 minutes. hCG (1µg) was then added and a further

incubation carried out at 37°C for 24 hrs. Supernatants were analysed for the presence

of IL-8 and VEGF by ELISA.



Migration of PBMCs towards cancer cells

Since hCG was previously shown to be capable of inducing the transmigration of

specific subsets of PBMCs, and since experiments reported earlier demonstrated the

reactivity of anti-hCG antibodies towards tumor cells, studies were carried out to

evaluate the capacity of COLO 205 cells to induce the migration of PBMCs. 2 x 106

COLO 205 cells were seeded in the lower chamber and 2 x 105 PBMC in the upper

chamber. As before, flow cytometry (using lineage-specific monoclonal antibodies to

human T, B and monocyte CD markers) was employed to assess the extent of

migration to the lower chamber after an incubation of 12 hr.

Results


Effect of hCG on IL-8, VEGF

Reverse transcriptase-PCR was carried out to assess the effects of hCG on VEGF and

IL-8 transcript levels. Analysis revealed that hCG was capable of inducing the

synthesis of VEGF and IL-8 mRNA from COLO 205, ChaGo and LLC cells (Figure

10A, B); secretion of the two molecules into the culture medium was also enhanced

(Figures 11, 12). Anti-hCG antibodies displayed a significant inhibitory influence on

these hCG-mediated effects; transcription and secretion were both down-modulated

by anti-hCG antibodies, whereas non-immune serum did not induce such decreases

(Figures 10-12).

Effect of hCG on the proliferation and transmigration of

endothelial cells

hCG enhanced cell growth/viability of human endothelial cells in a dose-dependent

manner in vitro (Figure 13A). Anti-hCG serum significantly inhibited this growth-

promoting effect, whereas normal serum had no effect (Figure 13B). Transmigration

assays were carried out to investigate if, in addition to have growth promoting

properties, hCG could also act as a chemotactic agent for endothelial cells. Indeed,

hCG was capable of inducing significantly enhanced migration over serum-free

medium not supplemented with the hormone. The enhanced migration was

significantly inhibited by anti-hCG antibodies, but not by non-immune serum (Figure

13C).

Effect of hCG on MMP-2, MMP-9 expression and on invasion

Reverse transcriptase-PCR analysis was employed to assess the effects of hCG on

MMP-2 and MMP-9 mRNA synthesis in COLO 205, ChaGo and LLC cells. hCG

treatment up-modulated mRNA expression of both molecules; respective PCR

products were detected at the expected sizes ( 600 bp for MMP-2 and 240 for

MMP-9). Anti-hCG antiserum inhibited hCG-induced increase in MMP transcription,

whereas non-immune serum has no effect (Figure 14A).

Zymogram analysis supported these findings. Gelatinase activity in cell supernatants

from all three cell lines was detected at 92 kDa (corresponding to the active form of

MMP-9) as well as at 72 kDa (corresponding to the active form of MMP-2). hCG up-

modulated MMP-2 and MMP-9 activity; in COLO 205 cells, the pro-form of MMP-9

Results


was also observed, particularly in cells stimulated with hCG. Whereas anti-hCG

antiserum inhibited the hCG-induced increase in MMP-2 and MMP-9 activity, non-

immune serum had no effect (Figure 14B).

As MMP-2 and MMP-9 appeared to be up-modulated by hCG, an in vitro invasion

assay was employed to assess whether the released enzymes were capable of causing

the destruction of model extracellular matrix proteins. hCG induced a significant

increase in the invasion of COLO 205, ChaGo and LLC tumor cells into a Matrigel

substrate. In all instances, invasion was almost completely inhibited when anti-hCG

antiserum was employed, an effect not seen when non-immune serum was used

(Figure 15).

Delineation of the biochemical pathways involved in the induction

of IL-8 and VEGF by hCG

To assess the potential signalling events involved in the angiogenic and chemotactic

events mediated by hCG, the effects of specific signalling inhibitors were examined.

