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
Role of hCG in chemo attraction, angiogenesis and invasion 40
Figure 3.1
Figure 3.1: Oxygen concentration gradient in tissues.
Introduction
Role of hCG in chemo attraction, angiogenesis and invasion 41
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
Role of hCG in chemo attraction, angiogenesis and invasion 42
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.
Introduction
Role of hCG in chemo attraction, angiogenesis and invasion 43
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
Role of hCG in chemo attraction, angiogenesis and invasion 44
Factor Role in Tumor Neovascularisation
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
Role of hCG in chemo attraction, angiogenesis and invasion 45
Factor Role in Tumor Neovascularisation
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
Introduction
Role of hCG in chemo attraction, angiogenesis and invasion 46
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
Role of hCG in chemo attraction, angiogenesis and invasion 47
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
Role of hCG in chemo attraction, angiogenesis and invasion 48
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
Role of hCG in chemo attraction, angiogenesis and invasion 49
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
Role of hCG in chemo attraction, angiogenesis and invasion 50
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.
Materials and methods
Role of hCG in chemo attraction, angiogenesis and invasion 51
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, Extension at 72
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’
Materials and methods
Role of hCG in chemo attraction, angiogenesis and invasion 52
Reverse Transcription: 500C for 30 min followed by a hold 95
0C for 15 min.
Denaturation at 940C for 1 min, Annealing at 60
0C for 1 min, Extension at 72
0C for 1
min, Final Extension at 720C for 10 min. Number of cycles: 35.
-actin (human): Forward 5’-AGATGACCCAGATCATGTTTGAGA-3’
Reverse 5’-CTAAGTCATAGTCCGCCTAGAAGC-3’
-actin (murine): Forward 5’- ATCCGTAAAGACCTCTATGC-3’
Reverse 5’- AACGCAGCTCAGTAACAGTC-3’
Reverse Transcription: 500C for 30 min followed by a hold 95
0C for 15 min.
Denaturation at 940C for 1 min, Annealing at 60
0C for 1 min, Extension at 72
0C for 1
min, Final Extension at 720C for 10 min. Number of cycles: 35.
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.
Materials and methods
Role of hCG in chemo attraction, angiogenesis and invasion 53
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
Materials and methods
Role of hCG in chemo attraction, angiogenesis and invasion 54
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.
Materials and methods
Role of hCG in chemo attraction, angiogenesis and invasion 55
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
Role of hCG in chemo attraction, angiogenesis and invasion 56
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
Role of hCG in chemo attraction, angiogenesis and invasion 57
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
Role of hCG in chemo attraction, angiogenesis and invasion 58
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
Role of hCG in chemo attraction, angiogenesis and invasion 59
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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