In COLO 205, ChaGo and LLC, PD98059 (an ERK-1/ERK- 2 kinase inhibitor),

markedly blocked hCG-induced IL-8 secretion, whereas H89 (a PKA inhibitor),

JNK1/2 (JNK inhibitor) and LY294002 (a PI3K inhibitor) did not (Figure 16).

PD98059 inhibited hCG-induced VEGF secretion from COLO 205 and ChaGo cells;

H89 effectively inhibited VEGF secretion from COLO 205 cells but were less

efficient in ChaGo cells. Interestingly, LY294002 significantly inhibit VEGF

secretion only in LLC cells (Figure 17). These results indicate that hCG may act via

the MAPK pathway to increase IL-8 secretion from COLO 205, ChaGo and LLC

cells. VEGF secretion from COLO 205 and ChaGo may be mediated by MAPK and

PKA and in LLC via the PI3K signalling pathway.

Assessment of the role of hCG as a chemotactic factor for PBMC

Experiments described above suggest that hCG can act as a growth promoter and

chemo attractant for endothelial cells, findings of direct relevance to tumor

progression. Given that non-transformed cells (particularly monocytes and fibroblasts)

have been shown to aid in the process of tumorogenesis, studies were then carried out

to assess whether PBMC too be induced to migrate under the influence of the

Results


hormone. A time-dependent increase in the number of migrating cells was observed

(Figure 18A).

JEG-3 cells are believed to exclusively secrete a hCG-H. hCG-H is considered critical

to the process of implantation and its role in tumorogenesis is suspected. The

transmigration of PBMC was assessed towards both JEG-3 supernatant (containing

hCG-H) and hCG (the latter diluted to the same concentration as hCG-H, 40ng/ml).

Migration of PBMC towards JEG-3 supernatant was markedly greater than towards

hCG. Anti-hCG antiserum (but not normal serum) decreased migration to background

levels in both instances, confirming that cellular movement was indeed hCG-induced.

Another indication of specificity was obtained by the observation that a combined

preparation of LH and FSH was unable to induce migration (Figure 18B).

Interestingly, supernatants from cell lines reactive towards anti-hCG antibodies

(ChaGO, COLO 205, LL2) were also able to induce the migration of PBMC, albeit

not to the same extent as supernatant from JEG-3 cells (Figure 18C); supernatant from

the cell line HepG2 (which was non-reactive to anti-hCG antibodies) was not. These

studies reveal that hCG, and particularly hCG-H, can act as a chemo attractant for

PBMC.

Migration of immune cells towards COLO 205 cells

The ability of COLO 205 cells (added to the lower chamber) to induce the migration

of PBMC (added to the upper chamber) was then assessed.

Various lineage-specific antibodies (to human CD3, CD4, CD8, CD14 and CD19

antigens) were employed to quantify migration; the reactivity of these antibodies

towards PBMC (Figure 19A) served as an assay control. Further, lack of the ability of

the antibodies to recognize COLO 205 was obviously crucial to the proper

interpretation of results, and was therefore verified; Figure 19B demonstrates that the

antibodies were incapable of binding COLO 205 cells.

After allowing for transmigration of PBMC, the lineage of cells remaining in the

upper chamber and those moving to the lower chamber was assessed by flow

cytometry. Results revealed that T cells and monocytes preferentially migrated to the

Results


lower chamber (containing COLO 205 cells), whereas B cells appeared to remain in

the upper chamber (Figure 19C, D).

Figure 10: The effect of hCG on (A) VEGF and (B) IL8 transcript levels in COLO205, ChaGO andLLC cells as determined by reverse transciptase-PCR analysis. The effects of concurrentincubation of hCG with anti-hCG serum or normal serum (NS) are also shown. Transcriptlevels for -actin served as control.

β-actin

VEGF

B

IL-8

β-actin

COLO 205

ChaGo

LLC

ChaGo

COLO 205

COLO 205

ChaGo

LLC

COLO 205

ChaGo

LLCLLC

- + + +

- + - -

- - - +

- + + +

- + - -

- - - +

A

hCGAnti-hCGNS

hCGAnti-hCGNS

Figure 11: The effect of hCG on the secretion of VEGF from (A) COLO205, (B) ChaGO and (C)LLC cells. The effects of concurrent incubation of hCG with anti-hCG serum or normal serum(NS) are also shown.

0

200

400

600

800

1000

0

500

1000

1500

2000

0

500

1000

1500

2000

hCGAnti-hCGNS

hCGAnti-hCGNS

hCGAnti-hCGNS

BA

C

- + + +

- - + -

- - - +

- + + +

- - + -

- - - +

- + + +

- - + -

- - - +

pg

/ml

pg

/ml

pg

/ml

0

500

1000

1500

2000

0

200

400

600

800

0

400

800

1200

1600

0

500

1000

1500

2000

2500

pg/

ml

pg/

ml

pg/

ml

pg/

ml

Figure 12: The effect of hCG on the secretion of IL8 from (A) HBMEC, (B) COLO205, (C) ChaGOand (D) LLC cells. The effects of concurrent incubation of hCG with anti-hCG serum or normalserum (NS) are also shown.

hCG

Anti-hCGNS

- + + +

- - + -

- - - +

hCG

Anti-hCGNS

- + + +

- - + -

- - - +

BA

C D

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7 *

Figure 13: (A) Influence of hCG on the viability/proliferation of HBMEC. (B) Influence of anti-hCG serum and normalserum (NS) on enhancement of cell viability/proliferation of HBMEC induced by 1 g/ml hCG. (p = 0.002 hCG vshCG+anti-hCG, 0.009 control vs hCG). (C) Effect of hCG on the transmigration of HBMEC. The influence of theconcurrent presence of anti-hCG serum and normal serum (NS) are also shown.

Ab

sorb

ance

(5

50

nm

)

Co

ntr

ol

hC

G+

An

ti-h

CG

hC

G+

NS

hC

G

BA

Mig

rati

ng

ce

lls

(x

10

- 4)

0

2

4

6

8

hCG

Anti-hCG

NS

- + + +

- - + -

- - - +

C hCG (ng)

0 200 400 600 800 1000 1200

Ab

so

rba

nc

e 5

50

nm

0.0

0.2

0.4

0.6

Figure 14: Influence of hCG on matrix metalloproteases. (A) RT-PCR analysis of hCG-induced effects onMMP-2 and MMP-9 transcripts from COLO205, ChaGO and LLC cells. -actin was employed as control. (B)Zymogram analysis of hCG-induced secretion of MMP-2 and MMP-9. In both assays, the effect of theconcurrent presence of anti-hCG serum or normal serum (NS) was also assessed.

1 2 3 4

MMP 9

MMP 2

MMP 9MMP 2

MMP 9MMP 2

COLO 205

ChaGo

LLC

hCG + + + -Anti-hCG - - + -NS + - - -

hCG - + + +

Anti-hCG - + - -

NS - - - +

MMP-9

COLO 205

ChaGo

LLC

MMP-2

COLO 205

ChaGo

LLC

COLO 205

ChaGo

LLC

β-actin

A B

COLO 205

ChaGo

LLC

Control hCG

hCG +

Anti-hCG hCG + NS

Figure 15: hCG-induced in vitro (Matrigel) invasion. The influence of anti-hCG antiserum andnormal serum (NS) on hCG-induced effects is also shown.

Figure 16: Analysis of the signaling pathways involved in hCG-induced IL8 secretion by (A)COLO 205, (B) ChaGo and (C) LLC cells. Secretion was assessed in the presence and absence ofvarious inhibitors as indicated.

Control

hCG

Ly294002+hCG

Ly294002

JNK 2+hCGJNK 2

PD98059+hCG

PD98059

H89+hCGH-89

0

500

1000

1500

2000

Control

hCG

Ly294002+hCG

Ly294002

JNK2+hCGJNK2

PD98059+hCG

PD98059

H89+hCG H890

1000

2000

3000

4000

5000

Control

hCG

Ly294002+hCG

Ly294002

JNK I2+hCGJNK 2

PD98059+hCG

PD98059

H89+hCG H890

500

1000

1500

2000

2500

A B

C

pg/

ml

pg/

ml

pg/

ml

Figure 17: Analysis of the signaling pathways involved in hCG-induced VEGF secretion by (A)COLO 205, (B) ChaGo and (C) LLC cells. Secretion was assessed in the presence and absence ofvarious inhibitors as indicated.

Control

hCG

Ly294002+hCG

Ly294002

JNK I2+hCGJNK2

PD98059+hCG

PD98059

H89+hCG H890

1000

2000

3000

4000

5000

Control

hCG

Ly294002+hCG

Ly294002

JNKI2+hCGJNK I2

PD98059+hCG

PD98059

H89+hCG0

2000

4000

6000

8000

10000

12000

14000

Control

hCG

Ly294002+hCG

Ly294002

JNK I2+hCGJNK I2

PD98059+hCG

PD98059

H89+hCG H890

2000

4000

6000

8000

10000

12000

14000

pg/

ml

pg/

ml

pg/

ml

A B

C

Figure 18: (A) Time-dependent chemotaxis of PBMC induced by hCG (1g/ml). (B) Chemotaxis of PBMC byJEG-3 supernatant (containing hCG-H), hCG and a preparation of LH+FSH. The influence of anti-hCGantibodies and normal serum (NS) on migration is also depicted. (C) Induction of transmigration of PBMCby supernatant of anti-hCG antibody-reactive and non-reactive cell lines.

Control

Medium ChaGo

COLO 205LL2

JEG-3

HEP-G20

5

10

15

20

25

30

Medium 1

Medium 2

JEG 3 sup

JEG 3 sup + anti-hCG

JEG 3 sup + NS

hCG

hCG + anti-hCG

hCG + NS

LH + FSH

LH + FSH + anti-hCG

0

5

10

15

20

25

30

0

5

10

15

20

25

30

Hours

Mig

rati

ng

ce

lls

(x1

0-4

)

6 12 24 48

Mig

rati

ng

ce

lls

(x1

0-4

)

Mig

rati

ng

ce

lls

(x

10

-4)

A B

C

Figure 19A: Flow cytometric analysis of PBMC. Reactivity towards antibodies to (ii) CD 3; (iii)CD 4; (iv) CD 8; (v) CD 14 and (vi) CD 19 are shown. (i) represents the negative control. Figuresabove the marker indicate the percentage of stained cells.

15.811.615.2

11.217.9

(i) (ii) (iii)

(iv) (v) (vi)

(i) (ii) (iii)

(iv) (v) (vi)

Figure 19B: Flow cytometric analysis of COLO 205 cells. Reactivity towards antibodies to(ii) CD 3; (iii) CD 4; (iv) CD 8; (v) CD 14 and (vi) CD 19 are shown. (i) represents the negativecontrol.

6.4

(i) (ii) (iii)

(iv) (v)

Figure 19C: Flow cytometric analysis of cells remaining in the upper chamber after migrationof PBMC towards COLO 205 cells. Reactivity towards antibodies to (ii) CD 3; (iii) CD 4; (iv) CD 8;(v) CD 14 and (vi) CD 19 are shown. (i) represents the negative control. Figures above themarker indicate the percentage of stained cells.

15.3

2.4

16.7

4.5

(i) (ii) (iii)

(iv) (v)

Figure 19D: Flow cytometric analysis of cells in the lower chamber after migration of PBMCtowards COLO 205 cells. Reactivity towards antibodies to (ii) CD 3; (iii) CD 4; (iv) CD 8; (v) CD 14and (vi) CD 19 are shown. (i) represents the negative control. Figures above the markerindicate the percentage of stained cells.

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